Enterovirus a species population broad-spectrum monoclonal antibody 1a11 and application thereof

Abstract

The application provides a broad-spectrum monoclonal antibody of enterovirus A population and application thereof, and belongs to the technical field of molecular biology and immune analysis. The monoclonal antibody comprises the following six CDR regions: the amino acid sequences of the CDR regions of the heavy chain are respectively shown in SEQ ID No. 1-SEQ ID No. 3; and the amino acid sequences of the CDR regions of the light chain are respectively shown in SEQ ID No. 4-SEQ ID No. 6. The application also provides a nucleotide fragment encoding the monoclonal antibody, a recombinant vector and an engineering cell line comprising the nucleotide fragment, an antibody conjugate and their uses. The monoclonal antibody is a non-neutralizing antibody of IgG1 subtype, can be combined with enterovirus A population viruses in a broad spectrum, and can be used for preparing a reagent or / and a kit for detecting enterovirus A population viruses, and for preparing a medicine for inhibiting, preventing or / and treating enterovirus A population viruses.

Description

Technical Field

[0001] This invention belongs to the fields of molecular biology and immunoassay technology, specifically relating to a broad-spectrum monoclonal antibody against enterovirus A and its applications. Background Technology

[0002] Enteroviruses belong to the Picornaviridae family and include 13 groups: enteroviruses A, B, C, D, E, F, G, H, I, and J, and rhinoviruses A, B, and C. Each group contains multiple serotypes or genotypes. In recent years, hand-foot-and-mouth disease (HFMD) caused by enteroviruses has shown characteristics of clustered outbreaks and cross-infection with multiple viruses. Enteroviruses of group A, such as CV-A2, CV-A4, CV-A5, CV-A6, CV-A10, CV-A16, and EV-A71, are the main pathogens causing HFMD in infants and young children. Therefore, research on monoclonal antibodies against enteroviruses is of significant value in virus detection, identification, and vaccine development. Previously reported broad-spectrum monoclonal antibodies against enteroviruses are few. Among them, the broad-spectrum monoclonal antibody 5-D8 / 1 can simultaneously recognize the VP1 region of the structural protein of Coxsackievirus A and B, poliovirus, and echovirus. However, monoclonal antibodies developed against the main pathogens of HFMD generally only bind specifically to one virus and lack broad-spectrum activity. Therefore, it is of great significance to develop monoclonal antibodies that can bind to enterovirus A in a broad spectrum. Summary of the Invention

[0003] This invention provides a broad-spectrum monoclonal antibody against enterovirus A. This monoclonal antibody is an IgG1 subtype non-neutralizing antibody that can bind to enterovirus A in a broad spectrum and is named 1A11.

[0004] Specifically, this monoclonal antibody contains the following six complementarity-determining regions (CDR regions):

[0005] (1) The amino acid sequence of the complementarity-determining region VHCDR1 of the heavy chain is shown in SEQ ID No. 1;

[0006] (2) The amino acid sequence of the complementarity-determining region VHCDR2 of the heavy chain is shown in SEQ ID No. 2;

[0007] (3) The amino acid sequence of the complementarity-determining region VHCDR3 of the heavy chain is shown in SEQ ID No. 3;

[0008] (4) The amino acid sequence of the complementarity-determining region VLCDR1 of the light chain is shown in SEQ ID No. 4;

[0009] (5) The amino acid sequence of the complementarity-determining region VLCDR2 of the light chain is shown in SEQ ID No. 5;

[0010] (6) The amino acid sequence of the complementarity-determining region VLCDR3 of the light chain is shown in SEQ ID No. 6.

[0011] The monoclonal antibody is an IgG1 type antibody, possessing binding activity but lacking neutralizing activity; it can bind broadly to enterovirus A. Specifically, the monoclonal antibody can bind to the VP3 region of the structural protein of enterovirus A, and has been identified as being able to bind to the linear epitope amino acid sequence P. 23 ILPGF 28 or / and a single amino acid mutant sequence P 23 ILPNF 28 Or / and other binding mutant sequences.

[0012] Preferably, the enterovirus A group includes CV-A2, CV-A3, CV-A4, CV-A5, CV-A6, CV-A7, CV-A8, CV-A10, CV-A12, CV-A14, CV-A16, EV-A71, EV-A76, EV-A89, EV-A90, EV-A91, EV-A92, EV-A114, EV-A119, EV-A120, EV-A121, SV19, SV43, SV46, and BA13.

[0013] More preferably, the enterovirus A group includes CV-A2, CV-A4, CV-A5, CV-A6, CV-A10, CV-A16, and EV-A71.

[0014] Preferably, the amino acid sequence of the heavy chain variable region of the monoclonal antibody is shown in SEQ ID No. 7.

