Recombinant chimeric antigen for lumpy skin disease virus, immunogenic composition comprising same, and use thereof

Abstract

The present application discloses a recombinant chimeric antigen for Lumpy skin disease virus, an immunogenic composition comprising same, and a use thereof. The recombinant chimeric antigen for Lumpy skin disease virus of the present application comprises two immunogens fused in tandem in a specific order: Lumpy skin disease virus ORF122 protein or an antigenic fragment thereof and Lumpy skin disease virus ORF060 protein or an antigenic fragment thereof. An immune response against two types of infectious viral particles, i.e. intracellular mature virion (IMV) and extracellular enveloped virion (EEV), can be elicited.

Description

A recombinant chimeric antigen of bovine nodular dermatitis virus, an immunogenic composition containing the same, and their applications.

[0001] Cross-referencing

[0002] This application claims priority to Chinese Patent Application No. 202411194804.X, filed on August 28, 2024, entitled “A Recombinant Chimeric Antigen of Bovine Nodular Dermatitis Virus, an Immunogenic Composition Containing the Same and Its Application”, the entire contents of which are incorporated herein by reference. Technical Field

[0003] This application belongs to the field of biomedicine, specifically relating to a bovine nodular dermatitis virus recombinant chimeric antigen, an immunogenic composition containing the same, and their applications. Background Technology

[0004] Bovine nodular skin disease (LSDV) is a systemic infectious disease in cattle caused by bovine nodular skin disease virus (LSDV). LSDV belongs to the family Poxviridae, subfamily Vertebraviirinae, and genus Capripoxvirus (CaPV). LSDV infection mainly manifests as high fever, edema of mucous membranes and skin, epidermal ulcers forming permanent scars, and systemic lymphadenitis in cattle. This leads to reduced milk production in dairy cows, emaciation, and impaired meat yield and quality. The permanent scarring caused by the infection also significantly reduces the quality and value of the hides, and in severe cases, it can be fatal (mortality rate of 1%-5%).

[0005] Bovine nodular dermatitis was first discovered in Yili, Xinjiang, my country on August 12, 2019. Since then, the disease has spread to many parts of my country. According to a report from the Ministry of Agriculture and Rural Affairs on July 15, 2020, outbreaks of bovine nodular dermatitis were confirmed in Changting, Fujian; Ganzhou, Jiangxi; Chaozhou, Guangdong; Huangshan, Anhui; and Jinhua, Zhejiang. By the end of 2023, the disease had broken out in multiple provinces, municipalities, and autonomous prefectures in my country, including Yunnan, Fujian, Shandong, Guangxi, Jiangxi, Guangdong, Chongqing, Inner Mongolia, Hong Kong, Zhejiang, Anhui, Ningxia, and Xinjiang. Currently, the disease has spread to neighboring countries such as India, Jordan, Iraq, Iran, Kazakhstan, Russia, Ukraine, Turkmenistan, Bulgaria, Georgia, Bahrain, and the United Arab Emirates. Due to frequent trade in livestock with neighboring countries and the continuous emergence of variant strains of the bovine nodular dermatitis virus, the incidence of the disease in my country has been increasing year by year, causing a serious impact on my country's livestock industry. Due to its significant impact on transboundary animal diseases and the severe economic losses it causes to cattle farming in many countries, bovine nodular dermatitis (BND) has been listed as a notifiable disease by the World Organization for Animal Health (WOAH). In 2022, my country's Ministry of Agriculture and Rural Affairs classified it as a Class II animal disease. Given the escalating BND outbreak in my country and neighboring countries, there is an urgent need to develop an effective vaccine to curb the spread of the virus.

[0006] Currently, all LSDV vaccines approved for use both domestically and internationally are viral vaccines, mainly including three types: LSDV live attenuated vaccines, goatpox live attenuated vaccines, and LSDV / goatpox inactivated vaccines. Among these, live attenuated LSDV vaccines (such as Vaccine-OBP vaccine produced by OBP, Lumpyvax vaccine produced by Merck, and Herbivac LS vaccine produced by Deltamune) are primarily used internationally to prevent LSDV infection. While these vaccines are highly effective, they carry a risk of secondary infection and have significant side effects. International studies have reported adverse reactions in cattle after Vaccine-OBP vaccination, finding that 12% (26 / 215) of cattle experienced swelling at the injection site, and 9% developed small lumps within 8-18 days post-vaccination. Data collected in Croatia showed that approximately 20% (25 / 120) of cattle experienced adverse reactions after prophylactic LSDV vaccination in high-risk areas. Furthermore, live attenuated vaccines also pose a potential biosafety risk due to vaccine strain mutations leading to a resurgence of virulence. In my country, the primary prevention of LSDV is vaccination with a 5-fold dose of live attenuated goatpox vaccine. Goatpox and LSDV both belong to the goatpoxvirus genus and offer some cross-protection, but the overall effectiveness is poor (morbidity rate after vaccination is 22.9%). Additionally, an inactivated goatpox virus vaccine has been approved for emergency evaluation. While inactivation improves vaccine safety, it significantly reduces its effectiveness. Furthermore, all of the aforementioned viral vaccines suffer from complex production conditions (requiring biosafety level 3), high production costs, and low production capacity.

[0007] Given the severity of the epidemic and the various shortcomings of existing vaccines, there is an urgent need to develop a new generation of vaccines with independent intellectual property rights, clear immunogenic components, well-defined mechanisms of action, safety, effectiveness, and rapid availability, in order to help control the disease.

[0008] LSDV is a type of DNA virus with a viral genome of approximately 150 kbp, encoding up to 156 viral proteins. Besides the complexity of its encoded viral proteins, the viral particles themselves are also quite complex, possessing two distinct types of infectious viral particles: intracellular mature virus (IMV) and extracellular enveloped virus (EEV). IMV has an envelope and exhibits greater stability than EEV, primarily involved in viral transmission between hosts. EEV, on the other hand, has a unique outer membrane structure and is mainly involved in viral diffusion within the host. Due to the different membrane structures, membrane components, and cellular infection mechanisms of IMV and EEV, their surface neutralizing antigens are also distinctly different. Summary of the Invention

[0009] Purpose of the invention

[0010] In view of the various drawbacks of existing vaccines, the purpose of this application is to provide a bovine nodular dermatitis virus recombinant chimeric antigen that can efficiently stimulate a specific immune response (e.g., produce protective antibodies) against bovine nodular dermatitis virus (LSDV), a related vaccine product, its preparation method, and its application; in addition, the vaccine product based on this recombinant chimeric antigen also has advantages such as safety, efficacy, clear immunogenic components and protective mechanisms, high production capacity, and low cost, thereby meeting the safety and production capacity requirements for large-scale emergency vaccination.

