Bovine dermoderatosis virus fusion antigen, products and uses thereof

Abstract

The application discloses a bovine papular dermatitis virus fusion antigen and a product and application thereof, and belongs to the field of immunological techniques. The technical problem to be solved is that the immunogenicity of the bovine papular dermatitis virus is low, and the immunogenicity is low. The technical solution is to provide a bovine papular dermatitis virus fusion antigen, wherein the fusion antigen comprises three antigen peptide segments derived from LSDV060 protein and LSDV122 protein of the bovine papular dermatitis virus, and the three antigen peptide segments are fused by head-to-tail splicing.

Description

Technical Field

[0001] This invention belongs to the field of immunology, and relates to bovine nodular dermatitis virus, specifically to bovine nodular dermatitis virus fusion antigen, its products, and applications. Background Technology

[0002] For understanding the technical content of this invention: Bovine nodular dermatitis, also known as bovine nodular rash or bovine skin bump disease, is an acute, subacute, and chronic infectious disease caused by bovine nodular skin disease virus (LSDV). LSDV can infect cattle of all breeds and ages. Clinical manifestations in affected cattle include fever, emaciation, swollen lymph nodes, swollen skin, localized nodules or ulcers. Necropsy reveals nodules of varying sizes on the mucous membranes of the skin, digestive tract, respiratory tract, and urogenital tract. Bovine nodular dermatitis can lead to emaciation, decreased milk production, and skin damage, and in severe cases, death. Monitoring data shows that bovine nodular dermatitis remains widespread in my country; therefore, the National Animal Disease Immunization Technical Guidelines recommend annual vaccination. Vaccination is an efficient and controllable measure for controlling animal diseases and is of great significance for the prevention and control of various animal diseases. Currently, the prevention and control of bovine nodular dermatitis mainly relies on attenuated goat pox virus, attenuated sheep pox virus, and live attenuated bovine nodular dermatitis vaccines. Currently, my country's goatpox attenuated live vaccine GTPV-AV41 has been approved for the prevention and control of bovine nodular dermatovirus (LSDV), but the protective effect of this vaccine against LSDV is still unclear and needs further evaluation. Attenuated live vaccines have some drawbacks, such as (1) potential biosafety risks such as strain reversion and viral recombination; and (2) side effects such as fever and decreased milk production. Therefore, it is very necessary to develop safer and more effective vaccines.

[0003] Relevant patent documents retrieved: This document, published in China (CN117229368A) on December 15, 2023, discloses a recombinant protein of bovine nodular dermatovirus P122, its encoding gene, recombinant vector, recombinant cell line, vaccine, and its applications, belonging to the field of animal vaccines and veterinary biological products technology. This invention utilizes the recombinant protein of bovine nodular dermatovirus P122 to prepare a subunit vaccine for the prevention and treatment of bovine nodular dermatovirus.

[0004] This document, published in China (CN117737088A) on March 22, 2024, discloses the preparation and application of circular RNA and vaccines for bovine nodular dermatitis virus (BND). The circular RNA encodes optimized LSDV122, LSDV141, LSDV117, and LSDV060 proteins of BND. The preferred circular RNA molecules of this invention increase the expression levels of the target proteins in bovine cells. Vaccines prepared using these circular RNA molecules successfully elicited high levels of antibodies and activated high-level cellular immune responses in cattle, providing a foundation for the preparation of RNA vaccines for the prevention and / or treatment of BND.

[0005] The prior art represented by the aforementioned documents has at least the following unresolved technical problems or defects: (1) Low immunogenicity: For example, attenuated sheep pox vaccines only provide partial cross-protection against wild-type bovine nodular dermatitis. In 2006–2007, when RM 65 (Ramyar) sheep pox vaccine was used in Israel to prevent bovine nodular dermatitis, only partial immunoprotective efficacy was observed. Compared to the side effects caused by attenuated bovine nodular dermatitis virus vaccines, sheep pox virus vaccines rarely cause side effects in vaccinated cattle. However, studies have shown that the use of high doses of sheep pox virus Yugoslavian RM65 vaccine may produce typical vaccine side effects, such as systemic skin lesions in cattle.

