Monoclonal antibody 2D11 capable of specifically recognizing CV-A5 virus and having neutralizing activity and application of monoclonal antibody 2D11

Abstract

The invention provides a monoclonal antibody capable of specifically recognizing a CV-A5 virus and having neutralizing activity and application of the monoclonal antibody. The monoclonal antibody is prepared from a CV-A5 virus antigen immunized mouse and splenocytes of the mouse through a cell fusion technology, and amino acid sequences of three CDR regions of a heavy chain variable region of the monoclonal antibody are sequentially shown as SEQ ID NO.1-3; the amino acid sequences of the three CDR regions of the light chain variable region are sequentially as shown in SEQ ID NO.4-6. The monoclonal antibody can be specifically combined with a CV-A5 virus, and is not combined with enteroviruses such as EV-A71, CV-A10, CV-A6, CV-A16 and the like. The monoclonal antibody targets conformational epitopes, has neutralizing activity, can specifically recognize CV-A5 viruses, is an ideal CV-A5 antigen detection antibody, and is beneficial to acceleration of the research and development process of hand-foot-mouth multivalent vaccines containing CV-A5 pathogens.

Description

Technical Field

[0001] The present invention belongs to the technical field of molecular biology, and in particular relates to a monoclonal antibody 2D11 that specifically recognizes CV-A5 virus and has neutralizing activity, and an application thereof. Background Art

[0002] The virus that causes hand, foot and mouth disease belongs to the Picornaviridae family ( Picornaviridae ) Enterovirus ( Enterovirus ), 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, EV-A71), echovirus ( Echoviruses Since the launch of the EV-A71 vaccine, hand, foot and mouth disease has shifted to a co-circulation trend among CV-A5, CV-A4, CV-A6, CV-A10, CV-A16, and EV-A71, which have become the six most prevalent serotypes. Multiple epidemiological studies have shown that different serotypes exhibit minimal or no cross-neutralizing activity, and the monovalent EV-A71 vaccine provides no protection against hand, foot and mouth disease caused by other serotypes.

[0003] Therefore, the development of a multivalent HFMD vaccine, including CV-A5, is crucial. Neutralizing antibodies are more effective in reflecting the antigenicity, immunogenicity, and function of the virus. Therefore, the development of neutralizing monoclonal antibodies that specifically recognize the structural protein of CV-A5 is crucial for quality control in vaccine development, clinical sample testing, and laboratory virus identification. Summary of the Invention

[0004] In view of this, the present invention provides a monoclonal antibody 2D11 that specifically recognizes CV-A5 virus and has neutralizing activity, and its application.

[0005] One of the objectives of the present invention is to provide: a monoclonal antibody 2D11 that specifically recognizes CV-A5 virus and has neutralizing activity, wherein the six CDR regions of the monoclonal antibody 2D11 are as follows: (1) The heavy chain CDR1 comprises the amino acid sequence shown in SEQ ID NO. 1; (2) the heavy chain CDR2 comprises the amino acid sequence shown in SEQ ID NO. 2; (3) the heavy chain CDR3 comprises the amino acid sequence shown in SEQ ID NO. 3; (4) the light chain CDR1 comprises the amino acid sequence shown in SEQ ID NO. 4; (5) the light chain CDR2 comprises the amino acid sequence shown in SEQ ID NO. 5; The light chain CDR3 contains the amino acid sequence shown in SEQ ID NO.6.

[0006] Furthermore, the heavy chain variable region of the monoclonal antibody 2D11 contains the amino acid sequence shown in SEQ ID NO.7, or a sequence with equivalent function formed after substitution, deletion or addition of one or more amino acids to the amino acid sequence shown in SEQ ID NO.7; and / or, the light chain variable region of the monoclonal antibody 2D11 contains the amino acid sequence shown in SEQ ID NO.9, or a sequence with equivalent function formed after substitution, deletion or addition of one or more amino acids to the amino acid sequence shown in SEQ ID NO.9.

[0007] Furthermore, the monoclonal antibody 2D11 is an IgG-type antibody.

[0008] Furthermore, the monoclonal antibody 2D11 targets a conformational epitope.

