Monoclonal antibody of BRVA virus VP7 protein and epitope and application thereof

Abstract

The invention relates to the technical field of biological medicine, in particular to a monoclonal antibody of BRVA virus VP7 protein and an epitope and application of the monoclonal antibody. The invention provides a hybridoma cell strain (preservation number: CCTCC NO: C202570). A monoclonal antibody prepared from the hybridoma cell strain can be combined with VP7 protein of G6, G8 and G10 type BRVA; the antigen epitope amino acid sequence of the monoclonal antibody is SEQ ID NO: 1. The monoclonal antibody and the antigen epitope peptide provided by the invention have broad-spectrum neutralizing activity, and provide a new strategy and tool for prevention and control of BRVA.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to a monoclonal antibody against VP7 protein of BRVA virus and 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 in newborn calves, resulting in significant economic losses. The overall infection rate of BRVA in cattle herds in my country is 46%, which is a serious threat. The genome of BRVA is a phased double-stranded RNA virus that is prone to mutation, and there are many different genotypes circulating. To date, 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 G6, G8, G10 and P [1], P [5], P

[11] genotypes are the most common. There are many types of BRVA GP type combinations, and the cross-protection of different genotype strains is poor, which brings great difficulties to disease prevention and control and vaccine development. At present, there are no broad-spectrum neutralizing antibodies or vaccines targeting multiple genotypes on the market in my country, and it is urgent to develop broad-spectrum vaccines targeting multiple genotypes.

[0003] The VP7 protein of BRVA is the main surface antigen that determines the G type of the virus. It 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 homology of the amino acid sequence of the VP7 protein of different G types of BRVA (68.0%~73.1%) (Li Fan, Zhou Fang, Yue Hua, et al. Progress in Molecular Biology of Group A Bovine Rotavirus [J]. Advances in Veterinary Medicine, 2017, 38(6):4.), and the presence of at least 4 highly conserved (more than 90%) peptide segments, it is difficult to identify 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 G6, G8, and G10 types of BRVA, and also confirms the existence of broad-spectrum neutralizing antigenic epitopes in VP7 protein.

[0005] The invention provides a hybridoma cell strain, and the deposit number of the hybridoma cell strain is CCTCC NO: C202570.

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

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

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

[0009] In some specific embodiments of the present invention, the amino acid sequence of the antigenic epitope to which the monoclonal antibody binds 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 as shown in SEQ ID NO:2 and a heavy chain as shown in SEQ ID NO:3.

[0012] SEQ ID NO:2:

[0013] QSPASSLSASVGETVTITCRASENIYSYLAWYQQKQGKSPQVLVYNAKXLPEGVPSRFSASGSGTQFSLKINSLQPEDFGSYYCQHHYGNPFTFGSGTKLEIKRADAAPTVS.

[0014] SEQ ID NO:3:

[0015] QSGPELKKPGETVKISCKASGYTFTTYAINWVKQAPGKGLKWMGWINTYTGEPTHDNDFKGRFAFSSETSASTAYLQINNLKNEDTATYFCTRGGRGPYFLYWGQGTLVTVSAAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSV TVPSSTWPSETVTCNVAHPASSTKVDKKIVPRDCGCKPCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQTQPREEQFNSTFRSSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTISIPPPKEQMAKDKVSLTCMITDFFPEDITVEWQWNGQ

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

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

[0018] (1) Molecular docking of the monoclonal antibody described above with the BRVA VP7 protein to predict the interaction region between the VP7 protein and the monoclonal antibody;

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

[0020] The present invention provides the use of the hybridoma cell line or the monoclonal antibody or the antigenic epitope peptide in the preparation of a product 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 hybridoma cell line or the monoclonal antibody or the antigenic epitope peptide described above.

[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 therapeutic that is able to provide protection against multiple different subtypes or strains of a pathogen.