[0015] Preferably, the amino acid sequence of the light chain variable region of the monoclonal antibody is shown in SEQ ID No. 8.

[0016] More preferably, the amino acid sequence of the heavy chain variable region of the monoclonal antibody is shown in SEQ ID No. 7, and the amino acid sequence of the light chain variable region of the monoclonal antibody is shown in SEQ ID No. 8.

[0017] The heavy chain of the monoclonal antibody is:

[0018] (1) A sequence with equivalent function formed by replacing, deleting, or adding one or more amino acids to the amino acid sequence shown in SEQ ID No. 7; or

[0019] (2) An amino acid sequence that has more than 95% homology with the amino acid sequence shown in SEQ ID No.7.

[0020] The light chain of the antibody is:

[0021] (1) A sequence with equivalent function formed by replacing, deleting, or adding one or more amino acids to the amino acid sequence shown in SEQ ID No. 8; or

[0022] (2) An amino acid sequence that has more than 95% homology with the amino acid sequence shown in SEQ ID No. 8.

[0023] The present invention also provides a nucleotide fragment encoding the monoclonal antibody.

[0024] Preferably, the nucleotide fragment comprises a nucleotide sequence, as shown in SEQ ID No. 9, encoding the variable region of the heavy chain of the monoclonal antibody.

[0025] Preferably, the nucleotide fragment comprises a nucleotide sequence, as shown in SEQ ID No. 10, encoding the variable region of the light chain of the monoclonal antibody.

[0026] More preferably, the nucleotide fragment comprises nucleotide sequences as shown in SEQ ID No. 9 and SEQ ID No. 10, wherein the nucleotide sequence shown in SEQ ID No. 9 encodes the heavy chain of the monoclonal antibody and the nucleotide sequence shown in SEQ ID No. 10 encodes the light chain of the monoclonal antibody.

[0027] This invention also provides a recombinant vector capable of expressing a protein with the function of the monoclonal antibody in recipient cells, wherein the recombinant vector contains the nucleotide fragment. The recombinant vector can be constructed by linking the nucleotide fragment of this invention to various expression vectors using conventional methods in the art. The expression vector can be any conventional vector in the art, such as a prokaryotic expression vector or a eukaryotic expression vector, as long as it can accommodate the nucleotide fragment. Preferably, the expression vector includes various plasmids, granules, bacteriophages, or viral vectors.

[0028] The present invention also provides an engineered cell line capable of stably expressing a protein having the function of the monoclonal antibody, the engineered cell line comprising the recombinant vector, or the engineered cell line having the nucleotide fragment integrated into its genome. The engineered cell line comprises prokaryotic cells (such as *Escherichia coli*) or eukaryotic cells (such as yeast, animal cells, etc.), preferably mammalian cells, such as 293 cells or CHO cells.

[0029] The present invention also provides an antibody-drug conjugate comprising an antibody portion and a conjugation portion; wherein the antibody portion is the monoclonal antibody, and the conjugation portion is selected from at least one of a detectable marker, a drug, a viral capsid protein, or a virus-like particle.

[0030] The monoclonal antibodies, recombinant vectors, engineered cell lines, and antibody conjugates provided by this invention can be used to prepare reagents and / or kits for detecting enterovirus A; they can also be used to prepare drugs for inhibiting, preventing, and treating enterovirus A.

[0031] Compared with existing technologies, the beneficial effects of this invention are as follows: The monoclonal antibody provided by this invention can bind to enterovirus group A viruses broadly, while essentially not binding to other enteroviruses besides enterovirus group A viruses. This monoclonal antibody can be used for basic experiments such as indirect immunofluorescence assay (IFA), Western blot (WB), and enzyme-linked immunosorbent assay (ELISA). This monoclonal antibody exhibits the same high binding titer against the main enterovirus group A viruses that cause hand-foot-mouth disease in infants and young children. This antibody has significant research and application value in the detection, identification, titration, vaccine antigen quantification, VP3 function development, and other applications based on antigen-antibody reactions of enterovirus group A viruses. Attached Figure Description

[0032] Figure 1 This is an SDS-PAGE image of the monoclonal antibody 1A11 prepared in Example 1;

[0033] Figure 2 This is a graph showing the results of the indirect immunofluorescence assay for monoclonal antibody 1A11 in Example 2;

[0034] Figure 3 This is an identification diagram of the CV-A5 structural protein region recognized by monoclonal antibody 1A11 in Example 2;

[0035] Figure 4 This is a graph showing the results of verifying the broad-spectrum binding of monoclonal antibody 1A11 to enterovirus A in Example 2;

[0036] Figure 5 This is a graph showing the results of the monoclonal antibody 1A11 binding titer detection in Example 2;

[0037] Figure 6 This is a preliminary localization result of the linear epitope of monoclonal antibody 1A11 in Example 2;

[0038] Figure 7 This is an amino acid sequence alignment diagram of monoclonal antibody 1A11 binding to the VP3 region of enterovirus A in Example 2;

[0039] Figure 8 This is a diagram showing the precise localization results of the linear epitope of monoclonal antibody 1A11 in Example 2. Detailed Implementation

[0040] The technical solution of the present invention will be clearly and completely described below with reference to the embodiments. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention. Any equivalent modifications or substitutions made by those skilled in the art based on the following embodiments are within the scope of protection of the present invention.