[0006] References: 1. Brenner J, et al: Appearance of skin lesions in cattle populations vaccinated against lumpy skin disease: statutory challenge. Vaccine 2009, 27(10):1500-1503. 2. Abutarbush SM, Tuppurainen ESM: Serological and clinical evaluation of the Yugoslavian RM65 sheep pox strain vaccine use in cattleagainst lumpy skin disease. Transbound Emerg Dis 2018, 65(6):1657-1663. (2) Low safety, specifically the risk of virulence reversion and the risk of recombinant virus.

[0007] References: 1.Sprygin A, et al. Evidence of recombination of vaccine strains of lumpy skin disease virus with field strains, causing disease. PLoS One. 2020May 13;15(5):e0232584. 2. Sendow I, et al. Molecular characterization of recombinant LSDVisolates from 2022 outbreak in Indonesia through phylogenetic networks and whole-genome SNP-based analysis. BMC Genomics. 2024 Mar 4;25(1):240. Other content that is useful for understanding, searching, and examining this invention: Fusion antigens are novel antigens created by splicing together two or more antigen fragments from different sources using gene fusion technology. Their core function is to elicit a broader or stronger immune response, and they are widely used in vaccine development and immunotherapy. Fusion antigens fuse key epitopes (core regions recognized by immune cells) of different antigens, allowing immune cells to recognize multiple targets simultaneously. For example, fusing viral structural proteins and immunogenic proteins ensures recognition specificity while enhancing immune stimulation. By fusing specific fragments (such as adjuvant sequences or signal peptides), the stability, delivery efficiency, or immunogenicity of antigens can be improved, addressing issues such as weak immune response and easy tolerance to single antigens. Fusion antigens are currently most widely used in infectious disease vaccines and tumor immunotherapy: 1) Vaccine development: This is the primary application area; 2) Infectious disease vaccines: For example, in COVID-19 vaccines, some candidate vaccines fuse the spike protein (S protein) of the SARS-CoV-2 virus with core antigens of other viruses to enhance the scope of immune protection; HPV vaccines also achieve protection against multiple HPV subtypes by fusing L1 proteins of different subtypes. 3) Tumor vaccines: These fuse tumor-specific antigens (TSA) with immune adjuvants (such as cytokine fragments), enabling precise targeting of tumors and active activation of immune cells to enhance anti-tumor immune responses; 4) Immunotherapy drugs: These serve as therapeutic antigens to activate the patient's own immune system to attack diseased cells. For example, in dendritic cell (DC) vaccines, loading fusion antigens allows DC cells to present antigens more efficiently, activating T cells. Summary of the Invention

[0008] The purpose of this invention is to provide: A fusion antigen targeting bovine nodular dermatitis virus, and related technologies, to address the technical problems of poor safety, difficulty in preparation, and low immunogenicity of bovine nodular dermatitis virus immunogenicity, or a combination thereof.

[0009] Terminology Explanation: Unless otherwise defined, all technical terms in this document have the same meanings as commonly understood by one of ordinary skill in the art to which the subject matter of the claims pertains. Unless otherwise stated, all patents, patent inventions, and publications cited in this document are incorporated herein by reference in their entirety. If multiple definitions exist for terms in this document, the definitions in this chapter shall prevail.

[0010] It should be understood that the above brief description and the following detailed description are exemplary and for illustrative purposes only, and do not limit the subject matter of the invention in any way. In this invention, the singular is used in conjunction with the plural unless otherwise specifically stated. It should also be noted that, unless otherwise stated, the use of “or” or “or” means “and / or”. Furthermore, the use of the term “comprising” and other forms such as “including,” “containing,” and “contains” are not limiting.

[0011] The definition of the standard chemical term can be found in the reference "Antibody Technology (Second Edition)," Science Press, 2nd edition, 2018.

[0012] Unless otherwise stated, conventional methods within the scope of the art, such as immunogenicity testing, antibody affinity testing (ELISA, Western Blot), etc., shall be used. Unless specifically defined, the use of all commercially available products used herein shall employ standard techniques. For example, they may be performed using the manufacturer's instructions for use with the kit, or in accordance with methods known in the art or the description of this invention. The techniques and methods described herein are generally performed according to conventional methods well known in the art, based on the descriptions in the various summary and more specific documents cited and discussed in this specification.

[0013] The term "peptide" refers to a compound formed by two or more amino acids linked together by peptide bonds. It is a substance intermediate between amino acids and proteins, with a relatively small molecular weight, typically composed of 2-100 amino acids. Peptides are widely distributed in living organisms and participate in various physiological activities, such as signal transduction and immune regulation.

