Hybridoma cell strain for resisting swine transmissible gastroenteritis virus S2 protein, monoclonal antibody, antigen epitope peptide recognized by monoclonal antibody and application of antigen epitope peptide

Abstract

The invention discloses a hybridoma cell strain for resisting swine transmissible gastroenteritis virus S2 protein, a monoclonal antibody, an antigen epitope peptide recognized by the monoclonal antibody and application of the monoclonal antibody, and belongs to the technical field of biology. The preservation number of the hybridoma cell strain is CCTCC (China Center For Type Culture Collection) NO: C2025108. The monoclonal antibody 12G8 is obtained by screening a TGEV S2 protein immune mouse, and the heavy chain subtype of the monoclonal antibody 12G8 is identified as IgG2b, and the light chain subtype of the monoclonal antibody 12G8 is identified as Kappa. The monoclonal antibody can specifically recognize TGEV, the amino acid sequence of the recognized minimum B cell epitope peptide is 1109-QGQALS-1114, and the epitope is located in the HR1 structural domain of TGEV S2 protein, is of an alpha helical structure and is highly conserved in different TGEV strains. The monoclonal antibody can be used for developing a diagnostic tool and a treatment strategy aiming at TGEV infection, and a new technical means is provided for clinical detection of the swine transmissible gastroenteritis virus.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and particularly to a hybridoma cell line against the S2 protein of transmissible gastroenteritis virus of pigs, a monoclonal antibody, an antigenic epitope peptide recognized thereby, and applications thereof. Background Art

[0002] Transmissible gastroenteritis virus (TGEV) is an alpha coronavirus belonging to the Coronaviridae family and the Coronavirinae subfamily, and is an important pathogen causing severe gastrointestinal diseases in piglets. After infecting piglets, the virus can cause clinical symptoms such as vomiting, diarrhea, dehydration and high mortality, causing serious economic losses to the pig-raising industry. TGEV was first identified in the United States in 1946 and has since spread widely in Asia and Europe, becoming a major pathogen in the pig-raising industry. In China, the virus was first detected in the 1960s and has since been endemic in pig farms in many provinces across the country. The severity of TGEV infection is often exacerbated by co-infection with other viral pathogens such as rotavirus and porcine epidemic diarrhea virus (PEDV), further increasing economic losses. Despite the progress of vaccines and biosecurity measures, TGEV still poses a continuous threat to the health of pigs, especially in areas with insufficient vaccine coverage or imperfect biosecurity measures.

[0003] The TGEV genome is approximately 28.5 kb and encodes multiple proteins, including ORF1a / b, spike protein (S), membrane protein (M), envelope protein (E), nucleoprotein (N), and non-structural proteins NS6, NS7, and NS7a. Among them, the S protein is a trimeric type I glycoprotein present on the surface of the coronavirus. During infection, host proteases cleave the S protein into two functional subunits: S1 and S2. The S1 subunit contains the receptor binding domain (RBD) and is responsible for the binding of the virus to the porcine aminopeptidase N receptor on the host cell; the S2 subunit mediates the fusion of the virus envelope with the host cell membrane. The S protein is a key mediator of virus entry and is also the main target of diagnostic methods. However, the high mutation rate in the S1 region limits its stability as a reliable diagnostic target. In contrast, the S2 subunit has a conserved structure, especially in the heptapeptide repeat regions (HR1 and HR2) involved in membrane fusion and virus entry. Due to its structural stability and functional importance, the S2 protein provides a promising target for the development of diagnostic tools and therapeutic interventions.

[0004] Monoclonal antibodies (mAbs) are of great value in virus diagnosis and treatment due to their excellent specificity and consistency. By targeting the conserved regions of the S2 protein, mAbs can provide promising solutions to address the limitations of S1 subunit reactions, facilitating the development of more powerful and reliable detection methods. Additionally, monoclonal antibodies, as powerful tools, contribute to determining the molecular mechanisms of TGEV entry and host immune responses. Despite their great potential, the development of S2-specific monoclonal antibodies remains understudied, highlighting a key gap in current research. Summary of the Invention

[0005] The object of the present invention is to provide a hybridoma cell line against the S2 protein of transmissible gastroenteritis virus of swine, a monoclonal antibody, an antigenic epitope peptide recognized thereby, and their applications, so as to solve the problems existing in the above-mentioned prior art. The monoclonal antibody 12G8 prepared by the present invention has a high titer, good specificity and reactivity, can specifically recognize TGEV, and has no cross-reaction with other common swine enteroviruses, and can be applied to laboratory and clinical detection.

