Method for using multi-epitope antigen to construct RNA vaccine for fipv

Abstract

Provided is a method for using a multi-epitope antigen to construct an RNA vaccine for feline infectious peritonitis virus (FIPV). The method relates to an isolated nucleic acid molecule. The nucleic acid molecule comprises: at least one of a first nucleic acid fragment, a second nucleic acid fragment, a third nucleic acid fragment, a fourth nucleic acid fragment and a fifth nucleic acid fragment, wherein the first nucleic acid fragment is derived from the N-terminal domain (NTD) of the N protein of the FIPV strain QS, the second nucleic acid fragment is derived from the C-terminal domain (CTD) of the N protein from the FIPV strain QS, the third nucleic acid fragment encodes the NSP12 protein of the FIPV strain QS, the fourth nucleic acid fragment is derived from the epitope HR2_4 of the S protein of the FIPV strain 79-1146, the fifth nucleic acid fragment is derived from the epitope HR2_11 of the S protein of the FIPV strain 79-1146, and the nucleic acid molecule is RNA.

Description

Method for constructing RNA vaccine targeting multi-epitope antigens of feline FIPV Technical Field

[0001] The present invention relates to the field of biotechnology, and specifically, to a method for constructing an RNA vaccine against the multi-epitope antigens of feline FIPV. More specifically, it relates to an isolated nucleic acid molecule, an expression vector, a recombinant virus, a liposome, a vaccine, a recombinant cell, and a method and use of constructing a feline infectious peritonitis virus vaccine. Background Art

[0002] Feline coronavirus (FCoV) belongs to the coronavirus family. Viruses in the Coronaviridae family are characterized by relatively large, round, enveloped, positive-strand RNA viruses with genomes ranging from 27 to 32 kb. These viruses encode replication polymerases, four structural proteins (S, M, N, and E), and several nonstructural proteins. The S protein (spike protein) is a key structural protein of coronaviruses. It forms the surface protrusions of these viruses and is a key factor in their infection. The S protein binds to receptors on host cells, allowing them to enter and infect. Furthermore, the S protein is a key antigen in many coronavirus vaccines. The M protein (membrane protein) is involved in the formation and localization of coronavirus particles. It spans the viral envelope and interacts with other proteins to form the structure of the virion. The N protein (nucleocapsid protein) encapsulates the viral RNA genome and is involved in viral gene replication and transcription. It also stimulates the host immune system to respond to the virus. E protein (Envelope protein) is a protein on the coronavirus envelope that can interact with M protein to form the structure of virus particles. It is also involved in the infection and assembly process of the virus.

[0003] According to relevant research on the new coronavirus, it was found that the N-terminus of the N protein has a conserved amino acid site that binds to nucleic acids, which is relatively conserved among coronaviruses and has multiple T cell or B cell epitopes; the C-terminus of the coronavirus's N protein can form a special secondary structure; and studies have reported that the area where T cell or B cell epitopes are concentrated mainly exists in the NSP12 non-structural protein; the two B cell epitopes HR2_4 and HR2_11 are derived from the SII region of FIPV.

[0004] Feline coronaviruses (FCoV) are categorized by biotype and pathogenicity as feline enteric coronavirus (FECV) and feline infectious peritonitis virus (FIPV). FECV is highly transmissible, infecting intestinal epithelial cells and causing little to no symptoms or only mild diarrhea. FIPV primarily infects feline monocytes and macrophages. Distinguishing FECV from FIPV based on genomic sequence is difficult. Although some studies have suggested that FIPV can be distinguished from FECV by amino acid mutations in the spike protein, these mutations have subsequently been found to be more correlated with tissue tropism.

[0005] The typical characteristics of FIPV are purulent granulomatous lesions in various tissues and organs, including the lungs, liver, spleen, omentum and brain. Infection of macrophages and monocytes is considered to be the key to the pathogenic mechanism. At the end of FIPV infection, a large decrease in T cells in peripheral and lymphoid tissues can be observed, and hypergammaglobulinemia is often present, indicating the presence of severe virus-induced immune disorders. Humoral immunity does not seem to have a protective effect and may lead to "early death syndrome". When S antibodies are present in sub-neutralizing titers, they can enhance the infection of target cells by binding to Fc receptors. Researchers have tried many times to develop FIPV vaccines, but most of them have failed. The main reason for the failure is the phenomenon of antibody-dependent enhancement (ADE) infection, which makes the antibodies unable to play an effective protective role. Currently, researchers are trying to control the infection and clearance of FIPV through cell-mediated immunity (CMI), but have not yet achieved good protection.

[0006] Therefore, there is an urgent need in this field to develop a vaccine against FIPV.

[0007] Summary of the Invention

[0008] This application is filed by the inventor based on the following findings:

[0009] Due to the long-term lack of new breakthroughs in the development of FIPV vaccines, FIPV infections almost always end in the death of cats.

[0010] To this end, in a first aspect, the present invention provides an isolated nucleic acid molecule. According to an embodiment of the present invention, the isolated nucleic acid molecule comprises at least one of a first nucleic acid fragment, a second nucleic acid fragment, a third nucleic acid fragment, a fourth nucleic acid fragment, and a fifth nucleic acid fragment; wherein the first nucleic acid fragment is derived from the N-terminus (NTD) of the N protein of the QS strain of feline infectious peritonitis virus; the second nucleic acid fragment is derived from the C-terminus (CTD) of the N protein of the QS strain of feline infectious peritonitis virus; the third nucleic acid fragment encodes the NSP12 protein of the QS strain of feline infectious peritonitis virus; the fourth nucleic acid fragment is derived from the epitope HR2_4 of the S protein of the 79-1146 strain of feline infectious peritonitis virus; and the fifth nucleic acid fragment is derived from the epitope HR2_11 of the S protein of the 79-1146 strain of feline infectious peritonitis virus; and the nucleic acid molecule is RNA. According to an embodiment of the present invention, the nucleic acid molecule expressing the feline infectious peritonitis virus can stimulate an animal's somatic cell-mediated immune response.