[0041] The method for preparing the purified CV-A5 antigen in step 1.1.1 of the example is as follows:

[0042] Take CV-A5 seed (7.68lg CCID) 50 The virus was inoculated into a ten-layer Vero cell factory at 0.001 MOI (multiple of infection) and cultured at 37°C for 48–72 hours. When the cytopathic effect reached 90%, the virus fluid was harvested. The virus harvest fluid was repeatedly frozen and thawed and centrifuged to remove cell debris. The supernatant was concentrated by ultrafiltration through a membrane with a pore size that could retain molecules up to 50 kDa. The concentrated product was centrifuged at 25,000 rpm for 4 hours with 20% (w / v) sucrose as a bottom layer. The precipitate was reconstituted with PBS buffer at pH 7.2 and centrifuged at 40,000 rpm for 4 hours with sucrose gradients of 15%, 25%, 35%, 45%, and 55% (w / v) to collect solid virus particles (FP) and hollow virus particles (EP). The FP and EP were centrifuged at 48,000 rpm for 18 hours with a 30% (w / v) CsCl density gradient to extract the target band and obtain purified CV-A5 virus particles as purified CV-A5 antigen.

[0043] The method for indirect ELISA detection of serum titer in step 1.1.2 of the embodiment is as follows:

[0044] P1. Prepare a 1 μg / mL coating solution with carbonate buffer (pH=9.6) for each CV-A5 virus purified particle and add it to an ELISA plate (100 μL / well). Incubate overnight at 4°C. After incubation, wash the plate three times with PBST (pH=7.4).

[0045] P2. Block with PBST containing 1% (w / v) BSA at pH 7.4 at 37°C for 1 hour, then discard the blocking solution;

[0046] P3. Dilute mouse serum tenfold (volume ratio), add 100 μL of diluted mouse serum to each well of the ELISA plate, incubate at 37°C for 1 h, and wash the plate 5 times with PBST at pH 7.4.

[0047] P4. Add 0.1 μg / mL of HRP-labeled goat anti-mouse IgG antibody to the ELISA plate, 100 μL / well, incubate at 37°C for 1 h, and wash the plate 5 times with PBST at pH 7.4.

[0048] P5. Add TMB (3,3',5,5'-tetramethylbenzidine) colorimetric solution and develop color at 37°C in the dark for 30 min. Terminate the reaction with 2 mol / L sulfuric acid and detect the absorbance at A450nm using an ELISA reader.

[0049] P6. Potency calculation method: the maximum dilution factor that is greater than 2.1 times the OD value of the negative well; negative wells are supplemented with negative mouse serum.

[0050] The method for indirect ELISA screening of hybridoma cells in step 1.1.4 of the embodiment is as follows:

[0051] Q1. Prepare a 1 μg / mL coating solution with carbonate buffer (pH=9.6) for each CV-A5 virus purified particle and add it to an ELISA plate (100 μL / well). Incubate overnight at 4°C. After incubation, wash the plate three times with PBST (pH=7.4).

[0052] Q2. Block with PBST containing 1% (w / v) BSA at pH 7.4 at 37°C for 1 hour, then discard the blocking solution.

[0053] Q3. Dilute the supernatant of the hybridoma cell culture medium to be tested by a 10-fold (volume ratio), add 100 μL of the diluted hybridoma cell culture medium supernatant to the microplate at 37°C for 1 h, and wash the plate 5 times with PBST at pH 7.4.

[0054] Q4. Add 0.1 μg / mL of HRP-labeled goat anti-mouse IgG antibody to the ELISA plate, 100 μL / well, incubate at 37°C for 1 h, and wash the plate 5 times with PBST at pH 7.4.

[0055] Q5. Add TMB colorimetric solution and develop color at 37℃ in the dark for 30 min. Terminate the reaction with 2 mol / L sulfuric acid and detect the absorbance at A450nm using an ELISA reader.

[0056] Q6. Potency calculation method: The maximum dilution factor that is greater than 2.1 times the OD value of the negative well; the negative well is treated with hybridoma cell culture medium as a control.

