Monoclonal antibody of bovine coronavirus s2 protein, antigen epitope and application thereof

Abstract

The present application relates to the technical field of biological medicine, in particular to a monoclonal antibody of bovine coronavirus S2 protein, antigen epitope and application thereof, and provides a monoclonal antibody of bovine coronavirus S2 protein, which is prepared from a hybridoma cell strain with a preservation number of CCTCC NO: C202510.The monoclonal antibody recognizes an antigen epitope with an amino acid sequence as shown in SEQ ID NO: 1.Experiments prove that the monoclonal antibody has neutralization activity, and the targeted epitope is a key neutralization epitope.The present application provides a new strategy and tool for the development of antibody drugs, vaccines and diagnostic detection of bovine coronavirus.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to a monoclonal antibody against bovine coronavirus S2 protein, its antigenic epitope, and its applications. Background Technology

[0002] Bovine coronavirus (BCoV) belongs to the order Nidovirales, family Coronaviridae, and genus Beta coronavirus. BCoV is a major pathogen causing diarrhea in newborn calves, winter dysentery in adult cattle, and respiratory diseases in cattle of all ages. First discovered in the United States in 1972, BCoV subsequently became a global pandemic, causing enormous economic losses to the world's cattle industry. Due to genetic variations in the S protein, BCoV's immune evasion ability has been enhanced, thus broadening its host range. BCoV not only poses a serious threat to the cattle industry but also poses potential risks to the health of various animals and humans through interspecies transmission.

[0003] The S protein of the BCoV virus, located on the viral envelope, is one of the main structural proteins responsible for viral infection and inducing a protective response in the host. It is a crucial target for studying virus-host interactions and vaccine development. The S protein consists of S1 and S2 subunits. The S1 subunit binds to host cell receptors and exhibits significant sequence homology variation, posing considerable challenges to disease control and vaccine development. The S2 subunit, involved in membrane fusion, has a highly conserved sequence, making it a potential target for vaccine research. Currently, there are no neutralizing antibodies or vaccines against the BCoV virus available in my country, highlighting the urgent need for the development of BCoV prevention and control products. Summary of the Invention

[0004] To address the aforementioned issues, this invention aims to provide a monoclonal antibody against bovine coronavirus S2 protein, along with its antigenic epitopes and applications, offering new strategies and tools for the development of antibody drugs and vaccines for bovine coronavirus, as well as for diagnostic testing.

[0005] In one aspect, the present invention provides a monoclonal antibody against bovine coronavirus S2 protein, which is prepared from a hybridoma cell line with accession number CCTCC NO: C202510.

[0006] In some embodiments, the monoclonal antibody is capable of recognizing the bovine coronavirus S2 protein antigenic epitope with an amino acid sequence as shown in SEQ ID NO: 1.

[0007] SEQ ID NO:1: 1075 LSRLDALE 1082 .

[0008] In some embodiments, the monoclonal antibody comprises a light chain variable region with an amino acid sequence as shown in SEQ ID NO: 2 and a heavy chain variable region with an amino acid sequence as shown in SEQ ID NO: 3.

[0009] SEQ ID NO:2:

[0010] IRERVTITCKASQDINNCLIWLQQKPGKSPKTLIYRANRLLDGVPSRFSGSGSGQDYSLTISSLEYEDMGIYYCLQYDEFPLTFGAGSKLELIRADAAPT VSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC.

[0011] SEQ ID NO:3:

[0012] RSRSGRELGSVKISCKTSGYTFTEYTVHWVKQSHGRSLEWIGGINPNNGGTGYNQKFMGKATLTVDKSSSTAYMEIRSLTSEDSAVYYCTSPTTYWGQGTTLTVSSAKTTPPSVYPLAPGCGDTTGSSVTLGCLVKGYFPESVTVTWNSGSLSSSVHTFPALLQSGLYTMSSSVTVPSSTWPSQTVTCSVAHPASNTEAC.

[0013] In one aspect, the present invention provides a hybridoma cell line, the hybridoma cell line having the accession number CCTCC NO: C202510.

[0014] In one aspect, the present invention provides an antigenic epitope peptide of bovine coronavirus S2 protein, the amino acid sequence of which is shown in SEQ ID NO: 1.

[0015] In one aspect, the present invention provides a bovine coronavirus S2 protein antigenic epitope fusion protein, the antigenic epitope fusion protein comprising the antigenic epitope peptide, the linker peptide and ferritin described in section 5, wherein the antigenic epitope peptide is linked to the ferritin via the linker peptide, the amino acid sequence of the linker peptide is GGGGS, and the ferritin is Helicobacter pylori ferritin.

[0016] In one aspect, the present invention provides a nucleic acid molecule that encodes the bovine coronavirus S2 protein antigenic epitope fusion protein, the nucleotide sequence of which is shown in SEQ ID NO: 5.

[0017] In one aspect, the present invention provides a bovine coronavirus vaccine comprising the said antigenic epitope peptide and / or the said antigenic epitope fusion protein.

[0018] In one aspect, the present invention provides a bovine coronavirus detection kit, the bovine coronavirus detection kit comprising the monoclonal antibody and / or the antigenic epitope peptide.

[0019] In one aspect, the present invention provides a method for identifying bovine coronavirus S2 protein antigenic epitopes, comprising the following steps:

[0020] (1) Molecular docking of the monoclonal antibody described above with bovine coronavirus S2 protein to predict the interaction region between S2 protein and the monoclonal antibody;

[0021] (2) Screen for epitope sequences with high antigenicity and verify them.