[0011] It should be noted that in the present application, the NTD, CTD, NSP12, HR2_4 or HR2_11 protein sequences of the wild-type FIPV virus can also be adaptively modified as needed to improve antigen expression and reduce the toxicity of the FIPV virus without affecting its three-dimensional structure and retaining its immunogenicity, thereby preparing a new type of FIPV virus vaccine. According to the sequence alignment results (Tables 1 and 2), the NTD protein has an amino acid sequence that is at least 91% homologous to SEQ ID NO: 1, and the NTD protein nucleic acid fragment has a nucleotide sequence that is at least 71% identical to SEQ ID NOs: 16 to 19; the CTD protein has an amino acid segment that is at least 92% homologous to SEQ ID NO: 2, and the CTD protein nucleic acid segment has a nucleotide sequence that is at least 72% identical to SEQ ID NOs: 20 to 23; the NSP12 protein has an amino acid segment that is at least 97% homologous to SEQ ID NO: 3, and the NSP12 protein nucleic acid segment has a nucleotide sequence that is at least 71% identical to SEQ ID NOs: 24 to 27; the HR2_4 protein has an amino acid segment that is at least 50% homologous to SEQ ID NO: 4, and the HR2_4 protein nucleic acid segment has a nucleotide sequence that is at least 50% identical to SEQ ID NOs: 28 to 31; the HR2_11 protein has an amino acid segment that is at least 95% homologous to SEQ ID NO: 5, and the HR2_11 protein nucleic acid segment has a nucleotide sequence that is at least 72% identical to SEQ ID NOs: 20 to 23. NO:32-35 Nucleotide sequences with at least 73% identity. FIPV virus vaccines are not particularly limited, as long as they can produce the modified FIPV virus NTD, CTD, NSP12, HR2_4 and / or HR2_11 protein receptor binding region in the organism, and have immunogenicity and can stimulate the organism to produce a corresponding immune response. Moreover, the isolated nucleic acid molecules can be used to stimulate the immune response of all animals that can be infected with feline infectious peritonitis virus, including but not limited to cats.

[0012] It should be noted that the linker (connecting peptide) is a flexible or rigid amino acid chain that acts as a link between two fusion proteins, such as 3×flag, EAAAK, GGGS, AAY, GPGPG, (GGGGS)n, etc. In this application, commonly used linker sequences in experiments can be applied to the embodiments of this application.

[0013] It should be noted that in this application, the NTD, CTD, NSP12, HR2_4, and HR2_11 are connected via a linker, but the connection method is not particularly limited. For example, the nucleic acid molecules can be connected in the form of NTD-linker-CTD-linker-NSP12-linker-HR2_4-linker-HR2_11, or NTD-linker-NSP12-linker-CTD-linker-HR2_4-linker-HR2_11, etc.

[0014] Table 1: Protein sequence homology among multiple substrains (%)

[0015] Table 2: Nucleic acid sequence identity among multiple substrains (%) Note: - means none.

[0016] According to an embodiment of the present invention, the isolated nucleic acid molecule may further include at least one of the following technical features:

[0017] According to an embodiment of the present invention, the nucleic acid fragments are connected or not connected.

[0018] According to an embodiment of the present invention, the nucleic acid fragments are connected by a linker. In some examples of the present application, the first to fifth nucleic acid fragments are connected by a linker as a nucleic acid molecule, which can stimulate an immune response mediated by animal somatic cells.

[0019] In some examples of this application, the nucleic acid fragments are not connected. In some examples of this application, any of the first to fifth nucleic acid fragments can stimulate an animal's somatic cell-mediated immune response. For example, immunizing a test animal with the first nucleic acid fragment alone can stimulate an animal's somatic cell-mediated immune response.

[0020] In other examples of the present application, the first to fifth nucleic acid fragments can be freely combined to stimulate an immune response mediated by animal somatic cells. For example, the first nucleic acid fragment and the second nucleic acid fragment are connected to immunize the test animal, which can also stimulate an immune response mediated by animal somatic cells. Among them, the combination method can be set based on actual experimental needs. In some examples of the present application, any combination method can achieve the effect of stimulating an immune response mediated by animal somatic cells.

[0021] According to an embodiment of the present invention, any one of the nucleic acid fragments comprises at least 15 amino acids. In some examples of this application, the inventors have found through extensive experimental verification that 15 amino acids from any nucleic acid fragment can stimulate an animal's somatic cell-mediated immune response.

[0022] In some examples of the present application, the 15 amino acids in any nucleic acid fragment can also be connected or not connected. Experimental verification shows that the effect of stimulating the immune response mediated by animal somatic cells can be achieved under the conditions of connection or not.

[0023] According to an embodiment of the present invention, the NTD protein has an amino acid sequence that is at least 91% homologous to SEQ ID NO: 1.

[0024] According to an embodiment of the present invention, the NTD protein has the amino acid sequence shown in SEQ ID NO:1.

[0025] According to an embodiment of the present invention, the CTD protein has an amino acid sequence that is at least 92% homologous to SEQ ID NO:2.

[0026] According to an embodiment of the present invention, the CTD protein has the amino acid sequence shown in SEQ ID NO: 2.

[0027] According to an embodiment of the present invention, the NSP12 protein has an amino acid sequence that is at least 97% homologous to SEQ ID NO: 3.

[0028] According to an embodiment of the present invention, the NSP12 protein has the amino acid sequence shown in SEQ ID NO:3.

[0029] According to an embodiment of the present invention, the HR2_4 protein has an amino acid sequence that is at least 50% homologous to SEQ ID NO:4.

[0030] According to an embodiment of the present invention, the HR2_4 protein has the amino acid sequence shown in SEQ ID NO:4.

[0031] According to an embodiment of the present invention, the HR2_11 protein has an amino acid sequence that is at least 95% homologous to SEQ ID NO:5.

[0032] According to an embodiment of the present invention, the HR2_11 protein has the amino acid sequence shown in SEQ ID NO:5.

[0033] According to an embodiment of the present invention, the first nucleic acid fragment has a nucleotide sequence that is at least 71% identical to any one of SEQ ID NOs: 16 to 19.

[0034] According to an embodiment of the present invention, the first nucleic acid fragment has a nucleotide sequence shown in SEQ ID NOs: 16 to 19.

[0035] According to an embodiment of the present invention, the second nucleic acid fragment has a nucleotide sequence that is at least 72% identical to any one of SEQ ID NOs: 20 to 23.

[0036] According to an embodiment of the present invention, the second nucleic acid fragment has a nucleotide sequence shown in SEQ ID NOs: 20 to 23.

[0037] According to an embodiment of the present invention, the third nucleic acid fragment has a nucleotide sequence that is at least 71% identical to any one of SEQ ID NOs: 24-27.

[0038] According to an embodiment of the present invention, the third nucleic acid fragment has the nucleotide sequence shown in SEQ ID NOs: 24 to 27.

