CN111234011B

CN111234011B - Hepatitis B virus neutralizing antibody B826 and application thereof

Info

Publication number: CN111234011B
Application number: CN201811442215.3A
Authority: CN
Inventors: 张林琦; 贲海静; 史宣玲
Original assignee: Tsinghua University
Current assignee: Tsinghua University
Priority date: 2018-11-29
Filing date: 2018-11-29
Publication date: 2022-01-11
Anticipated expiration: 2038-11-29
Also published as: CN111234011A

Abstract

The invention discloses a hepatitis B virus neutralizing antibody B826 and application thereof. The invention provides an IgG antibody, which is named as a monoclonal antibody B826 and consists of a light chain and a heavy chain; CDR1, CDR2 and CDR3 in the heavy chain variable region in the heavy chain are the 45 th-52 th amino acid residue, the 70 th-77 th amino acid residue and the 116 th-131 th amino acid residue from the N terminal of the sequence 1 in the sequence table in sequence; the CDR1, CDR2 and CDR3 in the light chain variable region in the light chain are the 43 th-54 th amino acid residue, the 72 th-74 th amino acid residue and the 111 th-119 th amino acid residue from the N terminal of the sequence 3 in the sequence table in sequence. The invention can be used for treating hepatitis B chronic infection patients and clinically replacing serum purified polyclonal antibody to prevent mother-infant transmission. The invention has important application prospect in treating and preventing hepatitis B virus infection.

Description

Hepatitis B virus neutralizing antibody B826 and application thereof

Technical Field

The invention relates to a hepatitis B virus neutralizing antibody B826 and application thereof.

Background

Hepatitis B Virus (HBV) infection is widespread throughout the world and is particularly severe in china. Hepatitis B virus belongs to a member of hepadnaviridae, and the infection of hepatitis B virus induces hepatitis, about 8000 ten thousand to 1 hundred million people in China have chronic hepatitis B virus infection, and about 2.4 hundred million chronic hepatitis B patients in the world. Hepatitis B virus infectors add up more if considered already recovered acute hepatitis B virus infectors. Research shows that 35-62% of Chinese people are infected by hepatitis B virus, 56-98% of sub-sahara south Africa people are infected by hepatitis B virus, about 20 hundred million people are infected by hepatitis B virus all over the world, and hepatitis B virus infection is a global sanitary problem.

Hepatitis B virus induces immune inflammation to damage the liver of a patient during the infection process. Most of Chinese hepatitis B patients are infected with hepatitis B virus by mother and infant transmission or children, most of the Chinese hepatitis B patients are infected with chronic hepatitis B virus, and the Chinese hepatitis B patients carry the hepatitis B virus throughout the life. The adult infected with hepatitis B virus can eliminate hepatitis B virus by means of autoimmune system, and is acute infection of hepatitis B virus. Hepatitis B virus infection causes acute and chronic hepatitis, cirrhosis and even liver cancer, the mechanism of inducing hepatitis is mainly that hepatitis B virus in liver cells induces liver inflammation of the immune system of the organism, the replication template covalent closed circular DNA (cccDNA) of the hepatitis B virus is used as a virus storage bank to be stably and timely supplemented and is very difficult to remove, and the replication of the hepatitis B virus induces liver inflammation in the continuous and repeated immune killing process.

At present, the hepatitis B virus vaccine has good prevention effect, can only control the replication of hepatitis B virus in clinical treatment, and can not cure hepatitis B virus infection. The hepatitis B vaccine controls the wide spread of hepatitis B virus in China and the mother-infant spread caused by the hepatitis B mother, clinically reduces the hepatitis B virus replication level of the hepatitis B mother during pregnancy through nucleoside medicaments such as entecavir and tenofovir, and comprehensively intervenes by measures of injecting Hepatitis B Immunoglobulin (HBIG) after the birth of a newborn and timely immunizing the hepatitis B vaccine, thereby effectively reducing the mother-infant spread of the hepatitis B virus. Although hepatitis B vaccines control the transmission of hepatitis B virus, the hepatitis B virus infection is not cured by clinical treatments such as nucleoside drugs and interferon for patients with a large number of infected hepatitis B viruses. The nucleoside medicine can effectively control the DNA replication of hepatitis B virus, but can not completely eliminate hepatitis B virus, and is difficult to reduce hepatitis B surface antigen, and hepatitis B virus still can relapse after a hepatitis B patient stops taking the medicine. The interferon has long treatment period and great side effect, can only act on a relatively small number of hepatitis B patients during the treatment period, has low cost performance of treatment effect, and basically treats hepatitis B infection by mainly taking nucleoside medicaments at present.

Disclosure of Invention

The invention aims to provide a hepatitis B virus neutralizing antibody B826 and application thereof.

