Monoclonal antibody for recognizing CV-A6 and application thereof

Abstract

The invention provides a monoclonal antibody for identifying CV-A6 and application thereof, the monoclonal antibody for identifying CV-A6 comprises a heavy chain complementarity determining region and a light chain complementarity determining region, the heavy chain complementarity determining region comprises VHCDR1, VHCDR2 and VHCDR3, the amino acid sequences of the VHCDR1, VHCDR2 and VHCDR3 are respectively shown as SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3; the light chain complementarity determining region comprises VLCDR1, VLCDR2 and VLCDR3, and the amino acid sequences of the VLCDR1, the VLCDR2 and the VLCDR3 are respectively shown as SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6. The monoclonal antibody provided by the invention not only can effectively identify the CV-A6 virus, but also has remarkable neutralizing activity, and can be used for quantitatively determining the CV-A6 virus.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and particularly relates to a monoclonal antibody that recognizes CV-A6 and its applications. Background Art

[0002] Hand, foot and mouth disease (HFMD) is caused by enteroviruses in the genus Enterovirus of the family Picornavirus. Common pathogenic viruses include types 2, 4, 5, 6, 7, 9, 10, 16, etc. in group A of Coxsackievirus, types 1, 2, 3, 4, 5, 6, 13, etc. in group B of Coxsackievirus, as well as Human Enterovirus 71 (EV-A71) and Echoviruses. Among them, EV-A71 and CV-A16 have always been considered the main pathogenic viruses of hand, foot and mouth disease.

[0003] However, with the approval of the EV-A71 inactivated vaccine in China in 2016, the incidence and mortality of EV-A71-related hand, foot and mouth disease have decreased significantly. Instead, infections with types 6 (CV-A6) and 10 (CV-A10) of group A of Coxsackievirus have become the main pathogens of hand, foot and mouth disease, and the infection rates of EV-A71 and CV-A16 have decreased significantly. This change reflects a significant shift in the virus epidemic spectrum of hand, foot and mouth disease. Similarly, in countries and regions where the EV-A71 vaccine has not been popularized, the proportion of hand, foot and mouth disease caused by non-EV-A71 enteroviruses has gradually increased. CV-A6 has become the most common pathogen and has a high proportion in both common cases and severe cases.

[0004] Research on CV-A6 is particularly important because conformational neutralizing antibodies can reflect the active ingredient content and structural function of virus vaccines. Therefore, developing neutralizing monoclonal antibodies against CV-A6, especially antibodies that can target its specific conformational epitopes, will contribute to quality control in vaccine research and development, clinical detection, and laboratory virus identification. In addition, the precise localization of virus neutralizing epitopes has important theoretical significance for the design and development of CV-A6 vaccines, providing key clues for further studying its immune mechanism and basic functions. These research results are not only crucial for improving the development efficiency of hand, foot and mouth disease vaccines, but also provide new directions for effectively controlling the spread of HFMD and clinical treatment. Summary of the Invention

[0005] In view of this, the present invention provides a monoclonal antibody that recognizes CV-A6 and its applications. It is a neutralizing antibody of the IgG2a subtype and can recognize CV-A6.

[0006] To achieve the above object, the present invention adopts the following technical solutions: In a first aspect, the present invention provides a monoclonal antibody that recognizes CV-A6, comprising a heavy-chain complementary determining region and a light-chain complementary determining region. The heavy-chain complementary determining region includes VHCDR1, VHCDR2, and VHCDR3. The amino acid sequence of VHCDR1 is as shown in SEQ ID NO:1, the amino acid sequence of VHCDR2 is as shown in SEQ ID NO:2, and the amino acid sequence of VHCDR3 is as shown in SEQ ID NO:3. The light-chain complementary determining region includes VLCDR1, VLCDR2, and VLCDR3. The amino acid sequence of VLCDR1 is as shown in SEQ ID NO:4, the amino acid sequence of VLCDR2 is as shown in SEQ ID NO:5, and the amino acid sequence of VLCDR2 is as shown in SEQ ID NO:6.

