A monoclonal antibody for recognizing coxsackievirus A2, A4 and A5 and its application

Abstract

The present invention relates to the technical fields of molecular biology and immunological analysis, and specifically to a broad-spectrum monoclonal antibody capable of recognizing coxsackievirus A2, A4, and A5, and its use. The monoclonal antibody comprises a heavy chain complementary determining region (CDR1) as set forth in SEQ ID NO.9, a heavy chain complementary determining region (CDR2) as set forth in SEQ ID NO.10, a heavy chain complementary determining region (CDR3) as set forth in SEQ ID NO.11, and a light chain complementary determining region (CDR1) as set forth in SEQ ID NO.12, a light chain complementary determining region (CDR2) as set forth in SEQ ID NO.13, and a light chain complementary determining region (CDR3) as set forth in SEQ ID NO.14. The monoclonal antibody specifically binds to the N-terminal 15 amino acids of the structural protein VP1 of coxsackievirus A2, A4, and A5, and can be used for clinical patient sample testing, laboratory antibody identification of virus isolates, and quantitative detection of coxsackievirus A2, A4, and A5 antigens in production intermediates and products.

Description

Technical Field

[0001] The present invention relates to the technical field of molecular biology and immunological analysis, and in particular to a broad-spectrum monoclonal antibody for recognizing Coxsackievirus A2, A4 and A5 (CV-A2, CV-A4 and CV-A5) and applications thereof. Background Art

[0002] Hand, foot, and mouth disease (HFMD) is a Class C infectious disease caused by various human enteroviruses. Symptoms include fever and a rash on the hands, feet, and mouth. It is most common in children under five years old. Severe cases can develop complications such as pulmonary edema and aseptic meningitis, and even death. In recent years, HFMD caused by enteroviruses has been characterized by clustered outbreaks and multi-virus cross-infection. Enterovirus A viruses, such as Coxsackieviruses (CV) A2, A4, A5, A6, A10, and A16, and Enterovirus (EV) A71, are the primary pathogens causing HFMD in infants and young children.

[0003] At present, only the monovalent EV-A71 whole-virus inactivated vaccine is on the market for the prevention of hand, foot and mouth disease. Coxsackievirus A group types 4, 2 and 5 (CV-A4, CV-A2, CV-A5) belong to the Picornaviridae family and the Enterovirus genus. They are also important pathogens that cause hand, foot and mouth disease, herpangina and acute flaccid paralysis. However, there are relatively few studies on them. Therefore, the study of specific or spectral monoclonal antibodies against other hand, foot and mouth disease entero-associated viruses such as CV-A4, CV-A2, CV-A5 is of great value for rapid virus detection in clinical samples, laboratory virus identification, functional research, quality control of vaccine development, and quantification of finished product antigens. Summary of the Invention

[0004] Based on this, one of the objects of the present invention is to provide a monoclonal antibody that recognizes Coxsackievirus A2, A4 and A5, wherein the monoclonal antibody has a heavy chain complementary determining region CDR1 shown in SEQ ID NO.9, a heavy chain complementary determining region CDR2 shown in SEQ ID NO.10, and a heavy chain complementary determining region CDR3 shown in SEQ ID NO.11, and a light chain complementary determining region CDR1 shown in SEQ ID NO.12, a light chain complementary determining region CDR2 shown in SEQ ID NO.13, and a light chain complementary determining region CDR3 shown in SEQ ID NO.14.

[0005] Preferably, the monoclonal antibody has a heavy chain with the amino acid sequence shown in SEQ ID NO.7; and / or, the monoclonal antibody has a light chain with the amino acid sequence shown in SEQ ID NO.8.

[0006] Preferably, the heavy chain amino acid sequence 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.7, or an amino acid sequence having more than 95% homology with the amino acid sequence shown in SEQ ID NO.7; and / or, the light chain amino acid sequence 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.8, or an amino acid sequence having more than 95% homology with the amino acid sequence shown in SEQ ID No.8.

[0007] This monoclonal antibody can broadly recognize Coxsackievirus A2, A4 and A5, and does not bind to other enteroviruses. It can be used in basic experiments such as indirect immunofluorescence, immunoblotting and enzyme-linked immunosorbent assay. It has the same antigen high titer binding to diseases such as hand, foot and mouth disease, herpangina and acute flaccid paralysis caused by Coxsackievirus A2, A4 and A5.

