CN112851809A - Antibody for resisting terminal glycosylated protein receptor and application thereof - Google Patents

Abstract

The invention discloses an antibody of an anti-terminal glycosylation protein receptor and application thereof, wherein the antibody comprises a light chain variable region and a heavy chain variable region; the CRD region sequence of the light chain variable region comprises light chain CDR1, light chain CDR2, light chain CDR 3; the CRD region sequence of the heavy chain variable region comprises heavy chain CDR1, heavy chain CDR2 and heavy chain CDR 3. The amino acid sequence of the heavy chain variable region of the antibody is shown in SEQ ID NO.1, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO. 2. The invention obtains a brand-new anti-human RAGE protein antibody by immunizing a mouse with recombinant sRAGE protein and screening by a hybridoma cell technology, wherein the antibody can inhibit the proliferation of cells expressing RAGE on the surface, relieve the inhibition of S100A4, S100A6 and A beta on the cell proliferation, and has the function of inhibiting and blocking the extracellular signal transmitted by RAGE on the cell into the cell.

Description

Antibody for resisting terminal glycosylated protein receptor and application thereof

Technical Field

The invention relates to the technical field of biomedicine, in particular to an antibody for resisting a terminal glycosylated protein receptor and application thereof.

Background

Receptor for Advanced Glycation Endproducts (RAGE) is a membrane protein belonging to the immunoglobulin superfamily. Human RAGE consists of 404 amino acids, each consisting of 3 parts of a larger extracellular segment (321 amino acid residues), a transmembrane segment (19 amino acid residues) and a short intracellular segment (41 amino acid residues). Amino acid sequence analysis indicates that RAGE is a novel member of the immunoglobulin superfamily, which is highly homologous to the amino acid sequences of MUC18 glycoprotein, Neural Cell Adhesion Molecule (NCAM), and the intracellular portion of CD20 in the immunoglobulin superfamily. Soluble RAGE (sRAGE), the RAGE extracellular domain, is a ligand binding site, and has an immunoglobulin-like structure with a V-type segment followed by two C-type segments, each containing a pair of conserved cysteine residues, and two N-coupled glycosylation sites, which are important for the stability of the RAGE molecular structure and the function of specific recognition ligands. The extracellular domain is followed by a transmembrane region and a highly negatively charged cytoplasmic tail. The intracellular segment, RAGE, which has high homology to the B cell activation marker CD20, is likely to bind to an intracytoplasmic signaling molecule after the ligand occupies the receptor, producing a cellular effect.

The patent documents US10550184B2, CN102089430A, CN102686611A, US20100143349a1, WO2008137552a2, etc. all describe anti-RAGE antibodies using different antibody variable region CDR sequences and different epitopes of the antibody binding antigen.

An article (Tekabe Y, Anthony T, Li Q, et al. treatment effect with anti-RAGE F (ab ') 2 anti-body improvises with limb angiogenisis and blood flow in Type 1 diabetic microorganism with left ferromagnetic arm stimulation. vascular medicine. 2015; 20(3):212-218.doi:10.1177/1358863X14568337) reported that treatment with anti-RAGE F (ab') 2antibody, which is murine rather than humanized, improved hindlimb angiogenesis and blood flow in Type 1 diabetic mice of the left femoral artery. An article (Yang Tekabe, Joane Luma, Qing Li, Ann Marie Schmidt, RavicChandra Ramasamy, Lynne L. Johnson, Imaging of Receptors for Advanced Glycation End Products in Experimental Myocardial Ischemia Reperfusion Injury, CC: Cardiovascular Imaging, Volume 5, Issue 1,2012, Pages 59-67, ISSN 1936-878X) reported visualization of Advanced glycosylation End product Receptors in Experimental Myocardial Ischemia Reperfusion Injury in animal models, where the anti-RAGE F (ab') 2antibody was a non-humanized murine antibody fragment. The anti-RAGE antibodies reported in the article (Demling, N., Ehrhardt, C., Kasper, M.et. al. promotion of cell adherence and dissemination: a novel function of RAGE, the high selective differentiation marker of human antibody epitope type I cells, cell Tissue Res 323, 475-. However, the above-mentioned existing antibodies produced from animal sera are not suitable for long-term treatment in humans.

Disclosure of Invention

Aiming at the defects of the prior art, the invention provides an antibody against a terminal glycosylation protein receptor and application thereof. The invention screens and clones a new antibody which can specifically bind to a Receptor of terminal glycosylated protein (RAGE). The light and heavy chains of the novel antibody have unique antigen-binding complementary-binding regions (CDRs) sequences, which are distinguished from the CDR regions of antibodies that have been found. The antibody obtained by the invention can be specifically combined with human RAGE protein on the surface of cells, and has the functions of inhibiting and blocking the extracellular signal transmission of RAGE on the cells into the cells.

The novel monoclonal antibodies produced by the present invention that bind to human RAGE, unlike the human RAGE-binding domains of the previously disclosed anti-human RAGE antibodies, inhibit the specific interaction of human RAGE with A β, S100-A4, S100-A6.

The purpose of the invention is realized by the following technical scheme:

in a first aspect, the present invention provides an antibody against a terminally glycosylated protein receptor,

comprises a light chain variable region and a heavy chain variable region; the CRD region sequence of the light chain variable region comprises light chain CDR1, light chain CDR2, light chain CDR 3; the amino acid sequence of the light chain CDR1 is shown in SEQ ID NO.11, the amino acid sequence of the light chain CDR2 is shown in SEQ ID NO.12, and the amino acid sequence of the light chain CDR3 is shown in SEQ ID NO. 13;

the CRD region sequence of the heavy chain variable region comprises heavy chain CDR1, heavy chain CDR2, heavy chain CDR 3; the amino acid sequence of the heavy chain CDR1 is shown in SEQ ID NO.5, the amino acid sequence of the heavy chain CDR2 is shown in SEQ ID NO.6, and the amino acid sequence of the heavy chain CDR3 is shown in SEQ ID NO. 7.

Preferably, the amino acid sequence of the heavy chain variable region of the antibody is shown as SEQ ID NO.1, and the amino acid sequence of the light chain variable region is shown as SEQ ID NO. 2;

or a mutant or humanized antibody comprising the light chain variable region and the heavy chain variable region as the basis, wherein the mutant comprises an amino acid sequence having homology of 50% or more with the light chain variable region and the heavy chain variable region sequence; the humanized antibody is obtained by mutating the non-CDR sequences in the light chain variable region and the heavy chain variable region.

Preferably, the base sequence of the heavy chain variable region of the antibody is shown in SEQ ID NO.3, and the base sequence of the light chain variable region is shown in SEQ ID NO. 4.

Preferably, the antibody further comprises a light chain constant region, a heavy chain constant region;

the amino acid sequence of the heavy chain constant region is shown as SEQ ID NO.30, and the amino acid sequence of the light chain constant region is shown as SEQ ID NO. 29.

The base sequence of the heavy chain constant region is shown as SEQ ID NO.32, and the base sequence of the light chain constant region is shown as SEQ ID NO. 31.

Preferably, the amino acid sequence of the heavy chain variable region of the humanized antibody is shown as SEQ ID NO.41, and the amino acid sequence of the light chain variable region is shown as SEQ ID NO. 42.

In a second aspect, the present invention provides a recombinant plasmid comprising the aforementioned antibody against a terminally glycosylated protein receptor.

In a third aspect, the present invention provides a method for producing an antibody against a terminally glycosylated protein receptor, comprising the steps of:

A. screening monoclonal hybridoma cells secreting anti-RAGE antibody and obtaining DNA fragments encoding the heavy chain variable region and the light chain variable region of the anti-human RAGE antibody;

B. and (3) respectively recombining the light chain variable region and the heavy chain variable region of the cloned anti-human RAGE antibody with the light chain constant region and the heavy chain constant region of a human IgG1 antibody to obtain the recombinant human RAGE antibody.

In a fourth aspect, the invention provides an application of an antibody against a terminal glycosylation protein receptor in preparing a composition for inhibiting stellate cell activation and hepatic fibrosis.

In a fifth aspect, the present invention provides the use of an antibody against a terminally glycosylated protein receptor for the preparation of a composition for the treatment of senile dementia.

In a sixth aspect, the invention provides an application of an antibody against a terminal glycosylation protein receptor in preparing S100A4 and S100A6 protein antagonists.

In a seventh aspect, the invention provides the use of an antibody against a terminally glycosylated protein receptor for the preparation of an antagonist of a β protein.

