Monoclonal antibody recognizing the N-terminus of CV-A4 VP1 protein and its application

Abstract

The present invention relates to the field of molecular biology, and in particular to a monoclonal antibody for recognizing CV-A4 and its application. The monoclonal antibody has heavy chain complementary determining regions CDR1 to CDR3 shown in SEQ ID NOs. 9 to 11, and light chain complementary determining regions CDR1 to CDR3 shown in SEQ ID NOs. 12 to 14; or has heavy chain complementary determining regions CDR1 to CDR3 shown in SEQ ID NOs. 19 to 21, and light chain complementary determining regions CDR1 to CDR3 shown in SEQ ID NOs. 22 to 24; or has heavy chain complementary determining regions CDR1 to CDR3 shown in SEQ ID NOs. 29 to 31, and light chain complementary determining regions CDR1 to CDR3 shown in SEQ ID NOs. 32 to 34, and can specifically recognize the N-terminus of the structural protein VP1 of CV-A4.

Description

Technical Field

[0001] The invention belongs to the field of molecular biology, and in particular relates to a monoclonal antibody capable of identifying the N-terminus of the VP1 protein of Coxsackievirus A group 4 (CV-A4) and an application thereof. Background Art

[0002] The enterovirus protein capsid is composed of four structural proteins, VP1, VP2, VP3 and VP4, and is symmetrically arranged in an icosahedral cubic shape. VP1, VP2 and VP3 form the outer shell, VP4 is located on the inner surface of the particle, and VP1 protein is located on the outermost layer of the virus particle. It has good immunogenicity and contains the most neutralizing epitopes. VP1 is mainly involved in immune response and host-cell receptor binding, and participates in the host's immune process. After VP1 binds to the cell receptor, it causes conformational changes and recognizes viral RNA to enter the cell. In addition, the amino acid and nucleotide sequences of VP1 are closely related to the neutralizing antigen typing, which can be used for virus identification and evolution analysis. The study of the self-cleavage mechanism of VP1 is very important for the study of the entire life cycle of the virus and the exploration of the process of conformational changes. At present, the diagnosis and vaccine development established for the enterovirus VP1 protein has important development potential. The discovery of mouse monoclonal antibodies or rabbit monoclonal antibodies (neutralizing and non-neutralizing types) that specifically recognize different regions of the VP1 protein will play an important role in promoting the basic research of the VP1 protein, protein function verification and the development of kits.

[0003] Coxsackievirus A4 (CV-A4) belongs to the Picornaviridae family and the Enterovirus genus. It mainly causes herpetic pharyngitis and hand, foot and mouth disease in children. In recent years, CV-A4 has repeatedly broken out in Fujian, Shandong, Sichuan, Yunnan and Jiangsu in my country, as well as in other countries such as Thailand, Australia and Italy. Although most CV-A4 infections are usually asymptomatic, they are still highly contagious. In clinical cases, a variety of diseases have appeared, ranging from mild symptoms (such as erythema, maculopapular rash) to severe symptoms (such as aseptic meningitis, myocarditis and acute flaccid paralysis). As the frequency of CV-A4 increases globally, new CV-A4 viruses have also begun to appear.

[0004] Therefore, the development of monoclonal antibodies that specifically recognize CV-A4 is of great significance for quality control of vaccine development, detection of clinical samples and laboratory virus identification, and research on the basic functions of CV-A4. Summary of the invention

[0005] Based on this, one of the objects of the present invention is to provide a monoclonal antibody capable of recognizing the N-terminus of CV-A4 VP1 protein, wherein the monoclonal antibody is 2E1A monoclonal antibody or 2D1B monoclonal antibody or 17C4C monoclonal antibody, wherein the 2E1A monoclonal antibody has heavy chain complementary determining regions CDR1 to CDR3 shown in SEQ ID NOs. 9 to 11, and light chain complementary determining regions CDR1 to CDR3 shown in SEQ ID NOs. 12 to 14;

[0006] The 2D1B monoclonal antibody has heavy chain complementary determining regions CDR1 to CDR3 shown in SEQ ID NOs. 19 to 21, and light chain complementary determining regions CDR1 to CDR3 shown in SEQ ID NOs. 22 to 24;

[0007] The 17C4C monoclonal antibody has the heavy chain complementary determining regions CDR1 to CDR3 shown in SEQ ID NOs. 29 to 31, and the light chain complementary determining regions CDR1 to CDR3 shown in SEQ ID NOs. 32 to 34.

[0008] This monoclonal antibody is different from the existing CV-A4 monoclonal antibody. It can specifically recognize the N-terminal 15 amino acids of the structural protein VP1 of CV-A4, and can be used for basic research on the intermediate function of the morphological conformational transition of CV-A4 virus particles and the role of the N-terminal amino acids in penetrating the cell membrane and releasing viral RNA into the cell.