The invention provides an IgG antibody, which is named as a monoclonal antibody B826 and consists of a light chain and a heavy chain; CDR1, CDR2 and CDR3 in the heavy chain variable region in the heavy chain are the 45 th-52 th amino acid residue, the 70 th-77 th amino acid residue and the 116 th-131 th amino acid residue from the N terminal of the sequence 1 in the sequence table in sequence; the CDR1, CDR2 and CDR3 in the light chain variable region in the light chain are the 43 th-54 th amino acid residue, the 72 th-74 th amino acid residue and the 111 th-119 th amino acid residue from the N terminal of the sequence 3 in the sequence table in sequence.

The heavy chain variable region consists of 20 th-142 th amino acid residues from the tail end of N of a sequence 1 in a sequence table.

The light chain variable region consists of 17 th to 129 th amino acid residues from the tail end of N of a sequence 3 in a sequence table.

The heavy chain is (a) or (b) as follows: (a) protein consisting of 20 th-472 th amino acid residues from the tail end of N in a sequence 1 of a sequence table; (b) a protein shown in a sequence 1 in a sequence table.

The light chain is (c) or (d) as follows: (c) protein consisting of 17 th to 236 th amino acid residues from the N tail end of the sequence 3 in the sequence table; (d) a protein shown in sequence 3 of the sequence table.

The gene encoding the IgG antibody also belongs to the protection scope of the invention.

The gene encoding the heavy chain is (1) or (2) or (3) as follows:

(1) a DNA molecule shown as nucleotide 972-2330 from the 5' end of the sequence 2 in the sequence table;

(2) a DNA molecule shown as the 915-2333 nucleotide from the 5' end of the sequence 2 in the sequence table;

(3) a DNA molecule shown in a sequence 2 of a sequence table.

The genes encoding the light chain are as follows (4) or (5) or (6):

(4) DNA molecule shown by the 963-1622 th nucleotide from the 5' end of the sequence 4 in the sequence table;

(5) DNA molecule shown by 915-1625 th nucleotide from 5' end of sequence 4 in the sequence table;

(6) DNA molecule shown in sequence 4 of the sequence table.

The invention also protects the application of any IgG antibody in preparing a medicament for inhibiting hepatitis B virus.

The invention also provides a medicament for inhibiting hepatitis B virus, and the active ingredient of the medicament is the IgG antibody.

The invention also protects the application of any IgG antibody in the preparation of a medicament for neutralizing hepatitis B virus.

The invention also provides a medicament for neutralizing hepatitis B virus, and the active ingredient of the medicament is the IgG antibody.

The invention also protects the application of any IgG antibody in the preparation of a medicament for preventing and/or treating hepatitis B.

The invention also provides a medicament for preventing and/or treating hepatitis B, which comprises the IgG antibody as an active ingredient.

Any one of the above hepatitis B viruses is a C genotype hepatitis B virus.

Any one of the hepatitis B viruses is hepatitis B caused by C genotype hepatitis B virus.

Hepatitis B virus of C genotype, i.e., the hepatitis B virus of which the genotype is C type.

The monoclonal antibody B826 does not aim at the a epitope of HBs small membrane protein (the a epitope is common to all hepatitis B virus genotypes), specifically identifies the most extensive hepatitis B virus genotype-C genotype hepatitis B virus in China, and is a specific neutralizing antibody of the hepatitis B virus C genotype. The a epitope mutation is a very important problem in clinic, and because the monoclonal antibody B826 can recognize a clinical mutant strain of the a epitope (such as a clinical mutant strain G145R) without aiming at the a epitope, the a epitope mutation can be clinically used for neutralizing the clinical mutant strain of the a epitope or can be combined with the a epitope monoclonal antibody to prevent the appearance of the mutant strain of the a epitope to enhance the treatment effect.

Compared with serum polyclonal antibody, the monoclonal antibody has great advantages in neutralizing activity and source convenience. The invention utilizes a single B cell cloning technology to separate the monoclonal antibody B826 from a hepatitis B vaccine immunized person, and can be used for treating hepatitis B chronic infection patients and clinically replacing serum purified polyclonal antibody to prevent mother-infant transmission. The invention has important application prospect in treating and preventing hepatitis B virus infection.

Drawings

FIG. 1 shows the result of step one in example 3.

FIG. 2 shows the results of step two of example 3.

FIG. 3 shows the results of example 4.