Compared with the prior art, the invention has the following beneficial effects:

1. the invention obtains a brand-new anti-human RAGE protein antibody A5 by immunizing a mouse through recombinant sRAGE protein and screening through a hybridoma cell technology; obtaining DNA sequences encoding the light and heavy chain variable regions of A5 by reverse transcription PCR; three methods of ELISA, flow cytometry and biofilm layer interference show that the A5 chimeric antibody has good affinity (KD ═ 2.15 × 10) for human RAGE (sRAGE)^-9M); further fusing the light chain and heavy chain variable regions of the murine antibody A5 to the constant region of the human IgG1 type antibody by using a genetic engineering technology to obtain a human-mouse chimeric antibody of anti-human RAGE; the constructed human-mouse chimeric antibody can inhibit the proliferation of cells expressing RAGE on the surface, relieve the inhibition effect of S100A4, S100A6 and A beta on the cell proliferation, and has the functions of inhibiting and blocking the transmission of extracellular signals to cells by RAGE on the cells.

2. The anti-RAGE antibodies obtained according to the present invention have the ability to treat RAGE-related diseases and disorders, preferably selected from sepsis, septic shock, listeriosis, inflammatory diseases including rheumatoid arthritis and psoriatic arthritis and bowel disease, cancer, arthritis, crohn's disease, chronic acute inflammatory diseases, cardiovascular diseases, erectile dysfunction, diabetes, diabetic complications, vasculitis, nephropathy, retinopathy, neuropathy, amyloidosis, atherosclerosis, peripheral vascular disease, myocardial infarction, congestive heart failure, diabetic retinopathy, diabetic neuropathy, diabetic nephropathy, and alzheimer's disease, especially diabetes and/or inflammatory disorders.

3. Because the RAGE protein and its ligand are closely related to liver fibrosis, when RAGE receptor expression is inhibited, RAGE activity is inhibited or RAGE receptor antagonist is added, the liver fibrosis progression in the body is inhibited, and the antibody provided by the present invention also has the potential to treat liver fibrosis.

Drawings

Other features, objects and advantages of the invention will become more apparent upon reading of the detailed description of non-limiting embodiments with reference to the following drawings:

FIG. 1 shows the results of measurement of the mouse immunotiter;

FIG. 2 is the result of the cloning of the anti-RAGE antibody gene;

FIG. 3 is a diagram showing the results of electrophoresis of PCR products of A5L1, A5H1, Y9L1 and Y9H 1;

FIG. 4 is a SDS-PAGE gel of the A5 chimeric antibody;

FIG. 5 shows the HPLC results of A5 chimeric antibody;

FIG. 6 shows the results of ELISA using A5 chimeric antibody;

FIG. 7 is a surface plasmon resonance sensor reaction signal for binding of A5 chimeric antibody to recombinant human sRAGE;

FIG. 8 is a CCK8 assay of the effect of A5 chimeric antibody on SH-SY5Y cell proliferation (. times.P <0.01,. times.P < 0.001);

FIG. 9 shows the change in the expression level of different groups of phosphorylated ERK1/2 proteins;

FIG. 10 is a CCK8 assay for the interaction between A5 chimeric antibody and S100A4/S100A6 (.: P < 0.05);

figure 11 is CCK8 assay for interaction between a5 chimeric antibody and a β (: P <0.05,: P < 0.01);

FIG. 12 is a SDS-PAGE gel of the humanized A5 single chain antibody;

FIG. 13 shows the results of humanized A5 single-chain antibody ELISA.

Detailed Description

The present invention will be described in detail with reference to specific examples. The following examples will assist those skilled in the art in further understanding the invention, but are not intended to limit the invention in any way. It should be noted that variations and modifications can be made by persons skilled in the art without departing from the spirit of the invention. All falling within the scope of the present invention.

The following examples are presented to prepare human murine chimeric antibodies by the following main steps:

1. immunization of mice

A male BALB/c mouse with the age of 6 weeks is taken as an immune object, recombinant human soluble RAGE protein is taken as an antigen, the first immunization is carried out after emulsification with Freund complete adjuvant, after 10 days, 2 nd, 3 rd and 4 th immunizations are respectively carried out after emulsification of Freund incomplete adjuvant and the antigen, and the interval of each immunization is 9 days. And 3 days after 4 th immunization, blood sampling is carried out to verify the immune effect. The titer of the anti-human RAGE antibody in the serum is more than 8000-10000 times of the dilution, and then the next experiment can be carried out.

2. Hybridoma fusion

Selecting the mice with the best immune effect, and after anesthesia, carrying out cervical vertebra dislocation and sacrifice and alcohol disinfection. Spleens were removed, splenocytes isolated, and fused with sterilized polyethylene glycol 4000(PEG-4000) mediated splenocytes and mouse ascites tumor cells (SP 2/0). When fused hybridoma cells were selectively cultured in HAT-containing medium (hypoxanthine (H), aminopterin (A) and thymidine (T) ], unsuccessfully fused spleen cells and SP2/0 cells could not survive.

3. Monoclonal cell screening

And (3) performing monoclonality on the fused hybridoma cells by adopting a limiting dilution method, screening monoclonal hybridoma cells secreting the anti-RAGE antibody by adopting an enzyme-linked immunosorbent assay (ELISA) technology after the monoclonal cells grow for 7 days, and cloning antibody genes after 2 rounds of screening are performed on positive cell strains to obtain monoclonal cells secreting the antibody.

4. Cloning of antibody genes

Culturing the screened monoclonal cells secreting the antibody, extracting total RNA of the cells by using a TRIzol reagent, respectively amplifying DNA fragments encoding the heavy chain variable region and the light chain variable region of the anti-human RAGE antibody in each monoclonal antibody cell strain by adopting a Reverse transcription polymerase chain reaction (RT-PCR) technology, sending the products to Shanghai bioengineering limited company for sequencing analysis, and translating the products into amino acid sequences according to amino acid codons after obtaining the gene sequences. 5. Acquisition and validation of recombinant anti-RAGE antibodies

The cloned light chain variable region and heavy chain variable region of the anti-human RAGE antibody are recombined with a light chain constant region and a heavy chain constant region corresponding to human IgG1 respectively, and are constructed on a pcDNA3.1/Myc-His (-) A vector to obtain a plasmid for expressing the mouse-human chimeric antibody. The constructed plasmid is introduced into human embryonic kidney cell line (HEK293T) cells for expression, and the binding capacity of the recombinant anti-RAGE antibody and the recombinant human RAGE protein is verified by an ELISA technology.

EXAMPLE 1 screening of mouse monoclonal antibodies

1. Immunization of mice

Human sRAGE protein was used as an antigen, and after fully emulsifying the protein with an equal volume of Freund's complete adjuvant for the first time according to a conventional immunization program, the mice of 6 weeks old male BALB/c were immunized by subcutaneous inoculation with a total of 5 mice immunized with 100. mu.g of protein per mouse. After 10 days, emulsifying by Freund's incomplete adjuvant and antigen, immunizing for 3 times every 9 days, continuously immunizing for 3 times, collecting tail vein blood 3-4 days after 4 th immunization to verify the immune effect, and measuring the antibody titer in the mouse by ELISA method. Mouse antibodies that bind to sRAGE were detected using an HRP-conjugated anti-mouse IgG (H + L) secondary antibody from Jackson immunoresearch, USA and a single component TMB from Soilebao, Beijing. The serum of 5 immunized mice is analyzed respectively, and the light absorption value measured after the serum is diluted 64000 times is still more than 2 times higher than that of the negative control serum, so that the successful immunization of the mice is shown, an antibody specifically binding with a RAGE extracellular structure is generated, and the requirements for developing a cell fusion experiment are met. The results of the analysis are shown in FIG. 1.

2. Mouse hybridoma fusion process

In order to obtain cell lines that can be cultured in vitro and continuously secrete antibodies, conventional mouse myeloma cells are fused with differentiated B lymphocytes in the spleen of an immunized mouse to produce hybridoma cell lines. The experimental materials included immunized Bab/c mice (mice from the center of laboratory animals of the academy of sciences, immunization was performed at Shanghai university of transportation, in the same manner as in step 1), unimmunized Bab/c mice (center of laboratory animals of the academy of sciences), myeloma cell line Sp2/0 (cell bank of the academy of sciences), 50% PEG4000 (Sigma reagent, Inc. of the USA, now brand name of Merck), 1640 serum-free medium (GE, Inc. of the USA), PBS, FBS (Silmer Feishl, Inc. of the USA, GIBCO), HAT (Sigma reagent, now brand name of Merch, USA). Other consumables were all purchased from corning, usa.

