CN113999306A

CN113999306A - Method for obtaining epitope antibody capable of recognizing spatial conformation

Info

Publication number: CN113999306A
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: 2022-02-01
Anticipated expiration: 2041-10-26
Also published as: CN113999306B

Abstract

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

Description

Method for obtaining epitope antibody capable of recognizing spatial conformation

Technical Field

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

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 generated by these methods remain challenging. Due to the nature of the protein antigen itself, antibodies obtained using the above methods 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 into type I antibodies (e.g., merovacizumab) and type II antibodies (e.g., obinutuzumab) according to the antibody binding epitope, and type II antibodies elicit significantly less complement killing than type I antibodies but have a stronger antibody-dependent cell killing ability and an ability to directly kill target cells that type I antibodies do not have. In comparison of clinical data for non-hodgkin's lymphoma, obituzumab significantly extended the progression-free survival of patients compared to merovacizumab. As another example, the anti-CD 22 antibodies M971 and Epratuzumab, due to the difference in binding epitopes, only the latter can elicit rapid internalization of the CD22 molecule. In conclusion, in the process of antibody screening, ensuring the diversity of the constructed antibody screening library is an important prerequisite for obtaining the antibody with biological activity. The nature of the antigen itself determines the preference of the binding epitope of the antibody obtained from the above method. The antibody-binding epitope may be a short peptide composed of amino acids arranged in a continuous manner (linear epitope) or a complex spatial conformation composed of amino acids arranged in a discontinuous manner (spatial epitope). Linear epitopes on antigens to which antibodies bind, such as polypeptides, are recognized predominantly by the histocompatibility complex (MHC) of T lymphocytes, while discrete, spatially conformational epitopes on antigens are recognized predominantly 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. Spatial conformational instability of antigens leads to inconsistency and irreproducibility in candidate drug screening. In general, for convenience of purification of protein antigens, it is necessary to fuse a purification tag (e.g., his tag, immunoglobulin Fc portion) 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 disrupt the spatial structure of the antigen; (2) these tags are immunogenic in animal immunization, requiring 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 usually required for optimization, which is cumbersome and laborious.

Disclosure of Invention

The invention aims to obtain an epitope antibody capable of recognizing the spatial conformation of an antigen protein.

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

(1) constructing a recombinant plasmid; the recombinant plasmid expresses a fusion protein formed by the antigen protein and the 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 an antigenic protein or an antigenic 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 the antibody for recognizing the space conformation epitope of the antigen protein.

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

In the step (1), the antigen protein may be CCN1 protein (protein ID is 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. 1 in the sequence 1, from the 5' end, the 1 st to 6 th positions are Kozak sequence, the 7 th to 1149 th positions are DNA encoding human CCN1 protein, and the 1150 th and 1296 th positions are DNA encoding GPI.

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

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

In the above method, when the antigenic protein is the CCN1 protein, the antibody recognizing the spatial conformation epitope of the CCN1 protein may be monoclonal antibody 15G 7. The monoclonal antibody 15G7 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 to CCN1 protein.

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

In the above application, the antibody for recognizing the spatial conformation epitope of the antigenic protein can be the monoclonal antibody 15G 7.

The invention also provides a monoclonal antibody 15G7, which specifically comprises 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 to CCN1 protein.

The invention also protects a nucleic acid molecule encoding the monoclonal antibody 15G 7. 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 protein or antigen protein fragments are displayed on the surface of the eukaryotic cells, and then the eukaryotic cells are adopted to immunize animals to obtain hybridoma cells; isolating the antibody from the hybridoma cells; the antibody is the antibody for recognizing the space conformation epitope of the antigen protein. The monoclonal antibody 15G7 which is the antibody for recognizing the space conformation epitope of the CCN1 protein and is obtained by adopting the method only binds to the natural CCN1 protein and does not bind to the denatured CCN1 protein, namely the CCN1 protein which can only specifically recognize 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 spatial conformation epitope. The invention has important application value.

Drawings

FIG. 1 shows the results of the detection in step three of 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 results of the test in example 4.

Detailed Description

The present invention is described in further detail below with reference to specific embodiments, which are given for the purpose of illustration only and are not intended to limit the scope of the invention. The examples provided below serve as a guide for further modifications by a person skilled in the art and do not constitute a limitation of the invention in any way.

