Monoclonal antibody and application thereof

Abstract

The invention provides a monoclonal antibody and application thereof, the monoclonal antibody is a 4D6 monoclonal antibody, the amino acid sequences of a heavy chain complementarity determining region VHCDR1, a complementarity determining region VHCDR2 and a complementarity determining region VHCDR3 of the 4D6 monoclonal antibody are respectively as shown in SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3, and the amino acid sequences of the VHCDR1, the VHCDR2 and the VHCDR3 are respectively as shown in SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3. A light chain complementarity determining region VLCDR1, a complementarity determining region VLCDR2 and a complementarity determining region VLCDR3 of the 4D6 monoclonal antibody, and amino acid sequences of the VLCDR1, the VLCDR2 and the VLCDR3 are shown as SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6. According to the invention, CVA5 can be specifically recognized, and other enteroviruses are not recognized.

Description

Technical Field

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

[0002] The virus that causes hand, foot and mouth disease belongs to the Picornavirus family and the Enterovirus genus, including Coxsackievirus A group 2, 4, 5, 6, 7, 9, 10, 16, etc., group B type 1, 2, 3, 4, 5, 6, 13, etc., enterovirus 71 (Human enterovirus 71, EV71), echoviruses, etc. Since the EV71 vaccine was launched, hand, foot and mouth disease has begun to change to a trend of co-epidemic of CVA5, CVA4, CVA6, CVA10, CVA16, and EV71, becoming the six most popular serotypes. According to multiple epidemiological studies, there is no or very little cross-neutralization activity between different serotypes, so the monovalent EV-A71 cannot protect against hand, foot and mouth disease caused by other serotypes. Therefore, it is very important to develop a multivalent hand, foot and mouth disease vaccine including CVA5. Conformational neutralizing antibodies can better reflect the content and function of the virus. Therefore, the development of neutralizing active 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 application thereof, which can specifically recognize CVA5 but not other enteroviruses.

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

[0005] 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.

[0006] 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, The amino acid sequence of the light chain variable region of the 4D6 monoclonal antibody is shown in SEQ ID NO: 8, specifically DIQMTQSSSSFSVSLGDRVSITCKASDDIYNRLAWYQLKPGNAPRLLISGATSLESGVPSRFSGSGSGKDYTLSITSLQTEDVATYYCQQYWSTYTFGGGTKLEIKR.

[0007] Preferably, the 4D6 monoclonal antibody is an IgM antibody.

[0008] Preferably, the 4D6 monoclonal antibody recognizes the 21st peptide segment of the CV-A5-VP1 linear epitope 155 VPPGAPVPTGRDTFQ 169 .

[0009] In a second aspect, the present invention provides a polynucleotide molecule encoding the monoclonal antibody.

[0010] Preferably, the nucleic acid molecule encoding the heavy chain variable region of the monoclonal antibody is any one of the following: Specifically, SEQ ID The nucleotide sequence shown in NO:9 is: GATGTACAGCTTCAGGAGTCAGGACCTGGCCTCGTGAAAACCTTCTCAGTCTCTGTCTCTCACCTGCCTCTGTCACTGGCTACTCCATCACCAGTGGTTATTACTGGAACTGGATCCGGCAGTTTCCAGGAAACAAACTGGAATGGATGGGCTACATAAGCTACGACGGTAGCA ATAACTACAACCCATCTCTCAAAAATCGAATCTCCATCACTCGTGACACATCTAAGAACCAGTTTTTCCTGAAGTTGAATTCTGTGACTACTGAGGACACAATTACATATTTCTGTGCAAGAGGGGGTACTGGGACGGGGTACTTTGACTACTGGGGCCAAGGCACCACTCTCACAGTCTCCTCA.

[0011] b. a nucleotide sequence complementary to the nucleotide sequence shown in SEQ ID NO: 9; c, a nucleotide sequence that encodes the same protein as the nucleotide sequences in a and b, but is different from them 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: e, having the nucleotide sequence shown in SEQ ID NO: 10; Specifically, SEQ ID The nucleotide sequence shown in NO:10 is GACATCCAGATGACACAATCTTCATCCTCCTTTTCTGTATCTCTAGGAGACAGAGTCAGCATTACTTGCAAGGCAAGTGACGACATATATAATCGGTTAGCCTGGTATCAGCTGAAACCAGGAAATGCTCCTAGGCTCTTAATATCTGGTGCAA CCAGTTTGGAAAGTGGGGTACCTTCAAGATTCAGTGGCAGTGGATCTGGAAAGGATTACACTCTCAGCATTACCAGTCTTCAGACTGAAGATGTTGCTACTTATTACTGTCAACAGTATTGGAGTACGTACACGTTCGGAGGGGGGACCAAACTGGAAATAAACGG.