[0039] According to an embodiment of the present invention, the fourth nucleic acid fragment has a nucleotide sequence that is at least 50% identical to any one of SEQ ID NOs: 28 to 31.

[0040] According to an embodiment of the present invention, the fourth nucleic acid fragment has the nucleotide sequence shown in SEQ ID NO: 28-31.

[0041] According to an embodiment of the present invention, the fifth nucleic acid fragment has a nucleotide sequence that is at least 73% identical to any one of SEQ ID NOs: 32 to 35.

[0042] According to an embodiment of the present invention, the fifth nucleic acid fragment has the nucleotide sequence shown in SEQ ID NOs: 32 to 35.

[0043] It should be noted that, in the present application, the homology of the amino acid sequence refers to the similarity between two amino acid sequences; the identity of the nucleotide sequence refers to the similarity between two nucleotide sequences.

[0044] According to an embodiment of the present invention, the nucleic acid molecule further includes a sixth nucleic acid segment encoding an MHC-I (major histocompatibility complex I) signal peptide sequence (MHC-I sp). According to an embodiment of the present invention, the purpose of adding the MHC-I signal peptide to the N-terminus of the antigen sequence is to enable ribosomes to attach to the endoplasmic reticulum membrane and guide protein transport within the cell.

[0045] According to an embodiment of the present invention, the signal peptide sequence of MHC-I does not contain a transmembrane region.

[0046] According to an embodiment of the present invention, the MHC-I signal peptide sequence has an amino acid sequence as shown in SEQ ID NO:6.

[0047] According to an embodiment of the present invention, the sixth nucleic acid fragment has the nucleotide sequence shown in SEQ ID NOs: 36 to 38.

[0048] According to an embodiment of the present invention, the sixth nucleic acid fragment is disposed at the 5' end of the nucleic acid molecule.

[0049] According to an embodiment of the present invention, the nucleic acid further comprises a seventh nucleic acid segment encoding a MITD (major histocompatibility complex class I molecule transport signal) sequence. According to an embodiment of the present invention, adding a MITD sequence to the C-terminus of the nucleic acid molecule can stimulate CD4 + T cells proliferate, inducing the production of more cytokines.

[0050] According to an embodiment of the present invention, the MITD sequence comprises a transmembrane region.

[0051] According to an embodiment of the present invention, the MITD sequence has the amino acid sequence shown in SEQ ID NO:7.

[0052] According to an embodiment of the present invention, the seventh nucleic acid fragment has the nucleotide sequence shown in SEQ ID NOs: 39 to 41.

[0053] According to an embodiment of the present invention, the seventh nucleic acid fragment is disposed at the 3' end of the nucleic acid molecule.

[0054] According to an embodiment of the present invention, the eighth nucleic acid fragment encodes the HBHA (heparin-binding hemagglutinin protein of Mycobacterium tuberculosis) adjuvant sequence. According to an embodiment of the present invention, HBHA has a strong immunostimulatory effect and can induce the maturation of DC cells, thereby planning CD4 + and CD8 + T cells, secrete IFN-γ, and induce T cell-mediated cytotoxicity.

[0055] According to an embodiment of the present invention, the HBHA sequence has the amino acid sequence shown in SEQ ID NO:8.

[0056] According to an embodiment of the present invention, the eighth nucleic acid fragment has the nucleotide sequence shown in SEQ ID NOs: 42 to 44.

[0057] According to an embodiment of the present invention, the ninth nucleic acid fragment encodes a PADRE (Pan HLA-DR reactive epitope) sequence; according to an embodiment of the present invention, PADRE is a "universal" 13-amino acid pan-HLA DR peptide epitope used to activate CD4+ T cells.

[0058] According to an embodiment of the present invention, the PADRE sequence has the amino acid sequence shown in SEQ ID NO:9.

[0059] According to an embodiment of the present invention, the ninth nucleic acid fragment has the nucleotide sequence shown in SEQ ID NOs: 45 to 47.

[0060] According to an embodiment of the present invention, the nucleic acid molecule further comprises a rigid or flexible linker sequence.

[0061] According to an embodiment of the present invention, the linker sequence has the amino acid sequence shown in SEQ ID NOs: 11 to 15.

[0062] According to an embodiment of the present invention, the linker sequence has the nucleotide sequence shown in SEQ ID NO: 51-70.

[0063] According to an embodiment of the present invention, the nucleic acid molecule is linear.

[0064] In a second aspect, the present invention provides an expression vector. According to an embodiment of the present invention, the expression vector carries the nucleic acid molecule described in the first aspect of the present invention. According to an embodiment of the present invention, the expression vector can be expressed in cells, bacteria, yeast, or feline organisms.

[0065] According to an embodiment of the present invention, the above-mentioned expression vector further includes at least one of the following technical features:

[0066] According to an embodiment of the present invention, the expression vector is a non-pathogenic viral vector.

[0067] According to an embodiment of the present invention, the non-pathogenic virus is selected from at least one of a retrovirus, a lentivirus, an adenovirus and an adeno-associated virus.

[0068] In a third aspect, the present invention provides a recombinant virus. According to an embodiment of the present invention, the recombinant virus carries the nucleic acid molecule described in the first aspect of the present invention. The recombinant virus containing the nucleic acid molecule described in the first aspect can be stably propagated in large quantities.

[0069] In a fourth aspect, the present invention provides a liposome. According to an embodiment of the present invention, the liposome comprises a liposome carrier and a nucleic acid fragment, wherein the nucleic acid fragment is as defined in the first aspect of the present invention. The liposome containing the liposome carrier and the nucleic acid fragment plays an important role in improving nucleic acid stability, cellular uptake, reducing toxic side effects, and improving delivery efficiency.

[0070] In its fifth aspect, the present invention provides a vaccine. According to embodiments of the present invention, the vaccine comprises the nucleic acid molecule described in the first aspect, the expression vector described in the second aspect, the recombinant virus described in the third aspect, or the liposome described in the fourth aspect. According to embodiments of the present invention, the aforementioned vaccines can efficiently activate cell-mediated immune responses in animals. Furthermore, the vaccine contains only proteins capable of activating cellular immune responses, thus avoiding toxic side effects and providing enhanced safety.

[0071] According to an embodiment of the present invention, the above vaccine may further include at least one of the following additional technical features:

[0072] According to an embodiment of the present invention, the vaccine includes at least one selected from RNA vaccine, DNA vaccine, protein recombinant vaccine, inactivated vaccine, attenuated vaccine, and viral vector vaccine.

