Monoclonal antibodies and their applications

Abstract

The present invention provides a monoclonal antibody and its application. The monoclonal antibody is the 4D6 monoclonal antibody, wherein the heavy chain complementary determining regions VHCDR1, VHCDR2, and VHCDR3 of the 4D6 monoclonal antibody have amino acid sequences as shown in SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3, respectively; and the light chain complementary determining regions VLCDR1, VLCDR2, and VLCDR3 of the 4D6 monoclonal antibody have amino acid sequences as shown in SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6. The present invention can specifically recognize CVA5 and does not recognize other enteroviruses.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and in particular relates to a monoclonal antibody and an application thereof. Background Art

[0002] The viruses that cause hand, foot, and mouth disease (HFMD) belong to the Picornavirus family, Enterovirus genus, and include coxsackievirus A group types 2, 4, 5, 6, 7, 9, 10, and 16; group B group types 1, 2, 3, 4, 5, 6, and 13; human enterovirus 71 (EV71); and echoviruses. Since the launch of the EV71 vaccine, HFMD has shifted to a co-circulation pattern among CVA5, CVA4, CVA6, CVA10, CVA16, and EV71, making them the six most prevalent serotypes. Multiple epidemiological studies have shown little or no cross-neutralization between serotypes, and therefore, a single-valent EV-A71 vaccine cannot protect against HFMD caused by other serotypes. Therefore, the development of a multivalent HFMD vaccine that includes CVA5 is crucial. Conformational neutralizing antibodies can better reflect the content and function of the virus. Therefore, the development of neutralizing monoclonal antibodies that target conformational epitopes and specifically recognize CVA5 is of great significance for the quality control of vaccine development, the detection of clinical samples and laboratory virus identification, and the study of the basic functions of CVA5. Summary of the Invention

[0003] In view of this, the present invention provides a monoclonal antibody and its application, which can specifically recognize CVA5 and does not recognize other enteroviruses.

[0004] In order to achieve the above object, the present invention adopts the following technical solutions:

[0005] In a first aspect, the present invention provides a monoclonal antibody, which is the 4D6 monoclonal antibody, wherein the heavy chain complementary determining region VHCDR1, complementary determining region VHCDR2 and complementary determining region VHCDR3 of the 4D6 monoclonal antibody have an amino acid sequence of VHCDR1 as shown in SEQ ID NO: 1, an amino acid sequence of VHCDR2 as shown in SEQ ID NO: 2, and an amino acid sequence of VHCDR3 as shown in SEQ ID NO: 3; the light chain complementary determining region VLCDR1, complementary determining region VLCDR2 and complementary determining region VLCDR3 of the 4D6 monoclonal antibody have an amino acid sequence of VLCDR1 as shown in SEQ ID NO: 4, an amino acid sequence of VLCDR2 as shown in SEQ ID NO: 5, and an amino acid sequence of VLCDR3 as shown in SEQ ID NO: 6.

[0006] Specifically, the amino acid sequence of the VHCDR1 is SGYYWN, the amino acid sequence of the VHCDR2 is YISYDGSNNYNPSLKN, and the amino acid sequence of the VHCDR3 is GGTGTGYFDY; the amino acid sequence of the VLCDR1 is KASDDIYNRLA, the amino acid sequence of the VLCDR2 is GATSLES, and the amino acid sequence of the VLCDR3 is QQYWSTYT.

[0007] Preferably, the amino acid sequence of the heavy chain variable region of the 4D6 monoclonal antibody is as shown in SEQ ID NO: 7, specifically: DVQLQESGPGLVKPSQSLSLTCSVTGYSITSGYYWNWIRQFPGNKLEWMGYISYDGSNNYNPSLKNRISITRDTSKNQFFLKLNSVTTEDTITYFCARGGTGTGYFDYWGQGTTLTVSS; and / or,

[0008] The amino acid sequence of the light chain variable region of the 4D6 monoclonal antibody is shown in SEQ ID NO: 8, specifically DIQMTQSSSSFSVSLGDRVSITCKASDDIYNRLAWYQLKPGNAPRLLISGATSLESGVPSRFSGSGSGKDYTLSITSLQTEDVATYYCQQYWSTYTFGGGTKLEIKR.