[0022] In one aspect, the present invention provides the use of the hybridoma cell line in the preparation of products for the prevention and / or treatment and / or detection and / or diagnosis of bovine coronavirus infection.

[0023] In one aspect, the present invention provides the use of the monoclonal antibody in the preparation of products for detecting and / or diagnosing and / or treating bovine coronavirus infection.

[0024] In one aspect, the present invention provides the use of the said antigenic epitope peptide in the preparation of products for the prevention of bovine coronavirus infection.

[0025] In this invention, the above-mentioned products include, but are not limited to, bovine coronavirus detection kits and bovine coronavirus vaccines.

[0026] In this invention, the term "monoclonal antibody" refers to an antibody derived from a substantially homologous group of antibodies, i.e., the individual antibodies constituting the group are identical and / or bind to the same epitopes, and such variants are typically present in trace amounts, except for possible variant antibodies (e.g., containing naturally occurring mutations or generated during the production of monoclonal antibody articles). Unlike polyclonal antibody articles, which typically comprise different antibodies targeting different determinants (epitopes), each monoclonal antibody in a monoclonal antibody article targets a single determinant on an antigen. Therefore, the modifier "monoclonal" indicates that the antibody is derived from a substantially homologous group of antibodies and should not be construed as requiring the antibody to be produced by any particular method. For example, monoclonal antibodies to be used according to the 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 human immunoglobulin loci, such methods and other exemplary methods for preparing monoclonal antibodies are described herein.

[0027] In this invention, the term "hybridoma cell" refers to a cell formed by fusing myeloma cells and B lymphocytes during the preparation of monoclonal antibodies, typically prepared through tumor cell culture.

[0028] In this invention, the term "marker" refers to a molecule that, after antibody binding, can indicate the location or amount of the antibody through color, chemical reaction, excitation light, mass spectrometry, etc. Examples include, for instance, alkaline phosphatase, peroxidase, luciferase, luciferin, fluorescent protein, isotopes, etc.

[0029] In this invention, "Enzyme-Linked Immunosorbent Assay (ELISA)" refers to a detection method that utilizes the characteristic that antibody molecules can specifically bind to antigen molecules to bind free impurity proteins and the target protein bound to a solid-phase carrier, and uses special labels for qualitative or quantitative analysis. Its principle is as follows: antigens or antibodies can be physically adsorbed onto the solid-phase surface while maintaining their immunological activity; antigens or antibodies can form enzyme conjugates with enzymes through covalent bonds, while maintaining their respective immunological or enzymatic activities; after the enzyme conjugate binds to the corresponding antigen or antibody, the occurrence of an immune reaction can be determined by the color reaction of the added substrate, and the intensity of the color reaction is directly proportional to the amount of the corresponding antigen or antibody in the sample. Depending on the substance to be detected and the available detection conditions, various types of detection methods can be designed; the double-antibody sandwich method is the most commonly used method for detecting antigens. The process involves adsorbing antiserum containing known antibodies into the wells of a microtiter plate, washing it once, adding the antigen to be tested, and if the two are specific, they will bind. Then, excess antibodies are washed away. An enzyme-linked antibody that specifically reacts with the antigen to be tested is added to form a "sandwich". The substrate of the enzyme is added. If a colored enzymatic digestion product is seen, it indicates the presence of the corresponding antigen.

[0030] In this invention, "plasmid" refers to a DNA molecule other than chromosomes (or nucleoids) in organisms such as bacteria, yeast, and actinomycetes. It exists in the cytoplasm or cell nucleus, has the ability to replicate autonomously, and can maintain a constant copy number in daughter cells and express the genetic information it carries.

[0031] In this invention, the term "vector" refers to a vector that can introduce a polynucleotide sequence (e.g., a foreign gene) into a host cell to transform the host and promote the expression (e.g., transcription and translation) of the introduced sequence. Vectors include plasmids, bacteriophage vectors, viral vectors, etc. Among them, a "viral vector" is a vector modified from a viral genome that introduces a foreign gene into a host cell through viral infection.

[0032] The advantages of this invention compared to existing technologies are as follows:

[0033] (1) This invention successfully obtained a hybridoma cell line that can stably secrete monoclonal antibodies against bovine coronavirus S2 protein, and prepared a monoclonal antibody with neutralizing activity from it. This antibody provides a core raw material for the development of specific detection reagents and antibody drugs for BCoV.

[0034] (2) This invention accurately identifies a novel linear antigenic epitope (SEQ ID NO:1) located on the bovine coronavirus S2 protein. This epitope is highly conserved in circulating strains and is a key target for stimulating broad-spectrum neutralizing antibodies, breaking through the bottleneck of the limited effectiveness of traditional vaccines and antibodies due to viral mutations.

[0035] (3) Based on the monoclonal antibody and antigenic epitope, the present invention can be directly used to develop a highly sensitive bovine coronavirus detection kit and a novel vaccine that can induce a broad-spectrum protective immune response, providing a new strategy and tool for the accurate diagnosis and effective prevention and control of bovine coronavirus. Attached Figure Description

[0036] Figure 1 PCR electrophoresis image of recombinant expression plasmid pET28a-S2 (M: DNA marker; 1: S2 recombinant plasmid);

[0037] Figure 2 For recombinant protein expression analysis (A: IPTG concentration screening, M: protein marker; lanes 1-5: 0.1 mM, 0.3 mM, 0.5 mM, 0.8 mM, 1.0 mM; B: induction time screening, M: protein marker; lanes 1-6: no induction, 2 h, 4 h, 6 h, 8 h, 12 h; C: identification of expression form, M: protein marker; lanes 1-2: soluble; lanes 3-4: inclusion bodies).

