Monoclonal antibody 7H7 capable of recognizing and neutralizing CV-A10 and its application

Abstract

The present invention provides a monoclonal antibody 7H7 capable of recognizing and neutralizing CV-A10 and its application, wherein the heavy chain variable region of the monoclonal antibody 7H7 includes complementary determining regions VHCDR1, VHCDR2, and VHCDR3, the amino acid sequence of VHCDR1 is shown in SEQ ID NO: 1, the amino acid sequence of VHCDR2 is shown in SEQ ID NO: 2, and the amino acid sequence of VHCDR3 is shown in SEQ ID NO: 3; the light chain variable region of the monoclonal antibody 7H7 includes complementary determining regions VLCDR1, VLCDR2, and VLCDR3, the amino acid sequence of VLCDR1 is shown in SEQ ID NO: 4, the amino acid sequence of VLCDR2 is shown in SEQ ID NO: 5, and the amino acid sequence of VLCDR3 is shown in SEQ ID NO: 6. The present invention can specifically recognize CV-A10 and does not recognize other enteroviruses.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and particularly relates to a monoclonal antibody 7H7 capable of recognizing and neutralizing CV-A10 and its application. Background Art

[0002] The viruses that cause hand, foot, and mouth disease (HFMD) belong to the Picornavirus family, Enterovirus genus. These include coxsackievirus A group 2, 4, 5, 6, 7, 9, 10, and 16, coxsackievirus B group 1, 2, 3, 4, 5, 6, and 13, enterovirus A group 71 (EV-A71), and echoviruses. Early studies identified EV-A71 and CV-A16 as the primary pathogens of HFMD. However, the spectrum of enterovirus pathogens of HFMD has shifted in recent years. Epidemiological data from recent years show that the incidence of HFMD caused by CV-A6 and CV-A10 has been increasing annually. Since 2012, CV-A10 has become one of the primary pathogens causing HFMD outbreaks in several cities and regions in my country, including Beijing, Guangdong, and Fujian. Therefore, CV-A10 has become another enteroviral pathogen that causes HFMD and deserves attention after EV-A71 and CV-A16.

[0003] Conformational neutralizing antibody testing can better reflect the content and function of the virus. Therefore, the development of neutralizing monoclonal antibodies that target conformational epitopes and specifically recognize CV-A10 is of great significance for quality control of vaccine development, testing of clinical samples, laboratory virus identification, and research on the basic functions of CV-A10. Summary of the Invention

[0004] In view of this, the present invention provides a monoclonal antibody 7H7 capable of recognizing and neutralizing CV-A10 and its application, which can specifically recognize CV-A10 and does not recognize other enteroviruses.

[0005] In order to achieve the above object, the present invention adopts the following technical solutions:

[0006] In a first aspect, the present invention provides a monoclonal antibody 7H7 capable of recognizing and neutralizing CV-A10, wherein the heavy chain variable region of the monoclonal antibody 7H7 capable of recognizing and neutralizing CV-A10 comprises a complementarity determining region VHCDR1, a complementarity determining region VHCDR2, and a complementarity determining region VHCDR3, wherein the amino acid sequence of the complementarity determining region VHCDR1 is shown in SEQ ID NO: 1, the amino acid sequence of the complementarity determining region VHCDR2 is shown in SEQ ID NO: 2, and the amino acid sequence of the complementarity determining region VHCDR3 is shown in SEQ ID NO: 3;

[0007] The light chain variable region of the monoclonal antibody 7H7 capable of recognizing and neutralizing CV-A10 includes a complementarity determining region VLCDR1, a complementarity determining region VLCDR2, and a complementarity determining region VLCDR3. The amino acid sequence of the complementarity determining region VLCDR1 is shown in SEQ ID NO: 4, the amino acid sequence of the complementarity determining region VLCDR2 is shown in SEQ ID NO: 5, and the amino acid sequence of the complementarity determining region VLCDR3 is shown in SEQ ID NO: 6.

[0008] Preferably, the amino acid sequence of the heavy chain variable region of the monoclonal antibody 7H7 capable of recognizing and neutralizing CV-A10 is as shown in SEQ ID NO.7; and / or, the amino acid sequence of the light chain variable region of the monoclonal antibody 7H7 capable of recognizing and neutralizing CV-A10 is as shown in SEQ ID NO.9.

[0009] Preferably, the monoclonal antibody is an IgM subtype antibody.

[0010] Preferably, the monoclonal antibody 7H7 capable of recognizing and neutralizing CV-A10 targets a conformational epitope.

[0011] In a second aspect, the present invention provides a polynucleotide molecule encoding the monoclonal antibody 7H7 capable of recognizing and neutralizing CV-A10.

