JP4171228B2 - Soluble type RAGE measurement method - Google Patents

Description

システム (Amersham Pharmacia) を用い、溶出液は230 nm, 280 nmおよび300 nmにおける吸光度を測定してモニターした。
【０１０５】
〔精製組換え可溶型RAGE蛋白とAGE との結合試験〕
可溶型RAGEのAGE 結合能を表面プラズモン共鳴法によりBIACORE(Biacore, Sweden)を用いて確認した。Sensor chip CM5 (Biacore) にアミノカップリング法を用いて精製可溶型RAGE蛋白を固定化した。グルコースとウシ血清アルブミン(BSA) を無菌的に37℃で12週間インキュベートして調製したAGE-BSA をBIACORE のマイクロ流路系にアナライトとして500 μg/mlの濃度で添加した。その結果、インラインレファランス（コントロールチップ）やイムノグロブリンを固定したチップには全く反応せず、可溶型RAGEを固定したセンサーチップとのみ特異的な結合を示した。
【０１０６】
実施例２：モノクローナル抗体の作成
(a) 免疫源
免疫に用いる抗原は、組換え可溶型RAGEをはじめ、配列表の配列番号:2のアミノ酸配列を基にデザインされた合成ペプチドを使用することができる。さらに、免疫に用いる抗原は、得られたcDNAを動物細胞発現ベクターに連結し、これをCHO 細胞、COS 細胞などで発現させて得られる組換え可溶型RAGEを使用することも可能である。これらの抗原タンパク質は、イオン交換、ゲル濾過またはそれ以外の各種クロマトグラフィーによって精製できる。
精製した免疫用抗原を一般的な方法で免疫し、抗体産生細胞を誘導、細胞融合によりハイブリドーマとして抗体産生細胞を得ることができる。さらに精製した免疫用抗原に対する反応性に基づいてクローニング、モノクローナル抗体産生ハイブリドーマとして株化できる。
また、可溶型RAGE特異的モノクローナル抗体を得るための免疫源としては、可溶型RAGEに特徴的なアミノ酸配列を持つハプテン化合成ペプチドが使用できる。例えば、C 末端にはGlu-Gly-Phe-Asp-Lys-Val-Arg-Glu-Ala-Glu-Asp-Ser-Pro-Gln-His-Met から選択された連続した少なくとも３個以上、好ましくは５個以上の連続したペプチド配列を含むことが特に好ましい。具体的にはCys-Glu-Gly-Phe-Asp-Lys-Val-Arg-Glu-Ala-Glu-Asp-Ser-Pro-Gln-His-Met (CEGFDKVREAEDSPQHM) などが免疫源として好適である。
【０１０７】
(b) 抗原ポリペプチドの調製
配列番号:2に記載したヒト可溶型RAGEのアミノ酸配列中より特徴的な配列を選択し、合成する。ペプチドはペプチド合成機（ペプチドシンセサイザー9600、MilliGen/Biosearch) を使用して、Fmoc-bop法で合成する。ポリペプチドのN 末端にはシステインを導入する。合成したペプチドはμBondasphere, C18カラム（Waters) を用いた高速液体クロマトグラフィーなどにより精製する。Cys-Glu-Gly-Phe-Asp-Lys-Val-Arg-Glu-Ala-Glu-Asp-Ser-Pro-Gln-His-Met 〔配列番号：７〕を合成し、以下の実験で使用した。
(c) ポリペプチドとBSA の複合体の調製
システイン残基を介してウシ血清アルブミン（BSA)と結合させ、抗原コンジュゲートとした。10.1mgのBSA を1mL の0.1Mリン酸緩衝液、pH7.0 に溶解したものと1.14mgのEMCS(N-(ε-maleimidecaproyloxy)-succinimide)を24.9μL のジメチルホルムアミドに溶解したものと混合し、30℃、30分間反応させ、ついで、上記の混合液を0.1Mリン酸緩衝液、pH7.0 で平衡化したSephadex G-25 （Pharmacia)ゲルカラム( 直径13mm、長さ120mm)でゲルろ過する。前記（b)で合成したポリペプチドを0.1Mリン酸緩衝液、pH7.0 に溶解し、マレイミド結合BSA に対しおおよそ50倍モル量を混合する。すなわち、ポリペプチドに対しマレイミド結合BSA を混合し、4 ℃、20時間インキュベートし、BSA-ポリペプチド複合体を調製する。得られるBSA-ポリペプチド複合体を0.1Mリン酸緩衝液、pH7.0 で希釈した後、150 μL ずつに分注して-30 ℃で凍結保存する。
【０１０８】
(d) 抗体産生細胞の調製
前記(c) で調製したBSA-ポリペプチド複合体を完全フロインドアジュバントと共に6 週令Balb/c雌マウスに腹腔内投与し、初回免疫した。おおよそ18日目に0.1Mリン酸緩衝液、pH7.5 に溶解したBSA-ポリペプチド複合体を初回免疫したマウスに腹腔内投与し、追加免疫する。さらにおおよそ52日目に0.1Mリン酸緩衝液、pH7.5 に溶解したBSA-ポリペプチド複合体を静脈内投与し、最終免疫とする。その4 日後に脾臓を摘出し、脾細胞懸濁液を調製する。
(e) 細胞融合
細胞融合には、以下の材料および方法を用いる。RPMI-1640 培地：RPMI-1640 （Flow Lab.)に重炭酸ナトリウム（24mM) 、ピルビン酸ナトリウム（1mM)、ペニシリンG カリウム（50U/ml) 、硫酸アミカシン（100 μg/ml) を加え、ドライアイスでpHを7.2 にし、0.2 μm 東洋メンブレンフィルターで除菌ろ過する。NS-1培地：上記RPMI-1640 培地に除菌ろ過したウシ胎児血清（FCS, M.A. Bioproducts)を15％（v/v)の濃度になるように加える。PEG-4000溶液：RPMI-1640 培地にポリエチレングリコール-4000 （PEG-4000, Merk＆Co.)を50％（w/w)になるように加えた無血清培地を調製する。
8-アザグアニン耐性ミエローマ細胞SP2 （SP2/0-Ag14) との融合は、Selected Method in culture immunology p351〜372 (ed. B. B. Mishell and S. N. Shiigi), W. H. Freeman and Company (1980)に記載のOiらの方法を若干改変して行う。
【０１０９】
前記(d) で調製した有核脾細胞（生細胞率100 ％) とミエローマ細胞（生細胞率100 ％) とをおおよそ5:1 〜10:1の比率で以下の手順で融合する。ポリペプチド免疫脾細胞懸濁液とミエローマ細胞をそれぞれRPMI1640培地で洗浄する。次に同じ培地に懸濁し、融合させるために有核脾細胞とミエローマ細胞を混合する。すなわち、おおよそ4.0 ×10⁸ 個の有核脾細胞に対しおおよそ8.0 ×10⁷ 個のミエローマ細胞を混合する。
次に、それぞれの細胞混合液を遠心分離により細胞を沈殿させ、上清を完全に吸引除去する。沈殿した細胞に37℃に加温した50%PEG-4000 含有RPMI-1640 培地（ミエローマ細胞がおおよそ3 ×10⁷ 個/mL となるよう体積を決定する) を滴下し、撹拌し、細胞を再懸濁、分散させる。次に添加した50%PEG-4000 含有RPMI-1640 培地の2 倍体積の37℃に加温したRPMI-1640 培地を滴下する。さらに添加した50%PEG-4000 含有RPMI-1640 培地の7 倍体積のRPMI-1640 培地を常に撹拌しながら滴下し、細胞を分散させる。これを遠心分離し、上清を完全に吸引除去する。次に、ミエローマ細胞がおおよそ3 ×10⁶ 個/mL となるように37℃に加温したNS-1培地を沈殿した細胞に速やかに加え、大きい細胞塊を注意深くピペッティングで分散する。さらに同培地を加えて希釈し、ポリスチレン製96穴マイクロウエルにウエル当りミエローマ細胞がおおよそ6.0 ×10⁵ 個となるように接種する。それぞれの細胞を加えた上記マイクロウエルを7 ％炭酸ガス/93 ％空気中で温度37℃、湿度100 ％で培養する。
【０１１０】
(f) 選択培地によるハイブリドーマの選択的増殖
(1)使用した培地は以下の通りである。
HAT 培地：前記(e) 項で述べたNS-1培地に更にヒポキサンチン（100 μM)、アミノプテリン（0.4 μM)およびチミジン（16μM)を加える。
HT培地：アミノプテリンを除去した以外は上記HAT 培地と同一組成のものである。
(2)前記(e) 項の培養開始後翌日（1 日目) 、細胞にパスツールピペットでHAT 培地2 滴（約0.1 ml) を加える。2 、3 、5 、8 日目に培地の半分（約0.1 ml) を新しいHAT 培地で置き換え、10日目に培地の半分を新しいHT培地で置き換える。ハイブリドーマの生育が肉眼にて認められた全ウエルについて固相- 抗体結合テスト法（ELISA)により陽性ウエルを調べる。まず、20mM炭酸緩衝液(pH9.6) で希釈した抗原ポリペプチドでポリスチレン製96穴プレートをコート(100ng/ ウエル) し、次に0.05％ Tween20含有PBS を用いて洗浄して未吸着のペプチドを除く。各ウエルにハイブリドーマの生育が確認されたウエルの培養上清0.1 mlを添加し、室温で約1 時間静置する。洗浄後、2 次抗体として西洋わさびペルオキシダーゼ（HRP)標識ヤギ抗マウス免疫グロブリン（Cappel) を加え、さらに室温で約1 時間静置する。洗浄後、基質である過酸化水素と3,3',5,5'-テトラメチルベンジジン(TMB) を加え発色させる。各ウエルに2N硫酸を加え発色反応を停止し、発色の程度をマイクロプレート用吸光度測定機（MRP-A4、東ソー) を用いて450nm の吸光度で測定する。
【０１１１】
(g) ハイブリドーマのクローニング
上記(f) 項で得られた抗原ペプチドに対する陽性ウエル中のハイブリドーマを、限界希釈法を用いてモノクローン化する。すなわち、NS-1培地1 ml当りフィーダーとしておおよそ10⁷ 個のマウス胸腺細胞を含むクローニング培地を調製し、96穴マイクロウエルにハイブリドーマをウエル当り5 個、1 個、0.5 個になるように希釈し、それぞれ36穴、36穴、24穴に加える。5 日目、12日目に全ウエルに約0.1 mlのNS-1培地を追加する。クローニング開始後肉眼的に十分なハイブリドーマの生育を認めたもの、コロニー形成陰性ウエルが50％以上である群について（f)項に記載したELISA を行う。調べた全ウエルが陽性でない場合、抗体陽性ウエル中のコロニー数が1 個のウエルを4 〜6 個選択し、再クローニングを行う。最終的にそれぞれのポリペプチドに対するモノクローナル抗体を産生するハイブリドーマを得る。モノクローナル抗体４種類を得た。
(h) モノクローナル抗体のクラス、サブクラスの決定
前述したELISA に従い、それぞれのポリペプチドをコートしたポリスチレン製96穴プレートに、前記(g) 項で得られたハイブリドーマの上清を加える。次にPBS で洗浄後、アイソタイプ特異的ウサギ抗マウスIgG 抗体（Zymed Lab.) を加える。PBS により洗浄後、西洋わさびペルオキシダーゼ標識ヤギ抗ウサギIgG （H ＋L)を加え、基質として過酸化水素および2,2'- アジノ- ジ（3-エチルベンゾチアゾリン酸) を用いてクラス、サブクラスを決定する。
【０１１２】
【表１】

【０１１３】
(i) ハイブリドーマの培養とモノクローナル抗体の精製
得られたハイブリドーマ細胞をNS-1培地で培養し、その上清からモノクローナル抗体を得ることができる。また、得られたハイブリドーマ10⁷ 個を予め1 週間前にプリスタンを腹腔内投与したマウス（Balb/c系、雌、6 週齢) に同じく腹腔内投与し、1 〜2 週間後、腹水中からも4 〜7mg/mlのモノクローナル抗体を含む腹水を得ることができる。得られた腹水を40％飽和硫酸アンモニウムで塩析後、IgG クラスの抗体をプロテインA アフィゲル（Bio-Rad)に吸着させ、0.1Mクエン酸緩衝液、pH5.0 で溶出することにより精製する。
【０１１４】
実施例３：ウエスタンブロッティング
ヒト可溶型RAGEを発現するCOS7細胞の培養上清9 μl 、精製組換えヒト全長型成熟RAGE（膜型RAGE) 750 ngおよびヒト肺抽出蛋白150 ngを還元条件下、10% SDS-PAGEで分離後、polyvinylidene difluoride (PVDF)膜（MILLIPORE ）に転写した。続いて1% BSA，5% スキムミルクおよび0.1% Tween 20 含有PBS （ブロッキングバッファー）を用いて室温で１時間ブロッキングした後、ヒト可溶型RAGE特異抗体を発現するハイブリドーマ細胞株269-1D10, 269-4C9, 269-6B12 あるいは269-9C2 の培養上清中、25℃で1 時間インキュベートした。それぞれの膜を0.1% Tween 20 含有PBS （0.1%PBS-T)で4 回洗浄し、結合した抗体をブロッキングバッファーで1:1,000 に希釈したHRP 結合ヒツジ抗マウスイムノグロブリン抗体(Amersham Pharmacia)と25℃、1 時間反応させた。反応後、膜を0.1% PBS-Tで4 回洗浄し、結合した抗体をEnhanced chemiluminescence (ECL, Amersham Pharmacia)により検出した。また陽性コントロールは、ヒト可溶型RAGE蛋白発現COS7細胞培養上清、ヒト組換え全長型成熟RAGE蛋白およびヒト肺抽出蛋白をそれぞれ3 μl 、250ng および50ng用い、ブロッキングバッファーで1:500 に希釈したウサギ抗ヒトRAGE抗血清およびブロッキングバッファーで1:2,000 に希釈したHRP 結合ヤギ抗ウサギIgG(H+L)抗体 (Zymed)により同様に処理し、ECL で検出した。結果を図７に示す。
M ：分子量マーカー
1, 4, 7, 10, 16:組換え膜型RAGE
2, 5, 8, 11, 17:組換え可溶型RAGE
3, 6, 9, 12, 18:ヒト肺抽質物（膜型RAGEを多く含む)
結果
各モノクローナル抗体は組換え膜型RAGEおよびヒト肺抽質物中膜型RAGEとは交差反応せず、可溶型RAGEと反応した。
【０１１５】
実施例４：サンドイッチEIA
下記の方法に従えば、実施例２で調製した抗可溶型RAGE抗体から少なくとも１種を選択し、抗RAGE抗体の2 種の適当な組み合わせによってヒト可溶型RAGEを特異的に検出・測定するサンドイッチEIA 系が構成できる。EIA 系は1 ステップ法、2 ステップ法のいずれも可能であり、標識抗体はFab'-HRPに限定されない。各反応緩衝液の組成や反応条件は測定の目的に応じて、短縮、延長など調整できる。また、標準品となるヒト可溶型RAGEは、組織培養上清、細胞培養上清または実施例２記載あるいはそれ以外の方法で発現した組換え体から精製することができる。精製にはイオン交換、ゲルろ過、抗ヒト可溶型RAGEモノクローナル抗体を用いたアフィニティークロマトグラフィーまたそれ以外の各種アフィニティークロマトグラフィーの組み合わせによって達成される。
(a) 標識抗体の調製
抗ヒト可溶型RAGEモノクローナル抗体を0.1M NaCl を含む0.1M酢酸緩衝液、pH4.2 に抗体量の2%(W/W) のペプシンを加え、37℃、24時間消化する。消化物に3M Tris-HCl 、pH7.5 を添加し、反応を停止する。0.1Mリン酸緩衝液、pH7.0 で平衡化したウルトロゲルAcA54 カラムによるゲルろ過でF(ab')₂ 画分を分取する。このF(ab')₂ 画分に最終濃度0.01M となるようにシステアミン塩酸塩を添加し、37℃、1.5 時間還元し、5mM EDTA含有0.1Mリン酸緩衝液、pH6.0 で平衡化したウルトロゲルAcA54 カラムによるゲルろ過でFab'画分を分取する。
上記の操作とは別にHRP を0.1Mリン酸緩衝液、pH7.0 に溶解、HRP の25倍モル量のEMCSをDMF 溶液として加え、30℃、30分間反応させる。これを0.1Mリン酸緩衝液、pH6.0 で平衡化したNICK-5カラム（Pharmacia)でゲルろ過しマレイミド標識HRP 画分を分取する。
Fab'画分とマレイミド標識HRP を等モルとなるように両画分を混合し 4℃、20時間反応させた後、Fab'の10倍モル量のN-エチルマレイミドで未反応のチオール基をブロックする。これを0.1Mリン酸緩衝液、pH6.5 で平衡化したウルトロゲルAcA54 カラムでゲルろ過し、Fab'-HRP標識抗体を分取する。これに0.1% BSAおよび0.001%クロルヘキシジンを添加して 4℃で保存する。
【０１１６】
(b) モノクローナル抗体結合担体の調製
抗ヒト可溶型RAGEモノクローナル抗体を0.1Mリン酸緩衝液、pH7.5 に溶解し、50μg/mLの濃度に調製する。このモノクローナル抗体溶液を96穴マイクロプレートにウエルあたり100 μL ずつ加え、 4℃、18時間静置する。モノクローナル抗体溶液を除去し、生理食塩液で1 回、0.05% Tween20 、0.1M NaCl 、5mM CaCl₂ 含有Tris-HCl緩衝液、pH8.0 で３回洗浄後、1% BSA、0.1M NaCl 、5mM CaCl₂ 含有Tris-HCl緩衝液、pH8.0 を加えブロッキングする。
（c)１ステップサンドイッチEIA 法
精製したヒト可溶型RAGE画分を標準抗原としてヒト可溶型RAGE定量用標準曲線を作成する。1% BSA、0.05% Brij35、0.05% Tween20 、0.1M NaCl 、5mM CaCl₂ 含有Tris-HCl緩衝液、pH8.0 で段階希釈した標準ヒト可溶型RAGEを60μL ずつ分注、それぞれに1% BSA、0.05% Brij35、0.05% Tween 20、0.1M NaCl 、5mM CaCl₂ 含有Tris-HCl緩衝液、pH8.0 で100ng/50μL に調製した標識抗体Fab'-HRPを60μL ずつ添加し十分混和する。調製した抗体結合マイクロプレートを0.05% Tween20 、0.1M NaCl 、5mM CaCl₂ 含有Tris-HCl緩衝液、pH8.0 で３回洗浄し、標準抗原と標識抗体混合液を100 μL/ウエルずつ添加する。室温で1 時間反応した後0.05% Tween20 、0.1M NaCl 、5mM CaCl₂ 含有Tris-HCl緩衝液、pH8.0 で３回洗浄する。次に、6%ジメチルホルムアミド、0.005%過酸化水素含有0.1 M 酢酸緩衝液（pH5.5)に溶解した0.01% 3,3',5,5'-テトラメチルベンジジンをウエルあたり100 μL 添加し、室温で20分間反応後、2N硫酸を100 μL 添加し反応を停止する。この反応混液の450 nmをマイクロプレートリーダーを用いて測定し、標準曲線を求める。
測定検体は、ヒト血清、脊髄液、血漿、関節液、尿および唾液などヒトに由来する体液成分、各種ヒト組織の抽出液、ヒト由来あるいは組換え体など各種培養細胞の細胞抽出液、培養上清などから調製される。それぞれの測定検体は、標準ヒト可溶型RAGEにかえて上記の1 ステップサンドイッチEIA に供し、標準ヒト可溶型RAGEと同時に反応を進行させる。測定検体から得られた吸光度を標準曲線にあてはめ、測定検体に含まれるヒト可溶型RAGEの量を算出する。
【０１１７】
実施例５：ヒト可溶型RAGEモノクローナル抗体の検定
ウエスターン ブロッティング (Western blotting)
ヒト成熟型RAGEタンパク過剰発現GEN-T 細胞抽出物（750 ngタンパク相当) 、ヒト可溶型RAGEタンパク過剰発現COS 7 細胞の培養上清 3μl 、ヒト肺抽出物（150 ngタンパク相当) およびRainbow Marker (ミオシン(myosin), フォスフォリラーゼ b (phosphorylase b), ウシ血清アルブミン(bovine serum albumin), 卵白アルブミン(ovalbumin), カルボニック アンヒドラーゼ(carbonic anhydrase), トリプシン インヒビター(trypsin inhibitor), リゾチーム(lysozyme) 各 3μg) (Amersham Pharmacia) を還元条件下、10% SDS-PAGEにより分離後、Immobilon transmembrane (MILLIPORE) に転写し、膜をPBS/0.1% Tween 20/5% スキムミルク(skim milk) 溶液中、室温で１時間緩やかに振とうした。次に膜をクローン番号(clone No.) 269-1D10, -4C9, -6B12 および-9C2の培養上清中、室温で１時間反応後、0.1% Tween 20/PBS 溶液で15分間、４回洗浄した。続いてPBS/0.1% Tween 20/ 5% スキムミルク(skim milk) 溶液で1,000 倍に希釈した抗マウスIg (Anti-mouse Ig), horseradish peroxidase linked whole antibody (from sheep) (Amersham Pharmacia)中、室温で１時間反応させ、PBS/0.1% Tween 20 溶液で15分間、４回洗浄した。その後、膜をECL Western Blotting Detection System (Amersham Pharmacia)で処理し、シグナルをＸ線フィルムで検出した。結果を図８に示す。図８中、M:分子量マーカー（各 3μg)、R:ヒト成熟型RAGE-Hisタグ タンパク、CM: COS 7 細胞過剰発現ヒト可溶型RAGEタンパク、及びL:ヒト肺抽出物を示す。いずれのモノクローナル抗体もヒト成熟型RAGEおよびヒト肺抽出物と反応せず、ヒト可溶型RAGEのみと反応した。
【０１１８】
実施例６：免疫沈降
1 mlのProtein G Sepharose (Amersham Pharmacia)を 200×g で１分間遠心し、上清を取り除いた後にTSA を加え同様の操作で３回樹脂を洗浄した。次に10,000回転で20分間遠心したヒト可溶型RAGE過剰発現COS 7 細胞の培養上清600 μl を加え室温で２時間穏やかに撹拌、 200×g で１分間遠心した上清を以下に用いた。上記同様TSA で洗浄した樹脂50μl に 100μl のPBS に溶解した可溶型RAGEモノクローナル抗体269-1D10, 269-9C2 あるいは正常マウスイムノグロブリンをそれぞれ50μg ずつ加え、ときどき撹拌しながら４℃で２時間放置した。次に 200×g で１分間遠心し、上清を捨てた後に上記培養上清を 150μl ずつ分注し、ときどき撹拌しながら４℃で２時間放置した。その後 200×g で１分間遠心し、上清を捨てたのち、 500μl のTSA を加え同様の操作で３回樹脂を洗浄した。最後に 500μl のTris-HCl pH 6.8 を加えて樹脂を洗浄した後、 200×g で１分間遠心し上清を捨てた。これにサンプルバッファー(sample buffer; 0.05 M Tris-HCl, pH 6.8/ 2% SDS/0.6% 2- メルカプトエタノール(2-mercaptoethanol)/10% グリセロール(glycerol)/0.005% ブロモフェノール ブルー(bromophenol blue)) 50μl を加え、100 ℃で３分間加熱し、 200×g で１分間遠心した上清のうち20μl を10% SDS-PAGEにより分離した。分離後、Immobilon transmembrane (MILLIPORE) に転写し、膜をPBS/0.1% Tween 20/5% スキムミルク(skim milk) 溶液中、室温で１時間緩やかに振とうした。次に膜を５μg/mlウサギ抗ヒト可溶型RAGEポリクローナル抗体/PBS/0.1% Tween 20/5% スキムミルク(skim milk) 溶液中室温で１時間反応後、0.1% Tween 20/PBS 溶液で15分間、４回洗浄した。続いてPBS/0.1% Tween 20/5% スキムミルク(skim milk) 溶液で1,000 倍に希釈した抗マウスIg (Anti-mouse Ig), horseradish peroxidase linked whole antibody (from sheep) (Amersham Pharmacia)中、室温で１時間反応させ、PBS/0.1% Tween 20 溶液で15分間、４回洗浄した。その後、膜をECL Western Blotting Detection System (Amersham Pharmacia)で処理し、シグナルをＸ線フィルムで検出した。結果を図９に示す。図９中、1:モノクローナル抗体269-1D10、2:モノクローナル抗体269-9C2 及び3:正常マウスイムノグロブリンを示す。抗可溶型RAGEモノクローナル抗体結合アフィニティーカラムはヒト可溶型RAGEをトラップした。なお、対照に用いたマウス正常イムノグロブリン結合アフィニティーカラムにおいてはヒト可溶型RAGEをトラップすることは出来なかった。
【０１１９】
実施例７：ヒト可溶型RAGEイムノアフィニティーカラムの作製
ディスポーザブル(disposable) PD-10カラム (Amersham Pharmacia) を用いてヒト可溶型RAGEモノクローナル抗体269-1D10の溶媒をカップリングバッファー(coupling buffer; 0.2 M NaHCO₃, 0.5 M NaCl, pH 8.3) に交換した。氷冷した1 mM塩酸6 mlで洗浄したHiTrap NHS-activated 1ml (Amersham Pharmacia) に上記ヒト可溶型RAGEモノクローナル抗体溶液1 mlを注入し、室温で30分間反応させた。その後3 mlのcoupling buffer をカラムに注入し、溶出液に1 mlの2M Glycine-HCl, pH2.0 を加え、A₂₈₀を測定しカップリング効率を算出した。カラムは6 mlのBuffer A (0.5 M ethanolamine, 0.5 M NaCl, pH8.3)、6 mlのBuffer B (0.1 M acetate, 0.5 M NaCl, pH 4.0)で順次洗浄した後、6 mlのBuffer Aを注入し、室温で30分間放置した。再び6 mlのBuffer B、6 mlのBuffer Aで順次カラムを洗浄し、最後に6 mlのBuffer Aで洗浄した。カラムは2 mlの保存溶液(0.05 M Na₂HPO₄, 0.1% NaN₃, pH7.0)を注入後４℃で保存した。上記操作はすべて流速 200μl/min で行った。
【０１２０】
実施例８：ヒト血清からの可溶型RAGEタンパクの分離
上記の方法に従って作製した可溶型RAGEイムノアフィニティーカラムをSMART systemに取り付け、5 mlのスタートバッファ(start buffer: 50 mM Tris-HCl, 150 mM NaCl, pH8.0)、5 mlの溶出バッファ(elution buffer: 100 mM グリシン(glycine)-HCl, 150 mM NaCl, pH2.5)で順次洗浄後、10 ml のスタートバッファ(start buffer)でカラムを平衡化した。次にMILLEX-GV 0.22μm Filter Unit (MILLIPORE) で濾過したヒト血清 2 ml にスタートバッファ(start buffer) 8 ml を加え、流速 100μl/min でカラムに供し、10 ml のスタートバッファ(start buffer)で洗浄した。続いて溶出液を 230 nm, 280 nm および 300 nm の紫外吸収によりモニターしながら 3 ml の溶出バッファ(elution buffer)で結合物を溶出させ 100μl ずつ分画した。得られたピーク画分に 0.9倍量のトリクロロ酢酸を加えて氷中で30分間放置後、15,000 rpmで20分間遠心、その後上清を捨てエタノール 750μl を加えて再び15,000 rpmで5 分間遠心した後、上清を捨て風乾した。得られた沈殿物を還元条件下、10% SDS-PAGEにより展開後、Immobilon transmembrane (MILLIPORE) に転写し、膜をPBS/0.1% Tween20/5% スキムミルク(skim milk) 溶液中、室温で１時間緩やかに振とうした。次に膜を 5μg/mlウサギ抗ヒト可溶型RAGEポリクローナル抗体/PBS/0.1% Tween 20/5% スキムミルク(skim milk) 溶液中、室温で１時間反応後、PBS/0.1% Tween 20 溶液で15分間、４回洗浄した。続いて膜を 1.5μg/ml anti-ウサギ IgG(H+L) HRP コンジュゲート (Zymed)/PBS/0.1% Tween20/5% スキムミルク(skim milk) 溶液により室温で１時間反応し、PBS/0.1% Tween 20 溶液で15分間、４回洗浄した。その後 ECL Western Blotting Detection System (Amersham Pharmacia) で膜を処理し、シグナルをＸ線フィルムで検出した。結果を図１０に示す。
抗可溶型RAGEモノクローナル抗体結合アフィニティーカラムでヒト血清中の可溶型RAGEをトラップし、そのトラップされたタンパクがヒトRAGEであることをウエスターン ブロット(western blot)により確認した。このことより、ヒト血清中にRAGEタンパクの C末端が欠如した可溶型RAGEが存在することを確認した。
【０１２１】
実施例９：可溶性RAGEのAGE 中和作用の検討
（AGE 誘導性VEGF発現亢進への可溶性RAGEの影響）
実験には 25 cm² フラスコでサブコンフルエント(subconfluent)まで培養した初代培養ヒト皮膚微小血管内皮細胞（Cascade Biologics, Inc., Portland, OR 、クラボウ, 大阪より購入) を用いた。細胞は新しい5% ウシ胎児血清(fetal bovine serum), 5 ng/ml 塩基性線維芽細胞成長因子(basic fibroblast growth factor), 10μg/mlヘパリン(heparin) 含有 Hu-Media MV2 培地(medium)(assay medium)で洗浄後、(1) 未添加、(2) 10μg/ml AGE2-BSA 添加、(3) 10μg/ml AGE2-BSA および25μg/ml精製可溶性RAGE添加、のassay mediumに置換し、37℃、４時間保温した。処理後、細胞より poly(A)⁺ RNA をQuickprep micro mRNA isolation kit (Amersham Pharmacia Biotech) により分離し、SuperScript One-Step RT-PCR キット (GIBCO BRL)を用いて、VEGF mRNA の検出を行った。 VEGF mRNA増幅に用いた poly(A)⁺ RNA 量は30 ng 、PCR サイクル数は30サイクルである。内部コントロールとして、β−アクチンを用いた (30ng、20サイクル) 。RT-PCR産物を、3%アガロースゲル電気泳動にて分離後、ナイロン膜へアルカリトランスファーし、³²P-標識したVEGF特異的オリゴヌクレオチドプローブでハイブリダイゼーションを行った。VEGF mRNA 増幅用プライマーおよびプローブは Nomura et al., J. Biol. Chem. 1995 と同一である。
結果を図１１に示す。初代培養ヒト皮膚微小血管内皮細胞におけるVEGF mRNA 発現レベルはAGE 添加により約２倍亢進するが、AGE と可溶型RAGEが共存すると、可溶型RAGEがドミナントネガティブに作用し、AGE によるVEGF mRNA レベルの亢進が防止された。
【０１２２】
実施例10：ヒト可溶型RAGEを特異的に測定するサンドイッチアッセイ
（測定系Ａ）
(a) モノクローナル抗体結合担体の調製
抗ヒト可溶型RAGEモノクローナル抗体278-13F11 を0.1Mリン酸緩衝液、pH7.5 に溶解し、25μg/mLの濃度に調製した。このモノクローナル抗体溶液を96穴マイクロプレートにウェルあたり100 μL ずつ加え、４℃、16〜24時間静置した。モノクローナル抗体溶液を除去し、精製水で１回洗浄後、1% BSA、0.15M NaCl、0.001%クロロヘキシジン含有 10mM リン酸緩衝液、pH7.0 を300 μL ずつ加え４℃、24時間以上静置しブロッキングした。
(b) ポリクローナル抗体の調製
合成ペプチド [配列番号：7]をMBS 法によりKLH にコンジュゲートし、ウサギに免疫し抗血清を得た。同合成ペプチドをカップリングしたアフィニティーカラムで抗血清を精製した。
【０１２３】
(c) ポリクローナル抗体を用いたサンドイッチEIA 法
精製したヒト可溶型RAGE画分を標準抗原として標準曲線を作製した（図12）。調製したモノクローナル抗体結合担体を 300μL の30mM NaCl 、0.01% Tween 20含有 5mM リン酸緩衝液、pH7.0 （洗浄液 A）で１回洗浄した。1% BSA、0.1M NaCl 、15% ブロックエース（大日本製薬）、35μg/mL HAMA 試薬含有 10mM リン酸緩衝液、pH7.0 （緩衝液 A）で1.7 μg/mLに調製したウサギ抗ヒト可溶型RAGE C末端ペプチドポリクローナル抗体を160 μL ずつ抗体結合プレートに加えた。さらに、調製した標準液または測定検体を40μL ずつ加え、マイクロプレートミキサーで十分に混合後、反応液の蒸発を防ぐためにシールを貼り４℃、16〜24時間静置した。洗浄液A でウェルあたり 300μL で４回洗浄し、緩衝液A で5000倍希釈した抗ウサギIgG ペルオキシダーゼ標識抗体（Amersham Pharmacia）を100 μL 加え、25℃、２時間静置した。洗浄液A でウェルあたり 300μL で４回洗浄し、0.1mg/mL 3, 3', 5, 5'-テトラメチルベンジジン、0.0075% 過酸化水素, 1% DMF含有 0.1M クエン酸緩衝液, pH4.0 を 100μL ずつ加え、25℃、30分間静置した。2N硫酸を 100μL ずつ添加し反応を停止し、この反応混液を波長450nm で測定し、標準曲線より検体の測定値を求めた。
【０１２４】
実施例11：ヒト可溶型RAGEを特異的に測定するサンドイッチアッセイ
（測定系Ｂ）
(a) モノクローナル抗体結合担体の調製
抗ヒト可溶型RAGE C末端ペプチドモノクローナル抗体269-1D10を0.1Mリン酸緩衝液、pH7.5 に溶解し、25μg/mLの濃度に調製した。このモノクローナル抗体溶液を96穴マイクロプレートにウェルあたり 100μL ずつ加え、４℃、16〜24時間静置する。モノクローナル抗体溶液を除去し、精製水で１回洗浄後、1% BSA、0.15M NaCl、0.001%クロロヘキシジン含有 10mM リン酸緩衝液、pH7.0 を 300μL ずつ加え４℃、24時間以上静置しブロッキングした。
(b) モノクローナル抗体を用いたサンドイッチEIA 法
精製したヒト可溶型RAGE画分を標準抗原として標準曲線を作製した（図13）。調製したモノクローナル抗体結合担体を 300μL の洗浄液A で１回洗浄した。緩衝液A で 1μg/mLに調製した酵素標識抗体液(278-3A6、IgG-HRP)を 160μL ずつ抗体結合プレートに加えた。さらに、調製した標準液または測定検体を40μL ずつ加え、マイクロプレートミキサーで十分に混合後、反応液の蒸発を防ぐためにシールを貼り４℃、16〜24時間静置した。洗浄液A でウェルあたり 300μL で４回洗浄し、0.1mg/mL 3, 3', 5, 5'-テトラメチルベンジジン、0.0075% 過酸化水素, 1% DMF含有0.1Mクエン酸緩衝液, pH4.0 を 100μL ずつ加え、25℃、30分間静置する。 2N 硫酸を 100μL ずつ添加し反応を停止し、この反応混液を波長450nm で測定し、標準曲線より検体の測定値を求めた。
【０１２５】
実施例12：ヒト可溶型及び膜型RAGEを特異的に測定するサンドイッチアッセイ
（測定系Ｃ）
(a) モノクローナル抗体結合担体の調製
抗ヒト可溶型RAGEモノクローナル抗体278-13F11 を0.1Mリン酸緩衝液、pH7.5 に溶解し、25μg/mLの濃度に調製した。このモノクローナル抗体溶液を96穴マイクロプレートにウェルあたり 100μL ずつ加え、４℃、16〜24時間静置する。モノクローナル抗体溶液を除去し、精製水で１回洗浄後、1% BSA、0.15M NaCl、0.001%クロロヘキシジン含有 10mM リン酸緩衝液、pH7.0 を 300μL ずつ加え４℃、24時間以上静置しブロッキングした。
(b) モノクローナル抗体を用いたサンドイッチEIA 法
精製したヒト可溶型RAGE画分を標準抗原として標準曲線を作製した（図14）。調製したモノクローナル抗体結合担体を 300μL の洗浄液A で１回洗浄した。緩衝液A で 1μg/mLに調製した酵素標識抗体液 (278-3A6 、IgG-HRP)を 100μL ずつ抗体結合プレートに加えた。さらに、調製した標準液または測定検体を20μL ずつ加え、マイクロプレートミキサーで十分に混合後、25℃、１〜２時間静置した。洗浄液A でウェルあたり 300μL で４回洗浄し、0.1mg/mL 3, 3', 5, 5'-テトラメチルベンジジン、0.0075% 過酸化水素, 1% DMF含有0.1Mクエン酸緩衝液, pH4.0 を 100μL ずつ加え、25℃、30分間静置した。 2N 硫酸を 100μL ずつ添加し反応を停止し、この反応混液を波長450nm で測定し、標準曲線より検体の測定値を求めた。
【０１２６】
〔各測定系の特異性〕
測定系Ａ、Ｂ、Ｃいずれもヒト可溶型RAGEを標準物質として0.1 〜6.4ng/mLの範囲で良好な標準曲線を描くことができた。
膜型RAGE過剰発現COS-7 細胞をコンフルエントまで培養後、培地を抜き取り、PBS で細胞層を洗浄した。その後、1% n-Octylglucoside, 0.1 M NaCl, 1 mM PMSF含有20 mM Tris-HCl, pH 7.4で細胞をlysis し、遠心した上清について、可溶型RAGEを標準物質としてＡ、Ｂ、Ｃそれぞれの測定系で測定した。測定系Ｃでの測定値は48ng/mL であったが、測定系ＡおよびＢではそれぞれ0ng/mL、2.7ng/mLであった（図15）。これらのことから、測定系Ｃは可溶型および膜型両方のRAGEを認識し、測定系ＡおよびＢは可溶型RAGEのみ認識することが確認された。
【０１２７】
〔測定系Ａを用いたヒト血中可溶型RAGEの測定〕
糖尿病罹病期間 7.8〜17.3年（平均12.5±2.6 年）、インスリン皮下自己注射療法のみで治療を受けているＩ型糖尿病患者血清及び血漿について、ヒト可溶型RAGEを特異的に測定する測定系Ａで測定した。
網膜症の進展程度は眼底所見をもとに、正常、単純網膜症に分類した。腎症については糖尿病腎症病期分類厚生省改定案に準じ、随時尿による尿中アルブミン・クレアチニン比30mg/g未満を腎症前期、30〜299 mg/gを早期腎症と判定した。なお、病期が前増殖性網膜症、増殖性網膜症および顕性腎症以上の患者試料と血清クレアチニン値2mg/dL以上の腎不全期の患者試料は除外した。網膜症、腎症ともに発症していない患者群（合併症未発症群）と腎症を発症していない単純網膜症患者群（単純網膜症患者群）および網膜症を発症していない早期腎症患者群（早期腎症患者群）間で測定系Ａを利用して血中可溶型RAGE量を測定し、統計的に解析した。
その結果、合併症未発症群（糖尿病罹病期間12.1±2.3 年）の可溶型RAGE量は単純網膜症患者群（糖尿病罹病期間13.1±2.6 年）、早期腎症発症患者群（糖尿病罹病期間11.7±2.5 年）と比較してそれぞれ有意(p<0.005, p<0.05) に高値を示した（図16）。
糖尿病合併症発症には個人差があり、とくに腎症では約30% が罹患感受性を示すことが知られている。今回の検討では、合併症未発症群の血中可溶型RAGE量が単純網膜症患者群、早期腎症患者群と比較して有意に高い値を示した。このことから糖尿病合併症発症時期における個体間の可溶型RAGE発現量の遺伝的差異が糖尿病合併症罹患感受性／抵抗性の決定因子の一つであると推察され、可溶型RAGEが糖尿病合併症の発生に対して保護的に機能している可能性が考えられた。すなわち、可溶型RAGEを定量することによる、糖尿病合併症罹患感受性、抵抗性の予測可能性が示唆された。
【０１２８】
【発明の効果】
天然型の可溶型RAGEを測定することが可能であり、AGE とRAGEの間の相互作用に起因する疾患の診断、糖尿病合併症の原因究明、診断・リスク予知などに有用である。ヒト可溶型RAGE蛋白に対するモノクローナル抗体を始めとした抗体などの活性物質を作製し、これを用いた当該タンパクの測定系を開発することが可能で、糖尿病合併症発症・進展のリスク予知などに役立つ。また、可溶型RAGEの産生を制御する化合物の開発も可能となるし、がんの転移、浸潤の診断などにも有用である。
本発明の可溶型RAGEは、生体内に存在する天然型ペプチド（内在性ペプチドあるいは内因性ペプチド）で、Ｃ末端部分の16個のアミノ酸残基においてRAGE蛋白質と異なっているものである。本発明の可溶型RAGEは、内在性に産生される天然のフォームなので、生理的なものであり、抗体ができる恐れが少なく、さらに経口可能な可溶型RAGE産生制御化合物及び薬剤などを開発して、そうした医薬により本発明の可溶型RAGEの発現を誘導できるようになれば、苦痛を与えることなしに、糖尿病患者を合併症から守ることなどができる。本発明により、内因性可溶型RAGEポリペプチド若しくはその塩、さらにはそれを基礎とした変異体、修飾体、誘導体などをデザインして得ることが可能となり、またそれらをコードする核酸、該核酸を有するベクター、該ベクターで形質転換された宿主細胞が提供でき、内因性可溶型RAGEに関連した疾患、例えば糖尿病合併症、老化に付随した各種疾患、アルツハイマー病、動脈硬化症、生体内タンパク質のグリケーション化に起因した疾患あるいは病気の発症及び／又は進展、及び腫瘍の浸潤又は拡散などの病的な状態あるいは症状の研究に役立つし、医薬品、診断薬、さらには遺伝子診断や遺伝子治療の途を開くと期待できる。
本発明は、前述の説明及び実施例に特に記載した以外も、実行できることは明らかである。上述の教示に鑑みて、本発明の多くの改変及び変形が可能であり、従ってそれらも本件添付の請求の範囲の範囲内のものである。
【０１２９】
【配列表】

【図面の簡単な説明】
【図１】 ヒトRAGE遺伝子の構造及びRAGE遺伝子転写産物のオルタナティブスプライシングによって生成される RAGE 蛋白の分子多様性を示す。 C端欠失可溶型が、本発明の可溶型RAGEに相当するものである。
【図２】 膜貫通型RAGE(fullRAGE)及び膜貫通ドメインを有するfullRAGE遺伝子より人工的に作製された可溶化型RAGEとよばれるもの(US5864018) と比較しての本発明の可溶型RAGEをコードする核酸(solubleRAGE) の塩基配列を示す。
【図３】 図２に続く fullRAGE 、US5864018 及びsolubleRAGE の塩基配列を示す。
【図４】 図３に続く fullRAGE 、US5864018 及びsolubleRAGE の塩基配列を示す。
【図５】 fullRAGE及びUS5864018 と比較しての本発明の可溶型RAGE(solubleRAGE) のアミノ酸配列を示す。
【図６】 sRAGE タンパクの10%SDS-PAGE 分離後の電気泳動写真を示す。１は細胞培養上清の銀染色像、２は精製蛋白の銀染色像を示す。
【図７】 本発明の抗可溶型RAGEモノクローナル抗体を使用してのウエスタンブロッティングの結果を示す。
【図８】 ハイブリドーマクローンの検定ウエスターン ブロッティング (western blotting) の結果の電気泳動写真を示す。
成熟型RAGE(R) 、可溶型RAGE過剰発現 COS 7細胞培養上清(CM)、ヒト肺抽出タンパク(L) および７種のタンパクを含む分子量マーカー(M) をSDS-PAGEで分離後、抗可溶型RAGEモノクローナル抗体産生ハイブリドーマ培養上清を用いウエスターン ブロッティングを行った。