[0009] Preferably, the 4D6 monoclonal antibody is an IgM antibody.

[0010] Preferably, the 4D6 monoclonal antibody recognizes the 21st peptide segment of the CV-A5-VP1 linear epitope 155 VPPGAPVPTGRDTFQ 169 .

[0011] In a second aspect, the present invention provides a polynucleotide molecule encoding the monoclonal antibody.

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

[0013] Specifically, SEQ ID The nucleotide sequence shown in NO:9 is: GATGTACAGCTTCAGGAGTCAGGACCTGGCCTCGTGAAAACCTTCTCAGTCTCTGTCTCTCACCTGCCTCTGTCACTGGCTACTCCATCACCAGTGGTTATTACTGGAACTGGATCCGGCAGTTTCCAGGAAACAAACTGGAATGGATGGGCTACATAAGCTACGACGGTAGCA ATAACTACAACCCATCTCTCAAAAATCGAATCTCCATCACTCGTGACACATCTAAGAACCAGTTTTTCCTGAAGTTGAATTCTGTGACTACTGAGGACACAATTACATATTTCTGTGCAAGAGGGGGTACTGGGACGGGGTACTTTGACTACTGGGGCCAAGGCACCACTCTCACAGTCTCCTCA.

[0014] b. a nucleotide sequence complementary to the nucleotide sequence shown in SEQ ID NO: 9;

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

[0016] and / or,

[0017] The nucleic acid molecule encoding the light chain variable region of the monoclonal antibody is any one of the following:

[0018] e. having the nucleotide sequence shown in SEQ ID NO: 10;

[0019] Specifically, SEQ ID The nucleotide sequence shown in NO:10 is GACATCCAGATGACACAATCTTCATCCTCCTTTTCTGTATCTCTAGGAGACAGAGTCAGCATTACTTGCAAGGCAAGTGACGACATATATAATCGGTTAGCCTGGTATCAGCTGAAACCAGGAAATGCTCCTAGGCTCTTAATATCTGGTGCAA CCAGTTTGGAAAGTGGGGTACCTTCAAGATTCAGTGGCAGTGGATCTGGAAAGGATTACACTCTCAGCATTACCAGTCTTCAGACTGAAGATGTTGCTACTTATTACTGTCAACAGTATTGGAGTACGTACACGTTCGGAGGGGGGACCAAACTGGAAATAAACGG.

[0020] f. a nucleotide sequence complementary to the nucleotide sequence shown in SEQ ID NO: 10;

[0021] g. A nucleotide sequence that encodes the same protein as the nucleotide sequence in e and f, but differs from them due to the degeneracy of the genetic code.

[0022] In a third aspect, the present invention provides an expression vector comprising a polynucleic acid molecule encoding the monoclonal antibody.

[0023] In a fourth aspect, the present invention provides a host cell containing the polynucleic acid molecule or the expression vector.

[0024] In a fifth aspect, the present invention provides a use of the monoclonal antibody or the monoclonal antibody encoded by the polynucleotide molecule in the preparation of a reagent or kit for detecting Coxsackievirus A5.

[0025] Finally, the present invention provides a use of the monoclonal antibody or the monoclonal antibody encoded by the polynucleotide molecule in the preparation of drugs for inhibiting, preventing and treating Coxsackievirus A5.

[0026] Compared with the prior art, the present invention has the following beneficial effects:

[0027] (1) The monoclonal antibody provided by the present invention is prepared by immunizing mice with Coxsackievirus group A type 5 (CVA5) virus and screening hybridoma cells. It is an IgM subtype neutralizing antibody that can specifically recognize CVA5 and does not recognize other enteroviruses.

