Development of a multi-antigen mRNA vaccine against feline FIPV

Abstract

The present invention provides a pharmaceutical formulation comprising a nucleic acid fragment, the nucleic acid fragment being mRNA, and comprising at least one of a first nucleic acid fragment, a second nucleic acid fragment, and a third nucleic acid fragment, wherein the first nucleic acid fragment encodes the M protein of feline infectious peritonitis virus, the second nucleic acid fragment encodes the N protein of feline infectious peritonitis virus, and the third nucleic acid fragment encodes the S, S_ec, or SII protein of feline infectious peritonitis virus, and the first and second nucleic acid fragments are or are not linked to the third nucleic acid fragment.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, specifically to the development of a multi-antigen mRNA vaccine against feline infectious peritonitis virus (FIPV), and more specifically to pharmaceutical preparations, isolated nucleic acid molecules, expression vectors, recombinant viruses, liposomes, vaccines, recombinant cells, methods for constructing and using feline infectious peritonitis virus vaccines, and methods for preventing or treating feline infectious peritonitis virus infections.

Background Art

[0002] Feline coronavirus FCoV belongs to the coronavirus family. Coronaviridae viruses are all relatively large, round, and characterized by being forward-chain RNA viruses with an envelope. Their genomes are between 27 and 32 kb and encode replication polymerase, four structural proteins (S protein, M protein, N protein, and E protein), and several non-structural proteins. The S protein (spike protein) is one of the important structural proteins of coronaviruses. It is responsible for forming the surface protrusions of these viruses and is also an important element for the infection of these viruses. The S protein binds to receptors on host cells, thereby allowing the virus to enter the host cell and cause infection. In addition, the S protein is an important antigen in many coronavirus vaccines. The SII region of the S protein has few epitopes that mediate antibody-dependent enhanced immune responses (ADE). The M protein (membrane protein) is involved in the formation and positioning of the coronavirus particle morphology. It is a protein that penetrates the viral envelope and plays a role in forming the structure of the viral particle by interacting with other proteins. The N protein (Nucleocapsid protein) coats the viral RNA genome and is involved in the replication and transcription of viral genes. At the same time, the N protein can induce the host immune system's response to the virus. The E protein (Envelope protein) is a protein on the envelope of the coronavirus and can interact with the M protein to form the structure of the viral particle, while also being involved in the infection and assembly processes of the virus.

[0003] Feline coronavirus (FCoV) is classified into feline enteric coronavirus (FECV) and feline infectious peritonitis virus (FIPV) based on biotype / pathogenicity. FECV has a very wide transmission range and infects intestinal epithelial cells, but is usually asymptomatic or causes only mild diarrhea. FIPV mainly infects feline monocytes and macrophages. Distinguishing between FECV and FIPV from genome sequences is difficult, and some studies have suggested that FIPV can be distinguished from FECV by spike protein amino mutations, although these mutations were later found to be more strongly associated with tissue preference.

[0004] Typical characteristics of FIPV include the development of pyogenic granulomatous lesions in various tissues and organs, including the lungs, liver, spleen, retina, and brain. Infection of macrophages and monocytes is considered key to the pathogenic mechanism. In the terminal stage of FIPV infection, a significant decrease in T cells in peripheral and lymphatic tissues and frequent hypergammaglobulinemia have been observed, suggesting the presence of severe immunodeficiency induced by the virus. Humoral immunity appears to be ineffective and may lead to "early death syndrome." When S antibodies are present at subneutralizing titers, they can enhance infection of target cells by binding to Fc receptors. Researchers have repeatedly attempted to develop FIPV vaccines based on humoral immunity, but many have failed. The main reason for failure is the existence of antibody-dependent enhancement (ADE) infection, where antibodies cannot exert an effective protective effect. Currently, researchers are attempting to control FIPV infection and elimination through cell-mediated immunity (CMI), but have not yet achieved good protective effects.

[0005] Therefore, research and development of vaccines against FIPV is urgently needed in this field. [Overview of the Initiative] [Problems that the invention aims to solve]

[0006] This application is submitted by the inventor based on the following problems and facts: Current research and development of FIPV vaccines has failed to yield any significant breakthroughs over the long term, and FIPV infections have mostly resulted in cat deaths.

[0007] Therefore, in a first aspect of the present invention, the present invention provides a pharmaceutical formulation. According to an embodiment of the present invention, the pharmaceutical formulation comprises a nucleic acid fragment, the nucleic acid fragment being mRNA, and comprising at least one of a first nucleic acid fragment, a second nucleic acid fragment, and a third nucleic acid fragment, wherein the first nucleic acid fragment encodes the M protein of feline infectious peritonitis virus, the second nucleic acid fragment encodes the N protein of feline infectious peritonitis virus, and the third nucleic acid fragment encodes the S, S_ec (S extracellular layer), or SII protein of feline infectious peritonitis virus, and the first and second nucleic acid fragments are ligated to or not ligated to the third nucleic acid fragment. According to an embodiment of the present invention, a pharmaceutical formulation of any or a combination of the expressed M, N, S, S_ec, or SII proteins of feline infectious peritonitis virus can stimulate an animal somatic cell-mediated immune response. [Means for solving the problem]

[0008] Furthermore, in this application, adaptive modification can be performed on the M, N, S, S_ec, or SII proteins of the wild-type FIPV virus as needed to reduce the toxicity of the FIPV virus while simultaneously maintaining its immunogenicity without affecting its three-dimensional structure, thereby preparing a novel FIPV virus vaccine. Based on the sequence comparison results (Tables 1 and 2), the M protein has an amino sequence with at least 89% homology to SEQ ID NO: 1, the nucleic acid fragment encoding the M protein has a nucleotide sequence with at least 67% homology to any sequence from SEQ ID NO: 4 to 7, the N protein has an amino sequence with at least 91% homology to SEQ ID NO: 2, and the nucleic acid fragment encoding the N protein has a nucleotide sequence with at least 70% homology to any sequence from SEQ ID NO: 8 to 11. The S protein has an amino sequence with at least 45% homology to SEQ ID NO: 3, and the nucleic acid fragment encoding the S protein has a nucleotide sequence with at least 51% homology to any sequence of SEQ ID NO: 12 to 15. The S_ec protein has an amino sequence with at least 43% homology to the aminos at positions 1 to 1374 of SEQ ID NO: 3, and the nucleic acid fragment encoding the S_ec protein has a nucleotide sequence with at least 51% homology to the nucleotides at positions 1 to 4122 of any sequence of SEQ ID NO: 12 to 15. The SII protein has an amino sequence with at least 62% homology to the aminos at positions 782 to 1433 of SEQ ID NO: 3, and the nucleic acid fragment encoding the SII protein has a nucleotide sequence with at least 57% homology to the nucleotides at positions 2344 to 4299 of any sequence of SEQ ID NO: 12 to 15. FIPV virus vaccines are not particularly limited, but are capable of generating receptor-binding domains of modified FIPV virus M, N, S, S_ec, or SII proteins in vivo and possess immunogenicity capable of inducing an immune response in animals.Furthermore, the pharmaceutical formulation is used to stimulate the immune response of all animals, including but not limited to cats, that can be infected with feline infectious peritonitis virus.

[0009] [Table 1]

[0010] [Table 2-1] [Table 2-2]

[0011] According to embodiments of the present invention, the pharmaceutical formulation may include at least one of the following additional technical features.

[0012] According to embodiments of the present invention, the first nucleic acid fragment and the second nucleic acid fragment are not linked to the third nucleic acid fragment. In some examples of the present application, mRNA prepared from isolated nucleic acid fragments has a good immune effect against FIPV as a vaccine and can significantly improve various physiological indicators and survival rates in cats.

[0013] According to embodiments of the present invention, the mass ratio of the first nucleic acid fragment to the second nucleic acid fragment is 10:1 to 1:10. Selectively, the mass ratio of the first nucleic acid fragment to the second nucleic acid fragment is 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 10:1, 9:1, 8:1, 7:1, 6:1, 5:1, 4:1, 3:1, or 2:1.

[0014] According to embodiments of the present invention, the mass ratio of the second nucleic acid fragment to the third nucleic acid fragment is 10:1 to 1:10. Selectively, the mass ratio of the second nucleic acid fragment to the third nucleic acid fragment is 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 10:1, 9:1, 8:1, 7:1, 6:1, 5:1, 4:1, 3:1, or 2:1.

[0015] According to one preferred embodiment of the present invention, the mass ratio of the first nucleic acid fragment, the second nucleic acid fragment, and the third nucleic acid fragment is 1:1:1. In some examples of the present application, when the mass ratio of the three unlinked nucleic acid fragments is 1:1:1, the prepared mRNA has a good immune effect against FIPV as a vaccine and can significantly improve various physiological indicators and survival rates in cats.

[0016] According to preferred embodiments of the present invention, the third nucleic acid fragment is selected from an SII protein. In some examples of the present application, an mRNA vaccine is prepared by selecting an SII protein as the third nucleic acid fragment, the first nucleic acid fragment, and the second nucleic acid fragment, which has a good immune effect against FIPV and can significantly improve various physiological indicators and survival rates in cats.

[0017] According to an embodiment of the present invention, the first nucleic acid fragment and the second nucleic acid fragment are linked to the third nucleic acid fragment.

[0018] According to embodiments of the present invention, the mass ratio of the first nucleic acid fragment to the second nucleic acid fragment is 10:1 to 1:10. Selectively, the mass ratio of the first nucleic acid fragment to the second nucleic acid fragment is 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 10:1, 9:1, 8:1, 7:1, 6:1, 5:1, 4:1, 3:1, or 2:1.

