Monoclonal antibody capable of binding to ev-a71 and use thereof

Abstract

A monoclonal antibody capable of binding to EV-A71 and the use thereof. The monoclonal antibody has heavy chain complementarity determining regions CDR1-CDR3 as shown in SEQ ID NOs: 1-3 and light chain complementarity determining regions CDR1-CDR3 as shown in SEQ ID NOs: 4-6, can specifically bind to a conformational epitope formed by amino acid at position 159 of structural protein VP2 and amino acid at position 81 of VP3 of EV-A71, thereby blocking the process of virus invasion into a host.

Description

A monoclonal antibody capable of binding to EV-A71 and its application Technical Field

[0001] The present invention belongs to the field of molecular biology, and in particular relates to a monoclonal antibody capable of binding to EV-A71 and an application thereof. Background Art

[0002] Enterovirus A71 (EV-A71), a member of the Picornaviridae family and the Enterovirus genus, is the primary pathogen causing hand, foot, and mouth disease in infants and young children. In recent years, EV-A71 has frequently broken out in numerous countries and regions in Asia, causing a variety of clinical illnesses, severe illness, and deaths. This has posed a serious threat and economic burden to public health worldwide, particularly in the Asia-Pacific region.

[0003] Antibody therapy and vaccination are currently the most effective antiviral treatments. Therefore, the development of monoclonal neutralizing antibodies against EV-A71 is of great significance for the development of therapeutic and / or preventive antibodies for EV-A71, as well as for the development of EV-A71 vaccines, clinical sample testing, and laboratory virus identification. The enterovirus protein capsid is composed of four structural proteins: VP1, VP2, VP3, and VP4. They exhibit icosahedral cubic symmetry, with VP1, VP2, and VP3 forming the outer shell, and VP4 located on the inner surface of the particle. Therefore, the antigenicity of EV-A71 is primarily determined by VP1, VP2, and VP3. VP1, VP2, and VP3 exist on the surface of the viral particle as a ring structure consisting of eight antiparallel β-sheets and connecting sheets. Currently discovered neutralizing antibody recognition sites are primarily concentrated in the GH loop formed by VP1 and VP3, and the EF loop of VP2.

[0004] The development of monoclonal antibodies targeting different recognition sites is of great significance for the treatment and research of enterovirus group A71.

[0005] Summary of the Invention

[0006] Based on this, the present invention provides a monoclonal antibody that can bind to EV-A71 VP2. This monoclonal antibody is different from the currently available monoclonal antibodies. It can specifically bind to the conformational epitope formed by amino acid 159 of EV-A71 VP2 and amino acid 81 of VP3, and amino acid 159 of VP2 is an unreported neutralization-related site on the surface of EV-A71 virus particles.

[0007] The present invention adopts the following technical solution: a monoclonal antibody capable of binding to EV-A71 VP2, wherein the monoclonal antibody has heavy chain complementary determining regions CDR1 to CDR3 shown in SEQ ID NOs: 1 to 3 and light chain complementary determining regions CDR1 to CDR3 shown in SEQ ID NOs: 4 to 6.

[0008] Furthermore, the amino acid sequence of the heavy chain variable region of the monoclonal antibody is shown in SEQ ID NO: 9, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 10.

[0009] Furthermore, the amino acid sequence of the heavy chain variable region of the monoclonal antibody is an amino acid sequence formed by replacing, deleting or adding one or more amino acid sequences to the amino acid sequence shown in SEQ ID NO: 9, or an amino acid sequence having more than 95% homology to the amino acid sequence shown in SEQ ID NO: 9, and the amino acid sequence of the light chain variable region is an amino acid sequence formed by replacing, deleting or adding one or more amino acid sequences to the amino acid sequence shown in SEQ ID NO: 10, or an amino acid sequence having more than 95% homology to the amino acid sequence shown in SEQ ID NO: 10.

[0010] The present invention also provides a polynucleotide molecule encoding the above monoclonal antibody.

[0011] Furthermore, the polynucleotide molecule has the nucleotide sequence shown in SEQ ID NO: 7 and / or SEQ ID NO: 8.

[0012] The present invention also provides a kit for detecting EV-A71 antigen, which comprises the above-mentioned monoclonal antibody.

[0013] The present invention also provides a vector or cell comprising the above polynucleotide molecule.