[0028] (2) The monoclonal antibody of the present invention can target conformational epitopes, recognize CVA5 hollow particles and solid particles, and has neutralizing activity. It can be used for the quantification of CVA5 virus and reflects the content and function of the virus, which is related to immunogenicity and is crucial for quality control of vaccine development.

[0029] (3) The monoclonal antibody of the present invention can be combined with a conjugate (horseradish peroxidase or fluorescein isothiocyanate, etc.) for direct or indirect detection and rapid diagnosis. For example, it can be used to develop detection reagents or kits for detecting clinical samples caused by CVA5 infection. It can also be used for laboratory identification of antibodies in clinical virus isolates. It can also be used to prepare quantitative detection reagents for the production of preventive vaccine intermediates and products containing CVA5 antigens. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 This is an SDS-PAGE image of the IgM light chain and heavy chain of the monoclonal antibody 4D6 provided in Example 3 of the present invention;

[0031] Figure 2 This is an identification diagram of the monoclonal antibody 4D6 provided in Example 4 of the present invention recognizing CVA5 virus particles;

[0032] Figure 3 This is the indirect immunofluorescence result of the monoclonal antibody 4D6 provided in Example 4 of the present invention in detecting CVA5-infected RD cells;

[0033] Figure 4 This is a graph showing the recognition ability of the monoclonal antibody 4D6 provided in Example 5 of the present invention for hollow particles and solid particles;

[0034] Figure 5 This is a diagram showing the preliminary localization results of the monoclonal antibody 4D6 VP1 peptide segment recognition provided in Example 5 of the present invention;

[0035] Figure 6 This is a diagram showing the localization results of the monoclonal antibody 4D6 provided in Example 5 of the present invention when both ends are truncated;

[0036] Figure 7 This is a graph showing the alanine scanning results of the localized peptide segment of the monoclonal antibody 4D6 provided in Example 5 of the present invention;

[0037] Figure 8 This is a graph showing the results of determining the median lethal dose of the challenge strain in mice provided in Example 6 of the present invention;

[0038] Figure 9 This is a graph showing the passive protection ability of the monoclonal antibody 4D6 provided in Example 6 of the present invention. DETAILED DESCRIPTION

[0039] 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.

[0040] 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.

[0041] 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.

[0042] The "monoclonal antibody" mentioned in the present invention refers to a preparation of an antibody molecule having a single molecular composition.

[0043] The "CDR region" or "CDR" mentioned in the present invention refers to the hypervariable region of the heavy chain of an immunoglobulin, which contains most of the amino acid residues responsible for the binding of an antibody to an antigen or its recognized epitope through affinity.

[0044] CVA5 is a strain disclosed in the prior art and recorded in the NCBI database.

[0045] Reagent Source:

[0046] Freund's complete and incomplete adjuvants: purchased from Sigma;

[0047] Female Balb / c mice were purchased from Wuhan Institute of Biological Products Co., Ltd.

[0048] Semi-solid HAT medium: purchased from Biolong Co., Ltd.

[0049] RD cells: provided by Wuhan Institute of Biological Products Co., Ltd.;

[0050] HRP-labeled goat anti-mouse IgM: purchased from Abcam.

[0051] Example 1 Preparation of monoclonal antibodies

[0052] This example provides a method for preparing a monoclonal antibody, which is as follows:

[0053] (1) Immunization: Mice were immunized with the CV-A5-3487-M14-611 mouse-adapted strain. After the mice recovered from the disease, they were boosted with the CV-A5-3487-M14-611 mouse-adapted strain 14 days later. At 14 days later, purified CV-A5-3487-M14-611 mouse-adapted strain FP particles (fluorescent protein particles) were administered with aluminum adjuvant. Blood was collected to detect serum antibody titers. Before fusion, the spleen was pulsed once, and then fusion of spleen lymphocytes and myeloma cells was performed.