Detailed Description

The following examples are given to facilitate a better understanding of the invention, but do not limit the invention. The experimental procedures in the following examples are conventional unless otherwise specified. The test materials used in the following examples were purchased from a conventional biochemical reagent store unless otherwise specified. The quantitative tests in the following examples, all set up three replicates and the results averaged. Unless otherwise specified, the PBS buffer used in the examples was 10mM PBS buffer at pH 7.2. PBST solution: 0.5% (volume percent) Triton X-100 in PBS buffer. Hepatitis b immunoglobulin solution (HBIG solution): beijing Tiantan organism with concentration of about 120 mg/ml. Unless otherwise specified, the cell culture conditions were 37 ℃ and 5% CO₂And (4) environment. JC126 vector contains HDV replicon, references to JC126 vector: li Ying et al, construction of hepatitis D virus replication packaging vector, China journal of modern medicine, 2017(11), pages 31-35. The expression plasmid pHBV1.5 for type A hepatitis B envelope protein is described in the following documents: bruss, V.and D.Ganem, The roll of envelope proteins in nanoparticles B virus assembly, Proc Natl Acad Sci U S A,1991.88(3): p.1059-63. The expression plasmid for type B hepatitis B envelope protein, pHBV1.18-B, is described in the following documents: hu, w., et al, CpG oligodeoxynucleotide inhibitors HBV reproduction in a dynamic information microorganism model, antibody Ther,2014. The expression plasmid of the type C hepatitis B envelope protein is pHBV1.18-C. The following documents are described: wang, X.J., et al, A simple and effective strategy for the de novo constraint of great-this-genome-length syndromes B viruses reptilities.J., Methods 2014.207: p.158-62. The expression plasmid of hepatitis B envelope protein type D, namely pCMV-HBV, is described in the following documents: li, J., et al, Inhibition of reproduction of videos by MyD88involves accessed prediction ofpregenomic RNA and nuclear retentions of pre-S/S RNAs.J Virol,2010.84(13): p.6387-99. HBs antigen: GENBANK ACCESSION NO: P30019.1, VRL 07-NOV-2018. HBV transgenic mice (high replication HBV transgenic mice): the following documents are described: liu Lu luster, etc., the research on the effect of a high-replication HBV transgenic mouse model on anti-hepatitis B virus drugs, and 99-102 of Chinese pathophysiology journal, 2007,23 (1). Rabbit anti-human hepatitis delta antigen polyclonal antibody (HDV delta antibody for short): beijing Zexi Yuan GmbH. The monoclonal antibody MERS-27 and the preparation method thereof are disclosed in patent 201310565893.X (publication No. CN 104628848A, publication No. CN 104628848B). The hepatitis B virus surface antigen diagnostic kit (HBsAg, cat # 30811010101) and the hepatitis B virus e antigen detection kit (HBeAg, cat # 30811010103) are both Shanghai Kehua products.

Example 1 discovery of antibodies

The monoclonal antibody of hepatitis B virus is searched from the blood of hepatitis B vaccine immunized persons by using single B cell clone, and then the obtained antibodies are compared in effect.

The method for cloning the hepatitis B small membrane protein specific B cell antibody gene by antigen bait flow sorting comprises the following steps: separating peripheral blood lymphocytes PBMC from peripheral blood of normal human body immunized by the hepatitis B vaccine, sorting memory B cells by magnetic beads, sorting cells specifically combined with hepatitis B small membrane protein from the memory B cells by a flow cytometer, cracking the cells, obtaining cDNA by RT-PCR, performing nested PCR by using antibody variable region specific primers, sequencing to obtain antibody genes, and further screening to confirm that the antibody is hepatitis B small membrane protein monoclonal antibody.

A monoclonal antibody (binding antibody) having a good effect was found, and this monoclonal antibody was designated as monoclonal antibody B826, and abbreviated as B826 antibody.

The monoclonal antibody B826 is an IgG antibody, the heavy chain is shown as a sequence 1 in a sequence table (the amino acid residues at the 1 st to 19 th positions form a signal peptide, the amino acid residues at the 20 th to 142 th positions form a variable region, the amino acid residues at the 143 st and the 472 th positions form a constant region; the CDR1, the CDR2 and the CDR3 are sequentially shown as the 45 th to 52 th positions, the 70 th to 77 th positions and the 116 th and the 131 th positions), and the light chain is shown as a sequence 3 in the sequence table (the amino acid residues at the 1 st to 16 th positions form the signal peptide, the amino acid residues at the 17 th to 129 th positions form the variable region, the amino acid residues at the 130 th and the 236 th positions form the constant region; and the CDR1, the CDR2 and the CDR3 are sequentially shown as the 43 th to 54 th positions, the 72 th to 74 th positions and the 111 th and the 119 th positions).

Example 2 preparation of monoclonal antibody B826

Firstly, preparing recombinant plasmid

Inserting the DNA molecule shown in the sequence 2 of the sequence table into pLB-simple Vector to obtain the recombinant plasmid. The recombinant plasmid has been verified by sequencing. This plasmid was in turn designated as heavy chain expression plasmid.