[0012] Preferably, the nucleic acid molecule encoding the heavy chain is any one of the following:

[0013] a, having the nucleotide sequence shown in SEQ ID NO.8;

[0014] b. a nucleotide sequence complementary to the nucleotide sequence shown in SEQ ID NO.8;

[0015] c. a nucleotide sequence that encodes the same protein as the nucleotide sequence in a and b but differs from them due to the degeneracy of the genetic code; and / or

[0016] The nucleic acid molecule encoding the light chain is any one of the following:

[0017] e. having the nucleotide sequence shown in SEQ ID NO.10;

[0018] f. a nucleotide sequence complementary to the nucleotide sequence shown in SEQ ID NO.10;

[0019] g. A nucleotide sequence that encodes the same protein as the nucleotide sequence in e and f, but differs from them due to the degeneracy of the genetic code.

[0020] In a third aspect, the present invention provides an expression vector comprising a nucleic acid molecule encoding the monoclonal antibody 7H7.

[0021] In a fourth aspect, the present invention provides a host cell containing the polynucleotide molecule or the expression vector.

[0022] In a fifth aspect, the present invention provides a use of the monoclonal antibody 7H7, or the monoclonal antibody 7H7 encoded by the polynucleotide molecule, in the preparation of a reagent or kit for detecting CV-A10.

[0023] In a sixth aspect, the present invention provides a use of the monoclonal antibody 7H7, or the monoclonal antibody 7H7 encoded by the polynucleotide molecule, in the preparation of drugs for inhibiting, preventing and treating CV-A10.

[0024] Compared with the prior art, the present invention has the following beneficial effects:

[0025] (1) The monoclonal antibody provided by the present invention is prepared by immunizing mice with purified Coxsackievirus group A type 10 (CV-A10) virus and screening hybridoma cells. It is a neutralizing antibody of the IgM subtype that can specifically recognize CV-A10 and does not recognize other enteroviruses.

[0026] (2) The monoclonal antibody of the present invention can be combined with a conjugate (horseradish peroxidase or fluorescein isothiocyanate, etc.) for direct or indirect detection and rapid diagnosis. For example, it can be used to develop detection reagents or kits for detecting clinical samples caused by CV-A10 infection. It can also be used for laboratory identification of antibodies of clinical virus isolates. It can also be used to prepare preventive vaccine production intermediates and products containing CV-A10 antigen quantitative detection reagents. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 This is an SDS-PAGE image of the IgM heavy chain and light chain of the monoclonal antibody 7H7 capable of recognizing and neutralizing CV-A10 provided in Example 3 of the present invention;

[0028] Figure 2The monoclonal antibody 7H7 provided in Example 4 of the present invention, which is capable of recognizing and neutralizing CV-A10, cannot recognize the corresponding WB image of reduced CV-A10;

[0029] Figure 3 Fluorescent images of experimental wells for different strains of the in vitro microneutralization experiment of the monoclonal antibody 7H7 capable of recognizing and neutralizing CV-A10 provided in Example 5 of the present invention;

[0030] Figure 4 This is an ELISA statistical chart of the monoclonal antibody 7H7 capable of recognizing and neutralizing CV-A10 provided in Example 6 of the present invention recognizing CV-A10 hollow particles and solid particles. DETAILED DESCRIPTION

[0031] The present invention will be further described in detail below with reference to specific embodiments so that those skilled in the art can understand the present invention more clearly.

[0032] The following embodiments are only used to illustrate the present invention, but are not used to limit the scope of the present invention. Based on the specific embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention.

[0033] In the examples of the present invention, unless otherwise specified, all raw material components are commercially available products well known to those skilled in the art; in the examples of the present invention, unless otherwise specified, the technical means used are conventional means well known to those skilled in the art.

[0034] The "monoclonal antibody" mentioned in the present invention refers to a preparation of an antibody molecule having a single molecular composition.

[0035] The "CDR region" or "CDR" mentioned in the present invention refers to the hypervariable regions of the heavy and light chains of immunoglobulins, which contain most of the amino acid residues responsible for binding through the affinity of the antibody to the antigen or its recognized epitope.

[0036] Reagent Source:

[0037] MA135 adjuvant: provided by Wuhan Institute of Biological Products Co., Ltd.;

[0038] Female BaLb / c mice were provided by Wuhan Institute of Biological Products Co., Ltd.

[0039] Mouse monoclonal antibody enzyme-labeled secondary antibody ready-to-use kit for Ig subclass identification (including light chain) (G1\G2a\G2b\G3\M\A\Kappa\Lambda): purchased from Suzhou Botelon Immunotechnology Co., Ltd.

[0040] RD cells: provided by Wuhan Institute of Biological Products Co., Ltd.;

[0041] HRP Conjugated AffiniPure Goat Anti-mouse IgM μ Chain: purchased from Wuhan Boster Bioengineering Co., Ltd.