[0006] To achieve the above object, the present invention provides the following solutions:

[0007] The present invention provides a hybridoma cell line against the S2 protein of transmissible gastroenteritis virus of swine. The classification and naming of the hybridoma cell line is Hybridoma cell line TGEV-S2-12G8; the preservation number is CCTCC NO: C2025108; the preservation time is April 2, 2025; the preservation unit is China Center for Type Culture Collection, and the preservation address is Wuhan University, Wuhan, China.

[0008] The present invention also provides a monoclonal antibody produced by the above-mentioned hybridoma cell line.

[0009] The present invention also provides a preparation method of the above-mentioned monoclonal antibody, including the step of secreting and producing the monoclonal antibody by using the above-mentioned hybridoma cell line.

[0010] The present invention also provides an antigenic epitope peptide recognized by the above-mentioned monoclonal antibody. The amino acid sequence of the antigenic epitope peptide is 1109 QGQALS 1114 .

[0011] The present invention also provides the application of the above-mentioned hybridoma cell line, the above-mentioned monoclonal antibody or the above-mentioned antigenic epitope peptide in any one of the following:

[0012] (1) Application in the preparation of reagents or kits for detecting the S2 protein of transmissible gastroenteritis virus of swine;

[0013] (2) Use in the preparation of reagents or kits for detecting transmissible gastroenteritis virus of pigs.

[0014] The present invention also provides the use of the above-mentioned hybridoma cell line, or the above-mentioned monoclonal antibody, or the above-mentioned antigenic epitope peptide in the preparation of drugs against transmissible gastroenteritis virus of pigs.

[0015] The present invention also provides the use of the above-mentioned hybridoma cell line, or the above-mentioned monoclonal antibody, or the above-mentioned antigenic epitope peptide in the preparation of drugs against transmissible gastroenteritis.

[0016] The present invention also provides a reagent or kit for detecting transmissible gastroenteritis virus of pigs, comprising the above-mentioned monoclonal antibody.

[0017] The present invention also provides a drug against transmissible gastroenteritis virus of pigs, comprising the above-mentioned monoclonal antibody.

[0018] The present invention discloses the following technical effects:

[0019] The present invention screened a monoclonal antibody 12G8 against the S2 protein of transmissible gastroenteritis virus of pigs. After identification, the heavy chain subtype is IgG2b and the light chain subtype is Kappa. The amino acid sequence of the antigenic epitope peptide recognized by it is 1109-QGQALS-1114. This epitope is located in the HR1 domain of the TGEV S2 protein and presents an α-helical structure. The monoclonal antibody 12G8 prepared by the present invention has a high titer, good specificity and reactivity, can specifically recognize TGEV, has no cross-reaction with other common porcine enteroviruses, and can be used to develop diagnostic tools and treatment strategies for TGEV infection, providing a new technical means for the clinical detection of transmissible gastroenteritis virus of pigs. Description of the Drawings

[0020] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required to be used in the embodiments. Obviously, the following described drawings are only some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on these drawings.

[0021] Figure 1 It is the amplification result diagram of the TGEV S2 gene fragment in the present invention and the identification result diagram of the bacterial liquid after the gene fragment is ligated to pET-32a; A: The amplification result of the TGEV S2 gene fragment, M is DNA Maker, 1 is the negative control, and 2 is the amplified fragment of the TGEV S2 gene fragment; B: The identification result of the bacterial liquid after the gene fragment is ligated to pET-32a, M is Maker, 1 is the negative control, 2 is the empty vector bacterial liquid, and 3 is the bacterial liquid after the gene fragment is ligated to pET-32a;

[0022] Figure 2 Expression diagram of His-tagged TGEV S2 recombinant protein in the present invention; A shows the detection result of the sample before purification of His-tagged TGEV S2 recombinant protein, and B shows the detection result of the sample after purification of His-tagged TGEV S2 recombinant protein;