[0012] f. a nucleotide sequence complementary to the nucleotide sequence shown in SEQ ID NO: 10; g. A nucleotide sequence that encodes the same protein as the nucleotide sequence in e and f, but is different from them due to the degeneracy of the genetic code.

[0013] In a third aspect, the present invention provides an expression vector comprising a polynucleic acid molecule encoding the monoclonal antibody.

[0014] In a fourth aspect, the present invention provides a host cell containing the polynucleic acid molecule or the expression vector.

[0015] 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 a kit for detecting Coxsackievirus A5.

[0016] 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.

[0017] Compared with the prior art, the present invention has the following beneficial effects: (1) The monoclonal antibody provided by the present invention is prepared by immunizing mice with Coxsackievirus group A type 5 (CVA5) virus to prepare and screen hybridoma cells. It is an IgM subtype neutralizing antibody that can specifically recognize CVA5 and does not recognize other enteroviruses.

[0018] (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 CVA5 virus quantification and reflects the content and function of the virus, which is related to immunogenicity and is crucial for quality control of vaccine development.

[0019] (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, 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 the production of preventive vaccines containing CVA5 antigens. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 This is the SDS-PAGE image of the IgM light chain and heavy chain of the monoclonal antibody 4D6 provided in Example 3 of the present invention; Figure 2 This is an identification diagram of the monoclonal antibody 4D6 provided in Example 4 of the present invention recognizing CVA5 virus particles; Figure 3This is a graph showing the indirect immunofluorescence results of the monoclonal antibody 4D6 provided in Example 4 of the present invention in detecting CVA5-infected RD cells; 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; 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; Figure 6 This is a diagram showing the localization results of the monoclonal antibody 4D6 provided in Example 5 of the present invention with truncated ends; Figure 7 This is a graph showing the alanine scanning results of the localization peptide segment of the monoclonal antibody 4D6 provided in Example 5 of the present invention; Figure 8 This is a graph showing the results of the determination of the median lethal dose of the challenge strain in mice provided in Example 6 of the present invention; Fig. 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

[0021] 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.

[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 the field without creative work belong to the protection scope 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] The "monoclonal antibody" mentioned in the present invention refers to a preparation of an antibody molecule having a single molecular composition.

[0025] 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 binding through the affinity of the antibody to the antigen or its recognized epitope.

[0026] CVA5 is a strain that has been disclosed in the prior art and is recorded in the NCBI database.

[0027] Source of reagents: Freund's complete and incomplete adjuvants: purchased from Sigma; Female Balb / c mice: purchased from Wuhan Institute of Biological Products Co., Ltd.; Semi-solid HAT medium: purchased from Biolong Co., Ltd.; RD cells: provided by Wuhan Institute of Biological Products Co., Ltd.; HRP-labeled goat anti-mouse IgM: purchased from Abcam.

[0028] Example 1 Preparation of monoclonal antibodies This example provides a method for preparing a monoclonal antibody, which is as follows: (1) Immunization: The mice were immunized with the CV-A5-3487-M14-611 mouse-adapted strain. After the mice became ill and recovered, they were boosted with the CV-A5-3487-M14-611 mouse-adapted strain 14 days later. The purified CV-A5-3487-M14-611 mouse-adapted strain FP particles (fluorescent protein particles) were used with aluminum adjuvant at 14 days intervals, and blood was collected to detect the titer of serum antibodies. Before fusion, the spleen was immunized once, and then the spleen lymphocytes and myeloma cells were fused.

[0029] (2) Screening: The supernatant of fused cells was screened using the ELISA method, specifically: 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; 100 μL / well of the sample to be tested was added, and incubated at 37°C for 1 hour. 100 μL / well of HRP-labeled goat anti-mouse IgM* (1:10000 dilution) was added, and incubated at 37°C for 1 hour. After washing the plate, substrate solutions A and B were added, both at 50 μL / well, and color was developed at 37°C in the dark for 15 minutes; 50 μL / well of stop solution was added, and A was read at a wavelength of 450 nm on an ELISA reader. 450nm value.

[0030] (3) Subcloning: Subclone the positive mother clone using the limiting dilution method, screen the subclones using ELISA, and expand the culture to prepare ascites.

