A monoclonal antibody against BRVA virus VP7 protein, its antigenic epitope and application

Abstract

The present invention relates to the field of biomedical technology, and particularly to a monoclonal antibody against the VP7 protein of BRVA virus, its antigenic epitope and application. The present invention provides a hybridoma cell line (deposit number: CCTCC NO: C202570), and the monoclonal antibody prepared therefrom can bind to the VP7 proteins of BRVA of genotypes G6, G8, and G10; the amino acid sequence of the antigenic epitope of the monoclonal antibody is SEQ ID NO: 1. The monoclonal antibody and antigenic epitope peptide of the present invention have broad-spectrum neutralizing activity, providing new strategies and tools for the prevention and control of BRVA.

Description

Technical Field

[0001] The present invention relates to the field of biological medicine technology, and particularly to a monoclonal antibody against the VP7 protein of BRVA virus, its antigenic epitope and application. Background Art

[0002] Group A Bovine rotavirus (BRVA) is an important pathogen that causes diarrhea and enteritis in cattle, especially causing severe diseases in neonatal calves, resulting in significant economic losses. The overall infection rate of BRVA in the cattle herd in China is 46%, and the harm is serious. The genome of BRVA is a segmented double-stranded RNA virus, which is prone to variation, and there are various different genotype strains prevalent. So far, 12 G types (G1-G3, G5, G6, G8, G10, G11, G15, G17, G21 and G24) and 11 P types (P[1], P[3], P[5-7], P

[11] , P

[14] , P

[17] , P

[21] , P

[29] and P

[33] ) have been found in bovine rotavirus, among which the G6, G8, G10 and P[1], P[5], P

[11] genotypes are the most common. There are many types of BRVA G-P combinations, and the cross-protectivity of different genotype strains is poor, which brings great difficulties to the prevention and control of diseases and the development of vaccines. At present, there is no broad-spectrum neutralizing antibody or vaccine against multiple genotypes on the market in China, and there is an urgent need to develop a broad-spectrum vaccine against multiple genotypes.

[0003] The VP7 protein of BRVA is the main surface antigen that determines the G type of the virus, contains neutralizing antigenic epitopes, and is a key target for inducing the host to produce neutralizing antibodies. However, due to the large variation in the amino acid sequence homology of the VP7 proteins of different G types of BRVA (68.0%-73.1%) (Li Fan, Zhou Fang, Yue Hua, et al. Research progress on the molecular biology of group A bovine rotavirus [J]. Progress in Veterinary Medicine, 2017, 38(6):4.), and there are at least 4 highly conserved (more than 90%) peptide segments, which increases the difficulty of identifying antigenic epitopes with broad-spectrum neutralizing activity. Summary of the Invention

[0004] In order to solve the above problems, the present invention intends to provide a monoclonal antibody with broad-spectrum neutralizing activity, which can effectively neutralize BRVA of G6, G8 and G10 types, and at the same time, the existence of broad-spectrum neutralizing antigenic epitopes in the VP7 protein is also confirmed.

[0005] The present invention provides a hybridoma cell line, and the preservation number of the hybridoma cell line: CCTCC NO: C202570.

[0006] The present invention provides a monoclonal antibody, which is prepared from the above-mentioned hybridoma cell line.

[0007] In the present invention, the antigen bound by the monoclonal antibody is the VP7 protein of the BRVA virus.

[0008] In some specific embodiments of the present invention, the strain types of the BRVA virus include G6, G8, and G10 types.

[0009] In some specific embodiments of the present invention, the amino acid sequence of the antigenic epitope bound by the monoclonal antibody includes the sequence shown in SEQ ID NO:1.

[0010] SEQ ID NO:1: 87 IEASNEIADTEWKNT 101 。

[0011] In some specific embodiments of the present invention, the antibody comprises a light chain shown in SEQ ID NO:2 and a heavy chain shown in SEQ ID NO:3.

[0012] SEQ ID NO:2:

[0013] QSPASLSASVGETVTITCRASENIYSYLAWYQQKQGKSPQVLVYNAKXLPEGVPSRFSASGSGTQFSLKINSLQPEDFGSYYCQHHYGNPFTFGSGTKLEIKRADAAPTVS.

[0014] SEQ ID NO:3:

[0015] QSGPELKKPGETVKISCKASGYTFTTYAINWVKQAPGKGLKWMGWINTYTGEPTHDNDFKGRFAFSSETSASTAYLQINNLKNEDTATYFCTRGGRGPYFLYWGQGTLVTVSAAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSTWPSETVTCNVAHPASSTKVDKKIVPRDCGCKPCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQTQPREEQFNSTFRSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTISIPPPKEQMAKDKVSLTCMITDFFPEDITVEWQWNGQ

[0016] The present invention provides an antigenic epitope peptide of the BRVA virus VP7 protein, and the amino acid sequence of the antigenic epitope of the BRVA virus VP7 protein is as shown in SEQ ID NO: 1.

[0017] The present invention provides a method for identifying the antigenic epitope of the BRVA virus VP7 protein, comprising the following steps:

[0018] (1) Performing molecular docking between the above-mentioned monoclonal antibody and the BRVA VP7 protein to predict the interaction region between the VP7 protein and the monoclonal antibody;

[0019] (2) Screening epitope sequences with high antigenicity and verifying them.

[0020] The present invention provides the application of the above-mentioned hybridoma cell line or the above-mentioned monoclonal antibody or the above-mentioned antigenic epitope peptide in the preparation of products for preventing and / or treating, and / or detecting, and / or diagnosing BRVA virus infection.