[0023] Figure 3 Purification result diagram of His-tagged TGEV S2 recombinant protein in the present invention; M is the protein molecular weight marker, 1 is the empty vector control, 2 is the S2 protein sample after induced expression, 3 is the purified sample of S2 protein inclusion body, and 4 is the concentrated sample of S2 protein;

[0024] Figure 4 ELISA identification result diagram of monoclonal antibody 12G8 in the present invention;

[0025] Figure 5 Specific identification (A) of monoclonal antibody 12G8 and Western blot diagram (B) of cross-reactivity analysis in the present invention;

[0026] Figure 6 IFA diagram of specific identification and cross-reactivity analysis of monoclonal antibody 12G8 in the present invention;

[0027] Figure 7 Subtype identification diagram of monoclonal antibody 12G8 in the present invention;

[0028] Figure 8 Potency identification diagram of monoclonal antibody 12G8 in the present invention; A: Indirect ELISA was used to detect the potency of purified monoclonal antibody 12G8 with TGEV S2 protein as the coating antigen, B: Indirect ELISA was used to detect the potency of purified monoclonal antibody 12G8 with TGEV as the coating antigen;

[0029] Figure 9SDS-PAGE diagrams for the identification of antigenic epitopes recognized by the monoclonal antibodies in the present invention; A: SDS-PAGE diagrams of domains S2-1, S2-2, S2-3, S2-4, S2-5 of TGEV S2 protein and control pET-32a; B: SDS-PAGE diagrams of domains S2-4-1, S2-4-2, S2-4-3, S2-4-4 of TGEV S2 protein and control pET-32a; C: SDS-PAGE diagrams of domains S2-4-2-1, S2-4-2-2, S2-4-2-3, S2-4-2-4 of TGEV S2 protein and control pET-32a; D: SDS-PAGE diagrams of domains S2-4-2-4-1, S2-4-2-4-2, S2-4-2-4-3, S2-4-2-4-4, S2-4-2-4-5, S2-4-2-4-6, S2-4-2-4-7, S2-4-2-4-8, S2-4-2-4-9, S2-4-2-4-10, S2-4-2-4-11, S2-4-2-4-12, S2-4-2-4-13 of TGEV S2 protein and control pET-32a;

[0030] Figure 10 Diagram for the localization of antigenic epitopes recognized by the monoclonal antibodies in the present invention;

[0031] Figure 11 Model diagram for predicting the spatial structure of antigenic epitopes in the present invention;

[0032] Figure 12 Conservativeness analysis of the antigenic epitope of monoclonal antibody 12G8 in the present invention among different strains of TGEV, porcine enteric coronaviruses, α-coronaviruses, and β / γ / δ-coronaviruses. Detailed implementation manners

[0033] Now, various exemplary implementation manners of the present invention will be described in detail. This detailed description should not be regarded as a limitation of the present invention, but should be understood as a more detailed description of certain aspects, characteristics, and implementation schemes of the present invention.

[0034] It should be understood that the terms described in the present invention are only for describing specific implementation manners and are not used to limit the present invention. Additionally, for the numerical ranges in the present invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any intermediate value within any stated value or stated range, as well as each smaller range between any other stated value or intermediate value within the stated range, is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded from the range.

[0035] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Although this invention only describes preferred methods and materials, any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of this invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials related to the said documents. In case of conflict with any incorporated document, the content of this specification shall prevail.

[0036] Without departing from the scope or spirit of this invention, various improvements and changes can be made to the specific embodiments of the description of this invention, which are obvious to those skilled in the art. Other embodiments obtained from the description of this invention are obvious to those skilled in the art. The description and examples of this invention are merely exemplary.

[0037] Regarding "comprising", "including", "having", "containing", etc. used herein, they are all open-ended terms, meaning including but not limited to.