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

[0025] In the present invention, the term "monoclonal antibody" means an antibody derived from a population of substantially homologous antibodies, that is, 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 produced during the production of monoclonal antibody products), such variants are usually present in trace amounts. Unlike polyclonal antibody products that generally include different antibodies for different determinants (epitopes), each monoclonal antibody of a monoclonal antibody product is directed to a single determinant on an antigen. Thus, the modifier "monoclonal" indicates that the antibody is derived from a substantially homologous antibody population, and should not be interpreted as requiring the antibody to be produced by any particular method. For example, the monoclonal antibody 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 comprising all or part of a human immunoglobulin locus, 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 fusion of a myeloma cell and a B lymphocyte in the process of preparing a monoclonal antibody, and is generally prepared by culturing tumor cells.

[0027] In the present invention, the term "label" refers to a molecule that can indicate the location or amount of an antibody by color, chemical reaction, excitation light, mass spectrometry, etc. after the antibody is bound. Examples include alkaline phosphatase, peroxidase, luciferase, fluorescein, fluorescent protein, isotope, etc.

[0028] In the present invention, "enzyme-linked immunosorbent assay (ELISA)" refers to a detection method that utilizes the characteristic that antibody molecules can specifically bind to antigen molecules, combines free impurities with the target protein bound to a solid phase carrier, and uses special markers to qualitatively or quantitatively analyze them. The principle is: antigens or antibodies can be physically adsorbed on the solid surface and maintain their immune activity; antigens or antibodies can form enzyme conjugates with enzymes through covalent bonds while maintaining their respective immune activity or enzyme activity; after the enzyme conjugates bind to the corresponding antigens or antibodies, the occurrence of the immune reaction can be determined by the color reaction of the added substrate, and the depth of the color reaction is proportional to the amount of the corresponding antigen or antibody in the specimen. Various types of detection methods can be designed according to the substance to be detected and the conditions for detection. The double antibody sandwich method is the most commonly used method for detecting antigens. The method is to adsorb the antiserum containing known antibodies into the small holes of the microtiter plate and wash it once; add the antigen to be tested. If the two are specific, they will bind and then wash away the excess antibody; add an enzyme-linked antibody that reacts specifically with the antigen to be tested to form a "sandwich"; add the substrate of the enzyme, and if a colored enzymatic hydrolysis product is seen, it indicates the presence of the corresponding antigen.

[0029] In the present invention, "plasmid" refers to a DNA molecule other than chromosomes (or nucleoids) in organisms such as bacteria, yeast and actinomycetes, which exists in the cytoplasm or nucleus and has the ability to replicate autonomously, so that it can maintain a constant copy number in daughter cells and express the genetic information it carries.

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

[0031] Compared with the prior art, the present invention has the following beneficial effects:

[0032] (1) Broad-spectrum neutralizing activity: There are limitations in the prevention and control of bovine group A rotavirus (BRVA), especially the difficulty in vaccine development to cover multiple genotypes. The monoclonal antibody of the present invention can effectively neutralize G6, G8, and G10 BRVA, confirming the existence of broad-spectrum neutralizing antigen epitopes in the VP7 protein, breaking through the limitation of traditional vaccines and antibodies against a single strain, and having a significant broad-spectrum protective effect.

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

[0034] (3) In the prior art, the detection and prevention methods of BRVA are relatively simple and lack 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 for the preparation of highly sensitive detection kits to achieve rapid and accurate diagnosis of BRVA infection. In addition, the products can also be used to prevent and treat BRVA infection, and have broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

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

[0036] Figure 2 For recombinant protein expression analysis (M: protein molecular weight standard; 1: total protein before IPTG induction; 2: 20 ℃ supernatant; 3: 20 ℃ precipitate; 4: 37 ℃ supernatant; 5: 37 ℃ precipitate);

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

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

[0039] Figure 5 Indirect immunofluorescence identification images of 13 hybridoma cell lines (AM: indirect immunofluorescence images of BRVA monoclonal antibodies A1-A13, respectively; N negative control);