[0008] A second object of the present invention is to protect the polynucleotide molecules encoding the monoclonal antibodies that recognize Coxsackievirus A2, A4 and A5 as claimed above.

[0009] Preferably, the polynucleotide molecule has the nucleotide sequence shown in SEQ ID NO.5; and / or, the polynucleotide molecule has the nucleotide sequence shown in SEQ ID NO.6.

[0010] A third object of the present invention is to protect a kit for detecting Coxsackievirus A2, A4 and A5, which comprises the above-mentioned monoclonal antibody or the monoclonal antibody encoded by the polynucleotide molecule.

[0011] A fourth object of the present invention is to protect the use of the above-mentioned monoclonal antibodies or monoclonal antibodies encoded by polynucleotide molecules in the preparation of reagents or kits for detecting single, two or three of Coxsackievirus A2, A4 and A5.

[0012] A fifth object of the present invention is to protect the use of the above-mentioned monoclonal antibodies or monoclonal antibodies encoded by polynucleotide molecules in the preparation of drugs for inhibiting, preventing, and treating single, dual, or triple coxsackieviruses A2, A4, and A5.

[0013] A sixth object of the present invention is to protect the use of the above-mentioned monoclonal antibodies or monoclonal antibodies encoded by polynucleotide molecules in the study of VP1 proteins of Coxsackievirus A2, A4 and A5 and changes in viral structural conformation, such as studying the function of intermediates in the morphological conformational transition of Coxsackievirus A2, A4 and A5 virus particles or the role of N-terminal amino acids in penetrating the cell membrane and releasing viral RNA into the cell.

[0014] The monoclonal antibody provided by the present invention is obtained by immunizing Balb / c mice with solid particles (FP) of coxsackievirus A group 4 (CV-A4), preparing and screening hybridoma cells, and then purifying them. The monoclonal antibody is a broad-spectrum monoclonal antibody that can specifically recognize coxsackievirus A group 2, 4, and 5 (CV-A2, CV-A4, and CV-A5), is an IgG2b subtype non-neutralizing antibody, and can specifically bind to the 2nd to 15th amino acids at the N-terminus of the structural protein VP1 of CV-A2, A4, and A5. Among them, the residues at positions 2 to 4 (DAI), 6 to 8 (DAI), and 10 (N) of the VP1 N-terminal amino acid represent the footprints for the monoclonal antibody to specifically bind to the VP1 N-terminal amino acid.

[0015] This monoclonal antibody can be combined with a conjugate (horseradish peroxidase or fluorescein isothiocyanate, etc.) for direct or indirect detection or rapid diagnosis. It has important research and application value in rapid and economical detection of clinical samples, laboratory identification, virus titration, antigen quantification of vaccine preparation intermediates and products, research and development of structural protein VP1 function and drug targets, and other applications based on antigen-antibody reactions. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 This is the reducing SDS-PAGE result of IgG light chain and heavy chain of monoclonal antibody 3G4G;

[0017] Figure 2 Indirect immunofluorescence assay for detecting CV-A2, A4, and A5 infected RD cells using monoclonal antibody 3G4G;

[0018] Figure 3 Identification diagram of monoclonal antibodies recognizing CV-A2, A4, and A5 structural protein VP1;

[0019] Figure 4 This is the result of monoclonal antibody 3G4G ELISA binding titer test;

[0020] Figure 5 Figure 3 is the linear epitope peptide library and preliminary positioning structure of the monoclonal antibody 3G4G. A is the result of the 3G4G monoclonal antibody recognizing the linear epitope peptide library of the full length of VP1 (positions 1-305), and B is the result of the 3G4G monoclonal antibody recognizing the amino acids 1-50 of the VP1 linear epitope peptide. DETAILED DESCRIPTION

[0021] 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.

[0022] 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.

[0023] 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.

[0024] Reagent Source:

[0025] Freund's complete adjuvant and incomplete adjuvant: purchased from Sigma;

[0026] Female BALB / c mice were purchased from Wuhan Institute of Biological Products Co., Ltd.

[0027] Isotyping Kit for Mouse Monoclonal Antibody: purchased from Beijing Sino Biological Technology Co., Ltd.