[0021] The present invention provides a product for preventing and / or treating, and / or detecting, and / or diagnosing BRVA virus infection, comprising the above-mentioned hybridoma cell line or the above-mentioned monoclonal antibody or the above-mentioned antigenic epitope peptide.

[0022] In the present invention, the above-mentioned products include, but are not limited to, BRVA virus detection kits and BRVA vaccines.

[0023] The term "broad-spectrum" is used to describe an antigenic epitope, vaccine or treatment method that can provide a protective effect against a variety of different subtypes or strains of pathogens.

[0024] In the present invention, the broad-spectrum neutralizing activity refers to being effective against at least BRVA strains of types G6, G8, and G10.

[0025] In the present invention, the term "monoclonal antibody" refers to an antibody obtained from a population of antibodies that are substantially homologous, i.e., each antibody constituting the population is identical and / or binds to the same epitope, except for possible variant antibodies (e.g., containing naturally occurring mutations or generated during the production of monoclonal antibody preparations), and such variants are generally present in trace amounts. Different from polyclonal antibody preparations that usually include different antibodies against different determinants (epitopes), each monoclonal antibody in a monoclonal antibody preparation is directed against a single determinant on the antigen. Thus, the modifier "monoclonal" indicates the characteristic of the antibody obtained from a substantially homologous population of antibodies and should not be construed as requiring the antibody to be produced by any specific method. For example, monoclonal antibodies to be used according to the present invention can be prepared by a variety of techniques, including but not limited to hybridoma methods, recombinant DNA methods, phage display methods, and methods using transgenic animals containing all or part of the human immunoglobulin locus, and such methods and other exemplary methods for preparing monoclonal antibodies are described herein.

[0026] In the present invention, the term "hybridoma cell" refers to a cell formed by fusing myeloma cells and B lymphocytes during the preparation of monoclonal antibodies, and is generally prepared by culturing the tumor cells.

[0027] In the present invention, the term "label" refers to a molecule that can indicate the position or amount of an antibody through color, chemical reaction, excitation light, mass spectrometry, etc. after the antibody binds. Examples include, but are not limited to: alkaline phosphatase, peroxidase, luciferase, fluorescein, fluorescent protein, isotopes, etc.

[0028] In the present invention, "enzyme-linked immunosorbent assay (ELISA)" refers to a detection method that utilizes the specific binding characteristic between antibody molecules and antigen molecules, binds free heteroproteins and the target protein bound to a solid-phase carrier, and performs qualitative or quantitative analysis on it using a special label. Its principle is as follows: Antigens or antibodies can be physically adsorbed on the solid surface and maintain their immunological activity; antigens or antibodies can form an enzyme conjugate with an enzyme through a covalent bond while maintaining their respective immunological activity or enzymatic activity; after the enzyme conjugate binds to the corresponding antigen or antibody, the occurrence of the immune reaction can be determined by the color reaction of adding a substrate, and the depth of the color reaction is proportional to the amount of the corresponding antigen or antibody in the specimen. According to the substance to be detected and the detection conditions, various different types of detection methods can be designed, and the double antibody sandwich method is the most commonly used method for detecting antigens. It is to adsorb the antiserum containing the known antibody in the small holes on a microtiter plate, wash it once; add the antigen to be detected, if the two are specific, binding will occur, and then the excess antibody is washed away; add the enzyme-linked antibody that specifically reacts with the antigen to be detected to form a "sandwich"; add the substrate of this enzyme, if a colored enzymatic product is seen to be produced, it indicates the presence of the corresponding antigen.

[0029] In the present invention, "plasmid" refers to a DNA molecule outside the chromosome (or nucleoid) in organisms such as bacteria, yeast, and actinomycetes, which exists in the cytoplasm or nucleus, has the ability of autonomous replication, enables it to maintain a constant copy number in daughter cells, and expresses the genetic information it carries.

[0030] In the present invention, the term "vector" refers to a vector through which a polynucleotide sequence (such as a foreign gene) can be introduced into a host cell to transform the host and promote the expression (such as transcription and translation) of the introduced sequence. Vectors include plasmids, phage vectors, viral vectors, etc. Among them, "viral vector" is a vector modified from a viral genome, which introduces a foreign gene into a host cell through viral infection.

[0031] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0032] (1) Broad-spectrum neutralizing activity: The prevention and control means for bovine group A rotavirus (BRVA) have limitations, especially it is difficult to cover multiple genotype strains in vaccine research and development. The monoclonal antibody of the present invention can effectively neutralize G6, G8, and G10 type BRVA, confirming the existence of broad-spectrum neutralizing antigenic epitopes in the VP7 protein, breaking through the limitation of traditional vaccines and antibodies for a single strain, and having a significant broad-spectrum protection effect.

[0033] (2) Precise identification of antigenic epitopes: Through molecular docking and antigenicity analysis, the present invention successfully identified the key antigenic epitope (SEQ ID NO: 1) of BRVA VP7 protein and verified its neutralizing activity in multiple strains. This precise epitope identification method overcomes the problems of low epitope screening efficiency and poor specificity in the prior art, providing a clear target for the research and development of vaccines and diagnostic reagents.