[0057] The main reagents used in the following examples are as follows:

[0058] Freund's complete adjuvant: purchased from Sigma;

[0059] Freund's incomplete adjuvant: purchased from Sigma;

[0060] Female BALB / c mice: purchased from Wuhan Institute of Biological Products Co., Ltd.;

[0061] Protein A affinity chromatography column: purchased from Cytiva;

[0062] Isotyping Kit for Mouse Monoclonal Antibody: Purchased from Beijing Isotech Scientific Co., Ltd.

[0063] RD cells: provided by Wuhan Institute of Biological Products Co., Ltd.

[0064] FITC-labeled goat anti-mouse IgG: purchased from ThermoFisher;

[0065] HRP-labeled goat anti-mouse IgG: purchased from Wuhan Boster Biological Engineering Co., Ltd.

[0066] HRP-labeled goat anti-rabbit IgG: purchased from Wuhan Boster Biological Engineering Co., Ltd.

[0067] Example 1

[0068] This embodiment provides methods for the preparation, purification, and basic experiments of monoclonal antibody 1A11, as detailed below:

[0069] 1.1 Preparation and purification steps of monoclonal antibody 1A11:

[0070] 1.1.1 Mix 100 μL of purified CV-A5 solid particle antigen containing 60 μg with an equal volume of Freund's adjuvant (Freund's complete adjuvant was used for the first immunization, and Freund's incomplete adjuvant was used for subsequent booster immunizations), and immunize female BALB / c mice (6-8 weeks old) by subcutaneous injection in the back and intraperitoneal injection. The injection dose is 60 μg / dose. After the first immunization, booster immunizations were given on days 14, 28 and 42.

[0071] 1.1.2. Blood was collected from the orbital cavity on day 52, serum was separated, and serum titer was detected by indirect ELISA;

[0072] 1.1.3 Select serum titers exceeding 1×10⁻⁶. 6 Mice were subjected to a shock immunization. On the third day after the shock immunization, spleen cells were collected from the mice. PEG-1500 (polyethylene glycol-1500, Sigma) was used to induce the fusion of spleen cells from female BALB / c mice with SP2 / 0 myeloma cells. The fusion ratio of mouse spleen cells to myeloma cells was controlled between (1:5) and (1:10). The fused cells were first screened using HAT medium.

[0073] 1.1.4. After initial screening, the antibody titer in the hybridoma cell supernatant was detected using an indirect ELISA method, and cells with a titer greater than 1×10⁻⁶ were selected. 4 Positive hybridoma cells were cloned and purified three times before being expanded into larger cultures;

[0074] 1.1.5. Preparation of ascites: Female aged Balb / c mice were pre-sensitized by intraperitoneal injection of 0.5 mL of Freund's incomplete adjuvant; 10 days later, 0.5 mL of 5×10⁻⁶ styrosine phosphate was injected intraperitoneally. 6 Hybridoma cells in logarithmic growth phase were collected at a rate of 1 / mL. Ascites was harvested via a first puncture when mice showed moderate abdominal distension. If the mice's health did not significantly decline after 48 hours, ascites was harvested again. After the second aspiration, the mice were euthanized and a third puncture was performed to harvest ascites. Each harvested ascites was centrifuged at 5000 rpm for 15 minutes, the supernatant was collected, filtered, and stored at -80℃. The pre-purified ascites solutions from multiple collections were combined and purified using a Protein A affinity chromatography column on an AKTA instrument to obtain the monoclonal antibody 1A11.

[0075] 1.2 Basic Experiments

[0076] 1.2.1 Detection of amino acid sequence in the variable region of monoclonal antibody 1A11