[0028] Fast Pure Cell / Tissue Total RNA Isolation Kit: purchased from Novozymes Biotech;

[0029] Goat anti-mouse IgG (H+L) Alexa Fluor 488 fluorescent secondary antibody: purchased from Thermo Fisher Scientific;

[0030] HRP-labeled goat anti-mouse IgG: purchased from Wuhan Boster Bioengineering Co., Ltd.;

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

[0032] Example 1 Material Preparation

[0033] CV-A4 (strain CV-A4 / R3179 / XY / 2017, see article for details: Tian Yuxuan, Wang Mengjun and Wang Wenhui et al. Preparation of CV-A4 monoclonal antibody and establishment and validation of candidate vaccine type-specific quantitative analysis method. Modern Immunology, 2023, 43(01):8-15.), CV-A2 (strain CVA2-1388-M14 / XY / CHN / 2017, NCBI database accession number MW846233), CV-A5 (strain CV-A5-3487-M14-XY-CHN-2017, NCBI database accession number MW079817) and Echo 11 virus culture: CV-A4, A2, A5 and Echo 11 viruses were inoculated into serum-free Vero cells respectively. When the cytopathic effect reached 90%, the viruses were harvested and packaged.

[0034] Preparation of CV-A4, A2, A5 and Echo 11 virus full particles (FP) and empty particles (EP): CV-A4, CV-A2, CV-A5 and Echo 11 (MOI = 0.001) were inoculated into ten-layer Vero cell factories and cultured at 37°C. Cytopathic effect (CPI) was observed. When the viral load (CPE) reached 90%, the virus solution was harvested. After repeated freeze-thawing, the virus harvest was centrifuged to remove cell debris. The cell supernatant was concentrated 10-fold by ultrafiltration through a 100 kD pore size filter and passed through a 25% (w / v) sucrose cushion and centrifuged at 103,745 g for 4 h. The viral proteins were solubilized in PBS buffer (pH 7.2). The virus was collected by density gradient centrifugation at 103,745 g for 4 h using 15%, 25%, 35%, 45%, and 55% (w / v) sucrose. Full particles (FP) and empty particles (EP) were obtained. FP and EP were subjected to cesium chloride density gradient centrifugation at 260,000 g for 24 h at 4°C, and opalescent bands were extracted to obtain purified CV-A4, CV-A2, CV-A5, and Echo 11 virus particles.

[0035] For the preparation of CV-A2 virus-like particles (VLPs), please refer to the literature Yu Yuting, Hu Gang, Luo Zhiyu, et al. Preparation, purification and identification of Coxsackievirus A2 virus-like particles. Chinese Journal of Biological Products, 2021, 34(07): 782-787.

[0036] Cultivation of CV-A6 (EP+FP), CV-A10 (EP+FP), CV-A16 (EP+FP), and EV-A71 (EP+FP) viruses and acquisition of virus particles: CV-A6, CV-A10, CV-A16, and EV-A71 (MOI=0.001) were inoculated into Vero cells, and the virus solution was harvested when the cytopathic effect (CPE) reached 90%. The virus harvest solution was concentrated by ultrafiltration through a 100 kD pore size, and the volume was concentrated. The virus concentrate was subjected to two column chromatography steps to obtain the virus stock solutions of CV-A6, CV-A10, CV-A16, and EV-A71, i.e., mixed virus particles EP+FP.

[0037] CV-A5 DP (Dense Particles): These are viral particles obtained after CV-A5 infection and cesium chloride density gradient centrifugation, which are different from EP and FP. These particles contain a partially cleaved VP1 protein, i.e., a complete VP1 protein and an incomplete VP1 protein, i.e., a partial cleavage of the first 1-40 amino acids of the N-terminus. Information about these particles has been published in the article Jin WP, Lu J, Zhang XY, et al. Efficacy of Coxsackievirus A5 Vaccine Candidates in an Actively Immunized Mouse Model. J Virol. 2021; 95(6): e01743-20. Model.

[0038] Anti-A4 FP mouse serum: immunize SPF-grade 6-8 week old Balb / c mice with CV-A4 FP antigen on days 0 and 14, and collect serum on day 28 to obtain anti-A4 FP mouse serum.

[0039] Normal mouse serum: Obtain SPF level 6-8 week old Balb / c mice and collect serum to obtain normal mouse serum.