[0014] The term "recombinant protein" refers to artificially synthesized genes introduced into host cells using genetic engineering techniques, enabling them to express target proteins. Recombinant proteins are widely used in the production of drugs, such as growth hormones, insulin, and interleukins. These drugs can treat a variety of diseases, such as diabetes, cancer, and immune system disorders. Recombinant proteins can also be used to prepare vaccines. By expressing specific antigenic proteins of pathogens, the immune system can be induced to produce antibodies, thereby enhancing the body's resistance to pathogens.

[0015] The terms “polypeptide,” “peptide,” and “protein” are used interchangeably herein to refer to polymers of amino acid residues. These terms apply to amino acid polymers in which one or more amino acid residues are modified residues or non-naturally occurring residues, such as artificial chemical analogs of the corresponding naturally occurring amino acids; these terms also apply to naturally occurring amino acid polymers.

[0016] The term "fusion polypeptide" or "chimeric polypeptide" refers to a polypeptide comprising two or more heterologous amino acid sequences and / or protein domains in a single, continuous polypeptide. In some embodiments, the two or more heterologous protein domains are covalently linked directly or indirectly (e.g., via a linker).

[0017] The term "ELISA" refers to enzyme-linked immunosorbent assay, which is the most widely used technique in enzyme immunoassay. The basic method of ELISA involves adsorbing known antigens or antibodies onto the surface of a solid-phase support (polystyrene microplate), allowing the enzyme-labeled antigen-antibody reaction to occur on the solid-phase surface, and then washing away the free components in the liquid phase. Commonly used ELISA methods include the double-antibody sandwich method and the indirect method; the former is used to detect large molecular antigens, and the latter is used to determine specific antibodies.

[0018] The terms “antigen,” “Ag,” or “antigen molecule” refer to molecules that elicit an immune response. An immune response may involve antibody production or activation of specific immune-active cells, or both. In some embodiments, an antigen is any macromolecule, including all proteins or peptides. In other embodiments, the antigen is derived from recombinant or genomic DNA.

[0019] To achieve the above objectives, the technical solution provided by the present invention is as follows: On one hand, the present invention provides a fusion antigen against bovine nodular dermatitis virus, the fusion antigen comprising antigenic peptide 1, antigenic peptide 2 and antigenic peptide 3, wherein the amino acid sequence of antigenic peptide 1 is SEQ ID NO.1, the amino acid sequence of antigenic peptide 2 is SEQ ID NO.2 and the amino acid sequence of antigenic peptide 3 is SEQ ID NO.3; and antigenic peptide 1, antigenic peptide 2 and antigenic peptide 3 are fused by end-to-end splicing to form the following structure: antigenic peptide 3-antigen peptide 1-antigen peptide 2-antigen peptide 3.

[0020] Preferably, the amino acid sequence of the fusion antigen is SEQ ID NO.4.

[0021] Preferably, a signal peptide sequence is added to the N-terminus of the fusion antigen.

[0022] Preferably, the signal peptide sequence has the amino acid sequence shown in SEQ ID NO.5 or SEQ ID NO.8.

[0023] Preferably, the C-terminus of the fusion antigen is further augmented with a tag and / or the endoplasmic reticulum resident signal KDEL.

[0024] Preferably, the tag is selected from His tag, GST tag, MBP tag, FLAG tag, SUMO tag, Strep-tag II tag, Twin-Strep-tag tag, MyC tag, HA tag, AviTag tag, HaloTag tag, EGFP tag, SNAP tag, V5 tag, thioredoxin tag, GFP tag, mCherry tag, protein C tag, BCCP tag, Spot-tag tag, Isopeptag, and SpyTag.

[0025] Preferably, the label is a FLAG label or a His label.

[0026] Specifically, the FLAG label is 3 The flag tag.

[0027] Specifically, the His tag is 8 The "him" tag.

[0028] Preferably, the fusion antigen is selected from the amino acid sequence shown in SEQ ID NO.6 or SEQ ID NO.9.

