Method for constructing circular RNA and vaccine aiming at cat FIPV

Abstract

The invention provides a method for constructing circular RNA (Ribonucleic Acid) and a vaccine aiming at the cat FIPV. The method relates to a pharmaceutical preparation, the pharmaceutical preparation comprises nucleic acid fragments, the nucleic acid fragments are circular RNA, and the nucleic acid fragments comprise a first nucleic acid fragment and a second nucleic acid fragment; the first nucleic acid fragment encodes M protein of feline infectious peritonitis virus; the second nucleic acid fragment encodes N protein of feline infectious peritonitis virus; the first nucleic acid fragment and the second nucleic acid fragment are connected or not connected.

Description

Methods for constructing circular RNA and vaccines against feline FIPV Technical Field

[0001] The present invention relates to the field of biotechnology, specifically, to a method for constructing circular RNA and a vaccine against feline FIPV antigens, and more specifically, to a pharmaceutical preparation, a method for preparing a pharmaceutical preparation, an isolated nucleic acid molecule, an expression vector, a recombinant virus, a liposome, a vaccine, a recombinant cell, a method for constructing a feline infectious peritonitis virus vaccine and its use, or a method for preventing or treating feline infectious peritonitis virus infection. Background Art

[0002] Feline coronavirus (FCoV) belongs to the coronavirus family. Viruses belonging to the Coronaviridae family are characterized by relatively large, round, enveloped, positive-strand RNA viruses with genomes ranging from 27 to 32 kb. They encode replication polymerases, four structural proteins (S protein, M protein, N protein, and E protein), 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 SII region of the S protein contains relatively few epitopes that mediate antibody-dependent enhancement (ADE). The M protein (membrane protein) is involved in the formation and localization of coronavirus particle morphology. It is a protein that 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. Furthermore, the N protein can induce a host immune response against 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] 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.

[0004] 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 that there is a severe virus-induced immune disorder. 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 based on humoral immunity, 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.

[0005] Therefore, there is an urgent need in this field to develop a vaccine against FIPV.

[0006] Summary of the Invention

[0007] This application is filed by the inventor based on the following findings:

[0008] 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.

[0009] To this end, in its first aspect, the present invention provides a pharmaceutical preparation. According to an embodiment of the present invention, the pharmaceutical preparation comprises: a nucleic acid segment, wherein the nucleic acid segment is a circular RNA, and the nucleic acid segment comprises a first nucleic acid segment and a second nucleic acid segment; the first nucleic acid segment encodes the M protein of the feline infectious peritonitis virus; the second nucleic acid segment encodes the N protein of the feline infectious peritonitis virus; and the first nucleic acid segment and the second nucleic acid segment may be linked or unlinked. According to an embodiment of the present invention, a pharmaceutical preparation expressing either the M protein or the N protein of the feline infectious peritonitis virus, or a combination thereof, can stimulate an animal's somatic cell-mediated immune response.

[0010] It should be noted that in the present application, the M or N protein of the wild-type FIPV virus can also be adaptively modified as needed to reduce the toxicity of the FIPV virus without affecting its three-dimensional structure, retaining its immunogenicity, and preparing a new type of FIPV virus vaccine. According to the sequence alignment results (Tables 1 and 2), the M protein has an amino acid 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 one of SEQ ID NOs: 4 to 7, the N protein has an amino acid 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 one of SEQ ID NOs: 8 to 11. The FIPV virus vaccine is not particularly limited, as long as the receptor binding region of the modified FIPV virus M or N protein can be produced in the organism, and it has immunogenicity and can induce an immune response in the animal body. Moreover, the pharmaceutical preparation 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.

[0011] According to an embodiment of the present invention, the above-mentioned pharmaceutical preparation may further include at least one of the following additional technical features:

[0012] According to an embodiment of the present invention, the first nucleic acid fragment is connected to the second nucleic acid fragment.

[0013] According to an embodiment of the present invention, the first nucleic acid fragment is not connected to the second nucleic acid fragment.

[0014] According to an embodiment of the present invention, the pharmaceutical preparation further includes a third nucleic acid fragment, which encodes S, S_ec (S protein extracellular region) or SII protein of feline infectious peritonitis virus.

[0015] It should be noted that, according to the sequence alignment results (Tables 1 and 2), the S protein has an amino acid sequence that is at least 45% homologous to SEQ ID NO: 3, and the nucleic acid fragment encoding the S protein has a nucleotide sequence that is at least 51% homologous to any one of SEQ ID NOs: 12 to 15. The S_ec protein has an amino acid sequence that is at least 43% homologous to amino acids 1 to 1374 of SEQ ID NO: 3, and the nucleic acid fragment encoding the S_ec protein has a nucleotide sequence that is at least 51% homologous to nucleotides 1 to 4122 of any one of SEQ ID NOs: 12 to 15. The SII protein has an amino acid sequence that is at least 62% homologous to amino acids 782 to 1433 of SEQ ID NO: 3, and the nucleic acid fragment encoding the SII protein has a nucleotide sequence that is at least 57% homologous to nucleotides 2344 to 4299 of any one of SEQ ID NOs: 12 to 15.

[0016] Table 1: Protein sequence homology among multiple substrains (%) Note: - means none.

[0017] Table 2: Homology of nucleic acid sequences of multiple substrains (%) Note: - means none.

[0018] According to an embodiment of the present invention, the first nucleic acid fragment, the second nucleic acid fragment and the third nucleic acid fragment are connected or not connected.