[0014] The present invention also provides the use of the above-mentioned monoclonal antibody or the monoclonal antibody encoded by the polynucleotide molecule in the preparation of drugs for inhibiting, preventing and treating EV-A71, as well as the use in the study of changes in the structural conformation of the EV-A71 virus.

[0015] Compared with the prior art, the monoclonal antibody provided by the present invention is prepared by immunizing BALB / c mice with solid particles of a purified EV-A71 neutralizing standard virus (523-07T strain) as an immunogen, preparing and screening hybridoma cells. It is an IgA subtype neutralizing antibody that can specifically recognize EV-A71 and does not recognize other enteroviruses including CV-A16, CV-A10, and CV-A6. In addition, the monoclonal antibody can be combined with a conjugate (horseradish peroxidase or fluorescein isothiocyanate, etc.) for direct or indirect detection and rapid diagnosis of EV-A71. For example, it can be used to develop a kit for quantitative detection of EV-A71 antigens, and can also be used for laboratory identification of antibodies for clinical virus isolates. It can also be used for the development of neutralizing antibody drugs for the prevention or treatment of EV-A71, as well as for related research on the mechanism of EV-A71 invasion of host cells and the antibody neutralization mechanism. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] FIG1 is an SDS PAGE image of the IgA light chain and heavy chain of the monoclonal neutralizing antibody 8A4;

[0017] Figure 2 shows the identification of monoclonal antibody subclasses (types);

[0018] FIG3 is an indirect immunofluorescence result of the monoclonal neutralizing antibody 8A4 detecting antigens in RD cells infected with EV-A71;

[0019] FIG4 is the identification of the binding specificity of the 8A4 monoclonal neutralizing antibody;

[0020] Figure 5 shows the amino acid sequence alignment of EV-A71 before and after the escape strain mutation;

[0021] Figure 6 shows the reverse genetics construction of EV-A71 strain neutralization titer assay;

[0022] FIG7 is a determination of the protective efficacy of 8A4 monoclonal neutralizing antibody. DETAILED DESCRIPTION

[0023] 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.

[0024] The following embodiments are only used to illustrate the present invention, but are not intended to limit the scope of the present invention. Based on the specific embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without making creative work are within the scope of protection of the present invention.

[0025] 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.

[0026] In the examples of the present invention, the raw materials used are all conventional commercially available products.

[0027] Reagent Source:

[0028] Freund's complete and incomplete adjuvants: purchased from Sigma;

[0029] Isotyping Kit for Mouse Monoclonal Antibody: purchased from Beijing Sino Biological Technology Co., Ltd.

[0030] Goat anti-mouse IgG (H+L) Alexa Fluor 488 fluorescent secondary antibody: purchased from Thermo Fisher Scientific;

[0031] HRP-labeled goat anti-mouse IgG: purchased from Wuhan Boster Bioengineering Co., Ltd.

[0032] EV-A71 virus: neutralization standard virus 523-07T strain, from China Food Inspection Institute, for details, see the article: [1] Mao Qunying, Guo Zengbing, Hao Chunsheng, et al. Screening and preparation of standard virus candidate strains for enterovirus 71 neutralizing antibody detection [J]. Chinese Journal of Biological Products, 2012, 25(06): 725-729. DOI: 10.13200 / j.cjb.2012.06.70.maoqy.005;

[0033] EV-A71-E: An escape mutant of EV-A71, derived from the neutralization standard virus 523-07T strain, serially passaged, and obtained from the Viral Vaccine Laboratory 1 of Wuhan Institute of Biological Products Co., Ltd.

[0034] EV71-Q1 strain: An EV71 clinical sample (isolate) extracted from a patient infected with hand, foot and mouth disease in Hubei Province, adapted and passaged on Vero cells, and then plaque purified.

[0035] EV-A71 vaccine strain: BJ vaccine strain (Wuhan Institute) JX025561;

[0036] CV-A16, CV-A6, and CV-A10 are all strains disclosed in the prior art and recorded in the NCBI database with accession numbers: KF924762.1, MW410845, and MW713443.1, respectively;

[0037] Nov, Japanese encephalitis, and rabies virus vaccines (referred to as rabies vaccines) were provided by the Viral Vaccine Research Laboratory I of Wuhan Institute of Biological Products Co., Ltd.

[0038] Example 1 Material Preparation

[0039] EV-A71 virus (neutralization standard virus 523-07T strain) was inoculated into serum-free Vero cells. When the cytopathic effect reached 90%, the virus was harvested and packaged.