In the sequence 2 of the sequence table, the 1 st to 666 th nucleotides are CMV promoters, the 915 nd and 971 st nucleotides are signal peptide coding regions, the 972 nd 1340 th nucleotides are heavy chain variable region coding regions, the 1341 st 2330 th nucleotides are heavy chain constant region coding regions, the 2331 st 2333 th nucleotides are stop codons, and the 2392 nd 2540 th nucleotides are polyA terminators. The DNA molecule shown in the sequence 2 of the sequence table expresses the heavy chain shown in the sequence 1 of the sequence table.

Inserting the DNA molecule shown in the sequence 4 of the sequence table into pLB-simple Vector to obtain the recombinant plasmid. The recombinant plasmid has been verified by sequencing. This plasmid was in turn designated as light chain expression plasmid.

In the sequence 4 of the sequence table, the 1 st to 666 th nucleotides are CMV promoters, the 915 nd and 962 th nucleotides are signal peptide coding regions, the 963 nd and 1301 th nucleotides are light chain variable region coding regions, the 1302 nd and 1622 th nucleotides are light chain constant region coding regions, the 1623 nd and 1625 th nucleotides are stop codons, and the 1632 nd and 1780 th nucleotides are polyA terminators. The DNA molecule shown in the sequence 4 of the sequence table expresses the light chain shown in the sequence 3 of the sequence table.

pLB-simple Vector is a component of pLB zero background quick connect kit with the biological cargo number of Tiangen VT206, http:// www.tiangen.com/? productive show/t1/6/id/308. html.

II, preparing the antibody

1. The 293T cells are cultured by adopting serum-free DMEM medium, the heavy chain expression plasmid and the light chain expression plasmid are cotransfected by virtue of PEI transfection reagent and cultured for 8 hours, then the culture medium is replaced by DMEM medium containing 2% fetal calf serum and cultured for 72 hours, then the cells are centrifuged at 4 ℃ and 4000rpm for 30min, and supernatant is collected.

2. And (3) taking the supernatant obtained in the step 1, and purifying by using an Econo-Pac polypropylene chromatographic column.

Polypropylene chromatography column (Biorad, cat # 7321010), 1.5X 12cm, bed volume about 20 ml.

The method comprises the following operation steps: mixing 300-500mL of supernatant and about 1mL of protein A beads (Thermo, with the stock number of 10006D), carrying out shaking culture at 4 ℃ for 10 hours, and then adding the whole system into a chromatographic column; washing with 60ml binding buffer solution; and thirdly, eluting with 30mL of elution buffer solution, and collecting the solution after passing through the column.

Binding buffer (ph 8.0): each liter contains 112.6g of glycine, 175.2g of sodium chloride and the balance of water.

Elution buffer (ph 3.0): every 500ml contains 7.5g of glycine and the balance of water.

3. And (3) taking the solution obtained in the step (2) after passing through the column, concentrating by using an ultrafiltration concentration tube, and replacing the system by PBS buffer solution to obtain an antibody solution with the antibody concentration of about 2mg/ml, wherein the antibody solution is named as B826 antibody solution.

Example 3 binding ability of antibody to hepatitis B Virus

First, ELISA detection

1. The microplate was removed and the coating solution was added for coating (50ng coating antigen/well).

The coating antigen is HBs antigen. The coating stock solution was prepared using PBS buffer as a solvent.

2. After completion of step 1, the microplate was removed, 200. mu.l of PBS buffer containing 2% BSA was added to each well, incubated at 37 ℃ for 2 hours for blocking, and then the supernatant was aspirated and washed twice with PBST solution.

3. After completing step 2, the microplate was taken, 200. mu.l of antibody diluent was added to each well, incubated at 37 ℃ for 1 hour, and then the supernatant was aspirated and washed twice with PBST solution. The antibody dilution was obtained by diluting the B826 antibody solution or HBIG solution or MERS-27 antibody solution prepared in example 2 with PBS buffer. Each antibody dilution was set with 5 replicate wells.

4. After completion of step 3, the microplate was taken, anti-human IgG-HRP (goat anti-human IgG-HRP, Promega, cat # W4038) was added, incubated at 37 ℃ for 1 hour, and then the supernatant was aspirated and washed 6 times with PBST solution.

5. And (4) after the step (4) is finished, adding a developing solution into the ELISA plate for developing, stopping developing at a proper time, and reading the absorbance value of 450 nm.

The results are shown in FIG. 1. The abscissa of fig. 1 shows the concentration of B826 antibody in the antibody dilution, the concentration of HBIG is 1000 times the concentration of B826 antibody, and the concentration of MERS-27 antibody is the same as that of B826 antibody. For example, when the concentration of B826 antibody in the antibody dilution was 0.1. mu.g/ml, the concentration of HBIG was 0.1mg/ml, and the concentration of MERS-27 antibody was 0.1. mu.g/ml. The results indicate that the B826 antibody can efficiently bind to HBs antigen.