[0019] According to an embodiment of the present invention, the mass ratio of the second nucleic acid fragment to the third nucleic acid fragment is 10:1 to 1:10. Optionally, the mass ratio of the second nucleic acid fragment to the third nucleic acid fragment is 1:1 or 1:2 or 1:3 or 1:4 or 1:5 or 1:6 or 1:7 or 1:8 or 1:9 or 1:10 or 10:1 or 9:1 or 8:1 or 7:1 or 6:1 or 5:1 or 4:1 or 3:1 or 2:1.

[0020] According to an embodiment of the present invention, the mass ratio of the first nucleic acid fragment, the second nucleic acid fragment, and the third nucleic acid fragment is 1:1:1.

[0021] According to an embodiment of the present invention, the first nucleic acid fragment is linked to the second nucleic acid fragment, the third nucleic acid fragment is not linked to the first nucleic acid fragment and the second nucleic acid fragment, or the first nucleic acid fragment is linked to the third nucleic acid fragment, and the second nucleic acid fragment is not linked to the first nucleic acid fragment and the third nucleic acid fragment, or the second nucleic acid fragment is linked to the third nucleic acid fragment, and the first nucleic acid fragment is not linked to the second nucleic acid fragment and the third nucleic acid fragment.

[0022] According to an embodiment of the present invention, the 3' end of the first nucleic acid fragment is linked to the 5' end of the second nucleic acid fragment, and the third nucleic acid fragment is not linked to the first nucleic acid fragment and the second nucleic acid fragment; or the 3' end of the second nucleic acid fragment is linked to the 5' end of the first nucleic acid fragment, and the third nucleic acid fragment is not linked to the first nucleic acid fragment and the second nucleic acid fragment; or the 3' end of the first nucleic acid fragment is linked to the 5' end of the third nucleic acid fragment, and the second nucleic acid fragment is not linked to the first nucleic acid fragment and the third nucleic acid fragment; or the 3' end of the third nucleic acid fragment is linked to the 5' end of the first nucleic acid fragment, and the second nucleic acid fragment is not linked to the first nucleic acid fragment and the third nucleic acid fragment; or the 3' end of the second nucleic acid fragment is linked to the 5' end of the third nucleic acid fragment, and the first nucleic acid fragment is not linked to the second nucleic acid fragment and the third nucleic acid fragment; or the 3' end of the third nucleic acid fragment is linked to the 5' end of the second nucleic acid fragment, and the first nucleic acid fragment is not linked to the second nucleic acid fragment and the third nucleic acid fragment.

[0023] According to an embodiment of the present invention, the 3' end of the first nucleic acid fragment is linked to the 5' end of the second nucleic acid fragment, the 3' end of the second nucleic acid fragment is linked to the 5' end of the third nucleic acid fragment; or the 3' end of the first nucleic acid fragment is linked to the 5' end of the third nucleic acid fragment, the 3' end of the third nucleic acid fragment is linked to the 5' end of the second nucleic acid fragment; or the 3' end of the second nucleic acid fragment is linked to the 5' end of the first nucleic acid fragment, the 3' end of the first nucleic acid fragment is linked to the 5' end of the third nucleic acid fragment; or the 3' end of the second nucleic acid fragment is linked to the 5' end of the third nucleic acid fragment, the 3' end of the third nucleic acid fragment is linked to the 5' end of the first nucleic acid fragment; or the 3' end of the third nucleic acid fragment is linked to the 5' end of the first nucleic acid fragment, the 3' end of the first nucleic acid fragment is linked to the 5' end of the second nucleic acid fragment; or the 3' end of the third nucleic acid fragment is linked to the 5' end of the second nucleic acid fragment, the 3' end of the second nucleic acid fragment is linked to the 5' end of the first nucleic acid fragment.

[0024] Specifically, the linking scheme of the M, N, S, S_ec, or SII proteins of the feline infectious peritonitis virus (FIPV) can be any combination.

[0025] According to an embodiment of the present invention, the M protein has the amino sequence shown in the amino sequence shown in SEQ ID NO: 1.

[0026] According to an embodiment of the present invention, the M protein has an amino sequence that has at least 89% homology to the amino sequence shown in SEQ ID NO: 1, and the amino at position 90 is Y, at position 102 is V, at position 120 is I, at position 144 is A, and at position 180 is L.

[0027] According to an embodiment of the present invention, the M protein has the amino sequence shown in SEQ ID NO: 1.

[0028] According to an embodiment of the present invention, the N protein has an amino sequence that has at least 91% homology to the amino sequence shown in SEQ ID NO: 2.

[0029] According to an embodiment of the present invention, the N protein has the amino sequence shown in SEQ ID NO: 2.

[0030] According to an embodiment of the present invention, the S protein has an amino sequence that has at least 45% homology to SEQ ID NO: 3.

[0031] According to an embodiment of the present invention, the S protein has an amino acid sequence having at least 45% homology to SEQ ID NO: 3, and the amino acid at position 515 is V, the amino acid at position 577 is Q, the amino acid at position 1385 is V, the amino acid at position 1386 is V, the amino acid at position 1397 is F, and the amino acid at position 1415 is I.

[0032] According to an embodiment of the present invention, the S protein has the amino sequence shown in SEQ ID NO: 3.

[0033] According to an embodiment of the present invention, the S_ec protein has an amino sequence that has at least 43% homology to the aminos at positions 1 to 1374 of the amino sequence shown in SEQ ID NO: 3.

[0034] According to an embodiment of the present invention, the S_ec protein has an amino sequence having at least 43% homology to the aminos at positions 1 to 1374 of the amino sequence shown in SEQ ID NO: 3, and the amino at position 515 is V and the amino at position 577 is Q.

[0035] According to an embodiment of the present invention, the S_ec protein has the amino sequence from position 1 to 1374 of SEQ ID NO: 3.

[0036] According to an embodiment of the present invention, the SII protein has an amino sequence that has at least 62% homology to the aminos at positions 782 to 1433 of the amino sequence shown in SEQ ID NO: 3.

[0037] According to an embodiment of the present invention, the SII protein has an amino sequence having at least 62% homology to the aminos at positions 782 to 1433 of the amino sequence shown in SEQ ID NO: 3, and the amino at position 1385 is V, the amino at position 1386 is V, the amino at position 1397 is F, and the amino at position 1415 is I.

[0038] According to an embodiment of the present invention, the SII protein has an amino sequence at positions 782-1433 of SEQ ID NO: 3.

[0039] According to an embodiment of the present invention, the first nucleic acid fragment has a nucleotide sequence that has at least 67% homology to any of the sequences SEQ ID NO: 4 to 7.

[0040] According to an embodiment of the present invention, the first nucleic acid fragment has the nucleotide sequence shown in SEQ ID NO: 4-7.

[0041] According to an embodiment of the present invention, the second nucleic acid fragment has a nucleotide sequence that has at least 70% homology to any of the sequences with SEQ ID NO: 8 to 11.

[0042] According to an embodiment of the present invention, the second nucleic acid fragment has the nucleotide sequence shown in SEQ ID NO: 8~11.

[0043] According to an embodiment of the present invention, the third nucleic acid fragment has a nucleotide sequence having at least 51% homology to the nucleotides at positions 1 to 4122 of any sequence with SEQ ID NO: 12 to 15.

[0044] According to an embodiment of the present invention, the third nucleic acid fragment has a nucleotide sequence from positions 1 to 4122 of SEQ ID NO: 12 to 15.

[0045] According to an embodiment of the present invention, the third nucleic acid fragment has a nucleotide sequence having at least 57% homology to the nucleotides at positions 2344 to 4299 of any sequence SEQ ID NO: 12 to 15.

[0046] According to an embodiment of the present invention, the third nucleic acid fragment has a nucleotide sequence from positions 2344 to 4299 of SEQ ID NO: 12 to 15.

[0047] According to an embodiment of the present invention, the third nucleic acid fragment has a nucleotide sequence that has at least 51% homology to any of the sequences SEQ ID NO: 12 to 15.

[0048] According to an embodiment of the present invention, the third nucleic acid fragment has the nucleotide sequence shown in SEQ ID NO: 12-15.

[0049] According to embodiments of the present invention, the pharmaceutical formulation further comprises a fourth nucleic acid fragment, the fourth nucleic acid fragment encoding a signal peptide sequence of MHCI (major histocompatibility complex I) or a sequence having a similar function to the signal peptide of MHCI. According to embodiments of the present invention, by adding the MHCI signal peptide to the N-terminus of the antigen sequence, ribosomes can be attached to the endoplasmic reticulum membrane, guiding the transport of proteins within the cell.

[0050] Furthermore, in this application, the sequence having a similar function to the signal peptide of MHCI can similarly attach ribosomes to the endoplasmic reticulum membrane and guide the transport of proteins within the cell.

[0051] According to embodiments of the present invention, the signal peptide sequence of the MHCI does not include a transmembrane region.

[0052] According to an embodiment of the present invention, the signal peptide sequence of the MHCI has the amino sequence shown in SEQ ID NO: 16.

[0053] According to an embodiment of the present invention, the fourth nucleic acid fragment has the nucleotide sequence shown in SEQ ID NO: 17-22.

[0054] According to an embodiment of the present invention, the fourth nucleic acid fragment is provided at the 5' end of the nucleic acid fragment or at the 5' end of the nucleic acid molecule.

[0055] The fourth nucleic acid fragment is provided upstream of the nucleic acid fragment or the nucleic acid molecule and is selectively located at the 5' end of the nucleic acid fragment or the 5' end of the nucleic acid molecule.