[0054] (2) Screening: The supernatant of the fused cells was screened using the ELISA method. Specifically, the CVA5-3487 antigen (all antigens were from Wuhan Institute of Biological Products Co., Ltd.) was diluted with pre-cooled pH = 7.2 0.05M carbonate buffer, coated on a 96-well microplate at a coating concentration of 1 μg / mL, incubated at 4°C overnight, and blocked with blocking solution (PBST-1% BSA) at 37°C for 1 hour; the sample to be tested was added at 100 μL / well and incubated at 37°C for 1 hour. HRP-labeled goat anti-mouse IgM* (1:10000 dilution) was added at 100 μL / well and incubated at 37°C for 1 hour. After washing the plate, substrate solution A and solution B were added at 50 μL / well, and the plate was developed in the dark at 37°C for 15 minutes; stop solution was added at 50 μL / well, and the plate was placed on an enzyme reader to read the A at a wavelength of 450 nm. 450nm value.

[0055] (3) Subcloning: The positive mother clones were subcloned by limiting dilution method, subclones were screened by ELISA method, and ascites was prepared by expanded culture.

[0056] (4) Antibody production and purification: Female Balb / c mice aged 6-8 weeks were selected and intraperitoneally injected with 0.5 mL of liquid paraffin per mouse. 7-10 days after the injection of liquid paraffin, hybridoma cells were intraperitoneally injected. When the abdomen of the mouse was distended and the mouse was on the verge of death, the mouse was killed by cervical dislocation. Ascites was aseptically extracted in a clean bench. Monoclonal antibodies in the ascites were purified using octanoic acid-ammonium sulfate.

[0057] Example 2 Sequence Analysis of Screened Monoclonal Antibodies

[0058] (1) The hybridoma cells that can secrete the monoclonal antibody 4D6 screened in Example 1 were inoculated into RPMI 1640 medium (Gibco) containing 20% ​​fetal bovine serum and cultured at 37°C.

[0059] (2) Extraction of cellular RNA

[0060] Under a clean bench, add 1 mL of Trizol reagent to the centrifuged cells, let them stand for 5 minutes, then add 2 mL of chloroform, shake vigorously for 15 seconds, let them stand at room temperature for 3 minutes, and then spin at 12,000 rpm for 15 minutes. Transfer the upper aqueous layer to a new EP tube, add 0.5 mL of isopropanol, and let them stand at room temperature for 10 minutes. Spin at 12,000 rpm for 10 minutes. Discard the supernatant, add 1 mL of 75% ethanol, and spin at 7,500 rpm for 5 minutes. Dry the pellet, and add 50 μL of double-distilled water. Assess purity and quantify by agarose gel electrophoresis, and store at -70°C until needed.

[0061] (3) Reverse transcription to prepare cDNA

[0062] 1 μL of total cell RNA, 6 μL of RNase-Free ddH2O, 0.5 μL of oligo dT Primer, 0.5 μL of PRIME ScriptRT Enzyme Mix I, 2 μL of 5x Prime Script Buffer, mix well, incubate at 37°C for 15 min, and then at 85°C for 5 s.

[0063] (4) Amplification of cDNA

[0064] The mouse IgM VH and VL primer libraries designed by MysBio were used to amplify the cDNAs described above. 10 μL of 5x PrimeStar Buffer, 4 μL of dNTPs, 1 μL of cDNA, 1 μL of upstream primer, 1 μL of downstream primer, and 0.5 μL of PrimeSTAR were added to 50 μL of water. PCR was performed using the following reaction conditions: incubation at 94°C for 5 minutes, denaturation at 94°C for 45 seconds, annealing at 63°C for 45 seconds, extension at 72°C for 1 minute, and 30 cycles followed by extension at 72°C for 10 minutes.

[0065] (5) Agarose gel electrophoresis and gel recovery

[0066] The above PCR products were subjected to agarose gel electrophoresis, the electrophoresis results were observed, and the amplified products with a molecular weight of 250-350 bp were sent for sequencing.