[0009] Preferably, the 2E1A monoclonal antibody has a heavy chain with an amino acid sequence as shown in SEQ ID NO.7; and / or, the 2E1A monoclonal antibody has a light chain with an amino acid sequence as shown in SEQ ID NO.8.

[0010] Preferably, the 2D1B monoclonal antibody has a heavy chain with an amino acid sequence as shown in SEQ ID NO.17; and / or, the 2D1B monoclonal antibody has a light chain with an amino acid sequence as shown in SEQ ID NO.18.

[0011] Preferably, the 17C4C monoclonal antibody has a heavy chain with an amino acid sequence as shown in SEQ ID NO.27; and / or, the 17C4C monoclonal antibody has a light chain with an amino acid sequence as shown in SEQ ID NO.28.

[0012] Preferably, the heavy chain amino acid sequence of the 2E1A 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 2E1A 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.

[0013] Preferably, the heavy chain amino acid sequence of the 2D1B 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.17, or an amino acid sequence having more than 95% homology with the amino acid sequence shown in SEQ ID NO.17; and / or, the light chain amino acid sequence of the 2D1B 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.18, or an amino acid sequence having more than 95% homology with the amino acid sequence shown in SEQ ID NO.18.

[0014] Preferably, the heavy chain amino acid sequence of the 17C4C 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.27, or an amino acid sequence having more than 95% homology with the amino acid sequence shown in SEQ ID NO.27; and / or, the light chain amino acid sequence of the 17C4C 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.28, or an amino acid sequence having more than 95% homology with the amino acid sequence shown in SEQ ID NO.28.

[0015] The second objective of the present invention is to protect the polynucleotide molecule encoding the monoclonal antibody that recognizes the N-terminus of CV-A4 VP1 protein.

[0016] Preferably, the polynucleotide molecule encoding the 2E1A monoclonal antibody has a nucleotide sequence shown in SEQ ID NO.5 and / or SEQ ID NO.6.

[0017] Preferably, the polynucleotide molecule encoding the 2D1B monoclonal antibody has a nucleotide sequence shown in SEQ ID NO.15 and / or SEQ ID NO.16.

[0018] Preferably, the polynucleotide molecule encoding the 17C4C monoclonal antibody has the nucleotide sequence shown in SEQ ID NO.25 and / or SEQ ID NO.26.

[0019] The third object of the present invention is to protect a kit for detecting Coxsackievirus A4, which comprises the above-mentioned monoclonal antibody or the monoclonal antibody encoded by the above-mentioned polynucleotide molecule.

[0020] A fourth object of the present invention is to protect the use of the above-mentioned monoclonal antibody or the monoclonal antibody encoded by the polynucleotide molecule in the preparation of a reagent or kit for detecting Coxsackievirus A4.

[0021] The fifth object of the present invention is to protect 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 Coxsackievirus A4.

[0022] The sixth object of the present invention is to protect the use of the above-mentioned monoclonal antibody or the monoclonal antibody encoded by the polynucleotide molecule in the study of VP1 protein of Coxsackievirus A4 and changes in viral structural conformation.

[0023] The monoclonal antibody provided by the present invention is prepared by immunizing mice with purified solid particles of Coxsackievirus group A type 4 (CV-A4) virus to prepare and screen hybridoma cells, and is an IgG2b type non-neutralizing antibody that can specifically recognize CV-A4 and does not recognize other enteroviruses.

[0024] The monoclonal antibody 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-A4 infection, and can also be used for laboratory identification of antibodies for clinical virus isolates. It can also be used to prepare quantitative detection reagents for CV-A4 antigen-containing intermediates and products for preventive vaccine production, as well as for research on the function of CV-A4 VP1 gene and the process of virus assembly and particle conformation changes. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 The SDS-PAGE images of IgG light and heavy chains of monoclonal antibodies 2E1A, 2D1B and 17C4C;

[0026] Figure 2 Identification diagram for monoclonal antibodies 2E1A, 2D1B and 17C4C recognizing CV-A4 structural protein VP1;

[0027] Figure 3 The results of indirect immunofluorescence detection of antigens in RD cells infected with CV-A4 by monoclonal antibodies 2E1A, 2D1B and 17C4C;

[0028] Figure 4 The linear epitope peptide library and preliminary positioning structure diagram of the peptide segment of monoclonal antibodies 2E1A, 2D1B and 17C4C, where A is the result of the linear epitope peptide library identifying the full length of VP1 (positions 1 to 305), and B is the result of identifying the 1st to 50th amino acids of the linear epitope peptide segment of VP1. DETAILED DESCRIPTION

[0029] The present invention is further described in detail below in conjunction with specific embodiments so that those skilled in the art can understand the present invention more clearly.