[0056] According to embodiments of the present invention, the pharmaceutical formulation further comprises a fifth nucleic acid fragment, the fifth nucleic acid fragment encoding an MITD (MHCI molecular transport signal or major histocompatibility complex I molecular transport signal) sequence or a sequence having a similar function to MITD. According to embodiments of the present invention, by adding the MITD sequence to the C-terminus of the nucleic acid molecule, CD4 +This can stimulate the proliferation of T cells and induce the production of more cellular factors.

[0057] In this application, the sequence having a function similar to the aforementioned MITD is similarly CD4. + It can stimulate the proliferation of T cells, induce the production of more cellular factors, and trigger an immune response in the animal body.

[0058] According to an embodiment of the present invention, the MITD sequence has the amino sequence shown in SEQ ID NO: 23.

[0059] According to an embodiment of the present invention, the fifth nucleic acid fragment has the nucleotide sequence shown in SEQ ID NO: 24-32.

[0060] According to embodiments of the present invention, the fifth nucleic acid fragment is provided at the 3' end of the nucleic acid fragment or at the 3' end of the nucleic acid molecule.

[0061] The fifth nucleic acid fragment is provided downstream of the nucleic acid fragment or the nucleic acid molecule, and is selectively provided at the 3' end of the nucleic acid fragment or the 3' end of the nucleic acid molecule.

[0062] According to embodiments of the present invention, the nucleic acid fragment or nucleic acid molecule is a linear molecule.

[0063] According to embodiments of the present invention, the nucleic acid fragment or nucleic acid molecule has the nucleotide sequence shown in Table 4.

[0064] According to embodiments of the present invention, the pharmaceutical formulation further comprises a drug carrier, the drug carrier comprising at least one of liposomes, exosomes, polymer carriers, viral carriers, and nanoparticles.

[0065] The term "drug carrier" refers to a carrier that does not cause significant irritation to the test animal and does not alter the biological activity and properties of the pharmaceutical preparation.

[0066] In a second aspect of the present invention, the present invention provides a method for preparing the pharmaceutical formulation described in the first aspect. According to an example of the present invention, the method includes the step of mixing a first nucleic acid fragment, a second nucleic acid fragment, and a third nucleic acid fragment in a predetermined ratio to obtain the pharmaceutical formulation. The first nucleic acid fragment encodes the M protein of feline infectious peritonitis virus, the second nucleic acid fragment encodes the N protein of feline infectious peritonitis virus, and the third nucleic acid fragment encodes the S, S_ec, or SII protein of feline infectious peritonitis virus, and the nucleic acid fragments are mRNA.

[0067] According to embodiments of the present invention, this method can be used to prepare pharmaceutical formulations for the prevention or treatment of related diseases caused by feline infectious peritonitis virus.

[0068] According to embodiments of the present invention, the mass ratio of the first nucleic acid fragment to the second nucleic acid fragment is 10:1 to 1:10. Selectively, the mass ratio of the first nucleic acid fragment to the second nucleic acid fragment is 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 10:1, 9:1, 8:1, 7:1, 6:1, 5:1, 4:1, 3:1, or 2:1.

[0069] According to embodiments of the present invention, the mass ratio of the second nucleic acid fragment to the third nucleic acid fragment is 10:1 to 1:10. Preferably, the mass ratio of the first nucleic acid fragment to the second nucleic acid fragment is 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 10:1, 9:1, 8:1, 7:1, 6:1, 5:1, 4:1, 3:1, or 2:1.

[0070] According to an embodiment of the present invention, the mass ratio of the first nucleic acid fragment, the second nucleic acid fragment, and the third nucleic acid fragment is 1:1:1.

[0071] In a third aspect of the present invention, the present invention provides an isolated nucleic acid molecule. According to an embodiment of the present invention, the nucleic acid molecule comprises a first nucleic acid fragment encoding the M protein of feline infectious peritonitis virus, a second nucleic acid fragment encoding the N protein of feline infectious peritonitis virus, and a third nucleic acid fragment encoding the S, S_ec, or SII protein of feline infectious peritonitis virus, wherein the first and second nucleic acid fragments are linked to the third nucleic acid fragment, and the nucleic acid molecule is mRNA. According to an embodiment of the present invention, a pharmaceutical formulation expressing the M, N, S, S_ec, or SII protein of feline infectious peritonitis virus can stimulate an animal somatic cell-mediated immune response.

[0072] Furthermore, in this application, adaptive modification can be performed on the M, N, S, S_ec, or SII proteins of the wild-type FIPV virus as needed to reduce the toxicity of the FIPV virus while simultaneously maintaining its immunogenicity without affecting its three-dimensional structure, thereby preparing a novel FIPV virus vaccine. Based on the sequence comparison results (Tables 1 and 2), the M protein has an amino sequence with at least 89% homology to SEQ ID NO: 1, the nucleic acid fragment encoding the M protein has a nucleotide sequence with at least 67% homology to any sequence from SEQ ID NO: 4 to 7, the N protein has an amino sequence with at least 91% homology to SEQ ID NO: 2, and the nucleic acid fragment encoding the N protein has a nucleotide sequence with at least 70% homology to any sequence from SEQ ID NO: 8 to 11. The S protein has an amino sequence with at least 45% homology to SEQ ID NO: 3, and the nucleic acid fragment encoding the S protein has a nucleotide sequence with at least 51% homology to any sequence of SEQ ID NO: 12 to 15. The S_ec protein has an amino sequence with at least 43% homology to the aminos at positions 1 to 1374 of SEQ ID NO: 3, and the nucleic acid fragment encoding the S_ec protein has a nucleotide sequence with at least 51% homology to the nucleotides at positions 1 to 4122 of any sequence of SEQ ID NO: 12 to 15. The SII protein has an amino sequence with at least 62% homology to the aminos at positions 782 to 1433 of SEQ ID NO: 3, and the nucleic acid fragment encoding the SII protein has a nucleotide sequence with at least 57% homology to the nucleotides at positions 2344 to 4299 of any sequence of SEQ ID NO: 12 to 15. FIPV virus vaccines are not particularly limited as long as they can generate receptor-binding domains of modified FIPV virus M, N, S, S_ec, or SII proteins in vivo and possess immunogenicity that can induce an immune response in animals.Furthermore, the pharmaceutical formulation is used to stimulate the immune response of all animals, including but not limited to cats, that can be infected with feline infectious peritonitis virus.

[0073] According to embodiments of the present invention, the nucleic acid molecule may include at least one of the following additional technical features.

[0074] According to embodiments of the present invention, the first nucleic acid fragment is linked to the second nucleic acid fragment, and the third nucleic acid fragment is not linked to the first nucleic acid fragment and the second nucleic acid fragment; or the first nucleic acid fragment is linked to the third nucleic acid fragment, and the second nucleic acid fragment is not linked to the first nucleic acid fragment and the third nucleic acid fragment; or the second nucleic acid fragment is linked to the third nucleic acid fragment, and the first nucleic acid fragment is not linked to the second nucleic acid fragment and the third nucleic acid fragment.

[0075] According to embodiments of the present invention, the 3' end of the first nucleic acid fragment is connected to the 5' end of the second nucleic acid fragment, and the third nucleic acid fragment is not connected to the first nucleic acid fragment and the second nucleic acid fragment, or the 3' end of the second nucleic acid fragment is connected to the 5' end of the first nucleic acid fragment, and the third nucleic acid fragment is not connected to the first nucleic acid fragment and the second nucleic acid fragment, or the 3' end of the first nucleic acid fragment is connected to the 5' end of the third nucleic acid fragment, and the second nucleic acid fragment is connected to the first nucleic acid fragment and the third nucleic acid fragment The nucleic acid fragment is not connected, or the 3' end of the third nucleic acid fragment is connected to the 5' end of the first nucleic acid fragment, and the second nucleic acid fragment is not connected to the first nucleic acid fragment and the third nucleic acid fragment, or the 3' end of the second nucleic acid fragment is connected to the 5' end of the third nucleic acid fragment, and the first nucleic acid fragment is not connected to the second nucleic acid fragment and the third nucleic acid fragment, or the 3' end of the third nucleic acid fragment is connected to the 5' end of the second nucleic acid fragment, and the first nucleic acid fragment is not connected to the second nucleic acid fragment and the third nucleic acid fragment.

[0076] According to embodiments of the present invention, the 3' end of the first nucleic acid fragment is connected to the 5' end of the second nucleic acid fragment, the 3' end of the second nucleic acid fragment is connected to the 5' end of the third nucleic acid fragment, or the 3' end of the first nucleic acid fragment is connected to the 5' end of the third nucleic acid fragment, the 3' end of the third nucleic acid fragment is connected to the 5' end of the second nucleic acid fragment, or the 3' end of the second nucleic acid fragment is connected to the 5' end of the first nucleic acid fragment, and the 3' end of the first nucleic acid fragment is connected to the 5' end of the third nucleic acid fragment. The 3' end of the second nucleic acid fragment is linked to the 5' end of the third nucleic acid fragment, the 3' end of the third nucleic acid fragment is linked to the 5' end of the first nucleic acid fragment, or the 3' end of the third nucleic acid fragment is linked to the 5' end of the first nucleic acid fragment, the 3' end of the first nucleic acid fragment is linked to the 5' end of the second nucleic acid fragment, or the 3' end of the third nucleic acid fragment is linked to the 5' end of the second nucleic acid fragment, and the 3' end of the second nucleic acid fragment is linked to the 5' end of the first nucleic acid fragment.

[0077] Specifically, the linking scheme of the M, N, S, S_ec, or SII proteins of the feline infectious peritonitis virus (FIPV) can be any combination.