[0073] According to an embodiment of the present invention, the vaccine is an RNA vaccine.

[0074] According to an embodiment of the present invention, the vaccine further comprises an adjuvant.

[0075] According to an embodiment of the present invention, the adjuvant includes TLR agonist, Mn 2+ At least one of.

[0076] According to an embodiment of the present invention, the TLR agonist includes at least one of HBHA, CpG, R837, MPLA and derivatives thereof.

[0077] In a sixth aspect, the present invention provides a recombinant cell. According to embodiments of the present invention, the recombinant cell carries the nucleic acid molecule described in the first aspect of the present invention, the expression vector described in the second aspect of the present invention, or the recombinant virus described in the third aspect of the present invention. According to embodiments of the present invention, the recombinant cell is used to package a virus carrying the nucleic acid molecule for use in preparing a nucleic acid vaccine to stimulate an immune response in the body.

[0078] In its seventh aspect, the present invention provides a method for constructing a feline infectious peritonitis virus vaccine. According to embodiments of the present invention, the method comprises introducing the nucleic acid molecule described in the first aspect of the present invention, the expression vector described in the second aspect, or the recombinant virus described in the third aspect into a recipient cell. The method according to embodiments of the present invention can package a virus carrying the nucleic acid molecule for use in preparing a nucleic acid vaccine. This method for constructing an infectious peritonitis virus vaccine is safe, simple, and highly effective.

[0079] According to an embodiment of the present invention, the above method further includes at least one of the following technical features:

[0080] According to an embodiment of the present invention, prior to introduction into the recipient cells, the method further includes encapsulating the nucleic acid, expression vector, or recombinant virus with an encapsulation vector. According to an embodiment of the present invention, encapsulating the nucleic acid, expression vector, or recombinant virus with an encapsulation vector can protect the vaccine components from external environmental interference that affects their potency. The encapsulation vector can also reduce the contact of the vaccine components with the external environment, thereby reducing the risk of contamination of the vaccine components and improving the safety of the vaccine. In addition, some encapsulation vectors also have the effect of enhancing the infectivity of the vaccine components, thereby enabling them to more effectively stimulate an immune response.

[0081] According to an embodiment of the present invention, the encapsulation carrier is selected from at least one of liposomes, polymer carriers, viral carriers, and nanoparticles.

[0082] According to a specific embodiment of the present invention, the carrier is a nanoparticle.

[0083] According to an embodiment of the present invention, the recipient cell is a CRFK cell, HEK293FT, HEK293T or BHK cell.

[0084] According to an embodiment of the present invention, the recipient cells are CRFK cells.

[0085] In an eighth aspect, the present invention provides a use of the nucleic acid molecule described in the first aspect, the expression vector described in the second aspect, the recombinant virus described in the third aspect, the liposome described in the fourth aspect, or the recombinant cell described in the sixth aspect in the preparation of a drug or vaccine. According to an embodiment of the present invention, the drug or vaccine is used to prevent or treat diseases associated with feline infectious peritonitis virus infection. According to an embodiment of the present invention, the drug or vaccine prepared based on the aforementioned nucleic acid molecule, expression vector, recombinant virus, or recombinant cell has high safety and can activate an animal cell-mediated immune response in a short period of time.

[0086] In the ninth aspect of the present invention, the present invention proposes a method for preventing or treating feline infectious peritonitis virus infection. According to an embodiment of the present invention, the method comprises: administering the nucleic acid molecule described in the first aspect of the present invention, the expression vector described in the second aspect, the recombinant virus described in the third aspect, the liposome described in the fourth aspect, the vaccine described in the fifth aspect, or the recombinant cell described in the sixth aspect to the test animal. According to an embodiment of the present invention, administering an effective dose of a pharmaceutical preparation, nucleic acid molecule, expression vector, recombinant virus, liposome, vaccine or recombinant cell to a test animal infected with FIPV can significantly improve the various physiological indicators and survival rate of the test animal. In addition, the aforementioned treatment methods have good immune effects on various epidemic strains of FIPV.

[0087] Herein, the term "effective dose" refers to an amount that can produce a function or activity on a subject animal and can be accepted by the subject animal.

[0088] The effective amount of the nucleic acid molecule, expression vector, recombinant virus, liposome, vaccine or recombinant cell of the present invention may vary depending on the mode of administration and the severity of FIPV infection in the test animal. The selection of the preferred effective amount can be determined by a person of ordinary skill in the art based on various factors (e.g., through clinical trials). Various factors include, but are not limited to: pharmacokinetic parameters of the active ingredient, such as bioavailability, metabolism, half-life, etc.; the severity of FIPV infection in the test animal, the weight of the test animal, the immune status of the test animal, the route of administration, etc. For example, depending on the urgency of the treatment situation, several divided doses may be administered daily, or the dose may be reduced proportionally.

[0089] According to an embodiment of the present invention, the above method may further include at least one of the following technical features:

[0090] According to an embodiment of the present invention, the test animal is selected from cats.

[0091] In the twelfth aspect of the present invention, the present invention proposes a use of the nucleic acid molecule described in the first aspect, the expression vector described in the second aspect, the recombinant virus described in the third aspect, the liposome described in the fourth aspect, the vaccine described in the fifth aspect or the recombinant cell described in the sixth aspect in preventing or treating feline infectious peritonitis virus infection. According to an embodiment of the present invention, administering an effective dose of nucleic acid molecules, expression vectors, recombinant viruses, liposomes, vaccines or recombinant cells to a test animal infected with FIPV can significantly improve the various physiological indicators and survival rates of the test animals. In addition, the aforementioned treatment methods have good immune effects on various epidemic strains of FIPV.

[0092] Additional aspects and advantages of the present invention will be set forth in part in the description which follows and, in part, will be obvious from the description which follows, or may be learned by practice of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0093] The above and / or additional aspects and advantages of the present invention will become apparent and readily understood from the following description of the embodiments with reference to the accompanying drawings, in which:

[0094] FIG1 is a result of detecting the expression of target mRNA encapsulated by LNP according to Example 1 of the present invention.

[0095] FIG2 shows the change in survival rate after immunization with the target mRNA packaged in LNP according to Example 2 of the present invention.

[0096] FIG3 is a median statistical result of ELISPOT detection stimulated by a multi-epitope antigen polypeptide library after immunization with the target mRNA packaged by LNP according to Example 3 of the present invention.