[0031] (4) Antibody production and purification: Select female Balb / c mice aged 6-8 weeks and inject liquid paraffin intraperitoneally, 0.5 mL per mouse. 7-10 days after the injection of liquid paraffin, inject hybridoma cells intraperitoneally. When the abdomen of the mouse is swollen and the mouse is on the verge of death, the mouse is killed by cervical dislocation, and the ascites is aseptically extracted in an ultra-clean workbench. Caprylic acid-ammonium sulfate is used to purify the ascites monoclonal antibody.

[0032] Example 2 Sequence analysis of screened monoclonal antibodies (1) The hybridoma cells that can secrete the monoclonal antibody 4D6 selected in Example 1 were inoculated into RPMI 1640 medium (Gibco) containing 20% ​​fetal bovine serum and cultured at 37°C.

[0033] (2) Extraction of cellular RNA Under the clean bench environment, add 1mL Trizol reagent to the centrifuged cells, let stand for 5min, add 2mL chloroform, shake vigorously for 15sec, let stand at room temperature for 3min, 12000rpm x 15min, transfer the upper water layer to a new EP tube, add 0.5mL isopropanol, let stand at room temperature for 10min. 12000rpm x 10min. Discard the supernatant, add 1mL 75% ethanol, 7500rpm x 5min, dry the precipitate, and add 50μL double distilled water. Agarose electrophoresis was used to identify the purity and quantify, and stored at -70℃ for future use.

[0034] (3) Reverse transcription to prepare cDNA 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 85°C for 5 s.

[0035] (4) Amplification of cDNA The mouse IgM VH VL primer library designed by MysBio was used to amplify the above cDNAs. 5x Prime Star Buffer 10μL, dNTP 4μL, cDNA 1μL, upstream primer 1μL, downstream primer 1μL, PrimeSTAR 0.5μL, add water to 50μL. PCR reaction was performed according to the following reaction conditions: incubation at 94℃ for 5min, denaturation at 94℃ for 45s, annealing at 63℃ for 45s, extension at 72℃ for 1min, and extension at 72℃ for 10min after 30 cycles.

[0036] (5) Agarose gel electrophoresis and gel recovery 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.

[0037] (6) Analysis of sequencing results The sequence was analyzed in the Kabat database to obtain the correct amino acid sequences of the light and heavy chain variable regions. Among them, the sequence of the heavy chain universal forward primer VH-F is: acggccagtgaattcmarctgcagsagtcwgg, the sequence of the reverse primer VH-R is: gattacgccaagctttgaggagacggtgaccg and the sequence of the light chain universal forward primer VL-F is: acggccagtgaattccgattgtkctsacycartctcca, the sequence of the reverse primer VL-R is: gattacgccaagcttcgttggatctccagcttg. Positive clones were obtained by screening and sequencing, and the sequence was analyzed in 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 region (complementarity determining region) and the light chain CDR' region (complementarity determining region) analyzed are as follows: 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; The amino acid sequence of the heavy chain complementarity determining region VHCDR3 is specifically: GGTGTGYFDY; The amino acid sequence of the complementarity determining region VLCDR1 of the light chain is specifically: KASDDIYNRLA; The amino acid sequence of the complementarity determining region of the light chain, VLCDR2, is specifically: GATSLES; The amino acid sequence of the complementarity determining region VLCDR3 of the light chain is specifically: QQYWSTYT.