[0078] According to an embodiment of the present invention, the M protein has the amino sequence shown in the amino sequence shown in SEQ ID NO: 1.

[0079] According to an embodiment of the present invention, the M protein has an amino sequence that has at least 89% homology to the amino sequence shown in SEQ ID NO: 1, and the amino at position 90 is Y, at position 102 is V, at position 120 is I, at position 144 is A, and at position 180 is L.

[0080] According to an embodiment of the present invention, the M protein has the amino sequence shown in SEQ ID NO: 1.

[0081] According to an embodiment of the present invention, the N protein has an amino sequence that has at least 91% homology to the amino sequence shown in SEQ ID NO: 2.

[0082] According to an embodiment of the present invention, the N protein has the amino sequence shown in SEQ ID NO: 2.

[0083] According to an embodiment of the present invention, the S protein has an amino sequence that has at least 45% homology to SEQ ID NO: 3.

[0084] According to an embodiment of the present invention, the S protein has an amino acid sequence having at least 45% homology to SEQ ID NO: 3, and the amino acid at position 515 is V, the amino acid at position 577 is Q, the amino acid at position 1385 is V, the amino acid at position 1386 is V, the amino acid at position 1397 is F, and the amino acid at position 1415 is I.

[0085] According to an embodiment of the present invention, the S protein has the amino sequence shown in SEQ ID NO: 3.

[0086] According to an embodiment of the present invention, the S_ec protein has an amino sequence that has at least 43% homology to the aminos at positions 1 to 1374 of the amino sequence shown in SEQ ID NO: 3.

[0087] According to an embodiment of the present invention, the S_ec protein has an amino sequence having at least 43% homology to the aminos at positions 1 to 1374 of the amino sequence shown in SEQ ID NO: 3, and the amino at position 515 is V and the amino at position 577 is Q.

[0088] According to an embodiment of the present invention, the S_ec protein has the amino sequence from position 1 to 1374 of SEQ ID NO: 3.

[0089] According to an embodiment of the present invention, the SII protein has an amino sequence that has at least 62% homology to the aminos at positions 782 to 1433 of the amino sequence shown in SEQ ID NO: 3.

[0090] According to an embodiment of the present invention, the SII protein has an amino sequence having at least 62% homology to the aminos at positions 782 to 1433 of the amino sequence shown in SEQ ID NO: 3, and the amino at position 1385 is V, the amino at position 1386 is V, the amino at position 1397 is F, and the amino at position 1415 is I.

[0091] According to an embodiment of the present invention, the SII protein has an amino sequence at positions 782-1433 of SEQ ID NO: 3.

[0092] According to an embodiment of the present invention, the first nucleic acid fragment has a nucleotide sequence that has at least 67% homology to any of the sequences SEQ ID NO: 4 to 7.

[0093] According to an embodiment of the present invention, the first nucleic acid fragment has the nucleotide sequence shown in SEQ ID NO: 4-7.

[0094] According to an embodiment of the present invention, the second nucleic acid fragment has a nucleotide sequence that has at least 70% homology to any of the sequences with SEQ ID NO: 8 to 11.

[0095] According to an embodiment of the present invention, the second nucleic acid fragment has the nucleotide sequence shown in SEQ ID NO: 8~11.

[0096] According to an embodiment of the present invention, the third nucleic acid fragment has a nucleotide sequence having at least 51% homology to the nucleotides at positions 1 to 4122 of any sequence with SEQ ID NO: 12 to 15.

[0097] According to an embodiment of the present invention, the third nucleic acid fragment has a nucleotide sequence from positions 1 to 4122 of SEQ ID NO: 12 to 15.

[0098] According to an embodiment of the present invention, the third nucleic acid fragment has a nucleotide sequence having at least 57% homology to the nucleotides at positions 2344 to 4299 of any sequence SEQ ID NO: 12 to 15.

[0099] According to an embodiment of the present invention, the third nucleic acid fragment has a nucleotide sequence from positions 2344 to 4299 of SEQ ID NO: 12 to 15.

[0100] According to an embodiment of the present invention, the third nucleic acid fragment has a nucleotide sequence that has at least 51% homology to any of the sequences SEQ ID NO: 12 to 15.

[0101] According to an embodiment of the present invention, the third nucleic acid fragment has the nucleotide sequence shown in SEQ ID NO: 12-15.

[0102] According to embodiments of the present invention, the nucleic acid molecule further comprises a fourth nucleic acid fragment, the fourth nucleic acid fragment encoding a signal peptide sequence of MHCI (major histocompatibility complex I) or a sequence having a similar function to the signal peptide of MHCI. According to embodiments of the present invention, by adding the MHCI signal peptide to the N-terminus of the antigen sequence, ribosomes can be attached to the endoplasmic reticulum membrane, guiding the transport of proteins within the cell.

[0103] Furthermore, in this application, sequences having a similar function to the signal peptide of MHCI similarly attach ribosomes to the endoplasmic reticulum membrane and guide intracellular protein transport.

[0104] According to embodiments of the present invention, the signal peptide sequence of the MHCI does not include a transmembrane region.

[0105] According to an embodiment of the present invention, the signal peptide sequence of the MHCI has the amino sequence shown in SEQ ID NO: 16.

[0106] According to an embodiment of the present invention, the fourth nucleic acid fragment has the nucleotide sequence shown in SEQ ID NO: 17-22.

[0107] According to an embodiment of the present invention, the fourth nucleic acid fragment is provided at the 5' end of the nucleic acid fragment or at the 5' end of the nucleic acid molecule.

[0108] Furthermore, the fourth nucleic acid fragment is provided upstream of the nucleic acid fragment or the nucleic acid molecule, and is selectively located at the 5' end of the nucleic acid fragment or the 5' end of the nucleic acid molecule.

[0109] According to embodiments of the present invention, the nucleic acid molecule further comprises a fifth nucleic acid fragment, the fifth nucleic acid fragment encoding an MITD (MHC I molecular transport signal or major histocompatibility complex I molecular transport signal) sequence or a sequence having a similar function to MITD. According to embodiments of the present invention, by adding an MITD sequence to the C-terminus of the nucleic acid molecule, CD4 + This can stimulate the proliferation of T cells and induce the production of more cellular factors.

[0110] In this application, the sequence having a function similar to the aforementioned MITD is similarly CD4. + It can stimulate the proliferation of T cells, induce the production of more cellular factors, and trigger an immune response in the animal body.

[0111] According to an embodiment of the present invention, the MITD sequence has the amino sequence shown in SEQ ID NO: 23.

[0112] According to an embodiment of the present invention, the fifth nucleic acid fragment has the nucleotide sequence shown in SEQ ID NO: 24-32.

[0113] According to embodiments of the present invention, the fifth nucleic acid fragment is provided at the 3' end of the nucleic acid fragment or at the 3' end of the nucleic acid molecule.

[0114] The fifth nucleic acid fragment is provided downstream of the nucleic acid fragment or the nucleic acid molecule, and is selectively provided at the 3' end of the nucleic acid fragment or the 3' end of the nucleic acid molecule.

[0115] According to embodiments of the present invention, the nucleic acid fragment or nucleic acid molecule is a linear molecule.

[0116] According to embodiments of the present invention, the nucleic acid fragment or nucleic acid molecule has the nucleotide sequence shown in Table 4.

[0117] In a fourth aspect of the present invention, the present invention provides an expression vector. According to an embodiment of the present invention, the expression vector supports a nucleic acid molecule described in a third aspect of the present invention. According to an embodiment of the present invention, the expression vector can be expressed in vivo in cells, bacteria, yeast, or felines.

[0118] According to embodiments of the present invention, the expression vector may include at least one of the following additional technical features.

[0119] According to embodiments of the present invention, the expression vector is a nonviral carrier.

[0120] In a fifth aspect of the present invention, the present invention provides a recombinant virus. According to an embodiment of the present invention, the recombinant virus carries a nucleic acid molecule described in the third aspect of the present invention. The recombinant virus containing the nucleic acid molecule described in the third aspect can be reproduced in large quantities, and the recombinant virus plays an important role in vaccine research and development.

[0121] In a sixth aspect of the present invention, the present invention provides liposomes. According to embodiments of the present invention, the liposome comprises a liposome carrier and a nucleic acid fragment, the nucleic acid fragment being limited by the first and third aspects of the present invention. The liposome comprising the liposome carrier and the nucleic acid fragment plays an important role in improving nucleic acid stability, cell uptake rate, reducing toxic side effects, and improving delivery efficiency.

[0122] In a seventh aspect of the present invention, the present invention provides a vaccine. According to an embodiment of the present invention, the vaccine comprises a pharmaceutical formulation according to the first aspect of the present invention, a nucleic acid molecule according to the third aspect, an expression vector according to the fourth aspect, a recombinant virus according to the fifth aspect, or a liposome according to the sixth aspect of the present invention. According to an embodiment of the present invention, by employing the vaccine described above, an animal cell-mediated immune response can be efficiently activated. Furthermore, the vaccine contains only proteins that activate the immune response of cells, thus avoiding the occurrence of toxic side effects and providing higher safety.

[0123] According to embodiments of the present invention, the vaccine may include at least one of the following additional technical features.

[0124] According to embodiments of the present invention, the vaccine further comprises an adjuvant.

[0125] According to embodiments of the present invention, the adjuvant is a TLR agonist, Mn 2+ It includes at least one of the following.

[0126] According to embodiments of the present invention, the TLR agonist comprises at least one of CpG, R837, MPLA, and its derivatives.