Second, immunofluorescence assay

The test plasmid is an A-type hepatitis B envelope protein expression plasmid or a B-type hepatitis B envelope protein expression plasmid or a C-type hepatitis B envelope protein expression plasmid or a D-type hepatitis B envelope protein expression plasmid.

The test antibody solution was the B826 antibody solution or MERS-27 antibody solution prepared in example 2.

1. Taking 6-well plate, placing alcohol-sterilized cover glass in each well before plate spreading, and adding about 10 per well⁶Huh7 cells were cultured for 15 hours.

2. After completion of step 1, the 6-well plate was taken, and 2. mu.g of the plasmid to be tested and 2. mu.g of PEI transfection reagent were added to each well and cultured for 72 hours.

3. After step 2, the coverslip in the 6-well plate was taken, washed 3 times with PBS buffer, then fixing for 15min by 4% paraformaldehyde solution, then washing for 3 times by PBS buffer solution, then the membrane was penetrated with PBST solution at room temperature for 20min, and then washed 3 times with PBS buffer, then adding goat serum dropwise, sealing at room temperature for 30min, adding primary antibody working solution (diluting the test antibody solution to 1000 times volume) dropwise, incubating at 4 deg.C for 15 hr, then washed 3 times with PBST solution, then added dropwise with a working solution of a fluorescent secondary antibody (goat anti-human IgG-Dylight 594, cat # CW0248, kang century) and incubated at 37 ℃ for 1h, then 3 times of immersion washing with PBST solution, then counterstaining with DAPI, then 4 times of washing with PBST solution, then sealing the film by using a sealing liquid containing an anti-fluorescence quenching agent, and collecting an analysis image under a laser confocal microscope.

The results are shown in FIG. 2. The result shows that the B826 antibody can be specifically combined with the membrane protein expressed by the hepatitis B virus of the C genotype, and is not combined with the membrane protein expressed by the hepatitis B virus of the A genotype, the membrane protein expressed by the hepatitis B virus of the B genotype and the membrane protein expressed by the hepatitis B virus of the D genotype.

Example 4 HDV infection and antibody neutralization assay

The test antibody solution was the B826 antibody solution or MERS-27 antibody solution or HBIG solution prepared in example 2. A blank control (NC) was set with an equal volume of PBS buffer instead of test antibody solution.

The test plasmid is C-type hepatitis B envelope protein expression plasmid or D-type hepatitis B envelope protein expression plasmid.

1. Huh7 cells were cultured to 80% -90% confluency in 10cm diameter dishes.

2. And (3) after the step 1 is completed, taking the culture dishes, adding 20 mu g of test plasmid, 10 mu g of JC126 vector and 30 mu g of PEI transfection reagent into each culture dish, culturing for 8 days, collecting supernatant, adding PEG8000 to enable the concentration of the PEG8000 in a system to be 5%, centrifuging to precipitate viruses, and dissolving by using a PMM culture medium to obtain the HDV virus solution.

3. HepG2-NTCP cells were seeded in 48-well plates and after approximately 5 hours of attachment, culture was continued for 16 hours by replacing with PMM medium.

4. After completion of step 3, the 48-well plate was taken out, the supernatant was aspirated and added with the virus solution (MOI ═ 200) prepared in step 2, the test antibody solution and PEG8000 (in the system, the final concentration of PEG8000 was 5%), cultured for 16 to 20 hours, then the supernatant was aspirated and discarded, the cells were washed 2 times with PBS buffer, and then cultured for 7 days with PMM medium (every two-day change).

In the system, the concentration of the B826 antibody was 0.5. mu.g/ml. In the system, the concentration of HBIG is 0.5mg/ml, which is 1000 times of the concentration of monoclonal antibody. In the system, the concentration of MERS-27 antibody was 0.5. mu.g/ml.

5. After completion of step 4, the 48-well plate was taken, washed 3 times with PBS buffer, then fixed with 4% paraformaldehyde solution for 15min, then washed 3 times with PBS buffer, then penetrated 20min at room temperature with PBST solution, then washed 3 times with PBS buffer, then goat serum was added dropwise and blocked at room temperature for 30min, then primary anti-working solution (primary anti-HDV δ antibody) was added dropwise and incubated at 4 ℃ for 15 hours, then washed 3 times with PBST solution, then fluorescent secondary anti-working solution (secondary anti-donkey anti-rabbit Alexa Fluor TM 594 labeled secondary antibody, cat # a-21207, Invitrogen usa) was added dropwise and incubated at 37 ℃ for 1h, then washed 3 times with PBST solution, then counterstained with DAPI for nuclei, then washed 4 times with PBST solution, then mounted with mounting solution containing an anti-fluorescent quencher, and analysis images were collected under a laser confocal microscope.

