CN113999306B

CN113999306B - Method for obtaining antibody for recognizing space conformation epitope

Info

Publication number: CN113999306B
Application number: CN202111247175.9A
Authority: CN
Inventors: 宋南; 林坜桁
Original assignee: Xinglian Pharmaceutical Suzhou Co ltd
Current assignee: Xinglian Pharmaceutical Suzhou Co ltd
Priority date: 2021-10-26
Filing date: 2021-10-26
Publication date: 2024-01-12
Anticipated expiration: 2041-10-26
Also published as: CN113999306A

Abstract

The invention discloses a method for obtaining an antibody for recognizing a space conformational epitope, which comprises the following steps: constructing a recombinant plasmid; the recombinant plasmid expresses fusion protein formed by antigen protein and surface anchoring protein; transferring the recombinant plasmid into eukaryotic cells to obtain recombinant eukaryotic cells; recombinant eukaryotic cell surface display antigen proteins or antigen protein fragments; immunizing an animal by adopting a recombinant eukaryotic cell to obtain a hybridoma cell; isolating antibodies from the hybridoma cells; the antibody is an antibody for recognizing the epitope of the antigen protein space conformation. The monoclonal antibody 15G7 which is obtained by the method and is used for recognizing the spatial conformational epitope of the CCN1 protein only binds to the natural CCN1 protein, and does not bind to the denatured CCN1 protein, namely only specifically recognizes the CCN1 protein in the natural conformation. The invention can obtain the antibody for recognizing the space conformation epitope and has important application value.

Description

Method for obtaining antibody for recognizing space conformation epitope

Technical Field

The invention belongs to the technical field of biology, and particularly relates to a method for obtaining an antibody for recognizing a space conformational epitope.

Background

Therapeutic antibodies have proven to be a powerful tool against human diseases. To date, most therapeutic antibodies on the market have been developed by hybridoma and phage display methods. However, the high affinity antibodies produced by these methods remain challenging. Due to the nature of the protein antigen itself, antibodies obtained using the above method may randomly recognize and bind to different structural regions of the antigen (antibody binding epitopes). Antibodies vary in biological activity in vivo. For example, anti-CD 20 antibodies are classified according to antibody binding epitopes into type I antibodies (e.g., me Luo Huashan antibodies) and type II antibodies (e.g., obrituximab), which have significantly reduced complement killing but greater antibody-dependent cell killing capacity than type I antibodies, and which do not have the ability to directly kill target cells. In clinical data comparison with non-hodgkin's lymphoma, obrituximab significantly prolonged the progression-free survival of patients compared to the mei Luo Huashan antibody. As another example, anti-CD 22 antibodies M971 and Epratuzumab, due to the difference in binding epitopes, only the latter can trigger rapid internalization of CD22 molecules. In summary, in the antibody screening process, ensuring the diversity of the constructed antibody screening library is an important premise for obtaining an antibody with biological activity. The nature of the antigen itself determines the preference for obtaining binding epitopes of antibodies from the above method. The antibody binding epitope may be a short peptide (linear epitope) composed of amino acids arranged in succession, or may be a complex spatial conformation (spatial epitope) formed by amino acids arranged in discontinuous fashion. Linear epitopes, e.g. polypeptides, on antigens bound by antibodies are recognized primarily by the histocompatibility complex (MHC) of T lymphocytes, while discontinuous spatial conformational epitopes on antigens are recognized primarily by B cells.

The generation of antibodies against conformational epitopes is challenging because the spatial conformational structure of the antigen must be well maintained in animal immunization and in vitro antibody screening. The conformational instability of the antigen leads to inconsistent and non-reproducible screening of candidate drugs. Generally, to facilitate purification of protein antigens, purification tags (e.g., his tag, immunoglobulin Fc portion) need to be fused to the antigen in advance at the time of antigen expression, but such a strategy has the following limitations: (1) The introduction of protein tags may destroy the spatial structure of the antigen; (2) These tags are immunogenic in animal immunization and require the introduction of additional antibody screening steps; (3) In order to efficiently harvest the antigen and maintain its spatial conformation during purification, conventional purification procedures are often required for optimization, which is cumbersome and laborious.

Disclosure of Invention

The object of the present invention is to obtain antibodies recognizing the conformational epitope in the space of an antigen protein.

The invention firstly protects a method for obtaining an epitope antibody for recognizing the space conformation of an antigen protein, which comprises the following steps:

(1) Constructing a recombinant plasmid; the recombinant plasmid expresses fusion protein formed by antigen protein and surface anchoring protein;

(2) Transferring the recombinant plasmid constructed in the step (1) into eukaryotic cells, and screening to obtain recombinant eukaryotic cells;

the surface of the recombinant eukaryotic cell displays antigen protein or antigen protein fragment;

(3) Immunizing an animal by adopting the recombinant eukaryotic cells obtained in the step (2) to obtain hybridoma cells;

(4) Isolating antibodies from the hybridoma cells obtained in step (3); the antibody is an antibody for recognizing the epitope of the antigen protein space conformation.

In the step (1), the surface anchoring protein may be a Glycosylphosphatidylinositol (GPI).

In the step (1), the antigen protein may be CCN1 protein (protein ID np_ 001545.2).

In the step (1), the recombinant plasmid contains a dihydrofolate reductase resistance gene.