[0011] Solution

[0012] To achieve the objectives of this application, the following technical solutions are provided:

[0013] In a first aspect, this application provides a recombinant chimeric antigen of bovine nodular dermatitis virus, said recombinant chimeric antigen being a single-chain fusion protein comprising bovine nodular dermatitis virus ORF122, ORF060 proteins or antigenic fragments thereof, and said recombinant chimeric antigen comprising an amino acid sequence arranged according to the following formula (I): A1-C1-B1-C2-A2-C3-B2 (I)

[0014] in:

[0015] A1 and A2 each independently represent the bovine nodular dermatosis virus ORF122 protein or its antigenic fragment, or an amino acid sequence that has at least 90%, 92%, 95%, 96%, 97%, 98%, or 99% identity with it and has the same or substantially the same immunogenicity as it.

[0016] B1 and B2 each independently represent the bovine nodular dermatosis virus ORF060 protein or its antigenic fragment, or an amino acid sequence that has at least 90%, 92%, 95%, 96%, 97%, 98%, or 99% identity with it and has the same or substantially the same immunogenicity.

[0017] C1, C2, and C3 are each independently either none or a concatenated sequence (GGGGS)n, where n is any integer between 1 and 10; and,

[0018] in,

[0019] A1 and A2 may be the same or different.

[0020] B1 and B2 may be the same or different.

[0021] C1, C2, and C3 may be the same or different.

[0022] In a feasible implementation, the bovine nodular skin disease virus ORF122, ORF060 proteins or antigenic fragments thereof are derived from Lumpy skin disease virus NI-2490.

[0023] In a further feasible embodiment, A1 and / or A2 each independently represent the amino acid sequence shown in SEQ ID NO:1, or the amino acid sequence shown in SEQ ID NO:1 plus a fragment extending 1-33 amino acids from it to the N-terminus of the ORF122 protein, or an amino acid sequence with the same or substantially the same immunogenicity obtained by substituting, deleting, or adding one or more amino acids to the above amino acid sequence; preferably, A1 represents the amino acid sequence shown in SEQ ID NO:1, or an amino acid sequence with the same or substantially the same immunogenicity obtained by substituting, deleting, or adding one or more amino acids to the amino acid sequence shown in SEQ ID NO:1; and / or, A2 represents the amino acid sequence shown in SEQ ID NO:2, or an amino acid sequence with the same or substantially the same immunogenicity obtained by substituting, deleting, or adding one or more amino acids to the amino acid sequence shown in SEQ ID NO:2.

[0024] In feasible embodiments, B1 and / or B2 each independently represent the amino acid sequence shown in SEQ ID NO:3, or the amino acid sequence shown in SEQ ID NO:3 plus a fragment extending 1-2 amino acids from it to the N-terminus of the ORF060 protein, or an amino acid sequence with the same or substantially the same immunogenicity obtained by substituting, deleting, or adding one or more amino acids to the above amino acid sequence; preferably, B1 and / or B2 represent the amino acid sequence shown in SEQ ID NO:3, or an amino acid sequence with the same or substantially the same immunogenicity obtained by substituting, deleting, or adding one or more amino acids to the amino acid sequence shown in SEQ ID NO:3.

[0025] In a preferred embodiment, A1 represents the amino acid sequence shown in SEQ ID NO:1, A2 represents the amino acid sequence shown in SEQ ID NO:2, B1 represents the amino acid sequence shown in SEQ ID NO:3, and B2 represents the amino acid sequence shown in SEQ ID NO:3; more preferably, C1, C2, and C3 are all absent; most preferably, the recombinant chimeric antigen comprises the amino acid sequence shown in SEQ ID NO:4.

[0026] Preferably, the N-terminus of the recombinant chimeric antigen further includes a signal peptide sequence; optionally, the signal peptide sequence is shown in SEQ ID NO:5.

[0027] Preferably, the C-terminus of the recombinant chimeric antigen further includes a tag sequence; optionally, the tag is selected from at least one of the Flag tag, His tag, MBP tag, HA tag, myc tag, GST tag, and SUMO tag, preferably the His tag.

[0028] In this application, to achieve immunoprotective effects against both EEV and IMV viral particles, a recombinant chimeric antigen was designed that simultaneously contains the neutralizing antigen ORF122 of EEV and the neutralizing antigen ORF060 of IMV. These two immunogens are encoded by the ORF122 and ORF060 genes of bovine nodular dermatitis virus, respectively, and are homologous to vaccinia virus A33R and L1R, respectively. The designed recombinant chimeric antigen can induce balanced and high-level specific antibodies against the two antigenic epitopes, achieving simultaneous immunoprotective effects against both viral particles with a single immunogen protein.

[0029] Secondly, this application provides a method for preparing a recombinant chimeric antigen of bovine nodular dermatitis virus as described in the first aspect above, the method comprising the following steps:

[0030] The Kozak sequence and the coding sequence of the signal peptide are added to the 5' end of the nucleotide sequence encoding the bovine nodular dermatitis virus recombinant chimeric antigen as described in the first aspect above, and the coding sequence of the histidine tag and the stop codon are added to the 3' end. The recombinant antigen is cloned and expressed, the correct recombinant is screened, and then it is transfected into expression system cells for expression. The cell culture supernatant is collected and the bovine nodular dermatitis virus recombinant chimeric antigen is isolated from it.

[0031] In one feasible implementation of the above preparation method, the cells of the expression system are mammalian cells, insect cells, yeast cells, or bacterial cells;

[0032] Optionally, the mammalian cells are HEK293T cells, 293F series cells, or CHO cells; more preferably, the 293F series cells are HEK293F cells, Freestyle293F cells, or Expi293F cells.

[0033] Optionally, the insect cells are sf9 cells, Hi5 cells, sf21 cells, or S2 cells;

[0034] Optionally, the yeast cells are Pichia pastoris cells or yeast cells modified therefrom;

[0035] Optionally, the bacterial cells are Escherichia coli cells.

[0036] Thirdly, this application provides a polynucleotide encoding a bovine nodular dermatitis virus recombinant chimeric antigen as described in the first aspect above.

[0037] The polynucleotide can be DNA or mRNA;

[0038] Preferably, the polynucleotide comprises a DNA sequence as shown in SEQ ID NO:6;

[0039] Preferably, the polynucleotide comprises an mRNA sequence as shown in SEQ ID NO:7.

[0040] Fourthly, this application provides a nucleic acid construct comprising a polynucleotide as described in the third aspect above, and optionally, at least one expression regulatory element operatively linked to the polynucleotide.

[0041] Fifthly, this application provides an expression vector comprising the nucleic acid construct as described in the fourth aspect above.

[0042] Sixthly, this application provides a host cell transformed or transfected with the polynucleotides as described in the third aspect above, the nucleic acid constructs as described in the fourth aspect above, or the expression vectors as described in the fifth aspect above.