[0127] In an eighth aspect of the present invention, the present invention provides recombinant cells. According to an example of the present invention, the recombinant cells carry a nucleic acid fragment, a nucleic acid molecule as described in the third aspect of the present invention, an expression vector as described in the fourth aspect of the present invention, or a recombinant virus as described in the fifth aspect of the present invention. The nucleic acid fragment comprises at least one of a first nucleic acid fragment, a second nucleic acid fragment, and a third nucleic acid fragment, wherein the first nucleic acid fragment encodes the M protein of feline infectious peritonitis virus, the second nucleic acid fragment encodes the N protein of feline infectious peritonitis virus, and the third nucleic acid fragment encodes the S, S_ec, or SII protein of feline infectious peritonitis virus, and the first nucleic acid fragment and the second nucleic acid fragment are ligated to or not ligated to the third nucleic acid fragment.

[0128] According to embodiments of the present invention, the recombinant cells are used to package a virus carrying the nucleic acid molecule, to prepare a nucleic acid vaccine to stimulate an immune response in an animal body, or to express at least one of the M, N, S, S_ec, or SII proteins of the FIPV virus.

[0129] In a ninth aspect of the present invention, the present invention provides a method for constructing a feline infectious peritonitis virus vaccine. According to an example of the present invention, the method comprises the step of introducing a nucleic acid fragment, a nucleic acid molecule according to a third aspect of the present invention, an expression vector according to a fourth aspect of the present invention, or a recombinant virus according to a fifth aspect of the present invention into a receptor cell. The nucleic acid fragment comprises at least one of a first nucleic acid fragment, a second nucleic acid fragment, and a third nucleic acid fragment, wherein the first nucleic acid fragment encodes the M protein of feline infectious peritonitis virus, the second nucleic acid fragment encodes the N protein of feline infectious peritonitis virus, and the third nucleic acid fragment encodes the S, S_ec, or SII protein of feline infectious peritonitis virus, and the first and second nucleic acid fragments are or are not linked to the third nucleic acid fragment.

[0130] The method according to the embodiments of the present invention can be used to package a virus carrying the nucleic acid molecule and to prepare a nucleic acid vaccine. The method for constructing an infectious peritonitis virus vaccine is safe, convenient, and efficient.

[0131] According to embodiments of the present invention, the method for constructing the above-mentioned feline infectious peritonitis virus vaccine may include at least one of the following additional technical features.

[0132] According to embodiments of the present invention, the method further comprises the step of coating the nucleic acid, expression vector, or recombinant virus using a coating carrier before introducing receptor cells.

[0133] According to embodiments of the present invention, the coated carrier is selected from at least one of liposomes, exosomes, polymer carriers, viral carriers, and nanoparticles.

[0134] According to embodiments of the present invention, the coating carrier is a nanoparticle. By selecting nanoparticles and coating mRNA, the mRNA can be protected from degradation and, by binding to the cell membrane, the delivery of mRNA into the cell can be promoted.

[0135] According to a specific embodiment of the present invention, the coated carrier is a lipid nanoparticle.

[0136] According to embodiments of the present invention, the receptor cells are CRFK cells, HEK293FT, HEK293T, BHK cells, or insect cells.

[0137] According to the embodiment of the present invention, the receptor cell is a CRFK cell. According to the embodiment of the present invention, the CRFK cell did not show a rejection reaction in the body of the test animal.

[0138] In a tenth aspect of the present invention, the present invention provides the use of the pharmaceutical formulation described in the first aspect of the present invention, the nucleic acid molecule described in the third aspect, the expression vector described in the fourth aspect, the recombinant virus described in the fifth aspect, and the liposome described in the sixth aspect in the preparation of a pharmaceutical or vaccine. According to an example of the present invention, the pharmaceutical or vaccine is used to prevent or treat a disease associated with feline infectious peritonitis virus infection. According to an example of the present invention, the pharmaceutical 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 time.

[0139] In an eleventh aspect of the present invention, the present invention provides a method for preventing or treating feline infectious peritonitis virus infection. According to an embodiment of the present invention, the method includes administering to a test animal a pharmaceutical formulation according to the first aspect of the present invention, a nucleic acid molecule according to the third aspect, an expression vector according to the fourth aspect, a recombinant virus according to the fifth aspect, a liposome according to the sixth aspect, a vaccine according to the seventh aspect, or a recombinant cell according to the eighth aspect. According to an embodiment of the present invention, administering an effective dose of a pharmaceutical formulation, nucleic acid molecule, expression vector, recombinant virus, liposome, vaccine, or recombinant cell to a test animal infected with FIPV can significantly improve various physiological indicators and survival rates of the test animal. The above-described treatment method shows good immunological effects against various circulating strains of FIPV.

[0140] In this specification, the term "effective dose" means the amount that can produce function or activity in a test animal and is acceptable to the test animal.

[0141] The effective dose of the pharmaceutical formulation, nucleic acid molecule, expression vector, recombinant virus, liposome, vaccine, or recombinant cell described in the present invention varies depending on the method of administration, the severity of FIPV infection in the test animal, and other factors. The selection of a preferred effective dose can be determined by those skilled in the art based on various factors (e.g., clinical trials). These factors include, but are not limited to, pharmacokinetic parameters of the active ingredient, such as bioavailability, metabolism, and half-life, the severity of FIPV infection in the test animal, the body weight of the test animal, the immune status of the test animal, and the route of administration. For example, depending on the urgency of the treatment situation, the dose may be administered in multiple divided doses per day, or the dose may be reduced proportionally.

[0142] According to embodiments of the present invention, the above method may include at least one of the following additional technical features.

[0143] According to an embodiment of the present invention, the test animal is selected from cats.

[0144] In a twelfth aspect of the present invention, the present invention proposes the use of the pharmaceutical formulation described in the first aspect, the nucleic acid molecule described in the third aspect, the expression vector described in the fourth aspect, the recombinant virus described in the fifth aspect, the liposome described in the sixth aspect, the vaccine described in the seventh aspect, or the recombinant cell described in the eighth aspect in the prevention or treatment of feline infectious peritonitis virus infection. According to examples of the present invention, administering an effective dose of the pharmaceutical formulation, nucleic acid molecule, expression vector, recombinant virus, liposome, vaccine, or recombinant cell to a test animal infected with FIPV can significantly improve various physiological indicators and survival rates of the test animal. Furthermore, the above-described treatment method shows good immunological effects against various circulating strains of FIPV.

[0145] Additional aspects and advantages of the present invention are shown in part in the following description, some of which become apparent from the following description or are understood through the practice of the present invention. [Brief explanation of the drawing]

[0146] The above and / or additional aspects and advantages of the present invention will become apparent and readily apparent from the description of the embodiments with reference to the following drawings. [Figure 1] This shows the expression detection results of M, N, and S proteins according to Example 1 of the present invention. [Figure 2] This is the result of detecting the expression of target mRNA coated with LNP according to Example 2 of the present invention. [Figure 3] This shows the change in viability after immunization of target mRNA coated with LNP according to Example 2 of the present invention, followed by a viral attack. [Figure 4] This is the result of detecting the expression of target mRNA coated with LNP according to Example 3 of the present invention. [Figure 5] This shows the change in viability after immunization of target mRNA coated with LNP according to Example 3 of the present invention, followed by a viral attack. [Figure 6] This shows the change in viability after immunization of target mRNA coated with LNP according to Example 4 of the present invention, followed by a viral attack. [Figure 7]This shows the change in viability after immunization of target mRNA coated with LNP according to Example 5 of the present invention, followed by a viral attack. [Modes for carrying out the invention]

[0147] Embodiments of the present invention shown in the drawings will be described in detail below. The embodiments described hereafter with reference to the drawings are illustrative and used solely to illustrate the present invention and should not be understood as limiting the present invention.

[0148] Furthermore, the terms “first” and “second” are used solely for descriptive purposes and should not be understood as indicating the number of technical features of relative importance, whether explicitly or implicitly. Therefore, features limited by “first” and “second” may explicitly or implicitly include at least one such feature. In this description, “multiple” means at least two, for example, two, three, etc., unless otherwise specified.

[0149] In this application, the terms "homology" and "identity" both refer to the similarity between amino or nucleotide sequences, and there is no specific distinction in their meaning.

[0150] Beneficial effects The mRNA vaccine for preventing feline infectious peritonitis described in the present invention involves constructing a carrier encoding at least one of the M, N, S, S_ec, or SII proteins of the FIPV virus, and then preparing the mRNA vaccine for preventing the FIPV virus with lipid nanoparticles (LNPs). The vaccine stimulates the test animal to induce a cellular immune response. The vaccine described in the present invention has advantages such as a simple preparation process, high safety, no toxic side effects, and the ability to be industrially produced, and sufficient protective effect can be achieved by using very small doses, making it superior to existing therapeutic methods in terms of safety and efficacy.

[0151] The arrangement relating to this application is shown in Table 3.