【図９】 可溶型RAGEモノクローナル抗体を用いた免疫沈降の結果の電気泳動写真を示す。
可溶型RAGEモノクローナル抗体269-1D10 (1), 269-9C2 (2) 或いは正常マウスイムノグロブリン(3) を結合させたprotein G Sepharose を用いて可溶型RAGE過剰発現 COS 7細胞培養上清から当該タンパクを免疫沈降後、SDS-PAGEで分離し、ウエスターン ブロッティング (western blotting) により可溶型RAGEを検出した。
【図１０】 ヒト血清中に存在する可溶型RAGEタンパクの検出のため、抗ヒト可溶型RAGEモノクローナル抗体を使用したイムノアフィニティーカラムクロマトグラフィーを行った結果の溶出パターンとそのピーク画分より得られた沈殿物の電気泳動写真を示す。
2.5 mgのヒト可溶型RAGEモノクローナル抗体269-1D10が結合したイムノアフィニティーカラムにヒト血清2 mlをアプライし、カラムクロマトグラフィーを行った。ピーク画分 (fraction no. 16 〜18) はTCA で処理し、沈殿物をSDS-PAGEにより展開、ウサギ抗ヒト可溶型RAGEポリクローナル抗体を用いてウエスターン ブロッティング (western blotting) を行った。
【図１１】 可溶型RAGEによるAGE 内皮作用の中和作用を示す。
初代培養ヒト皮膚微小血管内皮細胞を(1) 未添加、(2) 10μg/ml AGE2-BSA 添加、(3) 10μg/ml AGE2-BSA および25μg/ml精製可溶性RAGE添加培養液中で処理後、細胞より poly(A)⁺ RNA を分離しVEGF mRNA の検出を行った。
【図１２】 抗ヒト可溶型RAGE抗体を使用したヒト可溶型RAGE測定サンドイッチアッセイ系（測定系Ａ）で得られた可溶型RAGE画分（標準抗原）の標準曲線を示す。
【図１３】 抗ヒト可溶型RAGE抗体を使用したヒト可溶型RAGE測定サンドイッチアッセイ系（測定系Ｂ）で得られた可溶型RAGE画分（標準抗原）の標準曲線を示す。
【図１４】 抗ヒト可溶型RAGE抗体を使用したヒト可溶型及び膜型RAGE特異的測定サンドイッチアッセイ系（測定系Ｃ）で得られた可溶型RAGE画分（標準抗原）の標準曲線を示す。
【図１５】 膜型RAGE過剰発現COS-7 細胞のライゼート（上清）を測定系Ａ、Ｂ及びＣで測定した結果を示す。可溶型RAGEを標準物質として使用した。
【図１６】 Ｉ型糖尿病患者について、血中ヒト可溶型RAGEを測定系Ａで測定した結果を示す。[0001]
BACKGROUND OF THE INVENTION
The present invention relates to an antibody against a soluble RAGE polypeptide derived from human, and uses for screening, measurement test, diagnosis and treatment using the same.
[0002]
[Prior art]
In recent years, the number of diabetic patients has been increasing. According to statistics from the Ministry of Health and Welfare in 1998, the estimated affected population in Japan was 6.9 million, including the reserve army, 14 million. The prognosis and quality of life (QOL) of diabetic patients are directly influenced by vascular disorders or vascular complications in various parts of the body secondary to hyperglycemia, not primary insulin deficiency. Therefore, to elucidate the cause of diabetic complications and to elucidate how to overcome it is a national research subject that requires urgent solutions. As a result of searching for environmental factors and genetic factors related to the onset and progression of diabetic complications, the inventors have advanced glycation end products (AGE) that are formed and accumulated at an accelerated rate in the diabetic state as the former. As for the latter, the importance of cell surface-specific receptors (receptor for AGE, RAGE) that recognize and bind to AGE and downstream signal molecule group / effector gene group is important in vitro experiments using cultured vascular cells and trans It was clarified from experiments at the individual level using transgenic animals (J. Biol. Chem., 272, 8723-8730, 1997; 275, 27781-25790, 2000; J. Clin. Invest., 108, 261-268 , 2001).
[0003]
Conventionally, it has been known that there are susceptibility / resistance genetic factors in diabetic vascular complications, but the substance is still unknown.
AGE has been pointed out to be involved in various complications associated with diabetes and aging, and cell surface receptors such as receptors expressed on the cell surface such as monosite / macrophages, neurons, smooth muscle cells, and endothelial cells. It is also known to combine with AGE is thought to interact with these receptors (receptors) and exert various physiological and biological effects on living organisms and cells. AGE, for example, proliferates endothelial cells and increases permeability and thrombus formation. In addition, monosite / macrophage promotes the release of cytokines, and further promotes the release of various factors involved in cell proliferation, migration, and matrix synthesis. It is also suspected to be involved in the inflammatory response in the vessel wall.
[0004]
[Problems to be solved by the invention]
There is evidence that AGE interacts with its receptors (receptors) and exerts various physiological and biological effects on living organisms and cells, resulting in various diseases and illnesses. As it is being elucidated, it is necessary to clarify substances that affect the interaction between AGE and RAGE, and to enable the prevention, treatment, and diagnosis of various diseases and illnesses. .
[0005]
[Means for Solving the Problems]
Most recently, the present inventor Yamamoto has revealed that there is molecular diversity in RAGE proteins expressed in human vascular cells, which is due to alternative splicing of the RAGE gene transcript (see Fig. 1), and the major molecular species. One is a soluble RAGE protein, which is secreted extracellularly because it lacks a membrane-bound region, while it has the same extracellular domain as the mature membrane-bound protein, and has found that it can capture AGE. In fact, when recombinant human soluble RAGE protein was purified and binding experiments with AGE ligands, it was proved that it binds to various AGE fractions with high affinity. Therefore, if there is an individual difference in the level of expression of this soluble RAGE protein, patients with high blood levels of the protein are less likely to cause diabetic complications, and conversely, if the blood levels are low, complications are likely to occur. The possibility is assumed.
Thus, a monoclonal antibody against this human soluble RAGE protein is prepared, and a measurement system for the protein using the monoclonal antibody is developed to predict the risk of developing or progressing diabetic complications. The present invention provides an antibody that specifically immunoreacts with a novel soluble RAGE polypeptide or a salt thereof, and various uses thereof, for example, screening, diagnosis, or therapeutic use.
[0006]
The present invention
[1] Antibodies against:
(1) (A) soluble RAGE polypeptide or
(B) (i) having at least 60% homology with the amino acid sequence of the soluble RAGE and
(ii) (a) a soluble RAGE amino acid sequence having at least 5 to 347 consecutive amino acid residues,
(b) The transmembrane domain possessed by membrane-bound RAGE is deleted, and the amino acid sequence Glu of SEQ ID NO: 2 in the sequence listing is located on the C-terminal side. ³³² ~ Met ³⁴⁷ Having at least 1 to 16 consecutive amino acid residues,
(c) N-terminal amino acid sequence Met of SEQ ID NO: 2 in the sequence listing ¹ ~ Val ¹¹⁷ One having at least 1 to 117 consecutive amino acid residues and lacking the transmembrane domain of membrane-bound RAGE,
(d) having at least 1 to 117 consecutive amino acid residues out of 1 to 117 consecutive amino acid residues of the N-terminal amino acid sequence of membrane-bound RAGE and the C-terminal side thereof Contains the amino acid sequence Glu of SEQ ID NO: 2 in the Sequence Listing ³³² ~ Met ³⁴⁷ Having at least 1 to 16 consecutive amino acid residues, and
(e) Those having the amino acid sequence of SEQ ID NO: 2 in the sequence listing or those having substantially the same biological activity as that
A polypeptide selected from the group consisting of:
(2) Among the amino acid sequences represented by SEQ ID NO: 2 in the sequence listing,
(i) having at least 5 to 115 consecutive amino acid residues,
(ii) having at least 116 to 230 amino acid residues in succession;
(iii) having at least consecutive 231 to 347 amino acid residues,
(iv) having at least one or more consecutive amino acid residues of the first to 117th amino acid sequences;
(v) having at least one or more consecutive amino acid residues from the 332th to 347th amino acid sequences;
(vi) having the 19th to 347th amino acid sequence,
(vii) having the first to 347th amino acid sequence, and
(viii) having an amino acid sequence substantially equivalent to any one of them
A polypeptide selected from the group consisting of:
(3) Of the polypeptides of (1) or (2), active against diseases caused by changes in interaction between AGE and its receptor, soluble RAGE expression level and / or AGE capture activity Or a salt thereof, or
(4) the partial peptide of the polypeptide according to any one of (1) to (3) or a salt thereof;
[0007]
[2] An antibody that specifically immunoreacts with the polypeptide according to any one of (1) to (4) of [1] above or a salt thereof as a measuring reagent. 1) A method for immunological measurement of the polypeptide or a salt thereof according to any one of (4);
[3] A composition comprising an antibody that specifically immunoreacts with the polypeptide or a salt thereof according to any one of (1) to (4) above [1];
[4] The antibody according to [1] above, which comprises an antibody that specifically immunoreacts with the polypeptide according to any one of [1] to [4] above or a salt thereof. (4) an immunoassay reagent for the polypeptide or salt thereof according to any one of
[5] A pharmaceutical comprising the antibody according to [1] above;
[6] Diagnosis of disease caused by change in interaction between AGE and its receptor, expression level of soluble RAGE and / or change in AGE capture activity characterized by containing the antibody according to [1] above Agent;
[7] A screening method or screening kit for a compound that increases the production of soluble RAGE and prevents the onset and / or progression of diabetic complications using the antibody according to [1] above;
[8] Soluble RAGE in body fluids is detected using the antibody according to [1] above, and diabetic complications, various diseases associated with aging, Alzheimer's disease, arteriosclerosis, in vivo protein glycation A method for predicting the onset and / or progression of a disease or illness caused by cancer, tumor invasion or spread; and
[9] A soluble RAGE production control compound obtained by screening production of soluble RAGE using the antibody according to [1] above is provided.
[0008]
In another aspect, the invention provides:
[10] Amino acid sequence Glu of SEQ ID NO: 2 in the sequence listing ³³² ~ Met ³⁴⁷ An antibody that specifically immunoreacts with a polypeptide having at least 4 to 16 consecutive amino acid residues, or a salt thereof;
[11] The immunological measurement method for a polypeptide or a salt thereof according to any one of (1) to (4) above, wherein the antibody according to [10] is used as a measurement reagent;
[12] A composition comprising the antibody according to [10] above;
[13] An immunoassay reagent for the polypeptide or salt thereof according to any one of (1) to (4) above, which comprises the antibody according to [10] above;
[14] A pharmaceutical comprising the antibody according to [10] above;
[15] Diagnosis of a disease caused by an interaction between AGE and its receptor, an expression level of soluble RAGE and / or a change in AGE capture activity characterized by containing the antibody according to [10] above Agent;
[16] The antibody according to [1] or [10] above, which is a solid-phased antibody;
[17] The antibody according to [1] or [10] above, which is a labeled antibody;
[18] The antibody according to [1] or [10] above, which is a humanized antibody;
[19] Diseases caused by changes in interaction between AGE and its receptor, soluble RAGE expression level and / or AGE capture activity using the antibody according to [1], [10] or [18] Treatment and / or prevention of
[0009]
[20] Use of the antibody described in [1], [10], [16], [17] or [18] as an affinity probe for affinity chromatography;
[21] Diagnosis or detection of diseases or illnesses caused by diabetic complications, various diseases associated with aging, Alzheimer's disease, tumor invasion or spread, arteriosclerosis, glycation of in vivo proteins [2] Or the measurement method according to [11];
[22] Diabetes complications, various diseases associated with aging, Alzheimer's disease, tumor invasion or spread, arteriosclerosis, diseases or illnesses caused by glycation of proteins in the body [4] Or the reagent according to [13]; and
[23] For treatment and / or prevention of diseases or illnesses caused by diabetic complications, various diseases associated with aging, Alzheimer's disease, tumor invasion or spread, arteriosclerosis, glycation of in vivo proteins [ 5] or [14] The pharmaceutical according to [14] is provided.
In addition, the present invention provides antibodies that specifically immunoreact with the novel soluble RAGE, and uses thereof for screening, diagnosis, or therapeutic purposes. The present invention provides a genetic diagnostic technique related to soluble RAGE, such as the expression of a novel soluble RAGE that is considered to be a predisposing factor for determining resistance to and susceptibility to diabetes, cancer, Alzheimer's disease, etc. Provided is a gene therapy technique for genetic diagnosis of a polymorphism and further reducing the risk of morbidity of a related disease based on the diagnosis result.
[0010]
Other objects, features, excellence and aspects of the present invention will be apparent to those skilled in the art from the following description. However, it is understood that the description of the present specification, including the following description and the description of specific examples and the like, show preferred embodiments of the present invention and are presented only for explanation. I want. Various changes and / or modifications (or modifications) within the spirit and scope of the present invention disclosed herein will occur to those skilled in the art based on the following description and knowledge from other parts of the present specification. Will be readily apparent. All patent documents and references cited herein are cited for illustrative purposes and are not to be construed as a part of this specification. is there.
As used herein, the term “and / or” means that both (1) a joint connection and (2) a selective connection exist, for example “treatment and / or prevention”. In some cases, it is used to encompass both (1) treatment and prevention and (2) both treatment and prevention. In other cases, the term “and / or” is similarly used to include both (1) merged connections and (2) selective connections.
[0011]
DETAILED DESCRIPTION OF THE INVENTION
As used herein, the term “soluble RAGE” refers to a peptide related to the receptor for advanced glycation endproducts (RAGE) that is closely related to diabetic complications, and is a splicing variant of RAGE that does not have a transmembrane region. Refers to the novel peptide disclosed in. The soluble RAGE is a peptide consisting of 347 amino acid residues, and has a characteristic sequence GluGlyPheAspLysValArgGluAlaGluAspSerProGlnHisMet on the C-terminal side. It is characterized by the lack of a transmembrane domain present in The soluble RAGE has an advanced glycation endproducts (AGEs) binding activity, or has an inhibitory or inhibitory activity on the interaction between AGE and its receptor. Typically, the soluble RAGE of the present invention is a naturally occurring peptide (endogenous peptide or endogenous peptide) that exists in vivo and differs from the RAGE protein in 16 amino acid residues in the C-terminal part. Is. A typical soluble RAGE of the present invention includes a polypeptide encoded and produced by the DNA of SEQ ID NO: 1 in the sequence listing, for example, the amino acid sequence of SEQ ID NO: 2 in the sequence listing or substantially equivalent thereto. A polypeptide having an amino acid sequence can be mentioned. In addition, a typical soluble RAGE of the present invention has the amino acid sequence Glu of SEQ ID NO: 2 in the sequence listing. ³³² ~ Met ³⁴⁷ Having at least 1 to 16 consecutive amino acid residues on the C-terminal side thereof and having AGE binding activity, amino acid sequence Met of SEQ ID NO: 2 in the Sequence Listing ¹ ~ Val ¹¹⁷ Having at least 1 to 117 consecutive amino acid residues on the N-terminal side and having AGE binding activity, or those characteristics, and the amino acid sequence Tyr of SEQ ID NO: 2 in the Sequence Listing ¹¹⁸ ~ Gly ³³¹ And novel ones having at least 60% homology to the above.
[0012]
As used herein, the term “polypeptide” may refer to any polypeptide as described below. The basic structure of a polypeptide is well known and is described in numerous references and other publications in the art. In view of these, the term “polypeptide” as used herein refers to any peptide or any protein comprising two or more amino acids such that they are linked together by peptide bonds or modified peptide bonds. To do. As used herein, the term “polypeptide” is commonly referred to in the art as, for example, short chains, also referred to as peptides, oligopeptides or peptide oligomers, and proteins, commonly known in many forms. It may mean both of the long chains that are being made. Polypeptides often contain amino acids other than the 20 amino acids commonly found (naturally occurring amino acids: or amino acids encoded by a gene) (20 amino acids). It may be. Polypeptides also contain chemical chemistry well known to those skilled in the art, not only by natural processes, including terminal amino acid residues, in which many amino acid residues are translated and then processed and otherwise altered (or modified). It will be understood that the above-described polypeptide can be altered (modified) by genetic modification techniques. Many forms of modifications (modifications) made to the polypeptide are known, and they are described in detail in basic reference books and more detailed papers and numerous research literatures in the field. These are well known to those skilled in the art. Some particularly routine alterations / modifications include, for example, glycosylation, lipid binding, sulfation, γ-carboxylation of glutamic acid residues, hydroxylation and ADP-ribosylation, for example TE Creighton, Proteins- Structure and Molecular Properties, Second Edition, WH Freeman and Company, New York, (1993); BCJohnson (Ed.), Posttranslational Covalent Modification of Proteins, Academic Press, New York, (1983) (Wold, F., "Posttranslational Protein Modifications: Perspective and Prospects ", pp.1-12); Seifter et al.," Analysis for Protein Modifications and nonprotein cofactors ", Meth. Enzymol., 182: 626-646 (1990); Rattan et al.," Protein Synthesis: Posttranslational Modification and Aging ", Ann. NY Acad. Sci., 663: p.48-62 (1992).
[0013]
The “polypeptide” of the present invention specifically includes soluble RAGE and related polypeptides. Examples of the soluble RAGE and related polypeptides include those derived from humans, and those that are soluble and have AGE binding activity or lack the transmembrane domain present in transmembrane RAGE, for example, the transmembrane region And those having AGE binding activity, typically having the characteristic sequence GluGlyPheAspLysValArgGluAlaGluAspSerProGlnHisMet on the C-terminal side, more specifically, SEQ ID NO: 2 in the sequence listing Among the amino acid sequences represented, those having at least the 332th to 347th amino acid sequences, those having the 1st to 117th amino acid sequences, and those having the 19th to 347th amino acid sequences Having at least the first to 347 amino acid sequences, and at least 60% homology, preferably 70% or more homology, more preferably 80% or less. Homologies, preferably 85% or more, more preferably 90% or more, more preferably 95% or more, particularly preferably 97% or more, and AGE binding activity And those having an amino acid sequence having substantially equivalent biological activity such as inhibition or suppressive activity on the interaction between AGE and its receptor or equivalent antigenicity.