[0043] In a seventh aspect, this application provides the use of the bovine nodular dermatitis virus recombinant chimeric antigen as described in the first aspect above, the polynucleotide as described in the third aspect above, the nucleic acid construct as described in the fourth aspect above, the expression vector as described in the fifth aspect above, or the host cell as described in the sixth aspect above in the preparation of a medicament for the prevention and / or treatment of bovine nodular dermatitis virus infection.

[0044] Optionally, the drug is a vaccine, preferably a recombinant protein vaccine; more preferably, the recombinant protein vaccine uses an adjuvant selected from the following: aluminum adjuvant, MF59 adjuvant, MF59-like adjuvant, and ISA201 adjuvant;

[0045] Optionally, the vaccine may be in the form of a nasal spray, oral preparation, suppository, or parenteral preparation;

[0046] Preferably, the nasal spray is selected from aerosols, sprays, and powders;

[0047] Preferably, the oral preparation is selected from tablets, powders, pills, granules, soft / hard capsules, film-coated preparations, and ointments; more preferably, the tablets are sublingual tablets; more preferably, the granules are fine granules; more preferably, the powder is a powder; more preferably, the pills are small pills;

[0048] Preferably, the parenteral preparation is a transdermal preparation, ointment, plaster, topical liquid, or injectable preparation; more preferably, the injectable preparation is a push-in preparation.

[0049] Eighthly, this application provides a vaccine or immunogenic composition comprising a bovine nodular dermatitis virus recombinant chimeric antigen as described in the first aspect above, a polynucleotide as described in the third aspect above, a nucleic acid construct as described in the fourth aspect above, an expression vector as described in the fifth aspect above, or a host cell as described in the sixth aspect above, as well as physiologically acceptable mediators, adjuvants, excipients, carriers, and / or diluents.

[0050] In some preferred embodiments, the vaccine or immunogenic composition is a bovine nodular dermatitis virus recombinant protein vaccine, which includes the bovine nodular dermatitis virus recombinant chimeric antigen and adjuvant as described in the first aspect above;

[0051] Optionally, the adjuvant is one or more selected from the following adjuvants: aluminum adjuvant, MF59 adjuvant, MF59-like adjuvant, and ISA201 adjuvant.

[0052] In some other preferred embodiments, the vaccine or immunogenic composition is a bovine nodular dermatitis virus DNA vaccine, comprising:

[0053] (1) Eukaryotic expression vectors; and

[0054] (2) Constructing a DNA sequence encoding the bovine nodular dermatitis virus recombinant chimeric antigen as described in the first aspect above into the eukaryotic expression vector, preferably the DNA sequence shown in SEQ ID NO:6;

[0055] Optionally, the eukaryotic expression vector is selected from pGX0001, pVAX1, pCAGGS and pcDNA series vectors.

[0056] In some other preferred embodiments, the vaccine or immunogenic composition is a bovine nodular dermatitis virus mRNA vaccine, the mRNA vaccine comprising:

[0057] (I) An mRNA sequence encoding the bovine nodular dermatitis virus recombinant chimeric antigen as described in the first aspect above, preferably an mRNA sequence as shown in SEQ ID NO:7; and

[0058] (II) Lipid nanoparticles.

[0059] In some other preferred embodiments, the vaccine or immunogenic composition is a bovine nodular dermatitis virus-viral vector vaccine, comprising:

[0060] (1) Viral backbone vector; and

[0061] (2) Constructing a DNA sequence encoding the bovine nodular dermatitis virus recombinant chimeric antigen as described in the first aspect above into the viral backbone vector, preferably a DNA sequence as shown in SEQ ID NO:6;

[0062] Optionally, the viral backbone vector is selected from one or more of the following viral vectors: adenovirus vector, poxvirus vector, influenza virus vector, and adeno-associated virus vector.

[0063] In some other preferred embodiments, the vaccine or immunogenic composition is a bovine nodular dermatitis virus nanoparticle vaccine;

[0064] More preferably, the carrier of the nanoparticles is ferritin, and the bovine nodular dermatitis virus recombinant chimeric antigen as described in the first aspect above is covalently linked to ferritin and self-assembled into nanoparticles, so that the bovine nodular dermatitis virus recombinant chimeric antigen is present on the surface of the nanoparticles; more preferably, the bovine nodular dermatitis virus recombinant chimeric antigen and ferritin are linked by a linker, or covalently linked by a SpyTag / SpyCatcher linker system.

[0065] In a feasible implementation, the vaccine or immunogenic composition is in the form of a nasal spray, oral formulation, suppository, or parenteral formulation;

[0066] Preferably, the nasal spray is selected from aerosols, sprays, and powders;

[0067] Preferably, the oral formulation is selected from tablets, powders, pills, granules, soft / hard capsules, film-coated agents, and ointments;

[0068] More preferably, the tablet is a sublingual tablet;

[0069] More preferably, the granules are fine granules;

[0070] More preferably, the powder is a granule;

[0071] More preferably, the pills are small pills;

[0072] Preferably, the parenteral preparation is a transdermal preparation, ointment, plaster, topical liquid, or injectable preparation; more preferably, the injectable preparation is a push-in preparation.

[0073] Ninthly, this application provides a method for preventing and / or treating bovine nodular dermatitis virus infection, the method comprising: administering to a subject in need a preventive and / or therapeutically effective amount of the following substances: bovine nodular dermatitis virus recombinant chimeric antigen as described in the first aspect above, polynucleotides as described in the third aspect above, nucleic acid constructs as described in the fourth aspect above, expression vectors as described in the fifth aspect above, host cells as described in the sixth aspect above, and / or vaccines or immunogenic compositions as described in the eighth aspect above.

[0074] The "effective dose for prevention and / or treatment" may vary depending on the recipient, the organ involved, the symptoms, the method of administration, etc. It may be determined based on the veterinarian's judgment, taking into account factors such as the type of dosage form, the method of administration, the age and weight of the subject, and the subject's symptoms. Beneficial effects

[0075] The inventors of this application have designed a recombinant chimeric antigen against bovine nodular dermatitis virus, comprising two antigenic epitopes fused in tandem in a specific order: (1) bovine nodular dermatitis virus ORF122 protein or an antigenic fragment thereof (or their derived peptides), and (2) bovine nodular dermatitis virus ORF060 protein or an antigenic fragment thereof (or their derived peptides); wherein, ORF122 is a neutralizing antigen specific to extracellular enveloped viral particles (EEV), and ORF060 is a neutralizing antigen specific to intracellular mature viral particles (IMV); a vaccine containing both can elicit an immune response against the two infectious viral particles.

[0076] The recombinant chimeric antigen of this application can better stimulate the production of neutralizing antibodies compared to its corresponding mixed antigen, and both antigens contained herein retain their respective antigenicity. This design not only better stimulates neutralizing antibodies but also facilitates the establishment of subsequent vaccine production cell lines.