[0029] On the other hand, the present invention provides a recombinant protein to which at least one of the following is attached: an immunoglobulin Fc domain, a His tag, a GST tag, an MBP tag, a FLAG tag, a SUMO tag, a Strep-tag II tag, a Twin-Strep-tag tag, a MyC tag, a HA tag, an AviTag tag, a HaloTag tag, an EGFP tag, a SNAP tag, a V5 tag, a thioredoxin tag, a GFP tag, an mCherry tag, a protein C tag, a BCCP tag, a Spot-tag tag, an Isopeptag, a SpyTag, serum albumin, albumin-binding polypeptide, prealbumin, a C-terminal peptide, an elastin-like polypeptide, a cytokine, a single-chain antibody, a virus-like particle, a ligase, a kinase, a prodrug activating enzyme, a chemotherapeutic agent, a gold nanoparticle, or a liposome.

[0030] On the other hand, the present invention provides a nucleic acid molecule that encodes the above-mentioned fusion antigen or the above-mentioned recombinant protein.

[0031] Preferably, the nucleic acid molecule is selected from the nucleotide sequence shown in SEQ ID NO.7 or SEQ ID NO.10.

[0032] On the other hand, the present invention provides an expression vector containing the above-mentioned nucleic acid molecule, or the expression vector is used to express the above-mentioned fusion antigen, or the expression vector is used to express the above-mentioned recombinant protein.

[0033] Preferably, the expression vector is a eukaryotic expression vector or a prokaryotic expression vector; the eukaryotic expression vector includes any one or more of pHERC, pCAGGS, pFastBac, pEGFP-N1, pcDNA3.1, and pLVX-CMV-MCS-Linker-EGFP-IRES-Puro, and the prokaryotic expression vector includes any one or more of pET28a, pET32a, pGEX-4T, pMAL-p2x, and pMAL-c2X.

[0034] Preferably, the expression vector is pHERC or pCAGGS.

[0035] On the other hand, the present invention provides a cell that includes, expresses, or secretes the fusion antigen described above; or, the cell includes the recombinant protein described above; or, the cell includes the nucleic acid molecule described above; or, the cell includes the expression vector described above.

[0036] Preferably, the cells are eukaryotic cells or prokaryotic cells; the eukaryotic cells include any one or more of yeast, filamentous fungi, insect cells, mammalian cells, or plant cells; the prokaryotic cells include any one or more of Escherichia coli, Bacillus subtilis, lactic acid bacteria, Streptomyces, Proteus mirabilis, Corynebacterium glutamicum, thermophilic bacteria, cyanobacteria, and halophilic bacteria.

[0037] Preferably, the cell is a mammalian cell or a plant cell.

[0038] On the other hand, the present invention provides the use of the above-mentioned fusion antigen, the above-mentioned recombinant protein, the above-mentioned nucleic acid molecule, the above-mentioned expression vector or the above-mentioned cell in the preparation of bovine nodular dermatitis virus vaccine.

[0039] In another invention, the present invention provides an antibody prepared by means of the above-described fusion antigen, the above-described recombinant protein, the above-described nucleic acid molecule, the above-described expression vector, or the above-described cells.

[0040] On the other hand, the present invention provides the application of the above-mentioned antibody in the preparation of products for treating bovine nodular dermatitis.

[0041] The present invention has at least the following beneficial effects: The fusion antigen against bovine nodular dermatitis virus provided by this invention can be prepared in both tobacco and mammalian cells. The resulting bovine nodular dermatitis virus vaccine has high immunogenicity, good safety, rapid response, and production capacity, and has excellent clinical application prospects. Attached Figure Description

[0042] Figure 1 This is a schematic diagram of the fusion antigen structure in Example 1.

[0043] Figure 2 This is a schematic diagram of the structure constructed by expressing the fusion antigen in tobacco in Example 2.

[0044] Figure 3 This is a schematic diagram of the structure for the expression of the fusion antigen in mammalian cells in Example 3.

[0045] Figure 4 The images show the gel filtration chromatography elution curve and SDS-PAGE identification results of the fusion antigen expressed in the tobacco chassis in Example 4.

[0046] Figure 5 The gel filtration chromatography elution curve and SDS-PAGE identification results are shown for the fusion antigen expressed in the mammalian cell chassis in Example 5.

[0047] Figure 6 This is a schematic diagram of the mouse immunization procedure and serum sampling procedure in Example 6.

[0048] Figure 7 The titer of specific binding antibodies in the serum of immunized mice in Example 7 is shown; in the bar chart, the bars corresponding to d35 or d56 from left to right correspond to D060-122-3(B), D060-122-3(M), and control PBS, respectively.