[0014]
The human soluble RAGE of the present invention includes those that are not bound to the membrane, those that have a soluble AGE binding activity, those that lack the transmembrane domain present in the transmembrane RAGE, and those on the C-terminal side. Glu-Gly-Phe-Asp-Lys-Val-Arg-Glu-Ala-Glu-Asp-Ser-Pro-Gln-His-Met or a part of it and a kind of splicing variant of RAGE family And what is necessary is just to have a novel amino acid sequence. More preferably, as the peptide of the present invention, among those having an amino acid sequence having at least 60% homology with the RAGE family, the peptide of the present invention not only lacks a transmembrane domain but also has the amino acid sequence Glu of SEQ ID NO: 2 in the Sequence Listing. ³³² ~ Met ³⁴⁷ Of those having at least 1 to 16 consecutive amino acid residues, and in particular, of the amino acid sequence represented by SEQ ID NO: 2 in the sequence listing, at least the amino acid sequence of positions 332 to 347 Part, amino acid sequence part from position 1 to position 117 or a main part thereof (for example, consecutive amino acid residues including all or part of positions 332 to 347, or positions 1 to 347) 5 or more, preferably 10 or more, preferably 20 or more, more preferably 30 or more, more preferably 40 or more, more preferably 50 or more, more preferably 50 or more, 60 or more, more preferably 70 or more, preferably 80 or more, more preferably 90 or more, most preferably 100 or more, and preferably 110 or more). Typically, the peptide of the present invention is substantially the same as any one of the amino acid sequences represented by SEQ ID NO: 2 in the sequence listing, having at least the 19th to 347th amino acid sequences, and any one of them. Selected from the group consisting of those having substantially the same amino acid sequence. Furthermore, the peptide of the present invention may have a part or all of the amino acid sequence represented by SEQ ID NO: 2 in the sequence listing. Anything having such a sequence may be included.
[0015]
In the present specification, “homology” refers to each amino acid residue constituting the chain between two chains in a polypeptide sequence (or amino acid sequence) or polynucleotide sequence (or base sequence) or This means the amount (number) of things that can be determined to be the same in each other's matching relationship, and means the degree of sequence correlation between two polypeptide sequences or two polynucleotide sequences. is there. Homology can be easily calculated. Many methods for measuring homology between two polynucleotide or polypeptide sequences are known, and the term “homology” (also referred to as “identity”) is well known to those skilled in the art (eg, , Lesk, AM (Ed.), Computational Molecular Biology, Oxford University Press, New York, (1988); Smith, DW (Ed.), Biocomputing: Informatics and Genome Projects, Academic Press, New York, (1993); Grifin , AM & Grifin, HG (Ed.), Computer Analysis of Sequence Data: Part I, Human Press, New Jersey, (1994); von Heinje, G., Sequence Analysis in Molecular Biology, Academic Press, New York, (1987 Gribskov, M. & Devereux, J. (Ed.), Sequence Analysis Primer, M-Stockton Press, New York, (1991), etc.). Common methods used to measure the homology of two sequences include Martin, J. Bishop (Ed.), Guide to Huge Computers, Academic Press, San Diego, (1994); Carillo, H. & Lipman , D., SIAM J. Applied Math., 48: 1073 (1988), etc., but are not limited thereto. Preferred methods for measuring homology include those designed to obtain the largest match between the two sequences being tested. An example of such a method is one assembled as a computer program. Preferred computer programming methods for measuring homology between two sequences include the GCG program package (Devereux, J. et al., Nucleic Acids Research, 12 (1): 387 (1984)), BLASTP, BLASTN, FASTA (Atschul, SF et al., J. Molec. Biol., 215: 403 (1990)) and the like, but are not limited thereto, and methods known in the art can be used. .
[0016]
The nucleic acid encoding the soluble RAGE of the present invention typically contains a peptide represented by SEQ ID NO: 2 in the sequence listing and a base sequence encoding a part of the continuous amino acid sequence, for example, One containing at least positions 25-1068 of the base sequence represented by SEQ ID NO: 1 in the sequence listing, at least positions 136-1065 of the base sequence represented by SEQ ID NO: 1 in the sequence listing Containing a base sequence composed, and a base sequence composed of ATG at positions 25-27 to TGA at positions 1066-1068 from the base sequence represented by SEQ ID NO: 1 in the sequence listing (1066) -1068 stop codon TGA may be TAA or TAG), and contains a nucleotide sequence from position 25 to position 1065 of the nucleotide sequence represented by SEQ ID NO: 1 in the sequence listing The start codon (codon encoding Met) and Those that have been added to the stop codon, also, SEQ ID and the Sequence Listing has an amino acid sequence having at least 80% homology with a protein base sequence encodes: 2 amino acid sequence Glu ³³² ~ Met ³⁴⁷ It has at least 1 to 16 consecutive amino acid residues, and has AGE binding activity, inhibitory or inhibitory activity on the interaction between AGE and its receptor, or an antigenicity equivalent thereto, etc. Any base may be used as long as it contains a base sequence that has the same effect as that of a peptide having a biological activity equivalent to that of the peptide. Nucleic acids encoding the soluble RAGE are nucleic acids such as single-stranded DNA, double-stranded DNA, RNA, DNA: RNA hybrid, synthetic DNA, human genomic DNA, human genomic DNA library, human tissue / cell Either derived cDNA or synthetic DNA may be used. The base sequence of the nucleic acid encoding the soluble RAGE can be modified (for example, added, removed, substituted, etc.), and such modified ones can also be included. Furthermore, as described below, the nucleic acid of the present invention may encode the peptide of the present invention or a part thereof, and preferred examples include DNA. In addition, the “same-effect base sequence” refers to, for example, 5 or more consecutive base sequences of the base sequence of SEQ ID NO: 1 in the sequence listing, preferably 10 or more base sequences under stringent conditions. Preferred are those that hybridize with 15 or more base sequences, more preferably 20 or more base sequences, and encode an amino acid sequence substantially equivalent to the soluble RAGE.
[0017]
The DNA of the present invention containing the base sequence represented by SEQ ID NO: 1 in the sequence listing or the base sequence having the same effect as that can be obtained, for example, by the method shown below.
For example, J. Sambrook, EF Fritsch & T. Maniatis, "Molecular Cloning: A Laboratory Manual (2nd edition)", Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York (1989); DM Glover et al. Ed., "DNA Cloning", 2nd ed., Vol. 1 to 4, (The Practical Approach Series), IRL Press, Oxford University Press (1995); 1, Gene Research Method II ”, Tokyo Kagaku Doujin (1986); Edited by the Japanese Biochemical Society,“ New Chemistry Experiment Course 2, Nucleic Acid III (Recombinant DNA Technology) ”, Tokyo Kagaku Doujin (1992); R. Wu ed., "Methods in Enzymology", Vol. 68 (Recombinant DNA), Academic Press, New York (1980); R. Wu et al. Ed., "Methods in Enzymology", Vol. 100 (Recombinant DNA, Part B) & 101 (Recombinant DNA, Part C), Academic Press, New York (1983); R. Wu et al. Ed., "Methods in Enzymology", Vol. 153 (Recombinant DNA, Part D), 154 (Recombinant DNA, Part E ) & 155 (Recombinant DNA, Part F), Academic Press, New York (1987); JH Miller ed., "Methods in Enzymology", Vol. 204, Academic Press, New York (1991); R. Wu et al. Ed., "Methods in Enzymology", Vol. 218, Academic Press, New York (1993 ) Or the method described in the literature cited therein, or a method or modification method substantially similar thereto (the description contained therein is incorporated herein by reference) Included in the disclosure).
[0018]
Cloned human cDNA libraries such as various human tissue or cultured cells (especially human kidney, brain, pineal gland, pituitary gland, nerve cells, retina, retinal vascular cells, retinal nerves) Cells, thymus, blood vessels, endothelial cells, vascular smooth muscle cells, blood cells, macrophages, lymphocytes, testis, ovary, uterus, intestine, heart, liver, pancreas, small intestine, large intestine, gingival related cells, skin related cells, glomerulus Cells, tubule cells, connective tissue cells, etc.) AGE binding activity or interaction between AGE and its receptor using a suitable detection system for plasmids containing cDNA inserts obtained from cDNA libraries. Selection is carried out using at least one of the inhibitory or inhibitory activities against the above as an index. This search technique can be repeated. The nucleic acid thus identified is sequenced. In addition, appropriate primers are designed and synthesized based on the identified sequence, and in some cases, the cDNA sequence derived from the animal that is the origin of the identified sequence is used, and the target sequence is polymerase chain reaction. Amplify (polymerase chain reaction: PCR). The obtained DNA fragment is used as a probe to screen a human genomic DNA library or a human-derived cDNA library constructed from various human tissues or cultured cells, and a clone that hybridizes to the probe is selected. (C) It is also possible to determine the base sequence of the DNA insertion sequence and to determine and obtain a novel soluble RAGE and a DNA fragment having the relevant base sequence. If necessary, the inserted sequence of the DNA in the clone can be subcloned. Based on the DNA sequence considered to have a nucleic acid encoding the novel polypeptide thus analyzed, it is also possible to obtain a gene encoding the soluble RAGE and its related nucleotide sequence. is there. In addition, a sense primer and an antisense primer can be designed and synthesized based on the analyzed novel soluble RAGE and a gene sequence (DNA sequence) related thereto. The sense primer can preferably be synthesized by selecting from the exon site at the 5 ′ end of the coding sequence presumed to be the desired peptide analyzed, and the antisense primer is preferably the analyzed desired peptide It can be selected and synthesized from exon sites on the 3 ′ end side of the coding sequence presumed to be, and more preferably, it can be selected from other than the exon sites used for the synthesis of the sense primer.
[0019]
You may aim to obtain the full length of the cDNA at once, but use the analyzed exon sites (multiple exon sites) to design and synthesize multiple primers and design multiple PCRs. (If necessary, analyze the DNA fragment whose nucleotide sequence has been determined, determine the entire nucleotide sequence of the cDNA, and clone based on it), and obtain the desired cDNA from the obtained DNA fragment Can do. The primer is preferably an oligonucleotide composed of 5 or more bases, more preferably 10 or more bases, more preferably an oligonucleotide composed of 18 to 25 bases. Primers can be prepared by methods known in the art, and typically can be synthesized by a phosphodiester method, a phosphophotoester method, a phosphoramidite method, etc., for example, an automatic DNA synthesizer, for example, a model It can be synthesized using 381A DNA synthesizer (Applied Biosystems). It is also possible to amplify cDNA by performing PCR using a cDNA library and the above-mentioned sense primer and antisense primer. The nucleic acid is obtained by preparing a specific hybridization probe from the clone identified as described above, screening a human-derived DNA library, and selecting a clone that hybridizes to the probe. be able to. To label a probe or the like with a radioactive isotope or the like, a commercially available labeling kit such as a random prime DNA labeling kit (Boehringer Mannheim) can be used. For example, using a random-priming kit (Pharmacia LKB, Uppsala), etc. ³² A radioactive probe can be obtained by labeling with P] dCTP (Amersham) or the like.
[0020]
Furthermore, as a cDNA library used as a template, commercially available cDNA libraries derived from various tissues can be used directly, for example, cDNA libraries commercially available from Stratagene, Invitrogen, Clontech, etc. can be used. Typical examples include gene libraries prepared from the labeled DNA fragments and human tissues / cells, such as the human P1 artificial chromosome genomic library (Human Genome Mapping Resource Center), human brain cDNA libraries (for example, Clontech, etc. Hybridization is possible. A human brain cDNA library can be constructed, for example, in a phage such as λgt10, which can be obtained by infecting a host E. coli such as the E. coli C600hfl strain to form a plaque. If necessary, the inserted sequence of cDNA in the clone can be subcloned. Based on the determined nucleotide sequence, the target DNA can also be isolated.
The nucleotide sequence can be determined using the dideoxy method, for example, the M13 dideoxy method, Maxam-Gilbert method, etc., but a commercially available sequencing kit such as Taq dye primer cycle sequencing kit (Applied Biosystems), Sequenase v 2.0 kit or the like can be used, or an automatic nucleotide sequence determination device such as a fluorescent DNA sequencer device (for example, Model 373A, Applied Biosystems) can be used. Examples of the polymerase used in the dideoxy method include Klenow fragment of DNA polymerase I, AMV reverse transcriptase, Taq DNA polymerase, T7 DNA polymerase, and modified T7 DNA polymerase.
[0021]
In the present specification, “polymerase chain reaction” or “PCR” generally refers to a method as described in US Pat. No. 4,683,195, for example, as desired. It refers to a method for enzymatically amplifying a nucleotide sequence in vitro. In general, the PCR method includes repeating cycles such as primer extension synthesis using two oligonucleotide primers that can preferentially hybridize with a template nucleic acid. Typically, the primer used in the PCR method can be a primer complementary to the nucleotide sequence to be amplified in the template, for example, complementary to the nucleotide sequence to be amplified at both ends thereof. Those adjacent to the nucleotide sequence to be amplified can be preferably used.
The PCR reaction can be performed by a method known in the art, or a method or modification method substantially similar thereto. For example, R. Saiki, et al., Science, 230: 1350, 1985; R. Saiki, et al., Science, 239: 487, 1988; HA Erlich ed., PCR Technology, Stockton Press, 1989; DM Glover et al. ed., "DNA Cloning", 2nd ed., Vol. 1, (The Practical Approach Series ), IRL Press, Oxford University Press (1995); MA Innis et al. Ed., "PCR Protocols: a guide to methods and applications", Academic Press, New York (1990)); MJ McPherson, P. Quirke and GR Taylor (Ed.), PCR: a practical approach, IRL Press, Oxford (1991); MA Frohman et al., Proc. Natl. Acad. Sci. USA, 85, 8998-9002 (1988) Alternatively, it can be performed according to a modified or modified method. In addition, the PCR method can be performed using a commercially available kit suitable for the PCR method, and can also be performed according to a protocol clarified by the kit manufacturer or the kit vendor.
[0022]
In a typical case, for example, 1st strand DNA used as a template and a primer are mixed with 10 × reaction buffer (attached to Taq DNA polymerase), dNTPs (deoxynucleoside triphosphates dATP, dGTP, dCTP, dTTP). Mix), Taq DNA polymerase and deionized distilled water. The mixture is repeated 25-60 times under typical PCR cycle conditions using an automated thermal cycler such as GeneAmp 2400 PCR system, Perkin-Elmer / Cetus, etc. Depending on the number of times, it can be set to an appropriate number of times. PCR cycle conditions include, for example, denaturation 90-95 ° C. 5-100 seconds, annealing 40-60 ° C. 5-150 seconds, extension 65-75 ° C. 30-300 seconds, preferably denaturation 94 ° C. 15 seconds, annealing 58 A cycle of 15 ° C and extension 72 ° C for 45 seconds can be mentioned, and the reaction temperature and time for annealing can be appropriately selected by experiment, and the time for denaturation and extension reaction can also be estimated by the chain of the expected PCR product. An appropriate value can be selected according to the length. It is preferable to change the annealing reaction temperature according to the Tm value of the hybrid of the primer and template DNA. The extension reaction time is generally about 1 minute per 1000 bp chain length, but a shorter time can be selected in some cases.
[0023]
The obtained PCR product is usually subjected to 1-2% agarose gel electrophoresis, excised from the gel as a specific band, and DNA is extracted using a commercially available extraction kit such as gene clean kit (Bio 101). The extracted DNA is cleaved with an appropriate restriction enzyme, purified as necessary, and further phosphorylated at the 5 'end with T4 polynucleotide kinase as necessary, and then with an appropriate pUC vector such as pUC18. Ligation into a plasmid vector and transformation of appropriate competent cells. The cloned PCR product is analyzed for its nucleotide sequence.
In addition, by using a primer designed based on the base sequence of the 5 ′ end exon site analyzed among the exon sites of the gene, cDNA of the 5 ′ end side of the desired cDNA is obtained, Using the primers designed based on the nucleotide sequence of the 3 'end exon site analyzed among the exon sites of the gene, obtain the 3' end cDNA of the desired cDNA, and then if necessary These primers and the primers designed using the 5'-end cDNA and 3'-end cDNA base sequence information of the gene of interest are obtained from human tissues, particularly human brain tissues. A cDNA of the gene can also be obtained by amplifying by PCR using 1st strand cDNA prepared from the isolated mRNA by reverse transcriptase as a template.
[0024]
The 5′-end primer is selected so as to contain at least the start codon or can be amplified including the start codon, and the 3′-end primer contains at least a stop codon, or It is preferable to select such that it can be amplified including the stop codon. In obtaining cDNA of the full length of the gene, PCR can be carried out as described above, and preferable PCR cycle conditions include, for example, denaturation 92-95 ° C. 10-20 seconds, annealing 55-60 ° C. 10-30 And a cycle of 65 to 75 ° C. for 150 to 300 seconds, more preferably denaturation 94 ° C. for 15 seconds, annealing 58 ° C. for 15 seconds, and extension at 68 ° C. for 4 minutes.
The obtained PCR product is cloned in the same manner as described above, and its base sequence is analyzed and determined.
In addition, by designing primers based on the determined DNA base sequence and screening using these primers and various animal cell-derived cDNA libraries (eg, various human cell-derived cDNA libraries), Similarly, the target clone can be obtained. PCR amplification using these primers can be performed to obtain the target coding sequence-containing nucleic acid, novel genes, fragments thereof, and the like. Thus, a PCR product having a homology with soluble RAGE but having a novel sequence can also be searched.
[0025]
In the present specification, the “oligonucleotide” is a relatively short single-stranded or double-stranded polynucleotide, preferably a polydeoxynucleotide, and Angew. Chem. Int. Ed. Engl., Vol. 28 , p.716-734 (1989), for example, such as triester method, phosphite method, phosphoamidite method, phosphonate method, and the like. It is known that normal synthesis can be conveniently carried out on a modified solid support, for example using an automated synthesizer, which is commercially available. The oligonucleotide may contain one or more modified bases, for example, a non-naturally occurring base such as inosine or a tritylated base.
The obtained PCR product can be cloned, the base sequence of the obtained PCR product can be determined, and a novel soluble RAGE and a DNA fragment having a coding sequence related thereto can be obtained. In addition, by using this DNA fragment as a probe, various cDNA libraries can be screened and the target DNA can be isolated. For cloning PCR products, for example, p-Direct (Clontech), pCR-Script ^TM SK (+) (Stratagene), pGEM-T (Promega), pAmp ^TM Commercially available plasmid vectors such as (Gibco-BRL) can be used.
[0026]
In order to construct a cDNA library, it is necessary to obtain cDNA. This can be obtained, for example, as follows. MRNA is isolated from various human tissues or cultured cells. Isolation of mRNA can be performed by a method known in the art, or a method or modification substantially similar thereto, but J. Sambrook et al., “Molecular Cloning”, 2nd ed., Chapter 7, Cold. Spring Harbor Laboratory, Cold Spring Harbor, NY (1989); DM Glover et al. Ed., "DNA Cloning", 2nd ed., Vol. 1, (The Practical Approach Series), IRL Press, Oxford University Press (1995) L. Grossman et al. Ed., "Methods in Enzymology", Vol. 12, Part A & B, Academic Press, New York (1968); SL Berger et al. Ed., "Methods in Enzymology", Vol. 152, p.33 & p.215, Academic Press, New York (1987); Biochemistry, 18: 5294-5299, 1979, etc., such as the guanidine-cesium chloride method, the thiocyanate guanidine method, the phenol method, etc. Can be done by the method. Examples of kits used for mRNA isolation include those commercially available from Pharmacia, Stratagene, Gibco-BRL, and the like. If necessary, the total RNA obtained can be purified using an oligo (dT) -cellulose column, spin column, oligo (dT) -coupled magnetic beads, etc. ⁺ mRNA can be obtained.
[0027]
Using this mRNA and reverse transcriptase (RNA-dependent DNA polymerase), cDNA is prepared. In the reverse transcription reaction, an oligo (dT) primer can be used. An oligo (dT) primer having preferably 12 to 18 T residues can be used. When performing directional cloning, it is also preferable to use a synthetic oligonucleotide primer in which a restriction enzyme site is linked to the 5 ′ side of 12 to 18 T residues. Examples of such primers include Xba I oligo (dT) primer adapters. When a random hexamer primer is used, the possibility of obtaining the 5 ′ end of mRNA is increased, and this random hexamer primer can be used alone or mixed with an oligo (dT) primer. In the reverse transcription reaction, an RNase inhibitor such as RNasin (Boehringer Mannheim) can be added as necessary. cDNA synthesis using mRNA and reverse transcriptase can be carried out by a method known in the art or substantially the same method or modification, but H. Land et al., Nucleic Acids Res., 9: 2251, 1981; U. Gubler et al., Gene, 25: 263-269, 1983; SL Berger et al. Ed., "Methods in Enzymology", Vol. 152, p.307, Academic Press, New York (1987 ) And the like.
[0028]
Based on the obtained cDNA, a cDNA library can be constructed by using a phage vector or a plasmid vector. Other than using phage vectors, transformation of host cells such as E. coli can be performed using, for example, the calcium method, rubidium / calcium method, calcium / manganese method, TFB high-efficiency method, FSB frozen competent cell method, rapid colony method. It can be carried out by a method known in the art such as a method, electroporation, or a method substantially similar thereto (D. Hanahan, J. Mol. Biol., 166: 557, 1983, etc.). In order to isolate the target DNA, reverse transcription PCR (polymerase chain reaction coupled reverse transcription; RT-PCR) or RACE (rapid amplification of cDNA ends) can be applied. RACE was described, for example, in MA Innis et al. Ed., "PCR Protocols" (MA Frohman, "a guide to methods and applications"), pp. 28-38, Academic Press, New York (1990). Can be done according to the method. The RT-PCR product can be cloned into a plasmid vector and introduced into highly efficient competent cells.
Furthermore, a method capable of isolating and purifying mRNA from a minute amount of cells or tissues, for example, REX kit, United States Biochemical; Glass MAX ^TM Using commercially available kits such as RNA spin cartridge system, Gibco-BRL, reverse transcription of the obtained mRNA using oligo (dT) primer to synthesize 1st strand DNA, then to the 3 ′ end of 1st strand DNA After attaching a homopolymer tail (eg, G residue) or attaching an adapter to the DNA, the cDNA can also be PCR amplified using an oligo (dT) primer and an oligo (dC) primer or an adapter primer. . A suitable commercial kit for this is SuperScript ^TM pre-amplification system (Gibco-BRL); cDNA Cycle ^TM kit (Invitrogen).
[0029]
Hybridization is performed by transferring plaques formed by microorganisms that hold a predetermined inserted DNA to a membrane such as a nylon filter, and performing transformation, immobilization, washing, etc. as necessary. This is carried out by reacting the material transferred to the membrane with a labeled probe DNA fragment that has been modified as necessary in a hybridization buffer. The hybridization treatment is usually performed at about 35 ° C. to about 80 ° C., more preferably about 50 ° C. to about 65 ° C., for about 15 minutes to about 36 hours, more preferably about 1 hour to about 24 hours. The optimum conditions can be selected as appropriate. For example, the hybridization treatment is performed at about 55 ° C. for about 18 hours. The hybridization buffer can be selected from those commonly used in the art, and for example, Rapid hybridization buffer (Amersham) can be used. Examples of the modification treatment of the transferred film include a method using an alkali-denaturing solution, and it is preferable to treat with a neutralizing solution or a buffer solution after the treatment. The membrane is usually fixed at about 40 ° C. to about 100 ° C., more preferably about 70 ° C. to about 90 ° C., about 15 minutes to about 24 hours, more preferably about 1 hour to about 4 hours. Although it is carried out by baking, it can be carried out by appropriately selecting preferable conditions. For example, immobilization is performed by baking the filter at about 80 ° C. for about 2 hours. The transferred membrane can be washed by washing with a commonly used washing solution in the art, such as 50 mM Tris-HC1 buffer containing 1 M NaCl, 1 mM EDTA and 0.1% Sodium Dodecyl sulfate (SDS), pH 8.0. It can be carried out. A membrane such as a nylon filter can be selected from those commonly used in the art, and examples thereof include a nylon filter [Hybond-N, Amersham].
[0030]
The alkali-denaturing solution, neutralizing solution, and buffer solution can be selected from those commonly used in the field, and the alkali-denaturing solution contains, for example, 0.5M NaOH and 1.5M NaCl. Examples of the neutralizing solution include 1.5M NaCl-containing 0.5M Tris-HCl buffer solution, pH 8.0. Examples of the buffer solution include 2 × SSPE (0.36). M NaCl, 20 mM NaH ₂ PO _Four And 2 mM EDTA). Prior to the hybridization treatment, it is preferable to pre-hybridize the transferred film as necessary in order to prevent nonspecific hybridization reaction. This prehybridization treatment can be performed, for example, by using a prehybridization solution [50% formamide, 5 × Denhardt's solution (0.2% bovine serum albumin, 0.2% polyvinyl pyrrolidone), 5 × SSPE, 0.1% SDS, 100 μg / ml heat-denatured salmon. Sperm DNA] and the like, and the reaction is carried out at about 35 ° C. to about 50 ° C., preferably about 42 ° C., for about 4 to about 24 hours, preferably about 6 to about 8 hours. Those skilled in the art can repeat the experiment as appropriate to determine more preferable conditions. The labeled probe DNA fragment used for hybridization is denatured, for example, by heating at about 70 ° C. to about 100 ° C., preferably about 100 ° C., for about 1 minute to about 60 minutes, preferably about 5 minutes. it can. Hybridization can be carried out by a method known per se or a method analogous thereto, and the stringent conditions in this specification are, for example, about 15 to about 50 mM, preferably about 19 to about sodium concentration. The temperature is about 40 mM, more preferably about 19 to about 20 mM, and the temperature is about 35 to about 85 ° C., preferably about 50 to about 70 ° C., more preferably about 60 to about 65 ° C.
[0031]
After completion of hybridization, the filter is thoroughly washed to remove labeled probes other than labeled probe DNA fragments that have undergone a specific hybridization reaction. The filter can be washed by selecting from those commonly used in the field, for example, with 0.5% SSC (O.15M NaCl, 15 mM citric acid) solution containing 0.1% SDS. Can be implemented.
The hybridized plaque can be typically detected by autoradiography, but can be appropriately selected from methods used in the art and used for plaque detection. Plaques corresponding to the detected signal are collected in a suitable buffer, eg SM solution (100 mM NaCl and 10 mM MgSO _Four Suspended in 50 mM Tris-HCl buffer, pH 7.5), etc., and then this phage suspension is appropriately diluted to infect E. coli, and the resulting E. coli is cultured. The desired recombinant phage is obtained. If necessary, the above-described probe DNA can be repeatedly used for screening the target recombinant phage from a gene library or cDNA library by hybridization treatment. The target recombinant phage can be obtained by subjecting the cultured Escherichia coli to extraction treatment, centrifugation treatment, and the like.
[0032]
The obtained phage particles can be purified and separated by a method commonly used in the art. For example, glycerol gradient ultracentrifugation (Molecular cloning, a laboratory manual, ed. T. Maniatis, Cold Spring Harbor Laboratory , 2nd ed. 78, 1989). From the phage particles, DNA can be purified and separated by a method commonly used in the art. For example, the obtained phage can be treated with TM solution (10 mM MgSO _Four Suspend in 50 mM Tris-HCl buffer (pH 7.8), etc., and treat with DNase I and RNase A. Add 20 mM EDTA, 50 μg / ml Proteinase K and 0.5% SDS mixture, and add about 65 ° C, about 65 ° C. After incubating for 1 hour, this was phenol extracted and extracted with diethyl ether, and then DNA was precipitated by ethanol precipitation. Next, the obtained DNA was washed with 70% ethanol and dried, and then TE solution (10 mM EDTA-containing 10 mM Tris-HC1 buffer) was obtained. Obtained by dissolving in a liquid, pH 8.0). The target DNA can also be obtained in large quantities by subcloning. For example, subcloning can be performed using Escherichia coli as a host and a plasmid vector. The DNA obtained by such subcloning can also be purified and separated by methods such as centrifugation, phenol extraction, and ethanol precipitation in the same manner as described above.
[0033]
Thus, according to the present invention, a clone (for example, as a recombinant phage) containing the target DNA can be obtained. For example, the total length of the sequenced nucleotide sequence of the DNA insert isolated from the cloned recombinant phage is 1223 bp, and the sequence is represented by SEQ ID NO: 1 in the sequence listing. It is recognized that In the identified DNA sequence, the presence of an open reading frame encoding an estimated 347 amino acids is recognized, and the estimated amino acid sequence is recognized as shown in SEQ ID NO: 2 in the sequence listing. . This sequence is a splicing variant of RAGE and has a characteristic sequence Glu-Gly-Phe-Asp-Lys-Val-Arg-Glu-Ala-Glu-Asp-Ser-Pro-Gln- It has His-Met and lacks the transmembrane domain present in transmembrane RAGE. This putative protein is one of the novel human RAGEs and is referred to herein as “soluble RAGE”. It is clear that the soluble RAGE gene encodes a polypeptide belonging to the novel RAGE family, and all recombinant plasmids prepared using the soluble RAGE gene are novel recombinants. There is also a novel transformant or transfectant obtained by transforming or transfecting with the plasmid.
[0034]
A nucleic acid having all or part of the base sequence shown in SEQ ID NO: 1 in the sequence listing can also be obtained by chemical synthesis. In that case, the fragments may be chemically synthesized and joined together by an enzyme. In addition, a chemically synthesized fragment can be used as a primer or a probe as described above to obtain a target sequence. The primer used in the PCR method is not particularly limited as long as it can amplify a DNA fragment containing the above site. Typically, the primer is (a) an oligonucleotide having a base sequence corresponding to any region of the base sequence shown in SEQ ID NO: 1 in the sequence listing; and (b) SEQ ID NO: 1 in the sequence listing. An oligonucleotide having a complementary base sequence for any region of the indicated base sequence can be used, more preferably (1) 5 'of the base sequence shown in SEQ ID NO: 1 of the sequence listing An oligonucleotide having a base sequence corresponding to an arbitrary region on the end side, and (2) an oligonucleotide having a complementary base sequence to an arbitrary region on the 3 ′ end side of the base sequence shown in SEQ ID NO: 1 in the sequence listing Nucleotides can be used, for example, oligonucleotides having 5 or more bases, more preferably 10 or more bases, more preferably 15 or more bases, and 3 to 150, preferably 10 to 150 pieces, more preferred Or those containing 10 to 50, more preferably 15 to 35 nucleotides. The PCR conditions are not particularly limited, and may be known conditions that are usually performed. For example, the conditions described above can be selected by reference. In PCR, one cycle consisting of heat denaturation of a DNA strand, annealing of a primer, and synthesis of a complementary strand by a polymerase is repeated, for example, 10 to 50 times, preferably 20 to 35 times, more preferably 25 to 30 times. Done.
[0035]
The DNA fragment obtained in the present invention is incorporated into an appropriate vector as described in detail below, for example, a vector such as plasmid pEX, pMAMneo or pKG5, and an appropriate host cell as described in detail below, for example, E. coli. It can be expressed in yeast, CHO cells, COS cells and the like. The DNA fragment can be used as it is or as a DNA fragment to which an appropriate control sequence is added, or incorporated into an appropriate vector and introduced into an animal to express a soluble RAGE gene such as a soluble RAGE. A transgenic animal can be created. Examples of animals include mammals, such as mice, rats, rabbits, guinea pigs, and cows. Preferably, a transgenic animal can be prepared by introducing the DNA fragment into a fertilized egg of an animal such as a mouse.