[0038] Figure 3 SDS-PAGE analysis of purified bovine coronavirus S2 recombinant protein (M: protein marker; lanes 1-8: all are purified bovine coronavirus S2 protein).

[0039] Figure 4 Western blotting detection of bovine coronavirus S2 recombinant protein (M: protein marker; lane 1: bovine coronavirus S2 recombinant protein; lane 2: negative control);

[0040] Figure 5 Indirect immunofluorescence identification images of three hybridoma cell lines (1B8, 4B12 and 5E9) (MOCK: negative control);

[0041] Figure 6 The molecular model is 1B8_scFv;

[0042] Figure 7 Molecular docking diagram ( Figure 7 In After Effects: the green model at the top is the bovine coronavirus S2 model; the blue model at the bottom is 1B8_scFv; red represents the docking and interaction region.

[0043] Figure 8 This is a diagram illustrating the conservation of antigenic epitopes.

[0044] Figure 9 The pET28a-5-8aa-Fe plasmid map;

[0045] Figure 10 SDS-PAGE expression map of 5-8aa-Fe recombinant protein (M: protein marker; lane 1: negative control; lane 2: 5-8aa-Fe recombinant protein).

[0046] Figure 11 Western blot diagram for 5-8aa-Fe identification (M: protein marker; lane 1: 5-8aa-Fe recombinant protein; lane 2: negative control). Detailed Implementation

[0047] The technical solution of the present invention will be clearly and completely described below. Of course, the described embodiments are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention. It should be noted that any processes not specifically described in detail below are those that those skilled in the art can implement or understand by referring to the prior art. Reagents or instruments used without specifying the manufacturer are considered to be conventional products that can be purchased commercially.

[0048] The reagents and materials used in the examples are as follows:

[0049] HCT-8 and SP2 / 0 cells were preserved by the Animal Medicine Laboratory of Southwest University for Nationalities; bovine coronavirus strains (XHD7, GenBank accession number: OR621174.1; XHD2, GenBank accession number: OR596700.1; HXD7, GenBank accession number: OR612027.1; XHD3, GenBank accession number: OR621177.1) were preserved by the Animal Medicine Laboratory of Southwest University for Nationalities, and bovine coronavirus S2 recombinant protein 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. Plasmid miniprep kits were purchased from OMEGA Biotechnology Co., Ltd.; ultrasensitive ECL chemiluminescent substrates were 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 Bio-Sens Biotechnology Co., Ltd.; FITC-labeled goat anti-mouse IgG and BCA protein concentration assay kits were purchased from Boster Biological Engineering Co., Ltd.; His Cap 6FF nickel ion purification column was purchased from Changzhou Tiandi Renhe Biotechnology Co., Ltd.; and Montanide ISA 206 adjuvant was purchased from SEPPIC.

[0050] Hybridoma cell line 1B8 was deposited on October 28, 2025, at the China Center for Type Culture Collection (CCTCC), Wuhan University, Wuhan, China, with accession number CCTCC NO: C202510.

[0051] Example 1: Preparation of recombinant bovine coronavirus S2 protein

[0052] 1 Experimental Methods

[0053] 1.1 Construction and Identification of Bovine Coronavirus S2 Gene Expression System

[0054] Based on the codon preference of E. coli, the sequence of the domestically prevalent bovine coronavirus S2 strain (GenBank accession number: NP_150077.1) was optimized. An Nco I restriction enzyme site was added to the 5' end, and an Xho I restriction enzyme site was added to the 3' end. The addition of restriction enzyme sites on both sides is mainly for the accurate localization of restriction endonucleases during vector construction. The sequence was sent to Sangon Biotech (Shanghai) Co., Ltd. for synthesis. The synthesized sequence was ligated with the pET-28a(+) vector that had been double-digested with Nco I and Xho I. After ligation, the recombinant plasmid was transformed into E. coli TOP10 competent cells. Single colonies were picked, and after identification by colony PCR, the plasmids of positive clones were sent to Sangon Biotech (Shanghai) Co., Ltd. for sequencing.

[0055] Recombinant S2 amino acid sequence (SEQ ID NO:4):

[0056] SITTGYRFTNFEPFTVNSVNDSLEPVGGLYEIQIPSEFTIGNMEEFIQTSSPKVTIDCSAFVCGDYAACKSQLVEYGSFCDNINAILTEVNELLDTTQLQVANSLMNGVTLSTKLKDGVNFNVDDINFSPVLGCLGSDCNKVSSRSAI EDLLFSKVKLADVGFVEAYNNCTGGAEIRDLICVQSYNGIKVLPPLLSENQISGYTLAATSASLFPPWSAAAGVPFYLNVQYRINGIGVTMDVLSQNQKLIANAFNNALGAIQEGFDATNSALVKIQAVVNANAEALNNLLQQLSNRFG AISSSLQEILSRLDALEAQAQIDRLINGRLTALNAYVSQQLSDSTLVKFSAAQAMEKVNECVKSQSSRINFCGNGNHIISLVQNAPYGLYFIHFSYVPTKYVTAKVSPGLCIAGDRGIAPKSGYFVYVNNTWMFTGSGYYYPEPITGNN VVVMSTCAVNYTKAPDVMLNISTPNLPDFKEELDQWFKNQTSVAPDLSLDYINVTFLDLQDEMNRLQEAIKVLNQSYINLKDIGTYEYYVKWPWYVWLLIGFAGVAMLVLLFFICCCTGCGTSCFKKCGGCCDDYTGHQELVIKTSHED