[0040] CV-A5 VP1 rabbit serum: The antigen was expressed using conventional prokaryotic expression; that is, the expression vector pGEX-6P-1 containing the VP1 gene was transformed into DE3 E coli expression bacteria. After IPTG induction, the expression bacteria were disrupted and purified using a GST-tagged gravity column to obtain the immunogen. The antigen was injected into New Zealand white rabbits and immunized multiple times to obtain CV-A5 VP1 rabbit serum. Article: Jin Weiping, Lu Jia, Wu Jie, et al. Prokaryotic expression of Coxsackievirus group A type 5 VP1 protein and preparation of polyclonal antibodies. International Journal of Biologicals, 2020, 43(2): 53-57. This information has been published.

[0041] Example 2 Preparation of monoclonal antibodies

[0042] The present invention provides a method for preparing a monoclonal antibody, which is as follows:

[0043] S1: 60 μg of CV-A4 FP particles were mixed with Freund's complete adjuvant (V / V = 1:1). Boosters were administered with Freund's incomplete adjuvant mixed with antigens. Female Balb / c mice (6-8 weeks old) were immunized subcutaneously and intraperitoneally at the back with an injection volume of 500 μL / time. Mice were immunized on days 0, 14, 28, and 42. Serum titers of mice on day 52 were measured by indirect ELISA. Serum titers exceeding 1×10 6 The mice were subjected to shock immunization. The mouse spleen cells were collected on the third day of immunization and PEG 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: (5-10). The hybridoma cells with successful fusion were screened using HAT culture medium.

[0044] S2. The antibody titer of the culture supernatant of the successfully fused hybridoma cells was detected by indirect ELISA, and the titer was greater than 1×10 4 Positive hybridoma cells were cloned and purified three times, and then expanded 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.

[0045] Among them, the method of indirect ELISA screening hybridoma cells is:

[0046] The CV-A4 virus purified particles were prepared into 1 μg / mL coating solution with carbonate buffer at pH = 9.6 and added to the ELISA plate, 100 μL / well, and incubated at 4°C overnight. After the incubation, the plate was washed 5 times with PBST at pH = 7.4; the plate was washed with PBST containing 1% (W / V) bovine serum albumin at pH = 7.4. The plates were blocked with PBST containing (adenosine monophosphate (BSA)) at 37°C for 1 h, 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 an ELISA plate at 100 μL / well. The plates were incubated at 37°C for 1 h, and the plates were washed five times with PBST (pH 7.4). 0.1 μg / mL HRP-labeled goat anti-mouse IgG antibody (Thermo Fisher) was added to the ELISA plate at 100 μL / well. The plates were incubated at 37°C for 1 h, and the plates were washed five times with PBST (pH 7.4). TMB colorimetric solution was added and color was developed at 37°C in the dark for 30 min. The reaction was terminated with 2 M sulfuric acid, and the absorbance at 450 nm was measured 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 wells. Negative wells and hybridoma cell culture medium served as negative controls.

[0047] Example 3 Monoclonal Antibody Sequence Analysis

[0048] The hybridoma cells screened in Example 2 (capable of secreting the 3G4G monoclonal antibody) were inoculated into RPMI 1640 medium containing 10% fetal bovine serum and placed in an incubator at 37°C and 5% CO2. Total RNA was extracted using the Fast Pure Cell / Tissue Total RNA Isolation Kit and reverse transcribed using the Oligo dT primer in the TaKa Ra RimeScript II 1st Strand cDNA Synthesis Kit to obtain single-stranded cDNA. The heavy and light chain variable region genes of the monoclonal antibody 3G4G were amplified using a heavy chain universal primer pair and a light chain universal primer pair with sequences homologous to the cloning vector pUC-Kan. The purified PCR products were respectively cloned into the pUC-Kan vector (purchased from Nanjing GenScript Biotechnology Co., Ltd.). Positive clones were screened using amplification primers and then sequenced. The correctly aligned variable region amino acid sequences were analyzed using the Kabat database. The sequence of the universal forward primer VH-F of the heavy chain is SEQ ID NO.1: acggccagtgaattcmarctgcagsagtcwgg, and the sequence of the reverse primer VH-R is SEQ ID NO.2: gattacgccaagctttgaggagacggtgaccg; the sequence of the universal forward primer VL-F of the light chain is SEQ ID NO.3: acggccagtgaattccgattgtkctsacycartctcca, and the sequence of the reverse primer VL-R is SEQ ID NO.4: gattacgccaagcttcgttggatctccagcttg.