[0042] Example 1 Preparation of monoclonal antibodies

[0043] This example provides a method for preparing a monoclonal antibody, which is as follows:

[0044] (1) Immunization: BaLb / c mice were immunized with purified CV-A10 stock solution (stock solutions were obtained from Wuhan Institute of Biological Products Co., Ltd.) using four intraperitoneal immunizations, 14 days apart. The fourth immunization was mixed with MA135 adjuvant and CV-A10 stock solution. Blood was collected 14 days after the last immunization to detect serum antibody titers. Three days before fusion, the spleen was pulsed once, followed by fusion of spleen lymphocytes and myeloma cells.

[0045] (2) Screening: The supernatant of the fused cells was screened using ELISA and CV-A10-iLOV (recombinant CV-A10 with the reporter gene iLOV) fluorescence neutralization rapid screening, specifically:

[0046] First, ELISA was used to screen the fused cell wells containing monoclonal antibodies that could bind to CV-A10 in the supernatant. β-Propiolactone was mixed with CV-A10 stock solution at a ratio of 1:4000, stored at 4°C for 24 hours, and then transferred to 37°C for 2 hours to complete inactivation. The inactivated CV-A10 stock solution was used to coat a 96-well microplate at a coating concentration of 1 μg / mL, 100 μL / well, and coated at 4°C overnight. After washing the plate three times with PBST, a blocking solution (containing 1% After washing the plate three times with PBST, the sample to be tested was added at 100 μL / well and incubated at 37°C for 1 hour. After washing the plate three times with PBST, HRP-labeled goat anti-mouse IgG or HRP-labeled goat anti-mouse IgM (both diluted 1:10,000) was added at 100 μL / well and incubated at 37°C for 1 hour. After washing the plate three times with PBST, substrate solution A (acetic acid-sodium acetate buffer containing 0.6 g / L urea peroxide and 0.01 mol / L citric acid) and substrate solution B (citric acid buffer containing 0.8 g / L TMB (tetramethylbenzidine), 3% DMSO and 20% glycerol) were added at 50 μL / well each and developed at 37°C in the dark for 30 minutes. Stop solution was added at 50 μL / well and the plate was placed on a microplate reader and read at a wavelength of 450 nm. 450nm value.

[0047] The fusion cell wells that were positive for ELISA continued to be screened for CV-A10-iLOV fluorescence neutralization. The supernatant of the above fusion cell wells was diluted with MEM maintenance solution at a ratio of 1:8 and added to the first column of the 96-well plate, 100 μL / well, and 2 replicates were set up. 50 μL of dilution was added to each well of columns 2-12. The supernatant in column 1 was diluted 2-fold continuously to columns 12. After the last column was diluted and mixed, 50 μL was discarded. Dilute CV-A10-iLOV to 100 CCID 50 / 50μL, draw 50μL vertically and add it to the 96-well plate with diluted antibody as a neutralization plate, and place it in a 37℃ incubator for 2h. The diluted CV-A10-iLOV was diluted 10-fold to 10 CCID 50 / 50μL, 1 CCID 50 / 50μL, 0.1 CCID 50 / 50μL. Add 50μL of maintenance solution and 50μL of virus solution to each well (a total of 4 dilutions, namely 100, 10, 1, and 0.1 CCID 50 / 50 μL), 8 replicate wells for each dilution, as a back titration plate, placed at 4℃. After neutralization, the prepared RD cell suspension was diluted to 1×10 5 Plate the cells evenly at a density of 1000 cells / mL in a neutralization plate and a back titration plate. Incubate in a 5% CO2 incubator at 37°C for 18-4 hours, then observe under a fluorescence microscope to determine neutralization results. If a well shows a concentrated cluster of fluorescent spots, it indicates that the RD cells in that well have undergone pathological changes. If a well shows no fluorescence, it indicates that the RD cells in that well are effectively protected by the neutralizing monoclonal antibody.

[0048] (3) Subcloning: The mother clones with positive screening results were subcloned by limiting dilution method, and the subclones were screened by ELISA method. After further expansion of culture, ascites was prepared.

[0049] (4) Antibody production and purification: Female BALB / c mice aged 6-8 weeks were selected and intraperitoneally injected with 0.5 mL of liquid paraffin per mouse. 7-10 days after the injection of liquid paraffin, hybridoma cells were intraperitoneally injected. When the abdomen of the mouse was distended and the mouse was on the verge of death, it was euthanized by cervical dislocation. Ascites was aseptically extracted in a clean bench. Monoclonal antibodies in the ascites were purified by Protein L affinity chromatography.

[0050] Example 2 Sequence Analysis of Screened Monoclonal Antibodies

[0051] The hybridoma cells screened in Example 1 (capable of secreting the monoclonal antibody 7H7) were inoculated in RPMI 1640 medium (Gibco) supplemented with 20% fetal bovine serum and cultured at 37°C. Hybridoma cell RNA was extracted using the Vazyme FastPure Cell / Tissue Total RNA Isolation Kit, and then reverse transcribed using the Oligo dT primer in the TaKaRa RimeScript™ II 1st Strand cDNA Synthesis Kit to obtain cDNA. Universal primers for the heavy and light chain variable region genes of the mouse monoclonal antibody were synthesized, and the heavy and light chain variable region genes of 7H7 were amplified by nested PCR and sequenced. The sequenced sequences were analyzed in the abYsis database to obtain the correct amino acid sequences of the light and heavy chain variable regions.