[0057] 1.2 Induced expression of recombinant bovine coronavirus S2 protein

[0058] To induce the expression of bovine coronavirus S2 recombinant protein, engineered bacteria were induced at 20°C for different durations (2, 4, 6, 8, 12 h) with different concentrations of IPTG (0.1, 0.3, 0.5, 0.8, 1.0 mM) to determine the optimal induction conditions. Following induction under these optimal conditions, the bacterial culture was collected and centrifuged at 12000 r / min for 15 min at 4°C to collect the bacterial pellet. The pellet was resuspended in PBS and then sonicated in an ice bath (400 W, 2 s, 3 s interval, total 15 min). After sonication, the pellet was centrifuged again, and the supernatant and pellet were collected separately. The pellet was dissolved in 8 M urea solution. Finally, the supernatant and pellet fractions were analyzed by SDS-PAGE, and Coomassie brilliant blue staining and destaining were performed to identify the soluble expression of the recombinant protein.

[0059] 1.3 Purification of recombinant bovine coronavirus S2 protein

[0060] Crude protein was collected after induction, resuspended in PBS buffer, and dissolved thoroughly using an ultrasonicator. The precipitate was collected by centrifugation, dissolved in binding buffer, and then dissolved again by ultrasonication. The supernatant was collected by centrifugation. The crude protein was purified using a 5 mL HisCap 6FF nickel ion purification column. First, the nickel ion purification column was equilibrated: 5 mL of Ni-NTA was used to wash and equilibrate the column with 25 mL of binding buffer. Then, the crude protein was added to the nickel ion purification column, and the effluent was collected as the loading effluent. The nickel ion purification column was equilibrated again. Impurities were washed: the nickel ion purification column was washed with 25 mL of washing buffer, and the effluent was collected as the washing effluent. The target protein was eluted with 25 mL of elution buffer, and the effluent was collected. All collected effluents were analyzed by SDS-PAGE. Recombinant protein fractions showing high purity and a single target band after SDS-PAGE were dialyzed using refolding buffer, with the dialysate changed every 6 hours for a total of 5 dialyzes. After dialysis, the protein was concentrated to half its original volume using PEG20000 embedding, filtered through a 0.45 μm filter membrane, and aliquoted into 1.5 mL tubes (1 mL per tube). The tubes were then stored at -80 °C for later use. The concentration of recombinant bovine coronavirus S2 protein was determined using the BCA protein assay kit according to the kit's instructions.

[0061] 1.4 Western Blotting Identification of Recombinant Bovine Coronavirus S2 Protein

[0062] Recombinant bovine coronavirus S2 protein was subjected to SDS-PAGE electrophoresis, then transferred to a nitrocellulose membrane and incubated with 5% skim milk powder at 37°C on a shaker for 2 h. After washing three times with TBST for 15 min each time, primary antibody (1:1000 dilution of rabbit anti-bovine coronavirus S2 positive serum) was added and incubated overnight. After washing three times with TBST for 15 min each time, secondary antibody (1:5000 dilution of HRP-labeled goat anti-rabbit IgG) was added and incubated at 37°C on a shaker for 2 h. ECL was added for color development, and images were taken using a multifunctional stain-free Western blot system. The reactivity of the recombinant bovine coronavirus S2 protein was identified.

[0063] 2 Results Analysis

[0064] 2.1 Construction and Identification of Bovine Coronavirus S2 Gene Expression System

[0065] like Figure 1 As shown, the recombinant expression plasmid pET28a-S2 showed a specific band of approximately 1719 bp after PCR identification. The band size was consistent with the target fragment, indicating that the S2 gene had been successfully ligated into the expression vector.

[0066] 2.2 Expression of recombinant bovine coronavirus S2 protein

[0067] like Figure 2 As shown, after induction with different IPTG concentrations and induction times, the recombinant bacteria exhibited a target protein band at approximately 64 kDa. The target protein expression level was highest when induced at 20℃ with 0.5 mM IPTG for 12 h. Expression form identification results showed that the recombinant S2 protein was mainly expressed in the form of inclusion bodies.

[0068] 2.3 Purification and Concentration of Recombinant Bovine Coronavirus S2 Protein

[0069] like Figure 3 As shown, the recombinant bovine coronavirus S2 protein obtained by induced expression, after purification by nickel ion affinity chromatography, showed a clear target protein band at approximately 64 kDa with few contaminating protein bands, indicating that the recombinant bovine coronavirus S2 protein was effectively purified with good purification results. The concentration of the purified recombinant bovine coronavirus S2 protein was measured to be 0.8 mg / mL using a BCA protein concentration assay kit, making it suitable for subsequent animal immunization and monoclonal antibody preparation experiments.

[0070] 2.4 Reactivity Detection of Bovine Coronavirus S2 Recombinant Protein

[0071] like Figure 4As shown, after Western blotting, the purified bovine coronavirus S2 recombinant protein showed a specific reaction band at approximately 64 kDa, while no corresponding band was observed in the negative control. This indicates that the recombinant protein can be specifically recognized by rabbit anti-bovine coronavirus S2 positive serum and has reactivity.