[0097] FIG4 is a statistical result of ELISPOT detection of multi-epitope antigen polypeptide stimulation after immunization with LNP-encapsulated target mRNA according to Example 3 of the present invention.

[0098] FIG5 shows the change in survival rate after immunization with the target mRNA packaged in LNP according to Example 4 of the present invention. DETAILED DESCRIPTION

[0099] The embodiments of the present invention are described in detail below, and examples of the embodiments are shown in the accompanying drawings. The embodiments described below with reference to the accompanying drawings are exemplary and intended to be used to explain the present invention, but should not be understood as limiting the present invention.

[0100] Furthermore, the terms "first" and "second" are used for descriptive purposes only and should not be construed as indicating or implying relative importance or implicitly specifying the number of the technical features being referred to. Thus, a feature defined as "first" or "second" may explicitly or implicitly include at least one such feature. In the description of the present invention, "plurality" means at least two, such as two, three, etc., unless otherwise specifically defined.

[0101] In this application, unless otherwise specified, the term "non-pathogenic viral vector" refers to a class of viruses that can express target antigens and are non-pathogenic, and are usually used to prepare vaccines. These vectors are genetically modified and the gene sequence of the target antigen is added, which is then expressed and replicated by the viral vector. The advantages of using non-pathogenic viral vectors as vaccine vectors are that they can induce the immune system to produce a strong immune response, thereby stimulating the body's immune response to the target antigen; non-pathogenic viral vectors are usually designed to be unable to replicate, so they will not reproduce in the body and will not cause disease; compared with other traditional vaccine preparation methods, vaccines prepared with non-pathogenic viral vectors are more convenient and safer in production and storage, and have better stability and purity. Beneficial effects

[0102] The RNA vaccine for preventing feline infectious peritonitis described in the present invention is prepared by constructing a vector encoding multiple epitope antigens of the FIPV virus, and then preparing an RNA vaccine for preventing the FIPV virus through lipid nanoparticles (LNP). The vaccine can produce a strong immune response after immunizing cats. The vaccine described in the present invention uses the N-terminus, C-terminus, NSP12 protein sequence of the N protein of the FIPV virus and the HR2-4 and HR2-11 sequences of the S protein as the main components of the constructed nucleic acid molecule. The RNA vaccine expressed by the nucleic acid molecule has the advantages of simple preparation process, high safety, no toxic side effects, and industrial production; sufficient protection effect can be achieved using a very small dose, and it is superior to existing treatment methods in terms of safety and effectiveness.

[0103] The sequences involved in this application are shown in Table 3:

[0104] Table 4: Description of sequences involved in the examples of the present invention

[0105] Table 5: Sequence composition involved in the examples of the present invention Note: L stands for linker; - stands for none.

[0106] Table 6: Sequence composition involved in the embodiments of the present invention Note: L stands for linker; - stands for none.

[0107] It should be noted that the sequences of the products corresponding to the names in Table 5 or Table 6 are formed by connecting the corresponding sequences in "SEQ ID NO:" in the 5' to 3' direction. Taking d3 in Table 6 as an example, the sequence of MHC-I sp-HBHA-L-PADRE-L-NTD-L-CTD-L-NSP12-L-HR2_4-L-HR2_11-L-MITD is composed of the MHC-I sp sequence shown in SEQ ID NO:38, the HBHA sequence shown in SEQ ID NO:44, the L (linker) sequence shown in SEQ ID NO:53, the PADRE sequence shown in SEQ ID NO:47, the L (linker) sequence shown in SEQ ID NO:56, the NTD sequence shown in SEQ ID NO:19, the L (linker) sequence shown in SEQ ID NO:59, the CTD sequence shown in SEQ ID NO:23, the L (linker) sequence shown in SEQ ID NO:62, the NSP12 sequence shown in SEQ ID NO:27, the L (linker) sequence shown in SEQ ID NO:65, The HR2_4 sequence shown in NO:31, the L (linker) sequence shown in SEQ ID NO:67, the HR2_11 sequence shown in SEQ ID NO:35, the L (linker) sequence shown in SEQ ID NO:70, and the MITD sequence shown in SEQ ID NO:41 are connected.

[0108] In which, the 3' end of the sequence shown by SEQ ID NO:38 is connected to the 5' end of the sequence shown by SEQ ID NO:44, the 3' end of the gene sequence shown by SEQ ID NO:44 is connected to the 5' end of the sequence shown by SEQ ID NO:53, the 3' end of the gene sequence shown by SEQ ID NO:53 is connected to the 5' end of the sequence shown by SEQ ID NO:47, the 3' end of the gene sequence shown by SEQ ID NO:47 is connected to the 5' end of the sequence shown by SEQ ID NO:56, the 3' end of the sequence shown by SEQ ID NO:56 is connected to the 5' end of the sequence shown by SEQ ID NO:19, the 3' end of the gene sequence shown by SEQ ID NO:19 is connected to the 5' end of the sequence shown by SEQ ID NO:59, the 3' end of the gene sequence shown by SEQ ID NO:59 is connected to the 5' end of the sequence shown by SEQ ID NO:23, the 3' end of the gene sequence shown by SEQ ID NO:23 is connected to the 5' end of the sequence shown by SEQ ID NO:62, and the 3' end of the sequence shown by SEQ ID NO: The 5' end of the sequence shown in SEQ ID NO: 27 is connected to the 5' end of the sequence shown in SEQ ID NO: 65, the 3' end of the gene sequence shown in SEQ ID NO: 65 is connected to the 5' end of the sequence shown in SEQ ID NO: 31, the 3' end of the gene sequence shown in SEQ ID NO: 31 is connected to the 5' end of the sequence shown in SEQ ID NO: 67, the 3' end of the sequence shown in SEQ ID NO: 67 is connected to the 5' end of the sequence shown in SEQ ID NO: 35, the 3' end of the gene sequence shown in SEQ ID NO: 35 is connected to the 5' end of the sequence shown in SEQ ID NO: 70, and the 3' end of the gene sequence shown in SEQ ID NO: 70 is connected to the 5' end of the sequence shown in SEQ ID NO: 41. In the present application, the linker is not particularly limited, and the above linker can be selected, or other flexible peptide or rigid peptide amino acid sequences can be selected for connection according to experimental requirements.