[0038] Example 1

[0039] 1. Experimental materials

[0040] Viruses: TGEV HN-2012 strain (GenBank accession number: OP434397.1), PDCoV HNZK-02 strain (GenBank accession number: MH708123), PEDV (GenBank accession number: OK584017.1), PSV HNHB-01 strain (GenBank accession number: MN939541), MRV HN2019-01 (GenBank accession number: PV125521), PAstV5 HNPDS-01 strain (GenBank accession number: OQ781001) were isolated and preserved by the Laboratory of Animal Molecular Pathogenology, Henan Agricultural University;

[0041] Plasmids and cells: pET-32a(+) plasmid, ST cells, Vero cells and SP2 / 0 cells were all preserved by the Key Laboratory of Animal Pathogens and Biosafety, Ministry of Education; BALB / c mice were purchased from Henan Experimental Animal Center;

[0042] Mouse monoclonal antibody subtype identification kit was purchased from Wuhan Sanying Biotechnology Co., Ltd.

[0043] 2. Experimental methods

[0044] 2.1 Analyze the conservation of different domains of TGEV and select the antigenic dominant region of the S2 subunit

[0045] The antigenicity of the TGEV S protein (accession number OP434397.1) was analyzed using DNA Star software and AlphaFold3 software, and the predicted three-dimensional structure was visualized with PyMOL software. The SnapGene and ESPript software were used to perform multiple sequence alignment analysis on the S protein sequences of different subtypes of TGEV strains in China and other countries. The results showed that there were a large number of mutations in the RBD region, while the S2 subunit was highly conserved among the variant strains. Based on the results of the conservation analysis, the S2 subunit containing the S1 / S2 cleavage region, fusion peptide, HR1, and HR2 was selected as the antigenic dominant region for monoclonal antibody preparation.

[0046] 2.2 Construction and identification of the pET-32a-TGEV-S2 recombinant vector

[0047] The total RNA of TGEV was extracted using the Trizol method and transcribed into cDNA using a reverse transcription kit. According to the TGEV HN-2012 strain (GenBank accession number OP434397.1) published in GenBank, primers were designed to amplify the S2 domain, and PCR amplification was performed using the cDNA of TGEV as a template.

[0048] PCR amplification primers:

[0049] F: 5’-CTCGAGTGCGGCCGCAAGCTTTTTTACATAGGTTTC-3’ (SEQ ID NO.1);

[0050] R: 5’-GCTGATATCGGATCCGAATTCGATTTGTCAGTGCTA-3’ (SEQ ID NO.2).

[0051] Reaction program: 95°C for 5 min; 95°C for 1 min, 60°C for 30 s, 72°C for 90 s for a total of 30 cycles; 72°C for 10 min.

[0052] Reaction system (total 25 μL): 13 μL of Green Taq Mix, 9 μL of ddH2O, 0.5 μL each of F / R primers, and 2 μL of cDNA.

[0053] The TGEV S2 gene fragment was amplified, and the amplification result of the target gene was verified by agarose gel electrophoresis (the result is as Figure 1In A). The amplified target fragment was purified by gel extraction. The pET-32a(+) vector was digested with endonucleases EcoRI and XhoⅠ into a linear vector. Through homologous recombination ligation, it was transformed into Escherichia coli Rosetta. Single colonies were picked for colony PCR identification. The strains with positive identification results (pET-32a-TGEV-S2) were sent to Shangya Biotechnology Co., Ltd. for sequencing. The strains with correct sequences were reserved for subsequent experiments (the results are as shown in Figure 1 In B).

[0054] 2.3 Expression and purification of recombinant TGEV-S2 protein

[0055] The frozen pET-32a-TGEV-S2 expression bacterial strain was inoculated into liquid LB medium. Isopropyl-β-D-1-thiogalactopyranoside (IPTG) with a final concentration of 0.8 mmol / L was added to induce the expression of TGEV S2 recombinant protein at 16 °C for 20 h. Bacterial cells were collected, broken, and centrifuged to obtain the supernatant and precipitate. The expression of His-tagged fusion protein was analyzed by SDS-PAGE and Western Blot. It was found that the TGEV S2 protein was expressed in the form of inclusion bodies in bacteria (the results are as shown in Figure 2 ). Subsequently, after large-dose induction, the TGEV S2 protein in the form of inclusion bodies was purified. The precipitate was washed three times with inclusion body washing solution in an ice-water bath. The sample was washed and precipitated successively with 2 mol / L urea and 1 mol / L sodium chloride solution. The inclusion body protein was denatured with 8 mol / L urea and 0.5 M dithiothreitol at 4 °C for 2 h. 1 mM oxidized glutathione and 2 mM reduced glutathione were added to the denatured protein solution, and it was renatured in an ice bath environment for 2 h. Finally, the protein solution was dialyzed in 4 M urea for 12 h by gradient dilution method, and the supernatant protein was collected. The purification results are shown in Figure 3 .