[0009] The second object of the present invention is to provide: a nucleotide sequence encoding the above monoclonal antibody 2D11.

[0010] Furthermore, the nucleotide sequence encoding the heavy chain variable region of the monoclonal antibody 2D11 is any one of the following: a. having the nucleotide sequence shown in SEQ ID NO.8; b. a nucleotide sequence complementary to the nucleotide sequence shown in SEQ ID NO.8; c. a nucleotide sequence that encodes the same protein as the nucleotide sequences of a and b, but is different therefrom due to the degeneracy of the genetic code; and / or, the nucleotide sequence encoding the light chain variable region of the monoclonal antibody 2D11 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 sequences of e and f, but is different therefrom due to the degeneracy of the genetic code.

[0011] The third object of the present invention is to provide: an expression vector, the vector containing the above nucleotide sequence.

[0012] The fourth object of the present invention is to provide: a host cell, the host cell containing the above nucleic acid sequence, or containing the above expression vector.

[0013] A fifth object of the present invention is to provide an antibody conjugate, which comprises the above monoclonal antibody 2D11 or an antigen-binding fragment of the above monoclonal antibody 2D11 and a marker, and the marker is selected from one or more of enzyme labeling, biotin labeling, and chemiluminescent dye labeling.

[0014] A sixth object of the present invention is to provide the use of the above monoclonal antibody 2D11 in the preparation of a reagent or kit for detecting Coxsackievirus A5; and / or, the use of the above monoclonal antibody 2D11 in the preparation of a drug for inhibiting, preventing and treating Coxsackievirus A5.

[0015] Compared with the prior art, the beneficial effects of the present invention are as follows: (1) The monoclonal antibody provided by the present invention is prepared by immunizing mice with Coxsackievirus A group type 5 (CV-A5) virus and screening hybridoma cells. It is an IgG subtype neutralizing antibody (the neutralizing antibody titer is shown as 386, and the corresponding neutralizing concentration is 1.852 μg / mL), which can specifically recognize CV-A5 and does not recognize other enteroviruses.

[0016] (2) The monoclonal antibody provided by the present invention can be combined with a conjugate (such as horseradish peroxidase or fluorescein isothiocyanate) for direct or indirect detection and rapid diagnosis. For example, it can be used to develop a detection reagent or kit for clinical samples infected by CV-A5, and can also be used for the laboratory identification of antibodies of clinical virus isolates. At the same time, it can also be used to prepare intermediate products for preventive vaccine production and quantitative detection reagents for products containing CV-A5 antigen. After humanization transformation, it has certain therapeutic significance. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 It is the SDS-PAGE electrophoresis diagram of the IgG heavy chain and light chain of monoclonal antibody 2D11.

[0018] Figure 2 It is the corresponding WB image that monoclonal antibody 2D11 cannot recognize the reduced CV-A5.

[0019] Figure 3 It is the indirect immunofluorescence assay of monoclonal antibody 2D11 for detecting the antigen of CV-A5-infected RD cells. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0020] The following further describes the present invention in detail with reference to specific embodiments, so that those skilled in the art can understand the present invention more clearly. Unless otherwise specified, the technical means used in the following embodiments are all conventional means well known to those skilled in the art, and all reagent consumables are commercially available products.

[0021] Key reagents and their sources: 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 Bioworld Technology Co., Ltd.; RD cells: Provided by Wuhan Institute of Biological Products Co., Ltd.; Goat anti-mouse IgG (H+L) Alexa Fluor 488: Purchased from Thermo Fisher Scientific; HRP-labeled goat anti-mouse IgG: Purchased from Beyotime Biotechnology Co., Ltd.

[0022] Example 1 This example provides the preparation of monoclonal antibody 2D11, and the specific steps are as follows: (1) Immunization: Immunize mice with the CV-A5-3487-M14-611 mouse adapted strain (provided by Wuhan Institute of Biological Products Co., Ltd.). After the mice recover from the disease, mix the purified CV-A5-3487-M14-611 mouse adapted strain FP particles with aluminum adjuvant at an interval of 14 days, and collect blood to detect the serum antibody titer. Before fusion, boost the immunization of the spleen once, and then perform the fusion of spleen lymphocytes and myeloma cells.