[0067] (6) Analysis of sequencing results

[0068] The sequence was analyzed against the Kabat database to obtain the correct amino acid sequences of the light and heavy chain variable regions. The sequences of the universal forward primer (VH-F) for the heavy chain are: acggccagtgaattcmarctgcagsagtcwgg, and the reverse primer (VH-R) are: gattacgccaagctttgaggagacggtgaccg. The sequence of the universal forward primer (VL-F) for the light chain is: acggccagtgaattccgattgtkctsacycartctcca, and the reverse primer (VL-R) is: gattacgccaagcttcgttggatctccagcttg. Positive clones were screened and sequenced, and the sequence was analyzed against the Kabat database to obtain the correct amino acid sequences of the light and heavy chain variable regions. The sequences of the heavy chain CDR (complementarity determining region) and light chain CDR' (complementarity determining region) regions were analyzed as follows:

[0069] The amino acid sequence of the heavy chain complementarity determining region VHCDR1 is specifically: SGYYWN; the amino acid sequence of the heavy chain complementarity determining region VHCDR2 is specifically: YISYDGSNNYNPSLKN;

[0070] The amino acid sequence of the heavy chain complementarity determining region VHCDR3 is specifically: GGTGTGYFDY;

[0071] The amino acid sequence of the complementarity determining region VLCDR1 of the light chain is specifically: KASDDIYNRLA;

[0072] The amino acid sequence of the complementarity determining region VLCDR2 of the light chain is specifically: GATSLES;

[0073] The amino acid sequence of the complementarity determining region VLCDR3 of the light chain is specifically: QQYWSTYT.

[0074] The amino acid sequence of the heavy chain variable region is specifically:

[0075] DVQLQESGPGLVKPSQSLSLTCSVTGYSITSGYYWNWIRQFPGNKLEWMGYISYDGSNNYNPSLKNRISITRDTSKNQFFLKLNSVTTEDTITYFCARGGTGTGYFDYWGQGTTLTVSS;

[0076] The nucleotide sequence of the heavy chain variable region is specifically: GATGTACAGCTTCAGGAGTCAGGACCTGGCCTCGTGAAACCTTCTCAGTCTCTGTCTCTCACCTGCTCTGTCACTGGCTACTCCATCACCAGTGGTTATTACTGGAACTGGATCCGGCAGTTTCCAGGAAACAAACTGGAATGGATGGGCTACATAAGCTACGACGGTAGCAATAACTACAACCCATCTCTCAAAAATCGAATCTCCATCACTCGTGACACATCTAAGAACCAGTTTTTCCTGAAGTTGAATTCTGTGACTACTGAGGACACAATTACATATTTCTGTGCAAGAGGGGGTACTGGGACGGGGTACTTTGACTACTGGGGCCAAGGCACCACTCTCACAGTCTCCTCA;

[0077] The amino acid sequence of the light chain variable region is specifically:

[0078] DIQMTQSSSSFSVSLGDRVSITCKASDDIYNRLAWYQLKPGNAPRLLISGATSLESGVPSRFSGSGSGKDYTLSITSLQTEDVATYYCQQYWSTYTFGGGTKLEIKR;

[0079] The nucleotide sequence of the light chain variable region is specifically:

[0080] GACATCCAGATGACACAATCTTCATCCTCCTTTTCTGTATCTCTAGGAGACAGAGTCAGCATTACTTGCAAGGCAAGTGACGACATATATAATCGGTTAGCCTGGTATCAGCTGAAACCAGGAAATGCTCCTAGGCTCTTAATATCTGGTGCAACCAGTTT GGAAAGTGGGGGTACCTTCAAGATTCAGTGGCAGTGGATCTGGAAAGGATTACACTCTCAGCATTACCAGTCTTCAGACTGAAGATGTTGCTACTTATTACTGTCAACAGTATTGGAGTACGTACACGTTCGGAGGGGGGACCAAACTGGAAATAAAACGG.