The operation process is completed in a sterile operation table. The method comprises the following specific steps:

2.1 first, feeder cells are isolated, the specific steps are as follows: the non-immunized Bab/c mice were sacrificed by cervical dislocation, sterilized by immersion in 70% alcohol, and the skin of the abdomen of the mice was cut open (without cutting the muscle layer). 5ml of 1640 serum-free medium was aspirated by a 5ml syringe, injected into the abdomen of the mouse, and the body of the mouse was shaken to dissolve macrophages in the medium sufficiently. The medium was carefully aspirated back through the syringe, placed in a 15ml centrifuge tube at 1000rpm for 5 minutes, and the supernatant was discarded. Washed once with PBS, centrifuged and the supernatant discarded. Adding 40ml of 1640 containing HAT and 20% FBS, mixing, adding into 96-well plate at a rate of 100 ul/well, and adding CO at 37 deg.C₂Culturing in an incubator to obtain separated feeder cells for later use.

2.2 the mouse myeloma cell line Sp2/0 was then prepared and screened once with 8-N-guanine before fusion to ensure that all Sp2/0 cells participating in fusion were sensitive to HAT medium. The selected SP2/0 cells were cultured in 1640+ 10% FBS medium (prepared by adding 10% FBS to 1640 serum-free medium), passaged at a ratio of 1:3 every 2 to 3 days, and the cell coverage at the bottom of the flask was observed. The final passage is carried out 48 hours before the preparation of the fusion, and the cell density is increased to cover about 80% of the bottom of the bottle after the passage and is used for cell fusion experiments.

2.3 spleen lymphocytes per mouse were prepared using 8 flasks (size 75cm) of SP2/0 cells. Preparation of lymphocytes from mice immunized before fusion, mice immunized with sRAGE (i.e., the aforementioned immunized Bab/c mice, referred to as No.1 mice) were sacrificed by cervical dislocation, and sterilized by immersion in 70% ethanol. The upper abdomen was cut to the left back of the mouse, the spleen was isolated, the connective tissue on the spleen was removed, placed in a sterile plate, a small amount of PBS was added, the spleen was ground with two sterile slides frosted on one end, a small amount of PBS was added while grinding, and the ground spleen cells were washed into the plate. Then, all spleen cells in the plate are transferred into a 15ml centrifuge tube, an appropriate amount of PBS is added, the mixture is uniformly mixed, and after centrifugation, the mixture is washed once by the PBS for standby. All Sp2/0 cells prepared in step 2.2 were collected and washed once with PBS until use.

2.4 spleen cells and Sp2/0 cells treated in step 2.3 were mixed in a 50ml centrifuge tube, 30-40ml of Medium containing 1640+ 20% FBS (prepared by adding 20% FBS to 1640 serum-free Medium), centrifuged at 1000rpm for 3 minutes, and the supernatant was removed. The bottom of the centrifuge tube was tapped to suspend the cells in the bottom culture.

2.5 dissolving the PEG4000 which is packaged and sterilized under high pressure in water bath, adding 1640 serum-free culture medium with the same volume, and fully dissolving to prepare 50% PEG4000 solution.

2.6 put the centrifuge tube with cells from step 2.4 into a water bath at 40 deg.C and slowly drop 50% PEG4000 solution step by step. The specific step-by-step dropping step is as follows: 1) slowly dripping 1ml of 50% PEG4000 solution along the tube wall by a glass dropper, lasting for 2 minutes, and pausing for 30 seconds; 2) dropping 1ml of 1640 complete medium in the same manner as in step 1), for 1 minute, and repeating for 1 time; 3) dropping 1ml of 1640 complete culture medium by the same method of the step 1), lasting for 0.5 minute, and repeating for 1 time; 4) add 15ml of complete medium, centrifuge for 1 min, remove supernatant and tap the bottom.

2.7 finally, 25ml of complete medium containing HAT was added to the centrifuge tube treated in step 2.6, and the solution in the centrifuge tube was seeded into 96-well plates containing feeder cells of step 1.1 at 50. mu.l/well. Place 96-well plate in CO₂After 5 days in the cell incubator, the growth of the clones was observed and 50. mu.l/well of complete medium was added. When cell clones covered the well bottom 1/4, the culture supernatant was aspirated for antibody Elisa assay. Finally, 342 wells in total from 768 wells were observed to grow cell clones, and the fusion efficiency reached 44.5%. The results of Elisa assays showed 48 wells as positive, and 3 wells with high OD signal were selected for further screening。

3. Hybridoma cell selection and cloning

Cells from the 3 wells with the highest secreted antibody after fusion were selected for a second round of screening, diluted at a cell density of 0.8 cells/well in HT-containing 1640+ 10% FBS medium and seeded into feeder cells-containing 96-well plates at 5% CO₂Culturing at 37 deg.C under saturated humidity. The colonies were observed daily for growth using an inverted microscope and wells with only one colony growing were marked. When 1/3-1/2 full of the bottom of the wells are cloned and proliferated in a large amount, the culture solution antibody and the cells of the antibody positive wells are measured to obtain 20 preferable positive clone cells, which is shown in table 1.

TABLE 1

For the preferred antibody positive well cells, they were transferred to tissue culture flasks with feeder layer (i.e., feeder cells isolated for co-culture in step 2.1) and maintained after 2 passages, and subjected to limiting dilution once more and establishment of stable monoclonal strains to obtain monoclonal cytoplasms A5 and Y9.

4. Gene cloning of anti-human RAGE antibodies

After amplification of monoclonal cell lines A5 and Y9 expressing anti-RAGE antibodies, 1 x 10E6 cells were harvested, lysed with 1ml of TRIzol reagent (Thermofisiher, USA) and total RNA extracted from the cells. Total RNA of hybridoma cells (Y5, Y9) was used as a reverse transcription template, Antibody cloning primers were synthesized using a reverse transcription kit (gangbao bioengineering limited) and the technical route of the reference, and reverse transcription was performed to synthesize cDNA and PCR amplification was performed on DNA sequences encoding the light and heavy chain variable regions of the Antibody (Andrew Bradbury, Antibody Engineering, Springlinker press, 2010, pages 15-20). The DNA product shown in figure 2 is obtained and belongs to the complete sequence of the variable region of the encoding mouse antibody after DNA sequencing, wherein the amino acid sequence of the heavy chain variable region of A5 is shown as SEQ ID NO.1, and the amino acid sequence of the light chain variable region is shown as SEQ ID NO. 2. Analyzing the amino acid sequences of the heavy chain variable regions and the light chain variable regions of the antibodies A5 and Y9 to obtain that the amino acid sequences of CDR1, CDR2 and CDR3 of the heavy chain of the A5 antibody are respectively SEQ ID No.5, SEQ ID No.6 and SEQ ID No.7, the amino acid sequences of FR1, FR2 and FR3 of the heavy chain are respectively SEQ ID No.8, SEQ ID No.9 and SEQ ID No.10, the amino acid sequences of CDR1, CDR2 and CDR3 of the light chain of the A5 antibody are respectively SEQ ID No.11, SEQ ID No.12 and SEQ ID No.13, and the amino acid sequences of FR1, FR2 and FR3 of the light chain of the A5 antibody are respectively SEQ ID No.14, SEQ ID No.15 and SEQ ID No. 16; the amino acid sequences of CDR1, CDR2 and CDR3 of the heavy chain of the Y9 antibody are respectively SEQ ID NO.17, SEQ ID NO.18 and SEQ ID NO.19, the amino acid sequences of FR1, FR2 and FR3 of the heavy chain of the Y9 antibody are respectively SEQ ID NO.20, SEQ ID NO.21 and SEQ ID NO.22, and the amino acid sequences of CDR1, CDR2 and CDR3 of the light chain of the Y9 antibody are respectively SEQ ID NO.23, SEQ ID NO.24 and SEQ ID NO.25, and the amino acid sequences of FR1, FR2 and FR3 of the light chain of the Y9 antibody are respectively SEQ ID NO.26, SEQ ID NO.27 and SEQ ID NO.28, which are detailed in tables 2 and 3.