The soluble RAGE gene product can be confirmed using animal cells suitable for such as 293T cells and COS-1 cells transfected with the soluble RAGE gene. This foreign gene can be introduced into animal cells such as mammals by methods known in the art or substantially similar methods, such as the calcium phosphate method (for example, FL Graham et al., Virology). , 52: 456, 1973, etc.), DEAE-dextran method (eg, D. Warden et al., J. Gen. Virol., 3: 371, 1968), electroporation method (eg, E. Neumann et al. ., EMBO J, 1: 841, 1982, etc.), microinjection method, ribosome method, virus infection method, phage particle method and the like. Thus, the gene product produced by the animal cell transfected with the soluble RAGE gene can be analyzed.
[0036]
As a plasmid into which a soluble RAGE gene or the like (such as the DNA obtained in the present invention) is incorporated, a host cell commonly used in genetic engineering (for example, prokaryotic hosts such as Escherichia coli and Bacillus subtilis, yeast, CHO cells, COS cells) Any plasmid may be used as long as the DNA can be expressed in a eukaryotic host such as Sf21 or an insect cell host such as Sf21. Such sequences can include codons suitably modified for expression in, for example, a selected host cell, can include restriction enzyme sites, and can contain the desired gene. Control and facilitating sequences to facilitate expression, such as linkers and adapters useful for binding the target gene, and antibiotic resistance, metabolism control, selection, etc. Useful sequences (including those encoding hybrid proteins and fusion proteins) can be included.
Preferably, suitable plasmids such as plasmids hosted in E. coli are tryptophan promoter (trp), lactose promoter (lac), tryptophan lactose promoter (tac), lipoprotein promoter (lpp), λ phage P _L For plasmids with animal cells as the host, SV40 rate promoter, MMTV LTR promoter, RSV LTR promoter, CMV promoter, SRα promoter, etc. can be used. For yeasts with host cells, GAL1, GAL10 promoter, etc. can be used. .
[0037]
Examples of plasmids hosted in E. coli include pBR322, pUC18, pUC19, pUC118, pUC119, pSP64, pSP65, pTZ-18R / -18U, pTZ-19R / -19U, pGEM-3, pGEM-4, pGEM-3Z, pGEM-4Z, pGEM-5Zf (-), pBluescript KS ^TM , (Stratagene) and the like. Examples of plasmid vectors suitable for expression in E. coli include pAS, pKK223 (Pharmacia), pMC1403, pMC931, pKC30, and pRSET-B (Invitrogen). Examples of plasmids that use animal cells as hosts include SV40 vectors, polyoma virus vectors, vaccinia virus vectors, retrovirus vectors, etc., for example, pcD, pcD-SRα, CDM8, pCEV4, pME18S, pBC12BI, pSG5 (Stratagene) Etc. Examples of plasmids that use yeast as a host include YIp type vectors, YEp type vectors, YRp type vectors, and YCp type vectors. Examples thereof include pGPD-2. When the host cell is E. coli, examples of the host cell include those derived from E. coli K12 strain, such as NM533, XL1-Blue, C600, DH1, DH5, DH11S, DH12S, DH5α, DH10B, HB101, MC1061, JM109. STBL2 and B834 strains include BL21 (DE3) pLysS. When the host cell is an animal cell, for example, COS-7 cell, COS-1 cell, CV-1 cell derived from African green monkey fibroblast, COP cell derived from mouse fibroblast, MOP cell, WOP cell, Chinese hamster cell CHO cells derived from CHO DHFR ^- Cell, human HeLa cell, mouse cell-derived C127 cell, mouse cell-derived NIH 3T3 cell and the like. Insect cells include silkworm larvae or silkworm cultured cells, such as BM-N cells, using Bombyx mori nuclear polyhedrosis virus or a vector derived therefrom as a vector. Plant cells can also be used as host cells, and along with suitable vectors, they are widely known in the art.
[0038]
In the genetic engineering technique of the present invention, restriction enzymes, reverse transcriptases, and enzymes for modifying or converting DNA fragments into structures suitable for cloning are known or widely used in the art. Certain DNA modifying / degrading enzymes, DNA polymerases, terminal nucleotidyl transferases, DNA ligases, etc. can be used. Examples of restriction enzymes include RJ Roberts, Nucleic Acids Res., 13: r165, 1985; S. Linn et al. Ed. Nucleases, p. 109, Cold Spring Harbor Lab., Cold Spring Harbor, New York, 1982; RJ Roberts, D. Macelis, Nucleic Acids Res., 19: Suppl. 2077, 1991 and the like. Examples of the reverse transcriptase include reverse transcriptase derived from mouse Moloney leukemia virus (MMLV) and reverse transcriptase derived from chicken myeloblastosis virus (AMV). . As the reverse transcriptase, an RNase H deficient can be preferably used, and in particular, modified MMLV RT lacking RNase H activity can be preferably used, and further, one having high thermal stability is preferable. Suitable reverse transcriptases include MMLV RT (Gibco-BRL) and Superscript RT plus (Life Technologies).
[0039]
Examples of the DNA polymerase include Escherichia coli DNA polymerase, its derivative Klenow fragment, Escherichia coli phage T4 DNA polymerase, Escherichia coli phage T7 DNA polymerase, thermostable DNA polymerase, and the like. Examples of terminal nucleotidyl transferase include deoxynucleotides at the 3′-OH end described in R. Wu et al. Ed., “Methods in Enzymology”, Vol. 100, p. 96, Academic Press, New York (1983). and TdTase to which (dNMP) is added. Examples of DNA modifying / degrading enzymes include exonuclease and endonuclease. , Nuclease S1, Micrococcus nuclease and the like. Examples of the DNA ligase include E. coli DNA ligase, T4 DNA ligase and the like.
Suitable vectors for cloning DNA genes to construct a DNA library include plasmids, λ phage, cosmids, P1 phage, F factor, YAC, etc., preferably λ phage derived vectors, For example, Charon 4A, Charon 21A, λgt10, λgt11, λDASHII, λFIXII, λEMBL3, λZAPII ^TM (Stratagene).
[0040]
A transformant transformed with an expression vector containing a nucleic acid encoding the protein of the present invention is a cell having high expression ability stably by repeating cloning using an appropriate selection marker as necessary. Stocks can be obtained. For example, in a transformant using animal cells as host cells, when the dhfr gene is used as a selection marker, the MTX concentration is gradually increased and cultured, and a resistant strain is selected to encode the protein of the present invention. A cell line capable of amplifying DNA and obtaining higher expression can be obtained. The transformant of the present invention can be cultured under conditions that allow expression of the nucleic acid encoding the protein of the present invention, and the target product can be produced and accumulated. The transformant can be cultured in a medium commonly used in the art. For example, a liquid medium can be suitably used for a transformant having a prokaryotic host such as Escherichia coli or Bacillus subtilis or yeast as a host. The medium contains a carbon source, a nitrogen source, an inorganic substance and the like necessary for the growth of the transformant. Examples of the carbon source include glucose, dextrin, soluble starch, and sucrose. Examples of the nitrogen source include ammonium salts, nitrates, corn steep liquor, peptone, casein, meat extract, malt extract, soybean cake, and potato extract. Examples of inorganic or organic substances and inorganic substances include calcium chloride, sodium dihydrogen phosphate, magnesium chloride, calcium carbonate, and the like. In addition, yeast, vitamins, casamino acids, growth promoting factors and the like may be added. Moreover, in order to make a promoter work efficiently if necessary, a drug such as 3β-indolylacrylic acid can be added. The pH of the medium is preferably about 5-8.
[0041]
For example, Escherichia coli is usually cultured at about 15 to about 45 ° C. for about 3 to about 75 hours, and if necessary, aeration or agitation can be added. When culturing a transformant whose host is an animal cell, for example, a MEM medium containing about 5 to about 20% fetal bovine serum, a PRMI1640 medium, a DMEM medium, or the like is used. The pH is preferably from about 6 to about 8. Culture is usually performed at about 30 ° C. to about 40 ° C. for about 15 to about 72 hours, and aeration and agitation are added as necessary. When extracting from the above cultured cells, the cells or cells are collected by a known method after culturing, suspended in an appropriate buffer solution, and the cells or cells are destroyed by ultrasonic waves, lysozyme and / or freeze-thawing. After that, a method of obtaining a crude extract by centrifugation or filtration can be used as appropriate. A protein denaturant such as urea or guanidine hydrochloride or a surfactant such as Triton X-100 (trade name) or Tween-80 (trade name) may be added to the buffer. When the target product is secreted into the culture solution, after completion of the culture, the cells or cells and the supernatant are separated by a method known per se, and the supernatant is collected. The target product contained in the culture supernatant or the extract thus obtained can be purified by appropriately combining per se known separation and purification methods, for example, a salt such as ammonium sulfate precipitation method. Analysis, gel filtration using Sephadex, for example, ion exchange chromatography using a carrier having a diethylaminoethyl group or carboxymethyl group, for example, a carrier having a hydrophobic group such as a butyl group, an octyl group, or a phenyl group. It can be obtained by purification by the used hydrophobic chromatography method, dye gel chromatography method, electrophoresis method, dialysis, ultrafiltration method, affinity chromatography method, high performance liquid chromatography method or the like. Preferably, it can be purified and separated by treatment with polyacrylamide gel electrophoresis, affinity chromatography with a ligand or the like immobilized. Examples include gelatin-agarose affinity chromatography, heparin-agarose chromatography, and the like.
[0042]
Furthermore, by using a method commonly used in genetic engineering based on the gene sequence of the soluble RAGE according to the present invention, one or more amino acids are appropriately added to the amino acid sequence of the soluble RAGE. Corresponding proteins into which mutations such as substitutions, deletions, insertions, transfers or additions have been introduced can be produced. Such mutation / conversion / modification methods are described in the Japanese Biochemical Society, “Sequel Biochemistry Experiment Course 1, Genetic Research Method II”, p105 (Susumu Hirose), Tokyo Chemical Doujin (1986); Laboratory Lecture 2, Nucleic Acid III (Recombinant DNA Technology), p233 (Susumu Hirose), Tokyo Chemical Doujin (1992); R. Wu, L. Grossman, ed., "Methods in Enzymology", Vol. 154, p. 350 & p. 367, Academic Press, New York (1987); R. Wu, L. Grossman, ed., "Methods in Enzymology", Vol. 100, p. 457 & p. 468, Academic Press, New York ( 1983); JA Wells et al., Gene, 34: 315, 1985; T. Grundstroem et al., Nucleic Acids Res., 13: 3305, 1985; J. Taylor et al., Nucleic Acids Res., 13: 8765 , 1985; R. Wu ed., "Methods in Enzymology", Vol. 155, p. 568, Academic Press, New York (1987); AR Oliphant et al., Gene, 44: 177, 1986, etc. Is mentioned. For example, site-directed mutagenesis using synthetic oligonucleotides (site-directed mutagenesis) (Zoller et al., Nucl. Acids Res., 10: 6487, 1987; Carter et al., Nucl. Acids Res., 13: 4331, 1986), cassette mutagenesis (cassette mutagenesis: Wells et al., Gene, 34: 315, 1985), restriction site selection mutagenesis: Wells et al., Philos. Soc. London Ser A, 317: 415, 1986), alanine scanning (Cunningham & Wells, Science, 244: 1081-1085, 1989), PCR mutagenesis, Kunkel, dNTP [αS] (Eckstein) ), Methods such as region-directed mutagenesis using sulfurous acid or nitrous acid.
[0043]
Furthermore, the obtained protein of the present invention can be modified by a chemical method to modify the amino acid residues contained therein, or can be modified with peptidases such as pepsin, chymotrypsin, papain, bromelain, endopeptidase, and exopeptidase. It can be modified or partially decomposed into its derivatives. In the protein of the present invention, the C-terminus is usually carboxyl group (-COOH) or carboxylate (-COO ^- ), But the C-terminus is an amide (-CONH ₂ ) Or ester (-COOR). Here, R 1 in the ester is, for example, C such as methyl, ethyl, n-propyl, isopropyl or n-butyl. _1-6 Alkyl groups such as C, such as cyclopentyl, cyclohexyl, etc. _3-8 A cycloalkyl group, for example, C such as phenyl, α-naphthyl, etc. _6-12 Aryl groups such as phenyl-C such as benzyl, phenethyl and the like _1-2 Alkyl groups or α-naphthyl-C such as α-naphthylmethyl _1-2 C such as alkyl group _7-14 In addition to the aralkyl group, a pivaloyloxymethyl group that is widely used as an oral ester is used. When the protein of the present invention has a carboxyl group (or carboxylate) other than the C-terminus, the protein of the present invention also includes those in which the carboxyl group is amidated or esterified. As the ester in this case, for example, the above-mentioned C-terminal ester or the like is used.
[0044]
Further, in the protein of the present invention, the amino group of the N-terminal methionine residue in the above-described protein is a protecting group (for example, C-form such as formyl group, acetyl, etc.). _1-5 C such as alkyl-carbonyl group _1-6 Such as an acyl group), a glutamyl group produced by cleavage of the N-terminal side in vivo and pyroglutamylated, a substituent on the side chain of an amino acid in the molecule (for example, -OH, -COOH, Amino group, imidazole group, indole group, guanidino group, etc.) are suitable protecting groups (for example, C such as formyl group, acetyl group, etc.) _1-6 Examples thereof include those protected with an acyl group or the like, or complex proteins such as so-called glycoproteins to which sugar chains are bound. It can also be expressed as a fusion protein when it is produced by a genetic recombination method, and converted and processed into a protein that has substantially the same biological activity as natural soluble RAGE in vivo or in vitro. Good. Although fusion production methods commonly used in genetic engineering can be used, such fusion proteins can also be purified by affinity chromatography using the fusion part. These fusion proteins include those fused to a histidine tag, or β-galactosidase (β-gal), maltose binding protein (MBP), glutathione-S-transferase (GST), thioredoxin (TRX) or Cre Recombinase. Examples include those fused to amino acid sequences. Similarly, a polypeptide can be tagged with a heterogeneous epitope so that it can be purified by immunoaffinity chromatography using an antibody that specifically binds to the epitope. In a more suitable embodiment, the epitope tag includes, for example, AU5, c-Myc, CruzTag 09, CruzTag 22, CruzTag 41, Glu-Glu, HA, Ha.11, KT3, FLAG (registered trademark, Sigma-Aldrich ), Omni-probe, S-probe, T7, Lex A, V5, VP16, GAL4, VSV-G. (Field et al., Molecular and Cellular Biology, 8: pp. 2159-2165 (1988); Evan et al., Molecular and Cellular Biology, 5: pp. 3610-3616 (1985); Paborsky et al., Protein Engineering , 3 (6): pp. 547-553 (1990); Hopp et al., BioTechnology, 6: pp. 1204-1210 (1988); Martin et al., Science, 255: pp. 192-194 (1992) Skinner et al., J. Biol. Chem., 266: pp. 15163-15166 (1991); Lutz-Freyermuth et al., Proc. Natl. Acad. Sci. USA, 87: pp. 6393-6397 (1990) )Such) . A two-hybrid method using yeast can also be used. Further, the fusion protein may be one with a marker that becomes a detectable protein. In a more preferred embodiment, the detectable marker may be a biotin / streptavidin-based Biotin Avi Tag, a fluorescent substance, or the like. Examples of the fluorescent substance include a green fluorescent protein (GFP) derived from a luminescent jellyfish such as Aequorea victorea, and a modified variant (GFP variant) such as EGFP (Enhanced-humanized). GFP), rsGFP (red-shift GFP), yellow fluorescent protein (YFP), green fluorescent protein (GFP), cyan fluorescent protein (CFP), blue Blue fluorescent protein (BFP), Renilla reniformis-derived GFP, etc. (Miyawaki Atsushi edited by experimental medicine, separate post-genome experimental course 3-GFP and bio-easing, Yodosha (2000) ). Detection can also be performed using an antibody (including a monoclonal antibody and a fragment thereof) that specifically recognizes the fusion tag.
[0045]
In a preferred embodiment of the present invention, a marker sequence useful for suitably carrying out the purification, such as a fusion of hexa-histidine peptide, can be used. Expression and purification of such a fusion protein can be carried out using a commercially available kit suitable for it, and can also be carried out according to protocols established by the kit manufacturer or kit vendor. Modifications and alterations of protein structure are described in, for example, the Japanese Biochemical Society edited by “Neurochemistry Experiment Course 1, Protein VII, Protein Engineering”, Tokyo Kagaku Dojin (1993), or the method described therein or cited therein. It can be carried out in the manner described in the literature, or in a substantially similar manner thereto. In addition, as described below, the biological activity may include immunological activity, eg, antigenicity. Among the modifications and alterations, deamination, hydroxylation, phosphorylation, methylation, acetylation, ring opening, ring closure, changing the type of sugar chain contained in a different one, and increasing or decreasing the number of sugar chains contained In addition, substitution to a D-form amino acid residue may be used. These methods are known in the art (eg, TE Creighton, Proteins: Structure and Molecular Properties, pp. 79-86 WH Freeman & Co., San Francisco, USA (1983), etc.).
[0046]
Thus, the human-derived protein of the present invention may have one or more amino acid residues that differ from the natural ones in terms of identity, or one or more amino acid residues that differ in position from the natural ones. Good. The human-derived protein of the present invention has one or more amino acid residues unique to soluble RAGE (for example, 1 to 80, preferably 1 to 60, more preferably 1 to 40, more preferably 1). Deletion analogues lacking ~ 20, in particular 1-10 etc., one or more of the unique amino acid residues (eg 1-80, preferably 1-60, more preferably 1-40) 1 or more (for example, 1 to 80, preferably 1 to 60), more preferably 1 to 20, more preferably 1 to 20, particularly 1 to 10, etc. More preferably, 1 to 40, more preferably 1 to 20, particularly 1 to 10 amino acid residues are added. As long as the domain structure or ligand-binding ability that is characteristic of natural soluble RAGE is maintained, all of the above-mentioned mutants are included in the present invention. Further, it is considered that the soluble RAGE of the present invention may include those having a primary structure conformation or a part thereof that is substantially equivalent to the natural soluble RAGE. It may be included that has biological activity substantially equivalent to RAGE. It can also be one of naturally occurring variants. The human-derived protein of the present invention includes, for example, (1) an amino acid sequence represented by SEQ ID NO: 2 in the sequence listing, (1) a protein having an amino acid sequence of positions 19 to 347, (2) From the group consisting of those having an amino acid sequence from position 347 to position 347, and (3) having at least the amino acid sequence from position 38 to position 117 and having the amino acid sequence from position 332 to position 347 Examples include those having 60% homology or higher than 70% homology to the selected amino acid sequence, and more preferably those having 80% or 90% homology amino acid sequence. In particular, those lacking the transmembrane domain and those having the sequence GluGlyPheAspLysValArgGluAlaGluAspSerProGlnHisMet or a part thereof on the C-terminal side. A part of the human-derived protein of the present invention is a partial peptide of the human-derived protein (that is, a partial peptide of the protein), which is substantially equivalent to the soluble RAGE of the present invention. Any one may be used as long as it has a sufficient activity. For example, the partial peptide of the protein of the present invention is at least 5 or more, preferably 20 or more, more preferably 50 or more, more preferably 70 or more, among the constituent amino acid sequences of the soluble RAGE of the present invention. More preferably 100 or more, in some cases 200 or more peptides having an amino acid sequence, preferably they correspond to consecutive amino acid residues or, for example, SEQ ID NO: in the sequence listing : Among the amino acid sequences represented by 2, there are those having the same homology as described above with respect to the homology to the corresponding region.
[0047]
As used herein, “substantially equivalent” means that the activity of the protein, for example, inhibitory activity, physiological activity, and biological activity are substantially the same. Furthermore, the meaning of the term may include the case of having substantially the same activity, including the activity for the interaction between AGE and RAGE, For example, the activity of suppressing or inhibiting the binding activity to any one of AGEs, the binding activity to any one of AGEs of transmembrane RAGE, and the like can be mentioned. The substantially homogeneous activity means that the activities are qualitatively homogeneous, for example, physiologically, pharmacologically, or biologically homogeneous. For example, the activity such as the activity of inhibiting the binding of AGE to transmembrane RAGE is equivalent (for example, about 0.001 to about 1000 times, preferably about 0.01 to about 100 times, more preferably about 0.1 to about 20 times, It is preferably about 0.5 to about 2 times), but quantitative factors such as the degree of activity and the molecular weight of the protein may be different. Second, amino acid substitutions, deletions, or insertions often do not cause significant changes in the physiological or chemical properties of the polypeptide, in which case the substitutions, deletions, or insertions were made. A polypeptide will be substantially identical to one without such substitutions, deletions or insertions. Substantially identical substitutions of amino acids in the amino acid sequence can be selected from other amino acids of the class to which the amino acid belongs. For example, nonpolar (hydrophobic) amino acids include alanine, phenylalanine, leucine, isoleucine, valine, proline, tryptophan, methionine, etc., and polar (neutral) include glycine, serine, threonine, cysteine, tyrosine, Examples include asparagine and glutamine. Examples of positively charged amino acids (basic amino acids) include arginine, lysine, and histidine. Examples of negatively charged amino acids (acidic amino acids) include aspartic acid and glutamic acid. It is done.
[0048]
For the synthesis of the protein of the present invention and a part of the peptide, methods known in the peptide synthesis field, for example, chemical synthesis methods such as liquid phase synthesis method and solid phase synthesis method can be used. In such a method, for example, a resin for protein or peptide synthesis is used, and appropriately protected amino acids are sequentially bonded onto the desired amino acid sequence on the resin by various known condensation methods. For the condensation reaction, various activating reagents known per se are preferably used. As such reagents, for example, carbodiimides such as dicyclohexylcarbodiimide can be preferably used. When the product has a protecting group, the desired product can be obtained by removing the protecting group as appropriate.
When the protein of the present invention and a part of the peptide thereof are obtained as a free form, they can be converted into a salt by a method known per se or a method analogous thereto, and they can also be converted into a salt. When obtained, it can be converted into a free form or other salt by a method known per se or a method analogous thereto. The salt of the protein of the present invention and a part of the peptide is preferably physiologically acceptable or pharmaceutically acceptable, but is not limited thereto. Examples of such salts include salts with inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, such as acetic acid, formic acid, maleic acid, fumaric acid, succinic acid, citric acid, tartaric acid, malic acid, benzoic acid. And salts with organic acids such as acid, methanesulfonic acid, p-toluenesulfonic acid, and benzenesulfonic acid. Further, examples of the salt include ammonium salts such as salts with organic bases such as ethylamine, dimethylamine, trimethylamine, and hydroxyethylamine.
[0049]
Such soluble RAGE of the present invention and mutants, modifications, derivatives and the like thereof can be subjected to separation / purification treatment as described above. In the present invention, the terms “fragment”, “derivative” and “analog” are transcribed and unspliced or specifically spliced from the polypeptide of SEQ ID NO: 2, SEQ ID NO: 1. In the context of a polypeptide encoded by hnRNA or mRNA, or a polypeptide encoded by genomic DNA, such a polypeptide, when referred to as a “fragment”, “derivative” or “analog” thereof, Means a polypeptide having the same biological function or activity. Thus, analogs include proproteins that can be activated such that the proprotein portion is cleaved to produce an active mature polypeptide. The polypeptide of the present invention may be a recombinant polypeptide, a natural polypeptide or a synthetic polypeptide. In certain preferred embodiments, this is a recombinant polypeptide.
The soluble RAGE disclosed in the prior art has been artificially created by genetic manipulation from the RAGE gene having a transmembrane domain, whereas the soluble RAGE of the present invention. Is a naturally occurring peptide (endogenous peptide or endogenous peptide) that exists in vivo and differs from the RAGE protein in 16 amino acid residues at the C-terminal part. The soluble RAGE artificially created by genetic engineering disclosed in the prior art is not a naturally occurring form itself, so it is non-physiological and removed during the separation and purification process. There may be an immune response against contaminants derived from host insect cells that could not be removed, and it is necessary to administer from the outside of the body, but since it is a protein, it is impossible to do it orally, and it is impossible to administer it to veins, etc. There is a problem of burden on patients and the like, such as requiring injection and forcing the patient to suffer. On the other hand, the soluble RAGE of the present invention has an advantage in that it is a natural form produced endogenously, so it is physiological and less likely to produce antibodies. Thus, if such a pharmaceutical can induce the expression of the soluble RAGE of the present invention, it is possible to protect diabetic patients from complications without causing pain. Since the mutants, modifications, derivatives and the like of the soluble type RAGE of the present invention are derived from the soluble type RAGE of the present invention, the same advantages as the advantageous points of the soluble type RAGE can be expected.
[0050]
On the other hand, the present invention thus includes a DNA sequence encoding the above-described polypeptide, a soluble RAGE polypeptide having all or part of its natural properties, and a DNA sequence encoding an analog or derivative thereof. To do. The polynucleotide of the present invention is a mature protein obtained by adding an additional amino acid at the amino terminus or an additional amino acid at the carboxyl terminus, or a polypeptide endogenous to the mature protein (for example, when it has one or more polypeptide chains in a mature form). It can be one that encodes an amino acid. Such sequences may also play a role in the processing of the precursor to the mature form of the protein, such as facilitating protein transport and transport, extending or shortening the protein half-life. Or may be capable of manipulating the protein to facilitate its detection or production. In general, for example, additional amino acids are processed by cellular enzymes and removed from the mature protein. A precursor protein having a mature form polypeptide fused to one or more prosequences can be an inactive form polypeptide. When the prosequence is removed, such inactive precursors are usually activated. Some or all of the prosequences can be removed prior to activation. Such precursors are usually referred to as proproteins. The polypeptide of the present invention comprises a mature protein, a mature protein to which a leader sequence has been added (which can be referred to as a preprotein), and a precursor of a mature protein having one or more prosequences that are not leader sequences of the preprotein. Or a preproprotein that is a precursor of a proprotein having a leader sequence and one or more prosequences. The prosequence may also be removed at a processing stage that normally yields the active form polypeptide and the mature form polypeptide.
[0051]
Since the DNA sequence of the present invention provides information on the amino acid sequence of mammalian proteins that has not been known so far, the use of such information is also encompassed by the present invention. Such applications include, for example, the design of probes for the isolation and detection of genomic DNA and cDNA of mammals, particularly preferably humans, encoding soluble RAGE and related proteins.
The DNA sequences of the present invention are useful as probes for the isolation and detection of genomic DNA and cDNA, for example, mammals encoding soluble RAGE and related proteins, particularly preferably mice, rats and humans. The probe can optionally be provided with the labels listed in connection with the antibody. For gene isolation, PCR and PCR using reverse transcriptase (RT) (RT-PCR) can be used. Soluble RAGE cDNA and its related DNA are selected from the sequence region of amino acids deduced from the cloned and sequenced soluble RAGE cDNA sequence, and DNA primers are designed and chemically synthesized. The obtained DNA primer can be used for isolation, detection, etc. of soluble RAGE-related genes using PCR, RT-PCR, and other methods. For example, the expression of soluble RAGE mRNA in human tissues ⁺ It can be examined by Northern blot analysis for RNA. When the cDNA of the present invention is used as a probe, for example, detection of soluble RAGE mRNA expression in human tissues, soluble RAGE gene itself, etc. by Northern blotting, Southern blotting, in situ hybridization, etc. A role in the interaction between AGE and RAGE that can be measured and involved in many normal cellular processes, including intracellular protein metabolism in human tissues, activation of hormone precursors, and tissue matrix and bone modifications It can contribute to the development of research such as Alzheimer's disease, diabetic complications, arteriosclerosis, hyperlipidemia, allergic disease, inflammatory disease, neurodegenerative disease and cancer invasion / metastasis. It can also be used for genetic diagnosis of diseases associated with soluble RAGE. Such diagnosis can diagnose abnormalities of nucleic acids encoding the soluble RAGE and related proteins, such as damage, mutation, decreased expression, and excessive expression.
[0052]
According to the present invention, the gene diagnosis method (detection method) of the soluble RAGE of the present invention can be provided. In the genetic diagnosis method, (a) a step of obtaining a nucleic acid sample, (b) the nucleic acid sample obtained in step (a) is subjected to, for example, a PCR method, a nucleic acid amplification method using RNA polymerase, a strand displacement amplification method, etc. A step of obtaining a nucleic acid fragment amplified by a gene amplification, for example, a region including a mutation site that may be present in the soluble RAGE gene, and (c) a step of examining the presence of a mutation in the nucleic acid fragment of step (b) The aspect containing is mentioned. The region containing the mutation site to be amplified is not particularly limited as long as it is a region containing the mutation causing the disease in the base sequence of the soluble RAGE gene of the present invention, for example, Examples include a region containing a base at an arbitrary position in the base sequence shown in SEQ ID NO: 1 in the sequence listing. In the above step (c), an appropriate method can be selected from among detection methods for the presence of mutations known to those skilled in the art in the art, and is not particularly limited. For example, ASPCR (allele-specific It can be detected by examining the length of the DNA fragment obtained by the PCR method. The method for examining the DNA fragment length is not particularly limited, and can be performed using, for example, a fluorescent DNA sequencer. Examples of the mutation detection method used in this step include a method of detecting and examining restriction fragment length polymorphism (RFLP). For detection of mutation, for example, a known mutation detection method such as a hybridization method using an appropriate DNA fragment containing a mutation site as a probe, or an SSCP method (single-chain conformation polymorphism) may be used. . In accordance with the genetic diagnosis of the present invention, genetic diagnosis related to the soluble type RAGE of the present invention is possible. For example, the present invention is considered to be a predisposing factor for determining resistance to and susceptibility to diabetes, cancer, Alzheimer's disease, etc. It is possible to genetically diagnose the expression and polymorphism of soluble RAGE, and to perform gene therapy that lowers the risk of suffering from a related disease based on the diagnosis result.
[0053]
The soluble RAGE and its related proteins, fragments thereof, and nucleic acids including DNA (including mRNA and oligonucleotide) disclosed herein can be used alone or organically and further described below. It can be applied to genomics and proteomics techniques in combination with techniques (antisense methods, antibodies including monoclonal antibodies, transgenic animals, etc.) as appropriate. For example, the soluble RAGE mutant can also be used for functional analysis using a dominant negative effect. In addition, there are applications to RNAi (RNA interference) technology using double-stranded RNA (dsRNA). Thus, gene polymorphism analysis centering on single nucleotide polymorphisms (SNP), gene expression analysis using nucleic acid array and protein array, gene function analysis, protein-protein interaction analysis, related disease analysis, disease treatment Drug analysis can be performed. For example, in nucleic acid array technology, a cDNA library is used, or DNA obtained by PCR technology is placed on a substrate with a spotting device at high density, and sample analysis is performed using hybridization. The arraying is performed by attaching the DNA to each unique position of a substrate such as a glass slide, a silicon plate, or a plastic plate using a needle or a pin, or using an inkjet printing technique or the like. Can do. Data is acquired by observing signals obtained as a result of hybridization on the nucleic acid array. The signal may be obtained from a label such as a fluorescent dye (for example, Cy3, Cy5, BODIPY, FITC, Alexa Fluor dyes (trade name), Texas red (trade name), etc.). A laser scanner or the like can be used for detection, and the obtained data may be processed by a computer system having a program according to an appropriate algorithm. In addition, protein array technology may utilize tagged recombinant expressed protein products, including two-dimensional electrophoresis (2-DE), mass spectrometry (MS) including enzyme digestion fragments (including electrolysis). Includes technologies such as spray ionization (ESI), matrix-assisted laser desorption / ionization (MALDI), MALDI-TOF analyzer, ESI-3 quadrupole analyzer Staining techniques, isotope labeling and analysis, image processing techniques, etc. can be used. Therefore, the present invention may also include software, databases, etc. relating to the soluble RAGE obtained or used above and antibodies thereto.