[0081] Example 3 Purity and subtype identification

[0082] The purified monoclonal antibody 4D6 in Example 1 was taken and analyzed for purity by SDS-PAGE. The monoclonal antibody subtype was identified by mouse antibody subtype detection kit. Result analysis: SDS-PAGE results are shown in Figure 1 Under reducing conditions, two bands with molecular weights of approximately 70 kDa and 25 kDa were observed, corresponding to the heavy and light chains of the antibody, respectively. Isotype identification of monoclonal antibody 4D6 revealed IgM. The neutralizing antibody titer of monoclonal antibody 4D6, as determined by the microcytopathic effect inhibition assay, was 0.775 μg / mL.

[0083] Example 4 Functional Analysis

[0084] The functions of the screened 4D6 monoclonal antibodies were analyzed using indirect immunofluorescence assay and immunoblotting assay, as follows:

[0085] (1) Immunoblotting

[0086] The structural protein region of CVA5 recognized by the screened 4D6 monoclonal antibody was identified by immunoblotting as follows:

[0087] CVA5 stock solution was added to reducing and non-reducing loading buffers and heated at 100°C for 10 minutes for 4-20% SDS-PAGE. After electrophoresis, the membrane was transferred to a 0.45 μm nitrocellulose membrane using a rapid wet transfer apparatus (GenScript). After transfer, 2% BSA (W / V) in PBST blocking solution was added and blocked for 30 minutes. 4D6 was added and incubated at 37°C for 1 hour, followed by five washes with PBST. HRP-labeled goat anti-mouse IgM was added at a dilution of 1:10,000 and incubated at 37°C for 1 hour. The membrane was washed five times with PBST. Imaging was performed using a chemical exposure colorimeter. The antibody recognition results are shown in Figure 2. Figure 2 shown.

[0088] (2) Indirect immunofluorescence experiment

[0089] CVA5 cells were inoculated into 6-well plates with RD cells at a confluence of 95%. RD cells without virus infection served as a negative control and were cultured. After 24 h of incubation, the 6-well plates were incubated, the cell supernatant was discarded, and the cells were gently washed three times with 0.01 M PBS. 2 mL / well of 4% paraformaldehyde was added for fixation at room temperature for 1 h, and then washed five times with 0.01 M PBS for 5 min. 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 min, and then washed five times with 0.01 M PBS for 5 min. Blocking was performed with 2% BSA-PBST solution at room temperature for 1 h, and the blocking solution was discarded. 1 mL / well of 2 μg / mL monoclonal antibody 4D6 was added and incubated for 1 h at room temperature, and then washed five times with 0.01 M PBS for 5 min. 1 mL / well of 2 μg / mL goat anti-mouse fluorescent antibody IgM (H+L) was added. (Abcam), incubated at room temperature for 1 hour in the dark, washed with PBS, and added 1 ml / well of 5µg / ml DAPI solution (Biyuntian); observed and photographed under a fluorescence microscope. The negative control group was cells not infected with CVA5 and added with the corresponding antibody. Figure 3 shown.

[0090] from Figure 3 As can be seen from the figure, 4D6 monoclonal antibody can be used in indirect immunofluorescence experiments to recognize CVA5 antigen and can be used in antigen identification experiments.

[0091] Example 5 Linear epitope research

[0092] (1) Neutralizing antibody titer detection (in vitro microneutralization test)