[0077] The positive hybridoma cells selected in step 1.1.4 were seeded into RPMI 1640 medium containing 10% fetal bovine serum and cultured at 37°C with 5% CO2 for 72 hours. Total RNA was extracted from the cells using the TaKaRaMin iBEST Universal RNA Extraction Kit and analyzed using the TaKaRaRimeScript. TMcDNA was obtained by reverse transcription using OligodT primers from the 1st Strand cDNA Synthesis Kit. The variable region gene of monoclonal antibody 1A11 was amplified using a heavy chain universal primer pair with added homologous sequences to the cloning vector (SEQ ID No. 11 for forward primer VH-F: acggccagtgaattcmarctgcagsagtcwgg, SEQ ID No. 12 for reverse primer VH-R: gattacgccaagctttgaggagacggtgaccg) and a light chain universal primer pair (SEQ ID No. 13 for forward primer VL-F: acggccagtgaattccgattgtkctsacycartctcca, SEQ ID No. 14 for reverse primer VL-R: gattacgccaagcttcgttggatctccagcttg). The cDNA was then cloned into two pUC-Kan vectors, and the amino acid sequences of the variable region were obtained through sequencing. Sequence analysis was performed using the Kabat database. Sequencing was conducted by Sangon Biotech (Shanghai) Co., Ltd.The nucleotide sequence (SEQ ID No.9) of the heavy chain variable region of the measured monoclonal antibody 1A11 is as follows: aagctgcaggagtctggaggaggcttggtgcaacctggaagatccatgaaactctcctgtgttgtctctggattcactttctataactactggatgaactgggtccgccagtctccagggaagggtcttgagtggcttgctgaaattagattgaaatctaataattatgcaacacattatgcggagtctgtgaaagggaggttcaccatctcaagagatgattccaaaagtagtgtctacctgcaaatggacaacttaagagctgaagacactggcatttattactgtacccctgccagcccctttgcttactggggccaagggactctggtcaccgtctcctca; the nucleotide sequence (SEQ ID No.10) of the light chain variable region is as follows: attgtgctcactcaatctccagcactcatgtctgcatctctaggggaacgggtcaccatgacctgcactgccagctcaagtgtaagttccaattacttgcactggtaccggcagaagccaggctcctcccccaaagtctggatttatagcacatccaacctggcttctggagtcccagctcgcttcagtggcagtgggtctgggacctctttctctctcacaatcagcagcgtggaggctgaagatgctgccacttattactgccaccagtatcatcgttccccgtacccgttcggaggggggaccaagctggagatccaa。The nucleotide sequence of the heavy chain variable region (SEQ ID No. 9) encodes the following amino acid sequence (SEQ ID No. 7): KLQESGGGLVQPGRSMKLSCVVSGFTFYNYWMNWVRQSPGKGLEWLAEIRLKSNNYATHYAESVKGRFTISRDDSKSSVYLQMDNLRAEDTGIYYCTPASPFAYWGQGTLVTVSS; the nucleotide sequence of the light chain variable region (SEQ ID No. 10) encodes the following amino acid sequence (SEQ ID No. 8): IVLTQSPALMSASLGERVTMTCTASSSVSSNYLHWYRQKPGSSPKVWIYSTSNLASGVPARFSGSGSGTSFSLTISSVEAEDAATYYCHQYHRSPYPFGGGTKLEIQ. The sequences of the six CDR regions (complementarity-determining regions) identified are as follows: amino acid sequence of VHCDR1 (SEQ ID No. 1): NYWMN; amino acid sequence of VHCDR2 (SEQ ID No. 2): EIRLKSNNYATHYAESVKG; amino acid sequence of VHCDR3 (SEQ ID No. 3): ASPFAY; amino acid sequence of VLCDR1 (SEQ ID No. 4): TASSSVSSNYLH; amino acid sequence of VLCDR2 (SEQ ID No. 5): STSNLAS; amino acid sequence of VLCDR3 (SEQ ID No. 6): HQYHRSPYP.

[0078] The inventors discovered through experiments that the amino acid sequence of the heavy chain of the monoclonal antibody 1A11 is a sequence with equivalent function formed by substituting, deleting, or adding one or more amino acids to the amino acid sequence shown in SEQ ID No. 7; or an amino acid sequence with more than 95% homology to the amino acid sequence shown in SEQ ID No. 7. The amino acid sequence of the light chain of the monoclonal antibody 1A11 is a sequence with equivalent function formed by substituting, deleting, or adding one or more amino acids to the amino acid sequence shown in SEQ ID No. 8; or an amino acid sequence with more than 95% homology to the amino acid sequence shown in SEQ ID No. 8.

[0079] 1.2.2 Identification of Monoclonal Antibody 1A11 Subtypes

[0080] The purified monoclonal antibody 1A11 from step 1.1.5 was analyzed using 4–20% SDS-PAGE, and the monoclonal antibody isotype was identified using the Isotyping Kit for Mouse Monoclonal Antibody. The SDS-PAGE results are shown below. Figure 1 As shown, Figure 1 In the diagram, M represents the lane for the protein marker, 1 represents non-reducing SDS-PAGE electrophoresis, and 2 represents reducing SDS-PAGE electrophoresis. From... Figure 1 It can be seen that under non-reducing conditions, monoclonal antibody 1A11 shows a band with a molecular weight of approximately 150 kDa; under reducing conditions, monoclonal antibody 1A11 shows two bands with molecular weights of approximately 50 kDa and 25 kDa, corresponding to the heavy chain and light chain of the antibody, respectively.