[0077] Furthermore, the recombinant chimeric antigen of this application can induce the production of balanced and high-level specific antibodies against the two antigenic epitopes mentioned above.

[0078] Compared with existing bovine nodular dermatitis virus vaccines, the vaccine products based on the recombinant chimeric antigen of this application have the following advantages:

[0079] 1) Subunit vaccines have better safety, thus overcoming the safety issues of existing attenuated live virus vaccines; and, through practical verification of the subunit vaccine technology route, subunit vaccines have advantages over attenuated live virus vaccines in terms of lower production costs, rapid response, and production capacity support; and, through experimental verification, the bovine nodular dermatitis virus subunit vaccine of this application has good efficacy.

[0080] 2) The bovine nodular dermatitis virus (BNDV) vaccine utilizes its own antigenic sequence, which exhibits high specificity for BNDV. Currently, the vaccines used in my country to prevent BNDV are all goatpox attenuated live vaccines. Although goatpox virus and BNDV belong to the same genus, there are still some differences in their neutralizing antigenic sequences, and their protective effect against BNDV remains to be clarified. However, the BNDV vaccine proposed in this application is developed based on the antigenic epitopes of BNDV, and therefore, it has high specificity for the prevention and treatment of BNDV.

[0081] 3) Bovine nodular dermatitis virus contains a wide variety of proteins, most of which cannot elicit an effective antiviral immune response and are therefore ineffective. In addition, some viral proteins have immunosuppressive effects. Existing live virus vaccines cannot remove the above-mentioned ineffective and harmful components, thus posing risks and uncertainties in vaccination. However, the bovine nodular dermatitis virus vaccine of this application retains only two viral neutralizing antigens. Experimental data show that it can efficiently, stably and evenly elicit specific antibodies against these two antigens and can demonstrate a complete protective effect in mouse disease models.

[0082] 4) Compared with "cocktail" type multi-component vaccines prepared by mixing two antigen proteins, this application adopts a rational design approach based on the structure of the immunogen to rationally design the effective immune components of the two antigen proteins to achieve the simultaneous activation of specific antibodies targeting two viral particles by a single immunogen protein. This will greatly reduce the research and development and production costs of bovine nodular dermatitis subunit vaccines. Attached Figure Description

[0083] One or more embodiments are illustrated by way of example with reference to the accompanying drawings, and these illustrative examples are not intended to limit the embodiments. The term "illustrative" as used herein means "serving as an example, embodiment, or illustration." Any embodiment illustrated herein as "illustrative" is not necessarily to be construed as superior to or better than other embodiments.

[0084] Figure 1 shows the gel filtration chromatography elution curve and SDS-PAGE identification results of the immunogenic protein LSDV-DAM expressed in Example 2 of this application.

[0085] Figure 2 shows a schematic diagram of the mouse immunization strategy used in Example 3 of this application.

[0086] Figure 3 shows the specific binding antibody titers of each immunized mouse serum against antigens ORF122 (Figure 3A) and ORF060 (Figure 3B) detected in Example 4 of this application; where the vertical axis shows the serum antibody titer (Log10) and the horizontal axis shows each immunization group; ns indicates P > 0.05; * indicates P < 0.05, ** indicates P < 0.01, *** indicates P < 0.001, and **** indicates P < 0.0001.

[0087] Figure 4 shows a schematic diagram of the bovine immunization strategy used in Embodiment 5 of this application.

[0088] Figure 5 shows the specific binding antibody titers of each immunized bovine serum against the ORF122 (Figure 5A) and ORF060 (Figure 5B) antigens detected in Example 6 of this application; where the vertical axis shows the serum antibody titer (Log2) and the horizontal axis shows each immunization group; ns indicates P > 0.05; * indicates P < 0.05, ** indicates P < 0.01, *** indicates P < 0.001, and **** indicates P < 0.0001.

[0089] Figure 6 shows the ELISpot detection results of IFN-γ secreted by peripheral blood mononuclear lymphocytes of each immunized bovine group as described in Example 8 of this application; where the vertical axis shows the number of IFN-γ spots and the horizontal axis shows each immunization group. Detailed Implementation

[0090] To make the objectives, technical solutions, and advantages of the embodiments of this application clearer, the technical solutions in the embodiments of this application will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of this application, not all embodiments. Based on the embodiments in this application, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this application.

[0091] Furthermore, to better illustrate this application, numerous specific details are provided in the following detailed embodiments. Those skilled in the art should understand that this application can be implemented without certain specific details. In some embodiments, materials, elements, methods, and means well-known to those skilled in the art are not described in detail in order to highlight the main points of this application.

[0092] Unless otherwise expressly stated, throughout the specification and claims, the term "comprising" or its variations such as "including" or "comprises" shall be understood to include the stated elements or components without excluding other elements or other components.

[0093] Example 1: Immunogen Design

[0094] In this embodiment, as a representative example of this application, two ORF122 peptides (sequences shown in SEQ ID NO:1 and 2, respectively) and two ORF060 peptides (sequences shown in SEQ ID NO:3) are tandemly arranged in the order ORF122-ORF060-ORF122'-ORF060' from the N-terminus to the C-terminus. The resulting single-chain fusion peptide (hereinafter referred to as LSDV-DAM or DAM, its sequence is shown in SEQ ID NO:4) represents the recombinant chimeric antigen of this application. All the above peptide sequences are derived from the corresponding sequences of the bovine nodular skin disease virus isolate NI-2490, whose complete genome is registered in GenBank as NC_003027.

[0095] To express and purify LSDV-DAM, a signal peptide (sequence shown in SEQ ID NO:5) was added to the N-terminus of its amino acid sequence, and a 6×HIS tag was added to its C-terminus to form the complete LSDV-DAM construct. The sequence of the complete LSDV-DAM construct is as follows:

[0096] (SEQ ID NO:8, where the underlined part is the signal peptide, the bold part is the 6×HIS tag, and the italicized part is the single-chain fusion peptide LSDV-DAM as shown in SEQ ID NO:4).

[0097] In addition, for comparison, this embodiment also designed expression constructs of single immunogens ORF122 and ORF060 for subsequent experiments, and their amino acid sequences are as follows:

[0098] ORF122 construct (SEQ ID NO:9)

[0099] (The underlined part is the signal peptide, the bold part is the 6×HIS tag, and the middle part is the ORF122 peptide.)

[0100] ORF060 construct (SEQ ID NO:10)

[0101] (The underlined part is the signal peptide, the bold part is the 6×HIS tag, and the middle part is the ORF060 peptide as shown in SEQ ID NO:3).