[0034] (3) In the prior art, the detection and prevention and control means of BRVA are relatively single, lacking efficient and specific tools. The monoclonal antibodies and antigenic epitope peptides of the present invention can not only be used for vaccine development, but also be used to prepare highly sensitive detection kits to achieve rapid and accurate diagnosis of BRVA infection. In addition, the said products can also be used for the prevention and treatment of BRVA infection, having broad application prospects. Description of the Drawings

[0035] Figure 1 is the double digestion electrophoresis map of the recombinant expression plasmid pET28a-VP7 (the left lane is the DNA marker; the right lane is the VP7 recombinant plasmid);

[0036] Figure 2 is the analysis of recombinant protein expression (M: protein molecular weight standard; 1: total protein before IPTG induction; 2: supernatant at 20 °C; 3: precipitate at 20 °C; 4: supernatant at 37 °C; 5: precipitate at 37 °C);

[0037] Figure 3 is the SDS-PAGE analysis of the purified G6-type BRVA VP7 recombinant protein (M: protein marker; 1: G6-type BRVA VP7 protein);

[0038] Figure 4 is the Western blotting detection of the G6-type BRVA VP7 recombinant protein (M: protein marker; 1: G6-type BRVA VP7 protein);

[0039] Figure 5 is the indirect immunofluorescence identification map of 13 hybridoma cell lines (A-M: indirect immunofluorescence maps of BRVA monoclonal antibodies A1-A13 in sequence; N negative control);

[0040] Figure 6 is the molecular model A2_scFv;

[0041] Figure 7 is the molecular docking map (the upper blue-green model is A2_scFv; the lower is the BRVA VP7 trimer model; the yellow represents the docking interaction region);

[0042] Figure 8It is the plasmid map of pET28a-XXX_Fe;

[0043] Figure 9 It is the SDS-PAGE diagram (left: reaction with polyclonal antibody; right: reaction with monoclonal antibody). Specific implementation manners

[0044] The technical solutions of the present invention will be clearly and completely described below. Of course, the described embodiments are some embodiments of the present invention, rather than all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative efforts fall within the protection scope of the present invention. It should be noted that for the processes not specifically described in detail below, those skilled in the art can implement or understand them with reference to the prior art. The reagents or instruments used without indicating the manufacturer are regarded as conventional products that can be purchased commercially.

[0045] The reagents and materials used in the examples are as follows:

[0046] MA104 cells are preserved in the Animal Medicine Laboratory of Southwest Minzu University; the recombinant VP7 protein of G6 type BRVA is prepared in the Animal Medicine Laboratory of Southwest Minzu University; Escherichia coli BL21(DE3) competent cells are purchased from Beijing Qing Biotechnology Co., Ltd.; the plasmid miniprep kit is purchased from OMEGA Biological Company; the ultrasensitive ECL chemiluminescence substrate is purchased from Szebay Biotech Co., Ltd.; horseradish peroxidase (HRP)-labeled goat anti-rabbit IgG and HRP-labeled goat anti-mouse IgG are both purchased from Beijing Biosynthesis Biotechnology Co., Ltd.; fluorescein isothiocyanate (FITC)-labeled goat anti-rabbit IgG and BCA protein concentration assay kit are both purchased from Boster Biological Engineering Co., Ltd.; His Cap 6FF nickel ion purification column is purchased from Changzhou Tiandi Renhe Biotechnology Co., Ltd.; the animal immune adjuvant Montanide ISA 206 adjuvant is purchased from SEPPIC Company.

[0047] The preservation number of the hybridoma cell line A2: CCTCC NO: C202570, was preserved in the China Center for Type Culture Collection on March 25, 2025, address: Wuhan, China, Wuhan University; the preservation name is: Hybridoma cell line 8G11-B4-C8.

[0048] Example 1: Preparation of BRVA VP7 recombinant protein

[0049] 1 Experimental method

[0050] 1.1 Construction and identification of BRVA VP7 gene expression system

[0051] Refer to the VP7 gene sequence of the domestic prevalent strain RVA / Cow-tc / CHN / SDA2 / 2018 / G6P[1] (GenBank accession number: MN928491.1). The full length of the VP7 sequence is 1026 bp, encoding 326 amino acids. The expression sequence was screened according to the predicted antibody epitopes by biological information analysis software, and an 826-bp nucleotide sequence was selected for codon preference optimization to make its codon preference close to that of Escherichia coli. An Nde Ⅰ restriction enzyme site was added at its 5′-end, an Xho Ⅰ restriction enzyme site was added at its 3′-end, and a His tag was added at the N-terminus. The sequence was sent to Sangon Biotech (Shanghai) Co., Ltd. for synthesis. The synthesized sequence was ligated to the pET-28a(+) vector digested with NdeⅠ and Xho Ⅰ. After ligation, the recombinant plasmid was transformed into Escherichia coli TOP10 competent cells. Single colonies were picked and identified by colony PCR method. The positive clones were sent to Sangon Biotech (Shanghai) Co., Ltd. for sequencing.

[0052] Recombinant VP7 gene sequence (SEQ ID NO:4):