[0054]
In transferring the DNA obtained in the present invention (for example, DNA encoding soluble RAGE) to a target animal, it is bound as a DNA fragment or downstream of a promoter capable of expressing the DNA in animal cells. It is generally advantageous to use it. For example, when soluble RAGE DNA is introduced into a mouse, gene constructs bound to downstream of various promoters capable of expressing animal-derived soluble RAGE DNA having high homology with this are fertilized by the target animal. Gene-introduced (transgenic) mice that produce highly soluble RAGE can be produced by microinjection into eggs, such as mouse fertilized eggs. The mouse is not particularly limited to a pure mouse, for example, C57BL / 6, Balb / C, C3H, (C57BL / 6 × DBA / 2) F ₁ (BDF ₁ ) And the like. As this promoter, for example, a virus-derived promoter, a ubiquitous expression promoter such as metallothionein, and the like can be preferably used. In addition, when the soluble RAGE DNA is introduced, it can be recombined into a recombinant retrovirus and used. Preferably, a mouse fertilized egg into which the target DNA has been introduced can be grown, for example, using a foster mother mouse such as ICR.
Transfer of the DNA obtained by the present invention (for example, DNA encoding soluble RAGE) at the fertilized egg cell stage is ensured to be present in all germ cells and somatic cells of the subject animal. The presence of DNA encoding soluble RAGE in the germ cells of the produced animal after DNA transfer means that all of the progeny of the produced animal have DNA encoding the soluble RAGE in all the germ cells and somatic cells. Means that. The offspring of this type of animal that inherited the gene has the potential to express the soluble RAGE in all of its germ cells and somatic cells.
[0055]
The soluble RAGE DNA-introduced animal can be reared in a normal breeding environment as a DNA-bearing animal after confirming that the gene is stably retained by mating. Furthermore, by mating male and female animals carrying the target DNA, homozygous animals having the transgene on both homologous chromosomes are obtained, and by mating this male and female animals, all offspring will have the DNA. Can be bred to passage. The animal into which the soluble RAGE DNA has been introduced has high expression of the soluble RAGE protein, and thus is useful as an animal for screening for inhibitors against the soluble RAGE protein. . Further, it is useful as an antisense oligonucleotide capable of inhibiting the expression of soluble RAGE gene, for example, an animal for screening for antisense DNA and the like.
This transgenic animal can also be used as a cell source for tissue culture. For example, it is possible to analyze proteins related to the interaction between AGE and RAGE by directly analyzing DNA or RNA in the tissues of transgenic mice or analyzing proteins and tissues expressed by genes. it can. Cells of tissues that produce the soluble RAGE are cultured by standard tissue culture techniques, and using these cells, for example, blood cells such as brain, thymus, vascular endothelial cells, blood cells, testis, brain, intestines, kidneys, The function of cells derived from other tissues can be studied. In addition, by using the cells, it is possible to contribute to the development of medicines that enhance the functions of various tissues, for example. In addition, if there is a high-expressing cell line, soluble RAGE can be isolated and purified therefrom. Techniques related to transgenic mice include, for example, Brinster, RL, et al.,; Proc. Natl. Acad. Sci. USA, 82: 4438, 1985; Costantini, F. & Jaenisch, R. (eds): Genetic manipulation of the early mammalian embryo, Cold Spring Harbor Laboratory, 1985, or the like, a method described in the literature cited therein, or a modification thereof.
[0056]
A mutant mouse (knockout mouse) that has a mutation in the gene obtained in the present invention (for example, DNA encoding mouse soluble RAGE corresponding to soluble RAGE) and does not express mouse soluble RAGE at all is created. be able to. For example, targeting with a mutant gene in which a gene cassette consisting of a neo resistance gene-polyA addition signal is inserted in an exon that is located near the center of the genomic DNA of approximately 8 kb, including 4 kb before and after the translation initiation codon of the gene. A vector can be constructed. Examples of gene cassettes to be inserted include DT-A cassettes, tk cassettes, and lacZ cassettes in addition to neo-resistant gene cassettes. The targeting vector is opened in a linear form, introduced into established mouse embryonic stem cells (ES cells) by electroporation, and further cultured to select ES cells that have acquired neo resistance. ES cells can be prepared by selecting from mouse strains such as 129, C57BL / 6, F1 (C57BL / 6 × CBA) mice. ES cells that have acquired neo resistance are assumed to have undergone homologous recombination with a targeting vector inserted with a gene cassette in the mouse soluble RAGE gene region, and at least one mouse soluble RAGE gene allele is destroyed. Thus, mouse soluble RAGE cannot be expressed normally. For selection, an appropriate method is selected depending on the inserted gene cassette, and the introduction of the mutation can be confirmed using a method such as PCR, Southern hybridization or Northern hybridization.
[0057]
Transduced ES cells are injected into 8-cell embryos taken from C57BL / 6, BALB / c, ICR mice, etc., and cultured for 1 day, then transplanted to a foster parent such as ICR. Can be grown up to individuals. The born mouse is a chimeric mouse derived from a mutant ES cell and a normal host embryo, and the degree of ES cell-derived cells is determined by the individual's coat color. Therefore, it is desirable to combine ES cells and host embryos with different hair colors. The resulting chimeric mouse has a heterozygous mutation, and a homozygous mouse can be obtained by appropriately crossing these. The homozygous mutant mice thus obtained are disrupted only in the mouse soluble RAGE gene in all germ cells and somatic cells, do not express any mouse soluble RAGE gene, Has a similar expression system.
This knockout mouse is useful for analyzing the role of soluble RAGE in the life cycle of individuals such as development, growth, reproduction, aging and death and the function of soluble RAGE in each organ and tissue in comparison with normal mice It is. It can also be applied to drug development related to the interaction between AGE and RAGE. The knockout mouse can be used not only as a model animal but also as a cell source for tissue culture, and can be used for functional analysis of soluble RAGE at the cell level. For example, Mansour, SL, et al.,; Nature, 336: 348-352, 1988; Joyner, AL, ed .; Gene targeting, IRL Press, 1993; Shin Aizawa, Gene targeting Production of mutant mice using ES cells, methods described in documents such as Yodosha, 1995, or the methods described in the documents cited therein, and modifications thereof can be used.
[0058]
According to the present invention, an antisense oligonucleotide (nucleic acid) capable of inhibiting the expression of soluble RAGE gene is cloned on the basis of the nucleotide sequence information of the DNA encoding soluble RAGE that has been cloned or determined. Can be designed and synthesized. Such oligonucleotides (nucleic acids) can hybridize with mRNA of soluble RAGE gene, inhibit the function of the mRNA, or interact with soluble RAGE-related mRNA. It can regulate / control the expression of soluble RAGE gene. Oligonucleotides complementary to selected sequences of soluble RAGE-related genes and oligonucleotides that can specifically hybridize with soluble RAGE-related genes are soluble in vivo and in vitro. It is useful for regulating / controlling the expression of and for the treatment or diagnosis of diseases associated therewith. The term “corresponding” means homologous to or complementary to a specific sequence of nucleotides, base sequences or nucleic acids including genes. “Corresponding” between a nucleotide, base sequence or nucleic acid and peptide (protein) usually refers to the amino acid of the peptide (protein) in the direction derived from the nucleotide (nucleic acid) sequence or its complement. . 5 ′ end hairpin loop of the gene, 5 ′ end 6-base pair repeat, 5 ′ end untranslated region, polypeptide translation initiation codon, protein coding region, ORF translation initiation codon, 3 ′ end untranslated region, 3 ′ The end palindromic region and the 3 ′ end hairpin loop can be selected as preferred target regions, but any region within the gene can be selected as the target.
[0059]
The relationship between the nucleic acid of interest and the oligonucleotide complementary to at least a part of the region of interest means the relationship between the oligonucleotide that can hybridize to the object and that it is “antisense”. it can. Antisense oligonucleotides are polydeoxynucleotides containing 2-deoxy-D-ribose, polydeoxynucleotides containing D-ribose, and other types of polysides that are N-glycosides of purine or pyrimidine bases. Nucleotides or other polymers with a non-nucleotide backbone (eg, commercially available protein nucleic acids and synthetic sequence specific nucleic acid polymers) or other polymers containing special linkages, provided that the polymer is found in DNA or RNA And a nucleotide having a configuration allowing base attachment). They can be double-stranded DNA, single-stranded DNA, double-stranded RNA, single-stranded RNA, and also DNA: RNA hybrids, and also unmodified polynucleotides or unmodified oligonucleotides, as well as known modifications Of, for example, those with a label known in the art, capped, methylated, one or more natural nucleotides replaced with analogs, intramolecular nucleotide modifications Such as those having uncharged bonds (eg methylphosphonates, phosphotriesters, phosphoramidates, carbamates, etc.), charged bonds or sulfur-containing bonds (eg phosphorothioates, phosphorodithioates, etc.) What you have, such as proteins (nucleases, nuclease inhibitors, toxins, antibodies, signal peptides, poly-L- Lysine, etc.) and sugars (eg, monosaccharides), etc., having side chain groups, intercurrent compounds (eg, acridine, psoralen, etc.), chelate compounds (eg, metals, having radioactivity) It may be one containing a metal, boron, oxidizing metal, etc., one containing an alkylating agent, or one having a modified bond (for example, α-anomeric nucleic acid etc.). As used herein, “nucleoside”, “nucleotide” and “nucleic acid” may contain not only known purine and pyrimidine bases but also those having other modified heterocyclic bases. Such modifications may include methylated purines and pyrimidines, acylated purines and pyrimidines, or other heterocycles. Modified nucleosides and modified nucleotides may also be modified at the sugar moiety, eg, one or more hydroxyl groups are replaced by halogens or aliphatic groups, or converted to functional groups such as ethers, amines, etc. May have been.
[0060]
The antisense nucleic acid of the present invention is RNA, DNA, or a modified nucleic acid. Specific examples of the modified nucleic acid include, but are not limited to, nucleic acid sulfur derivatives and thiophosphate derivatives, and those resistant to degradation of polynucleoside amides and oligonucleoside amides. The antisense nucleic acid of the present invention can be preferably designed according to the following policy. That is, to make the antisense nucleic acid more stable in the cell, to increase the cell permeability of the antisense nucleic acid, to increase the affinity for the target sense strand, and to be antitoxic if toxic Make sense nucleic acids less toxic.
Many such modifications are known in the art, e.g. J. Kawakami et al., Pharm Tech Japan, 8: 247, 1992; 8: 395, 1992; ST Crooke et al. Ed., Antisense Research and Applications, CRC There are disclosures in Press, 1993, etc. The antisense nucleic acid of the present invention may be altered or contain modified sugars, bases and bonds, provided in special forms such as liposomes, microspheres, applied by gene therapy, It can be given in an added form. These additional forms include polycations such as polylysine that act to neutralize the charge of the phosphate group skeleton, lipids that enhance interaction with cell membranes and increase nucleic acid uptake ( For example, hydrophobic substances such as phospholipids and cholesterols). Preferred lipids to be added include cholesterol and derivatives thereof (for example, cholesteryl chloroformate, cholic acid, etc.). These can be attached to the 3 ′ end or 5 ′ end of the nucleic acid and can be attached via a base, sugar, or intramolecular nucleoside bond. Examples of the other group include a cap group specifically arranged at the 3 ′ end or 5 ′ end of a nucleic acid, which prevents degradation by nucleases such as exonuclease and RNase. Such capping groups include, but are not limited to, hydroxyl protecting groups known in the art, including glycols such as polyethylene glycol and tetraethylene glycol.
[0061]
The inhibitory activity of antisense nucleic acid can be examined using the transformant of the present invention, the in vivo or in vitro gene expression system of the present invention, or the in vivo or in vitro translation system of soluble RAGE. The nucleic acid can be applied to cells by various methods known per se.
The above-described research results of the present inventors have provided a method for obtaining a target soluble RAGE by transferring a soluble RAGE gene and a recombinant DNA molecule into a host and expressing the soluble RAGE. The Thus, according to the present invention, a recombinant or transfectant that substantially expresses a soluble RAGE gene, a method for producing the same, and a use thereof are also provided.
In another aspect, the present invention relates to a natural (native) soluble RAGE belonging to the RAGE family (particularly, an endogenous soluble RAGE), and has activity against the interaction between AGE and RAGE. (For example, it has the activity of inhibiting or inhibiting the binding of AGE to membrane-bound RAGE) and the amino acid sequence Glu of SEQ ID NO: 2 in the sequence listing is located on the C-terminal side. ³³² ~ Met ³⁴⁷ A protein or a salt thereof, which is a kind of a polypeptide having at least 1 to 16 consecutive amino acid residues, and has substantially the same activity as natural human soluble RAGE, Preferably, a polypeptide having at least a part or all of the protein having substantially the same activity or substantially the same primary structure conformation as soluble RAGE or a salt thereof, such as Escherichia coli It can relate to nucleic acids such as DNA and RNA that can be expressed in eukaryotes such as prokaryotes or mammalian cells. Such a nucleic acid, particularly DNA, includes (a) a sequence capable of encoding the amino acid sequence represented by SEQ ID NO: 2 in the sequence listing or a sequence complementary thereto, (b) the DNA sequence of (a) or a fragment thereof. It can be a sequence capable of hybridizing and (c) a sequence having a degenerate code capable of hybridizing to the sequence (a) or (b). Here, the hybridization conditions can be stringent conditions. Prokaryotic organisms such as Escherichia coli or eukaryotic organisms such as mammalian cells that can be transformed with such nucleic acids and express the polypeptide of the present invention also feature the present invention.
[0062]
Thus, typically, the object of the present invention is to use a soluble RAGE gene, a probe derived therefrom, or, if necessary, an inhibitor against soluble RAGE, to dissolve soluble RAGE in a test sample. Alternatively, it is an object of the present invention to provide an excellent method for detecting and fractionating and quantifying the gene, and further the production cell, and a reagent kit therefor. It is understood that the present invention includes all the reagents in the reagent kit capable of detecting and fractionating and quantifying such soluble RAGE or its gene, as well as production cells, in the embodiment. Furthermore, the object of the present invention is to detect soluble RAGE or its gene, and further to produce and differentiate quantitatively by using the above-mentioned method, so that intracellular protein metabolism, activation of hormone precursors, tissue matrix or bone The role of AGE and RAGE interactions involved in many normal cellular processes, such as alterations in diabetes, diabetic complications, arteriosclerosis, thrombosis, hyperlipidemia, Alzheimer's disease, allergic diseases, inflammatory diseases, It is an object of the present invention to provide a method, a reagent or a diagnostic agent capable of monitoring many diseases such as osteoporosis, neurodegenerative diseases and cancer invasion / metastasis. Therefore, the above reagents are used for various purposes such as medical / physiological fields, research / analysis / measurement of responses / symptoms / diseases caused by the interaction between AGE and RAGE, diagnosis, prevention, treatment, etc. It is understood that all that is included in that embodiment of the invention.
[0063]
The soluble RAGE of the present invention or a salt thereof has an activity with respect to the interaction between AGE and RAGE, for example, has an activity to suppress or inhibit the binding between AGE and transmembrane RAGE.・ It is considered to be an important factor in aging, adult pathological vascular disorder, and cancer. The protein is considered to be useful for the treatment of soluble RAGE-related dysfunction diseases such as soluble RAGE aplasia, soluble RAGE expression deficiency, and soluble RAGE gene deficiency. In other words, if a drug containing soluble RAGE, a mutant, a modified product, or a derivative is used, it is possible to bring a patient with a disease due to insufficient activity of soluble RAGE into a healthy state. . The polypeptide of the present invention is one of AGE activity inhibitors, and is useful as a medicament for the treatment and / or prevention of various diseases that occur when the expression level of the protein or the AGE activity inhibition activity is decreased. It is. In addition, the polypeptide of the present invention is useful as a medicament for treating and / or preventing various diseases that occur when AGE activity is enhanced. For example, in the case of a patient who does not have sufficient biological activity in cells due to a decrease or deficiency of soluble RAGE in the living body or who has abnormal symptoms, (A) (B) by administering a nucleic acid such as the DNA of the present invention to the patient to express the protein of the present invention in vivo, or (C) By transplanting a cell into which a nucleic acid such as the DNA of the present invention can be expressed in the patient, the symptom in the patient is improved by, for example, supplementing the protein of the present invention into the living body.
[0064]
A compound (agonist or promoter) or a salt thereof that promotes functions such as biological activity of the soluble RAGE of the present invention (for example, an activity that suppresses or inhibits the binding between AGE and transmembrane RAGE) Treatment of various diseases such as soluble RAGE dysfunction, diabetic complications, arteriosclerosis, thrombosis, hyperlipidemia, Alzheimer's disease, neurodegenerative disease, rheumatoid arthritis and cancer invasion / metastasis / Or can be used as a medicament useful as a preventive agent. On the other hand, a compound (antagonist or inhibitor) that inhibits functions such as biological activity of the soluble RAGE of the present invention (for example, an activity that suppresses or inhibits the binding between AGE and transmembrane RAGE) or the like The salt can be used as a medicament for treating and / or preventing various diseases such as hypersoluble RAGE dysfunction, diseases and illnesses caused by the interaction between AGE and RAGE, and cancer. For example, soluble RAGE has an action of suppressing or inhibiting the physiological action or biological activity of AGE, and is useful as a medicament for treating diseases caused by excessive AGE.
[0065]
Thus, the polypeptide such as the soluble RAGE of the present invention has functions such as the biological activity of the polypeptide such as the soluble RAGE of the present invention (for example, binding between AGE and transmembrane RAGE). It is useful as a reagent for screening a compound (agonist), a compound (antagonist) or a salt thereof that promotes an activity that suppresses or inhibits. Thus, a biology such as a protein such as the soluble RAGE of the present invention, a partial peptide thereof or a salt thereof using the polypeptide such as the soluble RAGE of the present invention, a part of the peptide or a salt thereof. A method for screening a compound (agonist), a compound (antagonist) or a salt thereof that promotes a function (such as an activity that suppresses or inhibits the physiological action or biological activity of AGE) is also provided.
In the screening, for example, (i) the protein of the present invention, a part of the peptide, or a salt thereof (which may contain a transformant expressing the protein, the same shall apply hereinafter) is contacted with a substrate such as AGE. And (ii) the case where the substrate and the test sample are contacted with the protein of the present invention, a part of the peptide, or a salt thereof. Specifically, in the above screening, the biological activity (for example, activity related to the interaction between AGE and RAGE) is measured and compared. The screening may be performed in the presence of membrane-bound RAGE.
Any substrate can be used as long as it can be a substrate such as AGE. For example, it can be selected from those used for the purpose of measuring the interaction between AGE and RAGE, and preferably, synthesized AGE or the like can be used. The substrate can be used as it is, but preferably a substrate labeled with a fluorescent, enzyme or radioactive substance such as fluorescein can be used.
[0066]
Examples of the test sample include proteins, peptides, non-peptidic compounds, synthetic compounds, fermentation products, plant extracts, tissue extracts such as animals, and cell extracts. Examples of test compounds used for test samples preferably include anti-AGE antibodies, anti-RAGE antibodies, AGE-RAGE binding inhibitors, compounds having inhibitory activity against protein glycation reactions, especially synthetic compounds, etc. You can leave. These compounds may be novel compounds or known compounds. The screening can be performed according to a usual method for measuring binding activity, and can be performed with reference to, for example, methods known in the art. In addition, various labels, buffer systems, and other appropriate reagents can be used, or the procedures described therein can be performed. The peptide used can be treated with an activator, or its precursor or latent form can be converted into an active form in advance. The measurement is usually performed in a buffer solution that does not adversely affect the reaction, such as a Tris-HCl buffer solution or a phosphate buffer solution, for example, at a pH of about 4 to about 10 (preferably, a pH of about 6 to about 8). It can be carried out. In these individual screenings, the normal conditions and procedures in each method are added to the ordinary technical considerations of those skilled in the art, and the soluble RAGE of the present invention or a polypeptide having substantially the same activity as the polypeptide or What is necessary is just to build the measurement system relevant to a peptide. Details of these general technical means can be referred to reviews, books, etc. (see, for example, Methods in Enzymology, Academic Press (USA)).
[0067]
The compound obtained by using the screening method or the screening kit of the present invention or a salt thereof is the above-mentioned test compound, for example, peptide, protein, non-peptide compound, synthetic compound, fermentation product, cell extract, plant extract, It is a compound selected from animal tissue extracts and the like and is a compound that promotes or inhibits the function of the protein of the present invention. Examples of the salt of the compound include pharmaceutically acceptable salts. Examples include salts with inorganic bases, salts with organic bases, salts with inorganic acids, salts with organic acids, salts with basic or acidic amino acids, and the like. Preferable examples of the salt with an inorganic base include alkali metal salts such as sodium salt and potassium salt, alkaline earth metal salts such as calcium salt and magnesium salt, aluminum salt and ammonium salt. Preferable examples of the salt with an organic base include, for example, trimethylamine, triethylamine, pyridine, picoline, 2,6-lutidine, ethanolamine, diethanolamine, triethanolamine, cyclohexylamine, dicyclohexylamine, N, N′-dibenzyl. And salts with ethylenediamine and the like. Preferable examples of the salt with inorganic acid include salts with hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid and the like. Preferable examples of salts with organic acids include formic acid, acetic acid, propionic acid, fumaric acid, oxalic acid, tartaric acid, maleic acid, citric acid, succinic acid, malic acid, methanesulfonic acid, benzenesulfonic acid, benzoic acid And salts with acids. Preferable examples of salts with basic amino acids include, for example, salts with arginine, lysine, ornithine, and preferable examples of salts with acidic amino acids include, for example, salts with aspartic acid, glutamic acid, and the like. Is mentioned.
[0068]
In the present specification, the term “antibody” may be used in a broad sense, and it may be a single monoclonal antibody against a desired soluble RAGE polypeptide and related peptide fragments or various epitopes. It may be an antibody composition with specificity and includes monovalent or multivalent antibodies as well as polyclonal and monoclonal antibodies, and further represents intact molecules and fragments and derivatives thereof. , F (ab ') ₂ , Fab 'and Fab fragments, and further chimeric or hybrid antibodies with at least two antigens or epitope binding sites, or bispecifics such as quadrome, triome, etc. Recombinant antibodies, interspecific hybrid antibodies, anti-idiotypic antibodies, and those that are chemically modified or processed to be considered as derivatives thereof, applying known cell fusion or hybridoma technology or antibody engineering, Antibodies obtained using semi-synthetic techniques, conventional techniques known from the point of view of antibody production, antibodies prepared using DNA recombination techniques, target antigens described and defined herein Antibodies which have neutralizing properties or binding properties with respect to the substance or target epitope may be included. Particularly preferred antibodies of the present invention are those that can specifically identify a naturally-occurring soluble RAGE polypeptide. For example, from the full-length RAGE polypeptide or the full-length RAGE polypeptide, the N-terminal side or the C-terminal side thereof. It can be recognized by distinguishing it from a known solubilized RAGE polypeptide produced by cleaving.
[0069]
Monoclonal antibodies produced against antigenic material are produced using any method that can provide for the production of antibody molecules by a series of cell lines in culture. The modifier “monoclonal” indicates the character of the antibody as being derived from a substantially homogeneous population of antibodies, and should not be construed as requiring production of the antibody by any particular method. Don't be. Individual monoclonal antibodies are those that contain a population of antibodies that are identical except that only a small amount of naturally occurring variants may be present. Monoclonal antibodies have a high specificity, which is directed against sites with a single antigenicity. In contrast to conventional (polyclonal) antibody preparations that typically contain different antibodies directed against different antigenic determinants (epitopes), each monoclonal antibody is a single antigen on that antigen. It is intended for determinants. In addition to its specificity, monoclonal antibodies are also superior in that they are synthesized by hybridoma culture and have no or little contamination with other immunoglobulins. Monoclonal antibodies include hybrid antibodies and recombinant antibodies. They can either replace the variable domain with a constant domain (eg, a humanized antibody) or replace the light chain with a heavy chain, regardless of its origin or type of immunoglobulin class or subclass, as long as it exhibits the desired biological activity. Can be obtained by replacement, replacement of one chain with another, or fusion with a heterogeneous protein (eg, US Pat. No. 4,816,567; Monoclonal Antibody Production Techniques and Applications, pp .79-97, Marcel Dekker, Inc., New York, 1987). Examples of suitable methods for producing monoclonal antibodies include the hybridoma method (G. Kohler and C. Milstein, Nature, 256, pp. 495-497 (1975)); the human B cell hybridoma method (Kozbor et al., Immunology). Today, 4, pp.72-79 (1983); Kozbor, J. Immunol., 133, pp.3001 (1984); Brodeur et al., Monoclonal Antibody Production Techniques and Applications, pp.51-63, Marcel Dekker, Inc., New York (1987); trioma method; EBV-hybridoma method (Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp. 77-96 (1985)) (human monoclonal antibody (Methods for production); U.S. Pat.No. 4,946,778 (Technology for production of single chain antibodies) and the following literature on antibodies:
[0070]
S. Biocca et al., EMBO J, 9, pp.101-108 (1990); RE Bird et al., Science, 242, pp.423-426 (1988); MA Boss et al., Nucl. Acids Res ., 12, pp. 3791-3806 (1984); J. Bukovsky et al., Hybridoma, 6, pp. 219-228 (1987); M. DAINO et al., Anal. Biochem., 166, pp. 223 -229 (1987); JS Huston et al., Proc. Natl. Acad. Sci. USA, 85, pp. 5879-5883 (1988); PT Jones et al., Nature, 321, pp. 522-525 (1986) JJ Langone et al. (Ed.), "Methods in Enzymology", Vol. 121 (Immunochemical Techniques, Part I: Hybridoma Technology and Monoclonal Antibodies), Academic Press, New York (1986); S. Morrison et al. , Proc. Natl. Acad. Sci. USA, 81, pp. 6851-6855 (1984); VT Oi et al., BioTechniques, 4, pp. 214-221 (1986); L. Riechmann et al., Nature, 332, pp.323-327 (1988); A. Tramontano et al., Proc. Natl. Acad. Sci. USA, 83, pp.6736-6740 (1986); C. Wood et al., Nature, 314, pp.446-449 (1985); Nature, 314, pp.452-454 (1985) or references cited in it (refer to it for descriptions in them) More included in this disclosure).
[0071]
As long as they exhibit the desired biological activity, the monoclonal antibodies according to the present invention have a corresponding sequence of an antibody in which a part of the heavy and / or light chain is derived from a specific species or belongs to a specific antibody class or subclass. While the same or homologous, the remainder of the chain is a “chimeric” antibody (immunoglobulin) that is identical or homologous to the corresponding sequence of an antibody derived from another species or belonging to another antibody class or subclass. In particular (US Pat. No. 4,816,567; Morrison et al., Proc. Natl. Acad. Sci. USA, 81, pp. 6851-6855 (1984)). Hereinafter, taking a monoclonal antibody as an example, the production of the antibody will be described in detail. The monoclonal antibody of the present invention may be a monoclonal antibody obtained by utilizing cell fusion technology using myeloma cells, and can be prepared, for example, by the following steps.
1. Preparation of immunogenic antigen
2. Immunization of animals with immunogenic antigens
3. Preparation of myeloma cells (myeloma cells)
4). Cell fusion between antibody-producing cells and myeloma cells
5. Selection and monocloning of hybridomas (fusion cells)
6). Production of monoclonal antibodies
[0072]
1. Preparation of immunogenic antigen
As described above, as described above, a soluble RAGE polypeptide or an isolated fragment derived therefrom can be used, but the amino acid sequence information of the determined soluble RAGE is used as the antigen. In addition, an appropriate oligopeptide can be chemically synthesized and used as an antigen. Typically, SEQ ID NO: 2
(1) an amino acid sequence of at least positions 332 to 347;
(2) amino acid sequence from position 1 to position 117;
(3) the amino acid sequence from position 19 to position 347; and
(4) Amino acid sequence from position 1 to position 347
Peptides having at least 5 consecutive amino acids among amino acid residues present in a region selected from the group consisting of:
The antigen can be used to immunize an animal by mixing it with an appropriate adjuvant as it is, but may be an immunogenic conjugate or the like. For example, the antigen used as an immunogen is a fragment of soluble RAGE, or a synthetic polypeptide fragment obtained by selecting a characteristic sequence region based on its amino acid sequence, designing the polypeptide, and chemically synthesizing it. It may be. In addition, the fragment can be combined with various carrier proteins via an appropriate condensing agent to form an immunogenic conjugate such as a hapten-protein, and this can be used to react only with a specific sequence (or only a specific sequence). Can also be used to design monoclonal antibodies. A cysteine residue or the like can be added in advance to the polypeptide to be designed so that an immunogenic conjugate can be easily prepared. In binding to carrier proteins, the carrier proteins can first be activated. For such activation, introduction of an activated linking group can be mentioned.
The activated linking group includes (1) activated ester or activated carboxyl group, such as nitrophenyl ester group, pentafluorophenyl ester group, 1-benzotriazole ester group, N-succinimide ester group, etc. A dithio group, such as a 2-pyridyldithio group, can be mentioned. Examples of carrier proteins include keyholes, limpets, hemocyanin (KLH), bovine serum albumin (BSA), ovalbumin, globulins, polylysine and other polypeptides, and bacterial cell components such as BCG.
[0073]
2. Immunization of animals with immunogenic antigens
Immunization can be carried out by methods known to those skilled in the art, for example, Shigeru Muramatsu, et al., Laboratory Biology Laboratory 14, Immunobiology, Maruzen Co., Ltd., 1985, Japan Biochemical Society, secondary biochemistry experiment Lecture 5, Immunobiochemical Research Method, Tokyo Chemical Doujin, 1986, Japan Biochemical Society, New Chemistry Laboratory 12, Molecular Immunology III, Antigen / Antibody / Complement, Tokyo Chemical Dojin, 1992, etc. It can be performed according to. Immunization is accomplished by injecting the mammal with the immunizing agent (optionally with adjuvant) one or more times. Typically, the immunizing agent and / or adjuvant is administered to a mammal by multiple subcutaneous injections or intraperitoneal injections. Examples of the immunizing agent include those containing the above antigen peptide or a related peptide fragment thereof. The immunizing agent may be used in the form of a conjugate with a protein known to be immunogenic in the mammal to be immunized (for example, the above carrier proteins). Examples of adjuvants include Freund's complete adjuvant, Ribi adjuvant, pertussis vaccine, BCG, lipid A, liposome, aluminum hydroxide, silica and the like. Immunization is performed using, for example, mice such as BALB / c, hamsters, and other suitable animals. The dose of the antigen is, for example, about 1 to 400 μg / animal for mice, and is generally injected intraperitoneally or subcutaneously into the host animal, and thereafter every 1 to 4 weeks, preferably every 1 to 2 weeks. The booster immunization is repeated about 2 to 10 times internally, subcutaneously, intravenously or intramuscularly. As a mouse for immunization, BALB / c mouse, F1 mouse of BALB / c mouse and other mouse, and the like can be used. If necessary, an antibody titer measurement system can be prepared and the antibody titer can be measured to confirm the degree of animal immunity. The antibody of the present invention may be obtained from the immunized animal thus obtained and includes, for example, antiserum, polyclonal antibody and the like.