[0030] 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 the field without creative work belong to the protection scope of the present invention.

[0031] 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.

[0032] Source of reagents:

[0033] Freund's complete and incomplete adjuvants: purchased from Sigma;

[0034] Female Balb / c mice: purchased from Wuhan Institute of Biological Products Co., Ltd.;

[0035] Isotyping Kit for Mouse Monoclonal Antibody: purchased from Beijing Sino Biological Technology Co., Ltd.;

[0036] RD cells: provided by Wuhan Institute of Biological Products Co., Ltd.;

[0037] goat anti-mouse IgG (H+L) Alexa Fluor 488 fluorescent secondary antibody (Thermo Fisher);

[0038] HRP-labeled goat anti-mouse IgG: purchased from Wuhan Boster Biotechnology Co., Ltd.

[0039] Example 1 Material Preparation

[0040] CV-A4 (strain is CV-A4 / R3179 / XY / 2017, see the 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 is CVA2-1388-M14 / XY / CHN / 2017, NCBI database accession number is MW846233), CV-A5 (strain is CV-A5-3487-M14-XY-CHN-2017, NCBI database accession number is MW079817) virus culture: CV-A4, A2 and A5 viruses were inoculated into serum-free Vero cells respectively, and the viruses were harvested and packaged when the cell pathological rate reached 90%.

[0041] Preparation of CV-A4, A2 and A5 virus solid particles (Full particle, FP) and empty particles (Empty particle, EP): CV-A4, CV-A2 and CV-A5 (MOI = 0.001) were inoculated into a ten-layer Vero cell factory, cultured at 37°C, and the virus solution was harvested when the cytopathic effect (CPE) reached 90%. The virus harvest solution was repeatedly frozen and thawed and centrifuged to remove cell debris. The cell supernatant was concentrated by ultrafiltration with a pore size of 100KD, concentrated 10 times, and cushioned with 25% (W / V) sucrose, centrifuged at 103745g for 4h, and the virus protein was dissolved with PBS buffer at pH = 7.2. The virus was collected by sucrose density gradient centrifugation at 15%, 25%, 35%, 45%, and 55% (W / V), and centrifuged at 103745g for 4h to obtain solid particles (Full particle, FP) and empty particles (Empty particle, EP). 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 virus particles of CV-A4, CV-A2 and CV-A5.

[0042] For the preparation of CV-A2 virus-like particles (VLPs), please refer to 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.

[0043] CV-A5 DP (DP, dense particles): Another virus particle obtained when CV-A5 is infected with the virus and subjected to cesium chloride density gradient centrifugation, which is different from EP and FP. The VP1 protein of this particle is partially cleaved, and there is an incomplete VP1 protein, that is, a part of the VP1 protein whose first 1-40 amino acids of the N-terminus are cleaved. The information of this particle has been disclosed 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.

[0044] Anti-A4 FP mouse serum: A4 FP antigen was intraperitoneally injected into Balb / c mice, immunized once every two weeks, for a total of two immunizations. On the 28th day after the first immunization, mouse serum was collected and aliquoted after centrifugation.

[0045] Example 2 Preparation of monoclonal antibodies

[0046] This example provides a method for preparing a monoclonal antibody, which is as follows:

[0047] S1, 60 μg of CV-A4 FP particles were mixed with Freund's complete adjuvant (V / V=1:1), and then Freund's complete adjuvant mixed with antigen was used for booster immunization. Female Balb / c mice (6-8 weeks old) were immunized subcutaneously and intraperitoneally at the back, with an injection volume of 500 μL / time, for a total of 6 injection points, and mice were immunized on days 0, 14, 28, and 42. The serum titer of mice on day 52 was detected by indirect ELISA, and the ELISA-bound serum titer exceeded 1×10 6 The mice were subjected to shock immunization, and the mouse spleen cells were collected on the 3rd day after the last immunization. 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 and myeloma cells was controlled at (1:5) to (1:10); the hybridoma cells with successful fusion were screened by HAT culture medium.

[0048] 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 ascites was prepared by expansion culture. Ascites was purified by three-step saturated ammonium sulfate method to obtain monoclonal antibodies, and the antibody subtype was identified using Isotyping Kit for Mouse Monoclonal Antibody.