TABLE 2

A5 heavy chain CDR region sequence		Sequence of
			CDR1	GFTFSSFG	SEQ ID NO.5
CDR2	ISGGSNVI	SEQ ID NO.6
			CDR3	VRNFRYNGSSLHYWYFDV	SEQ ID NO.7
FR region sequence of A5 heavy chain
			FR1	DVQLVESGGGLVQPGGSRKLSCAAS	SEQ ID NO.8
FR2	MHWVRQAPDKGLEWVAY	SEQ ID NO.9
			FR3	YYADTVEGRFTISRDNPKNTLFLQMTSLRSEDAAMYYC	SEQ ID NO.10
A5 light chain CDR region sequence
			CDR1	RHINVW	SEQ ID NO.11
CDR2	NAS	SEQ ID NO.12
			CDR3	LLGQSYPYS	SEQ ID NO.13
FR region sequence of A5 light chain
			FR1	DIRMNHAPSSLSASLVDTITISCHAS	SEQ ID NO.14
FR2	GTWYQQKPENIPKLLIY	SEQ ID NO.15
			FR3	RLHSGVPSRFSGNGFGTGFSLTINNLQPEDIATYYC	SEQ ID NO.16

TABLE 3

Example 2 construction of anti-human RAGE chimeric antibody expression plasmid

In this illustrative scheme, we assembled the variable region of murine antibody A5 and the constant region of human IgG1 type antibody (SEQ ID NO.29, SEQ ID NO.30) into a chimeric antibody for humanization of the antibody. The method comprises the following specific steps:

cloning a heavy chain variable region fragment (amino acid sequence SEQ ID NO.1, base sequence SEQ ID NO.3) encoding murine anti-RAGE antibody A5 with primer A5H1-V-F (5 '-3': CTAGTCTAGAATGGACTCCAGGCTCAAT, SEQ ID NO.33) and primer A5H1-V-R (5 '-3': CGCGCTGCTCACGGTTGAGGAGACGGTGACCGTGGTCCCTGC, SEQ ID NO.34) using the heavy chain variable region DNA encoding murine anti-human RAGE antibody A5 cloned in example 1 as a template; amplifying a nucleic acid fragment (an amino acid sequence SEQ ID NO.30 and a base sequence SEQ ID NO.32) of a human IgG1 type antibody heavy chain constant region by using a primer A5H1-C-F (5 '-3': GTCACCGTCTCCTCAACCGTGAGCAGCGCG, SEQ ID NO.35) and a primer A5H1-C-R (5 '-3': CCGGAATTCTCACTTCCCGGGGCTCAG, SEQ ID NO.36) by using a nucleic acid for coding a human IgG1 type antibody heavy chain as a template, carrying out PCR procedures of 94 ℃ for 10s, 55 ℃ for 10s and 72 ℃ for 10s and 30 cycles, and recovering the obtained PCR product by agarose gel electrophoresis for later use;

the overlapping PCR is used for connecting the heavy chain variable region nucleic acid sequence (SEQ ID NO.3) of the murine anti-RAGE antibody A5 obtained by amplification with the human Ig G1 heavy chain constant region nucleic acid sequence (SEQ ID NO.31), and the specific method comprises the following steps: the DNA fragment coding the heavy chain variable region of the A5 obtained above is mixed with the DNA fragment coding the heavy chain constant region of the human IgG1 antibody, the mixed DNA is used as a template, a primer A5H1-V-F and a primer A5H1-C-R are used as an upstream primer and a downstream primer for amplification, the PCR conditions are 94 ℃ for 10s, 55 ℃ for 10s, 72 ℃ for 10s and 30 cycles, the PCR detection result is shown in figure 3, the target fragment band size is consistent with the expectation, and the successful amplification of the target gene is shown.

The resulting product fragment was recovered by agarose gel electrophoresis, treated with XbaI and EcoRI, and ligated with pcDNA3.1(-) which was also treated with XbaI and EcoRI. The ligation product was used to transform E.coli DH 5. alpha. competent cells, and the transformed cells were plated on agar plates containing 50. mu.g/mL ampicillin and cultured overnight at 37 ℃. The single clone growing on the plate is picked up and cultured overnight in 5mL LB culture medium containing 50 mug/mL ampicillin by shaking, and the plasmid is extracted and sequenced, and the plasmid with correct sequencing result is named A5H1 and stored for standby.

Cloning a light chain variable region fragment (an amino acid sequence SEQ ID NO.2 and a base sequence SEQ ID NO.4) of an antibody A5 encoding the mouse anti-RAGE by using the cloned light chain variable region DNA encoding the mouse anti-human RAGE antibody as a template and using a primer A5L1-V-F (5 '-3': CTAGTCTAGAATGAGGGTCCTTGCTGAG, SEQ ID NO.37) and a primer A5L1-V-R (5 '-3': CCGGAATTCGGTCCGCTTGATCTCCAGCTTGGTCCCCCC, SEQ ID NO. 38); using a primer A5L1-C-F (5 '-3': CTAGTCTAGAGGGGGGACCAAGCTGGAGATCAAGCGGACC, SEQ ID NO.39) and a primer A5L1-C-R (5 '-3': CCGGAATTCTCAGCACTCGCCCCGGTT, SEQ ID NO.40) to code the nucleic acid of the light chain of the human IgG1 antibody as a template, amplifying the nucleic acid fragment (amino acid sequence SEQ ID NO.29 and base sequence SEQ ID NO.31) of the constant region of the light chain of the human IgG1 antibody, performing PCR at 94 ℃ for 10s, 55 ℃ for 10s and 72 ℃ for 10s for 30 cycles, and recovering the obtained PCR product by agarose gel electrophoresis for later use;

the overlapping PCR is used for connecting the light chain variable region nucleic acid sequence (SEQ ID NO.4) of the murine anti-RAGE antibody A5 obtained by amplification with the human Ig G1 light chain constant region nucleic acid sequence (SEQ ID NO.31), and the specific connecting method comprises the following steps: the DNA fragment encoding the light chain variable region of A5 obtained above was mixed with DNA encoding the light chain constant region of human IgG1 antibody, the mixed DNA was used as a template, and the primers A5L1-V-F and A5L1-V-R were used as upstream and downstream primers to amplify under PCR conditions of 94 ℃ for 10s, 55 ℃ for 10s, 72 ℃ for 10s, 30 cycles, and the PCR results are shown in FIG. 3, indicating that the band size of the target fragment matches the expected size, indicating that the target gene was successfully amplified.

The resulting product fragment was recovered by agarose gel electrophoresis, treated with XbaI and EcoRI, and ligated with pcDNA3.1(-) which was also treated with XbaI and EcoRI. The ligation product was used to transform E.coli DH 5. alpha. competent cells, and the transformed cells were plated on agar plates containing 50. mu.g/mL ampicillin and cultured overnight at 37 ℃. The single clone growing on the plate is picked up, shake-cultured overnight in 5mL LB culture medium containing 50 mug/mL ampicillin, and the plasmid is extracted and sequenced, and the plasmid with correct sequencing result is named A5L1 and stored for standby.

Example 3 expression of chimeric antibodies against human RAGE

In this exemplary embodiment, we used HEK293E cell transient expression for expression of human murine chimeric antibodies against human RAGE. The light chain expression plasmid A5L1 and the heavy chain expression plasmid A5H1 of the human murine chimeric antibody A5' of anti-human RAGE constructed in the step 1 are subjected to transient expression of HEK293E according to the mass ratio of the light-heavy chain plasmid of 3: 1. The method comprises the following specific steps:

3.1 day before transfection HEK293E cells were diluted to 1.5-2.5X 10 with fresh Gibco Freestyle 293 medium⁶Cells/ml, at 37 ℃, 120rpm, 5% CO₂Cultured for the next day of transfection.

3.2 day of transfection, according to 10 th day⁶The cells were treated with 0.5. mu.g of DNA, A5L1 and A5H1 mixed at a 3:1 mass ratio, DNA (DNA mixed with A5L1 and A5H 1) diluted to (40 ng/. mu.L) with Gibco Freestyle 293 medium, and DNA: PEI is added in a proportion of 1:5 (mass ratio), and the DNA-PEI complex after uniform mixing is incubated for 20min at room temperature for standby.

The HEK293E cells obtained in step 2.1 were harvested by centrifugation at 3.31000 rpm for 5min, washed 1 time in Gibco Freestyle 293 medium, harvested by centrifugation at 1000rpm for 5min, and resuspended in 200ml of Gibco Freestyle 293 medium to a cell density of 4X 10⁶Individual cells/ml were placed in new 1L shake flasks (Coming).

3.4 Add the incubated DNA-PEI complexes to the cells of step 2.3, 37 ℃, 110rpm, 5% CO₂Transfection was performed for 4 hours, followed by addition of an equal volume of SFX4HEK293 medium pre-warmed to 37 deg.C, addition of 100. mu.g/ml Geneticin (Gibco) and continuation of 37 deg.C, 130rpm, 5% CO₂Culturing until the cell viability is reduced to 50%, and collecting the sample. After the cell culture is centrifuged and the supernatant is collected, the cell culture is purified or the supernatant is collected and is stored at the temperature of minus 80 ℃ in a freezing way, and then the human-mouse chimeric antibody A5' of the anti-human RAGE is obtained.