[0102] Example 2: Expression and purification of immunogenic proteins

[0103] Expression and purification of the LSDV-DAM construct

[0104] The amino acid sequence of the LSDV-DAM construct designed in Example 1 was codon optimized to obtain the nucleotide sequence encoding the LSDV-DAM construct, as shown in SEQ ID NO:11. A Kozak sequence (GCCACC) was added to the 5' end of the nucleotide sequence and a translation stop codon was added to the 3' end. Then, a DNA fragment was artificially synthesized and cloned into the EcoRI and XhoI restriction sites of the pCAGGS vector to obtain the expression plasmid of the LSDV-DAM construct.

[0105] The LSDV-DAM expression plasmid was transfected into 293F cells for in vitro recombinant expression. Five to seven days post-transfection, cell supernatants containing the expressed immunogenic protein were collected. Protein purification was then performed; specifically, the cell supernatant containing the immunogenic protein was subjected to HisTrap chromatography. TM The sample was purified by HP(GE) crude purification, and then subjected to gel filtration chromatography (Superdex) column purification. TM 200pg Hiload TM The protein was further purified to 16 / 600 (GE), and finally, the purity and molecular weight of the protein were identified by SDS-PAGE.

[0106] The elution curve of gel filtration chromatography and the SDS-PAGE identification results of the elution peak are shown in Figure 1. Figure 1 shows that after two-step purification, high-purity LSDV-DAM protein with a molecular weight of ~70kDa can be obtained, which is in line with expectations.

[0107] Expression and purification of single immunogens ORF122 and ORF060

[0108] The amino acid sequences of the single immunogenic peptides ORF122 and ORF060 designed in Example 1 were codon-optimized to obtain their encoding nucleotide sequences, as shown in SEQ ID NO:12 and SEQ ID NO:13, respectively. 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 into the EcoRI and XhoI restriction sites of the pCAGGS vector to obtain expression plasmids of ORF122 and ORF060 constructs.

[0109] The expression and purification process of ORF122 and ORF060 was consistent with that of LSDV-DAM. The elution curves of gel filtration chromatography and the SDS-PAGE identification results of the elution peaks showed that high-purity ORF122 and ORF060 proteins with molecular weights of ~20 kDa could be obtained after two-step purification, which was in line with expectations.

[0110] Example 3: Mouse Immunization Experiment

[0111] To verify the immunoprotective efficacy of the recombinant chimeric antigen of this application, the inventors combined the immunogenic protein LSDV-DAM obtained in Example 2 with AddaVax. TM The adjuvant (purchased from InvivoGen, catalog number: vac-adx-10) was mixed and emulsified to prepare the LSDV-DAM recombinant protein vaccine (hereinafter referred to as "DAM" in the attached figure); the specific preparation method of the vaccine is as follows: 10 μg of immunogen protein was dissolved in 50 μL of PBS, and then adjuvant (50 μL of adjuvant) was added at a volume ratio of 1:1 to prepare a total volume of 100 μL of vaccine.

[0112] The mice were immunized with the vaccine prepared above according to the strategy shown in Figure 2; at the same time, the PBS immunization group was used as a negative control.

[0113] In the immunization experiment, all mice used were female BALB / c mice, aged 6-8 weeks, with an average weight of 15-20g. Ten mice were used in each experimental group. As shown in Figure 2, mice were immunized three times, on day 0, day 21, and day 42, with a dose of 10μg per mouse each time. The administration method was intramuscular injection, with 50μL injected into each leg, located in the thigh.

[0114] Blood was collected from the orbital sinus of mice in all groups 19 days after the first, second, and third immunizations (i.e., days 19, 40, and 61). Mouse serum was obtained by centrifugation at 1500 rpm for 10 minutes after allowing the blood to clot. The mouse serum was immediately aliquoted and stored at -80°C for subsequent ELISA detection of specific antibody titers and determination of live virus neutralizing capacity.

[0115] Example 4: ELISA assay to detect serum-specific antibody titers in vaccine-induced mice

[0116] Single immunogens ORF122 and ORF060 were diluted to 2 μg / mL with ELISA coating buffer (Solepro, C1050). 100 μL of the diluted immunogens were added to each well of a 96-well ELISA plate (Corning, 3590), and the plate was incubated overnight at 4°C. The coating buffer was discarded, and PBS was added to wash away any residual coating buffer. 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, the immunized mouse sera obtained in Example 3 were diluted with ELISA blocking buffer, starting from 200-fold and serially diluted 3-fold, with 11 dilutions per sample. After blocking, the blocking buffer was removed, and 100 μL of the diluted immunized mouse sera were added to the ELISA plate at each dilution. The plate was incubated at room temperature for 1 hour, followed by washing three times with PBST. Then, HRP-labeled goat anti-mouse secondary antibody (Abcam, ab6789) diluted 1:4000 with 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. After an appropriate reaction time, 2M hydrochloric acid was added to terminate the reaction. OD450 readings were measured on a microplate reader. An OD450 value greater than 2.5 times that of the negative control was considered positive. The highest dilution of the serum that was considered positive was defined as the serum antibody titer (Endpoint titer). When the reaction value at the lowest dilution was still less than 2.5 times that of the negative control, the titer of the sample was defined as half of the lowest dilution, i.e., Log10 = 1.

[0117] The results of the detection of specific antibody titers against the antigenic epitopes ORF122 and ORF060 in the serum of each immunized mouse are shown in Figures 3A and 3B, respectively. As can be seen from Figures 3A and 3B, the recombinant chimeric antigen LSDV-DAM of this application can simultaneously and evenly stimulate the production of specific antibodies against both antigenic epitopes. The specific antibody titers against both antigenic epitopes are significantly higher than those in the PBS group, and the serum antibody titers against ORF122 and ORF060 after three immunizations reach 10. 6.12 and 10 6.33 This is at a relatively high level among subunit vaccines, indicating good immunogenicity.

[0118] The above results indicate that the recombinant chimeric antigen LSDV-DAM of this application, as a single immunogen, can simultaneously elicit high levels of specific antibodies against two antigens in mice, and is therefore a potential LSDV recombinant subunit vaccine.

[0119] Example 5: Bovine Immunization Experiment

[0120] To verify the immunoprotective efficacy of the recombinant chimeric antigen of this application, the inventors mixed and emulsified the recombinant chimeric antigen LSDV-DAM obtained in Example 2 with ISA201 adjuvant (purchased from SEPPIC, product name Montanide ISA 201VG) to prepare the LSDV-DAM recombinant protein vaccine (hereinafter referred to as "DAM" in the attached figure); at the same time, a commercially available bovine nodular dermatitis inactivated vaccine (trade name: Mujiekang, hereinafter referred to as "AV-41" in the attached figure) produced by China Animal Husbandry Industry Co., Ltd. was used as a positive control.

[0121] The specific method for preparing the vaccine based on the recombinant chimeric antigen LSDV-DAM of this application is as follows: 300 μg of recombinant chimeric antigen LSDV-DAM is dissolved in 1 mL of PBS, and then adjuvant (1 mL adjuvant) is added at a volume ratio of 1:1 to obtain a vaccine with a total volume of 2 mL.