[0023] (2) Screening: Use the ELISA method to screen the supernatant of the fused cells. Specifically: Dilute the CV-A5 antigen with pre-cooled pH = 7.2, 0.05M carbonate buffer (all antigens are from Wuhan Institute of Biological Products Co., Ltd.), coat a 96-well microplate, and the coating concentration is 1 μg / mL. Incubate overnight at 4 °C. Use the blocking solution (PBST-1% BSA) to block for 1 h at 37 °C; Add the sample to be tested, 100 μL / well, and incubate at 37 °C for 1 h. Add 100 μL / well of HRP-labeled goat anti-mouse IgG (diluted 1:10000), and incubate at 37 °C for 1 h. After washing the plate, add 50 μL / well of substrate solution A and B solution, and develop color at 37 °C in the dark for 15 min; Add 50 μL / well of the stop solution, and read the A 450nm value at a wavelength of 450 nm using an enzyme-linked immunosorbent assay reader.

[0024] (3) Subcloning: Subclone the positive mother clone by the limiting dilution method, and use the ELISA method for subcloning screening, and expand the culture to prepare ascites.

[0025] (4)Antibody production and purification: Female Balb / c mice at 6 - 8 weeks old were selected and injected intraperitoneally with liquid paraffin, 0.5 mL per mouse. 7 - 10 days after the injection of liquid paraffin, hybridoma cells were injected intraperitoneally. When the abdomen of the mouse became enlarged and it was on the verge of death, the mouse was sacrificed by cervical dislocation, and ascites was aseptically extracted in a laminar flow hood. The ascites monoclonal antibody was purified using an AKTA protein purifier.

[0026] Example 2 In this example, sequence analysis was performed on the monoclonal antibody 2D11 prepared in Example 1, as follows: (1)Hybridoma cell culture The hybridoma cells screened in Example 1 that could secrete monoclonal antibody 2D11 were inoculated into RPMI 1640 medium (Gibco) containing 20% fetal bovine serum and cultured at 37°C.

[0027] (2)Cell RNA extraction Under the environment of a laminar flow hood, 1 mL of Trizol reagent was added to the centrifuged cells and allowed to stand for 5 min. Then 2 mL of chloroform was added, and the mixture was shaken vigorously for 15 s and allowed to stand at room temperature for 3 min. After centrifugation at 12000 rpm for 15 min, the upper aqueous layer was transferred to a new EP tube, 0.5 mL of isopropanol was added, and the mixture was allowed to stand at room temperature for 10 min. After centrifugation at 12000 rpm for 10 min, the supernatant was discarded, 1 mL of 75% ethanol was added, and after centrifugation at 7500 rpm for 5 min, the precipitate was dried and 50 μL of double-distilled water was added. The purity was identified by agarose electrophoresis and quantified, and it was stored at -70°C for later use.

[0028] (3)Reverse transcription to prepare cDNA 1 μL of total cellular RNA, 6 μL of RNase Free ddH2O, 0.5 μL of oligo dT Primer, 0.5 μL of PRIME Script RT Enzyme Mix I, and 2 μL of 5x Prime Script Buffer were mixed well. After extension at 37°C for 15 min, extension was carried out at 85°C for 5 s.

[0029] (4)Amplification of cDNA The mouse IgG VH VL primer library designed by Maisi Biology was used to amplify the above cDNA respectively. 10 μL of 5x Prime Star Buffer, 4 μL of dNTP, 1 μL of cDNA, 1 μL of upstream primer, 1 μL of downstream primer, 0.5 μL of PrimeSTAR, and water was added to make up to 50 μL. PCR reaction was carried out according to the following reaction conditions: incubation at 94°C for 5 min, denaturation at 94°C for 45 s, annealing at 63°C for 45 s, extension at 72°C for 1 min. After 30 cycles, extension was carried out at 72°C for 10 min.