In the step (1), the recombinant plasmid can be formed by connecting a fusion sequence shown in SEQ ID NO. 1 and a pOptiVEC-TOPO vector by using a TA cloning method. In SEQ ID NO. 1, from the 5' -end, the Kozak sequence is at positions 1-6, the DNA encoding human CCN1 protein is at positions 7-1149, and the DNA encoding GPI is at positions 1150-1296.

In the step (2), the eukaryotic cell may be Sp2/0-Ag14 cell. Sp2/0-Ag14 cells, i.e., wild-type Sp2/0-Ag14 cells (ATCC company product, catalog number CRL-1581).

In the step (2), the screening may be a methotrexate screening.

In the above method, when the antigen protein is CCN1 protein, the antibody recognizing the spatial conformational epitope of CCN1 protein may specifically be monoclonal antibody 15G7. The monoclonal antibody 15G7 consists in particular of a heavy chain and a light chain. The amino acid sequence of the heavy chain variable region is shown as SEQ ID NO. 3. The amino acid sequence of the light chain variable region is shown as SEQ ID NO. 5. The monoclonal antibody 15G7 binds CCN1 protein.

The application of the antigen protein space conformation epitope recognition antibody obtained by adopting any one of the methods in drug screening also belongs to the protection scope of the invention.

In the above application, the antibody recognizing the epitope in the spatial conformation of the antigen protein may be the monoclonal antibody 15G7.

The invention also protects a monoclonal antibody 15G7, which specifically consists of a heavy chain and a light chain. The amino acid sequence of the heavy chain variable region is shown as SEQ ID NO. 3. The amino acid sequence of the light chain variable region is shown as SEQ ID NO. 5. The monoclonal antibody 15G7 binds CCN1 protein.

The invention also protects a nucleic acid molecule encoding the monoclonal antibody 15G7. The nucleic acid molecule encoding the monoclonal antibody 15G7 may consist of a nucleic acid molecule encoding the heavy chain and a nucleic acid molecule encoding the light chain. The nucleotide sequence of the heavy chain variable region is shown as SEQ ID NO. 2. The nucleotide sequence of the light chain variable region is shown as SEQ ID NO. 4.

According to the invention, through engineering modification of eukaryotic cells, antigen proteins or antigen protein fragments are displayed on the surfaces of the eukaryotic cells, and then eukaryotic cells are adopted to immunize animals, so that hybridoma cells are obtained; isolating antibodies from the hybridoma cells; the antibody is an antibody for recognizing the epitope of the antigen protein space conformation. The monoclonal antibody 15G7 which is obtained by the method and is used for recognizing the spatial conformational epitope of the CCN1 protein only binds to the natural CCN1 protein, and does not bind to the denatured CCN1 protein, namely only specifically recognizes the CCN1 protein in the natural conformation. Compared with the traditional method, the method provided by the invention does not need an antigen purification step, and the prepared antibody is an antibody for recognizing the space conformational epitope. The invention has important application value.

Drawings

FIG. 1 shows the detection result of step three in example 1.

FIG. 2 shows the results of the test in example 2.

FIG. 3 shows the results of the test in example 3.

FIG. 4 shows the test results of example 4.

Detailed Description

The following detailed description of the invention is provided in connection with the accompanying drawings that are presented to illustrate the invention and not to limit the scope thereof. The examples provided below are intended as guidelines for further modifications by one of ordinary skill in the art and are not to be construed as limiting the invention in any way.

The experimental methods in the following examples, unless otherwise specified, are conventional methods, and are carried out according to techniques or conditions described in the literature in the field or according to the product specifications. Materials, reagents and the like used in the examples described below are commercially available unless otherwise specified.

CCN1 protein (protein ID np_ 001545.2) belongs to one of the products encoded by early response genes, whose expression is regulated by a number of pathological and physiological environments, such as cytokines, mechanical stretches, harmful toxins, etc. CCN1 has four domains, an insulin-like growth factor binding protein, a von willebrand factor C domain (von Willebrand factor type C domain, vWC), a platelet-like lectin module, and a carboxy-terminal module, respectively. Four domains can be respectively bound to different cell surface integrin receptors, and the diversity of receptor binding determines the abundant biological functions of CCN1 under physiological and pathological conditions, for example, the vWC domain of CCN1 can be bound with the αvβ3 integrin receptor to promote the adhesion, migration and proliferation of endothelial cells; binding to the αvβ5 integrin receptor can enhance phagocytosis by macrophages; binding to the α6β1 integrin receptor can lead to senescent apoptosis of fibroblasts. In addition, CCN1 also promotes cancer cell migration, aggravates lung tissue injury, and participates in physiological and pathological processes such as angiogenesis caused by hypoxia reaction. Therefore, the monoclonal antibodies aiming at different domains of CCN1 have potential clinical application prospect.

CCN1 proteins expressed and purified by eukaryotic cells are extremely susceptible to degradation when stored in neutral phosphate buffer or repeatedly frozen and thawed. CCN1 protein can be captured by using heparin columns, but heparin also captures many impurity proteins together, so that the method cannot specifically enrich CCN1 protein. Instability of the native structure of CCN1 in vitro creates great difficulties in screening antibodies recognizing the spatially conformational epitope CCN 1.