The experimental procedures in the following examples, unless otherwise indicated, are conventional and are carried out according to the techniques or conditions described in the literature in the field or according to the instructions of the products. Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

The CCN1 protein (protein ID NP _001545.2) is one of the products encoded by early response genes, and its expression is regulated by many pathological and physiological environments, such as cytokines, mechanical elongation, harmful toxins, etc. CCN1 has four domains, an insulin-like growth factor binding protein, a von Willebrand factor type C domain (vWC), a thrombospondin-like module, and a carboxy-terminal module. The four domains can be respectively combined with different integrin receptors on the cell surface, the diversity of the receptor combination determines the abundant biological functions of the CCN1 under physiological and pathological states, for example, the vWC domain of the CCN1 can be combined with the alphavbeta 3 integrin receptor to promote the adhesion, migration and proliferation of endothelial cells; binding to α v β 5 integrin receptors enhances phagocytosis by macrophages; binding to the α 6 β 1 integrin receptor can lead to apoptosis of fibroblasts with senescence. In addition, CCN1 also promotes cancer cell migration, aggravates lung tissue damage, and participates in physiological and pathological processes such as angiogenesis caused by hypoxia response. Therefore, monoclonal antibodies aiming at different domains of CCN1 have potential clinical application prospect.

The CCN1 protein expressed and purified by eukaryotic cells is extremely susceptible to degradation when stored in neutral phosphate buffer or repeatedly frozen and thawed. The use of heparin column can capture CCN1 protein, but heparin will also capture many impurity proteins, so this method can not specifically enrich CCN1 protein. The instability of the native structure of CCN1 in vitro has created great difficulty in screening for antibodies recognizing the conformational epitope CCN1 in space.

Example 1 obtaining of an anti-CCN 1 antibody recognizing the spatial conformational epitope CCN1 antibody

First, construction of CCN1-sp2/0 cell

1. The 3' end of the DNA sequence coding the CCN1 protein and the DNA sequence coding Glycophosphatidylinositol (GPI) are fused by molecular biology technology and then constructed into an expression vector containing a dihydrofolate reductase (DHFR) resistance gene, so as to obtain the recombinant plasmid. The method comprises the following specific steps:

(1) the DNA encoding human CCN1 protein (NM-001554) and the DNA encoding GPI were fused, followed by addition of Kozak sequence GCCACC at the 5' end to give the fusion sequence shown in SEQ ID NO: 1.

1 in the sequence 1, from the 5' end, the 1 st to 6 th positions are Kozak sequence, the 7 th to 1149 th positions are DNA encoding human CCN1 protein, and the 1150 th and 1296 th positions are DNA encoding GPI.

(2) According to pOptiVEC^TM-

The procedure of TACOCKING Kit (Thermo, Cat. 12744-One 017) comprises the steps of ligating the fusion sequence obtained in step (1) with pOptiVEC-TOPO vector by TA cloning method, and transforming One

Coli Competent cells (Thermo, cat # C404003), Plasmid was enriched and extracted using QIAGEN Plasmid Maxi Kit (QIAGEN, cat # 12163) to obtain recombinant Plasmid.

The pOptiVEC-TOPO vector is pOptiVEC^TM-

The elements of the TACOCKING Kit.

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

(1) mu.g of the recombinant plasmid obtained in step 1 was digested with PvUI enzyme (New England Biolabs, cat # R0150) at 37 ℃ for 12 hours to obtain a digested solution. The digestion solution contains linearized recombinant plasmids.

(2) And (3) after the step (1) is finished, adding isovolume saturated phenol into the digestive juice, mildly and fully mixing for 3min, then centrifuging for 10min at 5000g, and collecting the upper-layer water phase into a new centrifuge tube.

(3) And (3) after the step (2) is finished, adding equal volume of saturated phenol into the centrifuge tube, uniformly mixing, then centrifuging for 10min at 5000g, and collecting the upper-layer water phase into a new centrifuge tube.

(4) And (3) after the step (3) is finished, adding equal volume of phenol/chloroform (phenol and chloroform are mixed in equal volume), uniformly mixing, centrifuging for 10min at 5000g, and collecting the upper-layer water phase into a new centrifugal tube.

(5) And (4) after the step (4) is finished, adding equal volume of chloroform into the centrifuge tube, uniformly mixing, then centrifuging for 10min at 5000g, and collecting the upper aqueous phase into a new centrifuge tube.

(6) And (5) after the step (5) is finished, adding 1/10 volumes of pH5.2, 3M sodium acetate and 2.5 volumes of absolute ethyl alcohol into a centrifuge tube, slightly inverting and uniformly mixing, after floccules appear, centrifuging for 5min at 5000g, and collecting precipitates.