[0049] Among them, the method of indirect ELISA screening hybridoma cells is:

[0050] The purified CV-A4 virus particles were prepared with 1 μg / mL coating solution using carbonate buffer at pH = 9.6 and added to the ELISA plate, 100 μL / well, and incubated overnight at 4°C. After the incubation, the plate was washed 5 times with PBST at pH = 7.4; the plate was then incubated with PBST containing 1% (W / V) bovine serum albumin at pH = 7.4. albumin, BSA) in PBST at 37℃ for 1h, and discard the blocking solution; dilute the supernatant of the hybridoma cell culture fluid to be detected by ten-fold ratio (V / V), take the diluted hybridoma cell supernatant and add it to the ELISA plate, 100μL / well, incubate at 37℃ for 1h, and wash the plate 5 times with PBST at pH=7.4; add 0.1μg / mL HRP-labeled goat anti-mouse IgG antibody (Boster) to the ELISA plate, 100μL / well, incubate at 37℃ for 1h, and wash the plate 5 times with PBST at pH=7.4; add TMB colorimetric solution to color at 37℃ in the dark for 30min, terminate the reaction with 2M sulfuric acid, and detect the absorbance at 450nm with an ELISA reader; titer calculation method: the maximum dilution factor when it is greater than 2.1 times the OD value of the negative well; hybridoma cell culture medium is used as a negative control.

[0051] Example 3 Sequence analysis of the screened monoclonal antibodies

[0052] The hybridoma cells screened in Example 2 (capable of secreting monoclonal antibodies 2E1A, 2D1B and 17C4C, respectively) were inoculated in RPMI 1640 medium (Gibco) containing 20% ​​fetal bovine serum and cultured at 37°C. Total RNA was extracted using Fast PureCell / Tissue Total RNA Isolation Kit, and single-stranded cDNA was obtained by reverse transcription. The heavy chain and light chain variable region genes of monoclonal antibodies 2E1A, 2D1B and 17C4C were amplified by heavy chain universal primers with homologous sequences to the cloning vector pUC-Kan, and the purified PCR products were cloned into pUC-Kan vectors (purchased from Nanjing GenScript Biotechnology Co., Ltd.). Positive clones were obtained by screening and sequencing, and the sequenced sequences were sequenced in the Kabat database to obtain the correct light chain and heavy chain variable region amino acid sequences. Among them, the sequence of the heavy chain universal forward primer VH-F is SEQ ID NO.1: acggccagtgaattcmarctgcagsagtcwgg, the sequence of the reverse primer VH-R is SEQ ID NO.2: gattacgccaagctttgaggagacggtgaccg) and the sequence of the light chain universal forward primer VL-F is SEQ ID NO.3: acggccagtgaattccgattgtkctsacycartctcca, the sequence of the reverse primer VL-R is SEQ ID NO.4: gattacgccaagcttcgttggatctccagcttg.

[0053] The nucleotide sequence of the heavy chain variable region of the monoclonal antibody 2E1A determined was: ACTCGTGAACCTTCTCAGATTCTGTCCCTCACCTGTTCTGTCACTGGCGACTCCATCACCAGTGGTTACTGGAACTGGATCCGGAAATTCCCAGGGAATAAACTTGAGTATATGGGGTACATAGGCCACAGTGGTACCACTTACTACAATCCATCTCTCAAAAGTCGAGTCTCCATCACTCGAGACACATCCAAGAACCAGTACTTCCTGCAGTTGAATTCTGTGACTACTGAGGACACAGCCACATATTACTGTGCAAGATTGGGATATTACTCCGGTAGTAGCTACGACAAATGGGGCCAAGGCACCACTCTCACCGTCTCCTCA (SEQ ID NO.5);

[0054] The nucleotide sequence of the light chain variable region is: CCGATTGTTCTGACTCAATCTCCAGCT TCTTTGGCTGTGTCTCTAGGGCAGAGGGCCACCATATCCTGCAGAGCCAGTGAAACTGTTGATAGTTATGGCAATAGTTGTATGCACTGGTACCAGCAGAAATCAGGACAGCCACCCAAACTCCTCATCTATCGTGCATCCAACCTAGAATCTGGGATCC CTGCCAGGTTCAGTGGCATGGGTCTAGGACAGACTTCACCCTCACCATTAATCCTGTGGCGGCTGATGATGTTGCAACCTATTACTGTCAGCAAAGTAGTGAGGTTCCGTACACGTTCGGAGGGGGGACCAAGCTGGAGATCCAACGAAGCTAC(SEQID NO.6);

[0055] The amino acid sequence of the heavy chain variable region is: TREPSQILSLTCSVTGDSITSGYWNWIR KFPGNKLEYMGYIGHSGTTYYNPSLKSRVSITRDTSKNQYFLQLNSVTTE DTATYYCARLGYYSGSSYDKWGQGTTLTVSS (SEQ ID NO. 7);

[0056] The amino acid sequence of the light chain variable region is: PIVLTQSPASLAVSLGQRATISCRASETV DSYGNSCMHWYQQKSGQPPKLLIYRASNLESGIPARFSGSGSRTDFTLTIN PVAADDVATYYCQQSSEVPYTFGGGTKLEIQRSY (SEQ ID NO. 8);