[0049] The determined nucleotide sequence of the heavy chain variable region of monoclonal antibody 3G4G is as follows: GGCTTGC GGGAGTCAGGTGCTGAGCTTGTGAGGCCAGGGGCCTTAGTCAAGTTGTCCTGCAAAGCTTCTGGCTTCAACATTAAAGACTACTATATGCATTGGGTGAAGCAGAGGCCTGAACAGGGCCTGGAGTGGATTGGATGGATTGATCCTGAGAATGGTAATACTATATATGACCCGAAGTTCCAGGGCAAGGCCAGTATAACAGCAGACACATCCTCCAACACAGCCTACCTGCAGCTCAAAAGCCTGACATCTGAGGACACTGCCGTCTATTACTGTGCTCCCTTCTATGATGGTTTTTCCTGGTTTGCTAACTGGGGCCAGGGGACTCCGGTCACCGTCTCCTCA (SEQ ID NO. 5);

[0050] The nucleotide sequence of the light chain variable region is as follows: CCGATTGTGCTGACTCAGTCTCCAG CTTCCTTAGCTGTATCTCTGGGGCAGAGGGCCACCATCTCATACAGGGCCAGCAAAAGTGTCAGTACATCTGGCTATAGTTATATGCACTGGAACCAACAGAAACCAGGACAGCCACCCAGACTCCTCATCTATCTTGTATCCAACCTAGAATCTGGGGT CCCTGCCAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACCCTCAACATCCATCCTGTGGAGGAGGAGGATGCTGCAACCTATTACTGTCAGCACATTAGGGAGCTTCATGTGGAGTTCGGAGGGGGGACCAAGCTGGAGATCCAACGAAGCCTC(SEQ ID NO.6).

[0051] The amino acid sequence encoded by the nucleotide sequence of the heavy chain variable region is as follows: GLRESGAELVRPGA LVKLSCKASGFNIKDYYMHWVKQRPEQGLEWIGWIDPENGNTIYDPKFQ GKASITADTSSNTAYLQLKSLTSEDTAVYYCAPFYDGFSWFANWGQGTPV TVSS (SEQ ID NO. 7);

[0052] The amino acid sequence encoded by the nucleotide sequence of the light chain variable region is as follows: PIVLTQSPASLAVSL GQRATISYRASKSVSTSGYSYMHWNQQKPGQPPRLLIYLVSNLESGVPAR FSGSGSGTDFTLNIHPVEEEDAATYYCQHIRELHVEFGGGTKLEIQRSL (SEQ ID NO. 8).

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

[0054] The amino acid sequence of the heavy chain complementarity determining region VHCDR1 is: DYYMH (SEQ ID NO. 9); the amino acid sequence of the heavy chain complementarity determining region VHCDR2 is: WIDPENGNTIYDPKFQG (SEQ ID NO. 10);

[0055] The amino acid sequence of the heavy chain complementarity determining region VHCDR3 is: FYDGFSWFAN (SEQ ID NO.

[0056] The amino acid sequence of the complementarity determining region of the light chain, VLCDR1: RASKSVSTSGYSYMH (SEQ ID NO. 12);

[0057] The amino acid sequence of the complementarity determining region VLCDR2 of the light chain is: LVSNLES (SEQ ID NO. 13); the amino acid sequence of the complementarity determining region VLCDR3 of the light chain is: QHIRELHVE (SEQ ID NO. 14).

[0058] Example 4 Identification of subtypes of monoclonal antibody 3G4G

[0059] The monoclonal anti-3G4G purified in Example 2 was subjected to 4-20% SDS-PAGE. The results of the reduced SDS-PAGE assay are as follows: Figure 1 As shown, from Figure 1 It can be seen that the heavy chain and light chain of the 3G4G monoclonal antibody correspond to protein bands of 50kDa and 25kDa respectively, and the subtype is IgG2b, which is consistent with the expected results.