[0081] Example 2

[0082] This embodiment provides the method and results for functional analysis of monoclonal antibody 1A11, as detailed below:

[0083] 2.1 Indirect Immunofluorescence Assay (IFA) of Monoclonal Antibody 1A11

[0084] The indirect immunofluorescence assay for monoclonal antibody 1A11 includes the following steps:

[0085] 2.1.1. Inoculate CV-A5 virus into 24-well plates containing RD cells and culture for 24-28 hours. RD cells not inoculated with virus serve as a negative control.

[0086] 2.1.2 After the cells showed lesions, the cell supernatant was discarded, 4% paraformaldehyde was added to fix the cells at room temperature for 30 min, and the plate was washed 3 times with PBS at pH 7.2.

[0087] 2.1.3 Add PBS buffer containing 0.1% (v / v) Triton-X 100 at pH 7.2 and treat for 10 min, then wash the plate 3 times with PBS at pH 7.2;

[0088] 2.1.4 Block with PBS buffer containing 1% (w / v) BSA at pH 7.2 at 37°C for 1 h. Discard the blocking solution after blocking.

[0089] 2.1.5 Add 200 μL of 1 μg / mL monoclonal antibody 1A11 and incubate at 37°C for 1 h. Wash the plate three times with PBST at pH 7.4.

[0090] 2.1.6 Add 200 μL of FITC (fluorescein isothiocyanate)-labeled goat anti-mouse IgG at a concentration of 0.2 μg / mL, incubate at 37°C in the dark for 1 h, and wash the plate 3 times with PBST at pH 7.4.

[0091] 2.1.7 Observe and photograph using a fluorescence microscope. The results are as follows: Figure 2 As shown. From Figure 2 As can be seen, monoclonal antibody 1A11 can specifically bind to CV-A5 virus after RD cells are infected.

[0092] 2.2 Western Blotting (WB) Experiment

[0093] 2.2.1 Identification of the CV-A5 structural protein region by monoclonal antibody 1A11

[0094] The CV-A5 structural proteins include VP1, VP2, VP3, and VP4. Western blotting (WB) can preliminarily identify the structural protein regions where 1A11 binds. The specific steps are as follows:

[0095] 2.2.1.1 Take 16 μL of CV-A5 virus purification hollow particles (EP) and solid particles (FP) respectively, add 4 μL of 5×SDS-PAGE loading buffer containing 5% (v / v) β-mercaptoethanol, boil the samples at 100℃ for 10 minutes, and then load the samples using 4%–20% SDS-PAGE gel.

[0096] 2.2.1.2 After electrophoresis, use GenScript eBlot TM Transfer the membrane to a 0.45μm nitrocellulose (NC) membrane using the L1 rapid wet transfer apparatus;

[0097] 2.2.1.3 After transfer, block with PBST blocking buffer containing 1% (w / v) BSA at pH 7.4 for 1 h at 37°C;

[0098] 2.2.1.4 Add 10 mL of 0.2 μg / mL monoclonal antibody 1A11 or rabbit anti-CV-A5 polyclonal antibody serum diluted 1:5000 (v / v) and incubate overnight at 4°C. After incubation, wash the membrane 5 times with PBST at pH 7.4.

[0099] 2.2.1.5 Add 10 mL of 0.1 μg / mL HRP-labeled goat anti-mouse IgG or HRP (horseradish peroxidase)-labeled goat anti-rabbit IgG, and incubate at room temperature for 1 h; wash the membrane 5 times with PBST at pH 7.4;

[0100] 2.2.1.6. Add Millipore's Immobilon@WestenChemiluminescent HRPSubstrate, take photos using an Amersham ImageQuant 800, and the results are as follows. Figure 3 As shown. Figure 3 In the diagram, CVA5Rabbit PcAb represents rabbit anti-CV-A5 polyclonal antibody serum, 1A11 represents monoclonal antibody 1A11, M represents protein marker, Cell represents Vero cells, EP represents purified hollow CV-A5 virus particles, and FP represents purified solid CV-A5 virus particles. Figure 3 As can be seen, the monoclonal antibody 1A11 can specifically bind to the VP3 region of the structural protein of CV-A5 virus and recognize the antigenic epitope as a linear epitope.

[0101] 2.2.2 Verification of broad-spectrum binding of monoclonal antibody 1A11 to enterovirus A population.

[0102] To verify the broad-spectrum binding of monoclonal antibody 1A11 to enterovirus A, purified particles of the major enteroviruses A causing hand-foot-mouth disease in infants (CV-A2, CV-A4, CV-A5, CV-A6, CV-A10, CV-A16, and EV-A71) were selected for Western blotting (WB). Purified particles of enterovirus B (Echo-11), enterovirus C (Poliovirus III), and non-enterovirus (Rotavirus G8) were selected as WB controls. Specific experimental procedures are described in 2.2.1. The primary and secondary antibodies were 10 mL of 0.2 μg / mL monoclonal antibody 1A11 and 0.1 μg / mL HRP-labeled goat anti-mouse IgG, respectively.