[0040] Figure 6 is the molecular model A2_scFv;

[0041] Figure 7 It is a molecular docking diagram (the blue-green model on the top is A2_scFv; the model on the bottom is the BRVA VP7 trimer; the yellow represents the docking interaction area);

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

[0043] Fig. 9 The SDS-PAGE images are shown below (left: polyclonal antibody reaction; right: monoclonal antibody reaction). DETAILED DESCRIPTION

[0044] The technical scheme of the present invention will be clearly and completely described below. Of course, the described embodiments are part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments in the present invention, all other embodiments obtained by those skilled in the art without making creative work are within the scope of protection of the present invention. It should be noted that if there is a process that is not particularly described in detail below, it is all that those skilled in the art can realize or understand with reference to the prior art. If the manufacturer is not indicated for the reagents or instruments used, it is regarded as a conventional product that can be purchased commercially.

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

[0046] MA104 cells were maintained by the Animal Medicine Laboratory of Southwest University for Nationalities; the VP7 protein of recombinant G6-type BRVA was prepared by the Animal Medicine Laboratory of Southwest University for Nationalities; Escherichia coli BL21 (DE3) competent cells were purchased from Beijing Qing Biotechnology Co., Ltd.; the plasmid extraction kit was purchased from OMEGA Biotechnology Co., Ltd.; the ultrasensitive ECL chemiluminescent substrate was purchased from Sizhengbai Biotechnology Co., Ltd.; horseradish peroxidase (HRP)-labeled goat anti-rabbit IgG and HRP-labeled goat anti-mouse IgG were purchased from Beijing Bioson Biotechnology Co., Ltd.; fluorescein isothiocyanate (FITC)-labeled goat anti-rabbit IgG and BCA protein concentration determination kit were purchased from Boster Biotechnology Co., Ltd.; His Cap 6FF nickel ion purification column was purchased from Changzhou Tiandi Renhe Biotechnology Co., Ltd.; and animal immune adjuvant Montanide ISA 206 adjuvant was purchased from SEPPIC.

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

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

[0049] 1 Experimental methods

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

[0051] Referring to the VP7 gene sequence of the domestic epidemic strain RVA / Cow-tc / CHN / SDA2 / 2018 / G6P[1] (GenBank accession number: MN928491.1), the VP7 sequence is 1026 bp in length and encodes 326 amino acids. The expression sequence was screened based on the antibody epitope predicted by biological information analysis software, and the 826 bp nucleotide sequence was selected for codon preference optimization to make its codon preference close to that of Escherichia coli. The restriction site Nde Ⅰ was added to its 5′-end, the restriction site Xho Ⅰ was added to its 3′-end, and a His tag was added to the N-terminus. The sequence was sent to Sangon Biotech (Shanghai) Co., Ltd. for synthesis, and the synthesized sequence was connected to the pET-28a (+) vector that had been double-digested with Nde Ⅰ and Xho Ⅰ. After connection, the recombinant plasmid was transformed into Escherichia coli TOP10 competent cells, and single clones were picked and identified by colony PCR. 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] MGSSHHHHHHSSGLVPRGSHMVNLPITGSMDTAYANSTQSEPFLTSTLCLYYPVEASNEIADTEWKDTLSQLFLTKGWPTGSVYFKEYADIAAFSVEPQLYCDYNLVLMKYDSTQELDMSELADLILNEWLCNPMDITLYYYQQTD EANKWISMGSSCTVKVCPLNTQTLGIGCLITNPDTFETVATTEKLVITDVVDGVNHKLNVTTATCTIRNCKKLGPRENVAVIQVGGANVLDITADPTTTTPQTERMMRINWKKWWQVFYTVVDYVNQIIQTMSKRSRSLNSSAFYYRV

[0059] 1.2 Inducible expression of BRVA VP7 recombinant protein

[0060] IPTG was used to induce the expression of the recombinant protein. After induction, the bacteria were collected by centrifugation and resuspended in PBS 1:100 (W / V). After ultrasonic disruption for 20 minutes, the supernatant and precipitate were collected by centrifugation. The precipitate was dissolved with Binding buffer and the supernatant was collected by centrifugation. The collected supernatants were subjected to SDS-PAGE and the expression form of G6 BRVA VP7 recombinant protein was photographed and analyzed using a multifunctional stain-free protein blotting system.