[0052] The forward primer 5′MsVHE in the first round of heavy chain nested PCR was GGGAATTCGAGGTGCAGCTGCAGGAGTCTGG, and the reverse primers 3′Cμ outer, 3′Cγ1 outer, 3′Cγ2couter, 3′Cγ2b outer, 3′Cγ3 outer, and 3′Cα outer were GAGGGGGCTCTCGCAGGAGACGAGG, GGAAGGTGTGCACACCGCTGGAC, GGAAGGTGTGCACACCACTGGAC, GGAAGGTGTGCACACTGCTGGAC, AGACTGTGCGCACACCGCTGGAC, and AAAGTTCACGGTGGTTATATCC, respectively. The forward primer 5′MsVHE in the second round of heavy chain nested PCR was the same as that in the first round, and the reverse primers 3′Cμ inner, 3′Cγ1 inner, 3′Cγ2c inner, 3′Cγ2b inner, 3′Cγ3 inner, and 3′Cα outer were GAGGGGGCTCTCGCAGGAGACGAGG, GGAAGGTGTGCACACCGCTGGAC, GGAAGGTGTGCACACCACTGGAC, GGAAGGTGTGCACACTGCTGGAC, AGACTGTGCGCACACCGCTGGAC, and AAAGTTCACGGTGGTTATATCC. The inner sequences were AGGGGGAAGACATTTGGGAAGGAC, GCTCAGGGAAATAGCCCTTGAC, GCTCAGGGAAATAACCCTTGAC, ACTCAGGGAAGTAGCCCTTGAC, GCTCAGGGAAGTAGCCTTTGAC, and TGCCGAAAGGGAAGTAATCGTGAAT; the first round forward primer set of light chain nested PCR was 5′L-Vκ_3, 5′L-Vκ_4, 5′L-Vκ_5, 5′L-Vκ_6, 5′L-Vκ_6,8,9, 5′L-Vκ_14, 5′L The sequences of -Vκ_19 and 5′L-Vκ_20 were TGCTGCTGCTCTGGGTTCCAG, ATTWTCAGCTTCCTGCTAATC, TTTTGCTTTTCTGGATTYCAG, TCGTGTTKCTSTGGTTGTCTG, ATGGAATCACAGRCYCWGGT, TCTTGTTGCTCTGGTTYCCAG, CAGTTCCTGGGGCTCTTGTTG, and CTCACTAGCTCTTCTCCTC, and the sequence of the reverse primer 3′mCκ was GATGGTGGGAAGATGGATACAGTT;The sequence of the second round forward primer 5′mVkappa in the light chain nested PCR was GAYATTGTGMTSACMCARWCTMCA, and the sequences of the reverse primer set 3′BsiWI P-mJK01, 3′BsiWI P-mJK02, 3′BsiWI P-mJK03, and 3′BsiWI P-mJK04 were GCCACCGTACGTTTGATTTCCAGCTTGGTG, GCCACCGTACGTTTTATTTCCAGCTTGGTC, GCCACCGTACGTTTTATTTCCAACTTTGTC, and GCCACCGTACGTTTCAGCTCCAGCTTGGTC.

[0053] The sequences of the six CDR regions (complementarity determining regions) analyzed are as follows:

[0054] amino acid sequence of the heavy chain complementarity determining region VHCDR1: TYIMS (SEQ ID NO. 1);

[0055] The amino acid sequence of the heavy chain complementarity determining region VHCDR2: TISSGGVKTYYPDSVKG (SEQ ID NO. 2);

[0056] The amino acid sequence of the heavy chain complementarity determining region VHCDR3 is: SYGNYYWYFDV (SEQ ID NO. 3);

[0057] The amino acid sequence of the complementarity determining region of the light chain, VLCDR1: KASQDINSYLS (SEQ ID NO. 4);

[0058] The amino acid sequence of the complementarity determining region VLCDR2 of the light chain: RANRLVD (SEQ ID NO. 5);

[0059] The amino acid sequence of the complementarity determining region VLCDR3 of the light chain is: LQYDEFPYT (SEQ ID NO. 6).

[0060] The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO.7, and the nucleotide sequence is shown in SEQ ID NO.8.