[0072] Example 2: Preparation of neutralizing monoclonal antibodies against bovine coronavirus S2 protein

[0073] 1 Experimental Methods

[0074] 1.1 Preparation of Monoclonal Antibodies

[0075] After subcutaneously immunizing BALB / c mice with bovine coronavirus S2 recombinant antigen, mouse spleen cells were fused with SP2 / 0 cells, and hybridoma subcloning was performed three times. Positive clones were then screened using indirect ELISA, with OD... 450 nm A value greater than 3.0 is considered positive.

[0076] 1.2 Identification of Monoclonal Antibodies

[0077] Monoclonal antibodies were identified using indirect immunofluorescence. When HCT-8 cells reached over 90% confluence in 6-well plates, they were inoculated with bovine coronavirus, with negative control wells included. Acetone was added for fixation, and BSA was added to block cell surface receptors. Subsequently, culture supernatants or purified monoclonal antibodies from hybridoma cell lines 1B8, 4B12, and 5E9 were added as primary antibodies, followed by incubation and washing. Then, FITC-labeled goat anti-mouse IgG was added as a secondary antibody, followed by incubation, washing, and staining with DAPI. Finally, fluorescence signals were observed using an inverted fluorescence microscope.

[0078] 1.3 Determination of neutralizing activity of monoclonal antibodies

[0079] The neutralizing activity of monoclonal antibodies against different bovine coronaviruses (XHD7, XHD2, HXD7, and XHD3 strains) was determined using a micro-neutralization assay, and the neutralizing titer of the monoclonal antibodies was calculated. The neutralizing antibody titers of the three mAbs were determined using the micro-neutralization assay, and the procedure is as follows:

[0080] (1) mAb prepared by continuous 2-fold dilution with cell maintenance medium.

[0081] (2) Different dilutions of mAb were mixed with bovine coronavirus solution (200 TCID50). 50 Mix equal volumes (0.1 mL) and incubate at 37°C for 1 h.

[0082] (3) Take 0.2 mL of the mixture and add it to each of the 96-well cell culture plates, with 4 replicates for each dilution. Also, set up 4 wells of normal cells as a negative control and 4 wells of virus-positive control;

[0083] (4) Place the 96-well cell culture plate at 37°C for incubation, and observe and record the cell pathogenesis daily.

[0084] (5) Determine the serum dilution at which 50% of cells do not show cytopathic effects, and calculate the 50% antibody neutralizing titer using the Reed-Muench method.

[0085] 2 Results Analysis

[0086] 2.1 Establishment of hybridoma cell lines

[0087] After screening hybridoma cell lines by ELISA, three hybridoma cell lines that stably secrete antibodies were obtained. Their cell numbers are 1B8, 4B12, and 5E9, respectively. The ELISA screening results are shown in Table 1.

[0088] Table 1. ELISA screening results of hybridoma cell lines

[0089]

[0090] Note: "+" indicates that the hybridoma well / clone is positive by indirect ELISA; "-" indicates that the hybridoma well / clone is negative by ELISA.

[0091] 2.2 Indirect immunofluorescence identification

[0092] The results are as follows Figure 5 As shown, the antibodies secreted by the three hybridoma cell lines (1B8, 4B12, and 5E9) were all reactive to bovine coronavirus strains and exhibited specific fluorescence. The negative control, however, did not show fluorescence, thus validating the negative control designation.

[0093] 2.3 Determination of neutralizing activity of monoclonal antibodies

[0094] Neutralization assays showed that the monoclonal antibodies secreted by the three hybridoma cell lines were neutralizing against different bovine coronavirus strains. The 1B8 monoclonal antibody exhibited the highest neutralizing titer; therefore, strain 1B8 was selected for epitope identification and application development in subsequent studies.

[0095] Table 2. Neutralizing titers of monoclonal antibodies against different strains of bovine coronavirus.

[0096]

[0097] Example 3: Screening of bovine coronavirus S2 epitopes

[0098] 1 Experimental Methods

[0099] 1.1 Extraction, amplification, and construction of single-chain antibody (scFv) molecular models from antibody genes

[0100] Total mRNA was extracted from hybridoma cells 1B8, which produced monoclonal antibodies with high broad-spectrum neutralizing activity. The mRNA was reverse transcribed into cDNA using a reverse transcription kit. Specific primers were designed to amplify the genes encoding the variable regions (VL and VH) of the light and heavy chains of the monoclonal antibodies. Primer sequences are shown in Tables 3 and 4.

[0101] The light and heavy chain amino acid sequences were translated using software, and the obtained antibody gene sequence was compared with mouse immunoglobulin V in the GenBank database. H and V L After BLAST alignment of the gene sequence to confirm its correctness, the protein molecular model was simulated using the AlphaFold3 server (https: / / alphafoldserver.com) for subsequent analysis.

[0102] Table 3 Primers for the variable region of mouse antibody heavy chain

[0103]

[0104] Table 4 Primers for the variable region of mouse antibody light chain

[0105]

[0106] 1.2 Molecular docking to predict antigenic epitopes

[0107] Molecular docking was performed between 1B8 and the bovine coronavirus S2 protein molecule (GenBank accession number: MN928491.1) using the AlphaFold3 server to predict the interaction region between the S2 protein and the monoclonal antibody and identify possible epitope sequences. The epitope with the highest frequency of occurrence during molecular docking was selected for validation.