[0007] Preferably, the antibody comprises a heavy chain and a light chain. The amino acid sequence of the heavy-chain variable region is as shown in SEQ ID NO:7; and / or, the amino acid sequence of the light-chain variable region is as shown in SEQ ID NO:9.

[0008] Preferably, the monoclonal antibody is an IgG2a-type antibody; and / or, the antigenic epitope targeted by the monoclonal antibody is a conformational epitope.

[0009] In a second aspect, the present invention provides a nucleotide molecule encoding the monoclonal antibody that recognizes CV-A6.

[0010] Preferably, the nucleic acid molecule encoding the heavy-chain variable region is any one of the following: a. Having the nucleotide sequence as 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 nucleic acid molecule encoding the light-chain variable region is any one of the following: d. Having the nucleotide sequence as shown in SEQ ID NO:10; e. A nucleotide sequence complementary to the nucleotide sequence shown in SEQ ID NO:10; f. A nucleotide sequence that encodes the same protein as the nucleotide sequences of d and e but is different therefrom due to the degeneracy of the genetic code.

[0011] In a third aspect, the present invention provides a recombinant DNA expression vector comprising the polynucleotide molecule.

[0012] In a fourth aspect, the present invention provides a host cell containing the nucleotide molecule described above, or containing the expression vector described above.

[0013] In a fifth aspect, the present invention provides an antibody conjugate comprising the monoclonal antibody described above or an antigen-binding fragment thereof and a label.

[0014] In a sixth aspect, the present invention provides the use of the antibody described above, or a monoclonal antibody encoded by the nucleotide molecule described above, in the preparation of a reagent or kit for detecting CV-A6.

[0015] In a seventh aspect, the present invention provides the use of the antibody described above, or a monoclonal antibody encoded by the nucleotide molecule described above, in the preparation of reagents and drugs for diagnosing, inhibiting, preventing and treating diseases caused by CV-A6.

[0016] 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 significantly different from the existing CV-A6 monoclonal antibodies, and it binds to different neutralizing sites. This antibody can not only effectively recognize CV-A6 virus, but also has significant neutralizing activity and can be used for quantitative determination of CV-A6 virus. Its reactivity is closely related to the content and function of the virus, and is also closely associated with the immunogenicity of the virus. Therefore, this antibody has important application value in the process of vaccine research and development, especially in the quality control and immunogenicity evaluation of vaccines.

[0017] (2) The monoclonal antibody of the present invention can be combined with a conjugate (such as horseradish peroxidase or fluorescein isothiocyanate, etc.) 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-A6, and can also be used for laboratory identification of antibodies of clinical virus isolates. At the same time, it can also be used to prepare intermediate products for the production of preventive vaccines and quantitative detection reagents for products containing CV-A6 antigen. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 It is an SDS-PAGE diagram of the IgG light chain and heavy chain of the monoclonal antibody 3E11 provided in Example 4 of the present invention; Figure 2 It is an indirect immunofluorescence result diagram of the antigen after the monoclonal antibody 3E11 provided in Example 5 of the present invention detects CV-A6-infected RD cells; Figure 3 It is a diagram of the in vitro micro-neutralization experiment result of the monoclonal antibody 3E11 provided in Example 5 of the present invention and CV-A6; Figure 4The recognition ability results graph of monoclonal antibody 3E11 provided in Example 5 of the present invention for EP, FP, and Heated FP; Figure 5 The determination result graph of the in vivo protective efficacy of monoclonal antibody 3E11 provided in Example 6 of the present invention. Specific implementation manners

[0019] The following further elaborates on the present invention in conjunction with specific embodiments, so that those skilled in the art can understand the present invention more clearly.

[0020] The technical concept of the present invention is to provide a neutralizing monoclonal antibody that can recognize CV-A6.

[0021] The amino acid sequence of the heavy chain complementarity determining region VHCDR1 of the monoclonal antibody: SFGMH (SEQ ID NO.1); The amino acid sequence of the heavy chain complementarity determining region VHCDR2 of the monoclonal antibody: YISSGSSTLHYADTVKG (SEQ ID NO.2); The amino acid sequence of the heavy chain complementarity determining region VHCDR3 of the monoclonal antibody: KLPSLCYGLLGS (SEQ ID NO.3); The amino acid sequence of the light chain complementarity determining region VLCDR1 of the monoclonal antibody: KSSQSLEDSNGNTYLN (SEQ ID NO.4); The amino acid sequence of the light chain complementarity determining region VLCDR2 of the monoclonal antibody: RVSNRFS (SEQ ID NO.5); The amino acid sequence of the light chain complementarity determining region VLCDR3 of the monoclonal antibody: LQVTHVPWT (SEQ ID NO.6).