[0061] 1.3 Purification of BRVA VP7 recombinant protein

[0062] The crude protein after induction was collected, resuspended in PBS buffer, fully dissolved by ultrasonic disruptor, and centrifuged to collect the precipitate. The precipitate was dissolved by Binding buffer, fully dissolved by ultrasonic disruptor, and then the supernatant was collected by centrifugation. The crude protein was purified using 5 mL HisCap 6FF nickel ion purification column. First, the nickel ion purification column was balanced: 5 mL Ni-NTA was taken and the balanced purification column was washed with 25 mL Binding buffer; then the crude protein was added to the nickel ion purification column, and the outflow liquid of the nickel ion purification column was collected as the loading outflow liquid; the nickel ion purification column was balanced again; the impurity protein was washed: the nickel ion purification column was washed with 25 mL Washing buffer, and the outflow liquid was collected as the washing outflow liquid; the target protein was eluted: 25 mL Elution buffer was used to elute and the outflow liquid was collected. All the collected outflow liquids were detected by SDA-PAGE. After SDS-PAGE detection, the recombinant protein component with high purity and a single target band was dialyzed with renaturation buffer, and the dialysate was changed every 6 hours for a total of 5 times. The dialyzed protein was embedded and concentrated to 1 / 2 of the original protein volume using PEG20000, filtered with a 0.45 μm filter membrane, dispensed into 1.5 mL tubes, 1 mL per tube, and stored at -80 °C for later use. The concentration of BRVA VP7 recombinant protein was determined using the BCA protein concentration assay kit according to the kit instructions.

[0063] 1.4 Identification of BRVA VP7 recombinant protein by Western blotting

[0064] The BRVA VP7 recombinant protein was subjected to SDS-PAGE electrophoresis, and then transferred to a nitrocellulose membrane, incubated with 5% skim milk powder at 37°C for 2 h, washed three times with TBST for 15 min each time, and then incubated overnight with the primary antibody (1:1000-fold diluted rabbit anti-G6P[1] type BRVA positive serum), washed three times with TBST for 15 min each time, and then incubated with the secondary antibody (1:5000 diluted HRP-labeled goat anti-rabbit IgG) at 37°C for 2 h, developed with ECL, and photographed with a multifunctional stain-free western blotting system. The reactivity of the BRVA VP7 recombinant protein was identified.

[0065] 2 Results Analysis

[0066] 2.1 Construction and identification of BRVA VP7 gene expression system

[0067] like Figure 1 As shown, two bands, one dark and one light, appeared at approximately 826 bp and 5000 bp, and the fragment size was consistent with expectations. 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 bacteria induced by IPTG was detected by SDS-PAGE, and a specific band was found at about 33 kDa. The recombinant protein was expressed as inclusion bodies, and the expression level of the recombinant bacteria was higher at 37 ℃ than at 20 ℃ (see Figure 2 ).

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

[0071] The purified G6-type BRVA VP7 recombinant protein showed a single electrophoretic band by SDS-PAGE, with a size of approximately 33 kDa and a protein purity of more than 99% (see Figure 3 ). The protein concentration determined by BCA kit was 3 mg / mL, and 9.509 mg of purified recombinant protein was obtained per liter of bacterial liquid.

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

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

[0074] Example 2: Preparation of broadly neutralizing monoclonal antibodies against BRVA VP7 protein

[0075] 1 Experimental methods

[0076] 1.1 Preparation of monoclonal antibodies

[0077] After BALB / c mice were subcutaneously immunized with BRVA VP7 recombinant antigen, mouse spleen cells were fused with SP2 / 0 cells, hybridomas were cloned, and positive clones were screened by indirect ELISA. 450 ≥0.19 was considered positive.