[0108] 1.3 Conservation Analysis of Antigenic Epitopes

[0109] Bioinformatics software was used to perform in-depth comparative analysis of the identified antigenic epitope sequence. The amino acid sequence of this antigenic epitope was compared with all complete bovine coronavirus S2 protein gene sequences in the GenBank database to analyze the conservation of this antigenic epitope.

[0110] 2 Results Analysis

[0111] 2.1 Extraction, amplification, and construction of single-chain antibody (scFv) molecular models from antibody genes

[0112] The light chain V region gene (VL) and heavy chain V region gene (VH) were successfully amplified, yielding 1B8 mAb VL and VH genes. These genes were translated into amino acid sequences (SEQ ID NO:2 and SEQ ID NO:3, respectively), as shown in Table 5. A 1B8 mAb molecular model was constructed by tandemly connecting VL and VH. Figure 6 ).

[0113] Table 5. Amino acid sequences of VL / VH

[0114]

[0115] 2.2 Molecular docking prediction of epitopes

[0116] 1B8 mAb was molecularly docked with bovine coronavirus S2 protein molecules using the AlphaFold3 server protein docking program. Figure 7 Analysis revealed that the epitope sequence numbered E had the highest frequency (Table 6). This epitope was named 5-8aa, with the amino acid sequence LSRLDALE (SEQ ID NO:1), corresponding to positions 1075-1082 of the bovine coronavirus S2 protein.

[0117] Table 6

[0118]

[0119] 2.3 Conservatism Analysis of Epitopes

[0120] Antigenic epitopes identified in this invention 1075 LSRLDALE 1082 The antigenic epitope was found in all 478 S2 gene sequences in GenBank. The amino acid sequences of 470 S2 genes were 100% identical, with only 8 S2 genes showing a single amino acid mutation, resulting in a conservation rate of 98.32%. This indicates that the antigenic epitope is highly conserved. Figure 8 ).

[0121] Example 4: Identification of predictive epitope neutralization activity

[0122] 1 Experimental Methods

[0123] 1.1 Expression and purification of bovine coronavirus epitope tandem ferritin

[0124] 1.1.1 Construction of recombinant plasmids

[0125] tabletop 1075 LSRLDALE 1082The nucleotide sequence was optimized by linking a single copy of the plasmid to Helicobacter pylori ferritin via the GGGGS amino acid combination linker. EcoR I (gaattc) and Xho I (ctcgag) restriction enzyme sites were inserted at both ends, and the plasmid was then ligated into the pET28a(+) vector to obtain the recombinant plasmid, named pET28a-5-8aa-Fe.

[0126] 5-8aa-Fe optimized nucleotide sequence (SEQ ID NO:5): CTGAGCCGCCTGGATGCGCTGGAAGGCGGCGGCGGCAGCATGCTGAGCAAAGATATTATTAAACTGCTGAACGAACAGGTGAACAAAGAGATGCAGAGCAGCAACCTGTATATGAGCATGAGCAGCTGGT GCTATACCCATAGCCTGGATGGTGCCGGCCTGTTCCTGTTCGATCATGCGGCGGAAGAATATGAACATGCGAAAAAACTGATTATCTTTCTGAATGAAAACAACGTTCCGGTGCAGCTGACCAGCATTAGCGCCCCG GAACATAAATTCGAAGGCCTGACCCAGATTTTTCAGAAGGCGTATGAACATGAACAGCATATTTCAGAAAGCATTAACAACATTGTGGATCATGCGATTAAAAGCAAAGATCACGCCACCTTTAACTTTCTGCAGT GGTATGTGGCGGAACAGCACGAAGAAGAAGTACTGTTCAAAGATATTCTGGATAAAATTGAACTGATTGGCAACGAAAACCATGGCCTGTATCTGGCGGATCAGTACGTTAAAGGCATTGCGAAAAGCCGCAAAAGC

[0127] 1.1.2 Transformation

[0128] Take BL21(DE3) competent E. coli cells stored at -80℃ and slowly thaw them on ice. After thawing, add 2 μL of pET28a-5-8aa-Fe recombinant plasmid at a concentration of 100 ng / μL, mix well, and incubate on ice for 30 min. Then, place the cells in a 42℃ water bath for 60 s and immediately transfer them to ice for 2 min. Add 1 mL of LB liquid medium to an EP tube and place the competent cells in a 37℃ benchtop shaker at 200 rpm for 1 h. Centrifuge at 5000 rpm for 5 min, discard a portion of the supernatant, and keep 100 mL. Spread the supernatant on an LB plate (containing 50 μg / mL Kanamycin) and place it in a 37℃ benchtop incubator. Incubate upright for 30 min and then invert overnight.

[0129] 1.1.3 Screening for positive clones

[0130] Single colonies from LB agar plates were transferred to 5 mL of LB liquid and incubated overnight at 37°C on a benchtop shaker at 2000 rpm. 2 μL of the culture was used as a template for PCR amplification using universal primers for the T7 promoter, followed by gel electrophoresis. The bands were observed under a gel imaging system. Single colonies matching the target band were considered positive clones. The bacterial culture was sent to the Chengdu branch of Beijing Qingke Biotechnology Co., Ltd. for sequencing comparison; the sequencing results were consistent with the synthesis.