[0040] Preparation of EV-A71 virus full particles (FP) and empty particles (EP): EV-A71 (MOI = 0.001) was inoculated into ten-layer RD cell factories and cultured at 37°C. The virus liquid was harvested when the cytopathic effect (CPE) reached 90%. The virus harvested liquid was repeatedly frozen and thawed and centrifuged to remove cell debris. The cell supernatant was concentrated 10-fold by ultrafiltration through a 100 kDa pore size filter and passed through a 25% (w / v) sucrose basal layer and centrifuged at 103745g for 4 h. The viral proteins were solubilized in PBS buffer (pH = 7.2). The virus was collected by density gradient centrifugation at 15%, 25%, 35%, 45%, and 55% (w / v) sucrose and centrifuged at 103745g for 4 h to obtain full particles (FP) and empty particles (EP).

[0041] FP and EP were subjected to cesium chloride density gradient centrifugation at 260,000 g and 4° C. for 24 h, and the opalescent bands were extracted to obtain purified EV-A71 virus particles.

[0042] The methods for other virus solid particles and hollow particles are the same as above.

[0043] Example 2 Preparation of monoclonal antibodies

[0044] This example provides a method for preparing a monoclonal antibody, which is as follows:

[0045] S1: 100 μg of EV-A71 (neutralization standard virus strain 523-07T) FP particles were mixed with Freund's complete adjuvant (V / V = 1:1). Subsequently, Freund's incomplete adjuvant mixed with antigen was used for booster immunization. Female BALB / c mice (4-6 weeks old) were immunized by multiple subcutaneous injections or intraperitoneal injections at the back. The injection volume was 500 μL / time, with a total of 6 injection sites. Mice were immunized on days 0, 28, and 45. The titer of mouse serum on day 52 was determined by indirect ELISA. Sera with an ELISA binding titer exceeding 1 × 10 6 The mice were subjected to shock immunization. The mouse spleen cells were collected on the third day after the last immunization, and polyethylene glycol-1500 was used to induce the fusion of mouse spleen cells and SP2 / 0 myeloma cells. The fusion ratio of mouse spleen cells to myeloma cells was controlled at (1:5) to (1:10); the hybridoma cells with successful fusion were screened using HAT culture medium.

[0046] S2. The antibody titer of the culture supernatant of the successfully fused hybridoma cells was detected by indirect ELISA method, and the antibodies with ELISA binding titer greater than 1×10 4 Positive hybridoma cells were cloned and purified three times, and then expanded and cultured to prepare ascites. The ascites was purified using a three-step saturated ammonium sulfate method to obtain monoclonal antibodies, and the antibody isotype was identified using the Isotyping Kit for Mouse Monoclonal Antibody.

[0047] Among them, the indirect ELISA method for screening hybridoma cells is as follows: EV-A71 virus purified particles are prepared with 1 μg / mL coating solution in carbonate buffer at pH = 9.6, added to the ELISA plate, 100 μL / well, incubated at 4°C overnight, and washed 5 times with PBST at pH = 7.4; The plates were blocked with PBST containing (1% BSA) and 0.1% albumin at 37°C for 1 hour, and the blocking solution was discarded. The supernatant of the hybridoma cell culture fluid to be tested was diluted tenfold (V / V) and added to the ELISA plate at 100 μL / well. The plates were incubated at 37°C for 1 hour and washed 5 times with PBST (pH = 7.4). 0.1 μg / mL HRP-labeled goat anti-mouse IgG antibody (Boster) was added to the ELISA plate at 100 μL / well. The plates were incubated at 37°C for 1 hour and washed 5 times with PBST (pH = 7.4). TMB colorimetric solution was added to develop the color at 37°C in the dark for 30 minutes. The reaction was terminated with 2 M sulfuric acid, and the absorbance at 450 nm was detected using a microplate reader. The titer was calculated as the maximum dilution factor that was greater than 2.1 times the OD value of the negative well. Hybridoma cell culture medium was used as a negative control.