(7) Washing the precipitate collected in step (6) with 75% (v/v) ethanol aqueous solution, centrifuging for 3min at 5000g, and collecting the precipitate. The ethanol in the precipitate was fully volatilized to obtain a linearized recombinant plasmid.

(8) Take 0.5X 10⁶Sp2/0-Ag14 cells were resuspended in 250. mu.L of Hybridoma-SFM medium (Thermo, cat. 12045076) to obtain a cell suspension.

(9) And (4) 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 electric rotor for electric transfer (set voltage 180 volts, capacitance 960. mu.F, time constant 24msec), and after completion of electric transfer, the mixture was carefully transferred to a sterile EP tube and left on ice for 10 min.

(10) After the step (9) is finished, transferring the mixed solution to a Hybridoma-SFM culture medium, and culturing for 48h at 37 ℃; transferring to a Hybridoma-SFM medium containing 0.05. mu. mol of methotrexate, and culturing at 37 deg.C for 3-5 days; during the culture period, the cells were passaged every 3 to 5 days.

(11) Inoculating the cells obtained in step (10) to a 96-well plate and the average number of cells per well is not more than 1, and then adding a Hybridoma-SFM medium containing 0.1. mu. mol of methotrexate per well, and culturing at 37 ℃ for 21 days; the single clones in the selected well plate were transferred to a culture flask, and the surface display of CCN1 was confirmed by the method in step 3 by enlarging the culture with Hybridoma-SFM medium containing 0.1. mu. mol of methotrexate.

(12) Replacing the "cells obtained in step (10)" in step (11) with the cells obtained in step (11), increasing the concentration of methotrexate, and keeping the other steps unchanged, to obtain CCN1-sp2/0 cells (i.e., gradually increasing the screening concentration of methotrexate in the culture medium to increase the CCN1 expression level of the screened cells).

3. The A10 monoclonal antibody (commercial CCN1 monoclonal antibody, product of Santa Cruz Biotechnology, Inc., catalog No. sc-374129) was used to confirm that CCN1 was displayed on the sp2/0-Ag14 cell surface. The method comprises the following specific steps:

(1) taking a mixture containing 1 × 10⁶Centrifuging cell suspension of CCN1-sp2/0 cells at 300g, and collecting cell precipitates; the pellet was then washed 1 time with phosphate buffer (pH 7.4).

(2) Adding 0.5ml of A10 monoclonal antibody diluent into the cell sediment obtained in the step (1), incubating for 2h at room temperature, removing the supernatant, and washing the sediment 3 times by using phosphate buffer (pH7.4).

The reaction solution was purified using phosphate buffer (pH7.4) 1: diluting A10 monoclonal antibody by 1000 to obtain A10 monoclonal antibody diluent.

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

The reaction solution was purified using phosphate buffer (pH7.4) 1: the mouse secondary antibody was diluted with 5000 (Jackson ImmunoResearch Laboratories, Inc. Cat. 115-035-003) to obtain a mouse secondary antibody dilution.

(4) After completion of step (3), TMB reaction substrate (3,3 ', 5, 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 absorbance at 450nm was read.

CCN1-sp2/0 cells were replaced with sp2/0-Ag14 cells according to the above procedure, and other procedures were not changed, as a negative control.

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

Secondly, preparing hybridoma cells

1. 1mL of phosphate buffer was added to 3X 10⁷And (3) fully and uniformly mixing 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 emulsified with shaking 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 6-week-old Balb/C mice were taken, each subjected to the following procedures: on day 1 of the experiment, 200. mu.L of solution 1 (cell number injected 3X 10) was intraperitoneally injected⁶Ones) (first immunization); on the 14 th day of the experiment, 200. mu.L of solution 2 (the number of cells injected was 3X 10)⁶Two) (second immunization); on the 28 th day of the experiment, 200. mu.L of solution 2 (the number of cells injected was 3X 10)⁶Ones) (third immunization); on day 42 of the experiment, 100. mu.L of solution 3 (number of injected cells 1.5X 10) was injected into the tail vein⁶One).

4. 3 days after completion of step 3, the mice were sacrificed and their spleens were removed to prepare hybridoma cell clones by hybridoma preparation techniques. The method comprises the following specific steps:

(1) sera were collected from 5 mice 4 days before 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 the serum is detected, a mouse with the highest monoclonal antibody titer in the serum is selected to be killed at the 45 th day, and the spleen is taken.