[0109] The present invention will be described below with reference to specific examples. It should be noted that these examples are merely illustrative and do not limit the present invention in any way. Where specific techniques or conditions are not indicated in the examples, they are performed according to the techniques or conditions described in the literature in this area or according to the product specifications. Reagents or instruments used that do not indicate the manufacturer are conventional products that can be obtained commercially.

[0110] Example 1: Target sequence selection

[0111] According to an embodiment of the present invention, the mRNA sequence is synthesized in vitro and then encapsulated using lipid nanoparticles (LNP) and expressed in CRFK (cat kidney) cells and cat DC (dendritic cells). The sequence with the higher expression level is selected as the final target sequence for vaccine preparation.

[0112] The specific steps are as follows:

[0113] According to an embodiment of the present invention, RNA sequences were synthesized in vitro and expressed in CRFK (cat kidney) cells, and the sequences with the highest expression levels were selected as the final target sequences for vaccine preparation. The specific steps are as follows:

[0114] 1) Codon optimization of multi-epitope antigen sequences was performed using the cat universal codon library and the cat mesenteric lymph node codon library, with three optimizations for each antigen;

[0115] 2) Sequence synthesis (Source: Universal Bio) was used to construct a vector containing the feline 5'HBB-UTR (SEQ ID NO:71), 3'HBA-UTR (SEQ ID NO:72) and polyA sequences, with the target sequence inserted between the 5'UTR and 3'UTR.

[0116] 3) After in vitro transcription into mRNA, it was packaged using LNPs and transfected into CRFK cells and cat DC cells to screen for sequences that could be expressed at high levels in both cat-derived cell lines.

[0117] According to an embodiment of the present invention, lipid nanoparticles (LNP) are prepared. The specific steps are as follows:

[0118] 1) Preparation of lipid solution: The average molecular weight of the liposome system is approximately 620.62. To prepare a 12 mM lipid solution, weigh 42.61 mg of SM-102, 4.52 mg of PEG-DMG, 9.48 mg of DSPC, and 17.86 mg of Chol, dissolve in 10 mL of anhydrous ethanol, and filter through a 0.22 μm filter membrane.

[0119] 2) Dilute the target mRNA with citric acid buffer (pH 4), mix well, and use a rapid nanodrug preparation system (Mingtai) with a flow rate precondition of 1:3 (organic phase X volume (containing cationic lipids): aqueous phase Y volume (containing nucleic acids) = 1:3) to prepare an mRNA vaccine liposome solution. Immediately place it in 30 volumes of PBS and concentrate using a 15ml ultrafiltration tube with a cutoff of 100K at 3000rpm for 20min. Finally, use 600mM sucrose solution (prepared in PBS, filtered with a 0.22μm membrane) to store an equal volume of dilution. Store the sample at -20°C for later use.

[0120] The results are shown in FIG1 , and target sequences B1, C1, and D1 that can be highly expressed in both cell lines were successfully screened (sequence descriptions and compositions are shown in Tables 4 and 5 ).

[0121] Example 2: Vaccine potency verification

[0122] The effectiveness of the vaccine was evaluated by assessing changes in physiological indicators such as body temperature, body weight, and survival rate of the test animals after mRNA vaccine immunization. The LNP preparation method was the same as in Example 1.

[0123] According to an embodiment of the present invention, D1 was constructed between the cat-derived 5'HBB-UTR (SEQ ID NO: 71) and 3'HBA-UTR (SEQ ID NO: 72), transcribed into mRNA in vitro, and packaged with LNP. The sequence description and composition are shown in Tables 4 and 5.

[0124] According to an embodiment of the present invention, test animals meeting the test criteria are screened through physical examination and laboratory tests. Physical examination items include: body temperature and weight; screening items include: PCR detection, N and S binding antibodies, and neutralizing antibody tests. The specific experimental steps are as follows:

[0125] 1) Physical examination: Measure the kitten's body temperature and weight every day for 7 days before vaccination. The normal body temperature is around 38.5℃, and the weight of a one-year-old pet cat is around 3kg.

[0126] 2) Screening of FIPV-negative cats: Detect the 7ab gene of FIPV by PCR; detect the binding antibodies in cat serum by ELISA using N and S proteins as antigens; detect neutralizing antibodies to FIPV using pseudovirus neutralization experiments.

[0127] According to an embodiment of the present invention, the screened test animals were immunized with the target mRNA encapsulated in LNPs. The immunization procedure is as follows:

[0128] The first vaccination was performed on D0, the second vaccination was performed on D21, and the virus was challenged on D28 after the second vaccination; the challenge virus strain was QS-1146; 5 kittens / group.

[0129] LNP-encapsulated target mRNA was transfected into CRFK cells, harvested 24 hours later, and immunoblotting was performed to detect protein expression. The results, as shown in Figure 2, demonstrated normal expression. Following challenge with LNP-encapsulated target mRNA in each group, all five kittens in the PBS group developed fever and weight loss, and autopsies revealed typical feline infectious peritonitis. Physiological indicators of the kittens in the group immunized with multi-epitope antigen mRNA significantly improved, with a significant increase in survival rate, as shown in Figure 2. Only one kitten died after 21 days, and the survival rate remained at 80% by day 30.

[0130] The above results show that the mRNA vaccine expressing multiple epitope antigens exhibits good immune effect against FIPV.

[0131] Example 3: Epitope Validation

[0132] The effectiveness of each antigen epitope was evaluated by detecting the stimulation response of the PBMC of the test animals to each epitope after immunization with the multi-epitope antigen mRNA vaccine. The LNP preparation method and the test animal screening method were the same as those in Examples 1 and 2.

[0133] According to an embodiment of the present invention, D1 was constructed between the cat-derived 5'HBB-UTR (SEQ ID NO: 71) and 3'HBA-UTR (SEQ ID NO: 72), transcribed into mRNA in vitro, and packaged with LNP. The sequence description and composition are shown in Tables 4 and 5.

[0134] According to an embodiment of the present invention, the antigen epitope polypeptide library is synthesized, and the specific implementation steps are as follows:

[0135] Each epitope peptide sequence was window-cut with a window size of 15 amino acids and a step size of 7 amino acids, and each window peptide and the full-length peptide were synthesized (Sino Biological).

[0136] According to an embodiment of the present invention, PBMC of the test animal is extracted after immunization, stimulated with each epitope, and the response is detected. The specific implementation steps are as follows:

[0137] PBMCs were extracted from the serum of the test animals and added to activated IFN-γ coated plates. Negative stimulators PBS and positive stimulators PMA were added and incubated with each epitope peptide for 16-24 hours. The cells were counted and statistically analyzed using an ELISPOT detection kit (Dakoway).