[0048] Example 3 Sequence analysis of the monoclonal antibodies screened

[0049] The hybridoma cells screened in Example 2 were inoculated in RPMI 1640 medium (Gibco) supplemented with 20% fetal bovine serum and cultured at 37°C. Total RNA was extracted using the Fast Pure Cell / Tissue Total RNA Isolation Kit and reverse transcribed to obtain single-stranded cDNA. The heavy and light chain variable region genes of monoclonal antibody 8A4 were amplified using a heavy chain universal primer pair with sequences homologous to the cloning vector pUC-Kan. The purified PCR products were cloned into the pUC-Kan vector (purchased from Nanjing GenScript Biotechnology Co., Ltd.). Positive clones were screened and sequenced. The sequenced sequences were analyzed using the Kabat database to obtain the correct amino acid sequences of the light and heavy chain variable regions.

[0050] Among them, the sequence of the universal forward primer VH-F of the heavy chain is: GGGAATTCGAGGTGCAGCTGCAGGAGTCTGG, and the reverse primer VH-R is a mixture of the following sequences: AGGGGGCTCTCGCAGGAGACGA, GGAAGGTGTGCACACCGCTGGAC, GGAAGGTGTGCACACCACTGGAC, GGAAGGTGTGCACACTGCTGGAC, AGACTGTGCGCACACCGCTGGAC, GAAAGTTCACGGTGGTTATATCC;

[0051] The universal forward primer VL-F of the light chain is a mixture of the following sequences: TGCTGCTGCTCTGGGTTCCAG, ATTWTCAGCTTCCTGCTAATC, TTTTGCTTTTCTGGATTYCAG, TCGTGTTKCTSTGGTTGTCTG, ATGGAATCACAGRCYCWGGT, TCTTGTTGCTCTGGTTYCCAG, CAGTTCCTGGGGCTCTTGTTGTTC, CTCACTAGCTCTTCTCCTC, and the sequence of the reverse primer VL-R is GATGGTGGGAAGATGGATACAGTT.

[0052] The determined nucleotide sequence of the heavy chain variable region of monoclonal antibody 8A4 is: gtgcagcctggagggtcccggaaactctcctgtgcagcctctggattcactttcagtagctttggaatgcactgggttcgtcaggctccagagaaggggctggagtgggtcgcatacattagtagtggcagtagtaccCtcCactatgcagacacagtgaagggccgattcaccatctccagagacaatcccaagaacaccctgttcctgcaaatgaccagtctaaggtctgaggacacggccatgtattac (SEQ ID NO: 7);

[0053] The nucleotide sequence of the light chain variable region is: gacattgtgctgacacagtctcctgcttccttagctgtatctctggggcagagggccaccatctcatacagggccagcaaaACAGtcagtacatctgTctatagttatatgcactggaaccaacagaaaccaggacagccac ccaCactcctcatctatcttgtatccaacctagaatctggggcccctgccaggttcagtggcagtgggtctgggacagacttcaccctcaacatccatcctgtggaggaggaggatgctgcaacctattactgtcagcacattagggagctt(SEQ ID NO: 8);

[0054] The amino acid sequence of the heavy chain variable region is: VQPGGSRKLSCAASGFTFSSFGMHWVRQAPEKGLEWVAYISSGSSTLHYADTVKGRFTISRDNPKNTLFLQMTSLRSEDTAMYY (SEQ ID NO: 9);

[0055] The amino acid sequence of the light chain variable region is: DIVLTQSPASLAVSLGQRATISYRASKTVSTSVYSYMHWNQQKPGQPPRLLIYLVSNLEYGVPARFSGSGSGTDFTLNIHPVEEEDAATYYCQHIREL (SEQ ID NO: 10);

[0056] The sequences of the six CDR regions (complementarity determining regions) analyzed are as follows:

[0057] The amino acid sequence of the heavy chain complementarity determining region HCDR1 is: GFTFSSFG (SEQ ID NO: 1);

[0058] The amino acid sequence of the heavy chain complementarity determining region HCDR2 is: ISSGSSTL (SEQ ID NO: 2);

[0059] The amino acid sequence of the heavy chain complementarity determining region HCDR3 is SEDTAMYY (SEQ ID NO: 3);

[0060] amino acid sequence of the complementarity determining region LCDR1 of the light chain: KTVSTSVYSY (SEQ ID NO: 4);

[0061] amino acid sequence of the complementarity determining region LCDR2 of the light chain: LVS (SEQ ID NO: 5);

[0062] Amino acid sequence of the complementarity determining region LCDR3 of the light chain: QHIREL (SEQ ID NO: 6).