[0053] CATATGGTGAACCTGCCAATCACTGGTAGCATGGACACTGCTTACGCAAACTCTACTCAGTCTGAGCCATTCCTGACTTCTACCCTGTGCCTGTACTACCCTGTTGAAGCATCTAACGAGATCGCAGACACTGAGTGGAAGGACACTCTGTCTCAGCTGTTCCTGACTAAGGGTTGGCCGACTGGTTCTGTATACTTCAAGGAATACGCTGACATCGCAGCTTTCTCTGTGGAACCGCAACTGTACTGCGACTACAACCTGGTCCTGATGAAATACGACTCCACTCAGGAACTGGACATGTCCGAACTGGCTGATCTGATCCTGAACGAATGGCTGTGTAACCCGATGGATATCACCCTGTACTACTATCAGCAGACCGACGAAGCGAACAAATGGATCAGCATGGGTAGCTCCTGCACTGTCAAAGTGTGCCCGCTGAATACCCAAACCCTGGGTATTGGCTGTCTGATCACCAATCCGGATACGTTTGAAACCGTCGCGACCACCGAAAAACTGGTAATCACCGATGTTGTAGACGGCGTTAACCACAAACTGAACGTGACCACCGCCACGTGTACCATTCGTAATTGCAAAAAACTGGGCCCGCGTGAAAACGTTGCGGTTATCCAGGTAGGCGGCGCGAACGTTCTGGATATCACCGCCGATCCGACCACGACGCCGCAGACCGAACGTATGATGCGTATTAACTGGAAAAAATGGTGGCAGGTGTTTTATACCGTTGTTGATTATGTTAACCAGATTATTCAGACCATGTCCAAACGCAGCCGTTCCCTGAACTCCAGCGCGTTCTATTATCGCGTGTAACTCGAG

[0054] Amino acid sequence expressed (SEQ ID NO:5):

[0055] Protein Length=293

[0056] MW=33 kDa

[0057] Predicted pI=5.30

[0058] MGSSHHHHHHSSGLVPRGSHMVNLPITGSMDTAYANSTQSEPFLTSTLCLYYPVEASNEIADTEWKDTLSQLFLTKGWPTGSVYFKEYADIAAFSVEPQLYCDYNLVLMKYDSTQELDMSELADLILNEWLCNPMDITLYYYQQTDEANKWISMGSSCTVKVCPLNTQTLGIGCLITNPDTFETVATTEKLVITDVVDGVNHKLNVTTATCTIRNCKKLGPRENVAVIQVGGANVLDITADPTTTPQTERMMRINWKKWWQVFYTVVDYVNQIIQTMSKRSRSLNSSAFYYRV

[0059] 1.2 Induced expression of BRVA VP7 recombinant protein

[0060] The expression of recombinant protein was induced using IPTG. After induction, the bacteria were collected by centrifugation, resuspended in PBS at 1:100 (W / V), sonicated for 20 min, and then centrifuged to collect the supernatant and precipitate respectively. The precipitate was dissolved in Binding buffer and centrifuged to collect the supernatant. The collected supernatant was subjected to SDS-PAGE, and the expression form of BRVA VP7 recombinant protein of genotype G6 was analyzed by photographing using a multifunctional stain-free Western blotting system.

[0061] 1.3 Purification of BRVA VP7 recombinant protein

[0062] Collect the crude protein after induction, resuspend it with PBS buffer, and use an ultrasonic crusher to fully dissolve it. Centrifuge to collect the precipitate, dissolve the precipitate with Binding buffer, use an ultrasonic crusher to fully dissolve it, and then centrifuge to collect the supernatant. Purify the crude protein using a 5 mL HisCap 6FF nickel ion purification column. First, equilibrate the nickel ion purification column: Take 5 mL of Ni-NTA and wash and equilibrate the purification column with 25 mL of Binding buffer. Then, add the crude protein to the nickel ion purification column and collect the liquid flowing out of the nickel ion purification column as the loading effluent. Equilibrate the nickel ion purification column again. Wash the miscellaneous proteins: Wash the nickel ion purification column with 25 mL of Washing buffer and collect the flowing out liquid as the washing effluent. Elute the target protein: Elute and collect the flowing out liquid with 25 mL of Elution buffer. Detect all the collected flowing out liquids by SDA-PAGE. The recombinant protein component with high purity and a single target band after SDS-PAGE detection is dialyzed with the renaturation solution, changing the dialysis solution every 6 h for a total of 5 times. The dialyzed protein is embedded and concentrated to 1 / 2 of the original protein volume with PEG20000, filtered through a 0.45 μm filter membrane, aliquoted into 1.5 mL tubes, 1 mL per tube, and stored at -80 °C for later use. Use a BCA protein concentration assay kit to measure the concentration of the BRVA VP7 recombinant protein according to the kit instruction manual.

[0063] 1.4 Identification of BRVA VP7 Recombinant Protein by Western Blotting

[0064] Perform SDS-PAGE electrophoresis on the BRVA VP7 recombinant protein, and then transfer the BRVA VP7 recombinant protein to a nitrocellulose membrane. Incubate it with 5% skim milk powder on a shaker at 37 °C for 2 h. After washing three times with TBST for 15 min each time, add the primary antibody (rabbit anti-G6P[1] type BRVA positive serum diluted 1:1000 times) and incubate overnight. Wash three times with TBST for 15 min each time, add the secondary antibody (HRP-labeled goat anti-rabbit IgG diluted 1:5000), incubate on a shaker at 37 °C for 2 h, add ECL for color development, and take pictures with a multi-functional non-staining protein blotting system. Identify the reactivity of the BRVA VP7 recombinant protein.

[0065] 2 Result Analysis

[0066] 2.1 Construction and Identification of BRVA VP7 Gene Expression System

[0067] As Figure 1 shown, two bands, one dark and one bright, appeared at approximately 826 and 5000 bp. The fragment sizes were consistent with the expectations, and the sequencing results showed that the G6 type BRVA VP7 gene sequence was correctly inserted into pET-28a(+).

[0068] 2.2 Expression of BRVA VP7 recombinant protein

[0069] The product expressed by the recombinant bacterium induced by IPTG was detected by SDS-PAGE, and a specific band was observed at about 33 kDa. The recombinant protein was expressed as inclusion bodies, and the expression level of the recombinant bacterium was higher at 37 °C than at 20 °C (see Figure 2 ).