[0061] Among them, the sequence information of SEQ ID NO.7 is: ELSCAASGFTFSTYIMSWVRQTPEKRLEWVATISSGGVKTYYPDSVKGRFTISRDNAKNDLYLQMSSLRSEDTALYFCTRSYGNYYWYFDVWGAGTTVTVSS;

[0062] SEQ ID The sequence information of NO.8 is: GCATCTCTAGGAGAGAGTCACTATCACTTGCAAGGCGAGTCAGGACATTAATAGCTATTTAAGCTGGTTCCAGCAGAAACCAGGGAAATCCTAAGACCCTGATCTATCGTGCAAACAGATTGGTAGATGGGGT CCCATCAAGGTTCAGTGGCAGTGGATCTGGGCAAGATTTTTCTCTCACCATCAGCAGCCTGGAGTATGAAGATATGGGAATTTATTGTCTACAGTATGATGAGTTTCCGTACACGTTCGGAGGGGGGACCAAGCTGGAAATCAAA.

[0063] The amino acid sequence of the light chain variable region is shown in SEQ ID NO.9, and the nucleotide sequence is shown in SEQ ID NO.10.

[0064] Among them, the sequence information of SEQ ID NO.9 is: ASLGERVTITCKASQDINSYLSWFQQKPGKSPKTLIYRANRLVDGVPSRFSGSGSGQDFSLTISSLEYEDMGIYYCLQYDEFPYTFGGGTKLEIK;

[0065] SEQ ID The sequence information of NO.10 is: GCATCTCTAGGAGAGAGTCACTATCACTTGCAAGGCGAGTCAGGACATTAATAGCTATTTAAGCTGGTTCCAGCAGAAACCAGGGAAATCCTAAGACCCTGATCTATCGTGCAAACAGATTGGTAGATGGGGT CCCATCAAGGTTCAGTGGCAGTGGATCTGGGCAAGATTTTTCTCTCACCATCAGCAGCCTGGAGTATGAAGATATGGGAATTTATTGTCTACAGTATGATGAGTTTCCGTACACGTTCGGAGGGGGGACCAAGCTGGAAATCAAA.

[0066] Example 3 Identification of Purity, Subtype and In Vitro Neutralization Potency

[0067] The purified monoclonal antibody 7H7 in Example 1 was analyzed for antibody purity by SDS-PAGE (Coomassie Brilliant Blue staining), and the monoclonal antibody subtype was detected using an enzyme-labeled secondary antibody ready-to-use kit for mouse monoclonal antibody Ig subclass identification (purchased from Suzhou Botelon Immunotechnology Co., Ltd.), as follows:

[0068] (1) SDS-PAGE (Coomassie Brilliant Blue staining)

[0069] The protein loading buffer and 7H7 were diluted and mixed in proportion, heated at 95°C for 10 min, and briefly centrifuged to remove the supernatant for sample loading. SDS-PAGE was performed on a 4-20% (w / v) polyacrylamide gel, and Coomassie Brilliant Blue staining and destaining were performed using an eStain® protein stainer (purchased from Nanjing GenScript Biotechnology Co., Ltd.).

[0070] The results are as follows Figure 1 As shown, after Coomassie Brilliant Blue staining, the gel showed two bands with molecular weights of approximately 75 kDa and 25 kDa, corresponding to the heavy chain and light chain of the monoclonal antibody 7H7, respectively.

[0071] (2) Enzyme-labeled secondary antibody kit for mouse monoclonal antibody Ig subclass identification

[0072] Use the inactivated CV-A10 stock solution to coat a 96-well microplate at 100 μL / well and coat overnight at 4°C; after washing the plate three times with PBST, add blocking solution (PBST containing 1% BSA) at 100 μL / well and block at 37°C for 1 hour; after washing the plate three times with PBST, add 7H7 (concentration of 1 μg / mL) at 100 μL / well and incubate at 37°C for 1 hour; after washing the plate three times with PBST, add HRP-labeled typing secondary antibody at 100 μL / well and incubate at 37°C for 1 hour; after washing the plate three times with PBST, add substrate solution A and substrate solution B at 50 μL / well and develop color at 37°C in the dark for 30 minutes; add stop solution at 50 μL / well; place the plate on a microplate reader and read A at a wavelength of 450 nm. 450nm ELISA results showed that the subtype of monoclonal antibody 7H7 was IgM and the light chain was κ chain.

[0073] (3) In vitro microneutralization test (Nt-CPE)

[0074] Add 7H7 (4.27 mg / mL) to the first column of a 96-well plate at 100 μL / well. Set up two replicates and add 50 μL of diluent (MEM maintenance solution) to each well of columns 2-12. Serially dilute the supernatant in column 1 by 2-fold to columns 12. Discard 50 μL of the last column after dilution and mixing. Dilute CV-A10 to 100 CCID 50 / 50μL, draw 50μL vertically and add it to the 96-well plate with diluted antibody as a neutralization plate, and place it in a 37℃ incubator for 2h. The diluted CV-A10 was diluted 10-fold to 10CCID 50 / 50μL, 1 CCID 50 / 50μL, 0.1 CCID 50 / 50μL. Add 50μL of maintenance solution and 50μL of virus solution to each well (a total of 4 dilutions, namely 100, 10, 1, and 0.1 CCID 50 / 50μL), 8 replicate wells for each dilution, as a back titration plate, placed at 4℃. After neutralization, the prepared RD cell suspension was diluted to 1×10 5 The cells were evenly plated at a density of 1000 cells / mL in a neutralization plate and a back titration plate, cultured in a 5% CO2 incubator at 37°C for 24-48 hours, and then observed under a fluorescence microscope to determine the neutralization results.