[0022] The amino acid sequence of the heavy chain variable region of the monoclonal antibody is as shown in SEQ ID NO:7, and the nucleotide sequence is as shown in SEQ ID NO:8: The amino acid sequence information of SEQ ID NO:7 is: EVQLQESGGGLVQPGGSRKLSCAASGFTFSSFGMHWVRQAPEKGLEWVAYISSGSSTLHYADTVKGRFTISRDNPKNTLFLQMTSLRSEDTAMYYCARKLPSLCYGLLGSRNQGTLLLSQN (SEQ ID NO.7); The nucleotide sequence information of SEQ ID NO:8 is as follows: GAGGTGCAGCTGCAGGAGTCTGGGGGAGGCTTAGTGCAGCCTGGAGGGTCCCGGAAACTCTCCTGTGCAGCCTCTGGATTCACTTTCAGTAGCTTTGGAATGCACTGGGTTCGTCAGGCTCCAGAGAAGGGGCTGGAGTGGGTCGCATACATTAGTAGTGGCAGTAGTACCCTCCACTATGCAGACACAGTGAAGGGCCGATTCACCATCTCCAGAGACAATCCCAAGAACACCCTGTTCCTGCAAATGACCAGTCTAAGGTCTGAGGACACGGCCATGTATTACTGTGCAAGAAAACTACCCTCACTATGCTATGGACTACTGGGGTCAAGGAACCAAGGGACTCTGCTCCTCAGCCAAAAC (SEQ IDNO.8) The amino acid sequence of the variable region of the monoclonal antibody light chain is as shown in SEQ ID NO:9, and the nucleotide sequence is as shown in SEQ IDNO:10: The amino acid sequence information of SEQ ID NO:9 is as follows: SVVVETASISCKSSQSLEDSNGNTYLNWYLQKPGQSPQLLIYRVSNRFSGVLDRFSGSGSGTDFTLKISRVEAEDLGVYFCLQVTHVPWT FGGGTKLEIK (SEQ ID NO.9); The nucleotide sequence information of SEQ ID NO:10 is as follows: TCAGTCGTCGTGGAGACAGCCTCCATCTCTTGCAAGTCTAGTCAGAGCCTTGAAGACAGTAATGGAAACACCTATTTGAACTGGTACCTCCAGAAACCAGGCCAGTCTCCACAGCTCCTGATCTACAGGGTTTCCAACCGATTTTCTGGGGTCCTAGACAGGTTCAGTGGTAGTGGATCAGGGACAGATTTTACACTGAAAATCAGCAGAGTGGAGGCTGAAGATTTGGGAGTTTATTTCTGCCTCCAAGTTACACATGTCCCGTGGACGTTCGGTGGAGGCACCAAGCTGGAAATCAAA (SEQ ID NO :10).

[0023] The present invention will be further described in detail below in conjunction with specific embodiments, so that those skilled in the art can understand the present invention more clearly. The following embodiments are only used to illustrate the present invention, but not to limit the scope of the present invention. Based on the specific embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative work belong to the protection scope of the present invention. In the embodiments 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 embodiments of the present invention, unless specifically specified, the technical means used are conventional means well known to those skilled in the art.

[0024] Reagent source: Complete and incomplete Freund's adjuvant: Purchased from Sigma Company; Female Balb / c mice, RD cells, Solution A, Solution B, CV-A6 stock solution, CV-A6 challenge strain (CVA6-R10): Provided by Wuhan Institute of Biological Products Co., Ltd.; Isotyping Kit for Mouse Monoclonal Antibody: Purchased from Beijing Sino Biological Inc.; Goat anti-mouse IgG (H+L) Alexa Fluor 488: Purchased from Thermo Fisher Scientific; HRP-labeled goat anti-mouse IgG: Purchased from Wuhan Boster Biological Technology Co., Ltd.; CV-A6 is a strain disclosed in the prior art and is recorded in the NCBI database with the accession number: MW410845.