[0070] 2.3 Purification and concentration of BRVA VP7 recombinant protein

[0071] The purified recombinant protein of G6-type BRVA VP7 showed a single electrophoretic band by SDS-PAGE, with a size of about 33 kDa and a protein purity greater than 99% (see Figure 3 ). The protein concentration was determined to be 3 mg / mL by the BCA kit, and 9.509 mg of purified recombinant protein could be obtained from each liter of bacterial solution.

[0072] 2.4 Detection of the reactogenicity of BRVA VP7 recombinant protein

[0073] The results of Western Blotting showed that the recombinant protein of G6-type BRVA VP7 could bind to the rabbit anti-G6P[1]-type BRVA serum antibody, and a specific reaction band was visible at about 34 kDa (see Figure 4 ), confirming that the recombinant protein of G6-type BRVA VP7 had reactogenicity.

[0074] Example 2: Preparation of a broad-spectrum neutralizing monoclonal antibody against BRVA VP7 protein

[0075] 1 Experimental method

[0076] 1.1 Preparation of monoclonal antibody

[0077] After subcutaneous immunization of BALB / c mice with the BRVA VP7 recombinant antigen, the spleen cells of the mice were fused with SP2 / 0 cells, and then the hybridomas were cloned. The positive clones were screened by the indirect ELISA method, and OD 450 ≥0.19 was determined as positive.

[0078] 1.2 Identification of monoclonal antibody

[0079] The monoclonal antibody was identified by indirect immunofluorescence. When MA-104 cells grew to more than 90% in a 6-well plate, they could be infected, and negative control wells were set up. Acetone was added for fixation, BSA was added to block cell surface receptors, rabbit anti-BRVA VP6 recombinant protein positive serum was added as the primary antibody and FITC-labeled goat anti-rabbit IgG was added as the secondary antibody, and then DAPI was used for staining. Finally, the results were observed with an inverted microscope.

[0080] 1.3 Neutralization activity assay of monoclonal antibodies

[0081] The neutralization activity of monoclonal antibodies against three BRVA strains (G6P[1] strain (10-5.561TCID50 / 0.1 mL), G8P[1] strain (10-5.362TCID50 / 0.1 mL) and G10P

[11] strain (10-4.12TCID50 / 0.1 mL)) was determined using a microneutralization test, and the neutralizing titers of monoclonal antibodies against different strains were calculated. The anti-BRVA monoclonal antibody was diluted with DMEM nutrient solution in multiple ratios and added to a 96-well cell plate, with 3 replicates for each dilution and 0.025 mL for each well; the titrated virus stock solution was diluted to 200 TCID50 with nutrient solution and 0.025 mL was added to each well; after thorough mixing, the plate was placed in a 37°C cell culture incubator for 1 h; the plate was then treated and digested according to conventional methods to prepare a cell suspension, which was added to the 96-well plate after the above-mentioned treatment, 0.025 mL per well, and a positive and negative control well was set; after mixing, the plate was placed in a 37°C cell culture incubator for observation; the CPE of the cells was observed, and the 50% serum neutralization endpoint was calculated using the Reed-muech method. If the neutralization titer was greater than or equal to 1:4, it could be proven to have neutralizing activity.

[0082] 2 Results Analysis

[0083] 2.1 Establishment of hybridoma cell lines

[0084] After ELISA screening of hybridoma cell lines, 13 hybridoma cell lines that can stably secrete antibodies were obtained. They are numbered as follows: A1-A13. 450 See Table 1 for values.

[0085] Table 1 ELISA identification of OD of 13 hybridoma cell lines 450 value

[0086]

[0087] 2.2 Indirect immunofluorescence identification

[0088] The results are as follows Figure 5As shown, the antibodies secreted by 13 hybridoma cells were all reactive to the G6P[1] type BRVA strain, showing specific fluorescence. However, no fluorescence was observed in the negative control, indicating that the negative control was valid.

[0089] 2.3 Determination of the neutralizing activity of monoclonal antibodies

[0090] As shown in Table 2, the monoclonal antibodies A2 and A3 had good neutralizing activities against the G6, G8, and G10 type BRVA strains, with the lowest neutralizing activity reaching 1:150. However, although the monoclonal antibody of strain A3 showed high neutralizing titers against the G6, G8, and G10 type BRVA strains, further verification through cross-neutralization tests found that the neutralizing activity of strain A2 was more stable and had better broad-spectrum properties against different strains. Therefore, strain A2 was selected for epitope identification and application development in subsequent studies.

[0091] Table 2 Neutralizing activities of monoclonal antibodies

[0092]

[0093] Example 3: Screening of BRVA VP7 epitopes

[0094] 1 Experimental method

[0095] 1.1 Extraction, amplification of antibody genes and construction of single-chain antibody (scFv) molecular models

[0096] Total mRNA was extracted from the hybridoma cell A2 that produced broad-spectrum neutralizing monoclonal antibodies. The mRNA was reverse transcribed into cDNA using a reverse transcription kit. Specific primers FMusVL400 and RMusVL400 (the primer sequences were SEQ ID NO:6 and SEQ ID NO:7 respectively) were designed to amplify the genes encoding the variable regions of the light and heavy chains (VL and VH) of the monoclonal antibody. The primer sequences are shown in Table 3.

[0097] The amino acid sequences of the light and heavy chains were translated using MEGA7 software, and single-chain antibody (single-chain fragment variable, scFv) was constructed in series with Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Ser ((GGGGS)3) to simulate its protein molecular model for subsequent docking analysis.