[0093] After purification, dilute the 4D6 monoclonal antibody to 800µg / mL and then dilute it 1:8 in diluent (such as MEM maintenance medium). Add 100µL / well to column 1 of a 96-well plate. Set up in duplicate, and add 50µL of diluent to each well in columns 2-12. Serially dilute the monoclonal antibody in column 1 by two-fold to column 12. Mix the dilutions thoroughly and discard 50µL from the last column. Dilute the neutralizing toxin (monoclonal strain CVA5-3487) to 100 CCID50 / 50µL. Pipette 50µL vertically and add it to the diluted antibody-containing 96-well plate. Place the 96-well plate in a 37°C incubator for 2 hours to neutralize. Then, serially dilute the diluted neutralizing toxin in a 10-fold series to 10 CCID50 / 50µL, 1 CCID50 / 50µL, and 0.1 CCID50 / 50µL. Add 50µL of maintenance solution and 50µL of virus solution to each well (a total of 4 dilutions, namely 100, 10, 1, and 0.1CCID50 / 50µL), and replicate 8 wells for each dilution to form a back titration plate, which was placed at 4°C. After neutralization, the digested RD cells were plated at 1×10 5Plate the cells at a density of 100 μg / ml in a neutralization plate and a back titration plate. Incubate at 37°C, 5% CO2 for 5-7 days to determine the neutralization results.

[0094] (2) Identification of linear epitopes

[0095] Indirect ELISA was used to characterize the binding ability of monoclonal antibody 4D6 to synthetic structural protein overlapping peptides. This preliminarily determined the peptides that the antibody recognized as linear epitopes. Synthetic peptides overlapping CVA5 structural protein VP1 were synthesized; the peptide sequences are shown in Table 1. Synthetic peptides were diluted to 4 µg / mL in carbonate buffer, and 100 µL was added to each well of the microplate. The plates were incubated at 4°C overnight. The plates were washed five times. Monoclonal antibody 4D6 was diluted to 10 µg / mL as the primary antibody, incubated at 37°C for 1 hour, and washed five times. HRP-goat anti-mouse IgM was diluted to 0.1 µg / mL as the secondary antibody, incubated at 37°C for 1 hour, and washed five times. After adding the colorimetric solution, the plates were incubated in a water bath for 30 minutes, and the absorbance at 450 nm was measured using a microplate reader.

[0096] Table 1 Overlapping peptides of CV-A5 structural protein VP1

[0097]

[0098] Based on the initially identified linear epitope peptides, a series of truncated peptides were synthesized and coated at a concentration of 4µg / mL for indirect ELISA. A peptide with single alanine mutations was synthesized from the amino acid sequence recognized by 4D6. The peptides were coated at a concentration of 4µg / mL for indirect ELISA.

[0099] The results are as follows Figure 4 It shows that monoclonal antibody 4D6 recognizes the 21st peptide segment of the CV-A5-VP1 linear epitope 155 VPPGAPVPTGRDTFQ 169 The results are as follows Figure 5 It was shown that the amino acid recognition of eight amino acids was confirmed in the truncated peptide library 158 GAPVPTGR 165 By alanine scanning such as Figure 6-7 The results showed that G158, V161, G164, and R165 were further identified as the key sites for monoclonal antibody binding and neutralization.

[0100] Example 6 Application of Monoclonal 4D6

[0101] This example provides a method for neutralizing antibody protection in vivo, as follows:

[0102] The CV-A5-3487-611 strain was titrated at the cellular level to detect the titer. Five groups of 14-day-old KM mice were used, with 10 mice in each group. The strain after titer determination was diluted 10-fold and 300µL was intraperitoneally inoculated into mice to detect virulence. The blank group was injected with an equal volume of MEM. The mice were observed for 14 consecutive days and the disease progression was recorded. The virus titer was calculated (combined with Figure 8-9 ).

[0103] Five groups of 14-day-old KM mice were used, with 10 mice in each group. Each of the five groups of mice was intraperitoneally injected with 300 μL of 3.16×10 7 CCID50 CV-A5-3487-611; purified and characterized monoclonal antibody 4D6 was diluted in sterile PBS to different concentrations of 600µg / mL, 200µg / mL, 66.67µg / mL, and 22.22µg / mL. Two hours later, each mouse was injected with 100µL of the antibody at four different dilutions. The health of the mice was observed for 14 consecutive days, and survival was recorded.