Example 4 purification of human murine chimeric antibody A5' against human RAGE

The supernatant collected from transient expression of A5' in HEK293E obtained in step 2 was diluted with an equal volume of PBS (20mM PBS, 150mM NaCl, pH 6.8-7.4), applied to a Protein A (Protein A) affinity chromatography column equilibrated with PBS in advance, washed with 5 column volumes of PBS after the application, washed with 100mM citric acid buffer pH5.0 to remove impurities, and eluted with 100mM citric acid buffer pH3.0 to elute the antibody, and the eluate was collected in a 1.5mL EP tube, 800. mu.L/tube, and the collected eluate was immediately neutralized with 1M Tris-HCl buffer pH 9.0. The purification of the collected protein was checked by using reduced SDS-PAGE gel (FIG. 4), and the collection tubes containing the target protein were pooled. The A5' antibody from Protein A purification was concentrated in MILLIPORE Amicon Ultra (30MWCO) ultrafiltration tubes and the solution of solubilized Protein was replaced with PBS.

Antibody concentration was determined using BCA kit: a5': 3.51mg/ml (3ml), i.e., 400ml, of the transfection system gave 9.53mg of A5 'antibody (23.83 mg/L yield of A5' antibody). The protein is sterilized by filtration through a 0.22 μm sterile filter head, and then is dispensed into sterile EP tubes, 100 μ l/tube for subsequent testing or stored in a refrigerator at-80 ℃. The purity of the purified human-mouse chimeric antibody A5 'was confirmed by HPLC using a TSK G2000SWxl column, and the HPLC result is as shown in FIG. 5, which shows that the human-mouse chimeric antibody A5' has only one peak and the peak shape is sharp.

Example 5 analysis of the antigen binding Capacity of human murine chimeric antibody A5' against human RAGE

5.1 determination of antigen-antibody affinity by enzyme-linked immunosorbent assay (ELISA)

The specific binding capacity between the purified chimeric antibody A5' against human RAGE and antigen was tested by ELISA assay. The specific method comprises the following steps: dissolving 20 mu g of sRAGE recombinant protein (human source) by using 10ml of antigen coating solution, adding the solution into a polystyrene 96-well enzyme label plate, wherein each well is 100 mu l, and placing the plate in a refrigerator at 4 ℃ for overnight; the next day, pour out the liquid in the 96-well plate, wash 3 times with wash buffer; adding the sealing solution into 96-well plate (100 μ l per well), sealing at 37 deg.C for 0.5 hr, pouring out the sealing solution, and storing in 4 deg.C refrigerator; diluting the purified A5' antibody and nonspecific human IgG to 5 mu g/ml with PBS respectively, diluting for 13 times, adding into 96-well plate coated with sRAGE recombinant protein, adding 100 mu l into each well, and placing in an incubator at 37 deg.C for 1-2h with PBS as negative control; the liquid in the 96-well plate was decanted, washed 3 times with wash buffer and horseradish peroxidase (HRP) labeled antibody was added: horse Radish Peroxidase (HRP) labeled Donkey Anti-Human antibody (1: 10000, which is a commercial detection antibody, Affinipure Donkey Anti-Human IgG (H + L), Jackson ImmunoResearch, 709-; pouring out the liquid in the 96-well plate, and washing for 3 times by using the washing buffer; adding 100ul TMB substrate solution into each hole, and standing at room temperature in dark for 5-30 min; adding 50 mul of stop solution into each hole to stop the reaction; the absorbance at a wavelength of 450nm was measured with a microplate reader. The ELISA results are shown in fig. 6, indicating that human murine chimeric antibody a 5' has high affinity for recombinant human sRAGE recombinant protein.

5.2 measurement of affinity between A5' chimeric antibody and recombinant human sRAGE by biofilm interference technique

The assay instrument is fortebio Octet RED 96. And carrying out biotin labeling on the recombinant human sRAGE antigen by using a biotin labeling kit. The labeled recombinant human sRAGE antigen was diluted to 100nM, the A5' antibody was diluted to 300nM, and the 300nM solution was diluted to 200, 100, 50nM with PBS. The sensor was placed in PBS solution for 30min and pre-wetted for use. Adding PBS, recombinant human sRAGE antigen, equilibrium eluent, A5' antibody (50, 100, 200, 300nm), regeneration buffer and buffer into a corresponding 96-well plate in sequence, wherein the negative control comprises the same all wells except that four wells of the antibody are replaced by PBS; the running procedure was as follows:

1)Baseline1:120s

2)Loading:300s

3)Baseline2:120s

4)Association:300s

5)Dissociation:300s

6)Regeneration:15s

7)Neutralization:5s

8) regeneration and neutrallization cycles 3 times

9)Association:300s

10)Dissociation:300s

11) Regeneration and neutrallization cycles 3 times

12)Association:300s

13)Dissociation:300s

14) Regeneration and neutrallization cycles 3 times

15)Association:300s

16)Dissociation:300s

After data acquisition, data analysis software of the instrument is used for analyzing the data, and signals acquired by Baseline2 are used as the basisThe reference signal was subtracted from the line (sample blank and sensor blank double subtraction), the resulting data were subjected to cohort analysis and fitting, and the results are shown in fig. 7, giving a KD value of 2.15 × 10 for binding of the a 5' chimeric antibody to recombinant human sRAGE^-9M, R2 ═ 0.99. Indicating that the antigen and the antibody have good affinity.

5.3 flow cytometry determination of the affinity of the A5' chimeric antibody for human RAGE

A RAGE high expressing cell line was established for determining the affinity of the a 5' chimeric antibody for the RAGE receptor on the cell surface. RAGE-NIH3T3 cells stably expressing RAGE proteins were constructed using a lentiviral transfection method. The A5 'chimeric antibody is subjected to flow cytometry with RAGE-NIH3T3 cells and NIH3T3 cells, and the dosage of the A5' chimeric antibody is 1 mug, 2 mug and 5 mug. Flow cytometry results show that for NIH3T3 cells, the absorption peaks of the negative control group and the A5 'antibody group coincide, and when the antibody amount reaches 5 mu g, the absorption peaks do not shift, which indicates that no target protein combined with the A5' antibody exists on the NIH3T3 cells. For RAGE-NIH3T3 cells, the A5 'antibody caused a shift in the absorption peak and the absorption peaks at 1 μ g, 2 μ g, and 5 μ g doses coincided, indicating that at the 1 μ g dose, saturation of RAGE protein and A5' antibody on RAGE-NIH3T3 cells had been achieved. The flow cytometry measurement result shows that the antigen antibody has good affinity.

Example 6 inhibition of cell proliferation by human murine chimeric antibody A5' against human RAGE

6.1 examination of the effect of the human murine chimeric antibody A5' on the proliferation of RAGE-expressing human neuroblastoma cells SH-SY 5Y.

SH-SY5Y cells are firstly cultured in DMEM medium containing 10% FBS under the conditions of 37 ℃ and 5% CO₂And (4) saturated humidity. When the cell density reaches 80-90%, discarding the original cell culture medium, washing away the residual FBS in the culture dish by using 3-5 ml of PBS, digesting for 3min by using 1ml of 0.25% pancreatin at room temperature, and adding double times of complete culture medium to terminate digestion. Gently blow and beat into single cell suspension, and mix with 1:3 or 1: 4-ratio subcultures or inoculations into different size cell culture plates for subsequent experiments.

SH-SY5Y cells are evenly inoculated in a 96-well plate (3000 cells/well), and the culture medium is 10% FBS + DMEM; after culturing for 24 hours, the cell culture medium is replaced by 1% FBS + DMEM medium; after further culturing for 24 hours, the cell culture medium was aspirated, and FBS-free medium containing different concentrations of antibody a5 'was added, the antibody concentrations of a 5' were 0, 6.25, 12.5, 25, 50 μ g/ml; after 48h of antibody A5' addition, cell proliferation was measured using the CCK8 kit. The CCK8 results show that the a5 chimeric antibody inhibits SH-SY5Y cell proliferation, and the inhibition is gradually enhanced with increasing antibody dose. The inhibition effect of the A5' antibody on SH-SY5Y cell proliferation was significantly different at 25. mu.g/ml and 50. mu.g/ml doses (FIG. 8). (0vs 25. mu.g/ml: 100. + -. 6.73% vs 85.01. + -. 5.35%, P <0.01,0vs 50. mu.g/ml: 100. + -. 6.73% vs 79.64. + -. 7.94%, P <0.001) shows that the human murine chimeric antibody A5' is capable of inhibiting the proliferation of RAGE-expressing human neuroblastoma cells, SH-SY5Y cells.