[0122] The inactivated vaccine used as a positive control was administered according to the recommended method in the instructions, namely, intramuscular injection, 2 mL / head. The recommended immunization schedule is two doses, i.e., a booster immunization one month after the first immunization. However, to ensure that the immunization schedule is parallel to that of the vaccine in this application, the inactivated vaccine adopted the exact same immunization schedule as the vaccine in this application, i.e., immunization was carried out on days 0, 21, and 42, for a total of three immunizations. The specific antibody titers of serum after the first, second, and third immunizations were measured to characterize its immunoprotective effect.

[0123] Specifically, the immunization and blood collection procedures for cattle immunized with the vaccine and inactivated vaccine of this application are shown in Figure 4.

[0124] In the immunization experiment, all cattle used were female Holstein dairy cows aged 3-6 months with an average weight of 200 kg. Each group consisted of 5 cows. As shown in Figure 4, the cows were immunized on day 0, day 21, and day 42, for a total of 3 times, with each dose being 300 μg / head. The vaccination was administered via intramuscular injection at the neck site, with each cow receiving 2 mL per injection.

[0125] Blood was collected from the jugular vein of cattle in all groups 21 days before, after, and after the first, second, and third immunizations (i.e., days 0, 21, 42, and 63). Bovine serum was obtained by centrifugation at 1500 rpm for 10 minutes after allowing the blood to clot. The bovine serum was immediately aliquoted and stored at -80°C for subsequent ELISA detection of specific antibody titers and determination of live virus neutralizing capacity.

[0126] Example 6: ELISA assay for detecting serum-specific antibody titers induced in bovine vaccines

[0127] Single immunogens ORF122 and ORF060 were diluted to 2 μg / mL with ELISA coating buffer (Solepro, C1050). 100 μL of the diluted immunogens were added to each well of a 96-well ELISA plate (Corning, 3590), and the plate was incubated overnight at 4°C. The coating buffer was discarded, and PBS was added to wash away any residual coating buffer. 100 μL of ELISA blocking buffer (5% BSA prepared with PBST) was added, and the plate was incubated at room temperature for 1 hour for blocking. During the blocking process, the immunized bovine serum obtained in Example 5 was diluted with ELISA blocking buffer, starting with a 2-fold serial dilution, with at least 11 dilutions per sample. After blocking, the blocking buffer was removed, and 100 μL of the diluted bovine serum was added to the ELISA plate at each dilution. The plate was incubated at room temperature for 1 hour, followed by washing three times with PBST. Then, HRP-labeled rabbit anti-bovine secondary antibody (Bioss, bs-0326R-HRP) diluted 1:4000 with 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. After an appropriate reaction time, 2M hydrochloric acid was added to terminate the reaction. The OD450 reading was measured using a microplate reader. An OD450 value greater than 2.5 times that of the negative control was considered positive. The highest dilution of the serum deemed positive was defined as the serum antibody titer (Endpoint titer). When the reaction value at the lowest dilution was still less than 2.5 times that of the negative control, the titer of the sample was defined as half of the lowest dilution.

[0128] The results of the detection of specific antibody titers against the antigenic epitopes ORF122 and ORF060 in the serum of each immunized bovine serum are shown in Figures 5A and 5B, respectively. As shown in Figures 5A and 5B, the recombinant chimeric antigen LSDV-DAM of this application can simultaneously and evenly elicit specific antibodies against both antigenic epitopes. Specifically, the level of specific antibodies against ORF122 after the first immunization was significantly higher than that of the inactivated vaccine group (as a positive control), and slightly higher after the second and third immunizations (with no significant difference). The level of specific antibodies against ORF060 after the first, second, and third immunizations was significantly higher than that of the inactivated vaccine group (as a positive control). Furthermore, after the third immunization, the serum antibody titers against ORF122 and ORF060 reached 2... 8.7 and 2 10.7 It showed good immunogenicity.

[0129] The above results indicate that the recombinant chimeric antigen LSDV-DAM of this application, as a single immunogen, can simultaneously elicit high levels of specific antibodies against two antigens in cattle. Specifically, the level of specific antibodies against the ORF060 antigen was significantly higher than that of the marketed inactivated vaccine group after each immunization, while the level of specific antibodies against the ORF122 was either significantly higher than that of the marketed inactivated vaccine group (after the first immunization) or comparable to it (as mentioned above, slightly higher than the marketed inactivated vaccine group, but without significant difference) after each immunization. Therefore, the overall immunization effect of the recombinant chimeric antigen vaccine of this application is superior to that of the currently marketed LSDV vaccine, and it is a potential LSDV recombinant subunit vaccine.

[0130] Example 7: Evaluation of serum neutralizing antibody titers induced in cattle by vaccine

[0131] Bovine serum following the third immunization in Example 5 was inactivated at 56°C for 30 min. The serum was diluted using DMEM medium containing 2% fetal bovine serum (FBS), initially as a 2-fold dilution, followed by serial 2-fold dilutions up to a maximum of 16-fold. The TCID was determined using DMEM medium containing 2% FBS. 50 LSDV viral fluid diluted to 200 TCID 50 / 0.1mL. Add the diluted virus solution to the diluted serum at a 1:1 volume ratio and incubate the mixture at 37°C and 5% CO2 for 1 hour. Transfer 100μL of the serum-virus mixture to a 96-well cell culture plate containing Vero cells. Simultaneously, set up negative serum controls, virus controls, and normal cell controls, with 3 replicates for each dilution. Incubate the plate at 37°C and 5% CO2. Observe the cytopathic effect (CPE) daily until the cell state stabilizes, then calculate the virus neutralizing antibody titer in the serum using the Reed-Muench method. If the serum neutralizing antibody titer is lower than the limit of detection (4.0), the titer of the sample is defined as half of the limit of detection (2.0).

[0132] The results of the bovine serum neutralizing antibody titer test are shown in Table 1.

[0133] Table 1. Results of bovine serum neutralizing antibody titer detection

[0134] As shown in Table 1, the average serum neutralizing antibody titer in the DAM group was 6.8, and the average serum neutralizing antibody titer in the AV-41 group was 6.3. Neutralizing protective effects were detected in 4 out of 5 calves in both the DAM and AV-41 groups. The average neutralizing antibody titer of the 4 calves in the DAM group was 8.1, while that in the AV-41 group was 7.4. These neutralizing titer results indicate that the neutralizing protective effect provided by DAM is comparable to that of commercially available vaccines.

[0135] Example 8: Assessment of vaccine-induced bovine T-cell immune response by enzyme-linked immunospot assay (ELISpot)

[0136] To assess the antigen-specific cellular immune response induced by the candidate vaccine, the bovine interferon-gamma (IFN-γ) enzyme-linked immunospot (ELISpot) assay kit (Mabtech) was used according to the manufacturer's (MabTech) recommended procedures. Peripheral blood was collected from cattle that had completed the full immunization program 7 days after the last immunization, and peripheral blood mononuclear cells (PBMCs) were isolated for ELISpot analysis.