[0049] Figure 8 The titer of neutralizing antibody against live LSDV virus in the serum of immunized mice in Example 8. Detailed Implementation

[0050] Unless otherwise specified, all raw materials and reagents used in this invention were purchased from commercial suppliers, and experiments were conducted in accordance with the operating instructions. Unless otherwise specified, all instruments, equipment, and apparatus used in this invention are conventional instruments, equipment, and apparatus, and experiments were conducted in accordance with the operating instructions and the accompanying reagents.

[0051] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. Unless otherwise specified in the embodiments, conditions are performed under conventional conditions or conditions recommended by the manufacturer. All reagents or instruments without specified manufacturers are commercially available conventional products. Numerous specific details are provided in the following detailed embodiments to better illustrate the invention. The specific embodiments described herein are for illustrative purposes only and are not intended to constitute any limitation on the invention.

[0052] Data analysis and statistical analysis were performed using professional data processing software. One-way ANOVA was used for significance analysis, and P<0.05 was considered to indicate a significant difference.

[0053] Example 1: Design of fusion antigens derived from bovine nodular dermatitis virus (BND) proteins LSDV060 and LSDV122 In this embodiment, as a representative example of the present invention, bovine nodular dermatitis viruses LSDV060 and LSDV122 are designed as fusion antigens from the N-terminus to the C-terminus, forming the structure shown in D060-122-3, as follows. Figure 1 As shown, the immunogen consists of three peptide segments arranged in the order 060-122-122-060, and the resulting single-chain fusion peptide can form a single-chain tetrasubunit structure.

[0054] The following sequences are all derived from the nucleic acid sequence of bovine nodular dermatitis virus (BNV), whose complete genome is registered in GenBank as ON563414.3. In the constructed structure, the first peptide is the T101-F196 peptide of the 122 protein (its amino acid sequence is shown in SEQ ID NO.1), the second peptide is the L77-F196 peptide of the 122 protein (its amino acid sequence is shown in SEQ ID NO.2), and the third peptide is derived from the 060 protein, with its amino acid sequence shown in SEQ ID NO.3; thus, the amino acid sequence of D060-122-3 is shown in SEQ ID NO.4.

[0055] SEQ ID NO.1: TESELNVYRSCKGIVYSGYCYTFNSEPKSFNDAYDDCEKKNSELPSNNLMNDWISDYLDGTWGEDGNVLFKEKNQELEAIDISDEMRSYYCVRSFF; SEQ ID NO.2: LNQFKEHKPYFLKNPNPTTYSDDDTESELNVYRSCKGIVYSGYCYTFNSEPKSFNDAYDDCEKKNSELPSNNLMNDWISDYLDGTWGEDGNVLFKEKNQELEAIDISDEMRSYYCVRSFF; SEQ ID NO.3: MGAAASIQTTVNTLNEKISSKLEQTAEATAEAKCDIEIGSIVFRQNKGCNVTVKNLCSSKAESQLDAILKAATETYDSLTPDQKAYVPGLMTAALNIQTSVNTVVKDFETYVKQKCTSKSVIDNKLKIHNIFIDECAAPTGTTTNFEFINSGTSQGICAIKTLMDVTTKASTKFSPSQSSG; SEQ ID NO.4: .

[0056] Example 2: Construction of Immunogen Expression in Tobacco In the process of constructing the chimeric antigen, for the purpose of protein expression and purification in tobacco, a signal peptide sequence (as shown in SEQ ID NO. 5) was added to the N-terminus of the amino acid sequence shown in SEQ ID NO. 4, and a 3-peptide peptide was added to its C-terminus. Flag tags and endoplasmic reticulum residency signals KDEL, such as Figure 2 As shown, this forms the complete tobacco construct represented by SEQ ID NO.6: SEQ ID NO.5: MIVLSVGSASSSPIVVVFSVALLLFYFSETSLG; The complete construct D060-122-3(B) has the amino acid sequence SEQ ID NO.6: .

[0057] The amino acid sequence of the above-designed construct was optimized using tobacco codons to obtain the encoding nucleotide sequence shown in SEQ ID NO.7. A Kozak sequence (GCCACC) was added to the 5' end of these nucleotide sequences and a translation stop codon was added to the 3' end. Then, a DNA fragment was artificially synthesized and cloned between the two BsaI restriction sites of the pHERC vector to obtain a tobacco-derived expression plasmid.