[0025] Example 1 Preparation of CV-A6 virus solid particles and hollow particles This embodiment provides a method for preparing CV-A6 virus solid particles and hollow particles, which is as follows: Using the CV-A6 virus strain provided by Wuhan Institute of Biological Products Co., Ltd. as the research object. First, the virus solution is preliminarily purified through a clarification and ultrafiltration step. Subsequently, a 20% (W / V) sucrose solution is used for bottom cushion treatment, and the virus particles are concentrated and separated by centrifugation (141,000×g, 3 hours). The precipitate is resuspended overnight at 4°C in 200 μL of PBS buffer (pH 7.2) to ensure sufficient dissociation of the virus particles. The resuspended sample is mixed with PBS buffer and cesium chloride (CsCl) to prepare a CsCl solution with a concentration of 1.34 g / mL. Then, high-speed centrifugation (288,000×g, 20 - 22 hours) is used to further purify the virus. After centrifugation, the centrifuge tube is gently transferred to a dark room, and the whole tube is irradiated with a direct light source to observe the bands based on the turbidity and light phenomenon of protein precipitation. According to the light conditions and the distribution of the bands, two virus bands are extracted respectively.

[0026] Finally, the two extracted virus bands are identified by electron microscopy, and it is confirmed that they are two types of virus particles, hollow and solid, which are respectively labeled as CV-A6 FP (hollow particles) and CV-A6 EP (solid particles). This process provides an efficient experimental method for the purification and separation of CV-A6 virus, and lays a foundation for subsequent functional analysis and research.

[0027] Example 2 Preparation of Monoclonal Antibodies (1) Immunization: First, the purified inactivated CV-A6 virus solution is used as an antigen to immunize BALB / c mice through Freund's adjuvant. The immunization protocol is as follows: The mice are intraperitoneally injected four times at intervals of 14 days. Before cell fusion, a single intravenous injection of CV-A6 inactivated antigen is used for booster immunization. After immunization, the antibody titer is detected by collecting mouse serum to evaluate the immunization effect.

[0028] (2) Fusion and Screening: After immunization, hybridoma cells are obtained by fusing spleen lymphocytes and myeloma cells. The screening of the fused cells is carried out by the ELISA method, and the specific operation is as follows: Antigen Coating: Dilute the inactivated CV-A6 antigen with 0.05 M carbonate buffer to a concentration of 1 μg / mL, and coat it on a 96-well microplate overnight at 4°C; Blocking: Block with a blocking solution (PBST - 1% BSA) at 37°C for 1 hour to prevent non-specific binding;; Detection: Add the supernatant of the fused cells to be tested (100 μL / well) and incubate at 37°C for 1 hour; Secondary antibody reaction: Add 100 μL / well of HRP-labeled goat anti-mouse IgG (diluted 1:10,000), and continue to incubate at 37 °C for 1 hour; Color development: After washing the plate, add substrate solutions A and B (50 μL / well each), and develop color at 37 °C in the dark for 15 minutes. Then add the stop solution (50 μL / well), and read the OD 450 value at a wavelength of 450 nm using an ELISA reader.

[0029] Through this screening step, the positive hybridoma cell line 3E11 was obtained.

[0030] (3) Subcloning: Subclone the positive hybridoma cells using the limiting dilution method, screen out monoclonal cells with high affinity, and perform amplification culture. At the same time, use the ELISA method to screen for antibodies for each subclone.

[0031] (4) Ascites production and antibody purification: Ascites induction: Select female BALB / c mice aged 6 - 8 weeks, and induce ascites formation by intraperitoneal injection of liquid paraffin (0.5 mL per mouse). 7 - 10 days after injecting liquid paraffin, inject hybridoma cells intraperitoneally. When the mouse's abdomen swells and it is near death, sacrifice the mouse by cervical dislocation, and aseptically extract ascites under a laminar flow hood.