[0075] The neutralizing antibody titer of monoclonal antibody 7H7 determined by Nt-CPE was 1:4096.

[0076] Example 4 Functional Analysis

[0077] The functions of the screened monoclonal antibody 7H7 were analyzed by enzyme-linked immunosorbent assay (double antibody sandwich method) and immunoblotting assay, as follows:

[0078] (1) Enzyme-linked immunosorbent assay (double antibody sandwich method)

[0079] Purified CV-A10 rabbit polyclonal antibody (obtained by immunizing Japanese white rabbits with inactivated CV-A10 as an antigen by conventional methods) was diluted to 1:5000 with 0.05 mol / L phosphate buffer (pH 7.2) and coated on a 96-well microplate at 100 μL / well at 4°C overnight. The plate was washed three times with PBST and then blocked with 1% After washing the plate three times with PBST, 100 μL / well of CV-A10 stock solution was added and the plate was incubated at 37°C for 1 hour. After washing the plate three times with PBST, 100 μL / well of 7H7 (1 μg / mL) was added and the plate was incubated at 37°C for 1 hour. After washing the plate three times with PBST, 100 μL / well of HRP-labeled goat anti-mouse IgM (1:10,000 dilution) was added and the plate was incubated at 37°C for 1 hour. After washing the plate three times with PBST, 50 μL / well of substrate solution A (from Wuhan Institute of Biological Products Co., Ltd.) and 50 μL / well of substrate solution B (from Wuhan Institute of Biological Products Co., Ltd.) were added and the plate was developed at 37°C in the dark for 30 minutes. 50 μL / well of stop solution was added and the plate was placed on a microplate reader and read at a wavelength of 450 nm. 450nm value.

[0080] The results of the ELISA experimental group were positive, indicating that the monoclonal antibody 7H7 could bind to CV-A10.

[0081] (2) Immunoblotting

[0082] The screened monoclonal antibody 7H7 was used to identify the structural protein region of CV-A10 by immunoblotting. The steps are as follows:

[0083] The protein loading buffer and CV-A10 stock solution were diluted and mixed in proportion, heated at 95°C for 10 min, and the supernatant was collected after brief centrifugation for sample loading. SDS-PAGE was performed on a 4-20% (W / V) polyacrylamide gel, and the membrane was transferred to a 0.45 μm nitrocellulose membrane using an eBlotTM fast wet transfer instrument (purchased from Nanjing GenScript Biotechnology Co., Ltd.). After transfer, blocking solution (PBST containing 5% BSA) was added and blocked at 37°C for 1 h. Monoclonal antibody 7H7 (concentration of 100 μg / mL) was added and incubated at 37°C for 1 h. The membrane was washed with PBST five times. HRP-labeled goat anti-mouse IgM (1:10,000 dilution) was added and incubated at 37°C for 1 h. The membrane was washed with PBST five times. A chemical color development solution was added for color development and exposed to the imaging system.

[0084] The results are as follows Figure 2 As shown in FIG, under the condition that CV-A10 was reduced, the Western Blot results showed that there was no target band in the experimental group. Combined with the experimental results of Example (1), it was determined that the monoclonal antibody 7H7 targeted the conformational epitope.

[0085] Example 5 Conformational epitope study

[0086] This example provides a method for studying conformational epitopes and preliminarily identifies the conformational neutralizing epitope of CV-A10 recognized by monoclonal antibody 7H7. Monoclonal antibody 7H7 recognizes the C-terminus of the VP1 protein of CV-A10, as follows:

[0087] (1) Screening and plaque purification of CV-A10 strains that escape 7H7

[0088] Prepare a 24-well plate with RD cells at a confluency of 95%; dilute the CV-A10-iLOV virus solution to 100 CCID 501. 50 μL of monoclonal antibody 7H7 was diluted 2-fold from 100 μg / mL to 3.125 μg / mL. 50 μL of each serially diluted antibody was mixed with 200 μL of diluted CV-A10-iLOV in equal volumes and neutralized in a 37°C (v / v) 5% CO2 incubator for 2 hours. After neutralization, the cells were added to a 24-well plate containing a monolayer of RD cells. Cell control wells, antibody control wells, and virus control wells were set up. The cells were incubated in a 37°C 5% CO2 incubator for 24-48 hours and observed under a fluorescence microscope. If the experimental wells showed no fluorescence, the harvested solution was freeze-thawed three times, centrifuged at 4000 g for 10 minutes at 4°C, and the supernatant was aliquoted into 1.5 mL centrifuge tubes. The supernatant was replaced with the virus solution and the above steps were repeated. If a concentrated group of fluorescent spots appears in an experimental well, it indicates that the well may contain a CV-A10 strain that has escaped 7H7. The virus liquid harvested from the well is subjected to three rounds of plaque purification.