[0057] The sequences of the six CDR regions (complementarity determining regions) analyzed are as follows: amino acid sequence of the heavy chain complementarity determining region VHCDR1: SGYWN (SEQ ID NO. 9);

[0058] The amino acid sequence of the heavy chain complementarity determining region VHCDR2: YIGHSGTTYYNPSLKS (SEQ ID NO. 10);

[0059] The amino acid sequence of the heavy chain complementarity determining region VHCDR3: LGYYSGSSYDK (SEQ ID NO. 11);

[0060] The amino acid sequence of the complementarity determining region VLCDR1 of the light chain: RATISCRASETVDSYGNSCMH (SEQ ID NO. 12);

[0061] The amino acid sequence of the complementarity determining region VLCDR2 of the light chain: RASNLES (SEQ ID NO. 13);

[0062] The amino acid sequence of the complementarity determining region VLCDR3 of the light chain is: QQSSEVPYT (SEQ ID NO. 14).

[0063] The nucleotide sequence of the heavy chain variable region of the monoclonal antibody 2D1B determined was: CAGTCAGGACCTAGACTCGTGAAACCTTCTCAGATTCTGTCCCTCACCTGTTCTGTCACTGGCGACTCCATCACCAGTGGTTACTGGAACTGGATCCGGAAATTCCCAGGGAATAAACTTGAGTATATGGGGTACATAGGCCACAGTGGTACCACTTACTACAATCCATCTCTCAAAAGTCGAGTCTCCATCACTCGAGACACATCCAAGAACCAGTACTTCCTGCAGTTGAATTCTGTGACTACTGAGGACACAGCCACATATTACTGTGCAAGATTGGGATATTACTCCGGTAGTAGCTACGACAAATGGGGCCAAGGCACCACTCTCACCGTCTCTCAA (SEQ ID NO.15);

[0064] The nucleotide sequence of the light chain variable region is: CCGATTGTGCTGACTCAGTCTCCAGCTTCCTTAGCTGTATCTCTGGGGCAGAGGGCCACCATCTCATACAGGGCCAGCAAAAGTGTCAGTACATCTGGCTATAGTTATATGCACTGGAACCAACAGAAACCAGGACAGCCACCCAGACTCCTCATCTATCTTGTA TCCAACCTAGAATCTGGGGTCCCTGCCAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACCCTCAACATCCATCCTGTGGAGGAGGAGGATGCTGCAACCTATTACTGTCAGCACATTAGGAGCTACACGTCGAAGTTCGGGGGGGGAACAAAACTCGAGATCCAGAGGGCT(SEQ IDNO.16);

[0065] The amino acid sequence of the heavy chain variable region is: QSGPRLVKPSQILSLTCSVTGDSITSGYWNWIRKFPGNKLEYMGYIGHSGTTYYNPSLKSRVSITRDTSKNQYFLQLNSVTTEDTATYYCARLGYYSGSSYDKWGQGTTLTVSQ (SEQ ID NO. 17);

[0066] The amino acid sequence of the light chain variable region is: PIVLTQSPASLAVSLGQRATISYRASKSVSTSGYSYMHWNQQKPGQPPRLLIYLVSNLESGVPARFSGSGSGTDFTLNIHPVEEEDAATYYCQHIRSYTSKFGGGTKLEIQRA (SEQ ID NO. 18);

[0067] The sequences of the six CDR regions (complementarity determining regions) analyzed are as follows:

[0068] The amino acid sequence of the heavy chain complementarity determining region VHCDR1: SGYWN (SEQ ID NO. 19);

[0069] The amino acid sequence of the heavy chain complementarity determining region VHCDR2: YIGHSGTTYYNPSLKS (SEQ ID NO. 20);

[0070] The amino acid sequence of the heavy chain complementarity determining region VHCDR3: LGYYSGSSYDK (SEQ ID NO. 21);

[0071] The amino acid sequence of the complementary determining region VLCDR1 of the light chain: RASKSVSTSGYSYMH (SEQ ID NO. 22);

[0072] The amino acid sequence of the complementarity determining region VLCDR2 of the light chain: LVSNLES (SEQ ID NO. 23);

[0073] The amino acid sequence of the complementarity determining region VLCDR3 of the light chain is: QHIRSYTSK (SEQ ID NO. 24).