[0074]
3. Preparation of myeloma cells (myeloma cells)
The infinitely proliferative strain (tumor cell line) used for cell fusion can be selected from cell lines that do not produce immunoglobulins, such as P3-NS-1-Ag4-1 (NS-1, Eur. J. Immunol , 6: 511-519, 1976), SP-2 / 0-Ag14 (SP-2, Nature, 276: 269-270,1978), P3-X63-Ag8-U1 derived from mouse myeloma MOPC-21 cell line (P3U1, Curr. Topics Microbiol. Immunol., 81: 1-7, 1978), P3-X63-Ag8 (X63, Nature, 256: 495-497, 1975), P3-X63-Ag8-653 (653, J Immunol., 123: 1548-1550, 1979) can be used. 8-Azaguanine-resistant mouse myeloma cell line is prepared by adding antibiotics such as penicillin and amikacin, fetal bovine serum (FCS), etc. to cell culture medium such as Dulbecco's MEM medium (DMEM medium) and RPMI-1640 medium. It is subcultured in a medium supplemented with azaguanine (for example, 5-45 μg / ml), but it can be subcultured in a normal medium 2 to 5 days before cell fusion to prepare the required number of cell lines. The cell line used was prepared by thawing the cryopreserved strain completely at about 37 ° C, washing it with a normal medium such as RPMI-1640 medium three times or more, and then culturing in the normal medium to prepare the required number of cell lines. There may be.
[0075]
4). Cell fusion between antibody-producing cells and myeloma cells
2. In the animal immunized according to the above step, for example, a mouse, the spleen is removed 2 to 5 days after the final immunization, and then a spleen cell suspension is obtained. In addition to spleen cells, lymph node cells in various parts of the body can be obtained and used for cell fusion. The spleen cell suspension thus obtained and 3. The myeloma cell line obtained according to the above step is placed in a cell medium such as minimum essential medium (MEM medium), DMEM medium, RPMI-1640 medium, and a cell fusion agent such as polyethylene glycol is added. As the cell fusion agent, those known in various other fields can be used, and examples thereof include inactivated Sendai virus (HVJ: Hemagglutinating Virus of Japan). Preferably, for example, 0.5 to 2 ml of 30 to 60% polyethylene glycol can be added, polyethylene glycol having a molecular weight of 1,000 to 8,000 can be used, and polyethylene glycol having a molecular weight of 1,000 to 4,000 can be more preferably used. The concentration of polyethylene glycol in the fusion medium is preferably 30 to 60%, for example. If necessary, for example, a small amount of dimethyl sulfoxide or the like can be added to promote fusion. The ratio of spleen cells (lymphocytes): myeloma cell line used for the fusion is, for example, 1: 1 to 20: 1, more preferably 4: 1 to 7: 1.
The fusion reaction is performed for 1-10 minutes and then a cell culture medium such as RPMI-1640 medium is added. The fusion reaction treatment can be performed multiple times. After the fusion reaction treatment, the cells are separated by centrifugation or the like and then transferred to a selection medium.
[0076]
5. Selection and monocloning of hybridomas (fusion cells)
Examples of the selection medium include media such as FCS-containing MEM media and RPMI-1640 media containing hypoxanthine, aminopterin and thymidine, so-called HAT media. In general, the selective medium can be replaced by adding a volume equal to the volume dispensed to the culture plate the next day, and then replacing the HAT medium half by half every 1 to 3 days. You can also change this. On the 8th to 16th day after the fusion, the medium can be changed every 1 to 4 days with a so-called HT medium excluding aminopterin. For example, mouse thymocytes can be used as a feeder, which may be preferred.
The culture supernatant of the culture well in which the hybridoma grows can be measured using a measurement system such as radioimmunoassay (RIA), enzyme immunoassay (ELISA), or fluorescence immunoassay (FIA), or a fluorescence-induced cell separator (FACS). Then, screening is performed by using a predetermined fragment peptide as an antigen, or by measuring a target antibody using a labeled anti-mouse antibody.
The hybridoma producing the target antibody is cloned. Cloning can be done by picking up colonies in agar or by limiting dilution. More preferably, the limiting dilution method can be used. Cloning is preferably performed multiple times.
[0077]
6). Production of monoclonal antibodies
The obtained hybridoma strain can be cultured in an appropriate growth medium such as an FCS-containing MEM medium or RPMI-1640 medium, and a desired monoclonal antibody can be obtained from the medium supernatant. In order to obtain a large amount of antibody, ascites of the hybridoma can be mentioned. In this case, each hybridoma is transplanted into the abdominal cavity of a histocompatibility animal syngeneic with an animal derived from myeloma cells, or each hybridoma is transplanted into, for example, a nude mouse, and allowed to proliferate. The produced monoclonal antibody can be recovered and obtained. Prior to transplantation of the hybridoma, the animal can be administered intraperitoneally with mineral oil such as pristane (2,6,10,14-tetramethylpentadecane). After that treatment, the hybridoma is allowed to grow and ascites is collected. You can also Ascites fluid is used as it is, or conventionally known methods such as salting out such as ammonium sulfate precipitation, gel filtration by Sephadex, ion exchange chromatography, electrophoresis, dialysis, ultrafiltration, affinity chromatography They can be purified by high performance liquid chromatography and used as monoclonal antibodies. Preferably, the ascites containing the monoclonal antibody can be purified and separated by treatment with an anion exchange gel such as DEAE-Sepharose and an affinity column such as a protein A column after ammonium sulfate fractionation. Particularly preferably, affinity chromatography in which an antigen or antigen fragment (for example, a synthetic peptide, recombinant antigen protein or peptide, a site specifically recognized by an antibody, etc.) is immobilized, affinity chromatography in which protein A is immobilized, hydroxy Examples include apatite chromatography.
[0078]
In addition, transgenic mice or other organisms, such as other mammals, can be used to express antibodies such as humanized antibodies against the immunogenic polypeptide products of the invention.
It is also possible to determine the sequence of the antibody thus obtained in large quantities, or to produce an antibody by a gene recombination technique using a nucleic acid sequence encoding the antibody obtained from a hybridoma strain. The nucleic acid encoding the monoclonal antibody can be isolated and sequenced by conventional techniques such as using an oligonucleotide probe that can specifically bind to the gene encoding the heavy or light chain of the mouse antibody. . Once isolated, the DNA can be put into an expression vector as described above and then into host cells such as CHO and COS. The DNA can be modified, for example, by substituting a sequence encoding a human heavy chain or light chain constant region domain in place of the homogeneous mouse sequence (Morrison et al., Proc Natl. Acad. Sci. USA, 81: 6581, 1984). Thus, it is possible to prepare a chimeric antibody or a hybrid antibody having a desired binding specificity. In addition, the antibody can be modified by preparing a chimeric antibody or a hybrid antibody by applying a chemical protein synthesis technique including the use of a condensing agent as described below.
Humanized antibodies can be performed by techniques known in the art (eg, Jones et al., Nature, 321: pp. 522-525 (1986); Riechmann et al., Nature, 332: pp .323-327 (1988); Verhoeyen et al., Science, 239: pp.1534-1536 (1988)). Human monoclonal antibodies can also be performed by techniques known in the art, and human myeloma cells and human / mouse heteromyeloma cells for producing human monoclonal antibodies are known in the art (Kozbor, J. Immunol., 133, pp. 3001 (1984); Brodeur et al., Monoclonal Antibody Production Techniques and Applications, pp. 51-63, Marcel Dekker, Inc., New York (1987)). Methods for producing bispecific antibodies are also known in the art (Millstein et al., Nature, 305: pp. 537-539 (1983); WO93 / 08829; Traunecker et al., EMBO J., 10: pp.3655-3659 (1991); Suresh et al., "Methods in Enzymology", Vol. 121, pp.210 (1986)).
[0079]
Furthermore, Fab, Fab ', F (ab') obtained by treating these antibodies with enzymes such as trypsin, papain, pepsin and optionally reducing them ₂ Such an antibody fragment may be used.
The antibodies can be used in any known assay, such as competitive binding assays, direct and indirect sandwich assays, and immunoprecipitation assays (Zola, Monoclonal Antibodies: A Manual of Techniques, pp. 147-158 (CRC Press, Inc., 1987).
Any method known in the art can be used to conjugate each antibody to a detectable atomic group, such as David et al., Biochemistry, 13, 1014-1021 (1974); Pain et al, J. Immunol. Meth., 40: pp. 219-231 (1981); and “Methods in Enzymology”, Vol. 184, pp. 138-163 (1990). As an antibody for imparting a label, an IgG fraction, or a specific binding part Fab ′ obtained by reduction after digestion with pepsin can be used. Examples of the label in these cases include enzymes (peroxidase, alkaline phosphatase, β-D-galactosidase, etc.), chemical substances, fluorescent substances, and radioisotopes as described below.
[0080]
Detection and measurement in the present invention can be performed by immunostaining such as tissue or cell staining, immunoassay such as competitive immunoassay or non-competitive immunoassay, radioimmunoassay, ELISA, etc., and BF separation. The measurement can be performed with or without. Preferred are radioimmunoassay and enzyme immunoassay, and further include a sandwich type assay. For example, in the sandwich type assay, one is an antibody against the soluble RAGE of the present invention and its related peptide fragment, the other is an antibody against the C-terminal residue of the soluble RAGE, and one is detectably labeled. Other antibodies capable of recognizing the same antigen are immobilized on the solid phase. Incubation treatment is performed in order to sequentially react the sample, labeled antibody, and solid-phased antibody as necessary. After separating the unbound antibody, the labeled product is measured. The amount of label measured is proportional to the amount of antigen, ie soluble RAGE polypeptide fragment antigen. This assay is referred to as a simultaneous sandwich type assay, a forward sandwich type assay, a reverse sandwich type assay, or the like depending on the order of addition of the insolubilized antibody or the labeled antibody. For example, washing, stirring, shaking, filtration, or pre-extraction of antigen are appropriately employed in these measurement steps under specific circumstances. Other measurement conditions such as the concentration of a specific reagent, buffer, etc., temperature, or incubation time can be varied according to factors such as the concentration of antigen in the sample and the nature of the sample. A person skilled in the art can perform measurement by appropriately selecting optimum conditions effective for each measurement using a normal experimental method.
[0081]
Many carriers capable of immobilizing an antigen or antibody are known, and in the present invention, they can be appropriately selected and used. Various carriers used for antigen-antibody reactions and the like are known, and of course, in the present invention, these carriers can be selected and used. Particularly preferably used are, for example, glass, such as activated glass, porous glass, silica gel, silica-alumina, alumina, magnetized iron, magnetized alloy and other inorganic materials, polyethylene, polypropylene, polyvinyl chloride, polyfluoride Cross-linked albumin such as vinylidene, polyvinyl acetate, polymethacrylate, polystyrene, styrene-butadiene copolymer, polyacrylamide, cross-linked polyacrylamide, styrene-methacrylate copolymer, polyglycidyl methacrylate, acrolein-ethylene glycol dimethacrylate copolymer Natural or modified cellulose such as collagen, gelatin, dextran, agarose, cross-linked agarose, cellulose, microcrystalline cellulose, carboxymethylcellulose, cellulose acetate, Organic polymer materials such as polyamides such as dextran and nylon, polyurethane and polyepoxy resins, and those obtained by emulsion polymerization of them, cells, erythrocytes, etc. If necessary, functional groups with silane coupling agents etc. The thing which introduce | transduced is mentioned.
In addition, filter paper, beads, inner walls of test containers, such as test tubes, titer plates, titer wells, glass cells, synthetic resin cells such as synthetic resin cells, glass rods, synthetic material rods, thickened ends, etc. Alternatively, the surface of a solid substance (object) such as a thinned rod, a rod having a round protrusion at the end or a flat protrusion, a thin plate, or the like may be used.
[0082]
An antibody can be bound to these carriers, and preferably a monoclonal antibody that specifically reacts with the antigen obtained in the present invention can be bound. The binding between the carrier and those involved in the antigen-antibody reaction may be performed by a physical method such as adsorption, a chemical method using a condensing agent, an activated one, etc. It can be performed by a method using a chemical bonding reaction.
Labels include enzymes, enzyme substrates, enzyme inhibitors, prosthetic molecules, coenzymes, enzyme precursors, apoenzymes, fluorescent materials, dye materials, chemiluminescent compounds, luminescent materials, chromogenic materials, magnetic materials, metal particles such as gold Examples thereof include radioactive materials such as colloids. Examples of enzymes include oxidoreductases such as dehydrogenase, reductase, and oxidase, such as transferases that catalyze the transfer of amino groups, carboxyl groups, methyl groups, acyl groups, phosphate groups, and the like, such as ester bonds, Examples include hydrolase, lyase, isomerase, ligase and the like that hydrolyze glycoside bonds, ether bonds, peptide bonds, and the like. The enzyme can be used for detection by using a plurality of enzymes in combination. For example, enzymatic cycling can be used.
[0083]
Typical radioisotope labeling isotopes include [ ³² P], [ ¹²⁵ I], [ ¹³¹ I], [ ^Three H], [ ¹⁴ C], [ ³⁵ S].
Typical enzyme labels include peroxidase such as horseradish peroxidase, galactosidase such as E. coli β-D-galactosidase, maleate dehydrogenase, glucose-6-phosphate dehydrogenase, glucose oxidase, glucoamylase, acetylcholinesterase, catalase, bovine Examples thereof include alkaline phosphatase such as small intestine alkaline phosphatase and Escherichia coli alkaline phosphatase.
When alkaline phosphatase is used, umbelliferone derivatives such as 4-methylumbelliferyl phosphate, phosphorylated phenol derivatives such as nitrophenyl phosphate, enzymatic cycling systems using NADP, luciferin derivatives, dioxetane derivatives and other substrates It can be measured by the fluorescence, luminescence, etc. that occur when used. A luciferin or luciferase system can also be used. When catalase is used, it reacts with hydrogen peroxide to produce oxygen, so that the oxygen can be detected by an electrode or the like. The electrode may be a glass electrode, an ion electrode using a hardly soluble salt film, a liquid film type electrode, a polymer film electrode, or the like.
The enzyme label can be replaced with a biotin label and enzyme-labeled avidin (streptavidin). The label may use a plurality of different types of labels. In such cases, it may be possible to perform multiple measurements continuously or discontinuously and simultaneously or separately.
[0084]
In the present invention, 4-hydroxyphenylacetic acid, 1,2-phenylenediamine, tetramethylbenzidine and the like, horseradish peroxidase, umbelliferyl galactoside, nitrophenyl galactoside and the like, β-D-galactosidase, glucose- Combinations of enzyme reagents such as 6-phosphate and dehydrogenase can also be used. Quinol compounds such as hydroquinone, hydroxybenzoquinone and hydroxyanthraquinone, thiol compounds such as lipoic acid and glutathione, phenol derivatives and ferrocene derivatives can be used as enzymes. What can be formed can be used.
Examples of fluorescent substances or chemiluminescent compounds include fluorescein isothiocyanate, such as rhodamine derivatives such as rhodamine B isothiocyanate and tetramethylrhodamine isothiocyanate, dansyl chloride, dansyl fluoride, fluorescamine, phycobiliprotein, acridinium salt, lumiferin, luciferase And luminol such as equolin, imidazole, oxalate ester, rare earth chelate compound, and coumarin derivative.
The labeling can be performed using a reaction between a thiol group and a maleimide group, a reaction between a pyridyl disulfide group and a thiol group, a reaction between an amino group and an aldehyde group, etc., and can be easily performed by a known method or a person skilled in the art. It is possible to apply the method appropriately selected from methods that can be used in addition to methods modified by them. Further, a condensing agent that can be used for preparing the immunogenic complex, a condensing agent that can be used for binding to a carrier, and the like can be used.
[0085]
Examples of the condensing agent include formaldehyde, glutaraldehyde, hexamethylene diisocyanate, hexamethylene diisothiocyanate, N, N′-polymethylene bisiodoacetamide, N, N′-ethylene bismaleimide, ethylene glycol bissuccinimidyl succinate. , Bisdiazobenzidine, 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide, succinimidyl 3- (2-pyridyldithio) propionate (SPDP), N-succinimidyl 4- (N-maleimidomethyl) cyclohexane-1-carboxy (SMCC), N-sulfosuccinimidyl 4- (N-maleimidomethyl) cyclohexane-1-carboxylate, N-succinimidyl (4-iodoacetyl) aminobenzoate, N-succinimidyl 4- (1-maleimidophenyl) Butyrate, N- (ε-Maleimide cap Yloxy) succinimide (EMCS), iminothiolane, S-acetylmercaptosuccinic anhydride, methyl-3- (4'-dithiopyridyl) propionimidate, methyl-4-mercaptobutyrylimidate, methyl-3-mercapto Examples include propion imidate and N-succinimidyl-S-acetylmercaptoacetate.
[0086]
According to the measurement method of the present invention, a labeled antibody reagent such as a monoclonal antibody in which a substance to be measured is labeled with an enzyme and the antibody bound to a carrier can be reacted sequentially or simultaneously. . The order in which reagents are added depends on the type of carrier system selected. When using beads such as sensitized plastics, first place a labeled antibody reagent such as a monoclonal antibody labeled with an enzyme together with a specimen sample containing the substance to be measured in an appropriate test tube, and then Measurements can be made by adding beads such as sensitized plastic.
In the quantification method of the present invention, an immunological measurement method is used. In this case, as a solid phase carrier, polystyrene, polycarbonate, polypropylene or polyvinyl balls, microplates that adsorb proteins such as antibodies well are used. Various materials and forms such as sticks, microparticles or test tubes can be arbitrarily selected and used.
The measurement can be performed in an appropriate buffer system so as to maintain an optimum pH, for example, a pH of about 4 to about 9. Particularly suitable buffers include, for example, acetate buffer, citrate buffer, phosphate buffer, Tris buffer, triethanolamine buffer, borate buffer, glycine buffer, carbonate buffer, Tris-HCl. A buffering agent etc. are mentioned. The buffering agents can be mixed and used at any ratio. The antigen-antibody reaction is preferably performed at a temperature between about 0 ° C and about 60 ° C.
Incubation treatment of antibody reagents such as monoclonal antibodies labeled with enzymes, antibody reagents bound to carriers, and substances to be measured can be performed until equilibrium is reached, but the equilibrium of the antigen-antibody reaction has been achieved. The reaction can be stopped after a limited incubation by separating the solid and liquid phases at a much earlier time, and the extent of the presence of a label such as an enzyme in either the liquid phase or the solid phase can be determined. Can be measured. The measurement operation can be performed using an automated measuring device, and a luminescence detector, a photo detector or the like is used to detect and measure a display signal generated by converting a substrate by the action of an enzyme. You can also.
[0087]
In the antigen-antibody reaction, appropriate measures can be taken to stabilize the reagents used, substances to be measured, and labels such as enzymes, or to stabilize the antigen-antibody reaction itself. In addition, proteins, stabilizers, surfactants, chelating agents, etc. can be added to the incubation solution to eliminate non-specific reactions, reduce the inhibitory effect, or activate the measurement reaction. It can also be added. As the chelating agent, ethylenediaminetetraacetate (EDTA) is more preferable.
A blocking treatment for preventing a non-specific binding reaction commonly employed in the field or known to those skilled in the art may be performed, for example, normal serum proteins such as mammals, albumin, skim milk, milk fermentation substances , Collagen, gelatin and the like. As long as the purpose is to prevent non-specific binding reaction, these methods are not particularly limited and can be used.
As a sample to be measured by the measurement method of the present invention, any form of solution, colloidal solution, non-fluid sample, etc. can be used, but preferably a sample derived from a living organism such as thymus, testis, intestine, kidney, brain, breast cancer. , Ovarian cancer, colorectal cancer, blood, serum, plasma, joint fluid, cerebrospinal fluid, pancreatic fluid, bile fluid, saliva, amniotic fluid, urine, other body fluids, cell culture fluid, tissue culture fluid, tissue phomodulate, biopsy Samples, tissues, cells and the like can be mentioned.
When various analysis / quantification methods including these individual immunological measurement methods are applied to the measurement method of the present invention, special conditions, operations, and the like are not required to be set. A measurement system related to the target substance of the present invention or a substance having substantially the same activity may be constructed by adding ordinary technical considerations to those skilled in the art to the normal conditions and operation methods in each method. .
[0088]
For details of these general technical means, refer to reviews, books, etc. [For example, Hiroshi Irie, “Radioimmunoassay”, Kodansha, published in 1974; Hiroshi Irie, “Second radioimmunoassay” "Kodansha, published in 1979; edited by Eiji Ishikawa et al.," Enzyme Immunoassay ", Medical School, published in 1978; edited by Eiji Ishikawa et al.," Enzyme Immunoassay "(2nd edition), Medical School, Showa Published in 1957; edited by Eiji Ishikawa et al., “Enzyme Immunoassay” (3rd edition), Medical School, published in 1987; HV Vunakis et al. (Ed.), “Methods in Enzymology”, Vol. 70 (Immunochemical Techniques, Part A), Academic Press, New York (1980); JJ Langone et al. (Ed.), "Methods in Enzymology", Vol. 73 (Immunochemical Techniques, Part B), Academic Press, New York (1981) JJ Langone et al. (Ed.), "Methods in Enzymology", Vol. 74 (Immunochemical Techniques, Part C), Academic Press, New York (1981); JJ Langone et al. ed.), "Methods in Enzymology", Vol. 84 (Immunochemical Techniques, Part D: Selected Immunoassays), Academic Press, New York (1982); JJ Langone et al. (ed.), "Methods in Enzymology", Vol 92 (Immunochemical Techniques, Part E: Monoclonal Antibodies and General Immunoassay Methods), Academic Press, New York (1983); JJ Langone et al. (Ed.), "Methods in Enzymology", Vol. 121 (Immunochemical Techniques, Part I: Hybridoma Technology and Monoclonal Antibodies), Academic Press, New York (1986); JJ Langone et al. (Ed.), "Methods in Enzymology", Vol. 178 (Antibodies, Antigens, and Molecular Mimicry), Academic Press, New York (1989); M. Wilchek et al. (Ed.), "Methods in Enzymology", Vol. 184 (Avidin-Biotin Technology), Academic Press, New York (1990); JJ Langone et al. (Ed. ), "Methods in Enzymology", Vol. 203 (Molecular Design and Modeling: Concepts and Applications, Part B: Anibodies and Antigens, Nucleic Acids, Polysaccharides, and Drugs), Academic Press, New York (1991), etc. In cited literature (described in in them are included in the present disclosure by reference thereto)].
[0089]
Epitope mapping can also be performed using the anti-soluble RAGE antibody of the present invention, particularly a monoclonal antibody. If an antibody that recognizes each epitope is used, soluble RAGE and its related peptide fragments are detected and measured. be able to.
Antibodies against soluble RAGE and related peptide fragments can be detected and / or measured by suppressing or inhibiting the interaction between AGE and membrane-bound RAGE receptor by soluble RAGE, and by excess of AGE. It is useful for the detection and / or measurement of various physiologically active substances or physiological phenomena or biological phenomena that occur, and for research and development of factors and mechanisms that control soluble RAGE production. The antibody, particularly a monoclonal antibody, detects (i) a disorder, abnormality and / or disease associated with a tissue or cell caused by an interaction between AGE and RAGE, or (ii) a relationship between AGE and RAGE. Detection of cell tumorigenesis, cell migration, invasion, migration and / or metastasis due to interactions between them, or the possibility thereof, and (iii) disorders, abnormalities and / or associated with protein glycation reactions Or detect disease or its potential, (iv) measure the expression level of soluble RAGE, (v) detect and / or measure changes in AGE capture activity, (vi) soluble RAGE It is useful for searching for compounds that control production and / or (vii) detecting and / or measuring the activity of compounds that control the production of soluble RAGE. It is expected that it can be used to determine the extent of diabetic complications, tissue abnormalities, cancer mobility, invasiveness, chemotaxis and / or metastasis.
According to the present invention, the inhibitory or inhibitory activity of the interaction of AGE with membrane-bound RAGE by soluble RAGE is detected and / or measured, and a therapeutic agent for diabetes, a preventive / therapeutic agent for diabetic complications, tissue disease Used as an effect monitor for prophylactic / therapeutic agents, anti-inflammatory agents, anticancer agents, cancer metastasis inhibitors, arteriosclerosis therapeutic agents, Alzheimer's therapeutic agents, joint destruction therapeutic agents, antiallergic agents and / or immunosuppressive agents It becomes possible to do.
In addition, the present invention can provide a method for detecting and / or measuring a tissue / cell or protein abnormality phenomenon due to AGE and a reagent therefor.
[0090]
The active ingredient of the present invention [for example, (a) a soluble RAGE polypeptide, a part of the peptide or a salt thereof, a peptide related thereto, (b) the soluble RAGE or the soluble RAGE polypeptide Nucleic acids such as encoding DNA, (c) the antibody of the present invention, a partial fragment thereof (including a monoclonal antibody) or a derivative thereof, (d) a mutual relationship between AGE by soluble RAGE and transmembrane RAGE A compound or salt thereof that suppresses and / or inhibits a biological activity such as a phenomenon such as suppression or inhibition of action or a tissue or protein alteration / overproduction or degradation phenomenon, or a compound or salt thereof that controls the production of soluble RAGE (E) an antisense oligonucleotide for a nucleic acid such as the DNA of the present invention, (f) an active substance found using the present invention, etc.) as a pharmaceutical, for example, between AGE and RAGE These interaction inhibitors or salts thereof can be administered as a pharmaceutical composition or a pharmaceutical preparation, etc., usually alone or mixed with various pharmacologically acceptable adjuvants. Preferably, it is administered in the form of a pharmaceutical preparation suitable for use such as oral administration, topical administration, or parenteral administration, and depending on the purpose, any dosage form (including inhalation or rectal administration) is preferred. Good.
The active ingredient of the present invention includes diabetic complication agent, arteriosclerosis agent, antihyperlipidemic agent, antitumor agent (anticancer agent), tumor transfer inhibitor, thrombus formation inhibitor, Alzheimer treatment Can also be used in combination with agents, joint destruction treatment agents, anti-inflammatory agents and / or immunosuppressive agents, and they can be used without limitation as long as they have advantageous effects, such as those known in the art You can choose from.
[0091]
The parenteral dosage form may include topical, transdermal, intravenous, intramuscular, subcutaneous, intradermal or intraperitoneal administration, but can also be administered directly to the affected area. Is also preferred. Preferably, orally or parenterally to mammals including humans (eg, intracellular, tissue, intravenous, intramuscular, subcutaneous, intradermal, intraperitoneal, intrathoracic, intrathecal, instillation, injection Administration to the intestines, rectum, ear drops, eye drops, nose, teeth, skin and mucous membranes, etc.). Specific preparation preparation forms include solution preparations, dispersion preparations, semi-solid preparations, granular preparations, molded preparations, leachable preparations, etc., for example, tablets, coated tablets, sugar-coated preparations, Agents, troches, hard capsules, soft capsules, microcapsules, implants, powders, powders, granules, fine granules, injections, solutions, elixirs, emulsions, irrigation agents, syrups, Water solution, emulsion, suspension, liniment, lotion, aerosol, spray, inhalant, spray, ointment, plaster, patch, pasta, poultice, cream, oil, suppository ( For example, rectal suppositories), tinctures, skin preparations, eye drops, nasal drops, ear drops, coatings, infusions, liquids for injection, freeze-dried preparations, gel preparations, etc. Can be mentioned.
The pharmaceutical composition can be formulated according to a usual method. For example, as necessary, physiologically acceptable carriers, pharmaceutically acceptable carriers, adjuvant agents, excipients, excipients, diluents, flavoring agents, fragrances, sweeteners, vehicles, preservatives, stable Agent, binder, pH adjuster, buffer, surfactant, base, solvent, filler, extender, solubilizer, solubilizer, isotonic agent, emulsifier, suspending agent, dispersant , Thickeners, gelling agents, curing agents, absorbents, adhesives, elastic agents, plasticizers, disintegrating agents, propellants, preservatives, antioxidants, sunscreen agents, moisturizers, relaxation agents, antistatic agents, By using a soothing agent alone or in combination with the protein of the present invention and the like, it can be produced into a unit dosage form required for the practice of a generally accepted formulation.
Formulations suitable for parenteral use include sterile solutions or suspensions of the active ingredient with water or other pharmaceutically acceptable media such as injections. In general, water, saline, dextrose aqueous solution, other related sugar solutions, and glycols such as ethanol, propylene glycol, and polyethylene glycol are preferable liquid carriers for injections. When preparing an injection, the solution is prepared by a method known in the art using a carrier such as distilled water, Ringer's solution, physiological saline, an appropriate dispersing agent or wetting agent and suspending agent. Injectable forms such as suspensions and emulsions.
[0092]
Examples of aqueous solutions for injection include isotonic solutions containing physiological saline, glucose and other adjuvants (eg, D-sorbitol, D-mannitol, sodium chloride, etc.), and are pharmacologically acceptable. Suitable solubilizers such as alcohols (eg ethanol), polyalcohols (eg propylene glycol, polyethylene glycol etc.), nonionic surfactants (eg polysorbate 80) ^TM , HCO-50, etc.). Examples of the oily liquid include sesame oil and soybean oil. As a solubilizing agent, benzyl benzoate, benzyl alcohol and the like may be used in combination. Further, a buffer (for example, phosphate buffer, sodium acetate buffer, etc.) or a reagent for adjusting osmotic pressure, a soothing agent (for example, benzalkonium chloride, procaine, etc.), a stabilizer (for example, human) Serum albumin, polyethylene glycol, etc.), preservatives (eg, benzyl alcohol, phenol, etc.), antioxidants such as ascorbic acid, absorption promoters and the like. The prepared injection solution is usually filled in a suitable ampoule.
[0093]
For parenteral administration, a solution in a sterile pharmaceutically acceptable liquid such as water, ethanol or oil, with or without the addition of surfactants and other pharmaceutically acceptable auxiliaries, or Formulated in the form of a suspension. The oily vehicle or solvent used in the formulation includes natural, synthetic or semi-synthetic mono-, di- or triglycerides, natural, semi-synthetic or synthetic fats or fatty acids, such as peanut oil, corn oil, large Examples include vegetable oils such as bean oil and sesame oil. For example, this injection can be prepared so that it usually contains about 0.1 to 10% by weight of the compound of the present invention.