[0138] After immunizing the test animals with PBS and LNPs containing multi-epitope antigen mRNA vaccines, PBMCs were extracted from the test animals and stimulated with each antigen epitope peptide library. The stimulation response to the entire antigen epitope peptide library was detected, and the results are shown in Figure 3. HR2-4 and HR2-11 are shorter in length and have a correspondingly smaller peptide library size. However, they still produced detectable responses after stimulation with the PBMCs of the immunized test animals. For the antigen epitopes NTD, CTD, and NSP12, which have larger peptide libraries, the PBMCs of the test animals immunized with the multi-epitope antigen mRNA vaccine showed significant responses to stimulation with each antigen epitope peptide library compared to the PBS immunization control group. Using the entire peptide library to stimulate the PBMCs of the immunized test animals can obtain a response that is even stronger than when stimulated with each antigen epitope peptide library.

[0139] After immunizing the test animals with PBS and LNPs encapsulating multi-epitope antigen mRNA vaccines, PBMCs of the test animals were extracted and stimulated with a single peptide from each antigen epitope peptide library. The stimulation response to each antigen epitope peptide was detected, and the overall distribution of the stimulation response of the peptides in each antigen epitope peptide library was statistically analyzed. The results are shown in Figure 4. Consistent with the stimulation using the entire antigen epitope peptide library, the overall distribution of the stimulation response of the peptides in each antigen epitope peptide library showed a lower distribution of the stimulation response of the peptides in the HR2-4 and HR2-11 peptide libraries, while the distribution of the stimulation response of the peptides in the NTD, CTD, and NSP12 peptide libraries was higher. However, they all had stronger responses than the PBS immunized control group. Stimulating the PBMCs of the immunized test animals with the full-length peptides can obtain a significantly stronger response than that of the peptides in each antigen peptide library.

[0140] The above results show that each antigen epitope of the multi-epitope antigen mRNA vaccine can effectively activate the immune response.

[0141] Example 4: Validation of the potency of multi-epitope antigen mRNA vaccines against different epidemic strains

[0142] This example evaluated the effectiveness of a multi-epitope antigen mRNA vaccine against different circulating FIPV strains. The LNP preparation method, test animal screening method, immunization schedule, and evaluation method were the same as in Examples 1 and 2. The challenge strains were HF1902 and SH2211.

[0143] According to an embodiment of the present invention, D1 was constructed between the cat-derived 5'HBB-UTR (SEQ ID NO: 71) and 3'HBA-UTR (SEQ ID NO: 72), transcribed into mRNA in vitro, and packaged with LNP. The sequence description and composition are shown in Tables 4 and 5.

[0144] After immunization, the multi-epitope antigen mRNA vaccine can effectively improve the physiological indicators and survival rate of the test animals after challenge with different FIPV epidemic strains. The survival rate is shown in Figure 5. The survival rate of the control group dropped below 50% around 20 days after challenge with the two epidemic strains, while the survival rate of the experimental group remained at 100% until 30 days after challenge.

[0145] The above results show that the multi-epitope antigen mRNA vaccine exhibits good immune effects against different FIPV strains.

[0146] In the description of this specification, the reference terms "one embodiment", "some embodiments", "example", "specific example", or "some examples" mean that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representations of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described can be combined in any one or more embodiments or examples in a suitable manner. In addition, those skilled in the art can combine and combine different embodiments or examples described in this specification and features of different embodiments or examples without contradiction.

[0147] Although the embodiments of the present invention have been shown and described above, it will be understood that the above embodiments are illustrative and are not to be construed as limitations on the present invention. A person skilled in the art may change, modify, replace and modify the above embodiments within the scope of the present invention.

Claims

1. An isolated nucleic acid molecule, characterized in that include: at least one of the first nucleic acid fragment, the second nucleic acid fragment, the third nucleic acid fragment, the fourth nucleic acid fragment, and the fifth nucleic acid fragment; in, The first nucleic acid fragment is derived from the N-terminus (NTD) of the N protein of the QS strain of feline infectious peritonitis virus; The second nucleic acid fragment is derived from the C-terminus (CTD) of the N protein of the QS strain of feline infectious peritonitis virus; The third nucleic acid segment encodes the NSP12 protein of the QS strain of feline infectious peritonitis virus; The fourth nucleic acid fragment is derived from epitope HR2_4 of the S protein of feline infectious peritonitis virus 79-1146 strain; The fifth nucleic acid fragment is derived from the epitope HR2_11 of the S protein of the feline infectious peritonitis virus 79-1146 strain; The nucleic acid molecule is RNA.

2. The nucleic acid molecule according to claim 1, characterized in that The nucleic acid fragments are connected or not connected; Optionally, the nucleic acid fragments are connected via a linker; Optionally, any one of the nucleic acid fragments comprises at least 15 amino acids; Optionally, the NTD protein has an amino acid sequence that is at least 91% homologous to SEQ ID NO: 1; Optionally, the NTD protein has the amino acid sequence shown in SEQ ID NO: 1; Optionally, the CTD protein has an amino acid sequence that is at least 92% homologous to SEQ ID NO: 2; Optionally, the CTD protein has the amino acid sequence shown in SEQ ID NO: 2; Optionally, the NSP12 protein has an amino acid sequence that is at least 97% homologous to SEQ ID NO: 3; Optionally, the NSP12 protein has the amino acid sequence shown in SEQ ID NO: 3; Optionally, the HR2_4 protein has an amino acid sequence that is at least 50% homologous to SEQ ID NO: 4; Optionally, the HR2_4 protein has the amino acid sequence shown in SEQ ID NO: 4; Optionally, the HR2_11 protein has an amino acid sequence that is at least 95% homologous to SEQ ID NO: 5; Optionally, the HR2_11 protein has the amino acid sequence shown in SEQ ID NO: 5; Optionally, the first nucleic acid fragment has a nucleotide sequence that is at least 71% identical to any one of SEQ ID NOs: 16 to 19; Optionally, the first nucleic acid fragment has a nucleotide sequence shown in SEQ ID NOs: 16 to 19; Optionally, the second nucleic acid fragment has a nucleotide sequence that is at least 72% identical to any one of SEQ ID NOs: 20 to 23; Optionally, the second nucleic acid fragment has the nucleotide sequence shown in SEQ ID NOs: 20 to 23; Optionally, the third nucleic acid fragment has a nucleotide sequence that is at least 71% identical to any one of SEQ ID NOs: 24 to 27; Optionally, the third nucleic acid fragment has the nucleotide sequence shown in SEQ ID NOs: 24 to 27; Optionally, the fourth nucleic acid fragment has a nucleotide sequence that is at least 50% identical to any one of SEQ ID NOs: 28 to 31; Optionally, the fourth nucleic acid fragment has the nucleotide sequence shown in SEQ ID NOs: 28 to 31; Optionally, the fifth nucleic acid fragment has a nucleotide sequence that is at least 73% identical to any one of SEQ ID NOs: 32 to 35; Optionally, the fifth nucleic acid fragment has the nucleotide sequence shown in SEQ ID NOs: 32-35.