[0063] Example 4 Subtype Identification

[0064] Monoclonal antibody 8A4, screened and purified in Example 2, was analyzed using 4-20% SDS-PAGE. The results of reducing SDS-PAGE are shown in Figure 1. Cell supernatants from four monoclonal antibody strains with neutralizing activity were identified using a monoclonal antibody subclass identification kit, and the results are shown in Figure 2. Figures 1 and 2 indicate that the heavy and light chains of monoclonal antibody 8A4 are represented by protein bands of 50 kDa and 25 kDa, respectively, indicating an IgA subclass, consistent with expectations.

[0065] Example 5 Function and specificity analysis

[0066] The functions and properties of the screened 8A4 monoclonal neutralizing antibodies were analyzed using indirect immunofluorescence assay and enzyme-linked immunosorbent assay, respectively, as follows:

[0067] (1) Indirect immunofluorescence experiment

[0068] EV-A71 (neutralization standard virus 523-07T strain) was inoculated into 6-well plates with RD cell confluence of 95%, and RD cells without virus inoculation were used as negative control for culture. After culturing the 6-well plates for 24 hours, the cell supernatant was discarded, the plates were washed 3 times with 0.01M PBS, and fixed with 2mL / well 4% paraformaldehyde at room temperature for 1 hour, and washed 5 times / 5min with 0.01M PBS. 2mL / well 2% BSA-PBST (W / V) solution (containing 0.5% Triton-X 100, V / V) was added and permeabilized at room temperature for 30 minutes, and washed 5 times / 5min with 0.01M PBS. Blocked with 2% BSA-PBST solution at room temperature for 1 hour, the blocking solution was discarded. 1mL / well 2μg / mL monoclonal antibody 8A4 was added and incubated at room temperature for 1 hour, and 0.01M The cells were washed with PBS five times for 5 minutes each. 1 mL / well of 2 μg / mL goat anti-mouse fluorescent antibody IgG (H+L) (Thermo Fisher Scientific) was added and incubated at room temperature in the dark for 1 hour. After washing with PBS, 1 mL / well of 5 μg / mL DAPI solution (Biyuntian) was added. The cells were observed and photographed using a fluorescence microscope. The negative control group consisted of cells not infected with EV-A71 but treated with the corresponding antibody. The results are shown in Figure 3.

[0069] As can be seen from Figure 3, the 8A4 monoclonal antibody can be used in indirect immunofluorescence experiments to recognize EV-A71 antigens and can be used in antigen identification experiments.

[0070] (2) ELISA

[0071] The purified 8A4 monoclonal antibody was diluted to 2 μg / mL with carbonate buffer and added to a 96-well ELISA plate at 100 μL / well and incubated at 37°C for 1 hour; 300 μL of PBST buffer was added to each well for washing, and the plate was washed 5 times and patted dry; PBST blocking solution containing 1% BSA was added to the coated ELISA plate at 200 μL / well and incubated at 37°C for 1 hour; EV-A71 vaccine strain, CV-A16, CV-A10, CV-A6, Nov, Japanese encephalitis, and rabies virus vaccine (abbreviated as rabies vaccine) purified virus particles were diluted to 1 ng / μL with PBST diluent containing 1% BSA and added to 96-well plates respectively, and incubated at 37°C for 1 hour; 300 μL of PBST was added to each well. Wash with PBST buffer, repeat 5 times, and pat dry. Dilute HRP-8A4 with PBST containing 1% BSA at a dilution of 1:1000 and add it to the ELISA plate. Incubate at 37°C for 1 hour. Add 300 μL of PBST buffer to each well and wash, repeat 5 times, and pat dry. Add 50 μL of substrate solution A and solution B to each well of the ELISA plate, incubate at 37°C for 15 minutes. Add 50 μL of 2 mol / L sulfuric acid to each well of the ELISA plate to terminate the reaction, and measure the OD value using a microplate reader. 450 and OD 630 value.

[0072] Solution A was prepared as follows: Weigh 9.185g of NaAC·3H2O, 1.576g of citric acid, and 0.45g of urea peroxide, thoroughly dissolve them in water for injection, and dilute to 750mL. Adjust the pH to 5.0-5.5 with glacial acetic acid. Solution B was prepared as follows: Weigh 225.0mg of TMB and slowly add 11.25mL of DMSO dropwise while shaking. Once completely dissolved, weigh 1.395g of EDTA-Na2, 1.343g of Na2HPO4·12H2O, and 0.788g of citric acid, dissolve them in 500mL of water for injection, add 11.25mL of the prepared TMB solution and 75mL of glycerol, and dilute to 750mL with water for injection.