Example 1 obtaining antibodies recognizing the spatial conformational epitope CCN 1-anti-CCN 1 antibodies

1. Construction of CCN1-sp2/0 cells

1. The 3' -end of the DNA sequence encoding CCN1 protein and the DNA sequence encoding GPI are fused by molecular biology technology and then constructed into an expression vector containing dihydrofolate reductase (DHFR) resistance gene, thus obtaining recombinant plasmid. The method comprises the following specific steps:

(1) The DNA encoding the human CCN1 protein (NM_ 001554) and the DNA encoding GPI were fused, and then a Kozak sequence GCCACC was added to the 5' end to obtain a fusion sequence shown in SEQ ID NO. 1.

In SEQ ID NO. 1, from the 5' -end, the Kozak sequence is at positions 1-6, the DNA encoding human CCN1 protein is at positions 7-1149, and the DNA encoding GPI is at positions 1150-1296.

(2) According to pOptiVEC ^TM -Operating procedure of TACloning Kit (Thermo, cat. No. 12744-017), linking the fusion sequence obtained in step (1) with pOptiVEC-TOPO vector by TA cloning, and then transforming OneTOP10 complete E.coli Competent cells (Thermo, cat# C404003), plasmid enrichment and extraction using QIAGEN Plasmid Maxi Kit (QIAGEN, cat# 12163) yielded recombinant plasmids.

The pOptiVEC-TOPO vector is pOptiVEC ^TM -The components in the TACloning Kit.

2. The recombinant plasmid obtained in the step 1 was transferred into wild type Sp2/0-Ag14 cells (product of ATCC company, catalog number CRL-1581) by electric shock, and positive Sp2/0 cell clones (hereinafter referred to as CCN1-Sp2/0 cells) were obtained by screening with methotrexate. The method comprises the following specific steps:

(1) 20. Mu.g of the recombinant plasmid obtained in step 1 was added to PvUI enzyme (NewEngland BioLabs, cat. No. R0150) and digested at 37℃for 12 hours to obtain a digested solution. The digestion solution contains linearized recombinant plasmid.

(2) After the step (1) is completed, adding equal volume of saturated phenol into the digestive juice, gently and fully mixing for 3min, centrifuging for 10min at 5000g, and collecting the upper water phase into a new centrifuge tube.

(3) After the step (2) is completed, adding an equal volume of saturated phenol into the centrifuge tube, uniformly mixing, centrifuging for 10min at 5000g, and collecting an upper water phase into a new centrifuge tube.

(4) After step (3) is completed, adding equal volume of phenol/chloroform (equal volume of phenol and chloroform mixture) into the centrifuge tube, mixing well, centrifuging for 10min at 5000g, and collecting the upper water phase into a new centrifuge tube.

(5) After the step (4) is completed, adding equal volume of chloroform into the centrifuge tube, uniformly mixing, centrifuging for 10min at 5000g, and collecting an upper water phase into a new centrifuge tube.

(6) After the step (5) is completed, 1/10 volume of pH5.2, 3M sodium acetate and 2.5 times volume of absolute ethyl alcohol are added into a centrifuge tube, the mixture is gently inverted and uniformly mixed, and after floccules appear, 5000g is centrifuged for 5min, and sediment is collected.

(7) The precipitate collected in step (6) was washed with 75% (v/v) aqueous ethanol, and then centrifuged at 5000g for 3min, and the precipitate was collected. Ethanol in the precipitate is fully volatilized, and the linearized recombinant plasmid is obtained.

(8) Taking 0.5X10 ⁶ Sp2/0-Ag14 cells were resuspended in 250. Mu.L of Hybridoma-SFM medium (Thermo, cat. No. 12045076) to give a cell suspension.

(9) And (3) fully and uniformly mixing the linearized recombinant plasmid obtained in the step (7) and the cell suspension obtained in the step (8) to obtain a mixed solution. The mixture was carefully transferred to an electrorotor for electrotransfer (set voltage 180 volts, capacitance 960. Mu.F, time constant 24 msec), after completion of electrotransfer the mixture was carefully transferred to a sterile EP tube and placed on ice for 10min.

(10) After the step (9) is completed, the mixed solution is firstly transferred to a hybrid oma-SFM culture medium and is cultured for 48 hours at 37 ℃; transferring to a hybrid-SFM medium containing 0.05. Mu. Mol methotrexate, and culturing at 37deg.C for 3-5 days; during the culture, passage was performed every 3-5 days.

(11) Inoculating the cells obtained in the step (10) into a 96-well plate with the average number of cells in each well not exceeding 1, and then adding the hybrid oma-SFM culture medium containing 0.1 mu mol of methotrexate into each well, and culturing at 37 ℃ for 21 days; the single clone in the well plate was selected and transferred to a flask, and the surface display of CCN1 was confirmed by the method in step 3 by performing an expansion culture using a hybrid-SFM medium containing 0.1. Mu. Mol of methotrexate.

(12) The "cells obtained in step (10)" in step (11) are replaced with the cells obtained in step (11), the concentration of methotrexate is increased, and the other steps are unchanged, so that CCN1-sp2/0 cells are obtained (namely, the screening concentration of the methotrexate in the culture medium is gradually increased to increase the CCN1 expression level of the screened cells).