3. The nucleic acid molecule according to claim 1, characterized in that further comprising a sixth nucleic acid segment encoding a signal peptide sequence of MHC-I; Optionally, the signal peptide sequence of MHC-I does not contain a transmembrane region; Optionally, the MHC-I signal peptide sequence has the amino acid sequence shown in SEQ ID NO: 6; Optionally, the sixth nucleic acid fragment has the nucleotide sequence shown in SEQ ID NOs: 36 to 38; Optionally, the sixth nucleic acid fragment is placed at the 5' end of the nucleic acid molecule.

4. The nucleic acid molecule according to claim 1, characterized in that further comprising a seventh nucleic acid segment encoding a MITD sequence; Optionally, the MITD sequence comprises a transmembrane region; Optionally, the MITD sequence has the amino acid sequence shown in SEQ ID NO: 7; Optionally, the seventh nucleic acid fragment has the nucleotide sequence shown in SEQ ID NOs: 39 to 41; Optionally, the seventh nucleic acid fragment is placed at the 3' end of the nucleic acid molecule.

5. The nucleic acid molecule according to claim 1, characterized in that further comprising an eighth nucleic acid segment encoding an HBHA adjuvant sequence; Optionally, the HBHA sequence has the amino acid sequence shown in SEQ ID NO: 8; Optionally, the eighth nucleic acid fragment has the nucleotide sequence shown in SEQ ID NOs: 42-44.

6. The nucleic acid molecule according to claim 1, characterized in that Further comprising a ninth nucleic acid segment encoding a PADRE sequence.

7. The nucleic acid molecule according to claim 6, characterized in that The PADRE sequence has the amino acid sequence shown in SEQ ID NO:

9.

8. The nucleic acid molecule according to claim 6, characterized in that The ninth nucleic acid fragment has the nucleotide sequence shown in SEQ ID NOs: 45 to 47.

9. The nucleic acid molecule according to claim 1, characterized in that The nucleic acid molecule further comprises a rigid or flexible linker sequence; Optionally, the linker sequence has the amino acid sequence shown in SEQ ID NOs: 11 to 15; Optionally, the linker sequence has the nucleotide sequence shown in SEQ ID NOs: 51-70.

10. The nucleic acid molecule according to claim 1, characterized in that The nucleic acid molecule is linear.

11. An expression vector, characterized in that: Carrying the nucleic acid molecule according to any one of claims 1 to 10.

12. The expression vector according to claim 11, characterized in that The expression vector is a non-pathogenic viral vector.

13. The expression vector according to claim 12, characterized in that The non-pathogenic virus is selected from at least one of a retrovirus, a lentivirus, an adenovirus and an adeno-associated virus.

14. A recombinant virus carrying the nucleic acid molecule according to any one of claims 1 to 10.

15. A liposome, characterized in that The invention comprises a liposome carrier and a nucleic acid fragment, wherein the nucleic acid fragment is as defined in any one of claims 1 to 10.

16. A vaccine, characterized in that comprising the nucleic acid molecule according to any one of claims 1 to 10, the expression vector according to claims 11 to 13, the recombinant virus according to claim 14, or the liposome according to claim 15; Optionally, the vaccine comprises at least one selected from RNA vaccine, DNA vaccine, protein recombinant vaccine, inactivated vaccine, attenuated vaccine, and viral vector vaccine; Preferably, the vaccine is an RNA vaccine.

17. The vaccine according to claim 16, characterized in that further comprising an adjuvant; Optionally, the adjuvant includes a TLR agonist, Mn 2+ at least one of; Optionally, the TLR agonist comprises at least one of HBHA, CpG, R837, MPLA and derivatives thereof.

18. A recombinant cell, characterized in that Carrying the nucleic acid molecule according to any one of claims 1 to 10, the expression vector according to claims 11 to 13, or the recombinant virus according to claim 14.

19. A method for constructing a feline infectious peritonitis virus vaccine, characterized in that: include: The nucleic acid molecule according to any one of claims 1 to 10, the expression vector according to claims 11 to 13, or the recombinant virus according to claim 14 is introduced into a recipient cell.

20. The method according to claim 19, characterized in that Before introduction into the recipient cell, the method further comprises encapsulating the nucleic acid, expression vector or recombinant virus using an encapsulation vector; Optionally, the encapsulation carrier is selected from at least one of liposomes, exosomes, polymer carriers, viral vectors, and nanoparticles; Preferably, the encapsulating carrier is a nanoparticle.

21. The method according to claim 19, wherein The recipient cells are CRFK cells, HEK293FT, HEK293T or BHK cells; Preferably, the recipient cells are CRFK cells.

22. Use of the nucleic acid molecule according to any one of claims 1 to 10, the expression vector according to claims 11 to 13, the recombinant virus according to claim 14, the liposome according to claim 15 or the recombinant cell according to claim 18 in the preparation of a drug or vaccine, wherein the drug or vaccine is used to prevent or treat diseases related to feline infectious peritonitis virus infection.

23. A method for preventing or treating feline infectious peritonitis virus infection, characterized in that: include: The nucleic acid molecule according to any one of claims 1 to 10, the expression vector according to any one of claims 11 to 13, the recombinant virus according to claim 14, the liposome according to claim 15, the vaccine according to claim 16 or 17, or the recombinant cell according to claim 18 is administered to a test animal.

24. The method according to claim 23, wherein The test animals are selected from cats.

25. Use of the nucleic acid molecule according to any one of claims 1 to 10, the expression vector according to any one of claims 11 to 13, the recombinant virus according to claim 14, the liposome according to claim 15, the vaccine according to claim 16 or 17, or the recombinant cell according to claim 18 in preventing or treating feline infectious peritonitis virus infection.

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