[0030] (5)Agarose gel electrophoresis and gel extraction Perform agarose gel electrophoresis on the above PCR products, observe the electrophoresis results, and send the amplified products with molecular weights between 250 - 350 bp for sequencing. (6)Analyze the sequencing results Analyze the sequencing sequences in the Kabat database to obtain the correct amino acid sequences of the light chain and heavy chain variable regions. Among them, the sequence of the heavy chain universal forward primer VH - F: acggccagtgaattcmarctgcagsagtcwgg, and the sequence of the reverse primer VH - R: gattacgccaagctttgaggagacggtgaccg; the sequence of the light chain universal forward primer VL - F: acggccagtgaattccgattgtkctsacycartctcca, and the sequence of the reverse primer VL - R: gattacgccaagcttcgttggatctccagcttg. Screen for positive clones and perform sequencing, then analyze the sequencing sequences in the Kabat database to obtain the correct amino acid sequences of the light chain and heavy chain variable regions.

[0031] The sequences of the six CDR regions (complementary determining regions) analyzed are as follows: The amino acid sequence of the heavy chain complementary determining region VHCDR1: SYYIH (SEQ ID NO.1); The amino acid sequence of the heavy chain complementary determining region VHCDR2: WIYPGNVNTNYNENFKG (SEQ ID NO.2); The amino acid sequence of the heavy chain complementary determining region VHCDR3: STGWSPFDY (SEQ ID NO.3); The amino acid sequence of the light chain complementary determining region VLCDR1: RASENIYSHLA (SEQ ID NO.4); The amino acid sequence of the light chain complementary determining region VLCDR2: AARNLAD (SEQ ID NO.5); The amino acid sequence of the light chain complementary determining region VLCDR3: QHFWGTPWT (SEQ ID NO.6).

[0032] The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO.7, and the nucleotide sequence is shown in SEQ ID NO.8, specifically as follows: SEQ ID NO.7: GASVRISCKASGYIFTSYYIHWVKQRPGQGLEWIGWIYPGNVNTNYNENFKGKATLTADKSSSTAYMQLSSLTSEDSAVYFCARSTGWSPFDYWGQGTTLTVSS。

[0033] SEQ ID NO.8: GGGGCTTCAGTGAGGATATCCTGCAAGGCTTCTGGCTACATCTTCACAAGCTACTATATACACTGGGTGAAGCAGAGGCCTGGACAGGGACTTGAGTGGATTGGATGGATTTATCCTGGAAATGTTAATACTAATTACAATGAGAACTTCAAGGGCAAGGCCACACTGACTGCAGACAAATCCTCCAGCACAGCCTACATGCAGCTCAGCAGCCTGACCTCTGAGGACTCTGCGGTCTATTTCTGTGCAAGATCGACGGGATGGTCCCCTTTTGACTACTGGGGCCAAGGCACCACTCTCACAGTCTCCTCA。

[0034] The amino acid sequence of the light chain variable region is shown in SEQ ID NO.9, and the nucleotide sequence is shown in SEQ ID NO.10, as follows: SEQ ID NO.9: SFLTQSPASLSVSVGETVTITCRASENIYSHLAWYQQKQGKSPQLLVYAARNLADGVPSRFSGSGSGTQYSLKINRLQSEDFGTYYCQHFWGTPWTFGGGTKLEIQRSLA。

[0035] SEQ ID NO.10: AGTTTTCTTACCCAGTCTCCAGCCTCCCTATCTGTATCTGTGGGAGAAACTGTCACCATCACATGTCGAGCAAGTGAGAATATTTACAGTCATTTAGCATGGTATCAGCAGAAACAGGGAAAATCTCCTCAGCTCCTGGTCTATGCTGCAAGAAACTTAGCAGATGGTGTGCCATCAAGGTTCAGTGGCAGTGGATCAGGCACACAGTATTCCCTCAAGATCAACAGGCTGCAGTCTGAAGATTTTGGGACTTATTACTGTCAACATTTTTGGGGTACTCCGTGGACGTTCGGTGGAGGCACCAAGCTGGAGATCCAACGAAGCTTGGCG。

[0036] Example 3 In this example, the purity and subtype of the monoclonal antibody 2D11 prepared in Example 1 were identified. Among them, the antibody purity was analyzed by SDS-PAGE, and the subtype was identified by a mouse antibody subtype detection kit.