(2) After completion of step (1), mouse spleen cells were isolated into single cells and fused with sp2/0-Ag14 cells, and the obtained single clones were cultured in a multi-well culture plate, and the culture supernatant was collected and assayed for the content of anti-CCN 1 antibody.

(3) According to the results of the measurement, monoclonal antibodies were cultured in the amount secreted by the antibodies using Hybridoma-SFM medium according to the method of 2(11) of the first step. Repeating the step for 2-3 times, measuring the content of the anti-CCN 1 antibody each time, and selecting the monoclone with high secretion until finally obtaining the target clone with stable genome.

Thirdly, obtaining anti-CCN 1 antibody from hybridoma cell

A. The activity of anti-CCN 1 monoclonal antibody in hybridoma cell culture supernatant was determined by enzyme-linked immunosorbent assay. The method comprises the following specific steps:

1. 50 μ L of CCN1 protein solution was added to each well of a 96-well plate, incubated at room temperature for 6h and the solution discarded.

Fresh CCN1 protein was prepared by heparin column and made up into 10 μ g/mL CCN1 protein solution using phosphate buffer.

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

3. Adding culture supernatant of hybridoma cells, incubating at room temperature for 2h, removing the culture supernatant, and washing twice.

4. An anti-mouse FC fragment, horseradish peroxidase (product of Jackson ImmunoResearch Laboratories, Inc., catalog number 115-.

5. Adding TMB reaction substrate, reacting at room temperature for 10min, and reading the absorbance at 450 nm.

According to the above-mentioned method, the culture supernatant of hybridoma cells was replaced with SM03 (anti-human CD22 monoclonal antibody, concentration 0.4. mu.g/mL) (product of China antibody pharmaceuticals, Inc., product lot number Sm03-202-201701-b003-04-09), and the other steps were not changed, and used as a negative control.

The culture supernatant of hybridoma cells was replaced with A10 monoclonal antibody according to the above method, and all other steps were not changed, and used as a positive control.

The results of the experiment are shown in FIG. 1. The results showed that the feedback signal was strongest for hybridoma clone 15G 7. Thus, hybridoma clone 15G7 was selected for further analysis and characterization.

B. Obtaining anti-CCN 1 antibody

1. mRNA from hybridoma clone 15G7 was extracted with Trizol (Thermo, cat # 15596026) followed by SuperScript^TMIV Reverse transcription (Thermo, cat # 18090200) to obtain cDNA.

2. Using cDNA as 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' to obtain the variable region fragment of 15G7 heavy chain. Using cDNA as template, light chain primer 1: 5'-GACATTCAGCTGACCCAGTCTCCA-3' and light chain primer 2: 5'-GTTAGATCTCCAGCTTGGTCCC-3' was amplified by PCR to obtain a variable region fragment of 15G7 light chain.

The reaction procedure is as follows: 2min at 95 ℃; 35 cycles of 95 ℃ for 15s, 56 ℃ for 30s, and 68 ℃ for 1 min; 3min at 68 ℃; storing 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 module in the TA Cloning Kit (Thermo, cat 12744-.

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

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

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

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

4. The variable region fragment of the 15G7 heavy chain was fused to the constant region (IgG1 constant region) sequence of the human mAb to obtain the 15G7 chimeric mAb heavy chain. The variable region fragment of 15G7 light chain was fused to the constant region sequence of human mab (IgG1 constant region) to obtain the light chain of the 15G7 chimeric mab.

5. The DNA fragment between restriction enzymes XhoI and NotI in pcDNA3.1(+) vector (Thermo, cat. No. V87020) was replaced with the heavy chain of the 15G7 chimeric monoclonal antibody, the DNA fragment between restriction enzymes XbaI and NheI was replaced with the light chain of the 15G7 chimeric monoclonal antibody, and the other sequences were not changed, thus obtaining recombinant plasmid pcDNA3.1(+) -15G 7.

6. The recombinant plasmid pcDNA3.1(+) -15G7 is transfected into ExpicHO cell (Thermo, the cargo number is A29133), and the 15G7 monoclonal antibody, namely the anti-CCN 1 antibody is obtained by protein A affinity adsorption purification.

Example 2, 15G7 monoclonal antibody (i.e., anti-CCN 1 antibody obtained in example 1) binds to native, but not denatured, CCN1 protein

Protein imprinting was used to test the binding capacity of 15G7 monoclonal antibody to CCN1 protein. The method comprises the following specific steps:

1. taking a mixture containing 3X 10⁵Centrifuging cell suspension of CCN1-sp2/0 cells at 300g for 3min, and collecting cell precipitate; then washed with 3mL PBS buffer.