[0131] 1.1.4 Induced Expression of Recombinant Proteins

[0132] Prepare 100 mL of liquid LB medium and add 1 mL of *E. coli* BL21(DE3) expressing the pET28a-5-8aa-Fe expression vector at a ratio of 100:1. Incubate at 37°C on a benchtop shaker at 200 rpm. When OD... 450When the nm value reaches 0.6, take 10 15 mL EP tubes and dispense 10 mL of bacterial solution into each tube. Ten 15 mL tubes were divided into two groups (5 tubes per group). One group underwent low-temperature induction at 16℃ for 18 h. Different concentration gradients (0.1 mM, 0.3 mM, 0.5 mM, 0.8 mM, and 1 mM) of IPTG were added to each of the five tubes for induction. 2 mL of bacterial culture was collected every 6 h. After centrifugation at 5000 rpm for 5 min, the supernatant was discarded, and the bacterial sludge was resuspended in 500 μL of PBS buffer. 20 μL of the resuspended bacterial cells was then added, followed by the addition of 5 μL of SDS loading buffer at a 4:1 ratio, and the mixture was boiled for 6–8 min to prepare SDS-PAGE samples. The other group underwent incubation at 37℃ for 6 h. Similarly, different concentration gradients (0.1 mM, 0.3 mM, 0.5 mM, 0.8 mM, and 1 mM) of IPTG were added to each of the five tubes for induction. 2 mL of bacterial culture was collected every 6 h. After processing mL of bacterial culture, SDS-PAGE samples were prepared according to the above steps. SDS-PAGE was performed on a 12% separating gel, followed by Coomassie Brilliant Blue staining for 10 min, destaining five times for 5 min each time, and observation using a gel imaging system. The target protein was located at the protein marker 25 kDa, consistent with the expected size (25 kDa). The successfully expressed recombinant protein was named 5-8aa-Fe. ImageJ software analysis of the protein gel determined the optimal induction conditions based on grayscale values: IPTG concentration 0.1 mM, induction at 16℃ for 14 h.

[0133] 1.1.5 Validation of the expression form of recombinant protein

[0134] The induced bacterial cells were lysed twice. If the recombinant protein was present in the supernatant after the first lysis, it was considered to be expressed in a soluble form; if it was present in the supernatant after the second lysis, it was considered to be expressed in an inclusion body form. Using optimized recombinant protein expression conditions, 50 mL of bacterial culture was induced for expression. After centrifugation at 8000 rpm for 10 min, the supernatant was discarded, and the cells were resuspended in 15 mL of PBS. The cells were then lysed using an ultrasonic sonicator with the following program: power 400 W, time 10 min, ultrasonic on for 5 s, stop for 3 s. After lysis, the cells were centrifuged at 12000 rpm for 15 min, and the supernatant protein was collected for identification of the soluble expression form. The precipitate was resuspended in an equal volume (15 mL) of binding buffer and centrifuged at 12000 rpm for 15 min. The supernatant protein was collected for identification of the inclusion body expression form. The expression of 5-8aa-Fe induced by 0.1 mM IPTG at 16℃ was confirmed to be soluble.

[0135] 1.1.6 Purification of 5-8aa-Fe protein

[0136] Purification was performed using a 5 mL HisCap 6FF nickel ion purification column according to the instructions for nickel agarose affinity chromatography. The specific steps are as follows:

[0137] (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;

[0138] (2) After inducing large amounts of recombinant protein expression according to the conditions in 1.1.4, centrifuge at 12000 rpm for 15 min, collect bacterial sludge, resuspend in PBS at a ratio of 10:3, break up and centrifuge again to collect the supernatant;

[0139] (3) Rinse the HisCap6FF nickel ion purification column with 10 column volumes of pure water;

[0140] (4) Equilibrate the HisCap 6FF nickel ion purification column with 5 column volumes of Binding Buffer;

[0141] (5) After centrifugation in (3), the supernatant is filtered through a 0.45 μm pore size filter and then loaded onto a column;

[0142] (6) Equilibrate the HisCap 6FF nickel ion purification column with 5 column volumes of Binding Buffer;

[0143] (7) Wash the HisCap6FF nickel ion purification column with 10 column volumes of Washing Buffer to remove impurities;

[0144] (8) Elute the target protein in the HisCap6FF column with 5 column volumes of Elution Buffer to purify it by nickel ion removal;

[0145] (9) Collect the protein in the eluent and verify its purity using SDS-PAGE with a 12% separating gel.

[0146] 1.2 Activity analysis of 5-8aa-Fe protein

[0147] Using 5% skim milk as the blocking solution, the mixture was incubated at 37°C on a benchtop constant temperature shaker at 40 rpm for 2 hours with horizontal shaking, followed by washing three times with TBST (once every 5 min). Using rabbit-derived Anti-BCoV S2 protein polyclonal antibody and 1B8 mAb as primary antibodies, the mixture was incubated overnight at 4°C with slow horizontal shaking, followed by washing three times with TBST (once every 5 min). Using HRP-labeled goat anti-rabbit IgG and goat anti-mouse IgG as secondary antibodies, the mixture was incubated at 37°C with horizontal shaking for 2 hours, followed by washing three times with TBST (once every 5 min). Development was performed using a high-sensitivity ECL chemiluminescent substrate.