Preparations suitable for topical, eg oral, or rectal use include, for example, mouthwash, toothpaste, oral spray, inhalant, ointment, dental filler, dental coating, dental paste, suppository, etc. Is mentioned. The mouthwash and other dental agents are prepared by a conventional method using a pharmacologically acceptable carrier. As an oral spray and an inhalant, the compound of the present invention itself or a pharmacologically acceptable inert carrier can be dissolved in an aerosol or nebulizer solution, or can be administered to teeth as a fine powder for inhalation. The ointment is prepared by a conventional method by adding a commonly used base such as an ointment base (white petrolatum, paraffin, olive oil, Macrogol 400, Macrogol ointment, etc.) and the like.
[0094]
Drugs for topical application to teeth, skin can be formulated into solutions or suspensions in appropriately sterilized water or non-aqueous excipients. Additives include, for example, buffering agents such as sodium bisulfite or disodium edetate; antiseptics including antibacterial and antifungal agents such as acetic acid or phenylmercuric nitrate, benzalkonium chloride or chlorohexidine and hypromelrose A thickener is mentioned.
Suppositories are carriers well known in the art, preferably non-irritating suitable excipients such as polyethylene glycols, lanolin, cocoa butter, fatty acid triglycerides, etc., preferably solid at normal temperature but at the temperature of the intestinal tract. Although it is prepared by a conventional method using a liquid that melts in the rectum and releases a drug, it is usually prepared so as to contain about 0.1 to 95% by weight of the compound of the present invention. Depending on the excipient and concentration used, the drug can be suspended or dissolved in the excipient. Adjuvants such as local anesthetics, preservatives and buffering agents can be dissolved in the vehicle. Examples of preparations suitable for oral use include solid compositions such as tablets, pills, capsules, powders, granules, and troches, and liquid compositions such as liquids, syrups, and suspensions. Can be mentioned. When preparing the formulation, formulation adjuvants known in the art are used. Tablets and pills can also be manufactured with an enteric coating. When the dispensing unit form is a capsule, a liquid carrier such as fats and oils can be further contained in the above-mentioned type of material.
[0095]
Further, when the nucleic acid such as the DNA of the present invention is used as a therapeutic and / or prophylactic agent as described above, the nucleic acid can be used alone or used in the gene recombination technique as described above. It can be used by binding to an appropriate vector, for example, a virus-derived vector such as a retrovirus-derived vector. Nucleic acids such as the DNA of the present invention can be administered in the usual known manner, either as it is or with appropriate adjuvants or physiologically acceptable carriers so as to promote uptake into cells, for example. And can be used as a pharmaceutical composition or pharmaceutical preparation as described above. A method known as gene therapy can also be applied.
The active ingredient of the present invention can be administered over a wide range of dosages, and the dosage and number of administrations are determined according to the sex, age, weight, general health condition, diet, administration time, administration method, etc. Depending on the excretion rate, combination of drugs, and the extent of the condition being treated by the patient, these or other factors may be taken into account.
[0096]
In the manufacture of pharmaceuticals, the additives and preparation methods are described in, for example, the Japanese Pharmacopoeia Manual Editorial Committee, the 14th revised Japanese Pharmacopoeia Manual, published on June 27, 2001, Yodogawa Shoten Co., Ltd .; Ichibanase Nao et al. Drug Development Volume 12 (Formulation Base [I]), published on October 15, 1990, Yodogawa Shoten Co., Ltd .; Drug Development Volume 12 (Formulation Material [II]) Heisei Issuing on October 28, 2 years, referring to the description of Yodogawa Shoten Co., Ltd., etc., it is possible to select and apply as necessary from among them.
The active ingredient of the present invention is not particularly limited as long as it has a biological activity such as suppressing or inhibiting the activity of AGE, particularly the interaction between AGE and RAGE, but preferably has an advantageous effect. Things. The active ingredient of the present invention includes, for example, (a) soluble RAGE, a mutant polypeptide thereof, a partial peptide thereof or a salt thereof, (b) DNA encoding the soluble RAGE, soluble type Nucleic acid such as DNA encoding RAGE variant polypeptide, (c) antibody of the present invention, partial fragment thereof (including monoclonal antibody) or derivative thereof, (d) AGE and RAGE of soluble RAGE A compound having a beneficial effect on biological activity such as inhibiting or inhibiting the interaction between the two or a salt thereof.
[0097]
The active ingredient of the present invention is expected to be useful for suppressing or inhibiting changes in various tissues or cells resulting from the interaction between AGE and RAGE. The active ingredient is useful for suppressing the expression of AGE activity, and is useful for preventing or treating disorders, abnormalities and / or diseases caused by the interaction between AGE and RAGE. In addition, it is expected to be useful for the control, for example, suppression of migration, invasion, migration and / or metastasis of tumor cells involved in the interaction between AGE and RAGE.
Soluble RAGE and related peptides are useful for the prevention and / or suppression of malignant tumors, i.e., cancer migration, invasion and / or metastasis, and angiogenesis / neogenesis inhibitors, antitumor agents and / or Expected to be a cancer metastasis inhibitor. It is also useful for the prevention or treatment of AGE-related disorders, abnormalities and / or diseases of blood cells, diabetic complication treatment / prevention agent, arteriosclerosis treatment / prevention agent, thrombosis treatment / prevention agent, It can also be expected as an anti-inflammatory agent and / or an immunosuppressant. Furthermore, it can be expected as a therapeutic agent for Alzheimer, a therapeutic agent for joint destruction, and the like.
[0098]
Further, in the present invention, in the amino acid sequence of (a) soluble RAGE, a sequence ranging from the first amino acid residue to the 347th amino acid residue:
(b) a sequence ranging from the first amino acid residue to the 117th amino acid residue in the amino acid sequence of soluble RAGE: and
(c) Sequence in the range from the 332nd amino acid residue to the 347th amino acid residue in the amino acid sequence of soluble RAGE
It can be used to obtain a substance having an activity of suppressing or inhibiting the interaction between AGE and RAGE by applying a molecular design based on a substance selected from the group consisting of The substance thus obtained is also within the scope of the idea of the present invention and can be treated as the active ingredient of the present invention. Whether a particular feature is selected from the sequence, and (i) whether a pharmacophore is replaced with an isostere, or (ii) at least one of the constituent amino acid residues is replaced with a D-form amino acid residue (Iii) modify the side chain of the amino acid residue, (iv) place and link amino acid residues different from the amino acid residues present in the sequence, or (v) analyze the three-dimensional structure and mimic Designing the body, etc., can be carried out by utilizing the techniques adopted in this field (for example, Shinichi Suto, Volume 7 of Drug Development (Molecular Design), issued on June 25, 1990, Yodogawa Co., Ltd.) Bookstores and references and papers cited there). Some of these techniques include those described above.
In the specification and drawings, the terms are based on the meanings of terms used by the IUPAC-IUB Commission on Biochemical Nomenclature or conventionally used in the art.
The hybridoma producing the anti-soluble RAGE monoclonal antibody described in Example 2 (g) described later: 269-9C2 was obtained from February 22, 2001, 1st Central, 1st Street, Tsukuba City, Ibaraki Prefecture. Address notation: 1-3-3 Higashi, Tsukuba City, Ibaraki Pref. (National Institute of Advanced Industrial Science and Technology, International Patent Organism Depositary: IPOD) ) (Former name: National Institute of Advanced Industrial Science and Technology, National Institute of Advanced Industrial Science and Technology (NIBH)) and deposited (Accession No. FERM P-18218).
[0099]
【Example】
The present invention will be specifically described below with reference to examples. However, it should be understood that the present invention is not limited to these examples, and various embodiments based on the idea of the present specification are possible. is there.
All examples were performed or can be performed using standard techniques, except as otherwise described in detail, and are well known and routine to those skilled in the art. .
In the following examples, unless otherwise indicated, specific procedures and processing conditions are as follows for DNA cloning in J. Sambrook, EF Fritsch & T. Maniatis, “Molecular Cloning”, 2nd ed., Cold. Spring Harbor Laboratory, Cold Spring Harbor, NY (1989) and DM Glover et al. Ed., "DNA Cloning", 2nd ed., Vol. 1 to 4, (The Practical Approach Series), IRL Press, Oxford University Press ( 1995); Especially in PCR, HA Erlich ed., PCR Technology, Stockton Press, 1989; DM Glover et al. Ed., "DNA Cloning", 2nd ed., Vol. 1, (The Practical Approach Series), IRL Press, Oxford University Press (1995) and MA Innis et al. Ed., “PCR Protocols”, Academic Press, New York (1990), and use commercially available reagents or kits. If so, they use attached protocols (protocols) and attached chemicals.
As a reagent,
Tween 20: Bio-Rad
Skim milk: Morinaga Milk Industry
Trizma base: Sigma
Ethanolamine: Sigma
Other reagents were purchased from Wako Pure Chemical.
[0100]
Example 1
〔cell〕
Primary cultured human skin microvascular endothelial cells were obtained from Cascade Biologics, Inc. (Portland, OR) and poly (A) ⁺ Cells with passage numbers 5-10 were used for RNA purification.
[Polysome-derived poly (A) ⁺ (Separation of RNA)
Human skin microvascular endothelial cells cultured in a tissue culture flask were washed with ice-cold phosphate buffered saline and then scraped with a cell scraper. The cell suspension is centrifuged to collect the cells as a precipitate, and 0.25 M KCl, 10 mM MgCl ₂ , 1mM EDTA, 0.25M sucrose (RNase free), 0.1mM DTT, 2mM 4- (2-aminoethyl) -benzenesulfonyl fluoride, 1,000u / ml RNase inhibitor (Ambion, Inc., Austin, TX) After suspending in a buffer solution (pH 7.6), it was crushed with a Dounce homogenizer. In order to remove cell nuclei and mitochondria, the cell lysate was centrifuged at 12,000 xg for 15 minutes, and the supernatant was collected. The obtained supernatant was centrifuged at 100,000 × g for 60 minutes, and the polysome fraction was recovered as a precipitate. From the obtained polysome fraction, using the Quickprep micro mRNA isolation kit (Amersham Pharmacia Biotech), poly (A) ⁺ RNA was purified.
[0101]
[Separation of soluble RAGE cDNA]
Poly (A) derived from human skin microvascular endothelial cell polysome ⁺ RNA was used as a template to synthesize cDNA using oligo (dT) primer and AMV-derived reverse transcriptase, and primers corresponding to exon 1 and exon 11 (see Fig. 1) of the RAGE gene (SEQ ID NO: 3, 5 in the sequence listing) '-GCCAGGACCCTGGAAGGAAGCA-3'; SEQ ID NO: 4, 5'-CTGATGGATGGGATCTGTCTGTG-3 ') and TaKaRa La Taq polymerase were used to amplify RAGE cDNA. The amplified RAGE cDNA was inserted into pCR2.1 (Invitrogen) to transform E. coli XL1-Blue. Plasmid DNA was purified from the resulting 32 recombinant Escherichia coli colonies, and nucleotide sequences were determined using an ABI377 sequencer (Aplied Biosystems Inc) to obtain cDNA encoding soluble RAGE.
The nucleotide sequence of the cDNA encoding the soluble RAGE thus determined and the amino acid sequence encoded by the open reading frame of the sequence are shown in SEQ ID NO: 1 in the sequence listing. In addition, SEQ ID NO: 2 in the sequence listing shows the amino acid sequence of soluble RAGE. 2 to 4 show that the present invention is compared with a so-called recombinant solubilized RAGE (US5864018) artificially produced from transmembrane RAGE (fullRAGE) and RAGE cDNA having a transmembrane domain (US5864018). This shows the base sequence of nucleic acid encoding soluble RAGE (solubleRAGE). FIG. 5 shows the amino acid sequence of soluble RAGE (solubleRAGE) of the present invention compared with fullRAGE and US5864018. The soluble type RAGE (solubleRAGE) of the present invention is a native type (endogenous), and fullRAGE is a C-terminal portion of 16 amino acids (sequence Glu of SEQ ID NO: 2). ³³² ~ Met ³⁴⁷ ) Is different.
[0102]
[Preparation of soluble RAGE expression vector]
5'-primer with EcoRI recognition sequence (SEQ ID NO: 5, 5'-GAGAATTCGCCAGGACCCTGGAAGGAAGCA-3 ') and 3'-primer with XbaI recognition sequence (arrangement) using cDNA encoding soluble RAGE as template Amplification was performed using SEQ ID NO: 6, 5′-GATCTAGAGATTGTTGACCATCCCCCCAG-3 ′) in the column table. The amplified DNA was purified, digested with EcoRI and XbaI, and inserted into the EcoRI and XbaI sites of the animal cell expression vector pCI-neo vector (Stratagen). The expression vector DNA was purified with a plasmid purification kit from QIAGEN (Valencia, Calif.), And the nucleotide sequence was confirmed using an ABI377 sequencer (Aplied Biosystems Inc).
[0103]
[Introduction of soluble RAGE expression vector into COS 7 cells and isolation of stable transformant]
The soluble RAGE expression vector was introduced into COS 7 cells using Tfx-20 reagent (Promega Corp., Madison, Wis.). 48 hours after introduction of the expression vector, G418 (Geneticin) was added to the cell culture medium, and a plurality of G418-resistant colonies were obtained 2 weeks later. The obtained clones were cultured separately, and the cell extract and culture medium were analyzed by Western blot using an anti-human RAGE antibody, and clones that highly expressed soluble RAGE were selected.
[Transformant culture]
Using a 150 mm dish for tissue culture, soluble RAGE overexpressing COS7 cells in Dulbecco's modified Eagle's medium containing 5% fetal calf serum and 500 μg / ml G418 at 37 ° C, 5% CO ₂ After culturing to confluence under the conditions of ²⁺ / Mg ²⁺ -free), and the medium was completely removed. Subsequently, the cells were cultured in serum-free Dulbecco's modified Eagle medium for 48 hours. Thereafter, the culture medium was collected, and the supernatant obtained by centrifugation at 10,000 rpm for 20 minutes was filtered through a 0.22 μm membrane filter, and soluble RAGE was purified from the obtained treated medium according to the following method.
[0104]
[Purification of soluble RAGE protein]
4L of treatment medium was applied to HiTrap Heparin (column volume 3 x 5 ml, Amersham Pharmacia) equilibrated with 20 mM Tris-HCl (pH 7.4) at a flow rate of 3 ml / min, and 75 ml of 0.15 M NaCl containing 20 mM The column was washed with Tris-HCl (pH 7.4) at a flow rate of 2 ml / min. Subsequently, the heparin-binding substance bound to the column was eluted stepwise with 75 ml of 0.3 mM, 0.5 M and 1 M NaCl in 20 mM Tris-HCl pH7.4 at a flow rate of 2 ml / min. I drew it. The eluted soluble RAGE fraction was identified by Western blotting. Next, a soluble RAGE fraction diluted 5-fold with 50 mM sodium acetate buffer (pH 4.5) was added to a RESOURCE S 1 ml column (Amersham Pharmacia) equilibrated with 50 mM sodium acetate buffer (pH 4.5). Applying at a flow rate of 1 ml / min, the column was washed with 3 ml of 50 mM sodium acetate buffer containing 0.2 M NaCl at a flow rate of 1 ml / min. Next, using 5 ml of 50 mM sodium acetate buffer, apply a linear NaCl concentration gradient from 0.2 M to 1.0 M at a flow rate of 1 ml / min to elute the anion substances bound to the column sequentially. Fractionated one by one. The soluble RAGE fraction was determined by Western blotting. 2 ml of the soluble RAGE fraction obtained in PBS (Ca ²⁺ / Mg ²⁺ -free) equilibrated with HiTrap Desalting column (column volume 2 × 5 ml, Amersham Pharmacia) and PBS (Ca ²⁺ / Mg ²⁺ -free), and gel filtration was performed at a flow rate of 0.5 ml / min to collect a high molecular weight fraction. Thereafter, a soluble RAGE (sRAGE) concentrated solution was prepared by ultrafiltration using Centricon YM-3 (MILLIPORE) as necessary.
The purity of the purified sRAGE protein was evaluated by silver staining of the gel after separation of 10 ng protein equivalent by 10% SDS-PAGE. The result is shown in FIG. In FIG. 6, 1 is a silver stained image of the cell culture supernatant, and 2 is a silver stained image of the purified protein.
The above chromatograph is
[Expression 1]

Using the system (Amersham Pharmacia), the eluate was monitored by measuring the absorbance at 230 nm, 280 nm and 300 nm.
[0105]
[Binding test between purified recombinant soluble RAGE protein and AGE]
The AGE binding ability of soluble RAGE was confirmed by surface plasmon resonance using BIACORE (Biacore, Sweden). The purified soluble RAGE protein was immobilized on Sensor chip CM5 (Biacore) using the amino coupling method. AGE-BSA prepared by aseptically incubating glucose and bovine serum albumin (BSA) at 37 ° C. for 12 weeks was added to the BIACORE microchannel system as an analyte at a concentration of 500 μg / ml. As a result, it did not react at all with the inline reference (control chip) or the chip on which immunoglobulin was immobilized, and showed specific binding only with the sensor chip on which soluble RAGE was immobilized.
[0106]
Example 2: Preparation of monoclonal antibodies
(a) Immunogen
As an antigen used for immunization, a synthetic peptide designed based on the amino acid sequence of SEQ ID NO: 2 in the sequence listing, including recombinant soluble RAGE, can be used. Further, as the antigen used for immunization, recombinant soluble RAGE obtained by ligating the obtained cDNA to an animal cell expression vector and expressing it in CHO cells, COS cells, etc. can be used. These antigenic proteins can be purified by ion exchange, gel filtration or other various chromatography.
The purified antigen for immunization is immunized by a general method, antibody-producing cells are induced, and antibody-producing cells can be obtained as hybridomas by cell fusion. Furthermore, it can be established as a cloned and monoclonal antibody-producing hybridoma based on the reactivity with the purified antigen for immunization.
In addition, as an immunogen for obtaining a soluble RAGE-specific monoclonal antibody, a haptenized synthetic peptide having an amino acid sequence characteristic of soluble RAGE can be used. For example, at the C-terminus, at least three or more consecutively selected from Glu-Gly-Phe-Asp-Lys-Val-Arg-Glu-Ala-Glu-Asp-Ser-Pro-Gln-His-Met, preferably It is particularly preferred to contain 5 or more consecutive peptide sequences. Specifically, Cys-Glu-Gly-Phe-Asp-Lys-Val-Arg-Glu-Ala-Glu-Asp-Ser-Pro-Gln-His-Met (CEGFDKVREAEDSPQHM) is suitable as an immunogen.
[0107]
(b) Preparation of antigen polypeptide
A characteristic sequence is selected from the amino acid sequences of human soluble RAGE described in SEQ ID NO: 2 and synthesized. Peptides are synthesized by the Fmoc-bop method using a peptide synthesizer (peptide synthesizer 9600, MilliGen / Biosearch). Cysteine is introduced at the N-terminus of the polypeptide. The synthesized peptide is purified by high performance liquid chromatography using μBondasphere, C18 column (Waters). Cys-Glu-Gly-Phe-Asp-Lys-Val-Arg-Glu-Ala-Glu-Asp-Ser-Pro-Gln-His-Met [SEQ ID NO: 7] was synthesized and used in the following experiment.
(c) Preparation of polypeptide and BSA complex
It was combined with bovine serum albumin (BSA) via a cysteine residue to obtain an antigen conjugate. Mix 10.1 mg BSA dissolved in 1 mL 0.1 M phosphate buffer, pH 7.0 with 1.14 mg EMCS (N- (ε-maleimidecaproyloxy) -succinimide) dissolved in 24.9 μL dimethylformamide. The mixture is allowed to react at 30 ° C. for 30 minutes, and then the above mixture is subjected to gel filtration with a Sephadex G-25 (Pharmacia) gel column (diameter 13 mm, length 120 mm) equilibrated with 0.1 M phosphate buffer, pH 7.0. . The polypeptide synthesized in (b) is dissolved in 0.1 M phosphate buffer, pH 7.0, and mixed in an approximately 50-fold molar amount with respect to maleimide-bound BSA. That is, maleimide-bound BSA is mixed with the polypeptide and incubated at 4 ° C. for 20 hours to prepare a BSA-polypeptide complex. The resulting BSA-polypeptide complex is diluted with 0.1 M phosphate buffer, pH 7.0, then dispensed in 150 μL aliquots and stored frozen at -30 ° C.
[0108]
(d) Preparation of antibody-producing cells
The BSA-polypeptide complex prepared in the above (c) was intraperitoneally administered to 6-week-old Balb / c female mice together with complete Freund's adjuvant for initial immunization. On the 18th day, a BSA-polypeptide complex dissolved in 0.1 M phosphate buffer, pH 7.5 is intraperitoneally administered to the first immunized mouse and boosted. Further, on the approximately 52nd day, a BSA-polypeptide complex dissolved in 0.1 M phosphate buffer, pH 7.5 is intravenously administered to make the final immunization. Four days later, the spleen is removed and a spleen cell suspension is prepared.
(e) Cell fusion
The following materials and methods are used for cell fusion. RPMI-1640 medium: RPMI-1640 (Flow Lab.) With sodium bicarbonate (24 mM), sodium pyruvate (1 mM), penicillin G potassium (50 U / ml), amikacin sulfate (100 μg / ml) and dry ice Adjust the pH to 7.2 and filter with 0.2 μm Toyo Membrane Filter. NS-1 medium: Fetal bovine serum (FCS, MA Bioproducts) that has been sterilized and filtered to the above RPMI-1640 medium is added to a concentration of 15% (v / v). PEG-4000 solution: Prepare a serum-free medium in which polyethylene glycol-4000 (PEG-4000, Merk & Co.) is added to RPMI-1640 medium at 50% (w / w).
Fusion with 8-azaguanine-resistant myeloma cells SP2 (SP2 / 0-Ag14) was performed by Oi et al. Described in Selected Method in culture immunology p351-372 (ed. BB Mishell and SN Shiigi), WH Freeman and Company (1980). The method is slightly modified.
[0109]
The nucleated spleen cells (live cell rate 100%) and myeloma cells (live cell rate 100%) prepared in (d) are fused at a ratio of approximately 5: 1 to 10: 1 by the following procedure. The polypeptide-immunized spleen cell suspension and myeloma cells are each washed with RPMI1640 medium. Next, suspend nucleated splenocytes and myeloma cells for suspension in the same medium. That is, approximately 4.0 x 10 ⁸ Approximately 8.0 x 10 per nucleated splenocyte ⁷ Mix myeloma cells.
Next, each cell mixture is centrifuged to precipitate cells, and the supernatant is completely removed by aspiration. RPMI-1640 medium containing 50% PEG-4000 heated to 37 ° C on the precipitated cells (myeloma cells are approximately 3 × 10 ⁷ (The volume is determined so that the number of cells / mL is reached) is dropped, and the cells are resuspended and dispersed. Next, RPMI-1640 medium heated to 37 ° C., which is twice the volume of RPMI-1640 medium containing 50% PEG-4000, is added dropwise. Furthermore, RPMI-1640 medium in a volume 7 times that of the added 50% PEG-4000-containing RPMI-1640 medium is added dropwise with constant stirring to disperse the cells. This is centrifuged and the supernatant is completely aspirated off. Next, myeloma cells are roughly 3 x 10 ⁶ NS-1 medium warmed to 37 ° C / ml is immediately added to the precipitated cells, and the large cell mass is carefully dispersed by pipetting. Furthermore, add the same medium and dilute, and approximately 96 x 10 myeloma cells per well in a 96-well polystyrene well. ^Five Inoculate to be individual. The microwells to which the respective cells are added are cultured in 7% carbon dioxide / 93% air at a temperature of 37 ° C. and a humidity of 100%.
[0110]
(f) Selective growth of hybridomas on selective medium
(1) The culture media used are as follows.
HAT medium: Hypoxanthine (100 μM), aminopterin (0.4 μM) and thymidine (16 μM) are further added to the NS-1 medium described in (e) above.
HT medium: The same composition as the HAT medium except that aminopterin was removed.
(2) On the next day (1st day) after the start of the culture in (e) above, add 2 drops (about 0.1 ml) of HAT medium to the cells with a Pasteur pipette. On days 2, 3, 5, and 8, half of the medium (approximately 0.1 ml) is replaced with fresh HAT medium and on day 10 half of the medium is replaced with fresh HT medium. For all wells where growth of the hybridoma was observed with the naked eye, positive wells were examined by a solid phase-antibody binding test (ELISA). First, coat a 96-well polystyrene plate (100 ng / well) with the antigen polypeptide diluted in 20 mM carbonate buffer (pH 9.6), and then wash with PBS containing 0.05% Tween20 to remove unadsorbed peptide. except. Add 0.1 ml of culture supernatant from wells where growth of hybridoma has been confirmed to each well and let stand at room temperature for about 1 hour. After washing, horseradish peroxidase (HRP) -labeled goat anti-mouse immunoglobulin (Cappel) is added as a secondary antibody and allowed to stand at room temperature for about 1 hour. After washing, hydrogen peroxide as a substrate and 3,3 ′, 5,5′-tetramethylbenzidine (TMB) are added to cause color development. Add 2N sulfuric acid to each well to stop the color reaction, and measure the degree of color development at an absorbance of 450 nm using a microplate absorbance analyzer (MRP-A4, Tosoh).
[0111]
(g) Cloning of hybridoma
The hybridoma in the positive well for the antigenic peptide obtained in (f) above is monocloned using the limiting dilution method. That is, approximately 10 feeders per 1 ml of NS-1 medium. ⁷ A cloning medium containing mouse thymocytes is prepared, and the hybridomas are diluted to 96, 96, and 0.5 wells per well, and added to 36, 36, and 24 wells, respectively. On day 5 and 12, add approximately 0.1 ml NS-1 medium to all wells. The ELISA described in the section (f) is performed on the group in which sufficient hybridoma growth is observed macroscopically after the start of cloning and the colony formation negative well is 50% or more. If all the wells examined are not positive, select 4 to 6 wells with 1 colony in the antibody-positive well and repeat the cloning. Finally, a hybridoma producing a monoclonal antibody against each polypeptide is obtained. Four types of monoclonal antibodies were obtained.
(h) Determination of monoclonal antibody class and subclass
According to the ELISA described above, the hybridoma supernatant obtained in the above (g) is added to a polystyrene 96-well plate coated with each polypeptide. Next, after washing with PBS, an isotype-specific rabbit anti-mouse IgG antibody (Zymed Lab.) Is added. After washing with PBS, horseradish peroxidase-labeled goat anti-rabbit IgG (H + L) was added, and the class and subclass were determined using hydrogen peroxide and 2,2'-azino-di (3-ethylbenzothiazophosphate) as substrates. To do.
[0112]
[Table 1]

[0113]
(i) Hybridoma culture and monoclonal antibody purification
The obtained hybridoma cells are cultured in NS-1 medium, and monoclonal antibodies can be obtained from the supernatant. The obtained hybridoma 10 ⁷ The mice were also intraperitoneally administered to mice (Balb / c strain, female, 6 weeks of age) that received pristane intraperitoneally one week in advance. After 1-2 weeks, 4-7 mg / ml Ascites fluid containing antibodies can be obtained. The obtained ascites is salted out with 40% saturated ammonium sulfate, and then purified by adsorbing IgG class antibody onto Protein A Affigel (Bio-Rad) and eluting with 0.1 M citrate buffer, pH 5.0.
[0114]
Example 3: Western blotting
9 μl of culture supernatant of COS7 cells expressing human soluble RAGE, 750 ng of purified recombinant human full-length mature RAGE (membrane-type RAGE) and 150 ng of human lung extract protein under reducing conditions on 10% SDS-PAGE After separation, it was transferred to a polyvinylidene difluoride (PVDF) membrane (MILLIPORE). Subsequently, after blocking for 1 hour at room temperature with PBS containing 1% BSA, 5% skim milk and 0.1% Tween 20 (blocking buffer), hybridoma cell lines 269-1D10, 269- expressing human soluble RAGE-specific antibodies The culture supernatant of 4C9, 269-6B12 or 269-9C2 was incubated at 25 ° C. for 1 hour. Each membrane was washed 4 times with PBS containing 0.1% Tween 20 (0.1% PBS-T), and the bound antibody was diluted 1: 1,000 with blocking buffer to an HRP-conjugated sheep anti-mouse immunoglobulin antibody (Amersham Pharmacia) and 25. The reaction was carried out at ° C for 1 hour. After the reaction, the membrane was washed 4 times with 0.1% PBS-T, and the bound antibody was detected by Enhanced chemiluminescence (ECL, Amersham Pharmacia). The positive control was human soluble RAGE protein-expressing COS7 cell culture supernatant, human recombinant full-length mature RAGE protein and human lung extract protein 3 μl, 250 ng and 50 ng, respectively, diluted 1: 500 with blocking buffer The same treatment was carried out with rabbit anti-human RAGE antiserum and HRP-conjugated goat anti-rabbit IgG (H + L) antibody (Zymed) diluted 1: 2,000 with blocking buffer, and ECL was detected. The results are shown in FIG.
M: Molecular weight marker
1, 4, 7, 10, 16: Recombinant membrane type RAGE
2, 5, 8, 11, 17: Recombinant soluble RAGE
3, 6, 9, 12, 18: Human lung extract (including a lot of membrane RAGE)
result
Each monoclonal antibody did not cross-react with recombinant membrane type RAGE and human lung extract medium membrane type RAGE, but reacted with soluble type RAGE.
[0115]
Example 4: Sandwich EIA
According to the following method, at least one anti-soluble RAGE antibody prepared in Example 2 is selected, and human soluble RAGE is specifically detected and measured by an appropriate combination of two anti-RAGE antibodies. A sandwich EIA system can be configured. The EIA system can be either a one-step method or a two-step method, and the labeled antibody is not limited to Fab′-HRP. The composition and reaction conditions of each reaction buffer can be adjusted according to the purpose of measurement, such as shortening or extending. In addition, human soluble RAGE as a standard product can be purified from tissue culture supernatant, cell culture supernatant, or a recombinant expressed in Example 2 or other methods. Purification can be achieved by ion exchange, gel filtration, affinity chromatography using an anti-human soluble RAGE monoclonal antibody, or any other combination of affinity chromatography.
(a) Preparation of labeled antibody
Anti-human soluble RAGE monoclonal antibody is digested at 37 ° C. for 24 hours by adding 2% (W / W) of pepsin to 0.1 M acetate buffer solution, pH 4.2 containing 0.1 M NaCl. Add 3M Tris-HCl, pH 7.5 to the digest to stop the reaction. F (ab ') by gel filtration on Ultrogel AcA54 column equilibrated with 0.1M phosphate buffer, pH 7.0 ₂ Collect fractions. This F (ab ') ₂ Add cysteamine hydrochloride to the fraction to a final concentration of 0.01M, reduce it at 37 ° C for 1.5 hours, and perform gel filtration on a Ultrogel AcA54 column equilibrated with 0.1M phosphate buffer containing 5 mM EDTA, pH 6.0. Collect the Fab 'fraction.
Separately from the above procedure, dissolve HRP in 0.1M phosphate buffer, pH 7.0, add 25 times molar amount of EMCS as HRP as DMF solution, and react at 30 ° C for 30 minutes. This is subjected to gel filtration with a NICK-5 column (Pharmacia) equilibrated with 0.1 M phosphate buffer, pH 6.0 to fractionate the maleimide-labeled HRP fraction.