[0038] The amino acid sequence of the heavy chain variable region is specifically: DVQLQESGPGLVKPSQSLSLTCSVTGYSITSGYYWNWIRQFPGNKLEWMGYISYDGSNNYNPSLKNRISITRDTSKNQFFLKLNSVTTEDTITYFCARGGTGTGYFDYWGQGTTLTVSS; The nucleotide sequence of the heavy chain variable region is specifically: GATGTACAGCTTCAGGAGTCAGGACCTGGCCTCGTGAAACCTTCTCAGTCTCTGTCTCTCACCTGCTCTGTCACTGGCTACTCCATCACCAGTGGTTATTACTGGAACTGGATCCGGCAGTTTCCAGGAAACAAACTGGAATGGATGGGCTACATAAGCTACGACGGTAGCAATAACTACAACCCATCTCTCAAAAATCGAATCTCCATCACTCGTGACACATCTAAGAACCAGTTTTTCCTGAAGTTGAATTCTGTGACTACTGAGGACACAATTACATATTTCTGTGCAAGAGGGGGTACTGGGACGGGGTACTTTGACTACTGGGGCCAAGGCACCACTCTCACAGTCTCCTCA; The amino acid sequence of the light chain variable region is specifically: DIQMTQSSSSFSVSLGDRVSITCKASDDIYNRLAWYQLKPGNAPRLLISGATSLESGVPSRFSGSGSGKDYTLSITSLQTEDVATYYCQQYWSTYTFGGGTKLEIKR; The nucleotide sequence of the light chain variable region is specifically: GACATCCAGATGACACAATCTTCATCCTCCTTTTCTGTATCTCTAGGAGACAGAGTCAGCATTACTTGCAAGGCAAGTGACGACATATATAATCGGTTAGCCTGGTATCAGCTGAAACCAGGAAATGCTCCTAGGCTCTTAATATCTGGTGCAACCAGTTT GGAAAGTGGGGGTACCTTCAAGATTCAGTGGCAGTGGATCTGGAAAGGATTACACTCTCAGCATTACCAGTCTTCAGACTGAAGATGTTGCTACTTATTACTGTCAACAGTATTGGAGTACGTACACGTTCGGAGGGGGGACCAAACTGGAAATAAAACGG.

[0039] Example 3 Purity and subtype identification The purified monoclonal antibody 4D6 in Example 1 was taken and the antibody purity was analyzed 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 presented, corresponding to the heavy chain and light chain of the antibody, respectively. The subtype identification results of monoclonal antibody 4D6 showed that the subtype was IgM. The neutralizing antibody titer of monoclonal antibody 4D6 determined by microcytopathic inhibition assay was 0.775 μg / mL.

[0040] Example 4 Functional Analysis The functions of the screened 4D6 monoclonal antibody were analyzed by indirect immunofluorescence assay and immunoblotting assay, respectively, as follows: (1) Immunoblotting The structural protein region of CVA5 recognized by the screened 4D6 monoclonal antibody was identified by immunoblotting, and the steps were as follows: CVA5 stock solution was added to reducing and non-reducing loading buffer, heated at 100℃ for 10min, and 4-20% SDS-PAGE was performed. 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 30min. 4D6 was added, incubated at 37℃ for 1h, and washed 5 times with PBST. HRP-labeled goat anti-mouse IgM was added at a dilution of 1:10000, and incubated at 37℃ for 1h. PBST was washed 5 times. The imaging was performed using a chemical exposure colorimetric solution. The antibody recognition results are shown in Figure 2. Figure 2 shown.

[0041] (2) Indirect immunofluorescence experiment CVA5 was inoculated into 6-well plates with RD cell confluence of 95%, and RD cells without virus inoculation were used as negative control for culture. After culturing the 6-well plates for 24 hours, the cell supernatant was discarded, and the plates were 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, and the plates were permeabilized at room temperature for 30 minutes, 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 4D6 was added for incubation at room temperature for 1 hour, and 0.01M PBS was washed 5 times / 5min. 1mL / well of 2μg / mL goat anti-mouse fluorescent antibody IgM (H+L) was added. (Abcam), incubate at room temperature for 1 hour in the dark, wash with PBS, and add 1 ml / well of 5 µg / ml DAPI solution (Biyuntian); observe and take pictures under a fluorescence microscope. The negative control group was cells not infected with CVA5 and added with the corresponding antibodies. Figure 3 shown.

[0042] from Figure 3 It can be seen that 4D6 monoclonal antibody can be used in indirect immunofluorescence experiments to recognize CVA5 antigens and can be used in antigen identification experiments.

[0043] Example 5 Linear epitope research (1) Neutralizing antibody titer detection (in vitro microneutralization test) After purification, the 4D6 monoclonal antibody is diluted to 800µg / mL, and then diluted with a diluent (such as MEM maintenance solution) at a ratio of 1:8, and added to the first column of the 96-well plate, 100µL / well, set up 2 replicates, and add 50µL of diluent to each well of columns 2-12. The monoclonal antibody in column 1 is diluted 2-fold continuously to column 12, and 50µL is discarded after the last column is diluted and mixed. The neutralizing toxin (monoclonal strain CVA5-3487) is diluted to 100 CCID50 / 50µL, and 50µL is vertically suspended and added to the 96-well plate with diluted antibodies. The 96-well plate is placed in a 37℃ incubator for 2h. The diluted neutralizing toxin is diluted 10 times in a gradient to 10 CCID50 / 50µL, 1CCID50 / 50µL, and 0.1CCID50 / 50µL. Add 50 µL of maintenance solution and 50 µL of virus solution to each well (4 dilutions in total, 100, 10, 1, 0.1 CCID50 / 50 µL), 8 replicate wells for each dilution, as a back titration plate, and place at 4°C. After neutralization, the digested RD cells were plated at 1×10 5The cells were plated at a density of 100 / ml in a neutralization plate and a back titration plate. The neutralization results were determined at 37°C and 5% CO2 for 5-7 days.