[0060] Example 5 Functional Analysis of Monoclonal Antibody 3G4G

[0061] The screened monoclonal antibody 3G4G was analyzed using indirect immunofluorescence assay and immunoblotting assay, as follows:

[0062] (1) Indirect immunofluorescence experiment

[0063] CV-A2, CV-A4 and CV-A5 viruses were inoculated into 6-well plates of RD cells with a confluence of 95%. RD cells without virus inoculation served as negative controls and were placed in a 37°C, 5% CO2 incubator. After culturing the 6-well plates for 24 hours, the cell supernatant was discarded and 2 mL / well of 4% paraformaldehyde was added for fixation at room temperature for 1 hour and washed 5 times / 5 min with 0.01 M PBS. 2 mL / well of 2% BSA-PBST (W / V) solution (containing 0.5% Triton-X 100, V / V) was added for permeabilization at room temperature for 30 minutes and washed 5 times / 5 min with 0.01 M PBS. Blocking was performed with 2% BSA-PBST solution at room temperature for 1 hour and the blocking solution was discarded. 1 mL / well of 2 μg / mL monoclonal antibody 3G4G was added and incubated at room temperature for 1 hour and 0.01 M Wash with PBS 5 times / 5 min; add 1 mL / well, 2 μg / mL goat anti-mouse fluorescent antibody IgG (H+L) (Thermo Fisher), incubate at room temperature for 1 h in the dark, wash with 0.01 M PBS 5 times / 5 min, and add 1 mL / well 5 μg / mL DAPI solution (Biyuntian); observe and photograph with a fluorescence microscope. The results are as follows Figure 2 shown.

[0064] from Figure 2 It can be seen that monoclonal antibody 3G4G can broadly recognize CV-A2 / CV-A4 / CV-A5 virus particles.

[0065] (2) Immunoblotting experiments

[0066] Identification of the CV-A2, CV-A4, and CV-A5 structural protein regions recognized by the 3G4G monoclonal antibody. Enterovirus structural proteins are VP1, VP2, VP3, and VP4. Western blot analysis can be performed to determine the CV-A2, CV-A4, and CV-A5 structural protein regions recognized by 3G4G. The specific steps are as follows:

[0067] CV-A4 FP (FP, solid particles), CV-A2 FP, CV-A2 VLP, CV-A5 FP, CV-A5 DP (DP, dense particles) and Vero cell lysate were added to 4× SDS-PAGE loading buffer (Bio-rad), heated at 100°C for 10 min, and subjected to 4-20% SDS-PAGE. After electrophoresis, the membrane was transferred to a 0.45 μm nitrocellulose membrane using an eBlotTML1 fast wet transfer instrument (GenScript). After transfer, the membrane was blocked with 2% BSA (W / V) in PBST blocking solution at 37°C for 30 min. 10 mL of 0.3 μg / mL 3G4G and 1:10,000 (V / V) diluted CV-A5 VP1 rabbit serum were added, respectively, incubated at 37°C for 1 h, and washed 5 times / 5 min with 0.01 M PBST. 10 mL of HRP-labeled goat anti-mouse IgG or HRP-labeled goat anti-rabbit IgG was diluted 1:10000, incubated at 37°C for 45 min, and washed 5 times / 5 min with 0.01M PBST. The color development solution Immobilon western chemiluminescent HRP substrate (Millipore) was used for chemical exposure (Gene). The antibody recognition results were as shown in Figure 2. Figure 3 shown.

[0068] Depend on Figure 3 The 3G4G monoclonal antibody specifically binds to the VP1 region of the structural protein of the CV-A2, CV-A4, and CV-A5 viruses, indicating that the recognized antigenic epitope is a linear epitope. Studies have shown that the N-terminus of the VP1 protein of CV-A5 DP is cleaved, while the VP1 protein of CV-A5FP is intact. Therefore, 3G4G recognizes the N-terminal amino acids of the CV-A2, CV-A4, and CV-A5 VP1 proteins.

[0069] (3) Monoclonal antibody 3G4G binding titer detection

[0070] ELISA was used to determine the binding ability of monoclonal antibody 3G4G to enterovirus A swarm viruses CV-A2, CV-A4, CV-A5, CV-A6, CV-A10, CV-A16, and EV-A71, with enterovirus B swarm echovirus (Echo 11) serving as a control. The specific experimental steps are as follows:

[0071] CV-A2 (FP), CV-A4 (EP, FP), CV-A5 (FP), CV-A6 (EP+FP), CV-A10 (EP+FP), CV-A16 (EP+FP), EV-A71 (EP+FP), and Echo 11 (FP) purified particles were prepared into 1 μg / mL coating solution in carbonate buffer (pH = 9.6), added to the ELISA plate at 100 μL / well, and incubated at 4°C overnight. After the incubation, the plate was washed three times with PBST (pH = 7.4);

[0072] Block with PBST (pH 7.4) containing 1% (mass-volume ratio) BSA at 37°C for 1 h, and discard the blocking solution;