The results are shown in FIG. 3. In FIG. 3, HDV-C represents HDV virus packaged with type C hepatitis B envelope protein expression plasmid, and HDV-D represents HDV virus packaged with type D hepatitis B envelope protein expression plasmid. The Mers27 antibody failed to block infection by HDV virus packaged with the type C hepatitis b envelope protein expression plasmid and infection by HDV virus packaged with the type D hepatitis b envelope protein expression plasmid, compared to the blank control. Compared with a blank control, the B826 antibody can specifically block the infection of the HDV packaged by the C-type hepatitis B envelope plasmid and does not block the infection of the HDV packaged by the D-type hepatitis B envelope plasmid, which is consistent with an immunofluorescence result, and proves that the B826 antibody can only identify and neutralize the C genotype hepatitis B virus and is a C genotype specific neutralizing antibody. Meanwhile, HBs membrane protein a epitope exists in all genotype hepatitis B viruses, and the antigen epitope recognized by the B826 antibody is proved to be non-a epitope. HBIG blocks infection by HDV virus packaged with type C hepatitis b envelope plasmid and infection by HDV virus packaged with type D hepatitis b envelope plasmid compared to blank control.

SEQUENCE LISTING

<110> Qinghua university

<120> hepatitis B virus neutralizing antibody B826 and application thereof

<130> CGGNQAYX186106

<160> 4

<170> PatentIn version 3.5

<210> 1

<211> 472

<212> PRT

<213> Artificial sequence

<400> 1

Met Gly Trp Ser Cys Ile Ile Leu Phe Leu Val Ala Thr Ala Thr Gly

1 5 10 15

Val His Ser Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys

20 25 30

Pro Gly Ser Ser Met Lys Val Ser Cys Lys Ala Ser Gly Gly Ser Phe

35 40 45

Asn Asp Tyr Ala Ile Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu

50 55 60

Gln Trp Met Gly Gly Ile Val Pro Ile Val Gly Ala Pro Asn Tyr Ser

65 70 75 80

Gln Lys Phe Gln Asp Arg Ile Lys Leu Thr Ala Asp Val Ser Thr Asn

85 90 95

Thr Val Tyr Met Glu Leu Ala Ser Leu Lys Phe Asp Asp Thr Ala Val

100 105 110

Tyr Tyr Cys Ala Arg Ala Gly His Val Phe Arg Gly Val Thr Cys Gly

115 120 125

Leu Asp Ile Trp Gly Gln Gly Thr Met Val Thr Val Ser Thr Ala Ser

130 135 140

Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr

145 150 155 160

Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro

165 170 175

Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val

180 185 190

His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser

195 200 205

Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile

210 215 220

Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val

225 230 235 240

Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala

245 250 255

Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro

260 265 270

Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val

275 280 285

Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val

290 295 300

Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln

305 310 315 320

Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln

325 330 335

Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala

340 345 350

Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro

355 360 365

Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr

370 375 380

Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser

385 390 395 400

Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr

405 410 415

Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr

420 425 430

Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe

435 440 445

Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys

450 455 460

Ser Leu Ser Leu Ser Pro Gly Lys

465 470

<210> 2

<211> 2540

<212> DNA

<213> Artificial sequence

<400> 2

acattgatta ttgactagtt attaatagta atcaattacg gggtcattag ttcatagccc 60

atatatggag ttccgcgtta cataacttac ggtaaatggc ccgcctggct gaccgcccaa 120

cgacccccgc ccattgacgt caataatgac gtatgttccc atagtaacgc caatagggac 180

tttccattga cgtcaatggg tggagtattt acggtaaact gcccacttgg cagtacatca 240

agtgtatcat atgccaagta cgccccctat tgacgtcaat gacggtaaat ggcccgcctg 300