6.2 mechanism of proliferation of SH-SY5Y cells by A5

SH-SY5Y cells were seeded homogeneously in 6-well plates (200000 cells/well). The culture medium is as follows: 10% FBS + DMEM; after culturing for 24 hours, the cell culture medium is replaced by 1% FBS + DMEM medium; after starvation culturing the cells for 24 hours, the cell culture medium was aspirated, and FBS-free DMEM medium in which a 5' antibody (concentration 50 μ g/ml) was dissolved were added; after 2 hours, the cells in the 6-well plate were collected with cell scraping and PBS, centrifuged at 1000rpm for 3min, and the supernatant was discarded. Mu.l of 1% TritonX100 PBS cell lysate (containing 1mM PMSF, 1mM Na3VO4, 5mM NaF) was added to each well, placed on ice for 15min, and then centrifuged: 14000rpm, 4 ℃ and 10 min. Supernatants were collected, assayed for each histone concentration using the BCA kit, protein loading was quantified, and changes in ERK1/2 and p (phosphorylation) -ERK1/2 protein levels were analyzed by Western Blotting.

Western Blotting results (FIG. 9) show that SH-SY5Y cell p-ERK/ERK ratio is increased by adding A5 ' antibody (IMAGEJ software scanning gray scale), which indicates that the A5 ' antibody inhibits SH-SY5Y cell proliferation by increasing ERK1/2 protein phosphorylation level, and also indicates that the binding of A5 ' antibody to RAGE receptor can influence ERK1/2 phosphorylation level.

Example 7 interaction between human murine chimeric antibody A5' and S100A4 recombinant protein, S100A6 recombinant protein

The S100A4 and S100A6 proteins are ligands of RAGE. Both the S100A4 and S100A6 proteins activate RAGE signaling pathways, which in turn activate hepatic stellate cells and exacerbate the progression of liver fibrosis.

The interaction between the human murine chimeric antibody and the recombinant protein S100A4, S100A6 was examined in this exemplary protocol by examining the proliferation of SH-SY5Y cells, which are neuroblastoma cells.

SH-SY5Y cells were first cultured in DMEM medium containing 10% FBS at 37 ℃ and 5% CO2 saturated humidity. When the cell density reaches 80-90%, discarding the original cell culture medium, washing away the residual FBS in the culture dish by using 3-5 ml of PBS, digesting for 3min by using 1ml of 0.25% pancreatin at room temperature, and adding double times of complete culture medium to terminate digestion. Gently blow and beat into single cell suspension, and mix with 1:3 or 1: 4-ratio subcultures or inoculations into different size cell culture plates for subsequent experiments.

SH-SY5Y cells are evenly inoculated in a 96-well plate (3000 cells/well), and the culture medium is 10% FBS + DMEM; after culturing for 24 hours, the cell culture is changed into a 1% FBS + DMEM culture medium; after a further 24 hours of culture, the cell culture medium was aspirated, the different proteins were solubilized with 10% FBS medium and tested with the addition of chemicals: a) negative: PBS + DMEM; b) a5': 20 mu g/ml; c) S100A4: final concentration 25. mu.M/ml; d) S100A6, wherein the final concentration is 25 mu M/ml; e)

a5 '+ S100A4 final concentration A5' 20. mu.g/ml, S100A 425. mu.M/ml; f) a5 '+ S100A6 final concentration A5' 20. mu.g/ml, S100A 625. mu.M/ml; after 48h of antibody addition, cell proliferation was measured using the CCK8 kit. CCK8 results show that the S100A4 and S100A6 proteins both inhibit SH-SY5Y cell proliferation, and SH-SY5Y cell viability is remarkably improved after the A5' antibody (20 mu g/ml) is added (FIG. 10, S100A4 vs S100A4+ A5: 69.07 +/-6.34% vs 82.95 +/-4.31%, P <0.05, S100A6 vs S100A6+ A5: 37.17 +/-6.71% vs 51.12 +/-14.77%, and P <0.05)), so that the A5 antibody is an antagonist of S100A4 and S100A6, and interaction between S100A4, S100A6 and a RAGE receptor is prevented to a certain extent.

Example 8 interaction between human murine chimeric antibody A5' and recombinant A β protein

A beta induces tissue fibrosis, is closely connected with the occurrence of Alzheimer's disease, can induce SH-SY5Y cells to generate autophagy and induce apoptosis, and a RAGE receptor is one of the main modes for mediating the A beta to enter the cells. The interaction between the human murine chimeric antibody A5' and the recombinant A β protein was examined in this exemplary protocol by examining the proliferation of the neuroblastoma SH-SY5Y cells. Experimental protocol the same procedure as described above in step 6, and CCK8 results showed that a β inhibited cell activity. However, after the addition of A5 'antibody, cell viability gradually increased, with significant differences between the A5' antibody at 25. mu.g/ml and 50. mu.g/ml levels and the A β group alone (FIG. 11, A β vs. A β + 25. mu.g/ml A5: 61.96. + -. 4.74% vs 72.95. + -. 4.33%, P <0.05, A β vs. A β + 50. mu.g/ml A5: 61.96. + -. 4.74% vs. 76.30. + -. 4.98%, P < 0.01). The results show that the a 5' antibody is an antagonist of a β and prevents its entry into cells by binding to RAGE receptors.

Example 9 expression and purification of humanized Single chain antibody against human RAGE (hscFV)

The heavy chain and the light chain of the A5 antibody are respectively compared with a human IgG sequence database, a human IgG sequence with the highest amino acid sequence similarity is selected as a template, and the sequence of a non-CDR region in the variable region of the light chain of the A5 is mutated according to the human IgG sequence, so that the similarity between the sequence of the variable region of the A5 antibody and the corresponding sequence of the human IgG is improved. The amino acid sequence of the heavy chain variable region after mutation is shown in SEQ ID No.41, and the amino acid sequence of the light chain variable region is shown in SEQ ID No. 42. The humanized anti-RAGE single-chain antibody (hscFV) is constructed by gene synthesis by using the mutated sequence, the VL and the VH are connected by 3 repeated GGGGS peptide chains, and the synthetic gene sequence is shown in SEQ ID NO. 43. The synthetic hscFV expression sequence was constructed into a commercial expression vector pET28a (+) by a general method. The results of SDS-PAGE electrophoresis of the purified hscFV antibody obtained by expressing the hscFV antibody by inclusion body expression using Escherichia coli BL21(DE3) as a host strain and the conditions and expression method of the reference (Journal of Biotechnology,2000,77: 169-178) and by nickel affinity chromatography are shown in FIG. 12. Wherein lane 1: flow-through, 2: 20mM imidazole elution fraction, 3-4: 50mM imidazole elution fraction, 5-6: 100mM imidazole elution fraction, 7-8: 200mM imidazole elution fraction, 9-10: 500mM imidazole elution fraction.

Example 10 affinity of hscFV against RAGE for antigen

To address the question of whether the affinity of anti-human RAGE antibody A5 for human RAGE was altered following humanization, we determined the binding capacity of the single chain antibody hscFV of A5 for human RAGE. The RAGE protein was diluted to 2. mu.g/mL with coating buffer and coated overnight at 4 ℃. The coating solution was poured off, PBS was added at 300. mu.L/well, and after standing for 5 minutes, the solution was poured off and repeated three times. Add blocking solution (1% BSA) 100. mu.l per well, block overnight at 4 ℃ and discard blocking solution, wash 3 times with PBST and pat dry. Samples of hscFV were diluted in multiples and 100. mu.l of each sample was added to RAGE-coated wells in 3 replicate wells per sample. After incubation at 37 ℃ for 2 hours, the samples were discarded from the microplate and the washing was repeated 4 times for 5 minutes each. Add 1: 20000 freshly diluted HRP-mouse anti-His antibody (Protech Biotech USA) 100. mu.L, 37 ℃ incubation for 60 minutes, repeated washing 4 times, each time 5 minutes. 100 mul of single-component TMB color developing solution (Beijing Solebao biotech) is added into each hole, and the mixture is placed for 5 minutes at room temperature in the dark. 50ul of 2M sulfuric acid is added into each hole to terminate the reaction, and the absorbance at 450nm is read by an enzyme-linked immunosorbent assay. The results are shown in FIG. 13, indicating that the humanized modification of the A5 antibody still retains the properties of recognizing and binding to RAGE.