[0074] The nucleotide sequence of the heavy chain variable region of the monoclonal antibody 17C4C determined was: CGAGCGCGCAGTCTGGGATGTGCTGGCAGGCCTGGGGCTTCAGTGAGGATGTCCTGCAAGGCTTCTGACTACACCTTTAGCAGCTACTGGATGCACTGGGTAAAGCAGAGGCCTGGACAGGGTCTGGAATGGATTGGCGCTATTTATCCTGGAAATAGTGATACTAGCTACAACCAGAAGTTCAAGGGCAAGGCCAAACTGACTACAGTCATATCCACCAACACTGCCTACATGGAGCTCAGCAGCCTGACAAATGAGGACTCTGCGGTCTATTACTGTACAAGAATCTACGGTAGTAACCAGCGGTACTTCGATGTCTGGGGCGCAGGGACCACGGTCACCGTCTCCTCA (SEQ ID NO.25);

[0075] The nucleotide sequence of the light chain variable region is: GCTGATGTGCTATCAGTCTCAGCCATCCTGTCTGTGAGTCCAGGAGAAAGAGTCAGTTTCTCCTGCAGGGCCAGTCAGAGCATTGGCACAAACATACACTGGTATCAGCAAGGAGCAAATGGTTCTCCAAGGCTTCTCATAAAGTATGCTTCTGAGTC TATCTCTGGGATCCCTTCCAGGTTTAGTGGCAGTGGATCAGGGACAGATTTTACTCTTAGCATCAACAGTGTGGAGTCTGAAGATATTGCAGATTATTACTGTCAACAGAGTAATAGCTGGCCGTGGACGTTCGGTGGAGGCACCAAGCTGGAGATCCAACGAAGCTT(SEQ ID NO.26);

[0076] The amino acid sequence of the heavy chain variable region is: RARSLGCAGRPGASVRMSCKASDYTFSSYWMHWVKQRPGQGLEWIGAIYPGNSDTSYNQKFKGKAKLTTVISTNTAYMELSSLTNEDSAVYYCTRIYGSNQRYFDVWGAGTTVTVSS (SEQ ID NO. 27);

[0077] The amino acid sequence of the light chain variable region is: ADVLSVSAILSVSPGERVSFSCRASQSIGTNIHWYQQGANGSPRLLIKYASESISGIPSRFSGSGSGTDFTLSINSVESEDIADYYCQQSNSWPWTFGGGTKLEIQRSL (SEQ ID NO. 28);

[0078] The sequences of the six CDR regions (complementarity determining regions) analyzed are as follows:

[0079] The amino acid sequence of the heavy chain complementarity determining region VHCDR1: SYWMH (SEQ ID NO. 29);

[0080] The amino acid sequence of the heavy chain complementarity determining region VHCDR2: AIYPGNSDTSYNQKFKG (SEQ ID NO. 30);

[0081] The amino acid sequence of the heavy chain complementarity determining region VHCDR3: IYGSNQRYFDV (SEQ ID NO. 31);

[0082] The amino acid sequence of the complementary determining region VLCDR1 of the light chain is: RASQSIGTNIH (SEQ ID NO.32); the amino acid sequence of the complementary determining region VLCDR2 of the light chain is: YASESIS (SEQ ID NO.33); the amino acid sequence of the complementary determining region VLCDR3 of the light chain is: QQSNSWPWT (SEQ ID NO.34).

[0083] Example 4 Subtype Identification

[0084] The monoclonal antibodies 2E1A, 2D1B and 17C4C purified in Example 2 were analyzed by 4-20% SDS-PAGE. The results of reducing SDS-PAGE were as follows: Figure 1 As shown. Figure 1 It was found that the heavy chain and light chain of 2E1A, 2D1B and 17C4C monoclonal antibodies corresponded to protein bands of 50kDa and 25kDa respectively, and the subtype was IgG2b, which was consistent with the expected results.

[0085] Example 5 Functional Analysis

[0086] The functions of the three monoclonal antibodies 2E1A, 2D1B and 17C4C were analyzed by indirect immunofluorescence assay and immunoblotting assay, respectively, as follows:

[0087] (1) Immunoblotting

[0088] The enterovirus structural proteins are VP1, VP2, VP3 and VP4. The structural protein regions of CV-A4 recognized by the three monoclonal antibodies 2E1A, 2D1B and 17C4C were identified by immunoblotting experiments. The steps are as follows:

[0089] The same loading amount of 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 for 10 minutes for 4-20% SDS-PAGE; after electrophoresis, the membrane was transferred to a 0.45μm nitrocellulose membrane using a fast wet transfer instrument (GenScript); after transfer, 2% BSA (W / V) PBST blocking solution was added and blocked for 30 minutes; 10mL 0.3μg / mL 2E1A, 2D1B and 17C4C were added respectively, incubated at 37℃ for 1h, and washed 5 times with PBST; HRP-labeled goat anti-mouse IgG was added at a dilution of 1:10,000, incubated at 37℃ for 45min; washed 5 times with PBST; imaging was performed using chemical exposure colorimetric solution, and the antibody recognition results are shown in the figure. Figure 3 shown.

[0090] Depend on Figure 2 It can be seen that 2E1A, 2D1B and 17C4C monoclonal antibodies can be used in immunoblotting experiments, specifically binding to CV-A4 virus, but not to other viruses such as CV-A2 and CV-A5.