3. CCN1 was confirmed to be displayed on the sp2/0-Ag14 cell surface using a10 mab (commercial CCN1 mab, santa Cruz Biotechnology, product of inc. Company under the product catalog number sc-374129). The method comprises the following specific steps:

(1) Taking a composition containing 1×10 ⁶ Cell suspensions of individual CCN1-sp2/0 cells, centrifuged at 300g, and cell pellet collected; the precipitate was then washed 1 time with phosphate buffer (pH 7.4).

(2) To the cell pellet obtained in the step (1), 0.5ml of a10 mab diluent was added, incubated at room temperature for 2 hours, the supernatant was discarded, and the pellet was washed 3 times with phosphate buffer (ph 7.4).

Phosphate buffer (pH 7.4) 1: and (3) diluting the A10 monoclonal antibody by 1000 to obtain an A10 monoclonal antibody diluent.

(3) After completion of step (2), murine secondary antibody dilution was added, incubated at room temperature for 1h, the supernatant was discarded, and the pellet was washed 3 times with phosphate buffer (pH 7.4).

Phosphate buffer (pH 7.4) 1: the murine secondary antibody (Jackson ImmunoResearch Laboratories, inc. No. 115-035-003) was diluted at 5000 to give a murine secondary antibody diluent.

(4) After completion of step (3), TMB substrate (3, 3', 5' -tetramethylbenzidine, sigma-Aldrich, cat# T0440) was added and reacted at room temperature for 10min, after which the supernatant was transferred to a 96-well plate and the absorbance at 450nm was read.

According to the above procedure, CCN1-sp2/0 cells were replaced with sp2/0-Ag14 cells, and the other procedures were unchanged, as a negative control.

The results show that the absorbance at 450nm of CCN1-sp2/0 cells is significantly higher than the absorbance at 450nm of sp2/0-Ag14 cells.

2. Preparation of hybridoma cells

1. 1mL of phosphate buffer was added to 3X 10 ⁷ And fully and uniformly mixing the CCN1-sp2/0 cells to obtain a cell suspension.

2. 1mL of complete Freund's adjuvant was added to the cell suspension obtained in step 1, and the mixture was shaken and emulsified at 1500rpm to obtain solution 1. To the cell suspension obtained in step 1, 1mL of incomplete Freund's adjuvant was added to obtain solution 2. To the cell suspension obtained in step 1, 1mL of phosphate buffer was added to obtain solution 3.

3. 5 Balb/C mice of 6 weeks of age were taken, each of which was operated as follows: on day 1 of the experiment, 200. Mu.L of solution 1 (3X 10 cells were injected) ⁶ Individual) (first immunization); on experiment day 14, 200. Mu.L of solution 2 was intraperitoneally injected (the number of injected cells was 3X 10) ⁶ Second immunization); on experiment day 28, 200. Mu.L of solution 2 was intraperitoneally injected (the number of injected cells was 3X 10) ⁶ Third immunization); on experiment day 42, 100. Mu.L of solution 3 was injected into the tail vein (the number of injected cells was 1.5X10) ⁶ And (c) a).

4. After 3 days from completion of step 3, mice were sacrificed and spleens of the mice were taken, and hybridoma cell clones were prepared by hybridoma preparation technique. The method comprises the following specific steps:

(1) Serum was collected from 5 mice 4 days prior to the first immunization, 7 days after the second immunization, and 7 days after the third immunization, respectively. Then, the titer of the anti-human CCN1 antibody in serum is detected, and the mice with the highest titer in the blood list are selected and sacrificed on the 45 th day, and spleens are taken.

(2) After the step (1) is completed, spleen cells of the mice are separated into single cells and fused with sp2/0-Ag14 cells, the obtained single clones are cultured in a porous culture plate, and culture medium supernatants are collected and the content of the anti-CCN 1 antibodies is determined.

(3) According to the measurement result, monoclonal antibody secretion amount was cultured according to the method of step 2 (11) using the Hybridoma-SFM medium. This step was repeated 2-3 times, each time the content of anti-CCN 1 antibody was determined, and a high secretion amount of the monoclonal antibody was selected until the target clone with stable genome was finally obtained.

3. Obtaining anti-CCN 1 antibodies from hybridoma cells

A. The activity of anti-CCN 1 mab in hybridoma cell culture supernatants was determined using enzyme-linked immunosorbent assay. The specific steps are as follows:

1. mu.L of CCN1 protein solution was added to each well of the 96-well plate, and after incubation for 6 hours at room temperature, the solution was discarded.

Fresh CCN1 protein was prepared by heparin column and formulated as a10 μg/mL CCN1 protein solution using phosphate buffer.

2. Washing twice with phosphate buffer, blocking with phosphate buffer containing 3% BSA, and incubating at room temperature for 2h; the solution was then discarded and washed twice with phosphate buffer.

3. The culture supernatant of the hybridoma cells was added, incubated at room temperature for 2 hours, and then the culture supernatant was discarded and washed twice.

4. Anti-mouse FC fragment-horseradish peroxidase (Jackson ImmunoResearch Laboratories, inc. product, catalog number 115-036-003) was added, incubated at room temperature for 1h, discarded, and the plate was washed with phosphate buffer.