[0032] Protein A affinity chromatography purification: Equilibration: Equilibrate the chromatography column with 5 - 10 column volumes of PBS buffer (pH 7.0) at a flow rate of 5 mL / min.

[0033] Loading: Load 2 mL of pretreated ascites sample at a flow rate of 5 mL / min.

[0034] Flow-through: Elute with PBS buffer for 5 column volumes to remove impurities.

[0035] Elution: Elute the antibody using glycine buffer (pH 3.0) with an elution volume of 10 column volumes at a flow rate of 5 mL / min. When the baseline starts to rise and an elution peak appears, collect the eluate in a 15 mL centrifuge tube. Subsequently, adjust the pH of the eluate to 7.0 using 1 M Tris buffer (pH 9.2).

[0036] Washing and storage: After elution, continue to wash the column with 0.5 M sodium hydroxide for 10 column volumes at a flow rate of 10 mL / min. Finally, wash the column with 20% ethanol and store it at a flow rate of 10 mL / min.

[0037] In this example, the 3E11 monoclonal antibody was prepared and purified.

[0038] Sequence Analysis of Monoclonal Antibodies Screened in Example 3 The 3E11 hybridoma cells screened in Example 1 were inoculated into RPMI 1640 medium (Gibco) containing 20% fetal bovine serum and cultured under the conditions of 37°C and 5% CO2.

[0039] Using the TaKaRa RimeScript™ II 1st Strand cDNA Synthesis Kit, the total RNA extracted was reverse-transcribed with an Oligo dT primer to synthesize cDNA. This step can convert mRNA into a cDNA template for PCR amplification. Universal primers for the heavy chain and light chain were used to amplify the variable region genes of the heavy chain and light chain of the 3E11 monoclonal antibody by PCR. During the amplification process, primers with homologous sequences to the cloning vector pUC-Kan were used to ensure that the amplified gene fragments could be correctly inserted into the vector. The purified PCR products were separately cloned into the pUC-Kan vector, which was provided by Nanjing Genscript Biotech Co., Ltd. and had a high transformation efficiency and screening marker. The cloned vector was transformed into Escherichia coli (usually by chemical transformation or electroporation methods), and positive clones were obtained through antibiotic screening. Plasmid DNA was extracted from the positive clones, and the inserted heavy chain and light chain genes were sequenced. By comparing the sequencing results with the known sequences in the Kabat database, it was determined whether the amino acid sequences of the variable regions of the heavy chain and light chain were accurate to ensure that the correct antibody variable region sequences were obtained.

[0040] Among them, the sequence of the heavy chain universal forward primer VH-F is: ACGGCCAGTGAATTCMARCTGCAGSAGTCWGG; the reverse primer VH-R sequence: GATTACGCCAAGCTTTGAGGAGACGGTGACCg, the sequence of the light chain universal forward primer VL-F is: ACGGCCAGTGAATTCCGATTGTKCTSACYCARTCTCCA, and the reverse primer VL-R sequence: GATTACGCCAAGCTTCGTTGGATCTCCAGCTTG. Positive clones were obtained through screening and sequenced, and the sequenced sequences were analyzed in the Kabat database to obtain the correct amino acid sequences of the variable regions of the light chain and heavy chain.

[0041] The sequences analyzed from the sequencing results of the monoclonal antibody 3E11 are shown in SEQ ID NO:1-10.

[0042] Example 4 Purity and Subtype Identification First, the purified 3E11 monoclonal antibody in Example 1 was mixed evenly with a protein loading buffer in an appropriate proportion. The mixture was heated at 95 °C for 10 minutes to fully denature the antibody. After heating, it was briefly centrifuged to remove air bubbles and impurities, and the supernatant was taken for subsequent experiments. SDS-PAGE analysis was performed using a 4-20% polyacrylamide gel. After loading the samples, electrophoresis separation was carried out according to the conventional steps. After the electrophoresis was completed, the gel was stained with Coomassie Brilliant Blue and decolorized using an eStain® protein staining instrument (Nanjing Genscript Biotech Co., Ltd.) to reveal the heavy and light chains of the antibody. The SDS-PAGE results are shown in Figure 1 , showing two bands with molecular weights of approximately 50 kDa and 25 kDa under reducing conditions, corresponding to the heavy and light chains of the antibody respectively. This result indicates that the antibody can be effectively depolymerized into its constituent heavy and light chains under reducing conditions. A mouse antibody subtype detection kit was used to identify the subtype of the 3E11 monoclonal antibody. The experimental results showed that the subtype of the 3E11 monoclonal antibody was IgG2a.