[0098] Table 3

[0099]

[0100] 1.2 Molecular docking to predict antigenic epitopes

[0101] The A2_scFv was docked with the BRVA VP7 protein molecule (GenBank accession number: MN928491.1) using the ZDOCK rigid protein molecule docking program to predict the interaction region between the VP7 protein and the monoclonal antibody and determine the possible epitope sequences. The predicted epitopes were analyzed for antigenicity (http: / / www.ddg - pharmfac.net / vaxijen / VaxiJen / VaxiJen.html), and the epitopes with high antigenicity were selected for verification.

[0102] 2 Result Analysis

[0103] 2.1 Extraction, Amplification of Antibody Genes and Construction of Single - Chain Antibody (scFv) Molecular Model

[0104] The light - chain V - region gene (VL) was successfully amplified. Since the amplification of the heavy - chain V - region (VH) was difficult, metagenomic sequencing was carried out. The VL and VH genes of the A2 mAb were obtained and translated into amino acid sequences (SEQ ID NO:2 and SEQ ID NO:3 respectively), as shown in Table 4. VL and VH were concatenated with (GGGGS)3 and then the molecular model A2_scFv ( Figure 6 ) was constructed.

[0105] Table 4

[0106]

[0107] 2.2 Prediction of Epitopes by Molecular Docking

[0108] The A2_scFv was docked with the BRVA VP7 protein molecule using the ZDOCK rigid protein molecule docking program ( Figure 7 ). After predicting the epitopes and analyzing their antigenicity, it was found that the epitope sequence numbered 2 had high antigenicity (Table 5). This epitope was named XXX, and the amino acid sequence (SEQ ID NO:1) was 87 IEASNEIADTEWKNT 101 .

[0109] Table 5

[0110]

[0111] Example 4: Identification of the Broad - Spectrum Neutralizing Activity of Predicted Epitopes

[0112] 1 Experimental Method

[0113] 1.1 Expression and Purification of BRVA Antigenic Epitope - Tandem Ferritin

[0114] 1.1.1 Construction of Recombinant Plasmid

[0115] The epitope XXX was connected to Helicobacter pylori ferritin in a single-copy form through GGGGS, the nucleotide sequence was optimized, NcoⅠ and XhoⅠ restriction enzyme sites were inserted at both ends, and it was ligated to the pET28a(+) vector to obtain a recombinant plasmid named pET28a-XXX_Fe.

[0116] The optimized nucleotide sequence of XXX_Fe (SEQ ID NO:8):

[0117] ATCGAAGCATCTAACGAAATTGCGGACACTGAATGGAAGAACACTGGCGGTGGTATGCTGTCCAAAGACATCATCAAGCTGCTGAACGAACAGGTTAACAAGGAAATGAACAGCAGCAATCTGTACATGTCCATGTCCTCTTGGTGTTATACCCATTCTCTGGACGGTGCTGGCCTGTTTCTGTTCGACCATGCCGCAGAAGAATATGAGCACGCCAAAAAACTGATCATCTTCCTGAACGAAAACAACGTTCCGGTCCAGCTGACCTCTATTTCCGCCCCGGAACATAAATTCGAAGGCCTGACTCAGATCTTCCAGAAGGCTTATGAGCATGAACAGCATATTTCCGAATCCATCAACAACATTGTTGATCATGCTATCAAATCTAAAGACCACGCGACTTTCAATTTCCTGCAATGGTACGTTGCGGAACAGCATGAAGAAGAGGTCCTGTTCAAGGATATCCTGGACAAAATCGAACTGATTGGTAACGAGAACCACGGTCTGTATCTGGCTGATCAGTACGTCAAAGGTATCGCAAAATCTCGTAAGTCTCACCACCACCACCACCAC

[0118] 1.1.2 Transformation

[0119] Take the BL21(DE3) Escherichia coli competent cells stored at -80 °C and slowly thaw them on ice. After thawing, add 2 μL of the pET28a-XXX-Fe recombinant plasmid with a concentration of 100 ng / μL. After mixing, let it stand on ice for 30 min, then place it in a 42 °C constant temperature water bath for 60 s, and immediately transfer it to ice and let it stand for 2 min. Add 1 mL of LB liquid medium to the EP tube, and then place the competent cells in a 37 °C bench-top constant temperature shaker with a rotation speed set at 200 rpm for 1 h. Then centrifuge at a low speed of 5000 rpm for 5 min, discard a part of the supernatant, and leave 100 μL to coat on an LB plate (containing 50 μg / mL Kanamycin), and place it in a 37 °C bench-top incubator for 30 min with the lid facing up and then incubate it overnight with the lid facing down.

[0120] 1.1.3 Screening of positive clones

[0121] Pick a single colony on the LB plate and inoculate it into 5 mL of liquid LB, and place it in a 37 °C bench-top constant temperature shaker with a rotation speed set at 2000 rpm for overnight culture. Pipette 2 μL as a template, perform PCR amplification using the T7 promoter universal primer, and then perform gel electrophoresis. Observe the bands under the gel imaging system. The single colony with the target band is a positive clone. Send the bacterial solution to Beijing Tsingke Biotechnology Co., Ltd., Chengdu Branch for sequencing comparison, and the sequencing result is consistent with the synthesis.