[0078] 1.2 Identification of monoclonal antibodies

[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 the 13 hybridoma cells were all reactive to the G6P[1] type BRVA strain and produced specific fluorescence. However, the negative control did not produce fluorescence, so the negative control was established.

[0089] 2.3 Neutralization activity assay of monoclonal antibodies

[0090] The results are shown in Table 2. Both A2 and A3 monoclonal antibodies have good neutralizing activity against G6, G8, and G10 BRVA strains, with the lowest neutralizing activity reaching 1:150; however, although A3 monoclonal antibodies showed high neutralizing titers against G6, G8, and G10 BRVA strains, further verification through cross-neutralization experiments found that A2 strain had more stable neutralizing activity against different strains and better broad-spectrum, so A2 strain was selected for epitope identification and application development in subsequent studies.

[0091] Table 2 Neutralizing activity of monoclonal antibodies

[0092]

[0093] Example 3: Screening of BRVA VP7 epitopes

[0094] 1 Experimental methods

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

[0096] Total mRNA was extracted from hybridoma cell A2 that produces a broad-spectrum neutralizing monoclonal antibody. The mRNA was reverse transcribed into cDNA using a reverse transcription kit. Specific primers FMusVL400 and RMusVL400 (primer sequences are SEQ ID NO: 6 and SEQ ID NO: 7, respectively) were designed to amplify the genes encoding the variable regions (VL and VH) of the light and heavy chains 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 expressed as Gly-Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Ser (GGGGS) 3 ) were used to construct a single-chain antibody (scFv) in series and simulate its protein molecular model for subsequent docking analysis.

[0098] Table 3

[0099]

[0100] 1.2 Molecular docking to predict antigen epitopes

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

[0102] 2 Results Analysis

[0103] 2.1 Extraction and 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 heavy chain V region (VH) was difficult to amplify, metagenomic sequencing was performed to obtain the VL and VH genes of A2 mAb and translate them into amino acid sequences (SEQ ID NO: 2 and SEQ ID NO: 3, respectively), see Table 4. VL and VH were separated using (GGGGS) 3 After concatenation, the molecular model A2_scFv was constructed ( Figure 6 ).

[0105] Table 4

[0106]

[0107] 2.2 Molecular docking to predict epitopes

[0108] The A2_scFv and BRVA VP7 protein molecules were docked using the ZDOCK rigid protein molecular docking program ( Figure 7 ), after predicting the epitope and conducting antigenic analysis, it was found that the epitope sequence numbered 2 had high antigenicity (Table 5). The 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 epitopes predicted for broad-spectrum neutralizing activity

[0112] 1 Experimental methods

[0113] 1.1 Expression and purification of BRVA antigen epitope tandem ferritin

[0114] 1.1.1 Construction of recombinant plasmid

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

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

[0117] ATCGAAGCATCTAACGAAATTGCGGACACTGAATGGAAGAACACTGGCGGTGGTATGCTGTCCAAAGACATCATCAAGCTGCTGAACGAACAGGTTAACAAGGAAATGAACAGCAGCAATCTGTACATGTCCATGTCCTCTTG GTGTTATACCCATTCTCTGGACGGTGCTGGCCTGTTTCTGTTCGACCATGCCGCAGAAGAATATGAGCACGCCAAAAAACTGATCATCTTCCTGAACGAAAACAACGTTCCGGTCCAGCTGACCTCTATTTCCGCCCCGGAAC ATAAATTCGAAGGCCTGACTCAGATCTTCCAGAAGGCTTATGAGCATGAACAGCATATTTCCGAATCCATCAACAACATTGTTGATCATGCTATCAAATCTAAAGACCACGCGACTTTCAATTTCCTGCAATGGTACGTTGCG GAACAGCATGAAGAAGAGGTCCTGTTCAAGGATATCCTGGACAAAATCGAACTGATTGGTAACGAGAACCACGGTCTGTATCTGGCTGATCAGTACGTCAAAGGTATCGCAAAATCTCGTAAGTCTCACCACCACCACCACCAC