[0103] The verification results are as follows Figure 4 As shown, from Figure 4 As can be seen, monoclonal antibody 1A11 can specifically bind to the VP3 region of the structural proteins of enterovirus A group CV-A2, CV-A4, CV-A5, CV-A6, CV-A10, CV-A16, and EV-A71; but cannot bind to enterovirus B group Echo-11, enterovirus C group Poliovirus, or non-enterovirus Rotavirus.

[0104] 2.3 Detection of 1A11 binding titer of monoclonal antibody

[0105] The binding affinity of monoclonal antibody 1A11 to enteroviruses of group A (CV-A2, CV-A4, CV-A5, CV-A6, CV-A10, CV-A16, and EV-A71) was determined by ELISA. Enteroviruses of group B (Echo-11), enteroviruses of group C (Poliovirus), and non-enteroviruses (Rotavirus) served as controls. The specific experimental procedures are as follows:

[0106] 2.3.1 Prepare coating solutions of 1 μg / mL for each of the purified viral particles of CV-A2, CV-A4, CV-A5, CV-A6, CV-A10, CV-A16, EV-A71, Echo-11, Poliovirus, and Rotavirus using carbonate buffer at pH 9.6. Add the solutions to the microplates at 100 μL / well and incubate overnight at 4°C. After incubation, wash the plates three times with PBST at pH 7.4.

[0107] 2.3.2 Block with PBST containing 1% (w / v) BSA at pH 7.4 at 37°C for 1 h, then discard the blocking solution;

[0108] 2.3.3 Add primary antibody: Dilute 1 mg / mL monoclonal antibody 1A11 10 times (volume ratio), take 100 μL of the diluted antibody / well and add it to the microplate, incubate at 37℃ for 1 h, wash the plate 5 times with PBST at pH 7.4;

[0109] 2.3.4 Add secondary antibody: Add 0.1 μg / mL of HRP-labeled goat anti-mouse IgG antibody to the ELISA plate, 100 μL / well, incubate at 37℃ for 1 h, and wash the plate 5 times with PBST at pH 7.4;

[0110] 2.3.5. Add TMB colorimetric solution and develop color at 37°C in the dark for 30 min. Terminate the reaction with 2M sulfuric acid and measure the absorbance at A450nm using an ELISA reader.

[0111] 2.3.6. Potency calculation method: The maximum dilution factor that is greater than 2.1 times the OD value of the negative well. No primary antibody is added to the negative well.

[0112] Test results as follows Figure 5 As shown, according to Figure 5 It can be calculated that monoclonal antibody 1A11 has the same binding titer of 1×10⁻⁶ against CV-A2, CV-A4, CV-A5, CV-A6, CV-A10, CV-A16, and EV-A71. 6 .

[0113] 2.4 Linear Epitope Study of Monoclonal Antibody 1A11

[0114] 2.4.1 Preliminary localization of the linear epitope of monoclonal antibody 1A11

[0115] The amino acid sequence of the linear epitope of monoclonal antibody 1A11 was initially identified by ELISA, as follows:

[0116] 2.4.1.1 Synthesize the overlapping polypeptide in the VP3 region of the CV-A5 structural protein. The polypeptide sequence is shown in Table 1.

[0117] 2.4.1.2. The polypeptide was coated onto an ELISA plate at a concentration of 4 μg / mL, 100 μL / well;

[0118] 2.4.1.3 Perform ELISA experiment: The concentration of the primary antibody monoclonal antibody 1A11 is 1 μg / mL, the concentration of the secondary antibody HRP-labeled goat anti-mouse IgG is 0.1 μg / mL, and other steps are as described in 2.3.

[0119] Table 1. Overlapping peptides in the VP3 region of CV-A5 structural proteins

[0120]

[0121]

[0122] Preliminary results of linear epitope localization of monoclonal antibody 1A11 are shown below. Figure 6 and Figure 7 ,from Figure 6 and Figure 7 As can be seen, monoclonal antibody 1A11 can bind to peptide 3 of the structural protein VP3 of CV-A5 virus. By comparing the conserved amino acid sequences in this region of enterovirus A group viruses CV-A2, CV-A4, CV-A5, CV-A6, CV-A10, CV-A16, and EV-A71, it can be preliminarily determined that the recognition epitope of monoclonal antibody 1A11 is located at peptide S. 21 APILPGF 28 Inside.