[0058] SEQ ID NO.7:

[0059] Example 3: Construction of Immunogen Expression in Mammalian Cells For the purpose of protein expression and purification in mammalian cells, such as Figure 3 As shown, a signal peptide sequence (as shown in SEQ ID NO. 8) was added to the N-terminus of the amino acid sequence shown in SEQ ID NO. 4, and a six-histidine tag was added to its C-terminus to form the complete construct shown in SEQ ID NO. 9. The amino acid sequence of the designed construct was optimized for mammalian cell codons to obtain the encoding nucleotide sequence shown in SEQ ID NO. 10. A Kozak sequence (GCCACC) was added to the 5' end of these nucleotide sequences, and a translation stop codon was added to the 3' end. Then, these DNA fragments were artificially synthesized and cloned between the EcoRI and XhoI restriction sites of the pCAGGS vector to obtain a mammalian cell-derived expression plasmid.

[0060] SEQ ID NO.8: METDTLLLWVLLLWVPGSTGD; The complete D060-122-3(M) construct, whose amino acid sequence is SEQ ID NO.9: METDTLLLWVLLLWVPGSTGDMGAAASIQTTVNTLNEKISSKLEQTAEATAEAKCDIEIGSIVFRQNKGCNVTVKNLCSSKAESQLDAILKAATETYDSLTPDQKAYVPGLMTAALNIQTSVNTVVKDFETYVKQKCTSKSVIDNKLKIHNIFIDECAAPTGTTTNFEFINSGTSQGICAIKTLMDVTTKASTKFSPSQSSGTESELNVYRSCKGIVYSGYCYTFNSEPKSFNDAYDDCEKKNSELPSNNLMNDWISDYLDGTWGEDGNVLFKEKNQELEAIDISDEMRSYYCVRSFFLNQFKEHKPYFLKNPNPTTYSDDDTESELNVYRSCKGIVYSGYCYTFNSEPKSFNDAYDDCEKKNSELPSNNLMNDWISDYLDGTWGEDGNVLFKEKNQELEAIDISDEMRSYYCVRSFFMGAAASIQTTVNTLNEKISSKLEQTAEATAEAKCDIEIGSIVFRQNKGCNVTVKNLCSSKAESQLDAILKAATETYDSLTPDQKAYVPGLMTAALNIQTSVNTVVKDFETYVKQKCTSKSVIDNKLKIHNIFIDECAAPTGTTTNFEFINSGTSQGICAIKTLMDVTTKASTKFSPSQSSGHHHHHHHH; SEQ ID NO.10:

[0061] Example 4: Expression and purification of immunogen in tobacco The expression plasmid prepared in Example 2 was transformed into *Agrobacterium tumefaciens*, and tobacco leaves were infected. Leaves were harvested 3 days post-infection, and protein purification was then performed. Specifically, leaves containing immunogenic proteins were ground with liquid nitrogen and lysed with a buffer (20 mM Tris, 150 mM NaCl, 1% Triton X-100, protease inhibitor, pH 8.0). The supernatant was crudely purified by flag affinity chromatography, and then further purified by gel filtration chromatography using a Superdex 200 10 / 300GL (GE) column. Finally, protein purity and molecular weight were identified by SDS-PAGE. The elution curve of gel filtration chromatography and the results of SDS-PAGE are shown below. Figure 4 As shown, the results indicate that high-purity D060-122-3(B) protein can be obtained after two-step purification.

[0062] Example 5: Expression and purification of immunogens in mammalian cells The expression plasmids of the constructs prepared in Example 3 were transfected into 293F cells for in vitro recombinant expression. Five days after transfection, cell supernatants containing the expressed immunogenic protein were collected. Protein purification was then performed. Specifically, the cell supernatant containing the immunogenic protein was crudely purified by nickel affinity chromatography (HisTrap™ HP (GE)), and then further purified by gel filtration chromatography using a Superdex 200 10 / 300GL (GE) column. Finally, the protein purity and molecular weight were identified by SDS-PAGE. The elution curve of gel filtration chromatography and the results of SDS-PAGE identification are shown below. Figure 5 As shown, after two-step purification, high-purity recombinant protein can be obtained. The peak position of the fusion protein and SDS-PAGE show that it is ~70kDa, which is in line with expectations.