[0089] After plaque purification, a total of five single-genotype CV-A10-iLOV strains were obtained. Amino acid sequence alignment of their structural protein regions revealed that all five CV-A10-iLOV strains had a common mutation at the C-terminus of the VP1 protein in the structural protein region (Table 1).

[0090] Table 1 Differences in amino acid sequence alignment of structural protein regions of CV-A10 monoclonal strains

[0091]

[0092] (2) Detection of the potency of neutralizing antibody 7H7 against escape strains (in vitro microneutralization test)

[0093] Neutralization titer was determined for one of the monoclonal strains, CV-A10-iLOV-4822, which had the only common mutation. 7H7 (at a concentration of 100 μg / mL) was added to the first column of a 96-well plate at 100 μL / well. Two replicate wells were set up, and 50 μL of diluent (MEM maintenance solution) was added to each well of columns 2-12. The supernatant in column 1 was serially diluted 2-fold to columns 12. After the last column was diluted and mixed, 50 μL was discarded. CV-A10-iLOV-4822 was diluted to 100 CCID 50 / 50μL, draw 50μL vertically suspended into the 96-well plate with diluted antibody as a neutralization plate, and place it in a 37℃ incubator for 2h. Diluted CV-A10-iLOV-4822 was diluted 10-fold to 10 CCID 50 / 50μL, 1 CCID 50 / 50μL, 0.1 CCID 50 / 50μL. Add 50μL of maintenance solution and 50μL of virus solution to each well (a total of 4 dilutions, namely 100, 10, 1, and 0.1 CCID 50 / 50 μL), 8 replicate wells for each dilution, as a back titration plate, placed at 4℃. After neutralization, the prepared RD cell suspension was diluted to 1×10 5 The cells were evenly plated at a density of 1000 cells / mL in a neutralization plate and a back titration plate, cultured in a 5% CO2 incubator at 37°C for 24-48 hours, and then observed under a fluorescence microscope to determine the neutralization results.

[0094] The results are as follows Figure 3 As shown in Table 2, CV-A10-iLOV-4822 can escape the neutralization of the monoclonal antibody 7H7 (wells with an antibody concentration of not less than 50 μg / mL that are still not neutralized are judged to have escaped). Based on the neutralization results, the differences in the amino acid sequence of the structural protein region of the escape strain CV-A10-iLOV-4822 and the neutralizing strain CV-A10-iLOV were analyzed, and it was speculated that the significant decrease in the neutralization ability of 7H7 against the escape strain was related to the 291st amino acid of the structural protein VP1. Based on the CV-A10-iLOV monoclonal strain, a point mutation was made to this amino acid site (from serine to proline) to obtain the recombinant strain rCV-A10-iLOV-S1291P. The recombinant strain was subjected to the neutralizing antibody titer determination of this example (2), and the results are as follows. Figure 3 , as shown in Table 2. The above neutralization experimental results show that the point mutation S1291P can significantly affect the neutralization ability of monoclonal antibody 7H7 against CV-A10. The neutralization site of 7H7 is the spatial conformational epitope formed by the 291st amino acid at the C-terminus of the VP1 protein of CV-A10 particles.

[0095] Table 2 Neutralization titer results of monoclonal antibody 7H7 in vitro microneutralization test

[0096]

[0097] Example 6 Application of Monoclonal Antibody 7H7

[0098] This example provides a method for detecting CV-A10 particles using ELISA, as follows:

[0099] The purified CV-A10 hollow particles and solid particles were diluted 2-fold from 5 μg / mL to 4.8828125 ng / mL, coated on a 96-well microplate at 100 μL / well, and coated overnight at 4°C. After washing the plate three times with PBST, blocking solution (PBST containing 1% BSA) was added at 100 μL / well and blocked at 37°C for 1 hour. After washing the plate three times with PBST, 7H7 (concentration of 1 μg / mL) was added at 100 μL / well and incubated at 37°C for 1 hour. After washing the plate three times with PBST, HRP-labeled goat anti-mouse IgM (1:10000 dilution) was added at 100 μL / well and incubated at 37°C for 1 hour. After washing the plate three times with PBST, substrate solution A and substrate solution B were added at 50 μL / well, and color was developed at 37°C in the dark for 30 minutes. Stop solution was added at 50 μL / well. A was read on a microplate reader at a wavelength of 450 nm. 450nm value.

[0100] The results are as follows Figure 4 As shown in the figure, there is no significant difference in the binding ability of CV-A10 hollow particles (EP) and solid particles (FP) to monoclonal antibody 7H7 at the same concentration (A 450nm The values ​​were paired by concentration (t-test, P-value>0.05), so this method can well identify both CV-A10 hollow particles and solid particles.