gcattatgcc cagtacatga ccttatggga ctttcctact tggcagtaca tctacgtatt 360

agtcatcgct attaccatgg tgatgcggtt ttggcagtac atcaatgggc gtggatagcg 420

gtttgactca cggggatttc caagtctcca ccccattgac gtcaatggga gtttgttttg 480

gcaccaaaat caacgggact ttccaaaatg tcgtaacaac tccgccccat tgacgcaaat 540

gggcggtagg cgtgtacggt gggaggtcta tataagcaga gctcgtttag tgaaccgtca 600

gatcgcctgg agacgccatc cacgctgttt tgacctccat agaagacacc gggaccgatc 660

cagcctccgc ggccgggaac ggtgcattgg aacgcggatt ccccgtgcca agagtgacgt 720

aagtaccgcc tatagagtct ataggcccac ccccttggct tcgttagaac gcggctacaa 780

ttaatacata accttatgta tcatacacat acgatttagg tgacactata gaataacatc 840

cactttgcct ttctctccac aggtgtccac tcccaggtcc aactgcacct cggttctatc 900

gattgaattc caccatggga tggtcatgta tcatcctttt tctagtagca actgcaaccg 960

gtgtacattc tcaggtgcag ctggtgcagt ctggggctga ggtgaagaag cctgggtcct 1020

cgatgaaggt ctcatgcaag gcttctggag gctccttcaa cgactatgct atcaattggg 1080

tgcgacaggc ccctggacaa ggacttcagt ggatgggagg aatcgtccct atcgttggag 1140

ccccaaacta ctcacagaag ttccaggaca gaatcaaact taccgcggac gtgtcgacga 1200

acacagtcta catggagctg gccagcctga aatttgacga cacggccgtc tattactgtg 1260

cgagagccgg acatgtgttt cggggagtca cctgtggact agatatttgg ggccaaggga 1320

caatggtcac ggtctctaca gcgtcgacca agggcccatc ggtcttcccc ctggcaccct 1380

cctccaagag cacctctggg ggcacagcgg ccctgggctg cctggtcaag gactacttcc 1440

ccgaacctgt gacggtctcg tggaactcag gcgccctgac cagcggcgtg cacaccttcc 1500

cggctgtcct acagtcctca ggactctact ccctcagcag cgtggtgacc gtgccctcca 1560

gcagcttggg cacccagacc tacatctgca acgtgaatca caagcccagc aacaccaagg 1620

tggacaagag agttgagccc aaatcttgtg acaaaactca cacatgccca ccgtgcccag 1680

cacctgaact cctgggggga ccgtcagtct tcctcttccc cccaaaaccc aaggacaccc 1740

tcatgatctc ccggacccct gaggtcacat gcgtggtggt ggacgtgagc cacgaagacc 1800

ctgaggtcaa gttcaactgg tacgtggacg gcgtggaggt gcataatgcc aagacaaagc 1860

cgcgggagga gcagtacaac agcacgtacc gtgtggtcag cgtcctcacc gtcctgcacc 1920

aggactggct gaatggcaag gagtacaagt gcaaggtctc caacaaagcc ctcccagccc 1980

ccatcgagaa aaccatctcc aaagccaaag ggcagccccg agaaccacag gtgtacaccc 2040

tgcccccatc ccgggaggag atgaccaaga accaggtcag cctgacctgc ctggtcaaag 2100

gcttctatcc cagcgacatc gccgtggagt gggagagcaa tgggcagccg gagaacaact 2160

acaagaccac gcctcccgtg ctggactccg acggctcctt cttcctctat agcaagctca 2220

ccgtggacaa gagcaggtgg cagcagggga acgtcttctc atgctccgtg atgcatgagg 2280

ctctgcacaa ccactacacg cagaagagcc tctccctgtc cccgggtaaa tgagtgcgac 2340

ggccggcaag cccccgctcc ccgggctctc gcggtcgtac gaggaaagct tggccgccat 2400

ggcccaactt gtttattgca gcttataatg gttacaaata aagcaatagc atcacaaatt 2460

tcacaaataa agcatttttt tcactgcatt ctagttgtgg tttgtccaaa ctcatcaatg 2520

tatcttatca tgtctggatc 2540

<210> 3

<211> 236

<212> PRT

<213> Artificial sequence

<400> 3

Met Gly Trp Ser Cys Ile Ile Leu Phe Leu Val Ala Thr Ala Thr Gly

1 5 10 15

Glu Ile Val Leu Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly

20 25 30

Glu Arg Ala Thr Val Asn Cys Lys Ser Ser Gln Ser Leu Tyr Tyr Ser

35 40 45

Ser Asp Asn Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln

50 55 60

Pro Pro Arg Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val

65 70 75 80

Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr

85 90 95

Ile Ser Gly Leu Gln Ala Glu Asp Val Ala Met Tyr Phe Cys Gln Gln

100 105 110

Tyr Tyr Phe Ser Pro Leu Thr Phe Gly Gly Gly Thr Lys Val Glu Ile

115 120 125

Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp

130 135 140

Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn

145 150 155 160

Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu

165 170 175

Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp

180 185 190

Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr

195 200 205

Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser

210 215 220

Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

225 230 235

<210> 4

<211> 1780

<212> DNA

<213> Artificial sequence

<400> 4

acattgatta ttgactagtt attaatagta atcaattacg gggtcattag ttcatagccc 60

atatatggag ttccgcgtta cataacttac ggtaaatggc ccgcctggct gaccgcccaa 120

cgacccccgc ccattgacgt caataatgac gtatgttccc atagtaacgc caatagggac 180

tttccattga cgtcaatggg tggagtattt acggtaaact gcccacttgg cagtacatca 240