[0122] 1.1.4 Induced expression of recombinant protein

[0123] Prepare 100 mL of liquid LB medium, add 1 mL of Escherichia coli BL21(DE3) containing the pET28a-XXX-Fe expression vector at a ratio of 100:1, and place it in a 37 °C bench-top constant temperature shaker with a rotation speed set at 200 rpm for culture. When OD 450When the nm value reaches 0.6, take 10 15-mL EP tubes and aliquot 10 mL of the bacterial solution into each tube. Divide the 10 15-mL tubes into two groups (5 tubes / group). One group is induced for expression at 16 °C for 18 h. Add IPTG with different concentration gradients (0.1 mM, 0.30 mM, 0.5 mM, 0.8 mM, and 1 mM) to the 5 tubes for induction of expression. Collect 2 mL of the bacterial solution every 6 h. After centrifugation at 5000 rpm for 5 min, discard the supernatant. Resuspend the bacterial pellet with 500 μL of PBS buffer. Pipette 20 μL of the resuspended bacteria, add 5 μL of SDS loading buffer according to a 4:1 ratio, and boil for 6 - 8 min to prepare SDS-PAGE samples. The other group is cultured at 37 °C for 6 h. Similarly, add IPTG with different concentration gradients (0.1 mM, 0.30 mM, 0.5 mM, 0.8 mM, and 1 mM) to the 5 tubes for induction of expression. After collecting 2 mL of the bacterial solution at 6 h, prepare SDS-PAGE samples according to the above steps. After SDS-PAGE with a 12% separating gel, stain with Coomassie Brilliant Blue solution for 10 min, decolorize 5 times, 5 min each time, and then observe under a gel imaging system. The size position of the target protein is between 25 KDa and 30 KDa of the protein Maker, and its size conforms to the expected size (22 KDa). Name the successfully expressed recombinant protein XXX-Fe. Analyze the protein gel with ImageJ software. According to the gray value, determine that the optimal conditions for protein induction are an IPTG concentration of 0.5 mM and induction at 16 °C for 12 h.

[0124] 1.1.5 Verification of the expression form of the recombinant protein

[0125] Break the induced bacteria twice. If the recombinant protein is in the supernatant after the first break, it is expressed in a soluble form; if it is in the supernatant after the second break, it is expressed in an inclusion body form. Induce the expression of 50 mL of bacterial solution using the optimized recombinant protein expression conditions. After centrifugation at 8000 rpm for 10 min, discard the supernatant. Add 15 mL of PBS to resuspend. Break the bacteria using an ultrasonic disruption instrument. The program is: power 400 w, time 10 min, ultrasound on for 5 s, stop for 3 s. After the disruption, centrifuge at 12000 rpm for 15 min, collect the supernatant protein for identification of the soluble expression form; resuspend the precipitate after centrifugation with an equal volume (15 mL) of Binding buffer and centrifuge at 12000 rpm for 15 min, collect the supernatant protein for identification of the inclusion body expression form. After identification, XXX-Fe induced by 0.5 mM IPTG at 16 °C is expressed in a soluble form.

[0126] 1.1.6 Purification of XXX-Fe protein

[0127] Purify using a 5 mL HisCap6FF nickel ion purification column according to the usage instructions of nickel-agarose affinity chromatography protein purification. The specific steps are as follows:

[0128] (1) Prepare purification reagents: Binding Buffer = 50 mM Tris + 300 mM NaCl; Washing Buffer = 50 mM Tris + 300 mM NaCl + 20 mM imidazole; Elution Buffer = 50 mM Tris + 300 mM NaCl + 500 mM imidazole;

[0129] (2) After inducing the large-scale expression of the recombinant protein under the conditions in 1.1.4, centrifuge at 12,000 rpm for 15 min, collect the bacterial pellet, resuspend it in PBS at a ratio of 10:3, centrifuge again after disruption, and collect the supernatant;

[0130] (3) Wash the HisCap6FF nickel ion purification column with 10 column volumes of pure water;

[0131] (4) Equilibrate the HisCap6FF nickel ion purification column with 5 column volumes of Binding Buffer;

[0132] (5) Take the supernatant after centrifugation in (3), filter it through a 0.45 μm pore size filter, and load it onto the column;

[0133] (6) Equilibrate the HisCap6FF nickel ion purification column with 5 column volumes of Binding Buffer;

[0134] (7) Wash the HisCap6FF nickel ion purification column with 10 column volumes of Washing Buffer to remove impurities;

[0135] (8) Elute the target protein in the HisCap6FF nickel ion purification column with 5 column volumes of Elution Buffer;

[0136] (9) Collect the protein in the eluate and verify its purity using a 12% separating gel for SDS-PAGE.

[0137] 1.2 Activity analysis of XXX-Fe protein

[0138] Using 5% defatted milk as the blocking solution, place it in a tabletop constant temperature shaker at 37°C with a rotation speed of 40 rpm, and incubate with horizontal shaking for 2 h. Wash it 3 times with TBST (1 time every 5 min); use the polyclonal antibody against BRVA VP7 protein and A2 mAb as the primary antibodies respectively, and incubate with horizontal slow shaking at 4°C overnight. Wash it 3 times with TBST (1 time every 5 min); use HRP-labeled Goat Anti-rabbit IgG as the secondary antibody, incubate with horizontal shaking at 37°C for 2 h, and wash it 3 times with TBST (1 time every 5 min); use the enhanced chemiluminescence substrate ECL for development.

[0139] 1.3 Immunogenicity evaluation of recombinant protein

[0140] (1)Preparation of XXX_Fe vaccine

[0141] Take the purified XXX-Fe in 1.1 and mix and emulsify it with the animal immune adjuvant Montanide ISA 206 adjuvant at a ratio of 1:1. When emulsified until the mixture of recombinant protein and adjuvant does not separate layers and does not spread rapidly after dropping into clear water, it is considered completely emulsified.