[0091] (2) Indirect immunofluorescence experiment

[0092] CV-A4 was inoculated into a 6-well plate with a confluence of 95% RD cells, and RD cells not inoculated with the virus were used as negative control for culture; after culturing the 6-well plate for 24 hours, the cell supernatant was discarded, and the plate was gently washed with 0.01M PBS for 3 times, 2mL / well of 4% paraformaldehyde was added for fixation at room temperature for 1 hour, and 0.01M PBS was washed 5 times / 5min; 2mL / well of 2% BSA-PBST (W / V) solution (containing 0.5% Triton-X 100, V / V) was added, permeabilized at room temperature for 30min, and 0.01M PBS was washed 5 times / 5min; 2% BSA-PBST solution was used for blocking at room temperature for 1 hour, and the blocking solution was discarded; 1mL / well of 2μg / mL monoclonal antibody 2E1A was added and incubated at room temperature for 1 hour, and 0.01M Wash with PBS 5 times / 5min; add 1mL / well, 2μg / mL goat anti-mouse fluorescent antibody IgG (H+L) (Thermo Fisher), incubate at room temperature for 1h in the dark, wash with PBS, and add 1ml / well 5μg / ml DAPI solution (Biyuntian); observe and take pictures with a fluorescence microscope. The negative control group is cells not infected with CV-A4, and the corresponding antibodies are added. Figure 3 shown.

[0093] from Figure 3 It can be seen that 2E1A, 2D1B and 17C4C monoclonal antibodies can be used in indirect immunofluorescence experiments to recognize CV-A4 antigens and can be used in antigen identification experiments.

[0094] Example 6 Linear epitope study

[0095] The amino acid sequence of the linear epitope of the CV-A4 monoclonal antibody was preliminarily identified by ELISA. The method is as follows: Synthesize overlapping peptides in the VP1 region of the CV-A4 structural protein, covering the full length of VP1 of 305 amino acids. The peptide sequence is shown in Table 1:

[0096] Table 1 Overlapping peptide sequences in the VP1 region of the CV-A4 structural protein

[0097]

[0098]

[0099] 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 then added to the ELISA plate, 100 μL / well, and incubated overnight at 4°C. After the plate was washed three times with PBST of pH = 7.4; it was blocked with PBST of pH = 7.4 containing 1% (W / V) BSA at 37°C for 1 hour, and the blocking solution was discarded; 1 μg / ml of monoclonal antibody 2E1A was added ,2D1B and 17C4C were added to the ELISA plate (100 μL / well), incubated at 37°C for 1 hour, and washed 5 times with PBST; 0.1 μg / mL HRP-labeled goat anti-mouse IgG antibody was added to the ELISA plate, 100 μL / well, incubated at 37°C for 1 hour, and washed 5 times; TMB (3,3',5,5'-tetramethylbenzidine) colorimetric solution was added to develop color at 37°C in the dark for 30 minutes, and the reaction was terminated with 2M sulfuric acid, and the absorbance at 450nm was detected. The titer was calculated as the maximum dilution factor greater than 2.1 times the OD value of the negative well; anti-A4 FP mouse serum was used as the positive control, and normal mouse serum was used as the negative control.

[0100] The preliminary results of linear epitope mapping of monoclonal antibodies are shown in Figure 4 A and 4B, from Figure 4 As can be seen from A and 4B, monoclonal antibodies 2E1A, 2D1B and 17C4C can all bind to peptide segment 1 (i.e., the first 1-15 amino acids of VP1) of the structural protein VP1 of the CV-A4 virus.

[0101] The inventors have also found through experiments that the amino acid sequence of the heavy chain amino acid sequence of the 2E1A monoclonal antibody shown in SEQ ID NO.7, after replacement, deletion or addition of one or more amino acid sequences, or an amino acid sequence having more than 95% homology with the amino acid sequence shown in SEQ ID NO.7, has the same function as the sequence shown in SEQ ID NO.7; the amino acid sequence of the light chain amino acid sequence of the 2E1A monoclonal antibody shown in SEQ ID NO.8, after replacement, deletion or addition of one or more amino acid sequences, or an amino acid sequence having more than 95% homology with the amino acid sequence shown in SEQ ID NO.8, has the same function as the sequence shown in SEQ ID NO.8; the amino acid sequence of the heavy chain amino acid sequence of the 2D1B monoclonal antibody shown in SEQ ID NO.17, after replacement, deletion or addition of one or more amino acid sequences, or an amino acid sequence having more than 95% homology with the amino acid sequence shown in SEQ ID NO.17, has the same function as the sequence shown in SEQ ID NO.17; the amino acid sequence of the light chain amino acid sequence of the 2D1B monoclonal antibody shown in SEQ ID NO.18, after replacement, deletion or addition of one or more amino acid sequences, or an amino acid sequence having more than 95% homology with the amino acid sequence shown in SEQ ID NO. An amino acid sequence having 95% or more homology to the amino acid sequence shown in SEQ ID NO.18 has the same function as the sequence shown in SEQ ID NO.18; the heavy chain amino acid sequence of the 17C4C 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.27, or an amino acid sequence having 95% or more homology to the amino acid sequence shown in SEQ ID NO.27, and has the same function as the sequence shown in SEQ ID NO.27; the light chain amino acid sequence of the 17C4C 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.28, or an amino acid sequence having 95% or more homology to the amino acid sequence shown in SEQ ID NO.28, and has the same function as the sequence shown in SEQ ID NO.28.