[0118] 1.1.2 Conversion

[0119] Take the BL21 (DE3) Escherichia coli competent cells stored at -80℃ and slowly thaw them on ice. After thawing, add 2μL of pET28a-XXX-Fe recombinant plasmid with a concentration of 100 ng / μL, mix well, let it stand on ice for 30 min, place it in a 42℃ constant temperature water bath for 60 s, immediately transfer it to ice and let it stand for 2 min, add 1 mL LB liquid culture medium to the EP tube, place the competent cells on a 37℃ desktop constant temperature shaker, set the speed to 200 rpm for 1 h, centrifuge at 5000 rpm for 5 min, discard part of the supernatant, keep 100 mL and spread it on an LB plate (containing 50 μg / mL Kanamycin), place it in a 37℃ desktop incubator for upright culture for 30 min, and then invert and culture overnight.

[0120] 1.1.3 Screening of positive clones

[0121] Pick a single colony on the LB plate and put it in 5 mL of liquid LB, place it in a 37°C tabletop constant temperature shaker, and set the speed to 2000 rpm for overnight culture. Take 2 μL as a template, use T7 promoter universal primers for PCR amplification, and then perform gel electrophoresis. Observe the bands under the gel imaging system. The single colony that meets the target band is a positive clone. The bacterial solution is sent to the Chengdu Branch of Beijing Qingke Biotechnology Co., Ltd. for sequencing comparison. The sequencing results are consistent with the synthesis.

[0122] 1.1.4 Inducible 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, place in a 37°C tabletop constant temperature shaker, set the speed to 200 rpm, and culture until the OD 450When the nm value reaches 0.6, take 10 15 mL EP tubes and fill each with 10 mL of bacterial solution. Ten 15 mL tubes were divided into two groups (5 / group). One group was induced to express at 16°C for 18 h. Different concentration gradients of IPTG (0.1 mM, 0.30 mM, 0.5 mM, 0.8 mM and 1 mM) were added to the 5 test tubes for induction expression. 2 mL of bacterial solution was collected every 6 h. After centrifugation at 5000 rpm for 5 min, the supernatant was discarded, and the bacterial slurry was resuspended with 500 μL PBS buffer. 20 μL of the resuspended bacteria were aspirated, 5 μL SDS loading solution was added at a ratio of 4:1, and then boiled in boiling water for 6-8 min to prepare SDS-PAGE samples. The other group was cultured at 37 degrees for 6 h. Similarly, different concentration gradients of IPTG (0.1 mM, 0.30 mM, 0.5 mM, 0.8 mM and 1 mM) were added to the 5 test tubes for induction expression. 2 mL of bacterial solution was collected every 6 h. After 6 h, 20 μL of the resuspended bacteria were aspirated, and 5 μL of SDS loading solution was added at a ratio of 4:1. The samples were boiled in boiling water for 6-8 min to prepare SDS-PAGE samples. mL of bacterial solution was used to prepare SDS-PAGE samples according to the above steps; after SDS-PAGE with 12% separation gel, it was stained with Coomassie Brilliant Blue solution for 10 min, destained 5 times, and observed in the gel imaging system after 5 min each time. The size of the target protein was between 25KDa and 30KDa of the protein Maker, and its size was consistent with the expected size (22KDa). The successfully expressed recombinant protein was named XXX-Fe. The protein gel was analyzed by ImageJ software, and the optimal conditions for protein induction were determined to be IPTG concentration of 0.5mM and induction at 16℃ for 12 h according to the gray value.