agtgtatcat atgccaagta cgccccctat tgacgtcaat gacggtaaat ggcccgcctg 300

gcattatgcc cagtacatga ccttatggga ctttcctact tggcagtaca tctacgtatt 360

agtcatcgct attaccatgg tgatgcggtt ttggcagtac atcaatgggc gtggatagcg 420

gtttgactca cggggatttc caagtctcca ccccattgac gtcaatggga gtttgttttg 480

gcaccaaaat caacgggact ttccaaaatg tcgtaacaac tccgccccat tgacgcaaat 540

gggcggtagg cgtgtacggt gggaggtcta tataagcaga gctcgtttag tgaaccgtca 600

gatcgcctgg agacgccatc cacgctgttt tgacctccat agaagacacc gggaccgatc 660

cagcctccgc ggccgggaac ggtgcattgg aacgcggatt ccccgtgcca agagtgacgt 720

aagtaccgcc tatagagtct ataggcccac ccccttggct tcgttagaac gcggctacaa 780

ttaatacata accttatgta tcatacacat acgatttagg tgacactata gaataacatc 840

cactttgcct ttctctccac aggtgtccac tcccaggtcc aactgcacct cggttctatc 900

gattgaattc caccatggga tggtcatgta tcatcctttt tctagtagca actgcaaccg 960

gtgaaattgt gctgacacag tctccagact ccctggctgt gtctctgggc gagagggcca 1020

ccgtcaactg caagtccagc cagagtctgt actacagttc cgacaataag aactacttgg 1080

cttggtacca gcagaaacca ggccagcctc ctaggctgct catttactgg gcatctacgc 1140

gggaatctgg ggtccctgac cgattcagtg gcagcgggtc tgggacagat ttcactctca 1200

ccatcagcgg cctgcaggct gaagatgtgg cgatgtattt ctgtcagcaa tattatttta 1260

gtccgctcac tttcggcgga gggaccaagg tggagatcaa acgtacggtg gctgcaccat 1320

ctgtcttcat cttcccgcca tctgatgagc agttgaaatc tggaactgcc tctgttgtgt 1380

gcctgctgaa taacttctat cccagagagg ccaaagtaca gtggaaggtg gataacgccc 1440

tccaatcggg taactcccag gagagtgtca cagagcagga cagcaaggac agcacctaca 1500

gcctcagcag caccctgacg ctgagcaaag cagactacga gaaacacaaa gtctacgcct 1560

gcgaagtcac ccatcagggc ctgagctcgc ccgtcacaaa gagcttcaac aggggagagt 1620

gttagaagct tggccgccat ggcccaactt gtttattgca gcttataatg gttacaaata 1680

aagcaatagc atcacaaatt tcacaaataa agcatttttt tcactgcatt ctagttgtgg 1740

tttgtccaaa ctcatcaatg tatcttatca tgtctggatc 1780

Claims

1. An IgG antibody against hepatitis B virus of C genotype, consisting of a light chain and a heavy chain; CDR1, CDR2 and CDR3 in the heavy chain variable region in the heavy chain are the 45 th-52 th amino acid residue, the 70 th-77 th amino acid residue and the 116 th-131 th amino acid residue from the N terminal of the sequence 1 in the sequence table in sequence; the CDR1, CDR2 and CDR3 in the light chain variable region in the light chain are the 43 th-54 th amino acid residue, the 72 th-74 th amino acid residue and the 111 th-119 th amino acid residue from the N terminal of the sequence 3 in the sequence table in sequence.

2. The IgG antibody of claim 1, wherein:

the heavy chain variable region consists of 20 th-142 th amino acid residues from the tail end of N of a sequence 1 in a sequence table;

3. The IgG antibody of claim 2, wherein:

the heavy chain is (a) or (b) as follows: (a) protein consisting of 20 th-472 th amino acid residues from the tail end of N in a sequence 1 of a sequence table; (b) protein shown in a sequence 1 in a sequence table;

4. A gene encoding the IgG antibody of claim 3, wherein:

the gene encoding the heavy chain is (1) or (2) or (3) as follows:

(3) a DNA molecule shown in a sequence 2 of a sequence table;

the genes encoding the light chain are as follows (4) or (5) or (6):

(6) DNA molecule shown in sequence 4 of the sequence table.

5. Use of the IgG antibody of claim 1 or 2 or 3 in the manufacture of a medicament for inhibiting C genotype hepatitis b virus.

6. A medicament for inhibiting C genotype hepatitis b virus, which contains as an active ingredient the IgG antibody according to claim 1 or 2 or 3.

7. Use of an IgG antibody according to claim 1 or 2 or 3 in the manufacture of a medicament for neutralizing C genotype hepatitis b virus.

8. A medicament for neutralizing C genotype hepatitis b virus, which has as an active ingredient the IgG antibody of claim 1 or 2 or 3.

9. Use of an IgG antibody according to claim 1 or 2 or 3 for the manufacture of a medicament for the prevention and/or treatment of hepatitis b; the hepatitis B is caused by hepatitis B virus of C genotype.

10. A medicament for preventing and/or treating hepatitis b, which comprises as active ingredients the IgG antibody according to claim 1 or 2 or 3; the hepatitis B is caused by hepatitis B virus of C genotype.