5. TMB substrate was added and reacted at room temperature for 10min, after which the absorbance at 450nm was read.

According to the above method, the culture supernatant of the hybridoma cells was replaced with SM03 (anti-human CD22 monoclonal antibody, concentration of 0.4. Mu.g/mL) (product batch number of Sm03-202-201701-b003-04-09, product of China antibody pharmaceutical Co., ltd.) and the other steps were unchanged, as a negative control.

According to the above method, the culture supernatant of the hybridoma cells was replaced with a10 mab, and the other steps were unchanged, as a positive control.

The experimental results are shown in FIG. 1. The results showed that the feedback signal was strongest for hybridoma clone 15G7. Thus, hybridoma clone 15G7 was selected for further analysis and identification.

B. Obtaining anti-CCN 1 antibodies

1. mRNA of hybridoma clone 15G7 was extracted with Trizol (Thermo, cat. No. 15596026), followed by SuperScript ^TM IV Reverse Transcriptase (Thermo, cat 18090200) was subjected to reverse transcription to obtain cDNA.

2. Using cDNA as a template, heavy chain primer 1:5'-AGGTNMAKCTGCAGNAGTCLGG-3' (N is C or G, M is A or C, K is A or G, L is A or T) and heavy chain primer 2:5'-AGCTGGGAAGGTGTGCAC-3', and obtaining the variable region fragment of 15G7 heavy chain. Light chain primer 1 was used with cDNA as template: 5'-GACATTCAGCTGACCCAGTCTCCA-3' and light chain primer 2:5'-GTTAGATCTCCAGCTTGGTCCC-3', and obtaining the variable region fragment of the 15G7 light chain.

The reaction procedure is: 95 ℃ for 2min;95℃15s,56℃30s,68℃1min,35 cycles; 68 ℃ for 3min; preserving at 4 ℃.

3. The variable region fragment of the 15G7 heavy chain and the variable region fragment of the 15G7 light chain were cloned into pOptiVEC-TOPO vectors, respectively, followed by DNA sequencing.

The pOptiVEC-TOPO vector is pOptiVEC ^TM -The components in the TA Cloning Kit (Thermo, cat. No. 12744-017).

The nucleotide sequence of the 15G7 heavy chain variable region fragment is: cagGtcaaactgcaggagtctgggcctgagctggtgaggcctggggtctcagtgaagatttcctgcaagggttccggctacacattcactgattatgctatgcactgggtgaagcagagtcatgcaaagagtctagagtggattggagttattagtacttactctggtaatacaaactacaaccagaagtttaagggcaaggccacaatgactgtagacaaatcctccagcacagcctatatggaacttgccagattgacatctgaggattctgccatctattactgtgcaagacagctatggttacgacgcccctactatgctatggactactggggtcaaggaacctcagtcaccgtctcctca (SEQ ID NO: 2)

The amino acid sequence of the variable region fragment of the 15G7 heavy chain is: QVKLQESGPELVRPGVSVKISCKGSGYTFTDYAMHWVKQSHAKSLEWIGVISTYSGNTNYNQKFKGKATMTVDKSSSTAYMELARLTSEDSAIYYCARQLWLRRPYYAMDYWGQGTSVTVSS (SEQ ID NO: 3)

The nucleotide sequence of the variable region fragment of the 15G7 light chain is: gacattcagctgacccagtctccagcttctttggctgtgactctagggcagagagccaccatctcctgcagagccagtgaaagtgttgaacattatggcacaagttttatgcagtggtaccaacagaaaccaggacagtcacccaaactcatcatctatgttgcttccaacgtagaatctggggtccctgccaggtttagtggcagtgggtctgggacagacttcagcctcaacatccatcctgtggaggaggatgatattgcaatatatttctgtcagcaaagtaggaaggttccttccacgttcggaggggggaccaagctggagatcAAA (SEQ ID NO: 4)

The amino acid sequence of the variable region fragment of the 15G7 light chain is: DIQLTQSPASLAVTLGQRATISCRASESVEHYGTSFMQWYQQKPGQSPKLIIYVASNVESGVPARFSGSGSGTDFSLNIHPVEEDDIAIYFCQQSRKVPSTFGGGTKLEIK (SEQ ID NO: 5)

4. The variable region fragment of 15G7 heavy chain and the constant region (IgG 1 constant region) sequence of human monoclonal antibody are fused to obtain the heavy chain of 15G7 chimeric monoclonal antibody. The variable region fragment of the 15G7 light chain and the constant region sequence of human monoclonal antibody (IgG 1 constant region) were fused to obtain a light chain of 15G7 chimeric monoclonal antibody.

5. The small DNA fragment between restriction enzymes XhoI and NotI in pcDNA3.1 (+) vector (Thermo, cat. V87020) is replaced by the heavy chain of 15G7 chimeric mab, the small DNA fragment between restriction enzymes XbaI and NheI is replaced by the light chain of 15G7 chimeric mab, and other sequences are unchanged, thus obtaining recombinant plasmid pcDNA3.1 (+) -15G7.

6. The recombinant plasmid pcDNA3.1 (+) -15G7 was transfected into an ExpiCHO cell (Thermo, cat. No. A29133) and purified by protein A affinity adsorption to obtain 15G7 mab, i.e., the anti-CCN 1 antibody.