[0037] Result analysis: The results of SDS-PAGE are shown in Figure 1 , showing two bands with molecular weights of approximately 55 kDa and 25 kDa under reducing conditions, corresponding to the heavy and light chains of the IgG antibody, respectively. The subtype identification result of the monoclonal antibody 2D11 showed that the subtype was IgG2a.

[0038] Example 4 In this example, the functional analysis of the monoclonal antibody 2D11 prepared in Example 1 was carried out as follows: (1) Enzyme-linked immunosorbent assay (double antibody sandwich method) The purified CV-A5 rabbit polyclonal antibody (obtained by immunizing Japanese white rabbits with inactivated CV-A5 as an antigen using conventional methods) was diluted to 1:5000 with 0.05 mol / L phosphate buffer (pH 7.2), and 100 μL per well was used to coat a 96-well microplate, and the coating was carried out overnight at 4 °C; after washing the plate 3 times with PBST, a blocking solution (PBST containing 1% BSA) was added, 100 μL per well, and blocked at 37 °C for 1 h; after washing the plate 3 times with PBST, the CV-A5 stock solution was added, 100 μL per well, and incubated at 37 °C for 1 h; after washing the plate 3 times with PBST, 2D11 (concentration 1 μg / mL) was added, 100 μL per well, and incubated at 37 °C for 1 h; after washing the plate 3 times with PBST, HRP-labeled goat anti-mouse IgG (diluted 1:10000) was added, 100 μL per well, and incubated at 37 °C for 1 h; after washing the plate 3 times with PBST, substrate solution A (from Wuhan Institute of Biological Products Co., Ltd.) and substrate solution B (from Wuhan Institute of Biological Products Co., Ltd.) were added, both 50 μL per well, and developed color in the dark at 37 °C for 30 min; a termination solution was added, 50 μL per well; placed in an enzyme-linked immunosorbent assay instrument and read the A value at a wavelength of 450 nm. 450nm value.

[0039] The result of the ELISA experimental group was positive, indicating that the monoclonal antibody 2D11 could bind to CV-A5.

[0040] (2) Immunoblotting experiment The structural protein region of CV-A5 recognized by the screened 2D11 monoclonal antibody was identified by immunoblotting experiment. The steps were as follows: The CV-A5 stock solution was added to a reducing loading buffer, heated at 100 °C for 10 min for 4-20% SDS-PAGE; after electrophoresis, a rapid wet transfer instrument (GenScript) was used to transfer the membrane to a 0.45 μm nitrocellulose membrane; after the transfer was completed, a PBST blocking solution containing 2% BSA (W / V) was added and blocked for 30 min; 2D11 was added respectively and incubated at 37 °C for 1 h, and washed 5 times with PBST; HRP-labeled goat anti-mouse IgG was added, with a dilution of 1:10000, and incubated at 37 °C for 1 h; washed 5 times with PBST; imaged using a chemical exposure developing solution.

[0041] The results were as Figure 2 shown. Under the condition that CV-A5 was reduced, there were no meaningful bands in the experimental group of the Western Blot result. Combining the experimental results of this example (1), it was determined that the monoclonal antibody 2D11 targeted conformational epitopes.

[0042] (3) Indirect immunofluorescence experiment CV-A5 was inoculated into a 6-well plate with 95% confluence of RD cells. The RD cells without virus inoculation served as the negative control and were cultured. After culturing the 6-well plate for 24 h, the cell supernatant was discarded, and it was gently washed 3 times with 0.01 M PBS. 2 mL / well of 4% paraformaldehyde was added and fixed at room temperature for 1 h, followed by washing 5 times with 0.01 M PBS for 5 min each; 2 mL / well of 2% BSA-PBST (W / V) solution (containing 0.5% Triton-X 100, V / V) was added, and permeabilized at room temperature for 30 min, then washed 5 times with 0.01 M PBS for 5 min each; blocked 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 2D11 was added and incubated at room temperature for 1 h, followed by washing 5 times with 0.01 M PBS for 5 min each; 1 mL / well of 2 μg / mL goat anti-mouse fluorescent antibody IgG (H+L) (Abcam) was added, incubated in the dark at room temperature for 1 h, washed with PBS, and 1 mL / well of 5 μg / mL DAPI solution (Beyotime) was added; observed and photographed under a fluorescence microscope. The negative control group was cells not infected with CV-A5, and the corresponding antibodies were added. The results are as Figure 3 shown.