[0137] The 96-well Merck Millipore plates were coated with 10 μg / mL bovine IFN-γ antibody (Mabtech) and incubated overnight at 4°C. 200 μL of RPMI 1640 medium (Gibco) containing 10% fetal bovine serum (FBS) was added to each well for blocking at room temperature for 30 minutes. 3 × 10⁻⁶ ppm of the antibody was added to each well. 5 Each cell and a peptide library for stimulation (the peptide library covers the entire open reading frame of ORF122, ORF060, ORF074 and ORF117, and consists of 18-meric peptides with 10 amino acid overlaps between peptide segments, and the concentration of a single peptide is 2 μg / mL) were added to RPMI 1640 medium containing 10% FBS to a final volume of 150 μL / well. The culture plates were then incubated at 37°C in a 5% CO2 incubator. After 36 hours of incubation, the plate was washed five times with PBS. Then, 100 μL of the monoclonal antibody (diluted to a final concentration of 0.25 μg / mL in PBS containing 0.5% FCS) was added to each well, and the plate was incubated at room temperature for 2 hours. After washing five times with PBS, 100 μL of streptavidin-alkaline phosphatase (Streptavidin-ALP) diluted 1:1000 with PBS containing 0.5% FCS was added to each well, and the plate was incubated at room temperature for 1 hour. After washing five times with PBS, 100 μL of substrate (BCIP / NBT-plus) was added to each well. After 15 minutes of development, when spots were visible, the plate was thoroughly rinsed with deionized water and dried. The number of spots was counted and analyzed using an automated ELISpot plate reader and its accompanying image analysis software (Mabtech).

[0138] The results, shown in Figure 6, indicate that IFN-γ secretion was detected in calf peripheral blood mononuclear cells (PBMCs) of both the DAM and AV-41 groups after stimulation with the peptide library. Quantitative analysis showed that the average number of spots produced in the DAM group was 85.6, and the average number in the AV-41 group was 116.8, but there was no significant difference between the two groups. This ELISApot result demonstrates that DAM protein combined with ISA201 adjuvant can effectively stimulate PBMCs to secrete IFN-γ, confirming its ability to induce antigen-specific cellular immune responses, and that its ability to induce cellular immune responses is comparable to that of commercially available vaccines.

[0139] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of this application, and are not intended to limit them. Although this application has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of this application. Industrial applicability

[0140] The recombinant chimeric antigen against bovine nodular dermatitis virus provided in this application can elicit an immune response against two types of infectious viral particles: intracellular mature viral particles (IMV) and extracellular enveloped viral particles (EEV), thereby efficiently eliciting a specific immune protective effect against bovine nodular dermatitis virus. In addition, the bovine nodular dermatitis virus vaccine of this application also has good safety, rapid response and production capacity, and has excellent prospects for clinical application.

[0141] The sequences discussed in this article:

Claims

A bovine nodular dermatitis virus recombinant chimeric antigen, characterized in that, The recombinant chimeric antigen is a single-chain fusion protein comprising bovine nodular dermatitis virus ORF122, ORF060 proteins or antigenic fragments thereof, and the recombinant chimeric antigen comprises an amino acid sequence arranged according to formula (I): A1-C1-B1-C2-A2-C3-B2 (I) in: A1 and A2 each independently represent the bovine nodular dermatosis virus ORF122 protein or its antigenic fragment, or an amino acid sequence that has at least 90%, 92%, 95%, 96%, 97%, 98%, or 99% identity with it and has the same or substantially the same immunogenicity as it. B1 and B2 each independently represent the bovine nodular dermatosis virus ORF060 protein or its antigenic fragment, or an amino acid sequence that has at least 90%, 92%, 95%, 96%, 97%, 98%, or 99% identity with it and has the same or substantially the same immunogenicity. C1, C2, and C3 are each independently either none or a concatenated sequence (GGGGS)n, where n is any integer between 1 and 10; and, in, A1 and A2 may be the same or different. B1 and B2 may be the same or different. C1, C2, and C3 may be the same or different. The bovine nodular dermatitis virus recombinant chimeric antigen according to claim 1 is characterized in that, The ORF122 and ORF060 proteins or their antigenic fragments of bovine nodular skin disease virus are derived from Lumpy skin disease virus NI-2490; Optionally, A1 and / or A2 each independently represent the amino acid sequence shown in SEQ ID NO:1, or the amino acid sequence shown in SEQ ID NO:1 plus a fragment extending 1-33 amino acids from it to the N-terminus of the ORF122 protein, or an amino acid sequence with the same or substantially the same immunogenicity obtained by substituting, deleting, or adding one or more amino acids to the above amino acid sequence; preferably, A1 represents the amino acid sequence shown in SEQ ID NO:1, or an amino acid sequence with the same or substantially the same immunogenicity obtained by substituting, deleting, or adding one or more amino acids to the amino acid sequence shown in SEQ ID NO:1; and / or, A2 represents the amino acid sequence shown in SEQ ID NO:2, or an amino acid sequence with the same or substantially the same immunogenicity obtained by substituting, deleting, or adding one or more amino acids to the amino acid sequence shown in SEQ ID NO:2; And / or, B1 and / or B2 each independently represent the amino acid sequence shown in SEQ ID NO:3, or the amino acid sequence shown in SEQ ID NO:3 plus a fragment extending 1-2 amino acids from it to the N-terminus of the ORF060 protein, or an amino acid sequence with the same or substantially the same immunogenicity obtained by substituting, deleting or adding one or more amino acids to the above amino acid sequence; preferably, B1 and / or B2 represent the amino acid sequence shown in SEQ ID NO:3, or an amino acid sequence with the same or substantially the same immunogenicity obtained by substituting, deleting or adding one or more amino acids to the amino acid sequence shown in SEQ ID NO:

3. The bovine nodular dermatitis virus recombinant chimeric antigen according to claim 2 is characterized in that, A1 represents the amino acid sequence shown in SEQ ID NO:1, A2 represents the amino acid sequence shown in SEQ ID NO:2, B1 represents the amino acid sequence shown in SEQ ID NO:3, and B2 represents the amino acid sequence shown in SEQ ID NO:

3. Preferably, C1, C2, and C3 are all absent; More preferably, the recombinant chimeric antigen comprises the amino acid sequence shown in SEQ ID NO:

4. The bovine nodular dermatitis virus recombinant chimeric antigen according to any one of claims 1-3 is characterized in that, The N-terminus of the recombinant chimeric antigen further includes a signal peptide sequence; optionally, the signal peptide sequence is shown in SEQ ID NO:5; And / or, the C-terminus of the recombinant chimeric antigen further includes a tag sequence; optionally, the tag is selected from at least one of the Flag tag, His tag, MBP tag, HA tag, myc tag, GST tag, and SUMO tag, preferably the His tag. A method for preparing the bovine nodular dermatitis virus recombinant chimeric antigen as described in any one of claims 1-3, comprising the following steps: The nucleotide sequence encoding the bovine nodular dermatitis virus recombinant chimeric antigen as described in any one of claims 1-3 is coupled with a Kozak sequence and a signal peptide coding sequence at the 5' end, and a histidine tag coding sequence and a stop codon at the 3' end. The recombinant antigen is then cloned and expressed, and the correct recombinant is screened. The recombinant antigen is then transfected into expression system cells for expression. The cell culture supernatant is collected, and the bovine nodular dermatitis virus recombinant chimeric antigen is isolated from it. The preparation method according to claim 5 is characterized in that: The expression system cells are mammalian cells, insect cells, yeast cells, or bacterial cells; Optionally, the mammalian cells are HEK293T cells, 293F series cells, or CHO cells; more preferably, the 293F series cells are HEK293F cells, Freestyle293F cells, or Expi293F cells. Optionally, the insect cells are sf9 cells, Hi5 cells, sf21 cells, or S2 cells; Optionally, the yeast cells are Pichia pastoris cells or yeast cells modified therefrom; Optionally, the bacterial cells are Escherichia coli cells. A polynucleotide encoding a bovine nodular dermatitis virus recombinant chimeric antigen as described in any one of claims 1-4. The polynucleotide according to claim 7 is characterized in that: The polynucleotide is DNA or mRNA; Preferably, the polynucleotide comprises a DNA sequence as shown in SEQ ID NO:6; Preferably, the polynucleotide comprises an mRNA sequence as shown in SEQ ID NO:

7. A nucleic acid construct comprising a polynucleotide as described in claim 7 or 8, and optionally, at least one expression regulatory element operatively linked to said polynucleotide. An expression vector comprising the nucleic acid construct as described in claim 9. A host cell wherein the polynucleotide of claim 7 or 8, the nucleic acid construct of claim 9, or the expression vector of claim 10 is transformed or transfected. The use of the bovine nodular dermatovirus recombinant chimeric antigen as described in any one of claims 1-4, the polynucleotide as described in claim 7 or 8, the nucleic acid construct as described in claim 9, the expression vector as described in claim 10, or the host cell as described in claim 11 in the preparation of a medicament for the prevention and / or treatment of bovine nodular dermatovirus infection; Optionally, the drug is a vaccine, preferably a recombinant protein vaccine; more preferably, the recombinant protein vaccine uses an adjuvant selected from the following: aluminum adjuvant, MF59 adjuvant, MF59-like adjuvant, and ISA201 adjuvant; Optionally, the vaccine may be in the form of a nasal spray, oral preparation, suppository, or parenteral preparation; Preferably, the nasal spray is selected from aerosols, sprays, and powders; Preferably, the oral preparation is selected from tablets, powders, pills, granules, soft / hard capsules, film-coated preparations, and ointments; more preferably, the tablets are sublingual tablets; more preferably, the granules are fine granules; more preferably, the powder is a powder; more preferably, the pills are small pills; Preferably, the parenteral preparation is a transdermal preparation, ointment, plaster, topical liquid, or injectable preparation; more preferably, the injectable preparation is a push-in preparation. A vaccine or immunogenic composition comprising a bovine nodular dermatovirus recombinant chimeric antigen as described in any one of claims 1-4, a polynucleotide as described in claim 7 or 8, a nucleic acid construct as described in claim 9, an expression vector as described in claim 10, or a host cell as described in claim 11, and a physiologically acceptable medium, adjuvant, excipient, carrier, and / or diluent. The vaccine or immunogenic composition according to claim 13 is a bovine nodular dermatitis virus recombinant protein vaccine, comprising the bovine nodular dermatitis virus recombinant chimeric antigen and adjuvant as described in any one of claims 1-4; Optionally, the adjuvant is one or more selected from the following adjuvants: aluminum adjuvant, MF59 adjuvant, MF59-like adjuvant, and ISA201 adjuvant. The vaccine or immunogenic composition according to claim 13 is a bovine nodular dermatosis virus DNA vaccine, said DNA vaccine comprising: (1) Eukaryotic expression vector; and (2) Constructing a DNA sequence encoding the bovine nodular dermatitis virus recombinant chimeric antigen as described in any one of claims 1-4 into the eukaryotic expression vector, preferably the DNA sequence shown in SEQ ID NO:6; Optionally, the eukaryotic expression vector is selected from pGX0001, pVAX1, pCAGGS and pcDNA series vectors. The vaccine or immunogenic composition according to claim 13 is a bovine nodular dermatosis virus mRNA vaccine, said mRNA vaccine comprising: (I) An mRNA sequence encoding the bovine nodular dermatitis virus recombinant chimeric antigen as described in any one of claims 1-4, preferably the mRNA sequence shown in SEQ ID NO:7; and (II) Lipid nanoparticles. The vaccine or immunogenic composition according to claim 13, being a bovine nodular dermatitis virus-viral vector vaccine, comprises: (1) Viral backbone vector; and (2) A DNA sequence encoding the bovine nodular dermatitis virus recombinant chimeric antigen as described in any one of claims 1-4, preferably a DNA sequence as shown in SEQ ID NO:6, is constructed into the viral backbone vector; Optionally, the viral backbone vector is selected from one or more of the following viral vectors: adenovirus vector, poxvirus vector, influenza virus vector, and adeno-associated virus vector. The vaccine or immunogenic composition according to claim 13 is a bovine nodular dermatosis virus nanoparticle vaccine; Preferably, the carrier of the nanoparticles is ferritin, and the bovine nodular dermatitis virus recombinant chimeric antigen as described in any one of claims 1-4 is covalently linked to ferritin and self-assembled into nanoparticles, such that the bovine nodular dermatitis virus recombinant chimeric antigen is present on the surface of the nanoparticles; more preferably, the bovine nodular dermatitis virus recombinant chimeric antigen and ferritin are linked by a linker, or covalently linked by a SpyTag / SpyCatcher linker system. The vaccine or immunogenic composition according to any one of claims 13-18 is characterized in that, The vaccine or immunogenic composition is in the form of a nasal spray, oral formulation, suppository, or parenteral formulation; Preferably, the nasal spray is selected from aerosols, sprays, and powders; Preferably, the oral formulation is selected from tablets, powders, pills, granules, soft / hard capsules, film-coated agents, and ointments; More preferably, the tablet is a sublingual tablet; More preferably, the granules are fine granules; More preferably, the powder is a granule; More preferably, the pills are small pills; Preferably, the parenteral preparation is a transdermal preparation, ointment, plaster, topical liquid, or injectable preparation; more preferably, the injectable preparation is a push-in preparation.

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