Examples 2, 15G7 monoclonal antibodies (i.e., the anti-CCN 1 antibodies obtained in example 1) bind to native, but not denatured, CCN1 proteins

The binding capacity of 15G7 mab to CCN1 protein was tested using western blotting. The method comprises the following specific steps:

1. taking a sample containing 3×10 ⁵ Cell suspensions of individual CCN1-sp2/0 cells, centrifuged at 300g for 3min, and cell pellet collected; followed by washing with 3mL of PBS buffer.

2. After the completion of step 1, ice-cold RIPA lysis buffer was added and the mixture was lysed for 5min to obtain a lysate.

RIPA lysis buffer: the solvent and its concentration are 150mM NaCl (sigma), 5mM EDTA (sigma), 1% Triton X-100 (sigma), 1% sodAn ium deoxyplate (sigma) and 0.1% SDS (sigma), the solvent being pH7.4, 50mM Tris-HCl buffer (sigma); when in use, the Hall is added ^TM Protease (Hall) ^TM Protease was diluted 100-fold to working concentration) and phosphatase inhibitor (thermo Fisher, cat 78446) (Hall) ^TM Phosphatase inhibitors were diluted 100-fold to working concentration).

3. After step 2 is completed, the lysate andLDS sample buffer (Thermo, cat. NP 0008) was mixed in equal volumes to give mixed solution A. The mixed solution A contains CCN1 protein with natural conformation (called natural CCN1 protein for short).

4. After completion of step 2, the lysate was combined with 5% beta-mercaptoethanol (Sigma, cat# M6250) added in advanceMixing LDS sample buffer solution in equal volume to obtain a mixed solution B; and (3) treating the mixed solution B at 95 ℃ for 10min (the aim is to fully denature CCN1 protein) to obtain mixed solution A. The mixed solution C contains structurally denatured CCN1 protein (abbreviated as denatured CCN1 protein).

5. Loading the mixed solution A obtained in the step 3 or the mixed solution C obtained in the step 4 to 12.5% polyacrylamide gel, and carrying out electrophoresis for 2h under 100V voltage; then transferred to PVDF membrane (constant current 400mA,1.5 h) by electrotransfer; afterwards, the PVDF membrane is blocked with PBS buffer (blocking solution) containing 5% bovine serum albumin, overnight at 4 ℃;15G7 mab at a concentration of 1mg/mL was purified according to 1: diluting the volume ratio of 1000 by using a sealing solution, and then incubating the PVDF membrane under the room temperature condition; the PVDF membrane was washed 3 times with TBST buffer (Biorad, cat. No. BUF 028) for 5min each; adding 1:5000 blocking solution dilution of anti-human FC fragment horseradish peroxidase secondary antibody (Jacksonimmunore search, cat. No. 109-035-098), PVDF membrane incubated at room temperature for 1h; PVDF membrane was washed 1 time with TBST buffer (Biorad, cat. No. BUF 028) for 5min each.

6. After completion of step 5, pierce was added ^TM ECLWestern Blotting substrate (ThermoFisher, cat. No 32209), at room temperatureAfter 1min, the excess substrate solution was blotted with blotting paper.

7. After step 6 is completed, chemiluminescent images are acquired using darkroom development techniques. The results were analyzed using ImageLab software (Biorad).

15G7 mab was replaced with SM03 as a negative control according to the procedure described above.

The detection results are shown in FIG. 2. The results indicate that 15G7 mab does not bind to denatured CCN1 protein, but only to native CCN1 protein.

Examples 3, 15G7 monoclonal antibodies bind to CCN1-Sp2/0 cells and do not bind to Sp2/0-Ag14 cells

1. Taking a sample containing 3×10 ⁵ The cell suspension of CCN1-sp2/0 cells was centrifuged at 300g for 5min and the pellet was collected.

2. After completion of step 1, the precipitate was washed 2 times with a washing solution (phosphate buffer containing 1% bovine serum albumin). The method for each washing is as follows: the pellet was resuspended in 500. Mu.L of wash solution, mixed well upside down, centrifuged and the pellet collected.

3. After the step 2 is completed, fixing the precipitate with 4% paraformaldehyde for 5min, and collecting the precipitate; the precipitate was washed 2 times with washing solution. The method for each washing is as follows: the pellet was resuspended in 500. Mu.L of wash solution, mixed well upside down, centrifuged and the pellet collected.

4. After the step 3 is completed, the sediment is resuspended by a sealing solution (phosphate buffer containing 3% bovine serum albumin), and the sediment is collected after the sediment is left to stand for 20min at room temperature and centrifuged; the pellet was resuspended in 500. Mu.L of wash solution, mixed well upside down, centrifuged and the pellet collected.

5. After the step 4 is completed, the sediment is resuspended by 500 mu L of 15G7 monoclonal antibody diluent, and the sediment is stood for 30min at room temperature, centrifuged and collected; the precipitate was washed 2 times with washing solution. The method for each washing is as follows: the pellet was resuspended in 500. Mu.L of wash solution, mixed well upside down, centrifuged and the pellet collected.

15G7 mab dilution: diluting 15G7 monoclonal antibody with washing liquid; the concentration of 15G7 mab diluent was 1. Mu.g/ml or 0.5. Mu.g/ml.