[0063] Example 6: Mouse Immunization Experiment To verify the immunogenicity of the tobacco-derived and mammalian cell-derived recombinant fusion antigens of the present invention, the immunogenic proteins obtained in Examples 4 and 5 were respectively mixed and emulsified with ISA-201 adjuvant, and then... Figure 6 The strategy for immunizing mice was as follows. Specifically, the mouse immunization experiment was set up with an equimolar mixture of antigen and PBS as the negative control group. The mouse grouping and vaccine dosage are shown in Table 1. The immunogen and ISA-201 adjuvant were mixed and emulsified at a 1:1 volume ratio to prepare the vaccine. The mixed vaccine was used to immunize BALB / c mice, with 5 mice in each group. The mouse experimental procedure is as follows. Figure 6As shown: All mice were immunized three times by intramuscular injection at weeks 0, 3 and 6, with each injection volume of 100 μL. Two weeks after each immunization, blood was collected from the mice through the subocular venous plexus and serum was separated. The serum was frozen at 80°C for subsequent detection of specific antibodies and neutralizing titers.

[0064] Table 1. Mouse grouping and vaccine dosage

[0065] Example 7: ELISA assay for detecting vaccine-induced specific antibody titers LSDV060 and LSDV122 single proteins were diluted to 3 μg / mL with ELISA coating buffer, and 100 μL was added to each well of a 96-well ELISA plate (Coring, 3590). The plate was incubated overnight at 4°C. The coating buffer was discarded, and PBS was added to wash away any residual coating buffer. Then, 100 μL of ELISA blocking buffer (10% skim milk powder prepared with PBST) was added, and the plate was incubated at room temperature for 1 hour for blocking. During the blocking process, immunized mouse serum was diluted with ELISA blocking buffer, starting at 200-fold and serially diluted 3-fold, with 11 dilutions per sample. After blocking, the blocking buffer was removed, and 100 μL of immunized mouse serum diluted 10-fold with the blocking buffer was added to the ELISA plate. The plate was incubated at room temperature for 1 hour, followed by washing three times with PBST buffer. Then, HRP-labeled goat anti-mouse secondary antibody (Abcam, ab6789) diluted 1:4000 with the blocking buffer was added, and the plate was incubated at room temperature for 1 hour. The plate was then washed 5-6 times with PBST, and TMB chromogenic buffer was added for color development. The reaction was terminated by adding 2M hydrochloric acid after an appropriate reaction time. The OD450 reading was measured using a microplate reader. The antibody titer was defined as the highest dilution of serum with a reaction value greater than 2.1 times the negative control value. When the reaction value at the lowest dilution (limit of detection) was still less than 2.1 times the background value, the titer of the sample was defined as half of the lowest dilution.

[0066] The results are as follows Figure 7 As shown, throughout the immunization process, the level of specific antibodies in the serum of mice in each immunization group increased significantly with the increase of the number of immunizations, with the highest antibody level after 3 immunizations. This result indicates that the fusion antigen (D060-122-3(B) or D060-122-3(M)) provided by this invention can effectively induce an antibody response in mice. Furthermore, the antibody levels produced by immunization with tobacco-derived antigen D060-122-3(B) and mammalian cell-derived antigen D060-122-3(M) are similar, as shown in Table 2.

[0067] Table 2. Specific binding antibody titers in the serum of immunized mice

[0068] Example 8: Determination of the neutralizing capacity of live LSDV virus in immune serum The serum obtained in Example 6 was initially diluted 1:4, then serially diluted four times (2-fold), and each diluted with an equal volume of 200 TCID50 LSDV virus. After co-incubation at 37°C for 1 hour, 100 μl of the mixture was added to a 96-well plate with approximately 80% Vero cell coverage and cultured at 37°C for 72 hours. The neutralizing antibody titers in the serum of the corresponding immunized mice were calculated using the REED-Muench method by statistically analyzing the fluorescent LSDV. Figure 8 As shown in Table 3, both groups of vaccines can produce antibodies capable of neutralizing LSDV, indicating that both tobacco-derived vaccines and mammalian cell-derived vaccines can elicit better neutralizing antibodies.