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

RIPA lysis buffer: the solvent and the concentration thereof are 150mM NaCl (sigma), 5mM EDTA (sigma), 1% Triton X-100(sigma), 1% sodium deoxyholate (sigma) and 0.1% SDS (sigma), and the solvent is pH7.4, 50mM Tris-HCl buffer solution (sigma); when in use, Halt is required to be added^TMProtease (Halt)^TMProtease diluted 100-fold to working concentration) and phosphatase inhibitors (ThermoFisher, cat 78446) (Halt)^TMPhosphatase inhibitor was diluted 100-fold to working concentration).

3. After completion of step 2, the lysate is combined with

LDS sample buffer solution (Thermo, cat # NP0008) was mixed in equal volume to obtain mixed solution A. The mixed solution A contains CCN1 protein (natural CCN1 protein for short) in natural conformation.

4. After completion of step 2, the lysate was mixed with 5% beta-mercaptoethanol (Sigma, cat # M6250)

Mixing LDS sample buffer solution in equal volume to obtain mixed solution B; and (3) treating the mixed solution B at 95 ℃ for 10min (the aim is to fully denature CCN1 protein) to obtain a mixed solution A. The mixed solution C contains structural denatured CCN1 protein (denatured CCN1 protein for short).

5. Loading the mixed solution A obtained in the step (3) or the mixed solution C obtained in the step (4) to 12.5 percent polyacrylamide gel, and performing electrophoresis for 2 hours under the voltage of 100 volts; then transferred to a PVDF membrane by an electrotransfer instrument (constant current 400mA, 1.5 h); then blocking the PVDF membrane by PBS buffer solution (blocking solution) containing 5% bovine serum albumin, and standing overnight at 4 ℃; 15G7 mab at a concentration of 1mg/mL was administered as 1: diluting the mixed solution with a blocking solution at a volume ratio of 1000, and then incubating the PVDF membrane under a room temperature condition; PVDF membrane was washed 3 times for 5min with TBST buffer (Biorad, cat # BUF 028); adding 1: a 5000 blocking solution diluted anti-human FC fragment horseradish peroxidase secondary antibody (Jacksonimmunorsearch, Cat. No. 109-; PVDF membrane was washed 1 time 5min with TBST buffer (Biorad, cat # BUF 028).

6. After completing step 5, adding Pierce^TMECLWestern Blotting substrate (ThermoFisher, cat # 32209) was incubated at room temperature for 1min and excess substrate solution was blotted with absorbent paper.

7. After step 6 is completed, a chemiluminescent image is collected using darkroom development techniques. The results were analyzed using ImageLab software (Biorad).

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

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

Example 3, 15G7 monoclonal antibody bound to CCN1-Sp2/0 cells and not to Sp2/0-Ag14 cells

1. Taking a mixture containing 3X 10⁵A 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 pellet was washed 2 times with a washing solution (phosphate buffer containing 1% bovine serum albumin). The method of each washing is as follows: the pellet was resuspended in 500. mu.L of washing solution, mixed by inversion, centrifuged and the pellet collected.

3. After the step 2 is finished, fixing the precipitate for 5min by using 4% paraformaldehyde, and collecting the precipitate; the precipitate was washed 2 times with washing solution. The method of each washing is as follows: the pellet was resuspended in 500. mu.L of washing solution, mixed by inversion, centrifuged and the pellet collected.

4. After step 3, resuspending the precipitate with blocking solution (phosphate buffer containing 3% bovine serum albumin), standing at room temperature for 20min, centrifuging, and collecting the precipitate; the pellet was resuspended in 500. mu.L of washing solution, mixed by inversion, centrifuged and the pellet collected.

5. After the step 4 is finished, resuspending the precipitate with 500 μ L of 15G7 monoclonal antibody diluent, standing at room temperature for 30min, centrifuging, and collecting the precipitate; the precipitate was washed 2 times with washing solution. The method of each washing is as follows: the pellet was resuspended in 500. mu.L of washing solution, mixed by inversion, centrifuged and the pellet collected.

15G7 mab diluent: diluting 15G7 monoclonal antibody with washing solution; the concentration of the 15G7 monoclonal antibody diluent is 1 mu G/ml or 0.5 mu G/ml.