6. After completion of step 5, 1 was added to the precipitate: secondary antibody (Jackson ImmunoResearch, cat 609-605-213) diluted with 2000 times of washing solution, incubating at room temperature for 30min, centrifuging, and collecting precipitate; the precipitate was washed 2 times with washing solution. The method for each washing is as follows: the pellet was resuspended in 500. Mu.L of wash solution, mixed well upside down, centrifuged and the pellet collected.

7. After completion of step 6, the pellet was resuspended in phosphate buffer and then single cells were filtered into a flow centrifuge tube using a filter screen. The fluorescence intensity distribution of 10000 cells was analyzed on the computer.

15G7 mab was replaced with SM03 as described above, with the other steps unchanged, as a negative control.

According to the above method, CCN1-Sp2/0 cells were replaced with Sp2/0-Ag14 cells, and the other steps were unchanged.

The results are shown in FIG. 3 (Sp 2/0-CCN1-GP1 is CCN1-Sp2/0 cells, sp2/0 is Sp2/0-Ag14 cells). The results indicate that 15G7 mab can bind to CCN1-Sp2/0 cells but cannot bind to Sp2/0-Ag14 cells.

The above results indicate that the 15G7 mab was specific for the binding of CCN1 protein and did not react with other proteins on the Sp2/0-Ag14 cell surface.

EXAMPLE 4 binding of 15G7 mab to CCN1 protein native to the cell surface of CCN1-sp2/0

1. Taking a sample containing 3×10 ⁵ Centrifuging 300g of a cell suspension of CCN1-sp2/0 cells for 5min, and collecting a precipitate; the pellet was then resuspended in 500. Mu.L phosphate buffer to give a cell suspension.

2. And (2) dripping 10 mu L of the cell suspension obtained in the step (1) on a sterile clean slide, heating the slide at 95 ℃ for 5min by using a metal bath, and evaporating the liquid to obtain denatured CCN1-sp2/0 cells.

3. 10. Mu.L of the cell suspension obtained in the step 1 was dripped on a sterile clean slide, and the liquid was air-dried in an ultra clean bench to obtain undenatured CCN1-sp2/0 cells.

4. Cells on the slide (denatured CCN1-sp2/0 cells or undenatured CCN1-sp2/0 cells) were fixed with 4% paraformaldehyde for 10min at room temperature, after which the fixed cells were washed 3 times with phosphate buffer.

5. After completion of step 4, the cells on the slide were incubated in PBST buffer containing 1% bovine serum albumin for 2h at room temperature.

PBST buffer: phosphate buffer containing 0.1% tween 20.

6. After completion of step 5, the solution was discarded, 15G7 mab solution (obtained by diluting 15G7 mab with PBST buffer containing 1% bovine serum albumin) was added at a concentration of 1. Mu.g/mL, and incubated for 2h at room temperature; the solution was discarded and the slide was washed 3 times with phosphate buffer.

7. After the step 6 is completed, the solution is discarded, the secondary antibody solution is added, and the mixture is incubated for 1h at room temperature; the solution was discarded and the slide was washed 3 times with phosphate buffer.

Secondary antibody solution: PBST buffer with 1% bovine serum albumin was used according to 1: obtained by 2000-fold dilution of secondary antibody (Jackson ImmunoResearch, cat No. 609-605-213).

8. After completion of step 7, staining was performed using DAPI (thermosusher, cat No. D1304), and after staining at room temperature for 15min, the solution was discarded, and the slide was washed 3 times with phosphate buffer.

9. Observed using a fluorescence microscope and photographed.

The results are shown in FIG. 4 (first behavior undenatured CCN1-sp2/0 cells, second behavior denatured CCN1-sp2/0 cells, red 15G7, blue nuclei). The results show that the heating can destroy the natural structure of CCN1 protein, and 15G7 monoclonal antibody can only recognize undenatured CCN1-sp2/0 cells and can not recognize denatured CCN1-sp2/0 cells.

The above results indicate that 15G7 mab only specifically recognizes the native conformation of CCN1 protein.

The present invention is described in detail above. It will be apparent to those skilled in the art that the present invention can be practiced in a wide range of equivalent parameters, concentrations, and conditions without departing from the spirit and scope of the invention and without undue experimentation. While the invention has been described with respect to specific embodiments, it will be appreciated that the invention may be further modified. In general, this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains. The application of some of the basic features may be done in accordance with the scope of the claims that follow.

<110> apricot associated pharmaceutical Co., ltd

<120> a method for obtaining an antibody recognizing a spatially conformational epitope