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

[0125] The induced expression bacteria were broken twice. If the recombinant protein was in the supernatant after the first break, it was expressed in a soluble form; if it was in the supernatant after the second break, it was expressed in the form of inclusion bodies. Using the optimized recombinant protein expression conditions, 50 mL of bacterial solution was induced to express, and the supernatant was discarded after centrifugation at 8000 rpm for 10 min, and 15 mL of PBS was added for resuspending. The bacteria were broken by ultrasonic disruption instrument. The program was: power 400 w, time 10 min, ultrasound on 5 s, stop 3 s; after the disruption, centrifuge at 12000 rpm for 15 min, collect the supernatant protein for soluble expression form identification; add an equal volume (15 mL) of Binding buffer to the precipitate after centrifugation, resuspend it, and centrifuge it at 12000 rpm for 15 min, collect the supernatant protein for inclusion body expression form identification. It was identified that XXX-Fe induced by 0.5 mM IPTG at 16℃ was soluble expression.

[0126] 1.1.6 Purification of XXX-Fe protein

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

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

[0129] (2) After inducing the expression of recombinant protein in large quantities according to the conditions in 1.1.4, centrifuge at 12,000 rpm for 15 min, collect the bacterial sludge, resuspend it in PBS at a ratio of 10:3, break it, and centrifuge it again to 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) After centrifugation in step (3), the supernatant was filtered through a 0.45 μm pore size filter and loaded onto the column;

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

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

[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 by SDS-PAGE using 12% separation gel.

[0137] 1.2 Activity analysis of XXX-Fe protein

[0138] 5% skim milk was used as the blocking solution, and the cells were placed in a tabletop constant temperature shaker at 37°C and 40 rpm, incubated with horizontal shaking for 2 hours, and washed with TBST three times (1 time / 5 min); Anti-BRVA VP7 protein polyclonal antibody and A2 mAb were used as primary antibodies, incubated with slow horizontal shaking at 4°C overnight, and washed with TBST three times (1 time / 5 min); HRP-labeled Goat Anti-rabbit IgG was used as the secondary antibody, incubated with horizontal shaking at 37°C for 2 hours, and washed with TBST three times (1 time / 5 min); ultrasensitive ECL chemiluminescent substrate was used for development.

[0139] 1.3 Evaluation of immunogenicity of recombinant proteins

[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 in a ratio of 1:1. When the mixture of the recombinant protein and the adjuvant is emulsified until there is no stratification and it does not diffuse rapidly after being added dropwise into clean water, it is considered to be completely emulsified.

[0142] (2) Animal grouping and immunization

[0143] The prepared XXX_Fe vaccine was subcutaneously immunized at multiple points in New Zealand white rabbits at a dose of 200 μg / rabbit as the vaccine group, and the immunization with Montanide ISA 206 adjuvant was used as the control group (2 rabbits in each group, respectively marked as A and B). The animals were immunized 3 times with an interval of 14 days. During the experiment, the immune rabbit serum was collected once every 7 days.

[0144] (3) Indirect ELISA to detect rabbit serum antibody levels

[0145] VP7 protein was diluted to a concentration of 1 μg / mL with PBS solution, mixed with an oscillator, and 100 μL was added to each well. After coating at 4℃ for 16 h, the solution was discarded and washed three times with PBST (1 time / 3 min); 100 μL of 2% bovine serum albumin (BSA) was added to each well, blocked at 37℃ for 1 h, the solution was discarded, and washed three times with PBST (1 time / 3 min); the collected rabbit serum was used as the primary antibody, and the primary antibody was graded diluted with antibody diluent (PBS+0.05% Tween 20+1% BSA), 100 μL was added to each well, incubated at 37℃ for 1 h, the solution was discarded, and washed three times with PBST (1 time / 3 min); HRP-labeled goat anti-mouse IgG was diluted with antibody diluent at 1:5000, 100 μL was added to each well, incubated at 37℃ for 1 h, the solution was discarded, and washed three times with PBST (1 time / 3 min); TMB colorimetric solution was added, 100 uL was added to each well, and incubated at 37℃ in the dark for 10 After 10 min, add the stop solution to terminate the reaction and read the OD using a microplate reader. 450 Value, positive OD 450 Value / Negative OD 450 A value ≥ 2.1 was identified as positive.