6. After completion of step 5, 1: a secondary antibody (Jackson ImmunoResearch, cat. No. 609-605-213) diluted by 2000 times of the washing solution, incubated for 30min at room temperature, centrifuged, and the precipitate was collected; the precipitate was washed 2 times with washing solution. The method of each washing is as follows: the pellet was resuspended in 500. mu.L of washing solution, mixed by inversion, centrifuged and the pellet collected.

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

The 15G7 mab was replaced with SM03 as described above, and all other steps were unchanged as a negative control.

The CCN1-Sp2/0 cells were replaced with Sp2/0-Ag14 cells as described above, all other steps remaining unchanged.

The results are shown in FIG. 3(Sp2/0-CCN1-GP1 is CCN1-Sp2/0 cells, Sp2/0 is Sp2/0-Ag14 cells). The results show that 15G7 monoclonal antibody can be combined with CCN1-Sp2/0 cells, but can not be combined with Sp2/0-Ag14 cells.

The above results indicate that the 15G7 monoclonal antibody is specific for the binding of CCN1 protein and does not react with other proteins on the cell surface of Sp2/0-Ag 14.

Example 4 binding of 15G7 monoclonal antibody to CCN1-sp2/0 cell surface native CCN1 protein

1. Taking a mixture containing 3X 10⁵Centrifuging cell suspension of CCN1-sp2/0 cells at 300g for 5min, and collecting precipitate; the pellet was then resuspended in 500. mu.L of phosphate buffer to obtain a cell suspension.

2. And (3) dropwise adding 10 mu L of the cell suspension obtained in the step (1) onto a sterile and clean glass slide, heating the glass slide for 5min at 95 ℃ by using a metal bath, and evaporating the liquid to obtain the denatured CCN1-sp2/0 cells.

3. And (3) dropwise adding 10 mu L of the cell suspension obtained in the step 1 onto a sterile and clean glass slide, and air-drying the liquid in a super clean bench to obtain undenatured CCN1-sp2/0 cells.

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

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

PBST buffer: phosphate buffer containing 0.1% tween 20.

6. After step 5, the solution was discarded, and a 1. mu.g/mL solution of 15G7 monoclonal antibody (obtained by diluting 15G7 monoclonal antibody with 1% bovine serum albumin in PBST buffer) was added and incubated at room temperature for 2 hours; discard the solution and wash the slide 3 times with phosphate buffer.

7. After the step 6 is finished, discarding the solution, adding a second antibody solution, and incubating for 1h at room temperature; discard the solution and wash the slide 3 times with phosphate buffer.

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

8. After completion of step 7, staining was performed using DAPI (thermolfisher, cat # D1304), and after 15min at room temperature, the solution was discarded, and the slide was washed 3 times with phosphate buffer.

9. The observation was performed using a fluorescence microscope and photographed.

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

The results show that the 15G7 monoclonal antibody can only specifically recognize CCN1 protein in the natural conformation.

The present invention has been described in detail above. It will be apparent to those skilled in the art that the 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 reference to specific embodiments, it will be appreciated that the invention can 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 use of some of the essential features is possible within the scope of the claims attached below.

<110> apricot Union pharmaceutical industry (Suzhou) Co., Ltd

<120> a method for obtaining an antibody recognizing a spatial conformation 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

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<212> DNA

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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

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<213> Artificial sequence

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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

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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

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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 method for obtaining an antibody capable of recognizing the epitope in the spatial conformation of an antigenic protein, which comprises the following steps:

2. The method of claim 1, wherein: in the step (1), the surface anchoring protein is glycophosphatidylinositol.

3. The method of claim 1, wherein: in the step (2), the eukaryotic cells are Sp2/0-Ag14 cells.

4. The method of claim 1, wherein: in the step (1), the antigen protein is CCN1 protein.

5. The method of claim 1, wherein: in the step (1), the recombinant plasmid is formed by connecting a fusion sequence shown in SEQ ID NO. 1 and a pOptiVEC-TOPO vector by using a TA cloning method.

6. The method of claim 4, wherein: the antibody for recognizing the CCN1 protein space conformation epitope is a monoclonal antibody 15G 7;

the monoclonal antibody 15G7 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 to CCN1 protein.

7. The method of claim 1, wherein: in the step (2), the screening is methotrexate screening.

8. Use of an antibody recognizing a spatial conformational epitope of an antigenic protein, obtained by the method of any one of claims 1 to 7, for screening drugs.

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

the monoclonal antibody 15G7 binds to CCN1 protein.

10. A nucleic acid molecule encoding the monoclonal antibody 15G7 of claim 9, consisting 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.