<160> 5

<170> PatentIn version 3.5

<210> 1

<211> 1296

<212> DNA

<213> Artificial sequence

<400> 1

gccaccatga gctcccgcat cgccagggcg ctcgccttag tcgtcaccct tctccacttg 60

accaggctgg cgctctccac ctgccccgct gcctgccact gccccctgga ggcgcccaag 120

tgcgcgccgg gagtcgggct ggtccgggac ggctgcggct gctgtaaggt ctgcgccaag 180

cagctcaacg aggactgcag caaaacgcag ccctgcgacc acaccaaggg gctggaatgc 240

aacttcggcg ccagctccac cgctctgaag gggatctgca gagctcagtc agagggcaga 300

ccctgtgaat ataactccag aatctaccaa aacggggaaa gtttccagcc caactgtaaa 360

catcagtgca catgtattga tggcgccgtg ggctgcattc ctctgtgtcc ccaagaacta 420

tctctcccca acttgggctg tcccaaccct cggctggtca aagttaccgg gcagtgctgc 480

gaggagtggg tctgtgacga ggatagtatc aaggacccca tggaggacca ggacggcctc 540

cttggcaagg agctgggatt cgatgcctcc gaggtggagt tgacgagaaa caatgaattg 600

attgcagttg gaaaaggcag ctcactgaag cggctccctg tttttggaat ggagcctcgc 660

atcctataca accctttaca aggccagaaa tgtattgttc aaacaacttc atggtcccag 720

tgctcaaaga cctgtggaac tggtatctcc acacgagtta ccaatgacaa ccctgagtgc 780

cgccttgtga aagaaacccg gatttgtgag gtgcggcctt gtggacagcc agtgtacagc 840

agcctgaaaa agggcaagaa atgcagcaag accaagaaat cccccgaacc agtcaggttt 900

acttacgctg gatgtttgag tgtgaagaaa taccggccca agtactgcgg ttcctgcgtg 960

gacggccgat gctgcacgcc ccagctgacc aggactgtga agatgcggtt ccgctgcgaa 1020

gatggggaga cattttccaa gaacgtcatg atgatccagt cctgcaaatg caactacaac 1080

tgcccgcatg ccaatgaagc agcgtttccc ttctacaggc tgttcaatga cattcacaaa 1140

tttagggacg cagagcccaa atcttgtgac aaaactcaca cctgcccacc gtgcccactg 1200

accaccagcg gcattgtgac catgagccat caggcgctgg gctttaccct gaccggcctg 1260

ctgggcaccc tggtgaccat gggcctgctg acctga 1296

<210> 2

<211> 366

<212> DNA

<213> Artificial sequence

<400> 2

caggtcaaac tgcaggagtc tgggcctgag ctggtgaggc ctggggtctc agtgaagatt 60

tcctgcaagg gttccggcta cacattcact gattatgcta tgcactgggt gaagcagagt 120

catgcaaaga gtctagagtg gattggagtt attagtactt actctggtaa tacaaactac 180

aaccagaagt ttaagggcaa ggccacaatg actgtagaca aatcctccag cacagcctat 240

atggaacttg ccagattgac atctgaggat tctgccatct attactgtgc aagacagcta 300

tggttacgac gcccctacta tgctatggac tactggggtc aaggaacctc agtcaccgtc 360

tcctca 366

<210> 3

<211> 122

<212> PRT

<213> Artificial sequence

<400> 3

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

1 5 10 15

Ser Val Lys Ile Ser Cys Lys Gly Ser Gly Tyr Thr Phe Thr Asp Tyr

20 25 30

Ala Met His Trp Val Lys Gln Ser His Ala Lys Ser Leu Glu Trp Ile

35 40 45

Gly Val Ile Ser Thr Tyr Ser Gly Asn Thr Asn Tyr Asn Gln Lys Phe

50 55 60

Lys Gly Lys Ala Thr Met Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr

65 70 75 80

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

85 90 95

Ala Arg Gln Leu Trp Leu Arg Arg Pro Tyr Tyr Ala Met Asp Tyr Trp

100 105 110

Gly Gln Gly Thr Ser Val Thr Val Ser Ser

115 120

<210> 4

<211> 333

<212> DNA

<213> Artificial sequence

<400> 4

gacattcagc tgacccagtc tccagcttct ttggctgtga ctctagggca gagagccacc 60

atctcctgca gagccagtga aagtgttgaa cattatggca caagttttat gcagtggtac 120

caacagaaac caggacagtc acccaaactc atcatctatg ttgcttccaa cgtagaatct 180

ggggtccctg ccaggtttag tggcagtggg tctgggacag acttcagcct caacatccat 240

cctgtggagg aggatgatat tgcaatatat ttctgtcagc aaagtaggaa ggttccttcc 300

acgttcggag gggggaccaa gctggagatc aaa 333

<210> 5

<211> 111

<212> PRT

<213> Artificial sequence

<400> 5

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

1 5 10 15

Gln Arg Ala Thr Ile Ser Cys Arg Ala Ser Glu Ser Val Glu His Tyr

20 25 30

Gly Thr Ser Phe Met Gln Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro

35 40 45

Lys Leu Ile Ile Tyr Val Ala Ser Asn Val Glu Ser Gly Val Pro Ala

50 55 60

Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Ser Leu Asn Ile His

65 70 75 80

Pro Val Glu Glu Asp Asp Ile Ala Ile Tyr Phe Cys Gln Gln Ser Arg

85 90 95

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

100 105 110

Claims

1. A monoclonal antibody 15G7 consisting of a heavy chain and a light chain;

the amino acid sequence of the variable region of the heavy chain is shown as SEQ ID NO. 3;

the amino acid sequence of the variable region of the light chain is shown as SEQ ID NO. 5;

the monoclonal antibody 15G7 binds CCN1 protein.

2. A nucleic acid molecule encoding the monoclonal antibody 15G7 of claim 1, consisting of a nucleic acid molecule encoding the heavy chain and a nucleic acid molecule encoding the light chain;

the nucleotide sequence of the variable region of the heavy chain is shown as SEQ ID NO. 2;

the nucleotide sequence of the variable region of the light chain is shown as SEQ ID NO. 4.