[0073] Add primary antibody: dilute 1 mg / mL monoclonal antibody 3G4G tenfold (volume ratio), add 100 μL / well of the diluted antibody to the ELISA plate, incubate at 37°C for 1 hour, and wash the plate five times with PBST (pH 7.4);

[0074] Add secondary antibody: add 0.1 μg / mL HRP-labeled goat anti-mouse IgG antibody (purchased from Wuhan Boster Biotechnology Co., Ltd.) to the ELISA plate, 100 μL / well, incubate at 37°C for 1 h, and wash the plate 5 times with PBST (pH = 7.4);

[0075] TMB colorimetric solution was added and color was developed at 37°C in the dark for 30 min. The reaction was terminated with 2 M sulfuric acid and the absorbance at A450 nm was measured using a microplate reader.

[0076] Titer calculation method: the maximum dilution factor that is 2.1 times greater than the OD value of the negative well. The negative control is the addition of secondary antibody only without 3G4G.

[0077] Test results such as Figure 4 As shown, according to Figure 4 It can be calculated that monoclonal antibody 3G4G has the same binding titer to CV-A2, CV-A4, and CV-A5. Among them, the positive control is CV-A4 FP mouse serum, which can specifically recognize CV-A4-EP and CV-A4-FP.

[0078] Example 6 Study on the linear epitope of monoclonal antibody 3G4G

[0079] (1) Preliminary mapping of the monoclonal antibody 3G4G linear epitope

[0080] The amino acid sequence of the linear epitope of the CV-A4 monoclonal antibody was preliminarily identified by ELISA. The method is as follows: overlapping peptides of the VP1 region of the CV-A4 structural protein were synthesized, covering the full length of VP1 (305 amino acids). The peptide sequences are shown in Table 1 below:

[0081] Table 1 Overlapping peptide sequences in the VP1 region of the CV-A4 structural protein

[0082]

[0083]

[0084] Preliminary positioning was performed using an indirect ELISA experiment: the peptide was prepared at a concentration of 4 μg / mL with a carbonate buffer solution of pH 9.6 to prepare a coating solution of 1 μg / mL, and added to the ELISA plate, 100 μL / well, and incubated at 4°C overnight. After the incubation, the plate was washed three times with PBST of pH 7.4; the plate was blocked with PBST containing 1% (W / V) BSA at pH 7.4 at 37°C for 1 hour, and the blocking solution was discarded; the monoclonal antibody was diluted tenfold (V / V), and 1 μg / ml of monoclonal antibody 3G4G, normal mouse serum, and anti-A4 were taken. FP mouse serum, 100 μL / well, was added to the ELISA plate, incubated at 37°C for 1 hour, and washed five times with PBST (pH = 7.4); 0.1 μg / mL HRP-labeled goat anti-mouse IgG antibody (purchased from Wuhan Boster Biotechnology Co., Ltd.) was added to the ELISA plate, 100 μL / well, incubated at 37°C for 1 hour, and washed five times with PBST (pH = 7.4); TMB (3,3',5,5'-tetramethylbenzidine) colorimetric solution was added and color was developed 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 measured using a microplate reader;

[0085] Titer calculation method: the maximum dilution factor that is 2.1 times greater than the OD value of the negative well; negative mouse serum is the negative control.

[0086] The preliminary results of linear epitope mapping of monoclonal antibodies are shown in Figure 5 A and 5B, from Figure 5 As can be seen from A and 5B, the monoclonal antibody can bind to peptide segment 1 of the CV-A4 structural protein VP1 (i.e., the first 1-15 amino acids of VP1).

[0087] (2) Precise localization of the monoclonal antibody 3G4G linear epitope

[0088] In order to further narrow the localization range of monoclonal antibody binding peptides, a single peptide was coated in the wells of a 96-well plate to identify the specific binding peptide. To further determine the binding residue, a set of single amino acid substitutions from X (any amino acid except G) to G were synthesized, with a total of 14 single-point mutation peptides, as shown in Table 2, covering the first 15 amino acids of the VP1 N-terminus. This was used to further locate the residue responsible for the binding of a single monoclonal antibody. The coating concentration range was 5-10 μg / mL, with 100 μL / well synthetic peptide solution. The subsequent ELISA steps were the same as the preliminary localization method described above. The results are shown in Table 2:

[0089] Table 2 Precise positioning of the key binding sites of monoclonal antibody 3G4G

[0090]

[0091] Among them, + and - indicate positive or negative ELISA results.