[0043] It can be seen from Figure 3 this that the monoclonal antibody 2D11 can be used for indirect immunofluorescence assay to recognize CV-A5 antigen and can be used for antigen discrimination assay.

[0044] Example 5 This example focuses on the study of the key conformational epitope sites targeted by the monoclonal antibody 2D11 prepared in Example 1, as follows: (1) Neutralizing antibody titer detection (in vitro micro-neutralization test) The purified monoclonal antibody 2D11 was diluted to 800 μg / mL, and then diluted at a ratio of 1:8 with a diluent (such as MEM maintenance solution), added to the first column of a 96-well plate, 100 μL / well, with 2 replicates set. 50 μL of diluent was added to each well of columns 2-12. The monoclonal antibody in the first column was serially diluted 2-fold to column 12, and 50 μL was discarded after mixing well in the last column. The neutralizing virus (monoclonal strain CV-A5-3487-M14-611) was diluted to 100 CCID 50 / 50 μL, and 50 μL was pipetted vertically and suspended into the 96-well plate with the diluted antibody. The 96-well plate was placed in a 37 °C incubator for neutralization for 2 h. The diluted neutralizing virus was serially diluted 10-fold to 10 CCID 50 / 50 μL, 1 CCID 50 / 50 μl, 0.1 CCID 50 / 50 μL. Add 50 μL of maintenance fluid and 50 μL of virus solution to each well (a total of 4 dilutions, namely 100, 10, 1, 0.1 CCID50 / 50 μL), with 8 replicates for each dilution, serving as the back-titration plate, and place it at 4°C. After neutralization, seed the digested RD cells into the neutralization plate and the back-titration plate at a density of 1×10 5 cells / mL. Incubate at 37°C and 5% CO2 for 5 - 7 days to determine the neutralization result. The neutralizing antibody titer of monoclonal antibody 2D11 measured by the micro-cytopathic effect inhibition method is shown to be 386, corresponding to a neutralizing concentration of 1.852 μg / mL.

[0045] (2)Screening of CV-A5 strains escaping from 2D11 and plaque purification Prepare a 96-well plate with 95% confluence of RD cells; dilute the CV-A5 virus solution to 1000 CCID 50 / 50 μL and place it in a 1.5 mL centrifuge tube; serially dilute monoclonal antibody 2D11 from 100 μg / mL by 2-fold to 7.408 μg / mL (4-fold neutralizing concentration); mix 50 μL of the serially diluted antibody with 50 μL of the diluted CV-A5 in equal volume, and place it in a 5% CO2 incubator at 37°C for 2 h of neutralization; after neutralization, add it to the 96-well plate with a monolayer of RD cells, and set up cell control wells, antibody control wells, and virus control wells, and culture in a 5% CO2 incubator at 37°C for 3 - 4 days, then observe under a microscope. If the experimental wells show cytopathic effects, freeze-thaw the harvest fluid 3 times, centrifuge at 4000 g at 4°C for 10 min, take the supernatant and aliquot it into 1.5 mL centrifuge tubes, and replace the virus solution with the supernatant and repeat the above steps. If cytopathic effects still appear after 3 - 6 rounds, it indicates that there may be a CV-A5 strain escaping from 2D11 in this well, and plaque purify the virus solution harvested from this well for 3 rounds.

[0046] After plaque purification, a total of 5 single-genotype CV-A5 strains were obtained. Align the amino acid sequences of their structural protein regions, and it can be seen that all 16 CV-A5 strains have 3 common mutations at VP2, VP3, and VP1 in the structural protein region (see Table 1).