[0102] It is necessary to point out that the above embodiments are limited to further elaboration and explanation of the technical solution of the present invention, and are not further limitations of the technical solution of the present invention. The method of the present invention is only a preferred implementation scheme, and is not used to limit the protection scope of the present invention. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A monoclonal antibody that recognizes the N-terminus of CV-A4VP1 protein, characterized in that: The monoclonal antibody is 2E1A monoclonal antibody, 2D1B monoclonal antibody or 17C4C monoclonal antibody, wherein The 2E1A monoclonal antibody has heavy chain complementary determining regions CDR1 to CDR3 shown in SEQ ID NOs. 9 to 11, and light chain complementary determining regions CDR1 to CDR3 shown in SEQ ID NOs. 12 to 14; The 2D1B monoclonal antibody has heavy chain complementary determining regions CDR1 to CDR3 shown in SEQ ID NOs. 19 to 21, and light chain complementary determining regions CDR1 to CDR3 shown in SEQ ID NOs. 22 to 24; The 17C4C monoclonal antibody has the heavy chain complementary determining regions CDR1 to CDR3 shown in SEQ ID NOs. 29 to 31, and the light chain complementary determining regions CDR1 to CDR3 shown in SEQ ID NOs. 32 to 34.

2. The monoclonal antibody that recognizes the N-terminus of CV-A4VP1 protein according to claim 1, characterized in that: The 2E1A monoclonal antibody has a heavy chain with an amino acid sequence as shown in SEQ ID NO.7; and / or, the 2E1A monoclonal antibody has a light chain with an amino acid sequence as shown in SEQ ID NO.8; and / or, The 2D1B monoclonal antibody has a heavy chain with an amino acid sequence as shown in SEQ ID NO.17; and / or, the 2D1B monoclonal antibody has a light chain with an amino acid sequence as shown in SEQ ID NO.18; and / or, The 17C4C monoclonal antibody has a heavy chain with an amino acid sequence as shown in SEQ ID NO.27; and / or, the 17C4C monoclonal antibody has a light chain with an amino acid sequence as shown in SEQ ID NO.

28.

3. The monoclonal antibody that recognizes the N-terminus of CV-A4VP1 protein according to claim 2, characterized in that: The heavy chain amino acid sequence of the 2E1A monoclonal antibody is an amino acid sequence formed by replacing, deleting or adding one or more amino acid sequences of 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 2E1A monoclonal antibody is an amino acid sequence formed by replacing, deleting or adding one or more amino acid sequences of 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; and / or The heavy chain amino acid sequence of the 2D1B 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.17, or an amino acid sequence having more than 95% homology with the amino acid sequence shown in SEQ ID NO.17; and / or, the light chain amino acid sequence of the 2D1B 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.18, or an amino acid sequence having more than 95% homology with the amino acid sequence shown in SEQ ID NO.18; and / or The heavy chain amino acid sequence of the 17C4C 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.27, or an amino acid sequence having more than 95% homology with the amino acid sequence shown in SEQ ID NO.27; and / or, the light chain amino acid sequence of the 17C4C 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.28, or an amino acid sequence having more than 95% homology with the amino acid sequence shown in SEQ ID NO.

28.

4. A polynucleotide molecule encoding the monoclonal antibody that recognizes the N-terminus of CV-A4VP1 protein according to claim 1 or 2.

5. The polynucleotide molecule according to claim 4, characterized in that The polynucleotide molecule encoding the 2E1A monoclonal antibody has the nucleotide sequence shown in SEQ ID NO.5 and / or SEQ ID NO.6; and / or The polynucleotide molecule encoding the 2D1B monoclonal antibody has the nucleotide sequence shown in SEQ ID NO.15 and / or SEQ ID NO.16; and / or The polynucleotide molecule encoding the 17C4C monoclonal antibody has the nucleotide sequence shown in SEQ ID NO.25 and / or SEQ ID NO.

26.

6. A kit for detecting Coxsackievirus A4, 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. 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 reagent or kit for detecting Coxsackievirus A4.

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