[0092] As shown in Table 2, amino acids 2 to 15 at the N-terminus of VP1 (VP1-2 to -15) were mutated from their original residues to glycine, i.e., VP1-2D-G indicates that the D at position 2 was mutated to G, while the first glycine was retained. These synthetic peptides containing single amino acid changes, as shown in the table, carry a single point mutation (G) and are coated into microtiter plates for detection. For the 3G4G monoclonal antibody to recognize CV-A4, CV-A2, and CV-A5, residues at positions 2-4 (DAI), 6-8 (DAI), and 10 (N) are essential. The conserved residues at the N-terminus of CV-A4, A2, and A5 VP1, i.e., 2-4 (DAI), 6-8 (DAI), and 10 (N), form the essential footprint for 3G4G monoclonal antibody binding.

[0093] (3) Amino acid sequence alignment of linear epitopes of monoclonal antibodies

[0094] Compared with other serotypes of the Coxsackievirus group A tested, amino acid sequence alignment of the seven serotypes tested showed that the first 15 residues of VP1 in CV-A2, CV-A4 and CV-A5 had a high degree of identity (see Table 3 for details). The specificity of the monoclonal antibody 3G4G was determined by footprints involving multiple residences, which explained the presence of cross-reactive epitopes and type-specific epitopes of CV-A2, CV-A4 and CV-A5 in this region. Several substitutions in this region can affect the binding of the monoclonal antibody to CV-A6, CV-A10, CV-A16 and EV-A71.

[0095] Table 3 Alignment of the N-terminal amino acid sequences of enterovirus CV-A2, A4, A5, A6, A10, A16 and EV-A71 VP1

[0096]

[0097] The bases with _ represent conserved sites, and the monoclonal antibody binding footprints are marked in yellow, which is the same color as in Table 2.

[0098] Through experiments, the inventors further found that the amino acid sequence of the heavy chain of the monoclonal antibody 3G4G, as represented by SEQ ID No. 7, has equivalent functions as a new sequence formed by replacing, deleting or adding one or more amino acids, or an amino acid sequence having more than 95% homology to the amino acid sequence represented by SEQ ID No. 7; and the amino acid sequence of the light chain, as represented by SEQ ID No. 8, has equivalent functions as a new sequence formed by replacing, deleting or adding one or more amino acids, or an amino acid sequence having more than 95% homology to the amino acid sequence represented by SEQ ID No. 8.

[0099] 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 that recognizes Coxsackievirus A2, A4, and A5, characterized in that: The monoclonal antibody has a heavy chain complementary determining region CDR1 as shown in SEQ ID NO.9, a heavy chain complementary determining region CDR2 as shown in SEQ ID NO.10, a heavy chain complementary determining region CDR3 as shown in SEQ ID NO.11, and The light chain complementary determining region CDR1 is shown in SEQ ID NO.12, the light chain complementary determining region CDR2 is shown in SEQ ID NO.13, and the light chain complementary determining region CDR3 is shown in SEQ ID NO.

14.

2. The monoclonal antibody for recognizing Coxsackievirus A2, A4 and A5 according to claim 1, characterized in that The monoclonal antibody has a heavy chain with an amino acid sequence shown in SEQ ID NO. 7; and / or, the monoclonal antibody has a light chain with an amino acid sequence shown in SEQ ID NO.

8.

3. The monoclonal antibody for recognizing Coxsackievirus A2, A4 and A5 according to claim 2, characterized in that The heavy chain amino acid sequence 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.7, or an amino acid sequence having more than 95% homology to the amino acid sequence shown in SEQ ID NO.7; and / or the light chain amino acid sequence 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.8, or an amino acid sequence having more than 95% homology to the amino acid sequence shown in SEQ ID No.

8.

4. A polynucleotide molecule encoding the monoclonal antibody recognizing Coxsackievirus A2, A4 and A5 according to claim 1 or 2.

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

6.

6. A kit for detecting Coxsackievirus A2, A4 and A5, characterized in that: The kit comprises the monoclonal antibody according to claim 1 or 2 or 3.

7. Use of the monoclonal antibody according to claim 1, 2 or 3 in the preparation of a reagent or kit for detecting one, two or three of Coxsackievirus A2, A4 and A5.

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