[0043] Example 5 Functional Analysis (1) Indirect immunofluorescence assay CV-A6 was inoculated into a 6-well plate with 95% confluent RD cells. The RD cells without virus inoculation were used as negative controls and cultured. After culturing the 6-well plate for 24 h, the cell supernatant was discarded, and the cells were gently washed 3 times with PBS, then fixed with 2 mL of 4% paraformaldehyde at room temperature for 1 h, and washed 3 times with PBS for 5 min each. The cells were permeabilized with 2 mL of PBST solution containing 0.5% Triton-X 100 at room temperature for 30 minutes, and then the cells were washed 3 times with PBS for 5 minutes each. Next, PBST containing 2% BSA was used as a blocking solution and blocked at room temperature for 1 hour, and the blocking solution was discarded. 1 mL of the 3E11 monoclonal antibody (concentration 2 μg / mL) was added and incubated at room temperature for 1 hour, and the cells were washed 3 times for 5 minutes each. Subsequently, 1 mL of Alexa Fluor 488-labeled goat anti-mouse IgG (concentration 2 μg / mL) was added and incubated in the dark at room temperature for 1 hour, and washed 3 times again. 1 mL of DAPI solution (concentration 5 μg / mL) was added for nuclear staining. After washing 3 times, the cells were observed and photographed under a fluorescence microscope. The negative control group used RD cells without CV-A6 inoculation and added the corresponding antibodies.

[0044] The results are as shown in Figure 2 . The monoclonal antibody 3E11 can be used in indirect immunofluorescence assays to recognize CV-A6 antigens and can be used for antigen discrimination experiments.

[0045] (2) Neutralization activity identification Dilute the purified 3E11 monoclonal antibody to 100 μg / mL, add it to the first column of a 96-well plate, add 100 μL to each well, and set up 8 replicates. Then serially dilute the antibody in the first column 2-fold. Dilute the harvested fluids of different CV-A6 strains to 100 CCID50 / 50 μL, and take 50 μL and add it to the diluted antibody solution. Place the 96-well plate in an incubator at 37 °C and incubate for 2 hours for neutralization. Gradient dilute the virus solution 10-fold, and finally dilute it to 100, 10, 1, 0.1 CCID50 / 50 μL. Set up 8 replicates for each dilution. Add 50 μL of MEM maintenance solution and 50 μL of the diluted virus solution to the back-titration plate. Then inoculate the digested RD cells (at a density of 1×10 5 cells / mL) into the neutralization plate and the back-titration plate. Place the cell culture plates in an incubator at 37 °C and 5% CO2 and culture for 4 days.

[0046] The test results of the neutralization activity of the monoclonal antibody 3E11 against CV-A6 are as Figure 3 shown. The monoclonal antibody 3E11 can effectively neutralize the infection of CV-A6 virus, verifying its neutralization activity.

[0047] (3) Identification of the binding activity against CV-A6 EP, FP and Heated FP Dilute EP, FP of CV-A6 and FP after heat treatment (FP treated at 56 °C for 15 minutes) to 1 μg / mL respectively, and coat them onto an ELISA plate with 0.05 M carbonate buffer (pH 7.2), 100 μL per well, overnight at 4 °C. After washing the plate 3 times, add 200 μL of blocking solution (PBST containing 1% BSA) for blocking, and incubate at 37 °C for 1 hour. After discarding the blocking solution, continue to incubate the 3E11 monoclonal antibody (diluted 1:1000), 100 μL per well, at 37 °C for 1 hour. Wash the plate 5 times, add HRP-labeled goat anti-mouse IgG (diluted 1:10000), 100 μL per well, and incubate at 37 °C for 1 h; wash again with PBST 5 times, add 50 μL each of substrate solution A and B, and incubate at 37 °C for 15 min; add 2 M sulfuric acid to each well at 50 μL to terminate the reaction, and read the OD 450 nm value at a wavelength of 450 nm using an ELISA reader.