[0073] The results are shown in Figure 4. As can be seen from Figure 4, the 8A4 monoclonal antibody only specifically recognizes the EV-A71 antigen and has good specificity and can be used for antigen identification experiments.

[0074] Example 6 Conformational epitope study

[0075] Previous experiments have demonstrated that 8A4 is a neutralizing monoclonal antibody that recognizes a conformational epitope. This conformational epitope was identified by screening an EV-A71 immune escape strain (EV-A71-E, sourced from Wuhan Institute of Biological Products Co., Ltd.) that targets 8A4. By comparing the nucleic acid and amino acid sequence differences between the escape strain and the parent strain (523-07T), the relevant neutralizing epitope of EV-A71 recognized by the 8A4 monoclonal neutralizing antibody was identified. The experimental steps are as follows:

[0076] Cell preparation: Digest RD cells and prepare 1×10 5 / mL cell suspension, 1mL / well was plated in a 96-well plate;

[0077] Antibody incubation: Mix 500 μL of virus dilution (100 TCID50 / 50 μL) and 500 μL of a certain concentration of neutralizing antibody (100 times the TCID50 titer endpoint) and incubate at 37°C for 2 h.

[0078] Escape screening: After the incubation is completed, discard the culture medium in the 96-well plate, rinse twice with PBS, and inoculate the incubated virus and monoclonal antibody mixture into a 96-well plate (or 24-well plate). After incubation at 37°C for 1 hour, wash 3 times with PBS to remove unadsorbed virus particles and add 1 mL of MEM maintenance solution (with a certain concentration of monoclonal antibody). After 3 to 5 days, observe whether the cells have lesions; if there are no lesions, continue to passage and repeat the above steps; if lesions appear, perform plate purification, cover with 1.2% agar (containing 10-fold neutralizing titer concentration of neutralizing antibodies), culture at 37°C for 3 days, stain with 0.1% neutral red (uninfected cells can be stained with neutral red, and plaques appear white), and pick spots;

[0079] At the same time, the pre-screening EV-A71 parent strain (523-07T) was plaque-purified to select a certain number of monoclonal samples. These pre- and post-escape monoclonal samples were inoculated into cultured RD cells, and viral RNA was extracted from the harvested viral fluid. After reverse transcription into cDNA, full-length sequencing was performed. The sites with amino acid differences after sequence alignment are shown in Figure 5 . In Figure 5 , RA, RB, RC, RD, RE, RF, RG, and RH represent the non-escape strains identified in the second round of screening, while R-EM-E, R-EM-N, R-EM-O, R-EM-P, R-EM-Q, R-EM-R, R-EM-S, and R-EM-T represent the escape strains identified based on the non-escape strains.

[0080] As can be seen in Figure 5, compared with the parent strain, the escaped strains commonly exhibited mutations at amino acid position 159 of VP2 and amino acid position 81 of VP3. It is preliminarily hypothesized that amino acid positions 159 and 81 of VP3 of EV-A71 VP2 are the binding sites for the 8A4 monoclonal neutralizing antibody.

[0081] Using reverse genetics, single mutant strains with single-point mutations in VP2-159 and VP3-81 (EV-A71-VP2, EV-A71-VP3) and double mutant strains with mutations in both VP2-159 and VP3-81 (EV-A71-VP23) were constructed based on the mother strain. The 8A4 neutralization titer was identified together with the mother strain, and the titer results are shown in Figure 6. The results showed that only the double-point mutation strain had a titer greater than 84.10 μg / mL and would undergo immune escape. The VP2-159 single-point mutation strain had a titer of 1.314 μg / mL, and the VP3-81 single-point mutation strain had a titer of 1.314 μg / mL, while the mother strain had a titer of 0.1640 μg / mL, and neither would undergo immune escape. This result proves that both sites are key neutralization sites.

[0082] Example 7 Passive protection experiment

[0083] To evaluate the protective ability of mAb 8A4, different concentrations of mAb 8A4 (1256 μg / mL, 314 μg / mL, 157 μg / mL, and 78.5 μg / mL) were mixed with equal volumes of a lethal dose of EV71-Q1 virus (from Wuhan Institute of Biological Products Co., Ltd.) and then neutralized at 37°C for 2 h before intraperitoneal injection into one-day-old ICR mice. Each mouse was injected with 100 μL of the virus-antibody mixture (containing 1.6 × 10 6 The mice were observed for 14 consecutive days, and the health status and survival rate of the mice were recorded every day.