Both fractions were mixed so that the Fab ′ fraction and maleimide-labeled HRP were equimolar and reacted at 4 ° C. for 20 hours, and then unreacted thiol groups were removed with N-ethylmaleimide in a 10-fold molar amount of Fab ′. To block. This is subjected to gel filtration with a Ultrogel AcA54 column equilibrated with 0.1 M phosphate buffer, pH 6.5, and the Fab′-HRP labeled antibody is collected. Add 0.1% BSA and 0.001% chlorhexidine to this and store at 4 ° C.
[0116]
(b) Preparation of monoclonal antibody binding carrier
Dissolve anti-human soluble RAGE monoclonal antibody in 0.1 M phosphate buffer, pH 7.5, and adjust to a concentration of 50 μg / mL. Add 100 μL of this monoclonal antibody solution to a 96-well microplate per well and let stand at 4 ° C. for 18 hours. Remove the monoclonal antibody solution and once with physiological saline, 0.05% Tween20, 0.1M NaCl, 5mM CaCl ₂ 1% BSA, 0.1 M NaCl, 5 mM CaCl after washing 3 times with Tris-HCl buffer, pH 8.0 ₂ Block by adding Tris-HCl buffer, pH 8.0.
(C) One-step sandwich EIA method
A standard curve for quantification of human soluble RAGE is prepared using the purified human soluble RAGE fraction as a standard antigen. 1% BSA, 0.05% Brij35, 0.05% Tween20, 0.1M NaCl, 5mM CaCl ₂ Disperse 60 μL of standard human soluble RAGE serially diluted with Tris-HCl buffer, pH 8.0, 1% BSA, 0.05% Brij35, 0.05% Tween 20, 0.1M NaCl, 5 mM CaCl ₂ Add 60 μL of labeled antibody Fab′-HRP prepared to 100 ng / 50 μL in Tris-HCl buffer solution, pH 8.0, and mix well. Prepare antibody-bound microplates with 0.05% Tween20, 0.1M NaCl, 5mM CaCl ₂ Wash 3 times with Tris-HCl buffer, pH 8.0, and add 100 μL / well of standard antigen and labeled antibody mixture. After 1 hour reaction at room temperature, 0.05% Tween20, 0.1M NaCl, 5mM CaCl ₂ Wash 3 times with Tris-HCl buffer, pH 8.0. Next, add 100 μL of 0.01% 3,3 ', 5,5'-tetramethylbenzidine dissolved in 0.1 M acetic acid buffer (pH 5.5) containing 6% dimethylformamide and 0.005% hydrogen peroxide, After 20 minutes of reaction at room temperature, add 100 μL of 2N sulfuric acid to stop the reaction. Measure 450 nm of this reaction mixture using a microplate reader to obtain a standard curve.
Samples to be measured are human serum, spinal fluid, plasma, joint fluid, urine and saliva, body fluid components derived from humans, various human tissue extracts, cell extracts of various cultured cells such as human or recombinant cells, Prepared from Qing et al. Each measurement sample is subjected to the above-mentioned one-step sandwich EIA instead of the standard human soluble RAGE, and the reaction is allowed to proceed simultaneously with the standard human soluble RAGE. The absorbance obtained from the measurement sample is applied to a standard curve, and the amount of human soluble RAGE contained in the measurement sample is calculated.
[0117]
Example 5: Assay of human soluble RAGE monoclonal antibody
Western blotting
Human mature RAGE protein overexpressing GEN-T cell extract (750 ng protein equivalent), human soluble RAGE protein overexpressing COS 7 cell culture supernatant 3 μl, human lung extract (150 ng protein equivalent) and Rainbow Marker (Myosin, phosphorylase b, bovine serum albumin, ovalbumin, carbonic anhydrase, trypsin inhibitor, lysozyme 3 μg each) ) (Amersham Pharmacia) was separated by 10% SDS-PAGE under reducing conditions, transferred to Immobilon transmembrane (MILLIPORE), and the membrane was transferred to PBS / 0.1% Tween 20/5% skim milk solution at room temperature. Shake gently over time. Next, after the membrane was reacted at room temperature for 1 hour in the culture supernatant of clone numbers 269-1D10, -4C9, -6B12 and -9C2, it was washed 4 times with 0.1% Tween 20 / PBS solution for 15 minutes. did. Subsequently, in anti-mouse Ig (Anti-mouse Ig), horseradish peroxidase linked whole antibody (from sheep) (Amersham Pharmacia) diluted 1,000-fold with PBS / 0.1% Tween 20/5% skim milk solution at room temperature. The mixture was allowed to react for 1 hour and washed 4 times with a PBS / 0.1% Tween 20 solution for 15 minutes. Thereafter, the membrane was treated with ECL Western Blotting Detection System (Amersham Pharmacia), and the signal was detected with X-ray film. The results are shown in FIG. In FIG. 8, M: molecular weight marker (each 3 μg), R: human mature RAGE-His tag protein, CM: COS 7 cell overexpressing human soluble RAGE protein, and L: human lung extract. None of the monoclonal antibodies reacted with human mature RAGE and human lung extract, but reacted only with human soluble RAGE.
[0118]
Example 6: Immunoprecipitation
1 ml of Protein G Sepharose (Amersham Pharmacia) was centrifuged at 200 × g for 1 minute, the supernatant was removed, TSA was added, and the resin was washed three times in the same manner. Next, 600 μl of culture supernatant of human soluble RAGE overexpressing COS 7 cells centrifuged at 10,000 rpm for 20 minutes was added, gently stirred at room temperature for 2 hours, and centrifuged at 200 × g for 1 minute. . 50 μg each of soluble RAGE monoclonal antibodies 269-1D10, 269-9C2 or normal mouse immunoglobulin dissolved in 100 μl of PBS was added to 50 μl of resin washed with TSA as above, and allowed to stand at 4 ° C. for 2 hours with occasional stirring. . Next, the mixture was centrifuged at 200 × g for 1 minute, and the supernatant was discarded. Then, 150 μl of the culture supernatant was dispensed and left at 4 ° C. for 2 hours with occasional stirring. After centrifugation at 200 × g for 1 minute and discarding the supernatant, 500 μl of TSA was added and the resin was washed three times in the same manner. Finally, 500 μl of Tris-HCl pH 6.8 was added to wash the resin, and then centrifuged at 200 × g for 1 minute, and the supernatant was discarded. Sample buffer (0.05 M Tris-HCl, pH 6.8 / 2% SDS / 0.6% 2-mercaptoethanol / 10% glycerol / 0.005% bromophenol blue) 50 μl was added, heated at 100 ° C. for 3 minutes, and 20 μl of the supernatant centrifuged at 200 × g for 1 minute was separated by 10% SDS-PAGE. After separation, the membrane was transferred to an Immobilon transmembrane (MILLIPORE), and the membrane was gently shaken in a PBS / 0.1% Tween 20/5% skim milk solution at room temperature for 1 hour. Next, the membrane was reacted in a 5 μg / ml rabbit anti-human soluble RAGE polyclonal antibody / PBS / 0.1% Tween 20/5% skim milk solution at room temperature for 1 hour, and then in a 0.1% Tween 20 / PBS solution for 15 minutes. Washed 4 times. Subsequently, in anti-mouse Ig (Anti-mouse Ig), horseradish peroxidase linked whole antibody (from sheep) (Amersham Pharmacia) diluted 1,000-fold with PBS / 0.1% Tween 20/5% skim milk solution at room temperature. The mixture was allowed to react for 1 hour and washed 4 times with a PBS / 0.1% Tween 20 solution for 15 minutes. Thereafter, the membrane was treated with ECL Western Blotting Detection System (Amersham Pharmacia), and the signal was detected with X-ray film. The results are shown in FIG. In FIG. 9, 1: monoclonal antibody 269-1D10, 2: monoclonal antibody 269-9C2, and 3: normal mouse immunoglobulin are shown. Anti-soluble RAGE monoclonal antibody binding affinity column trapped human soluble RAGE. The mouse normal immunoglobulin binding affinity column used as a control could not trap human soluble RAGE.
[0119]
Example 7: Preparation of human soluble RAGE immunoaffinity column
Dissolvable PD-10 column (Amersham Pharmacia) is used to couple the solvent of human soluble RAGE monoclonal antibody 269-1D10 to a coupling buffer (0.2 M NaHCO _Three , 0.5 M NaCl, pH 8.3). 1 ml of the above human soluble RAGE monoclonal antibody solution was injected into 1 ml of HiTrap NHS-activated (Amersham Pharmacia) washed with 6 ml of ice-cold 1 mM hydrochloric acid and allowed to react at room temperature for 30 minutes. Then inject 3 ml of coupling buffer into the column, add 1 ml of 2M Glycine-HCl, pH 2.0 to the eluate, and add A ₂₈₀ And the coupling efficiency was calculated. The column was washed sequentially with 6 ml Buffer A (0.5 M ethanolamine, 0.5 M NaCl, pH 8.3) and 6 ml Buffer B (0.1 M acetate, 0.5 M NaCl, pH 4.0), and then 6 ml Buffer A was added. Poured and left at room temperature for 30 minutes. The column was again washed sequentially with 6 ml Buffer B and 6 ml Buffer A, and finally with 6 ml Buffer A. The column is 2 ml stock solution (0.05 M Na ₂ HPO _Four , 0.1% NaN _Three , pH 7.0) was stored at 4 ° C. after injection. All the above operations were performed at a flow rate of 200 μl / min.
[0120]
Example 8: Separation of soluble RAGE protein from human serum
A soluble RAGE immunoaffinity column prepared according to the above method was attached to the SMART system, 5 ml start buffer (start buffer: 50 mM Tris-HCl, 150 mM NaCl, pH 8.0), 5 ml elution buffer (elution buffer: After washing sequentially with 100 mM glycine-HCl, 150 mM NaCl, pH 2.5), the column was equilibrated with 10 ml start buffer. Next, 8 ml of start buffer (start buffer) is added to 2 ml of human serum filtered with MILLEX-GV 0.22 μm Filter Unit (MILLIPORE), applied to the column at a flow rate of 100 μl / min, and 10 ml of start buffer (start buffer). Washed. Subsequently, while monitoring the eluate by ultraviolet absorption at 230 nm, 280 nm, and 300 nm, the bound product was eluted with 3 ml of elution buffer, and 100 μl was fractionated. 0.9 times the amount of trichloroacetic acid was added to the obtained peak fraction, left in ice for 30 minutes, centrifuged at 15,000 rpm for 20 minutes, then the supernatant was discarded and 750 μl of ethanol was added and centrifuged again at 15,000 rpm for 5 minutes. The supernatant was discarded and air-dried. The resulting precipitate was developed by 10% SDS-PAGE under reducing conditions, transferred to Immobilon transmembrane (MILLIPORE), and the membrane was transferred to PBS / 0.1% Tween20 / 5% skim milk solution at room temperature for 1 hour. Shake gently. Next, the membrane was reacted in a 5 μg / ml rabbit anti-human soluble RAGE polyclonal antibody / PBS / 0.1% Tween 20/5% skim milk solution at room temperature for 1 hour, and then with a PBS / 0.1% Tween 20 solution. Washed 4 times for 4 minutes. Subsequently, the membrane was reacted with 1.5 μg / ml anti-rabbit IgG (H + L) HRP conjugate (Zymed) /PBS/0.1% Tween20 / 5% skim milk solution at room temperature for 1 hour, and PBS / 0.1% Washed 4 times with Tween 20 solution for 15 minutes. Thereafter, the membrane was treated with ECL Western Blotting Detection System (Amersham Pharmacia), and the signal was detected with X-ray film. The results are shown in FIG.
The soluble RAGE in human serum was trapped with an anti-soluble RAGE monoclonal antibody-binding affinity column, and it was confirmed by Western blot that the trapped protein was human RAGE. From this, it was confirmed that soluble RAGE lacking the C-terminus of RAGE protein exists in human serum.
[0121]
Example 9: Examination of AGE neutralization of soluble RAGE
(Effect of soluble RAGE on AGE-induced enhancement of VEGF expression)
25 cm for the experiment ² Primary cultured human skin microvascular endothelial cells (purchased from Cascade Biologics, Inc., Portland, OR, Kurabo, Osaka) cultured to subconfluent in a flask were used. Cells are fresh 5% fetal bovine serum, 5 ng / ml basic fibroblast growth factor, 10 μg / ml heparin containing Hu-Media MV2 medium (assay medium) and then replaced with assay medium (1) not added, (2) added 10 μg / ml AGE2-BSA, (3) added 10 μg / ml AGE2-BSA and 25 μg / ml purified soluble RAGE, 37 ° C Incubated for 4 hours. After treatment, poly (A) from cells ⁺ RNA was isolated using Quickprep micro mRNA isolation kit (Amersham Pharmacia Biotech), and VEGF mRNA was detected using SuperScript One-Step RT-PCR kit (GIBCO BRL). Poly (A) used for VEGF mRNA amplification ⁺ The amount of RNA is 30 ng and the number of PCR cycles is 30. Β-actin was used as an internal control (30 ng, 20 cycles). After separating RT-PCR products by 3% agarose gel electrophoresis, alkaline transfer to nylon membrane, ³² Hybridization was performed with a P-labeled VEGF specific oligonucleotide probe. Primers and probes for VEGF mRNA amplification are the same as Nomura et al., J. Biol. Chem. 1995.
The results are shown in FIG. The level of VEGF mRNA expression in primary cultured human skin microvascular endothelial cells increases approximately 2-fold by the addition of AGE, but when AGE and soluble RAGE coexist, soluble RAGE acts as a dominant negative, and AGE mRNA levels by AGE Was prevented.
[0122]
Example 10: Sandwich assay for specifically measuring human soluble RAGE
(Measurement system A)
(a) Preparation of monoclonal antibody-binding carrier
Anti-human soluble RAGE monoclonal antibody 278-13F11 was dissolved in 0.1 M phosphate buffer, pH 7.5, and prepared to a concentration of 25 μg / mL. This monoclonal antibody solution was added to a 96-well microplate at 100 μL per well and allowed to stand at 4 ° C. for 16-24 hours. Remove the monoclonal antibody solution, wash once with purified water, add 300 μL of 1 mM BSA, 0.15 M NaCl, 0.001% chlorohexidine in 10 mM phosphate buffer, pH 7.0, and leave at 4 ° C. for 24 hours or longer. Blocked.
(b) Preparation of polyclonal antibody
Synthetic peptide [SEQ ID NO: 7] was conjugated to KLH by MBS method, and rabbit was immunized to obtain antiserum. The antiserum was purified using an affinity column coupled with the same synthetic peptide.
[0123]
(c) Sandwich EIA method using polyclonal antibody
A standard curve was prepared using the purified human soluble RAGE fraction as a standard antigen (FIG. 12). The prepared monoclonal antibody-binding carrier was washed once with 300 μL of 5 mM phosphate buffer containing 30 mM NaCl and 0.01% Tween 20, pH 7.0 (washing solution A). 1% BSA, 0.1 M NaCl, 15% Block Ace (Dainippon Pharmaceutical), 35 μg / mL HAMA reagent-containing 10 mM phosphate buffer, pH 7.0 (Buffer A) adjusted to 1.7 μg / mL 160 μL of soluble RAGE C-terminal peptide polyclonal antibody was added to each antibody-binding plate. Furthermore, 40 μL of the prepared standard solution or measurement sample was added and mixed well with a microplate mixer, and then a seal was attached to prevent the reaction solution from evaporating and left at 4 ° C. for 16 to 24 hours. Washing was performed 4 times with 300 μL per well of Washing Solution A, and 100 μL of anti-rabbit IgG peroxidase-labeled antibody (Amersham Pharmacia) diluted 5000-fold with Buffer A was added and allowed to stand at 25 ° C. for 2 hours. Wash 4 times with Wash Solution A, 300 μL per well, 0.1 mg / mL 3, 3 ', 5, 5'-tetramethylbenzidine, 0.0075% hydrogen peroxide, 1% DMF in 0.1M citrate buffer, pH 4.0 Was added in 100 μL aliquots and allowed to stand at 25 ° C. for 30 minutes. The reaction was stopped by adding 100 μL of 2N sulfuric acid, the reaction mixture was measured at a wavelength of 450 nm, and the measured value of the specimen was obtained from a standard curve.
[0124]
Example 11: Sandwich assay for specifically measuring human soluble RAGE
(Measurement system B)
(a) Preparation of monoclonal antibody-binding carrier
Anti-human soluble RAGE C-terminal peptide monoclonal antibody 269-1D10 was dissolved in 0.1 M phosphate buffer, pH 7.5, and prepared to a concentration of 25 μg / mL. Add 100 μL of this monoclonal antibody solution per well to a 96-well microplate and let stand at 4 ° C. for 16-24 hours. Remove the monoclonal antibody solution, wash once with purified water, add 1% BSA, 0.15 M NaCl, 0.001% chlorohexidine in 10 mM phosphate buffer, pH 7.0, 300 μL at a time, and leave at 4 ° C. for 24 hours or longer to block. did.
(b) Sandwich EIA method using monoclonal antibody
A standard curve was prepared using the purified human soluble RAGE fraction as a standard antigen (FIG. 13). The prepared monoclonal antibody-binding carrier was washed once with 300 μL of washing solution A. 160 μL of enzyme-labeled antibody solution (278-3A6, IgG-HRP) prepared to 1 μg / mL with buffer A was added to the antibody-binding plate. Furthermore, 40 μL of the prepared standard solution or measurement sample was added and mixed well with a microplate mixer, and then a seal was attached to prevent the reaction solution from evaporating and left at 4 ° C. for 16 to 24 hours. Wash 4 times with Wash Solution A at 300 μL per well, 0.1 mg / mL 3, 3 ', 5, 5'-tetramethylbenzidine, 0.0075% hydrogen peroxide, 0.1M citrate buffer with 1% DMF, pH 4.0 Add 100 μL each and leave at 25 ° C for 30 minutes. The reaction was stopped by adding 100 μL of 2N sulfuric acid, the reaction mixture was measured at a wavelength of 450 nm, and the measured value of the specimen was obtained from a standard curve.
[0125]
Example 12: Sandwich assay specifically measuring human soluble and membrane RAGE
(Measurement system C)
(a) Preparation of monoclonal antibody-binding carrier
Anti-human soluble RAGE monoclonal antibody 278-13F11 was dissolved in 0.1 M phosphate buffer, pH 7.5, and prepared to a concentration of 25 μg / mL. Add 100 μL of this monoclonal antibody solution per well to a 96-well microplate and let stand at 4 ° C. for 16-24 hours. Remove the monoclonal antibody solution, wash once with purified water, add 1% BSA, 0.15 M NaCl, 0.001% chlorohexidine in 10 mM phosphate buffer, pH 7.0, 300 μL at a time, and leave at 4 ° C. for 24 hours or longer to block. did.
(b) Sandwich EIA method using monoclonal antibody
A standard curve was prepared using the purified human soluble RAGE fraction as a standard antigen (FIG. 14). The prepared monoclonal antibody-binding carrier was washed once with 300 μL of washing solution A. 100 μL of enzyme-labeled antibody solution (278-3A6, IgG-HRP) prepared to 1 μg / mL with buffer A was added to the antibody-binding plate. Furthermore, 20 μL of the prepared standard solution or measurement sample was added, and after mixing well with a microplate mixer, the mixture was allowed to stand at 25 ° C. for 1 to 2 hours. Wash 4 times with Wash Solution A at 300 μL per well, 0.1 mg / mL 3, 3 ', 5, 5'-tetramethylbenzidine, 0.0075% hydrogen peroxide, 0.1M citrate buffer with 1% DMF, pH 4.0 Was added in 100 μL aliquots and allowed to stand at 25 ° C. for 30 minutes. The reaction was stopped by adding 100 μL of 2N sulfuric acid, the reaction mixture was measured at a wavelength of 450 nm, and the measured value of the specimen was obtained from a standard curve.
[0126]
[Specificity of each measurement system]
In all of measurement systems A, B, and C, a good standard curve could be drawn in the range of 0.1 to 6.4 ng / mL using human soluble RAGE as a standard substance.
Membrane-type RAGE overexpressing COS-7 cells were cultured to confluence, the medium was removed, and the cell layer was washed with PBS. Thereafter, the cells were lysed with 20 mM Tris-HCl, pH 7.4 containing 1% n-Octylglucoside, 0.1 M NaCl, 1 mM PMSF, and the centrifuged supernatants were respectively A, B, and C using soluble RAGE as a standard substance. The measurement system of The measurement value in the measurement system C was 48 ng / mL, but in the measurement systems A and B, they were 0 ng / mL and 2.7 ng / mL, respectively (FIG. 15). From these, it was confirmed that the measurement system C recognizes both soluble type and membrane type RAGE, and the measurement systems A and B recognize only soluble type RAGE.
[0127]
[Measurement of soluble RAGE in human blood using measurement system A]
Diabetes disease duration 7.8-17.3 years (average 12.5 ± 2.6 years), measurement system A that specifically measures human soluble RAGE in type I diabetic sera and plasma treated only with subcutaneous insulin self-injection therapy Measured with
The degree of progression of retinopathy was classified into normal and simple retinopathy based on the fundus findings. As for nephropathy, the urinary albumin / creatinine ratio by urine was determined to be less than 30 mg / g as early stage nephropathy and 30-299 mg / g as early stage nephropathy according to the revision plan of the Ministry of Health and Welfare. Patient samples with preproliferative retinopathy, proliferative retinopathy and overt nephropathy and patients with renal failure with serum creatinine value of 2 mg / dL or higher were excluded. Patients with no retinopathy and nephropathy (no complication group), patients with simple retinopathy without nephropathy (simple retinopathy patient group), and early nephropathy without retinopathy The amount of soluble RAGE in the blood was measured using a measurement system A between patient groups (early nephropathy patient group) and statistically analyzed.
As a result, the amount of soluble RAGE in the group without complications (diabetes morbidity 12.1 ± 2.3 years) was as follows: patients with simple retinopathy (diabetes morbidity 13.1 ± 2.6 years), patients with early nephropathy (diabetes morbidity 11.7 years) (P + 2.5 years) and significant (p <0.005, p A high value was observed at <0.05) (Fig. 16).
There are individual differences in the onset of diabetic complications, and it is known that about 30%, especially in nephropathy, is susceptible. In this study, the amount of soluble RAGE in the blood in the complication-free group was significantly higher than that in the simple retinopathy patient group and the early nephropathy patient group. This suggests that the genetic difference in the expression of soluble RAGE among individuals during the onset of diabetic complications is one of the determinants of susceptibility / resistance to diabetic complications. The possibility of functioning protectively against the occurrence of the disease was considered. That is, the possibility of predicting the susceptibility and resistance to diabetic complications by quantifying soluble RAGE was suggested.
[0128]
【The invention's effect】
It is possible to measure natural soluble RAGE and is useful for diagnosis of diseases caused by the interaction between AGE and RAGE, investigation of the cause of diabetic complications, diagnosis and risk prediction. It is possible to create an active substance such as a monoclonal antibody against human soluble RAGE protein and to develop a measurement system for the protein using this, for predicting the risk of developing or developing diabetic complications. Useful. In addition, it is possible to develop a compound that controls the production of soluble RAGE, and it is useful for diagnosis of cancer metastasis and invasion.
The soluble RAGE of the present invention is a naturally occurring peptide (endogenous peptide or endogenous peptide) that exists in vivo, and differs from the RAGE protein in 16 amino acid residues at the C-terminal portion. The soluble RAGE of the present invention is a natural form produced endogenously, so it is physiological, has a low risk of forming an antibody, and has developed an orally soluble soluble RAGE production control compound and drug. Then, if the expression of the soluble RAGE of the present invention can be induced by such a medicine, it is possible to protect diabetic patients from complications without causing pain. INDUSTRIAL APPLICABILITY According to the present invention, it is possible to design and obtain an endogenous soluble RAGE polypeptide or a salt thereof, as well as mutants, modifications, derivatives and the like based on the endogenous soluble RAGE polypeptide, and nucleic acids encoding them, the nucleic acids A host cell transformed with the vector, a disease associated with endogenous soluble RAGE, such as diabetic complications, various diseases associated with aging, Alzheimer's disease, arteriosclerosis, in vivo protein It is useful for studying the onset and / or progression of diseases or illnesses caused by glycation of drugs, and pathological conditions or symptoms such as tumor invasion or spread. It can be expected to open the way.
It will be apparent that the invention may be practiced otherwise than as particularly described in the foregoing description and examples. Many modifications and variations of the present invention are possible in light of the above teachings, and thus are within the scope of the claims appended hereto.
[0129]
[Sequence Listing]

[Brief description of the drawings]
FIG. 1 shows the structure of the human RAGE gene and the molecular diversity of the RAGE protein produced by alternative splicing of the RAGE gene transcript. The C-terminal deletion soluble type corresponds to the soluble type RAGE of the present invention.
[Fig. 2] The soluble RAGE of the present invention is compared with a transmembrane RAGE (fullRAGE) and a so-called solubilized RAGE (US5864018) artificially produced from a fullRAGE gene having a transmembrane domain. Shows the base sequence of the encoding nucleic acid (solubleRAGE).
FIG. 3 shows the base sequences of fullRAGE, US5864018 and solubleRAGE following FIG.
FIG. 4 shows the base sequences of fullRAGE, US5864018 and solubleRAGE following FIG.
FIG. 5 shows the amino acid sequence of soluble RAGE of the present invention compared to fullRAGE and US5864018.
FIG. 6 shows an electrophoresis photograph after 10% SDS-PAGE separation of sRAGE protein. 1 is a silver-stained image of the cell culture supernatant, and 2 is a silver-stained image of the purified protein.
FIG. 7 shows the results of Western blotting using the anti-soluble RAGE monoclonal antibody of the present invention.
FIG. 8 shows an electrophoretogram of the results of Western blotting of hybridoma clones.
After separating mature RAGE (R), soluble RAGE overexpressing COS 7 cell culture supernatant (CM), human lung extract protein (L) and molecular weight marker (M) containing 7 kinds of proteins by SDS-PAGE, Western blotting was performed using the supernatant of the hybridoma culture producing the anti-soluble RAGE monoclonal antibody.
FIG. 9 shows an electrophoresis photograph of the result of immunoprecipitation using a soluble RAGE monoclonal antibody.
Soluble RAGE monoclonal antibody 269-1D10 (1), 269-9C2 (2) or protein G Sepharose conjugated with normal mouse immunoglobulin (3) The protein was immunoprecipitated, separated by SDS-PAGE, and soluble RAGE was detected by Western blotting.
[Fig. 10] Obtained from the elution pattern and peak fraction obtained as a result of immunoaffinity column chromatography using anti-human soluble RAGE monoclonal antibody to detect soluble RAGE protein present in human serum. An electrophoretic photograph of the resulting precipitate is shown.
2 ml of human serum was applied to an immunoaffinity column bound with 2.5 mg of human soluble RAGE monoclonal antibody 269-1D10, and column chromatography was performed. The peak fraction (fraction no. 16 to 18) was treated with TCA, the precipitate was developed by SDS-PAGE, and Western blotting was performed using a rabbit anti-human soluble RAGE polyclonal antibody.
FIG. 11 shows neutralizing action of AGE endothelial action by soluble RAGE.
After treatment of primary cultured human skin microvascular endothelial cells in (1) not added, (2) 10 μg / ml AGE2-BSA added, (3) 10 μg / ml AGE2-BSA and 25 μg / ml purified soluble RAGE added culture medium, Poly (A) than cells ⁺ RNA was isolated and VEGF mRNA was detected.
FIG. 12 shows a standard curve of a soluble RAGE fraction (standard antigen) obtained by a human soluble RAGE measurement sandwich assay system (measurement system A) using an anti-human soluble RAGE antibody.
FIG. 13 shows a standard curve of a soluble RAGE fraction (standard antigen) obtained by a human soluble RAGE measurement sandwich assay system (measurement system B) using an anti-human soluble RAGE antibody.
FIG. 14: Standard curve of soluble RAGE fraction (standard antigen) obtained by sandwich assay system (measurement system C) specific for human soluble type and membrane type RAGE using anti-human soluble type RAGE antibody Indicates.
FIG. 15 shows the results of measuring lysates (supernatant) of membrane-type RAGE overexpressing COS-7 cells with measurement systems A, B and C. Soluble RAGE was used as a standard substance.
FIG. 16 shows the results of measuring human soluble RAGE in blood using measurement system A for type I diabetic patients.

Claims (5)

(1) A soluble protein comprising the following amino acid sequence (i) or (ii) and having an activity of binding to an advanced glycation endproducts ( AGE ):
(i) Amino acid sequence of SEQ ID NO: 2 in the sequence listing
(ii) having at least 7 to 16 consecutive amino acid sequences of amino acid numbers 332 to 347 of SEQ ID NO: 2 in the sequence listing, and a part of the amino acids of the amino acid sequence of (i) above being deleted, Among the amino acid numbers 332 to 347 of SEQ ID NO: 2 in the sequence listing, which is an antibody against an amino acid sequence which is substituted or added, and as a result, has no more than 10 amino acids mutated from the amino acid sequence of (i) above An antibody that specifically immunoreacts with at least 7 to 16 consecutive amino acid sequences, or
(2) An antibody against a peptide comprising a continuous amino acid sequence of at least 7 to 16 of amino acid numbers 332 to 347 of SEQ ID NO: 2 in the Sequence Listing. The antibody according to claim 1 is used as a measurement reagent.
(1) A soluble protein or salt thereof comprising the following amino acid sequence (i) or (ii) and having an activity of binding to a late glycation reaction product:
(i) Amino acid sequence of SEQ ID NO: 2 in the sequence listing
(ii) having at least 7 to 16 consecutive amino acid sequences of amino acid numbers 332 to 347 of SEQ ID NO: 2 in the sequence listing, and a part of the amino acids of the amino acid sequence of (i) above being deleted, An amino acid sequence in which the number of amino acids that have been substituted or added and consequently mutated from the amino acid sequence of (i) above is within 10;
Or
(2) A method for immunologically measuring a peptide consisting of at least 7 to 16 consecutive amino acid sequences of amino acid numbers 332 to 347 of SEQ ID NO: 2 in the sequence listing or a salt thereof.  A composition comprising the antibody according to claim 1. The antibody according to claim 1 is included.
(1) A soluble protein or salt thereof comprising the following amino acid sequence (i) or (ii) and having an activity of binding to a late glycation reaction product:
(i) Amino acid sequence of SEQ ID NO: 2 in the sequence listing
(ii) having at least 7 to 16 consecutive amino acid sequences of amino acid numbers 332 to 347 of SEQ ID NO: 2 in the sequence listing, and a part of the amino acids of the amino acid sequence of (i) above being deleted, An amino acid sequence in which the number of amino acids that have been substituted or added and consequently mutated from the amino acid sequence of (i) above is within 10;
Or
(2) An immunoassay reagent for a peptide consisting of at least 7 to 16 consecutive amino acid sequences of amino acid numbers 332 to 347 of SEQ ID NO: 2 in the sequence listing or a salt thereof.  An interaction between the late saccharification reaction product and its receptor, the expression level of a soluble protein having an activity of binding to the late saccharification reaction product, comprising the antibody according to claim 1 and / or Alternatively, a diagnostic agent for a disease caused by a change in capture activity of late saccharification reaction product.

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