[0152] [Table 3-1] [Table 3-2] [Table 3-3] [Table 3-4] [Table 3-5] [Table 3-6] [Table 3-7] [Table 3-8] [Table 3-9] [Table 3-10] [Table 3-11] [Table 3-12] [Table 3-13] [Table 3-14] [Table 3-15] [Table 3-16] Table 3-17 Table 3-18 Table 3-19 Table 3-20 Table 3-21 Table 3-22 Table 3-23 Table 3-24 Table 3-25 Table 3-26 Table 3-27

[0153] Table 4-1 Table 4-2

[0154] Table 5

[0155] The 3' end of the signal peptide 1 sequence shown in SEQ ID NO:20 is ligated to the 5' end of the tag peptide 1 sequence shown in SEQ ID NO:36, the 3' end of the tag peptide 1 sequence shown in SEQ ID NO:36 is ligated to the 5' end of the gene 1 sequence shown in SEQ ID NO:7, the 3' end of the gene 1 sequence shown in SEQ ID NO:7 is ligated to the 5' end of the MITD 1 sequence shown in SEQ ID NO:24, the 3' end of the MITD 1 sequence shown in SEQ ID NO:24 is ligated to the 5' end of the linked peptide sequence shown in SEQ ID NO:47, the 3' end of the linked peptide sequence shown in SEQ ID NO:47 is ligated to the 5' end of the tag peptide 2 sequence shown in SEQ ID NO:38, and the 3' end of the tag peptide 2 sequence shown in SEQ ID NO:38 is ligated to the 5' end of the gene 2 sequence shown in SEQ ID NO:9.

[0156] The present invention will be described below with reference to specific examples, which are illustrative and do not limit the invention in any way. Where specific techniques or conditions are not shown in the examples, the invention must be carried out in accordance with the techniques or conditions described in the literature in the art or in accordance with the product specifications. Where the manufacturer of the reagents or equipment used is not indicated, they are conventional products available commercially.

[0157] Example 1: Selection of the target sequence

[0158] According to an embodiment of the present invention, mRNA sequences were synthesized in vitro and expressed in CRFK (feline kidney) cells and feline DC (dendritic) cells. The sequence with the highest expression level was selected as the final target sequence, and a vaccine was prepared. The specific steps are as follows. 1) Codon optimization was performed on the M, N, and S antigen sequences using the feline universal codon library and the feline mesenteric lymph node codon library, and three sequences were optimized for each antigen. 2) After sequence synthesis (derived from Universal Bio), a support structure is constructed. The support structure contains cat-derived 5' HBB-UTR (SEQ ID NO: 44), 3' HBA-UTR (SEQ ID NO: 45) sequences and polyA, and the target sequence is constructed between the 5'UTR and 3'UTR. 3) After transcribing mRNA in vitro, the mRNA was transfected into CRFK cells and feline DC (dendritic) cells using a transfection reagent, and sequences with high expression levels were screened in the two feline source cell lines.

[0159] The results are shown in Figure 1, and we successfully screened for target antigen sequences M6 (SEQ ID NO: 7), N2 (SEQ ID NO: 9), and S2 (SEQ ID NO: 13) that could be highly expressed in two cell types.

[0160] Example 2: Vaccine titer verification

[0161] The embodiment of the present invention evaluates the effectiveness of a vaccine by evaluating changes in physiological indicators such as body temperature and body weight, as well as survival rate, after mRNA vaccine immunization and subsequent viral attack on test animals.

[0162] According to an embodiment of the present invention, sp-HA-M6-MITD, sp-His-N2, and sp-SII2-MITD were constructed between a feline source 5' HBB-UTR (SEQ ID NO: 44) and a 3' HBA-UTR (SEQ ID NO: 45), transcribed into mRNA in vitro, and the sequences are shown in Table 4. According to the embodiments of the present invention, lipid nanoparticles (LNPs) were prepared. The specific steps are as follows: 1) Preparation of lipid solution: The average molecular weight of the liposome system is approximately 620.62. A 12 mM lipid solution was prepared, and SM-102: 42.61 mg, PEG-DMG: 4.52 mg, DSPC: 9.48 mg, and Chol: 17.86 mg were added. After dissolving in 10 mL of anhydrous ethanol, the solution was filtered through a 0.22 μm filter membrane. 2) Diluting the target mRNA with citrate buffer (pH 4) and mixing uniformly, a rapid nanopharmaceutical preparation system (Meitai) was used to prepare the mRNA vaccine liposome solution by setting the flow rate preconditions to 1:3 (organic phase X volume (containing cationic lipids): aqueous phase Y volume (containing nucleic acids) = 1:3). This solution was then placed in 30 times the volume of PBS, concentrated in a 15 ml ultrafiltration tube with a cutoff volume of 100 kDa, and centrifuged at 3000 rpm for 20 minutes. Finally, the solution was stored in a 600 mM sucrose solution (PBS prepared, filtered through a 0.22 μm filter membrane) and diluted by equal volume. The sample was stored at -20°C and prepared for use. According to embodiments of the present invention, test animals meeting the test criteria were screened by physical examination and laboratory examination. Physical examination items included body temperature and weight, and screening items included PCR detection, N and S bound antibody testing, and neutralizing antibody testing. The specific experimental steps are as follows. 1) Physical examination: For 7 days prior to immunization, the kittens' body temperature and weight were measured daily. The normal body temperature was approximately 38.5°C, and the weight of a 1-year-old pet cat was approximately 3 kg. 2) Screening of FIPV-negative cats: The FIPV 7ab gene was detected by PCR, and the N and S proteins were used as antigens by ELISA to detect bound antibodies in cat serum. Neutralizing antibodies against FIPV were detected using pseudovirus neutralization experiments.

[0163] According to an embodiment of the present invention, test animals screened with target mRNA coated with LNPs were immunized. The immunization program was as follows: The D0 virus was first-stage, the D21 virus was second-stage, and after the second-stage virus was second-stage, the D28 virus attack was carried out. The toxic strain used in the virus attack was QS-1146, and it involved 5 kittens per group.

[0164] Target mRNA coated with LNP was transfected into CRFK cells, collected after 24 hours, and immunoblotting was performed to detect protein expression. The results are shown in Figure 2, and all cells were expressed normally. After immune viral attack on the LNP-coated target mRNA in each group, all five kittens in the PBS group successively developed symptoms of fever and weight loss. Autopsy revealed that these were typical feline transmissible abdominal lesions. The physiological indicators of the kittens were clearly improved in the immune groups immunized with mRNA expressing M, N, and SII antigens and their combinations. All kittens in the M+N+SII group were normal. The survival rates are shown in Figure 3. mRNA expressing each of the M, N, and SII antigens improved the survival rate after viral attack in all cases after immunization. Multi-antigen combinations further improved the survival rate after viral attack, with the survival rate after viral attack reaching 100% in the M+N+SII group.

[0165] The results above indicate that mRNA expressing the M, N, and SII antigens, as well as combinations thereof, has a good immune effect against FIPV as a vaccine, significantly improving various physiological indicators and survival rates, with the M+N+SII combination being the most effective.

[0166] Example 3: Verification of titer of multi-antigen mRNA vaccines using different linking methods

[0167] Examples of the present invention evaluate the efficacy of multi-antigen mRNA vaccines with different linkage schemes. The LNP preparation and expression verification methods, test animal screening methods, immunization programs, and evaluation methods are the same as in Example 2.

[0168] According to embodiments of the present invention, sp-HA-M6-MITD, sp-His-N2, sp-HA-M-MITD-2A-His-N, and sp-HA-M-GS-N-MITD were constructed between a feline source 5' HBB-UTR (SEQ ID NO: 44) and a 3' HBA-UTR (SEQ ID NO: 45), transcribed in vitro into mRNA, and the sequences are shown in Tables 4 and 5. The expression verification results after LNP coating the target mRNA are shown in Figure 4, and in all cases, normal expression was achieved. After immunoviral attack, all multi-antigen mRNA vaccines with different ligation methods were able to effectively improve the physiological indicators and survival rates of the test animals after viral attack. The survival rates are shown in Figure 5, and there were no significant differences between the different ligation methods.

[0169] These results demonstrate that both isolated single-antigen mRNA vaccines and multi-antigen mRNA vaccines linked via different linking peptides can exhibit favorable immune effects against FIPV.

[0170] Example 4: Verification of multi-antigen mRNA vaccine titers with different proportions

[0171] The embodiments of the present invention evaluate the efficacy of multi-antigen mRNA vaccines in different proportions. The LNP preparation method, test animal screening method, immunization program, and evaluation method are the same as in Example 2.

[0172] According to an embodiment of the present invention, sp-HA-M6-MITD, sp-His-N2, and sp-SII2-MITD were constructed between a feline source 5' HBB-UTR (SEQ ID NO: 44) and a 3' HBA-UTR (SEQ ID NO: 45), respectively, and transcribed in vitro into mRNA. The sequences are shown in Table 4. After immune viral attack, all multi-antigen mRNA vaccines with different proportions effectively improved the physiological indicators and survival rates of the test animals after viral attack, as shown in Figure 6.

[0173] These results demonstrate that multi-antigen mRNA vaccines with different proportions can all show good immune efficacy against FIPV.

[0174] Example 5: Verification of multi-antigen mRNA vaccine titers of different circulating strains

[0175] The present invention's examples evaluate the efficacy of a multi-antigen mRNA vaccine against different FIPV epidemic strains. The LNP preparation method, test animal screening method, immunization program, and evaluation method are the same as in Example 2. The virulent strains used for viral attack were HF1902 and SH2211.

[0176] According to an embodiment of the present invention, sp-HA-M6-MITD, sp-His-N2, and sp-SII2-MITD were constructed between a feline source 5' HBB-UTR (SEQ ID NO: 44) and a 3' HBA-UTR (SEQ ID NO: 45), respectively, and transcribed in vitro into mRNA. The sequences are shown in Table 4. After immune viral attack, the multi-antigen mRNA vaccine effectively improved physiological indicators and survival rates in test animals against different FIPV epidemic strains, as shown in Figure 7.

[0177] These results demonstrate that multi-antigen mRNA vaccines can show good immune efficacy against different FIPV epidemic strains.