[0148] 1.3 Immunogenicity evaluation of recombinant proteins

[0149] (1) Preparation of 5-8aa-Fe vaccine

[0150] Take the purified 5-8aa-Fe from 1.1 and mix and emulsify it with the animal immune adjuvant Montanide ISA 206 in a 1:1 ratio. The emulsification is complete when the mixture of recombinant protein and adjuvant does not separate into layers and does not spread rapidly after adding water.

[0151] (2) Animal grouping and immunization

[0152] The prepared 5-8aa-Fe vaccine was administered subcutaneously to New Zealand White rabbits at multiple sites at a dose of 200 μg / rabbit as the vaccine group, and immunized with Montanide ISA 206 adjuvant as the control group (2 rabbits per group, designated A and B respectively). The animals were immunized three times, with an interval of 14 days between immunizations. Serum from the immunized rabbits was collected every 7 days during the experiment.

[0153] (3) Neutralization test to detect neutralizing antibody levels in rabbit serum

[0154] The collected serum from immunized rabbits was used to detect bovine coronavirus neutralizing antibodies. The specific procedure was as follows: HCT-8 cells confluent in cell culture flasks were passaged into 96-well plates and incubated statically at 37°C for 24–48 h until a monolayer of cells was formed. Rabbit serum was then inactivated at 56°C for 30 min and serially diluted 2-fold with serum-free and antibiotic-free DMEM medium. The diluted serum was then mixed with 200 TCID45 solutions. 50 The mixture of bovine coronavirus was incubated at 37°C for 1 h. The 96-well plate containing HCT-8 cells was removed, the culture medium was discarded, and the plate was washed twice with Hanks. The incubated mixed culture medium was added to the 96-well plate at 100 μL per well and incubated at 37°C. The plate was observed every 12 h, and the lesion condition of each well was recorded. The titer of neutralizing antibodies in rabbit serum was calculated according to the Reed-Muech method.

[0155] 2 Results Analysis

[0156] 2.1 Expression and purification of bovine coronavirus epitope tandem ferritin

[0157] like Figure 9 As shown, the 5-8aa gene was fused with the ferritin gene via EcoRI / XhoI double digestion and inserted into the pET28a(+) vector using a GGGGS linker, successfully constructing the pET28a-5-8aa-Fe plasmid. Figure 10 As shown, the 5-8aa-Fe protein was successfully expressed after IPTG induction. The presence of the 5-8aa-Fe protein in the eluent indicates successful purification. Protein concentration was determined using a BCA kit, and the yield was 5 mg per 300 mL of bacterial culture after induction.

[0158] 2.2 Activity analysis of 5-8aa-Fe protein

[0159] like Figure 11 As shown, the 5-8aa-Fe recombinant protein exhibited a specific reaction band, while the negative control did not show a corresponding specific band. These results indicate that the prepared 5-8aa-Fe recombinant protein can be specifically recognized by the corresponding antibody and possesses antigenic reactivity.

[0160] 2.3 Immunogenicity evaluation of recombinant proteins

[0161] As shown in Table 7, low levels of antibodies began to appear 14 days after the first immunization, and peak antibody levels were reached 7 days after the third immunization. The neutralizing titer against bovine coronavirus could reach as high as 1:64.

[0162] Table 7

[0163]

[0164] Note: "—" indicates that neutralizing antibodies were not detected.

[0165] This article uses specific examples to illustrate the principles and implementation methods of this application. The description of the above embodiments is only for the purpose of helping to understand the technical solutions and core ideas of this application. Those skilled in the art should understand that they can still modify the technical solutions described in the foregoing embodiments or make equivalent substitutions for some of the technical features. However, these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of this application.

Claims

1. A monoclonal antibody against bovine coronavirus S2 protein, characterized in that, The monoclonal antibody was prepared from a hybridoma cell line with accession number CCTCC NO: C202510.

2. The monoclonal antibody according to claim 1, characterized in that, The monoclonal antibody can recognize the bovine coronavirus S2 protein antigenic epitope with an amino acid sequence as shown in SEQ ID NO:

1.

3. The monoclonal antibody according to claim 1 or 2, characterized in that, The monoclonal antibody comprises a light chain variable region with an amino acid sequence as shown in SEQ ID NO: 2 and a heavy chain variable region with an amino acid sequence as shown in SEQ ID NO:

3.

4. A hybridoma cell line, characterized in that, The hybridoma cell line has the accession number CCTCC NO: C202510.

5. An antigenic epitope peptide of bovine coronavirus S2 protein, characterized in that, The amino acid sequence of the antigenic epitope peptide is shown in SEQ ID NO:

1.

6. A bovine coronavirus S2 protein epitope fusion protein, characterized in that, The antigenic epitope fusion protein comprises the antigenic epitope peptide, the linker peptide, and ferritin as described in claim 5, wherein the antigenic epitope peptide is linked to the ferritin via the linker peptide, the amino acid sequence of the linker peptide is GGGGS, and the ferritin is Helicobacter pylori ferritin.

7. A nucleic acid molecule, characterized in that, The nucleic acid molecule encodes the bovine coronavirus S2 protein antigenic epitope fusion protein of claim 6, and the nucleotide sequence of the nucleic acid molecule is shown in SEQ ID NO:

5.

8. A bovine coronavirus vaccine, characterized in that, It comprises the antigenic epitope peptide of claim 5 or the antigenic epitope fusion protein of claim 6.

9. A bovine coronavirus detection kit, characterized in that, It comprises the monoclonal antibody according to any one of claims 1 to 3 or the antigenic epitope peptide according to claim 5.

Images