[0044] (2) Identification of linear epitopes The binding ability of monoclonal antibody 4D6 to the synthetic structural protein overlapping peptides was identified by indirect ELISA experiment, and the peptides where the antibody recognized the linear epitope were preliminarily determined. The CVA5 structural protein VP1 overlapping peptides were synthesized, and the peptide sequences are shown in Table 1. The synthetic peptides were prepared into 4µg / mL dilutions with carbonate buffer, and 100µL was added to each well of the ELISA plate and allowed to stand overnight at 4°C. The plate was washed 5 times, and the monoclonal antibody 4D6 was diluted to 10µg / mL as the primary antibody, incubated at 37°C for 1h, and washed 5 times. HRP-sheep anti-mouse IgM was diluted to 0.1µg / mL as the secondary antibody, incubated at 37°C for 1h, and washed 5 times. After adding the color developing solution, the water bath was incubated for 30min, and the absorbance at 450nm was detected by an ELISA reader.

[0045] Table 1 Overlapping peptides of CV-A5 structural protein VP1

[0046] A series of truncated peptides were synthesized based on the linear epitope peptides initially identified, and the peptides were plated at a concentration of 4µg / mL, and then indirect ELISA experiments were performed. The amino acid sequence recognized by 4D6 was synthesized into peptides with single alanine mutations, and the peptides were plated at a concentration of 4µg / mL, and then indirect ELISA experiments were performed.

[0047] 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 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.

[0048] Example 6 Application of Monoclonal 4D6 This example provides a method for neutralizing antibody protection levels in vivo, as follows: The CV-A5-3487-611 strain was titrated at the cell level to detect the titer. Five groups of 14-day-old KM mice were used, with 10 mice in each group. The strain after the titer was determined was diluted 10 times in a gradient manner, and 300µL was intraperitoneally inoculated into mice to detect the virulence. The blank group was injected with an equal volume of MEM. The disease status of the mice was observed for 14 consecutive days and recorded. The virus titer was calculated (combined with Figure 8-9 ).

[0049] 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; The purified and identified monoclonal antibody 4D6 was diluted to different concentrations of 600µg / mL, 200µg / mL, 66.67µg / mL, and 22.22µg / mL using sterile PBS. After 2 hours, each mouse was injected with 100μL of the antibody of 4 different dilutions. The health status of the mice was observed for 14 consecutive days, and the survival rate was recorded.

[0050] In the passive protection test of mice, a lethal dose of CV-A5-M14-611 virus solution (44LD50) was mixed and intraperitoneally injected into 14-day-old KM mice. Two hours later, different concentrations of monoclonal antibody 4D6 were intraperitoneally injected. When the injection dose was 20µg / mouse, monoclonal antibody 4D6 could achieve a 100% protection rate. When the virus amount was 3.16×10 7 At CCID50, the ED50 of mAb 4D6 against CV-A5-3487-M14-611 was 15.5µg.

[0051] The specific raw materials not described in the present invention are all existing materials and can be directly purchased from the market.

[0052] The above are only preferred embodiments of the present invention and are not intended to limit the protection scope of the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A monoclonal antibody, 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 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 according to claim 1, characterized in that The 4D6 monoclonal antibody is an IgM antibody.

4. The monoclonal antibody 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 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 is any one of the following: a, having the nucleotide sequence shown in SEQ ID NO:9; b. a nucleotide sequence complementary to the nucleotide sequence shown in SEQ ID NO: 9; c, a nucleotide sequence that encodes the same protein as the nucleotide sequences in a and b, but is different from them 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: e, having the nucleotide sequence shown in SEQ ID NO: 10; f. a nucleotide sequence complementary to the nucleotide sequence shown in SEQ ID NO: 10; g. A nucleotide sequence that encodes the same protein as the nucleotide sequence in e and f, but is different from them 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 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 drugs for inhibiting, preventing and treating Coxsackievirus A5.

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