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

[0147] The serum collected from the immunized rabbits for 14 days after the second immunization was tested for neutralizing antibodies of type G6, G8, and G10 BRVA. The specific operation was as follows: the MA104 cells that filled the cell culture bottle were subcultured to a 96-well plate, and cultured at 37°C for 24-48 hours. When the cells grew into a monolayer, the rabbit serum was inactivated at 56°C for 30 minutes, and the serum was diluted 2-fold with DMEM medium without serum and double antibodies, and then mixed with 200 TCID 50 The 96-well plate containing MA104 cells was taken out, the liquid was discarded and washed twice with Hanks, and the mixed culture medium after infection was added to the 96-well plate, 100 μL per well, and cultured at 37°C. It was observed every 12 h, the pathological changes in each well were recorded, and the titer of the neutralizing antibody in the serum at 2 immunization days 14 was calculated according to the Reed-Muech method.

[0148] 2 Results Analysis

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

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

[0151] 2.2 Activity analysis of XXX-Fe protein

[0152] like Fig. 9 As shown, XXX-Fc protein was reactive with both Anti-BRVA VP7 protein polyclonal antibody and A2 mAb.

[0153] 2.3 Evaluation of immunogenicity of recombinant proteins

[0154] As shown in Table 6, low levels of antibodies began to be produced 7 days after immunization 1, and the peak antibody level was reached 14 days after immunization 2. The neutralization titers of G6, G8, and G10 BRVA were 1:2, respectively. 6 , 1:2 5 , 1:2 6 .

[0155] Table 6

[0156]

[0157] This document uses specific examples to illustrate the principles and implementation methods of the present application. The description of the above embodiments is only used to help understand the technical solution and its core idea of ​​the present application. Ordinary technicians in this field should understand that they can still modify the technical solutions recorded in the aforementioned embodiments, or replace some of the technical features therein with equivalents; and these modifications or replacements do not make the essence of the corresponding technical solution deviate from the scope of the technical solution of the embodiments of the present application.

Claims

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

2. A monoclonal antibody, characterized in that: The monoclonal antibody is prepared by the hybridoma cell line according to claim 1.

3. The monoclonal antibody according to claim 2, characterized in that The antigen bound by the monoclonal antibody is VP7 protein of BRVA virus.

4. The monoclonal antibody according to claim 3, characterized in that The strain types of the BRVA virus include G6, G8 and G10.

5. The monoclonal antibody according to claim 2, characterized in that The amino acid sequence of the antigen epitope to which the monoclonal antibody binds includes the sequence shown in SEQ ID NO:

1.

6. The monoclonal antibody according to claim 4, characterized in that The antibody comprises a light chain as shown in SEQ ID NO:2 and a heavy chain as shown in SEQ ID NO:

3.

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

1.

8. A method for identifying antigenic epitopes of VP7 protein of BRVA virus, characterized in that: The following steps are involved: (1) molecular docking the monoclonal antibody according to any one of claims 2 to 6 with the BRVA VP7 protein to predict the interaction region between the VP7 protein and the monoclonal antibody; (2) Screening and verifying epitope sequences with high antigenicity.

9. Use of the hybridoma cell line according to claim 1, the monoclonal antibody according to any one of claims 2 to 6, or the antigenic epitope peptide according to claim 7 in the preparation of a product for preventing and / or treating, and / or detecting, and / or diagnosing BRVA virus infection.

10. A product for preventing and / or treating, and / or detecting, and / or diagnosing BRVA virus infection, characterized in that: The method comprises the hybridoma cell line according to claim 1, the monoclonal antibody according to any one of claims 2 to 6, or the antigen epitope peptide according to claim 7.

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