[0056] 2.4 Screening of positive hybridoma cell lines

[0057] Three BALB / c mice were immunized with purified recombinant TGEV S2 protein three times at two-week intervals. The first immunization was at a dose of 100 μg per mouse, emulsified with an equal volume of Freund's complete adjuvant and the protein. The second and third immunization doses were 100 μg per mouse, emulsified with an equal volume of Freund's incomplete adjuvant. The immunization route was intraperitoneal injection. Three days before fusion, a booster immunization was performed at a dose of 50 μg per mouse without adjuvant, by intraperitoneal injection. When the serum titer was relatively high, splenocytes from the mouse with the highest serum titer were fused with myeloma cells (SP2 / 0). After culturing with HAT selective medium, indirect ELISA was performed using the recombinant TGEV S2 protein as the coating antigen according to the kit instructions to screen for positive hybridoma cell lines. After subcloning to single cells by the limiting dilution method and then expanding the culture, a monoclonal antibody cell line that stably secretes was finally obtained, named 12G8. Indirect ELISA was performed using the TGEV S2 protein and TGEV as the coating antigens to verify the reactivity of the monoclonal cell line 12G8 (the results are shown in Figure 4 ).

[0058] This hybridoma cell line has been deposited with the China Center for Type Culture Collection. The deposit information is as follows:

[0059] Hybridoma cell line TGEV-S2-12G8, deposit number: CCTCC NO: C2025108, deposit date: April 2, 2025, depository: China Center for Type Culture Collection, deposit address: Wuhan University, Wuhan, China.

[0060] 2.5 Preparation and purification of monoclonal antibody ascites

[0061] Healthy BALB / c mice were selected and 500 μL of liquid paraffin was injected intraperitoneally one week before injecting the cells. The hybridoma cells were injected into the abdominal cavity of the mice. When the abdomen of the mice swelled, ascites was extracted, centrifuged to remove excess adipose tissue, and frozen at -40 °C for storage. The monoclonal antibody in the ascites was purified by the ammonium octanoate-ammonium sulfate precipitation method. The steps are as follows:

[0062] (1) Slowly add 2 volumes of 0.06 M acetate buffer to 1 volume of ascites at a ratio of 1:2, and stir and mix well.

[0063] (2) Slowly add ammonium octanoate while stirring at a ratio of 33 μL of ammonium octanoate per milliliter of ascites, let stand at 4 °C for 2 h, centrifuge at 12000 r / min for 30 min, and take the supernatant.

[0064] (3) Add 1 / 10 volume of 0.01 M PBS, and adjust the pH value to 7.4 with 1 M NaOH. Slowly add saturated sodium sulfate (pH = 7.4) with stirring until the final concentration of ammonium sulfate is 45%. Let it stand at 4°C for 12 - 16 h.

[0065] (4) Centrifuge at 12,000 r / min for 30 min at 4°C, discard the supernatant, and resuspend the precipitate with 0.01 M PBS.

[0066] (5) Add the resuspended solution into a dialysis bag, place it in 0.01 M PBS, and dialyze at 4°C for 12 - 16 h to remove salts. Determine the concentration of the purified antibody, aliquot and store it frozen at -80°C.

[0067] 2.6 Identification of the specificity and cross-reactivity of monoclonal antibodies

[0068] 2.6.1 Identification of the specificity and cross-reactivity of monoclonal antibodies by Western Blot

[0069] Respectively inoculate the TGEV HN-2012 strain, PDCoV HNZK-02 strain, PSV HNHB-01 strain, MRV HN2019-01 strain, PAstV5 HNPDS-01 strain on ST cells and PEDV on Vero cells. The infection dose is MOI = 1. After cytopathic effect, lyse the cells with RIPA lysis buffer at 4°C for 30 min. After electrophoresis and membrane transfer, block with 5% non-fat milk for 2 h. The primary antibody is the monoclonal antibody 12G8 diluted 1:1000, and the secondary antibody is the HRP-labeled goat anti-mouse secondary antibody diluted 1:2000. Use the ECL chemiluminescence color development solution for color development, and take pictures with a GE AI600 imager. The results are as Figure 5 shown. The monoclonal antibody 12G8 prepared by the present invention has good specificity and reactivity, can specifically recognize TGEV, and has no cross-reactivity with other common porcine enteroviruses, and can be applied to laboratory and clinical detection.