[0123] 2.4.2 Precise localization of linear epitopes of monoclonal antibody 1A11

[0124] Based on the initially identified linear epitope peptide SAPILPGF, a series of truncated peptides were synthesized, along with the mutant peptide S of EV-A71 at this linear epitope. 21 APILPNF 28 The peptide was coated onto a plate at a concentration of 4 μg / mL, and then an ELISA experiment was performed. The ELISA experimental method is as described in 2.4.1.

[0125] The precise localization results of the linear epitope of monoclonal antibody 1A11 are shown in the figure. Figure 8 ,from Figure 8 As can be seen from the experiment, S was found through truncated peptides.21 APILPGF 28 S in sequence 21 A 22 Two amino acids had no effect on the binding activity of monoclonal antibody 1A11, therefore it can be determined that monoclonal antibody 1A11 can recognize the linear epitope of the antigen with precision down to a six-amino acid sequence. 23 ILPGF 28 Simultaneously, the monoclonal antibody 1A11 can also recognize the single amino acid variant sequence P of EV-A71 at this linear epitope. 23 ILPNF 28 .

[0126] 2.5 Alignment of linear epitope amino acid sequences of monoclonal antibody 1A11

[0127] In the NCBI Enterovirus database (taxid: 12059), a BLAST comparison was performed on the linear epitope PILPGF and the mutant sequence PILPNF recognized by monoclonal antibody 1A11. The proportion of this sequence in different populations of Enterovirus was statistically analyzed. BLAST comparison analysis revealed that the amino acid sequence of Enterovirus A population viral protein contains the sequence corresponding to P... 23 ILPGF 28 and mutation sequence P 23 ILPNF 28 The proportion of viral sequences with identical sequences exceeded 99.5% (3250 out of 3259 sequences were identical), and all identical sequences appeared at the N-terminus of the VP3 region of the structural protein. No amino acid sequences of enteroviruses other than Enterovirus A were identical to PILPGF or PILPNF, and sequences with only one amino acid mutation as a match to PILPGF accounted for less than 0.1%. Therefore, based on the above identification and sequence alignment analysis, monoclonal antibody 1A11 was identified as a broad-spectrum monoclonal antibody specific to Enterovirus A, capable of broadly recognizing multiple serotypes and genotypes of Enterovirus A.

[0128] The above description is merely a preferred embodiment of the present invention and is not intended to limit the scope of protection of the present invention. Various modifications and variations can be made to the present invention by any person skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A broad-spectrum monoclonal antibody against enterovirus A, characterized in that, The monoclonal antibody contains the following six complementarity-determining regions: (1) The amino acid sequence of the complementarity-determining region VHCDR1 of the heavy chain is shown in SEQ ID No. 1; (2) The amino acid sequence of the complementarity-determining region VHCDR2 of the heavy chain is shown in SEQ ID No. 2; (3) The amino acid sequence of the complementarity-determining region VHCDR3 of the heavy chain is shown in SEQ ID No. 3; (4) The amino acid sequence of the complementarity-determining region VLCDR1 of the light chain is shown in SEQ ID No. 4; (5) The amino acid sequence of the complementarity-determining region VLCDR2 of the light chain is shown in SEQ ID No. 5; (6) The amino acid sequence of the complementarity-determining region VLCDR3 of the light chain is shown in SEQ ID No.

6.

2. The enterovirus A population broad-spectrum monoclonal antibody according to claim 1, characterized in that, The amino acid sequence of the heavy chain variable region of the monoclonal antibody is shown in SEQ ID No. 7, and / or the amino acid sequence of the light chain variable region of the monoclonal antibody is shown in SEQ ID No.

8.

3. A broad-spectrum monoclonal antibody against enterovirus A according to claim 1 or 2, characterized in that, The monoclonal antibody is an IgG1 type antibody.

4. A nucleic acid fragment, characterized in that, The nucleic acid fragment encodes the monoclonal antibody of claim 1 or 2, or the nucleic acid fragment encodes the monoclonal antibody of claim 3.

5. The nucleic acid fragment according to claim 4, characterized in that, The nucleic acid fragment contains a nucleotide sequence encoding the variable region of the heavy chain of the monoclonal antibody as shown in SEQ ID No. 9, or / and the nucleic acid fragment contains a nucleotide sequence encoding the variable region of the light chain of the monoclonal antibody as shown in SEQ ID No.

10.

6. A recombinant vector, characterized in that, The recombinant vector comprises the nucleic acid fragment as described in claim 4 or claim 5.

7. An engineered cell line, characterized in that, The engineered cell line comprises the recombinant vector as described in claim 6, or the genome of the engineered cell line integrates the nucleic acid fragment as described in claim 4 or claim 5.

8. The use of the monoclonal antibody according to any one of claims 1 to 3, the recombinant vector according to claim 6, or the engineered cell line according to claim 7 in the preparation of reagents and / or kits for detecting enterovirus A.

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