The invention has many applications, and the above description is only a preferred embodiment of the invention. It should be noted that the above examples are only for illustrating the present invention, and are not intended to limit the scope of the present invention. It will be apparent to those skilled in the art that various modifications can be made without departing from the principles of the invention and these modifications are to be considered within the scope of the invention.

Sequence listing

<110> Shanghai university of transportation

<120> antibody against terminal glycosylated protein receptor and application thereof

<130> KAG45787

<160> 43

<170> SIPOSequenceListing 1.0

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Asp Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Arg Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Phe

20 25 30

Gly Met His Trp Val Arg Gln Ala Pro Asp Lys Gly Leu Glu Trp Val

35 40 45

Ala Tyr Ile Ser Gly Gly Ser Asn Val Ile Tyr Tyr Ala Asp Thr Val

50 55 60

Glu Gly Arg Phe Thr Ile Ser Arg Asp Asn Pro Lys Asn Thr Leu Phe

65 70 75 80

Leu Gln Met Thr Ser Leu Arg Ser Glu Asp Ala Ala Met Tyr Tyr Cys

85 90 95

Val Arg Asn Phe Arg Tyr Asn Gly Ser Ser Leu His Tyr Trp Tyr Phe

100 105 110

Asp Val Trp Gly Ala Gly Thr Thr Val Thr Val Ser Ser

115 120 125

<210> 2

<211> 104

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

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Asp Ile Gln Met Asn Gln Ser Pro Ser Ser Leu Ser Ala Ser Leu Gly

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Asp Thr Ile Thr Ile Thr Cys His Ala Ser His His Ile Asn Val Trp

20 25 30

Val Thr Trp Tyr Gln Gln Lys Pro Gly Asn Ile Pro Lys Leu Leu Ile

35 40 45

Tyr Lys Ala Ser Lys Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Phe Gly Thr Gly Phe Ser Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Ile Ala Thr Tyr Tyr Cys Gln Gln Gly Gln Ser Tyr Pro Tyr

85 90 95

Thr Phe Gly Gly Gly Thr Lys Leu

100

<210> 3

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<212> DNA

<213> Artificial Sequence (Artificial Sequence)

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gatgtgcagc tggtggagtc tgggggaggc ttagtgcagc ctggagggtc ccggaaactc 60

tcctgtgcag cctctggatt cactttcagt agctttggaa tgcactgggt tcgtcaggct 120

ccagacaagg ggctggagtg ggtcgcatac attagtggtg gcagtaatgt catctactat 180

gcagacacag tggagggccg attcaccatc tccagagaca atcccaagaa caccctgttc 240

ctgcaaatga ccagtctaag gtctgaggac gcggccatgt actattgtgt aagaaacttc 300

cgttacaacg gtagtagcct tcactactgg tacttcgatg tctggggcgc agggaccacg 360

gtcaccgtct cctcag 376

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gacatccaga tgaatcagtc tccatccagt ctgtctgcat cccttggaga cacaattacc 60

atcacttgcc atgccagtca tcacattaat gtttgggtaa cctggtacca gcagaaacca 120

ggaaatattc ctaaactttt gatctataag gcttccaagt tgcactcagg cgtcccatca 180

aggtttagtg gcagtggatt tggaacaggt ttctcattaa ccatcagcag cctgcagcct 240

gaagacattg ccacttacta ctgtcaacag ggtcaaagtt atccgtacac gttcggaggg 300

gggaccaagc tgga 314

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Gly Phe Thr Phe Ser Ser Phe Gly

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Ile Ser Gly Gly Ser Asn Val Ile

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Val Arg Asn Phe Arg Tyr Asn Gly Ser Ser Leu His Tyr Trp Tyr Phe

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Asp Val

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Asp Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

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Ser Arg Lys Leu Ser Cys Ala Ala Ser

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Met His Trp Val Arg Gln Ala Pro Asp Lys Gly Leu Glu Trp Val Ala

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Tyr

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<211> 38

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Tyr Tyr Ala Asp Thr Val Glu Gly Arg Phe Thr Ile Ser Arg Asp Asn

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Pro Lys Asn Thr Leu Phe Leu Gln Met Thr Ser Leu Arg Ser Glu Asp

20 25 30

Ala Ala Met Tyr Tyr Cys

35

<210> 11

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<213> Artificial Sequence (Artificial Sequence)

<400> 11

Arg His Ile Asn Val Trp

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<210> 12

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<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 12

Asn Ala Ser

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<210> 13

<211> 9

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

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Leu Leu Gly Gln Ser Tyr Pro Tyr Ser

1 5

<210> 14

<211> 26

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

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Asp Ile Arg Met Asn His Ala Pro Ser Ser Leu Ser Ala Ser Leu Val

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Asp Thr Ile Thr Ile Ser Cys His Ala Ser

20 25

<210> 15

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<213> Artificial Sequence (Artificial Sequence)

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Gly Thr Trp Tyr Gln Gln Lys Pro Glu Asn Ile Pro Lys Leu Leu Ile

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Tyr

<210> 16

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<212> PRT

<213> Artificial Sequence (Artificial Sequence)

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Arg Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly Asn Gly Phe Gly

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Thr Gly Phe Ser Leu Thr Ile Asn Asn Leu Gln Pro Glu Asp Ile Ala

20 25 30

Thr Tyr Tyr Cys

35

<210> 17

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<213> Artificial Sequence (Artificial Sequence)

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Gly Phe Thr Phe Ser Asp Phe Tyr

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<210> 18

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<212> PRT

<213> Artificial Sequence (Artificial Sequence)

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Ser Arg Asp Lys Ala Asn Asp Tyr Thr Thr

1 5 10

<210> 19

<211> 14

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<213> Artificial Sequence (Artificial Sequence)

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Ala Arg Asp Ala Tyr Tyr Gly Asn Tyr Val Lys Phe Ala Tyr

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<210> 20

<211> 25

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

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Glu Val Lys Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

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Ser Leu Arg Leu Ser Cys Ala Thr Ser

20 25

<210> 21

<211> 17

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

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Met Glu Trp Val Arg Gln Pro Pro Gly Lys Arg Leu Glu Trp Ile Ala

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Ala

<210> 22

<211> 38

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

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Glu Tyr Ser Ala Ser Val Lys Gly Arg Phe Ile Val Ser Arg Asp Thr

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Ser Gln Ser Ile Leu Tyr Leu Gln Met Asn Ala Leu Arg Ala Glu Asp

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Thr Ala Ile Tyr Tyr Cys

35

<210> 23

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<213> Artificial Sequence (Artificial Sequence)

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Gln Asp Ile Asn Lys Phe

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<213> Artificial Sequence (Artificial Sequence)

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Tyr Thr Ser

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<210> 25

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<213> Artificial Sequence (Artificial Sequence)

<400> 25

Leu Gln Tyr Asp Asn Leu Arg Thr

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<210> 26

<211> 26

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

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Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Leu Gly

1 5 10 15

Gly Asn Val Thr Ile Thr Cys Lys Ala Ser

20 25

<210> 27

<211> 17

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 27

Ile Ala Trp His Gln Tyr Lys Pro Gly Lys Gly Pro Arg Leu Leu Ile

1 5 10 15

His

<210> 28

<211> 36

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

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Thr Leu Gln Pro Gly Ile Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly

1 5 10 15

Arg Asp Tyr Ser Phe Ser Ile Ser Asn Leu Glu Pro Glu Asp Ile Ala

20 25 30

Thr Tyr Tyr Cys

35

<210> 29

<211> 106

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 29

Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln

1 5 10 15

Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr

20 25 30

Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

100 105

<210> 30

<211> 330

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 30

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr

65 70 75 80

Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys

85 90 95

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

100 105 110

Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn

195 200 205

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

210 215 220

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

225 230 235 240

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

245 250 255

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

260 265 270

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

275 280 285

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

290 295 300

Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr

305 310 315 320

Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

325 330

<210> 31

<211> 318

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 31

actgttgctg ctccatctgt ttttattttt ccaccatctg atgaacaact taaatctgga 60

actgcttctg ttgtttgtct tcttaataat ttttatccaa gagaagctaa agttcaatgg 120

aaagttgata atgctcttca atctggaaat tctcaagaat ctgttactga acaagattct 180

aaagattcta cttattctct ttcttctact cttactcttt ctaaagctga ttatgaaaaa 240

cataaagttt atgcttgtga agttactcat caaggacttt cttctccagt tactaaatct 300

tttaatagag gagaatgt 318

<210> 32

<211> 993

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 32

gcctccacca agggcccatc ggtcttcccc ctggcaccct cctccaagag cacctctggg 60

ggcacagcgg ccctgggctg cctggtcaag gactacttcc ccgaaccggt gacggtgtcg 120

tggaactcag gcgccctgac cagcggcgtg cacaccttcc cggctgtcct acagtcctca 180

ggactctact ccctcagcag cgtggtgacc gtgccctcca gcagcttggg cacccagacc 240

tacatctgca acgtgaatca caagcccagc aacaccaagg tggacaagaa agttgagccc 300

aaatcttgtg acaaaactca cacatgccca ccgtgcccag cacctgaact cctgggggga 360

ccgtcagtct tcctcttccc cccaaaaccc aaggacaccc tcatgatctc ccggacccct 420

gaggtcacat gcgtggtggt ggacgtgagc cacgaagacc ctgaggtcaa gttcaactgg 480

tacgtggacg gcgtggaggt gcataatgcc aagacaaagc cgcgggagga gcagtacaac 540

agcacgtacc gggtggtcag cgtcctcacc gtcctgcacc aggactggct gaatggcaag 600

gagtacaagt gcaaggtctc caacaaagcc ctcccagccc ccatcgagaa aaccatctcc 660

aaagccaaag ggcagccccg agaaccacag gtgtacaccc tgcccccatc ccgggatgag 720

ctgaccaaga accaggtcag cctgacctgc ctggtcaaag gcttctatcc cagcgacatc 780

gccgtggagt gggagagcaa tgggcagccg gagaacaact acaagaccac gcctcccgtg 840

ctggactccg acggctcctt cttcctctac agcaagctca ccgtggacaa gagcaggtgg 900

cagcagggga acgtcttctc atgctccgtg atgcatgagg ctctgcacaa ccactacacg 960

cagaagagcc tctccctgtc tccgggtaaa tga 993

<210> 33

<211> 28

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 33

ctagtctaga atggactcca ggctcaat 28

<210> 34

<211> 42

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 34

cgcgctgctc acggttgagg agacggtgac cgtggtccct gc 42

<210> 35

<211> 30

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 35

gtcaccgtct cctcaaccgt gagcagcgcg 30

<210> 36

<211> 27

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 36

ccggaattct cacttcccgg ggctcag 27

<210> 37

<211> 28

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 37

ctagtctaga atgagggtcc ttgctgag 28

<210> 38

<211> 39

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 38

ccggaattcg gtccgcttga tctccagctt ggtcccccc 39

<210> 39

<211> 40

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 39

ctagtctaga ggggggacca agctggagat caagcggacc 40

<210> 40

<211> 27

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 40

ccggaattct cagcactcgc cccggtt 27

<210> 41

<211> 127

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 41

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Phe

20 25 30

Gly Met His Trp Val Arg Gln Ala Pro Asp Lys Gly Leu Glu Trp Val

35 40 45

Ala Tyr Ile Ser Gly Gly Ser Asn Val Ile Tyr Tyr Ala Asp Ser Val

50 55 60

Glu Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Val Arg Asn Phe Arg Tyr Asn Gly Ser Ser Leu His Tyr Trp Tyr Phe

100 105 110

Asp Val Trp Gly Ala Gly Thr Thr Val Thr Val Ser Ser Leu Glu

115 120 125

<210> 42

<211> 104

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 42

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

1 5 10 15

Asp Arg Val Thr Ile Thr Cys His Ala Ser His His Ile Asn Val Trp

20 25 30

Val Thr Trp Tyr Gln Gln Lys Pro Gly Lys Val Pro Lys Leu Leu Ile

35 40 45

Tyr Lys Ala Ser Lys Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Val Ala Thr Tyr Tyr Cys Gln Gln Gly Gln Ser Tyr Pro Tyr

85 90 95

Thr Phe Gly Gly Gly Thr Lys Leu

100

<210> 43

<211> 732

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 43

gacatccaga tgacccagtc tccgtcttct ctgtctgctt ctgttggtga ccgtgttacc 60

atcacctgcc acgcttctca ccacatcaac gtttgggtta cctggtacca gcagaaaccg 120

ggtaaagttc cgaaactgct gatctacaaa gcttctaaac tgcactctgg tgttccgtct 180

cgtttctctg gttctggttc tggtaccgac ttcaccctga ccatctcttc tctgcagccg 240

gaagacgttg ctacctacta ctgccagcag ggtcagtctt acccgtacac cttcggtggt 300

ggtaccaaac tgggtggtgg tggttctggt ggtggtggtt ctggtggtgg tggttctgaa 360

gttcagctgg ttgaatctgg tggtggtctg gttcagccgg gtggttctct gcgtctgtct 420

tgcgctgctt ctggtttcac cttctcttct ttcggtatgc actgggttcg tcaggctccg 480

gacaaaggtc tggaatgggt tgcttacatc tctggtggtt ctaacgttat ctactacgct 540

gactctgttg aaggtcgttt caccatctct cgtgacaacg ctaaaaactc tctgtacctg 600

cagatgaact ctctgcgtgc tgaagacacc gctgtttact actgcgttcg taacttccgt 660

tacaacggtt cttctctgca ctactggtac ttcgacgttt ggggtgctgg taccaccgtt 720

accgtttctt ct 732

Claims (10)

1. An antibody against a terminally glycosylated protein receptor, comprising a light chain variable region and a heavy chain variable region; the CRD region sequence of the light chain variable region comprises light chain CDR1, light chain CDR2, light chain CDR 3; the amino acid sequence of the light chain CDR1 is shown in SEQ ID NO.11, the amino acid sequence of the light chain CDR2 is shown in SEQ ID NO.12, and the amino acid sequence of the light chain CDR3 is shown in SEQ ID NO. 13;

the CRD region sequence of the heavy chain variable region comprises heavy chain CDR1, heavy chain CDR2, heavy chain CDR 3; the amino acid sequence of the heavy chain CDR1 is shown in SEQ ID NO.5, the amino acid sequence of the heavy chain CDR2 is shown in SEQ ID NO.6, and the amino acid sequence of the heavy chain CDR3 is shown in SEQ ID NO. 7.

2. The antibody against the terminally glycosylated protein receptor of claim 1, wherein the heavy chain variable region of the antibody has the amino acid sequence shown in SEQ ID No.1 and the light chain variable region has the amino acid sequence shown in SEQ ID No. 2;

or a mutant or humanized antibody comprising the light chain variable region and the heavy chain variable region as the basis, wherein the mutant comprises an amino acid sequence having homology of 50% or more with the light chain variable region and the heavy chain variable region sequence; the humanized antibody is obtained by mutating the non-CDR sequences in the light chain variable region and the heavy chain variable region.

3. The anti-terminally glycosylated protein receptor antibody of claim 1, wherein the antibody further comprises a light chain constant region, a heavy chain constant region;

the amino acid sequence of the heavy chain constant region is shown as SEQ ID NO.30, and the amino acid sequence of the light chain constant region is shown as SEQ ID NO. 29.

4. The antibody against the terminally glycosylated protein receptor of claim 2, wherein the humanized antibody has the heavy chain variable region amino acid sequence of SEQ ID No.41 and the light chain variable region amino acid sequence of SEQ ID No. 42.

5. A recombinant plasmid comprising the anti-terminally glycosylated protein receptor antibody of claim 1.

6. A method for producing an antibody against a terminally glycosylated protein receptor according to claim 1, comprising the steps of:

A. screening monoclonal hybridoma cells secreting anti-RAGE antibody and obtaining DNA fragments encoding the heavy chain variable region and the light chain variable region of the anti-human RAGE antibody;

B. and (3) respectively recombining the light chain variable region and the heavy chain variable region of the cloned anti-human RAGE antibody with the light chain constant region and the heavy chain constant region of a human IgG1 antibody to obtain the recombinant human RAGE antibody.

7. Use of an anti-terminally glycosylated protein receptor antibody according to claim 1 for the preparation of a composition for inhibiting stellate cell activation and liver fibrosis.

8. Use of an antibody against a terminally glycosylated protein receptor according to claim 1 for the preparation of a composition for the treatment of senile dementia.

9. Use of an anti-terminally glycosylated protein receptor antibody according to claim 1 for the preparation of an antagonist of the S100a4, S100a6 protein.

10. Use of an anti-terminally glycosylated protein receptor antibody according to claim 1 for the preparation of an antagonist of a β protein.

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