[0070] 2.6.2 Identification of the specificity and cross-reactivity of monoclonal antibodies by IFA

[0071] On ST cells, inoculate the TGEV HN-2012 strain, PDCoV HNZK-02 strain, PSV HNHB-01 strain, MRV, PAstV5 HNPDS-01 strain respectively, and inoculate PEDV on Vero cells. The infection dose is MOI = 1. After cytopathic effect, fix with absolute ethanol at 4°C for 8 h. For the TGEV group and the cell control group, use the TGEV monoclonal antibody 12G8 (diluted 1:200) as the primary antibody, and FITC-labeled goat anti-mouse IgG (diluted 1:200) as the secondary antibody; for the positive control group, use the mouse monoclonal antibody corresponding to the strain (diluted 1:200) as the primary antibody, and FITC-labeled goat anti-mouse IgG (diluted 1:200) as the secondary antibody. Incubate the antibody, wash with PBST, then add the mounting medium containing DAPI, and observe and photograph the results with an inverted fluorescence microscope as Figure 6 shown: The monoclonal antibody 12G8 prepared by the present invention has good specificity and reactivity, can specifically recognize TGEV, and has no cross-reaction with other common porcine enteroviruses, and can be applied to laboratory and clinical detection.

[0072] 2.7 Identification of monoclonal antibody subtype and determination of titer

[0073] Identify the subtype of the monoclonal antibody 12G8 according to the steps on the Wuhan Sanying Mouse Monoclonal Antibody Subtype Identification Kit. The steps are as follows:

[0074] (1) Dilute the purified antibody to 50 ng / mL with PBST, and add 50 μL per well to the enzyme-linked immunosorbent assay (ELISA) plate.

[0075] (2) Add 1× goat anti-mouse IgM + IgG-HRP at 50 μL per well to the ELISA plate.

[0076] (3) Incubate at room temperature for 1 h.

[0077] (4) Wash the plate 3 times with 1× PBST.

[0078] (5) After preparing the developing solution according to A liquid:B liquid = 1:100, add 100 μL per well.

[0079] (6) Develop color at room temperature in the dark for 15 min.

[0080] (7) Add 100 μL of stop solution per well, and read the OD 450nm value. The well corresponding to the highest OD value is the corresponding subtype.

[0081] The results are as Figure 7 shown. The heavy chain subtype of the monoclonal antibody 12G8 is IgG2b, and the light chain subtype is Kappa.

[0082] The titer of the purified monoclonal antibody 12G8 was detected by indirect ELISA using the TGEV S2 protein and TGEV as coating antigens, and the results were as follows Figure 8 shown, and the titer of the monoclonal antibody 12G8 was 1:25600.

[0083] 2.8 Identification of the antigenic epitope recognized by the monoclonal antibody

[0084] Using the TGEV S2 recombinant plasmid as a template, PCR amplification was carried out using primers and cloned into the pET-32a vector to express 5 cross-overlapping S2 truncated proteins. Each truncated region contained different domains of the TGEV S2 protein, which were named S2-1, S2-2, S2-3, S2-4, and S2-5 respectively. After identification by SDS-PAGE and Western Blot, as shown in Figure 9 A below, it was found that the monoclonal antibody 12G8 reacted with the S2-4 truncated protein. In the present invention, the S2-4 fragment was further divided into four regions, S2-4-1 to S2-4-4. After identification by SDS-PAGE and Western Blot, as shown in Figure 9 B below, it was found that the 12G8 monoclonal antibody only reacted with the S2-4-2 fragment. Subsequently, the S2-4-2 region was subdivided into five overlapping fragments, each containing 15 amino acids. After identification by SDS-PAGE and Western Blot, as shown in Figure 9 C below, it was confirmed that the 12G8 monoclonal antibody only recognized the S2-4-2-4 and S2-4-2-5 fragments. Finally, the determined fragment was divided into 13 overlapping peptide segments, each containing 10 amino acids, by the single amino acid displacement strategy. SDS-PAGE and Western Blot results, as shown in Figure 9 D below showed that the 12G8 monoclonal antibody specifically recognized the S2-4-2-4-6 to S2-4-2-4-10 truncated peptide segments, confirming that the amino acid sequence 1109-QGQALS-1114 in the TGEV S protein was the recognition epitope of the 12G8 monoclonal antibody.