[0069] Table 3. Neutralizing antibody titers against live LSDV virus in immunized mouse serum

[0070] Finally, it should be noted that the above content is only used to illustrate the technical solution of the present invention, and is not intended to limit the scope of protection of the present invention. Simple modifications or equivalent substitutions made by those skilled in the art to the technical solution of the present invention do not depart from the essence and scope of the technical solution of the present invention.

Claims

1. A fusion antigen against bovine nodular dermatitis virus, characterized in that, The fusion antigen is composed of antigenic peptide 1, antigenic peptide 2, antigenic peptide 3, a signal peptide, a tag, and an endoplasmic reticulum resident signal KDEL, or the fusion antigen is composed of antigenic peptide 1, antigenic peptide 2, antigenic peptide 3, a signal peptide, and a tag. The amino acid sequence of antigenic peptide 1 is SEQ ID NO. 1, the amino acid sequence of antigenic peptide 2 is SEQ ID NO. 2, and the amino acid sequence of antigenic peptide 3 is SEQ ID NO.

3. Furthermore, antigenic peptide 1, antigenic peptide 2, and antigenic peptide 3 are fused by end-to-end splicing to form the following structure: antigenic peptide 3-antigen peptide 1-antigen peptide 2-antigen peptide 3.

2. The fusion antigen according to claim 1, characterized in that, The signal peptide sequence has the amino acid sequence shown in SEQ ID NO.5 or SEQ ID NO.

8.

3. The fusion antigen according to claim 1, wherein the tag is selected from His tag, GST tag, MBP tag, FLAG tag, SUMO tag, Strep-tag II tag, Twin-Strep-tag tag, MyC tag, HA tag, AviTag tag, HaloTag tag, EGFP tag, SNAP tag, V5 tag, thioredoxin tag, GFP tag, mCherry tag, protein C tag, BCCP tag, Spot-tag tag, Isopeptag, and SpyTag.

4. The fusion antigen according to claim 3, wherein the tag is a FLAG tag or a His tag.

5. The fusion antigen according to claim 4, wherein the FLAG tag is 3 The flag tag.

6. The fusion antigen according to claim 5, wherein the His tag is 8 The "him" tag.

7. The fusion antigen according to claim 1, characterized in that, The fusion antigen is selected from the amino acid sequence shown in SEQ ID NO.6 or SEQ ID NO.

9.

8. A nucleic acid molecule, characterized in that, The nucleic acid molecule encodes the fusion antigen according to any one of claims 1-7.

9. The nucleic acid molecule according to claim 8, characterized in that, The nucleic acid molecule is selected from the nucleotide sequence shown in SEQ ID NO.7 or SEQ ID NO.

10.

10. An expression carrier, characterized in that, The expression vector is used to express the fusion antigen according to any one of claims 1-7.

11. The expression vector according to claim 10, characterized in that, The expression vector is a eukaryotic expression vector or a prokaryotic expression vector; the eukaryotic expression vector includes any one or more of pHERC, pCAGGS, pFastBac, pEGFP-N1, pcDNA3.1 and pLVX-CMV-MCS-Linker-EGFP-IRES-Puro, and the prokaryotic expression vector includes any one or more of pET28a, pET32a, pGEX-4T, pMAL-p2x and pMAL-c2X.

12. The expression vector according to claim 11, characterized in that, The expression vector is pHERC or pCAGGS.

13. A cell characterized in that, The cell comprises, expresses, or secretes the fusion antigen according to any one of claims 1-7; or, the cell comprises the nucleic acid molecule according to any one of claims 8-9; or, the cell comprises the expression vector according to any one of claims 10-12.

14. The cell according to claim 13, characterized in that, The cells are eukaryotic or prokaryotic cells; the eukaryotic cells include any one or more of yeast, filamentous fungi, insect cells, mammalian cells, or plant cells; the prokaryotic cells include any one or more of Escherichia coli, Bacillus subtilis, lactic acid bacteria, Streptomyces, Proteus mirabilis, Corynebacterium glutamicum, thermophilic bacteria, cyanobacteria, and halophilic bacteria.

15. The cell according to claim 14, characterized in that, The cells are mammalian cells or plant cells.

16. The use of the fusion antigen according to any one of claims 1-7, the nucleic acid molecule according to any one of claims 8-9, the expression vector according to any one of claims 10-12, or the cell according to any one of claims 13-15 in the preparation of a bovine nodular dermatosis virus vaccine.

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