[0178] In this specification, any description referring to terms such as “one example,” “several examples,” “example,” “specific example,” or “several examples” means that a particular feature, structure, material, or property described with reference to that example is included in at least one example of this disclosure. In this specification, the general expressions of the above terms do not necessarily apply to the same example. In addition, any particular feature, structure, material, or property described may be incorporated in an appropriate manner in any one or more examples. Furthermore, those skilled in the art can combine and combine the various examples and features relating to the various examples described herein without contradiction.

[0179] Although embodiments of the present invention have been presented and described, these embodiments are illustrative and should not be understood as limiting the disclosure. Those skilled in the art will understand that various changes, modifications, substitutions, and variations are possible with respect to the embodiments within the scope of the disclosure.

Claims

1. It is a pharmaceutical preparation, The material comprises a nucleic acid fragment, wherein the nucleic acid fragment is mRNA and comprises at least one of a first nucleic acid fragment, a second nucleic acid fragment, and a third nucleic acid fragment. The first nucleic acid fragment encodes the M protein of feline infectious peritonitis virus, The second nucleic acid fragment encodes the N protein of feline infectious peritonitis virus, The third nucleic acid fragment encodes the S, S_ec, or SII protein of feline infectious peritonitis virus. A pharmaceutical formulation characterized in that the first nucleic acid fragment and the second nucleic acid fragment are linked to or not linked to the third nucleic acid fragment.

2. The pharmaceutical formulation according to claim 1, characterized in that the first nucleic acid fragment and the second nucleic acid fragment are not linked to the third nucleic acid fragment.

3. The pharmaceutical formulation according to claim 1, characterized in that the first nucleic acid fragment and the second nucleic acid fragment are linked to the third nucleic acid fragment.

4. The pharmaceutical formulation according to claim 2 or 3, characterized in that the mass ratio of the first nucleic acid fragment to the second nucleic acid fragment is 10:1 to 1:

10.

5. The pharmaceutical preparation according to claim 4, characterized in that the mass ratio of the second nucleic acid fragment to the third nucleic acid fragment is 10:1 to 1:

10.

6. The pharmaceutical formulation according to claim 5, characterized in that the mass ratio of the first nucleic acid fragment, the second nucleic acid fragment, and the third nucleic acid fragment is 1:1:

1.

7. The pharmaceutical formulation according to claim 1, characterized in that the third nucleic acid fragment is selected from an SII protein.

8. A method for preparing a pharmaceutical preparation according to any one of claims 1 to 7, The process includes the step of mixing a first nucleic acid fragment, a second nucleic acid fragment, and a third nucleic acid fragment in a predetermined ratio to obtain the pharmaceutical preparation, The first nucleic acid fragment encodes the M protein of feline infectious peritonitis virus, The second nucleic acid fragment encodes the N protein of feline infectious peritonitis virus, The third nucleic acid fragment encodes the S, S_ec, or SII protein of feline infectious peritonitis virus. A method characterized in that the first nucleic acid fragment, the second nucleic acid fragment, and the third nucleic acid fragment are mRNA.

9. The method according to the 8th method, characterized in that the mass ratio of the first nucleic acid fragment to the second nucleic acid fragment is 10:1 to 1:

10.

10. The method according to 9, characterized in that the mass ratio of the second nucleic acid fragment to the third nucleic acid fragment is 10:1 to 1:

10.

11. The method according to 10, characterized in that the mass ratio of the first nucleic acid fragment, the second nucleic acid fragment, and the third nucleic acid fragment is 1:1:

1.

12. Isolated nucleic acid molecules, The first nucleic acid fragment encoding the M protein of feline infectious peritonitis virus, The second nucleic acid fragment encoding the N protein of feline infectious peritonitis virus, A third nucleic acid fragment encoding the S, S_ec, or SII protein of feline infectious peritonitis virus, The first nucleic acid fragment and the second nucleic acid fragment are linked to the third nucleic acid fragment. An isolated nucleic acid molecule characterized in that the nucleic acid molecule is mRNA.

13. The first nucleic acid fragment is linked to the second nucleic acid fragment, and the third nucleic acid fragment is not linked to the first and second nucleic acid fragments, or The first nucleic acid fragment is linked to the third nucleic acid fragment, and the second nucleic acid fragment is not linked to the first nucleic acid fragment and the third nucleic acid fragment, or The second nucleic acid fragment is linked to the third nucleic acid fragment, and the first nucleic acid fragment is not linked to the second and third nucleic acid fragments, or The 3' end of the first nucleic acid fragment is connected to the 5' end of the second nucleic acid fragment, and the third nucleic acid fragment is not connected to the first nucleic acid fragment or the second nucleic acid fragment, or The 3' end of the second nucleic acid fragment is connected to the 5' end of the first nucleic acid fragment, and the third nucleic acid fragment is not connected to the first nucleic acid fragment or the second nucleic acid fragment. The 3' end of the first nucleic acid fragment is connected to the 5' end of the third nucleic acid fragment, and the second nucleic acid fragment is not connected to the first nucleic acid fragment and the third nucleic acid fragment, or The 3' end of the third nucleic acid fragment is connected to the 5' end of the first nucleic acid fragment, and the second nucleic acid fragment is not connected to the first nucleic acid fragment or the third nucleic acid fragment, or The 3' end of the second nucleic acid fragment is linked to the 5' end of the third nucleic acid fragment, and the first nucleic acid fragment is not linked to the second and third nucleic acid fragments, or The 3' end of the third nucleic acid fragment is connected to the 5' end of the second nucleic acid fragment, and the first nucleic acid fragment is not connected to the second nucleic acid fragment or the third nucleic acid fragment. The 3' end of the first nucleic acid fragment is connected to the 5' end of the second nucleic acid fragment, and the 3' end of the second nucleic acid fragment is connected to the 5' end of the third nucleic acid fragment, or The 3' end of the first nucleic acid fragment is connected to the 5' end of the third nucleic acid fragment, and the 3' end of the third nucleic acid fragment is connected to the 5' end of the second nucleic acid fragment, or The 3' end of the second nucleic acid fragment is connected to the 5' end of the first nucleic acid fragment, and the 3' end of the first nucleic acid fragment is connected to the 5' end of the third nucleic acid fragment, or The 3' end of the second nucleic acid fragment is connected to the 5' end of the third nucleic acid fragment, and the 3' end of the third nucleic acid fragment is connected to the 5' end of the first nucleic acid fragment, or The 3' end of the third nucleic acid fragment is connected to the 5' end of the first nucleic acid fragment, and the 3' end of the first nucleic acid fragment is connected to the 5' end of the second nucleic acid fragment, or The pharmaceutical formulation according to claim 2 or the nucleic acid molecule according to claim 12, characterized in that the 3' end of the third nucleic acid fragment is connected to the 5' end of the second nucleic acid fragment, and the 3' end of the second nucleic acid fragment is connected to the 5' end of the first nucleic acid fragment.

14. The pharmaceutical formulation according to claim 1 or the nucleic acid molecule according to claim 12, characterized in that the M protein has an amino sequence having at least 89% homology with the amino sequence shown in SEQ ID NO:

1.

15. The pharmaceutical preparation or nucleic acid molecule according to claim 14, characterized in that the M protein has an amino sequence having at least 89% homology to the amino sequence shown in SEQ ID NO: 1, and the amino at position 90 is Y, at position 102 is V, at position 120 is I, at position 144 is A, and at position 180 is L.

16. The pharmaceutical preparation or nucleic acid molecule according to claim 15, characterized in that the M protein has the amino sequence shown in SEQ ID NO:

1.

17. The pharmaceutical formulation according to claim 1 or the nucleic acid molecule according to claim 12, characterized in that the N protein has an amino sequence having at least 91% homology with the amino sequence shown in SEQ ID NO:

2.

18. The pharmaceutical preparation or nucleic acid molecule according to claim 17, characterized in that the N protein has the amino sequence shown in SEQ ID NO:

2.

19. The pharmaceutical formulation according to claim 1 or the nucleic acid molecule according to claim 12, characterized in that the S protein has an amino sequence having at least 45% homology with SEQ ID NO:

3.

20. The pharmaceutical preparation or nucleic acid molecule according to claim 19, characterized in that the S protein has an amino sequence having at least 45% homology with SEQ ID NO: 3, and the amino at position 515 is V, the amino at position 577 is Q, the amino at position 1385 is V, the amino at position 1386 is V, the amino at position 1397 is F, and the amino at position 1415 is I.

21. The pharmaceutical preparation or nucleic acid molecule according to claim 20, characterized in that the S protein has the amino sequence shown in SEQ ID NO:

3.

22. The pharmaceutical formulation according to claim 1 or the nucleic acid molecule according to claim 12, characterized in that the S_ec protein has an amino sequence having at least 43% homology to the aminos at positions 1 to 1374 of the amino sequence shown in SEQ ID NO:

3.

23. The pharmaceutical preparation or nucleic acid molecule according to claim 22, characterized in that the S_ec protein has an amino sequence having at least 43% homology to the aminos at positions 1 to 1374 of the amino sequence shown in SEQ ID NO: 3, and the amino at position 515 is V and the amino at position 577 is Q.

24. The pharmaceutical preparation or nucleic acid molecule according to claim 23, characterized in that the S_ec protein has an amino sequence at positions 1 to 1374 of SEQ ID NO:

3.

25. The pharmaceutical formulation according to claim 1 or the nucleic acid molecule according to claim 12, characterized in that the SII protein has an amino sequence having at least 62% homology to the aminos at positions 782 to 1433 of the amino sequence shown in SEQ ID NO:

3.