[0048] The test of the binding activity of the monoclonal antibody 3E11 against CV-A6 EP, FP, Heated FP results are as Figure 4 shown. The monoclonal antibody 3E11 can specifically bind to the EP, FP and FP after heat treatment antigens of CV-A6, indicating its good antigen-binding activity.

[0049] Example 6 Therapeutic Immunoprotection To evaluate the protective ability of monoclonal antibody 3E11 in mice, a lethal dose of CV-A6 challenge strain (CVA6-R10) was intraperitoneally injected into 7-day-old Kunming mice at a dose of 100 μL per mouse. Four hours later, different concentrations of monoclonal antibody 3E11 (400 μg / mL, 80 μg / mL, 16 μg / mL, 3.2 μg / mL, 0.64 μg / mL) were intraperitoneally injected into the above mice at a dose of 100 μL per mouse, and the mice were continuously observed for 14 days, and the survival rate of the mice was recorded every day.

[0050] The determination results of the protective efficacy of monoclonal antibody 3E11 in mice are as Figure 5 shown. When the dose of monoclonal antibody was 0.32 μg / mouse, all mice survived during the 14-day observation period, showing a 100% protection rate. This result indicates that monoclonal antibody 3E11 has significant in vivo protective efficacy.

[0051] Further experimental analysis found that after one or more amino acids in the heavy chain amino acid sequence (SEQ ID NO.7) and light chain amino acid sequence (SEQ ID NO.8) of monoclonal antibody 3E11 were substituted, deleted or added, if the new amino acid sequence had more than 95% homology with the original sequence, the new sequence still retained the same function as the original sequence. In other words, amino acid sequences highly homologous to SEQ ID NO.7 and SEQ ID NO.8 can still exert the same biological function as monoclonal antibody 3E11. The results of this in vivo protection experiment indicate that monoclonal antibody 3E11 has a significant protective effect in mice. In addition, the analysis of the amino acid sequence of monoclonal antibody 3E11 further shows that changes in the amino acid sequences of its heavy chain and light chain within a certain range do not affect its function, providing potential sequence variability support for its future immunotherapy.

[0052] It is necessary to point out here that the above embodiments are only for further elaboration and illustration of the technical solutions of the present invention, and are not further limitations on the technical solutions of the present invention. The method of the present invention is only a preferred implementation, and is not used to limit the protection scope of the present invention. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

[0053] In the present invention, if no specific raw materials are described, they are all existing substances and can be directly purchased from the market.

[0054] Gene sequence SEQ ID NO.1 SFGMH.

[0055] SEQ ID NO.2 YISSGSSTLHYADTVKG。

[0056] SEQ ID NO.3 KLPSLCYGLLGS。

[0057] SEQ ID NO.4 KSSQSLEDSNGNTYLN。

[0058] SEQ ID NO.5 RVSNRFS。

[0059] SEQ ID NO.6 LQVTHVPWT。

[0060] SEQ ID NO:7 EVQLQESGGGLVQPGGSRKLSCAASGFTFSSFGMHWVRQAPEKGLEWVAYISSGSSTLHYADTVKGRFTISRDNPKNTLFLQMTSLRSEDTAMYYCARKLPSLCYGLLGSRNQGTLLLSQN。

[0061] SEQ ID NO:8 GAGGTGCAGCTGCAGGAGTCTGGGGGAGGCTTAGTGCAGCCTGGAGGGTCCCGGAAACTCTCCTGTGCAGCCTCTGGATTCACTTTCAGTAGCTTTGGAATGCACTGGGTTCGTCAGGCTCCAGAGAAGGGGCTGGAGTGGGTCGCATACATTAGTAGTGGCAGTAGTACCCTCCACTATGCAGACACAGTGAAGGGCCGATTCACCATCTCCAGAGACAATCCCAAGAACACCCTGTTCCTGCAAATGACCAGTCTAAGGTCTGAGGACACGGCCATGTATTACTGTGCAAGAAAACTACCCTCACTATGCTATGGACTACTGGGGTCAAGGAACCAAGGGACTCTGCTCCTCAGCCAAAAC。