[0047] Table 1 Amino acid sequence differences in the structural proteins of cloned strains

[0048] Note: “\” indicates the same amino acid as CV-A5-wt (3)Detection of the titer of neutralizing antibody 2D11 against the escape strain (in vitro micro-neutralization assay) Neutralization titers were determined for a monoclonal strain CV-A5-3 that had and only had this common mutation. Add 2D11 (concentration 100 μg / mL) to the first column of a 96-well plate, 100 μL per well, set up 2 replicates, and add 50 μL of dilution solution (MEM maintenance solution) to each well in columns 2 - 12. Continuously dilute the supernatant in the first column 2-fold to column 12, and discard 50 μL after mixing the last column. Dilute CV-A5-3 to 100 CCID 50 / 50 μL, pipette 50 μL vertically and add it to the 96-well plate with diluted antibody, as the neutralization plate, and incubate it in a 37 °C incubator for 2 h. Dilute the diluted CV-A5-3 in a 10-fold gradient to 10 CCID 50 / 50 μL, 1 CCID 50 / 50 μL, 0.1 CCID 50 / 50 μL. Add 50 μL of maintenance solution and 50 μL of virus solution to each well (a total of 4 dilution degrees, namely 100, 10, 1, 0.1 CCID 50 / 50 μL), with 8 replicates for each dilution degree, as the back-titration plate, and place it at 4 °C. After neutralization, spread the prepared RD cell suspension evenly onto the neutralization plate and the back-titration plate at a density of 1×10 5 cells / mL, culture it in a 5% CO2 incubator at 37 °C for 3 - 4 days, and then observe and determine the neutralization result under a microscope.

[0049] The results are shown in Table 2. CV-A5-3 showed escape against monoclonal antibody 2D11 (wells with antibody concentration higher than 50 μg / mL not being neutralized is considered escape). Based on the neutralization results, analyze the differences in the amino acid sequences of the structural protein regions between the escape strain CV-A5-3 and the neutralization strain CV-A5-wt, and speculate that the significant decrease in the neutralization ability of 2D11 against this virus is related to the amino acids at positions 76 on VP2, 179 on VP3, and 288 on VP1 of the structural protein. Mutations were made at these three amino acid sites on the basis of the monoclonal strain to obtain recombinant strains rCVA5, rCVA5-K2076R, rCVA5-E3179K, and rCVA5-F1288S.

[0050] Furthermore, the recombinant strains were subjected to the neutralizing antibody titer determination in this Example (3), and the results are shown in Table 2: The results prove that the mutation of K2076R can significantly affect the neutralization ability of monoclonal antibody 2D11 against CV-A5, and the neutralization site recognized by monoclonal antibody 2D11 may be the conformational epitope including the amino acid at position 76 of CV-A5 virus VP2.

[0051] Table 2 Neutralization verification results of monoclonal antibody 2D11 escape site mutant strains

[0052] Example 6 This example focuses on the application of the monoclonal antibody 2D11 prepared in Example 1, which is as follows: Coat a 96-well microplate with the stock solutions of CV-A5, CV-A6, CV-A10, EV-A71, CV-A4, CV-16 and the cell supernatant lysate, 100 μL per well, overnight at 4°C; after washing the plate 3 times with PBST, add the blocking solution (PBST containing 1% BSA), 100 μL per well, block at 37°C for 1 h; after washing the plate 3 times with PBST, add 2D11 (at a concentration of 1 μg / mL), 100 μL per well, incubate at 37°C for 1 h; after washing the plate 3 times with PBST, add HRP-labeled goat anti-mouse IgG (diluted 1:10000), 100 μL per well, incubate at 37°C for 1 h; after washing the plate 3 times with PBST, add substrate solution A and substrate solution B, both 50 μL per well, develop color at 37°C in the dark for 30 min; add the stop solution, 50 μL per well; place it in an enzyme-linked immunosorbent assay (ELISA) reader and read the A 450nm value at a wavelength of 450 nm.

[0053] As shown in Table 3 of the results, after 2D11 binds to the above-mentioned virus strains, only the ratio of CV-A5 to the negative control is greater than 2.1, showing a positive result, and the ratios of other virus strains to the negative control are all lower than 2.1. Therefore, 2D11 can specifically recognize the CV-A5 virus.