[0142] (2)Animal grouping and immunization

[0143] Subcutaneously immunize New Zealand white rabbits at multiple points with the prepared XXX_Fe vaccine at a dose of 200 μg / rabbit as the vaccine group, and use the immunized Montanide ISA 206 adjuvant as the control group (2 rabbits in each group, denoted as A and B respectively). Immunize the animals 3 times in total, with an immunization interval of 14 days, and collect the immune rabbit sera once every 7 days during the experiment.

[0144] (3)Indirect ELISA for detecting rabbit serum antibody levels

[0145] Dilute the VP7 protein with PBS solution to a concentration of 1 μg / mL. After mixing well with a shaker, add 100 μL per well and coat at 4°C for 16 h. Then discard the liquid and wash three times with PBST (1 time every 3 min); add 100 μL of 2% bovine serum albumin (BSA) per well, block at 37°C for 1 h, discard the liquid, and wash three times with PBST (1 time every 3 min); use the collected rabbit serum as the primary antibody, and dilute the primary antibody with antibody diluent (PBS + 0.05% Tween 20 + 1% BSA) in gradient. Then add 100 μL per well, incubate at 37°C for 1 h, discard the liquid, and wash three times with PBST (1 time every 3 min); dilute the HRP-labeled goat anti-mouse IgG with antibody diluent at a ratio of 1:5000, add 100 μL per well, incubate at 37°C for 1 h, discard the liquid, and wash three times with PBST (1 time every 3 min); add TMB chromogenic solution, 100 μL per well, incubate at 37°C in the dark for 10 min, then add the termination solution to terminate the reaction, and use an enzyme-linked immunosorbent assay reader to read the OD 450 value. The positive OD 450 value / negative OD 450 value ≥ 2.1 is identified as positive.

[0146] (4)Neutralization experiment to detect the neutralizing antibody level in rabbit serum

[0147] Detect the neutralizing antibodies against BRVA of G6, G8, and G10 types in the serum collected from the immunized rabbits on the 14th day after the second immunization. The specific operation is as follows: Passage the MA104 cells in the confluent cell culture flask to a 96-well plate and incubate statically at 37°C for 24 - 48 h until they grow into a monolayer of cells. Inactivate the rabbit serum at 56°C for 30 min, and perform 2-fold serial dilution with DMEM medium without serum and antibiotics. Mix it with 200 TCID 50 of BRVA and incubate in a 37°C incubator for 1 h; Take out the 96-well plate containing MA104 cells, discard the liquid, wash twice with Hanks, and add the mixed culture medium after incubation to the 96-well plate, 100 μL per well. Incubate statically at 37°C and observe once every 12 h, record the lesion situation of each well, and calculate the titer of the neutralizing antibody in the serum on the 14th day after the second immunization according to the Reed-Muench method.

[0148] 2 Result analysis

[0149] 2.1 Expression and purification of the tandem ferritin of BRVA antigen epitopes

[0150] As Figure 8As shown in the figure, the epitope XXX and the ferritin gene were fused and inserted into the pET28a(+) vector by double digestion with NcoⅠ / XhoⅠ using the GGGGS linker, and the pET28a-XXX_Fe plasmid was successfully constructed. After purification by nickel column, the XXX-Fe protein was present in the eluate, indicating that the XXX-Fe protein was successfully purified. After measuring the protein concentration of the purified protein using the BCA kit, in terms of yield, 10 mg could be obtained from every 300 mL of bacterial solution after induction.

[0151] 2.2 Activity analysis of XXX-Fe protein

[0152] As Figure 9 shown, the XXX-Fe protein was reactive with both the polyclonal antibody against Anti-BRVA VP7 protein and A2 mAb.

[0153] 2.3 Immunogenicity evaluation of the recombinant protein

[0154] As shown in Table 6, low-level antibodies began to be produced 7 days after the first immunization, and the antibody level reached its peak 14 days after the second immunization. The neutralization titers against BRVA of genotypes G6, G8, and G10 could reach 1:2 6 、1:2 5 、1:2 6 .

[0155] Table 6

[0156]

[0157] In this article, specific examples were used to elaborate on the principle and implementation mode of this application. The description of the above embodiments is only used to help understand the technical solution and its core idea of this application; those of ordinary skill in the art should understand that they can still modify the technical solutions recorded in the foregoing embodiments, or perform equivalent replacements for some of the technical features; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the scope of the technical solutions of the embodiments of this application.

Claims

1. A hybridoma cell line, characterized in that, The preservation number of the hybridoma cell line: CCTCC NO: C202570.

2. A monoclonal antibody, characterized in that, The monoclonal antibody is prepared from the hybridoma cell line described in claim 1.

3. An antigenic epitope peptide of BRVA virus VP7 protein, characterized in that, The amino acid sequence of the antigenic epitope peptide of the BRVA virus VP7 protein is shown in SEQ ID NO:

1.

4. A method for identifying the antigenic epitopes of the VP7 protein of the BRVA virus, characterized in that, Comprising the following steps: (1) Performing molecular docking on the monoclonal antibody described in claim 2 and the BRVA VP7 protein to predict the interaction region between the VP7 protein and the monoclonal antibody; (2) Screening and validating epitope sequences with high antigenicity.

5. Use of the hybridoma cell line described in claim 1 in the preparation of products for preventing and / or treating and / or detecting and / or diagnosing BRVA virus infection.

6. Use of the monoclonal antibody described in claim 2 in the preparation of products for detecting and / or diagnosing and / or treating BRVA virus infection.

7. Use of the antigenic epitope peptide described in claim 3 in the preparation of products for preventing BRVA virus infection.

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