[0085] To more intuitively and clearly show the antigenic epitope identification process, according to the WB identification results, a schematic diagram of monoclonal antibody identification was drawn (the results are shown in Figure 10 ). The red area is the HR1 domain of the S2 protein, and the blue area is the region recognized by the monoclonal antibody 12G8.

[0086] Table 1 Primer sequences of each truncated gene of the TGEV S2 protein

[0087]

[0088]

[0089]

[0090]

[0091] 2.9 Prediction of the Spatial Structure of Antigen Epitopes

[0092] The AlphaFold3 software (DeepMind, version 3.0) was used to predict the three-dimensional structure of the TGEV S protein (accession number NP_058424.1), and the PyMOL software (version 2.5.0) was used for visualization analysis and editing. Based on the prediction model, the spatial structure localization and analysis of the epitope (aa 1109-QGQALS-1114) recognized by the monoclonal antibody 12G8 were carried out. The results showed that the antigen epitope recognized by the monoclonal antibody 12G8 was located in the α-helix region between two loop structures of the HR1 domain of the TGEV S2 protein (the results are shown in Figure 11 ). To further analyze the conservation of the epitope, the SnapGene software was used to align the S protein sequences of different subtypes of TGEV strains and porcine coronaviruses, and the ggmsa package (Bioconductor, version 1.10.0) of the ESPript (IGBMC) and Rstudio software was used for the visualization of multiple sequence alignments to clearly show the conserved characteristics of the monoclonal antibody 12G8 epitope in TGEV strains and other porcine coronaviruses (the results are shown in Figure 12 ).

[0093] The above-described embodiments are only descriptions of the preferred modes of the present invention and do not limit the scope of the present invention. Without departing from the design spirit of the present invention, various deformations and improvements made by those of ordinary skill in the art to the technical solutions of the present invention shall fall within the protection scope determined by the claims of the present invention.

Claims

1. A hybridoma cell line against the S2 protein of transmissible gastroenteritis virus of swine, characterized in that, The preservation number of the hybridoma cell line is CCTCC NO: C2025108.

2. A monoclonal antibody, characterized in that, It is produced by the hybridoma cell line described in claim 1.

3. A method for preparing a monoclonal antibody as described in claim 2, characterized in that, It includes the step of secreting and producing the monoclonal antibody by using the hybridoma cell line described in claim 1.

4. An antigenic epitope peptide recognized by the monoclonal antibody according to claim 2, characterized in that, The amino acid sequence of the antigenic epitope peptide is 1109 QGQALS 1114 .

5. The application of the hybridoma cell line described in claim 1, or the monoclonal antibody described in claim 2, or the antigenic epitope peptide described in claim 4 in any of the following: (1) The application in the preparation of a reagent or kit for detecting the S2 protein of transmissible gastroenteritis virus of pigs; (2) The application in the preparation of a reagent or kit for detecting transmissible gastroenteritis virus of pigs.

6. The application of the hybridoma cell line described in claim 1, or the monoclonal antibody described in claim 2, or the antigenic epitope peptide described in claim 4 in the preparation of a drug against transmissible gastroenteritis virus of pigs.

7. The application of the hybridoma cell line described in claim 1, or the monoclonal antibody described in claim 2, or the antigenic epitope peptide described in claim 4 in the preparation of a drug against transmissible gastroenteritis of pigs.

8. A reagent or kit for detecting transmissible gastroenteritis virus of swine, characterized in that, It includes the monoclonal antibody described in claim 2.

9. A drug against transmissible gastroenteritis virus of pigs, characterized in that, It includes the monoclonal antibody described in claim 2.

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