[0062] SEQ ID NO:9 SVVVETASISCKSSQSLEDSNGNTYLNWYLQKPGQSPQLLIYRVSNRFSGVLDRFSGSGSGTDFTLKISRVEAEDLGVYFCLQVTHVPWT FGGGTKLEIK。

[0063] SEQ ID NO:10 TCAGTCGTCGTGGAGACAGCCTCCATCTCTTGCAAGTCTAGTCAGAGCCTTGAAGACAGTAATGGAAACACCTATTTGAACTGGTACCTCCAGAAACCAGGCCAGTCTCCACAGCTCCTGATCTACAGGGTTTCCAACCGATTTTCTGGGGTCCTAGACAGGTTCAGTGGTAGTGGATCAGGGACAGATTTTACACTGAAAATCAGCAGAGTGGAGGCTGAAGATTTGGGAGTTTATTTCTGCCTCCAAGTTACACATGTCCCGTGGACGTTCGGTGGAGGCACCAAGCTGGAAATCAAA。

Claims

1. A monoclonal antibody that recognizes CV-A6, characterized in that, Comprising: Heavy chain complementarity determining regions, including VHCDR1, VHCDR2 and VHCDR3, wherein the amino acid sequence of VHCDR1 is as shown in SEQ ID NO:1, the amino acid sequence of VHCDR2 is as shown in SEQ ID NO:2, and the amino acid sequence of VHCDR3 is as shown in SEQ ID NO:3; and, Light chain complementarity determining regions, including VLCDR1, VLCDR2 and VLCDR3, wherein the amino acid sequence of VLCDR1 is as shown in SEQ ID NO:4, the amino acid sequence of VLCDR2 is as shown in SEQ ID NO:5, and the amino acid sequence of VLCDR2 is as shown in SEQ ID NO:

6.

2. The monoclonal antibody for identifying CV-A6 according to claim 1, wherein the antibody comprises a heavy chain and a light chain, and is characterized in that, The amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO:7; and / or, The amino acid sequence of the light chain variable region is as shown in SEQ ID NO:

9.

3. The monoclonal antibody for identifying CV-A6 according to claim 1, characterized in that, The monoclonal antibody is an IgG2a type antibody; and / or, The antigenic epitope targeted by the monoclonal antibody is a conformational epitope.

4. A nucleotide molecule encoding the monoclonal antibody that recognizes CV-A6 as claimed in claim 1 or 2.

5. The nucleotide molecule according to claim 4, wherein The nucleic acid molecule encoding the heavy chain variable region is any one of the following: a. Having the nucleotide sequence as shown in SEQ ID NO:8; b. A nucleotide sequence complementary to the nucleotide sequence as 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 nucleic acid molecule encoding the light chain variable region is any one of the following: d. Having the nucleotide sequence as shown in SEQ ID NO:10; e. A nucleotide sequence complementary to the nucleotide sequence as shown in SEQ ID NO:10; f. A nucleotide sequence that encodes the same protein as the nucleotide sequences of d and e but is different therefrom due to the degeneracy of the genetic code.

6. A recombinant DNA expression vector comprising the polynucleotide molecule as claimed in claim 4.

7. A host cell, characterized in that, Containing the nucleotide molecule as claimed in claim 4 or 5, or containing the expression vector as claimed in claim 6.

8. An antibody conjugate, characterized in that, Comprising the monoclonal antibody as claimed in any one of claims 1-3 or its antigen-binding fragment and a label.

9. Use of the monoclonal antibody that recognizes CV-A6 as claimed in any one of claims 1-3, or the monoclonal antibody encoded by the nucleotide molecule as claimed in claim 4 or 5, in the preparation of a reagent or kit for detecting CV-A6.

10. Use of the monoclonal antibody that recognizes CV-A6 as claimed in any one of claims 1-3, or the monoclonal antibody encoded by the nucleotide molecule as claimed in claim 4 or 5, in the preparation of a reagent and drug for diagnosing, inhibiting, preventing and treating diseases caused by CV-A6.

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