26. The pharmaceutical preparation or nucleic acid molecule according to claim 25, characterized in that the SII protein has an amino sequence having at least 62% homology to the aminos at positions 782 to 1433 of the amino sequence shown in SEQ ID NO: 3, and the amino at position 1385 is V, the amino at position 1386 is V, the amino at position 1397 is F, and the amino at position 1415 is I.

27. The pharmaceutical preparation or nucleic acid molecule according to claim 26, characterized in that the SII protein has an amino sequence at positions 782 to 1433 of SEQ ID NO:

3.

28. The pharmaceutical formulation according to claim 1 or the nucleic acid molecule according to claim 12, characterized in that the first nucleic acid fragment has a nucleotide sequence having at least 67% homology with any of the sequences of SEQ ID NO: 4 to 7.

29. The pharmaceutical preparation or nucleic acid molecule according to claim 28, characterized in that the first nucleic acid fragment has the nucleotide sequence shown in SEQ ID NO: 4 to 7.

30. The pharmaceutical formulation according to claim 1 or the nucleic acid molecule according to claim 12, characterized in that the second nucleic acid fragment has a nucleotide sequence having at least 70% homology with any sequence of SEQ ID NO: 8 to 11.

31. The pharmaceutical preparation or nucleic acid molecule according to claim 30, characterized in that the second nucleic acid fragment has the nucleotide sequence shown in SEQ ID NO: 8 to 11.

32. The pharmaceutical formulation according to claim 1 or the nucleic acid molecule according to claim 12, characterized in that the third nucleic acid fragment has a nucleotide sequence having at least 51% homology with the nucleotides at positions 1 to 4122 of any sequence SEQ ID NO: 12 to 15.

33. The pharmaceutical formulation or nucleic acid molecule according to claim 32, characterized in that the third nucleic acid fragment has a nucleotide sequence from position 1 to 4122 of SEQ ID NO: 12 to 15.

34. The pharmaceutical formulation or nucleic acid molecule according to claim 33, characterized in that the third nucleic acid fragment has a nucleotide sequence having at least 57% homology with the nucleotides at positions 2344 to 4299 of any of the sequences of SEQ ID NO: 12 to 15.

35. The pharmaceutical preparation or nucleic acid molecule according to claim 34, characterized in that the third nucleic acid fragment has a nucleotide sequence from positions 2344 to 4299 of SEQ ID NO: 12 to 15.

36. The pharmaceutical formulation or nucleic acid molecule according to claim 35, characterized in that the third nucleic acid fragment has a nucleotide sequence having at least 51% homology with any of the sequences of SEQ ID NO: 12 to 15.

37. The pharmaceutical preparation or nucleic acid molecule according to claim 36, characterized in that the third nucleic acid fragment has the nucleotide sequence shown in SEQ ID NO: 12 to 15.

38. The pharmaceutical formulation according to claim 1 or the nucleic acid molecule according to claim 12, further comprising a fourth nucleic acid fragment, wherein the fourth nucleic acid fragment encodes an MHCI signal peptide sequence or a sequence having a similar function to an MHCI signal peptide.

39. The pharmaceutical formulation or nucleic acid molecule according to claim 38, characterized in that the signal peptide sequence of the MHCI does not contain a transmembrane region.

40. The pharmaceutical preparation or nucleic acid molecule according to claim 38, characterized in that the signal peptide sequence of the MHCI has the amino sequence shown in SEQ ID NO:

16.

41. The pharmaceutical preparation or nucleic acid molecule according to claim 38, characterized in that the fourth nucleic acid fragment has the nucleotide sequence shown in SEQ ID NO: 17 to 22.

42. The pharmaceutical formulation according to claim 2 or 38, or the nucleic acid molecule according to claim 12 or 38, characterized in that the fifth nucleic acid fragment is provided at the 5' end of the nucleic acid fragment or at the 5' end of the nucleic acid molecule.

43. The pharmaceutical formulation according to claim 1 or the nucleic acid molecule according to claim 12, further comprising a fifth nucleic acid fragment, wherein the fifth nucleic acid fragment encodes an MITD sequence or a sequence having a function similar to MITD.

44. The pharmaceutical preparation or nucleic acid molecule according to claim 43, characterized in that the MITD has the amino sequence shown in SEQ ID NO:

23.

45. The pharmaceutical preparation or nucleic acid molecule according to claim 38, characterized in that the fifth nucleic acid fragment has the nucleotide sequence shown in SEQ ID NO: 24 to 32.

46. The pharmaceutical formulation according to claim 2 or 43, or the nucleic acid molecule according to claim 12 or 43, characterized in that the fifth nucleic acid fragment is provided at the 3' end of the nucleic acid fragment or at the 3' end of the nucleic acid molecule.

47. The pharmaceutical formulation according to claim 1 or the nucleic acid molecule according to claim 12, characterized in that the nucleic acid fragment or nucleic acid molecule is a linear molecule.

48. The pharmaceutical preparation or nucleic acid molecule according to claim 47, characterized in that the nucleic acid fragment or nucleic acid molecule has the nucleotide sequence shown in Table 4.

49. The pharmaceutical formulation according to claim 1, further comprising a drug carrier, wherein the drug carrier comprises at least one of liposomes, exosomes, polymer carriers, viral carriers, and nanoparticles.

50. An expression vector characterized by supporting a nucleic acid molecule as described in any one of claims 12 to 48.

51. The expression vector according to claim 50, characterized in that the expression vector is a nonviral carrier.

52. A recombinant virus characterized by carrying a nucleic acid molecule as described in any one of claims 12 to 48.

53. A liposome comprising a liposome carrier and a nucleic acid fragment, wherein the nucleic acid fragment is limited by any one of claims 1 to 7 or 13 to 48.

54. A vaccine comprising a pharmaceutical preparation according to any one of claims 1 to 7 or 13 to 49, a nucleic acid molecule according to any one of claims 12 to 48, an expression vector according to any one of claims 50 to 51, a recombinant virus according to claim 52, or a liposome according to claim 53.

55. The vaccine according to claim 54, further comprising an adjuvant.

56. The aforementioned adjuvant is a TLR agonist, Mn ２＋ The vaccine according to claim 55, characterized by comprising at least one of the following.

57. The vaccine according to claim 56, characterized in that the TLR agonist comprises at least one of CpG, R837, MPLA, and its derivatives.

58. Recombinant cells carrying a nucleic acid fragment, a nucleic acid molecule according to any one of claims 12 to 48, an expression vector according to any one of claims 50 to 51, or a recombinant virus according to claim 52, The nucleic acid fragment comprises at least one of a first nucleic acid fragment, a second nucleic acid fragment, and a third nucleic acid fragment. The first nucleic acid fragment encodes the M protein of feline infectious peritonitis virus, The second nucleic acid fragment encodes the N protein of feline infectious peritonitis virus, The third nucleic acid fragment encodes the S, S_ec, or SII protein of feline infectious peritonitis virus. Recombinant cells characterized in that the first nucleic acid fragment and the second nucleic acid fragment are linked to or not linked to the third nucleic acid fragment.

59. A method for constructing a feline infectious peritonitis virus vaccine, The process includes the step of introducing a nucleic acid fragment, a nucleic acid molecule according to any one of claims 12 to 48, an expression vector according to any one of claims 50 to 51, and a recombinant virus according to claim 52 into a receptor cell. The nucleic acid fragment comprises at least one of the first nucleic acid fragment, the second nucleic acid fragment, and the third nucleic acid fragment. The first nucleic acid fragment encodes the M protein of feline infectious peritonitis virus, The second nucleic acid fragment encodes the N protein of feline infectious peritonitis virus, The third nucleic acid fragment encodes the S, S_ec, or SII protein of feline infectious peritonitis virus. A method for constructing a feline infectious peritonitis virus vaccine, characterized in that the first nucleic acid fragment and the second nucleic acid fragment are linked to or not linked to the third nucleic acid fragment.

60. The method according to 59, further comprising the step of coating the nucleic acid, expression vector, or recombinant virus using a coating carrier before introducing it into receptor cells.

61. The method according to 60, characterized in that the coated carrier is selected from at least one of liposomes, exosomes, polymer carriers, viral carriers, and nanoparticles.

62. The method according to 61, characterized in that the coating carrier is a liposome.

63. The method according to 59, characterized in that the receptor cells are CRFK cells, HEK293FT, HEK293T, BHK cells, and insect cells.

64. The method according to 63, characterized in that the receptor cells are CRFK cells.

65. Use of a pharmaceutical formulation according to any one of claims 1 to 7, 13 to 49, a nucleic acid molecule according to any one of claims 12 to 48, an expression vector according to any one of claims 50 to 51, a recombinant virus according to claim 52, or a liposome according to claim 53 in the preparation of a pharmaceutical or vaccine for the prevention or treatment of a disease associated with feline infectious peritonitis virus infection.

66. A method for preventing or treating feline infectious peritonitis virus infection, comprising the step of administering to a test animal a pharmaceutical preparation according to any one of claims 1 to 7 or 13 to 49, a nucleic acid molecule according to any one of claims 12 to 48, an expression vector according to any one of claims 50 to 51, a recombinant virus according to claim 52, a liposome according to claim 53, a vaccine according to any one of claims 54 to 57, or a recombinant cell according to claim 58.

67. The method according to 66, characterized in that the animal is selected from cats.

68. Use of a pharmaceutical preparation according to any one of claims 1 to 7 or 13 to 49, a nucleic acid molecule according to any one of claims 12 to 48, an expression vector according to any one of claims 50 to 51, a recombinant virus according to claim 52, a liposome according to claim 53, a vaccine according to any one of claims 54 to 57, or a recombinant cell according to claim 58, in the prevention or treatment of feline infectious peritonitis virus infection.

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