[0054] Table 3 ELISA detection of group enteroviruses (partial) and 2D11

[0055] Note: "-" indicates that it cannot bind to 2D11, and "+" indicates that it can bind to 2D11 The inventors also found through experiments that the amino acid sequence formed by replacing, deleting or adding one or more amino acid sequences in the amino acid sequence shown in SEQ ID NO.7 of the heavy chain amino acid sequence of the monoclonal antibody 2D11 or the 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 formed by replacing, deleting or adding one or more amino acid sequences in the amino acid sequence shown in SEQ ID NO.9 of the light chain amino acid sequence of the monoclonal antibody 2D11 or the amino acid sequence having more than 95% homology with the amino acid sequence shown in SEQ ID NO.9 has the same function as the sequence shown in SEQ ID NO.9.

[0056] In the present invention, the specific raw materials not described are all existing substances and can be directly purchased from the market.

[0057] The above are only the 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 principle of the present invention shall be included within the protection scope of the present invention.

Claims

1. A monoclonal antibody 2D11 that specifically recognizes CV-A5 virus and has neutralizing activity, characterized in that, The six CDR regions of the monoclonal antibody 2D11 are specifically as follows: (1) The heavy chain CDR1 contains the amino acid sequence shown in SEQ ID NO.1; (2) The heavy chain CDR2 contains the amino acid sequence shown in SEQ ID NO.2; (3) The heavy chain CDR3 contains the amino acid sequence shown in SEQ ID NO.3; (4) The light chain CDR1 contains the amino acid sequence shown in SEQ ID NO.4; (5) The light chain CDR2 contains the amino acid sequence shown in SEQ ID NO.5; (6) The light chain CDR3 contains the amino acid sequence shown in SEQ ID NO.

6.

2. The monoclonal antibody 2D11 according to claim 1, wherein The heavy chain variable region of the monoclonal antibody 2D11 contains the amino acid sequence shown in SEQ ID NO.7, or a sequence with equivalent function formed by substitution, deletion or addition of one or more amino acids to the amino acid sequence shown in SEQ ID NO.7; and / or, the light chain variable region of the monoclonal antibody 2D11 contains the amino acid sequence shown in SEQ ID NO.9, or a sequence with equivalent function formed by substitution, deletion or addition of one or more amino acids to the amino acid sequence shown in SEQ ID NO.

9.

3. The monoclonal antibody 2D11 according to claim 1, wherein The monoclonal antibody 2D11 is an IgG-type antibody.

4. The monoclonal antibody 2D11 according to claim 1, characterized in that, The monoclonal antibody 2D11 targets a conformational epitope.

5. The nucleotide sequence encoding the monoclonal antibody 2D11 according to any one of claims 1-4.

6. The nucleotide sequence according to claim 5, wherein The nucleotide sequence encoding the heavy chain variable region of the monoclonal antibody 2D11 is any one of the following: a. Having the nucleotide sequence shown in SEQ ID NO.8; b. The complementary nucleotide sequence to the nucleotide sequence shown in SEQ ID NO.8; c. A nucleotide sequence that encodes the same protein as the nucleotide sequences of a and b, but is different from them due to the degeneracy of the genetic code; and / or, the nucleotide sequence encoding the light chain variable region of the monoclonal antibody 2D11 is any one of the following: e. Having the nucleotide sequence shown in SEQ ID NO.10; f. The 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 sequences of e and f, but is different from them due to the degeneracy of the genetic code.

7. An expression vector, characterized in that, Containing the nucleotide sequence according to claim 5 or 6.

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

9. An antibody conjugate, characterized in that, The antibody conjugate contains the monoclonal antibody 2D11 according to any one of claims 1-4 or an antigen-binding fragment of the monoclonal antibody 2D11 and a label, and the label is selected from one or more of enzyme labeling, biotin labeling, and chemiluminescent dye labeling.

10. The use of the monoclonal antibody 2D11 according to any one of claims 1-4 in the preparation of a reagent or kit for detecting Coxsackievirus A5; and / or, the use of the monoclonal antibody 2D11 in the preparation of a drug for inhibiting, preventing and treating Coxsackievirus A5.

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