[0084] The results are shown in Figure 7. It can be seen from Figure 7 that all mice died within 5 days in the virus control group and when the monoclonal antibody dose was 3.925μg / mouse; when the monoclonal antibody dose was 7.85μg / mouse, the mouse survival rate was 37.5%; when the monoclonal antibody dose was 15.7μg / mouse, the mouse survival rate was 62.5%; when the monoclonal antibody dose was 62.8μg / mouse, all mice survived during the 14-day observation period, showing a 100% survival rate. At this time, the protein concentration of the monoclonal antibody was 1256μg / ml and the injection dose was 62.8μg / mouse, indicating that at an injection dose of 62.8μg / mouse, the monoclonal antibody 8A4 can achieve a 100% protection rate against the EV71-Q1 strain. According to SPASS software analysis, when the EV71-Q1 virus load was 1.6×10 6 TCID 50 When the ED of mAb 8A4 against EV71-Q1 was 50It is 11.5μg.

[0085] The inventors also discovered through experiments that the heavy chain amino acid sequence of the 8A4 monoclonal neutralizing antibody, wherein the amino acid sequence represented by SEQ ID NO: 9 is replaced by, deleted by, or added with one or more amino acid sequences, or an amino acid sequence having 95% or more homology to the amino acid sequence represented by SEQ ID NO: 9, has the same function as the sequence represented by SEQ ID NO: 9; and the light chain amino acid sequence of the 8A4 monoclonal neutralizing antibody, wherein the amino acid sequence represented by SEQ ID NO: 10 is replaced by, deleted by, or added with one or more amino acid sequences, or an amino acid sequence having 95% or more homology to the amino acid sequence represented by SEQ ID NO: 10, has the same function as the sequence represented by SEQ ID NO: 10.

[0086] It is important to note that the above embodiments are intended only to further illustrate and describe the technical solutions of the present invention and are not intended to further limit the technical solutions of the present invention. The methods of the present invention are merely preferred implementations and are not intended to limit the scope of protection of the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention shall be included within the scope of protection of the present invention.

Claims

1. A monoclonal antibody capable of binding to EV-A71, characterized in that: The monoclonal antibody has heavy chain complementary determining regions CDR1 to CDR3 shown in SEQ ID NOs: 1 to 3 and light chain complementary determining regions CDR1 to CDR3 shown in SEQ ID NOs: 4 to 6.

2. The monoclonal antibody according to claim 1, characterized in that The amino acid sequence of the heavy chain variable region of the monoclonal antibody is shown in SEQ ID NO: 9, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:

10.

3. The monoclonal antibody according to claim 1, characterized in that The amino acid sequence of the heavy chain variable region of the monoclonal antibody is an amino acid sequence formed by replacing, deleting or adding one or more amino acid sequences to the amino acid sequence shown in SEQ ID NO: 9, or an amino acid sequence having more than 95% homology to the amino acid sequence shown in SEQ ID NO: 9, and the amino acid sequence of the light chain variable region is an amino acid sequence formed by replacing, deleting or adding one or more amino acid sequences to the amino acid sequence shown in SEQ ID NO: 10, or an amino acid sequence having more than 95% homology to the amino acid sequence shown in SEQ ID NO:

10.

4. A polynucleotide molecule encoding the monoclonal antibody according to claim 1, 2 or 3.

5. The polynucleotide molecule according to claim 4, characterized in that The polynucleotide molecule has the nucleotide sequence shown in SEQ ID NO: 7 and / or SEQ ID NO:

8.

6. A kit for EV-A71 antigen detection, characterized in that: The kit comprises the monoclonal antibody according to claim 1 or 2 or 3 or the monoclonal antibody encoded by the polynucleotide molecule according to claim 4 or 5.

7. A vector or cell comprising the polynucleotide molecule of claim 4 or 5.

8. Use of the monoclonal antibody according to claim 1, 2 or 3, or the monoclonal antibody encoded by the polynucleotide molecule according to claim 4 or 5, in the preparation of a drug for inhibiting, preventing and treating EV-A71.

9. Use of the monoclonal antibody according to claim 1, 2 or 3, or the monoclonal antibody encoded by the polynucleotide molecule according to claim 4 or 5, in the study of structural conformational changes of EV-A71 virus.

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