[0104] In the passive protection test in mice, a lethal dose of CV-A5-M14-611 virus (44LD50) was mixed and intraperitoneally injected into 14-day-old KM mice. Two hours later, different concentrations of monoclonal antibody 4D6 were intraperitoneally injected. At a dose of 20µg / mouse, monoclonal antibody 4D6 achieved a 100% protection rate. At a viral load of 3.16×10 7 At CCID50, the ED50 of mAb 4D6 against CV-A5-3487-M14-611 was 15.5µg.

[0105] The raw materials not specifically described in the present invention are all existing materials that can be directly purchased from the market.

[0106] The above is only a preferred embodiment of the present invention and is not intended to limit the scope of protection of the present invention. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A monoclonal antibody that specifically recognizes Coxsackievirus A5, characterized in that: The monoclonal antibody is the 4D6 monoclonal antibody, wherein the heavy chain complementary determining region VHCDR1, complementary determining region VHCDR2 and complementary determining region VHCDR3 of the 4D6 monoclonal antibody, the amino acid sequence of VHCDR1 is shown in SEQ ID NO: 1, the amino acid sequence of VHCDR2 is shown in SEQ ID NO: 2, and the amino acid sequence of VHCDR3 is shown in SEQ ID NO: 3; the light chain complementary determining region VLCDR1, complementary determining region VLCDR2 and complementary determining region VLCDR3 of the 4D6 monoclonal antibody, the amino acid sequence of VLCDR1 is shown in SEQ ID NO: 4, the amino acid sequence of VLCDR2 is shown in SEQ ID NO: 5, and the amino acid sequence of VLCDR3 is shown in SEQ ID NO:

6.

2. The monoclonal antibody that specifically recognizes Coxsackievirus A5 according to claim 1, characterized in that The amino acid sequence of the heavy chain variable region of the 4D6 monoclonal antibody is shown in SEQ ID NO: 7; and / or, The amino acid sequence of the light chain variable region of the 4D6 monoclonal antibody is shown in SEQ ID NO:

8.

3. The monoclonal antibody that specifically recognizes Coxsackievirus A5 according to claim 1, characterized in that The 4D6 monoclonal antibody is an IgM antibody.

4. The monoclonal antibody that specifically recognizes Coxsackievirus A5 according to claim 1, characterized in that The 4D6 monoclonal antibody recognizes the 21st peptide segment of the CV-A5-VP1 linear epitope 155 VPPGAPVPTGRDTFQ 169 .

5. A polynucleotide molecule encoding the monoclonal antibody that specifically recognizes Coxsackievirus A5 according to any one of claims 1 to 4.

6. The polynucleotide molecule according to claim 5, characterized in that The nucleic acid molecule encoding the heavy chain variable region of the monoclonal antibody that specifically recognizes Coxsackievirus A5 is any one of the following: a, having the nucleotide sequence shown in SEQ ID NO: 9; b. A nucleotide sequence that encodes the same protein as the nucleotide sequence in a but differs from it due to the degeneracy of the genetic code; and / or, The nucleic acid molecule encoding the light chain variable region of the monoclonal antibody is any one of the following: c. having the nucleotide sequence shown in SEQ ID NO: 10; d. A nucleotide sequence that encodes the same protein as the nucleotide sequence in c but differs from it due to the degeneracy of the genetic code.

7. An expression vector, characterized in that The invention comprises a polynucleic acid molecule encoding the monoclonal antibody specifically recognizing Coxsackievirus A5 according to any one of claims 1 to 4.

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

9. Use of the monoclonal antibody according to any one of claims 1 to 4 or the monoclonal antibody encoded by the polynucleotide molecule according to claim 5 or 6 in the preparation of a reagent or kit for detecting Coxsackievirus A5.

10. Use of the monoclonal antibody according to any one of claims 1 to 4 or the monoclonal antibody encoded by the polynucleotide molecule according to claim 5 or 6 in the preparation of a medicament for inhibiting, preventing and treating Coxsackievirus A5.

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