[0101] The inventors also discovered through experiments that an amino acid sequence formed by replacing, deleting, or adding one or more amino acid sequences to the amino acid sequence set forth in SEQ ID NO. 7 of the heavy chain amino acid sequence of monoclonal antibody 7H7, or an amino acid sequence having more than 95% homology to the amino acid sequence set forth in SEQ ID NO. 7, has the same function as the sequence set forth in SEQ ID NO. 7; and an amino acid sequence formed by replacing, deleting, or adding one or more amino acid sequences to the amino acid sequence set forth in SEQ ID NO. 9 of the light chain amino acid sequence of monoclonal antibody 7H7, or an amino acid sequence having more than 95% homology to the amino acid sequence set forth in SEQ ID NO. 9, has the same function as the sequence set forth in SEQ ID NO. 9.

[0102] The raw materials not specifically described in the present invention are all existing materials that can be directly purchased from the market.

[0103] The above is only a preferred embodiment of the present invention and is not intended to limit the scope of protection of the present invention. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A monoclonal antibody 7H7 capable of recognizing and neutralizing CV-A10, characterized in that: Monoclonal antibody 7H7 comprises a heavy chain variable region and a light chain variable region. The heavy chain variable region of the monoclonal antibody 7H7 capable of recognizing and neutralizing CV-A10 comprises a complementarity determining region VHCDR1, a complementarity determining region VHCDR2, and a complementarity determining region VHCDR3. The amino acid sequence of the complementarity determining region VHCDR1 is shown in SEQ ID NO: 1, the amino acid sequence of the complementarity determining region VHCDR2 is shown in SEQ ID NO: 2, and the amino acid sequence of the complementarity determining region VHCDR3 is shown in SEQ ID NO:

3. The light chain variable region of the monoclonal antibody 7H7 capable of recognizing and neutralizing CV-A10 includes a complementarity determining region VLCDR1, a complementarity determining region VLCDR2, and a complementarity determining region VLCDR3. The amino acid sequence of the complementarity determining region VLCDR1 is shown in SEQ ID NO: 4, the amino acid sequence of the complementarity determining region VLCDR2 is shown in SEQ ID NO: 5, and the amino acid sequence of the complementarity determining region VLCDR3 is shown in SEQ ID NO:

6.

2. The monoclonal antibody 7H7 capable of recognizing and neutralizing CV-A10 according to claim 1, characterized in that The amino acid sequence of the heavy chain variable region of the monoclonal antibody 7H7 capable of recognizing and neutralizing CV-A10 is shown in SEQ ID NO.7; and / or, the amino acid sequence of the light chain variable region of the monoclonal antibody 7H7 capable of recognizing and neutralizing CV-A10 is shown in SEQ ID NO.

9.

3. The monoclonal antibody 7H7 capable of recognizing and neutralizing CV-A10 according to claim 2, characterized in that The monoclonal antibody is an IgM subtype antibody.

4. The monoclonal antibody 7H7 capable of recognizing and neutralizing CV-A10 according to claim 2, characterized in that The monoclonal antibody 7H7, which is capable of recognizing and neutralizing CV-A10, targets a conformational epitope.

5. A polynucleotide molecule encoding the monoclonal antibody 7H7 capable of recognizing and neutralizing CV-A10 according to any one of claims 1 to 4.

6. The polynucleotide molecule according to claim 5, characterized in that The nucleic acid molecule encoding the heavy chain is any one of the following: a, having the nucleotide sequence shown in SEQ ID NO.8; b. a nucleotide sequence complementary to the nucleotide sequence shown in SEQ ID NO.8; c. a nucleotide sequence encoding the same protein as the nucleotide sequence in a and b but differing therefrom due to the degeneracy of the genetic code; and, The nucleic acid molecule encoding the light chain is any one of the following: e. having the nucleotide sequence shown in SEQ ID NO.10; f. a nucleotide sequence complementary to the nucleotide sequence shown in SEQ ID NO.10; g. A nucleotide sequence that encodes the same protein as the nucleotide sequence in e and f, but differs from them due to the degeneracy of the genetic code.

7. An expression vector, characterized in that Contains a nucleic acid molecule encoding the monoclonal antibody 7H7 capable of recognizing and neutralizing CV-A10 according to any one of claims 1 to 4.

8. A host cell, characterized in that Containing the polynucleotide molecule according to claim 5 or 6, or containing the expression vector according to claim 7.

9. Use of the monoclonal antibody 7H7 capable of recognizing and neutralizing CV-A10 according to any one of claims 1 to 4, or the monoclonal antibody 7H7 capable of recognizing and neutralizing CV-A10 encoded by the polynucleotide molecule according to claim 5 or 6, in the preparation of a reagent or kit for detecting CV-A10.

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