[0019] According to an embodiment 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. Alternatively, the mass ratio of the first nucleic acid fragment to the second 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. In some preferred examples of the present application, the mass ratio of the first nucleic acid fragment to the second nucleic acid fragment is 1:1.

[0020] 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. Alternatively, 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.

[0021] 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. In some examples of the present application, when the mass ratio of the three non-connected nucleic acid fragments is 1:1:1, the mRNA prepared as a vaccine has a good immune effect against FIPV and can significantly improve various physiological indicators and survival rates of cats.

[0022] According to an embodiment of the present invention, the first nucleic acid fragment, the second nucleic acid fragment and the third nucleic acid fragment are not connected.

[0023] According to an embodiment of the present invention, the first nucleic acid fragment, the second nucleic acid fragment and the third nucleic acid fragment are connected.

[0024] According to an embodiment of the present invention, the first nucleic acid fragment is connected to 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 first nucleic acid fragment is connected to 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 second nucleic acid fragment is connected to 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.

[0025] According to an embodiment 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 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 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.

[0026] According to an embodiment 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 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 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;

[0027] Specifically, the connection mode of the M, N, S, S_ec or SII proteins of the feline infectious peritonitis virus (FIPV) can be arbitrarily combined.

[0028] In some examples of the present application, it is preferred that the M, N, and SII proteins of feline infectious peritonitis virus (FIPV) are linked or not.

[0029] In other examples of the present application, it is preferred that the M, N, and SII proteins of feline infectious peritonitis virus (FIPV) are not connected. Among them, whether it is a separated single-antigen circular RNA vaccine as a pharmaceutical composition or a multi-antigen circular RNA vaccine connected by different connecting peptides, it can show a good immune effect on FIPV. In addition, the single-antigen circular RNA vaccine separated in the present application as a pharmaceutical composition or a multi-antigen circular RNA vaccine connected by different connecting peptides shows a good immune effect on different FIPV epidemic strains.

[0030] According to an embodiment of the present invention, the M protein has an amino acid sequence that is at least 89% homologous to the amino acid sequence shown in SEQ ID NO: 1.

[0031] According to an embodiment of the present invention, the M protein has an amino acid sequence with at least 89% homology to SEQ ID NO: 1, and the 90th amino acid is Y, the 102nd is V, the 120th is I, the 144th is A, and the 180th is L.

[0032] According to an embodiment of the present invention, the M protein has the amino acid sequence shown in SEQ ID NO: 1.

[0033] According to an embodiment of the present invention, the N protein has an amino acid sequence that is at least 91% homologous to the amino acid sequence shown in SEQ ID NO:2.

[0034] According to an embodiment of the present invention, the N protein has the amino acid sequence shown in SEQ ID NO: 2.

[0035] According to an embodiment of the present invention, the S protein has an amino acid sequence that is at least 45% homologous to the amino acid sequence shown in SEQ ID NO:3.

[0036] According to an embodiment of the present invention, the S protein has an amino acid sequence with at least 45% homology to SEQ ID NO: 3, and the 515th amino acid is V, the 577th amino acid is Q, the 1385th amino acid is V, the 1386th amino acid is V, the 1397th amino acid is F, and the 1415th amino acid is I.

[0037] According to an embodiment of the present invention, the S protein has the amino acid sequence shown in SEQ ID NO:3.

[0038] According to an embodiment of the present invention, the S_ec protein has an amino acid sequence that is at least 43% homologous to amino acids 1 to 1374 of the amino acid sequence shown in SEQ ID NO:3.

[0039] According to an embodiment of the present invention, the S_ec protein has an amino acid sequence with at least 43% homology between amino acids 1 to 1374 and SEQ ID NO: 3, and the amino acid at position 515 is V and the amino acid at position 577 is Q.

[0040] According to an embodiment of the present invention, the S_ec protein has the amino acid sequence at positions 1 to 1374 of SEQ ID NO: 3.

[0041] According to an embodiment of the present invention, the SII protein has an amino acid sequence that is at least 62% homologous to amino acids 782 to 1433 of the amino acid sequence shown in SEQ ID NO: 3.

[0042] According to an embodiment of the present invention, the SII protein has an amino acid sequence with at least 62% homology between amino acids 782 to 1433 and SEQ ID NO: 3, and 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.

[0043] According to an embodiment of the present invention, the SII protein has the amino acid sequence at positions 782 to 1433 of SEQ ID NO: 3.

[0044] According to an embodiment of the present invention, the first nucleic acid fragment has a nucleotide sequence that is at least 67% homologous to any one of SEQ ID NOs: 4 to 7.

[0045] According to an embodiment of the present invention, the first nucleic acid fragment has a nucleotide sequence shown in SEQ ID NOs: 4 to 7.

[0046] According to an embodiment of the present invention, the second nucleic acid fragment has a nucleotide sequence that is at least 70% homologous to any one of SEQ ID NOs: 8 to 11.

[0047] According to an embodiment of the present invention, the second nucleic acid fragment has a nucleotide sequence shown in SEQ ID NOs: 8 to 11.

[0048] According to an embodiment of the present invention, the third nucleic acid fragment has a nucleotide sequence that is at least 51% homologous to nucleotides 1 to 4122 of any one of SEQ ID NOs: 12 to 15.

[0049] According to an embodiment of the present invention, the third nucleic acid fragment has a nucleotide sequence at positions 1 to 4122 of SEQ ID NOs: 12 to 15.

[0050] According to an embodiment of the present invention, the third nucleic acid fragment has a nucleotide sequence that is at least 57% homologous to nucleotides 2344 to 4299 of any one of SEQ ID NOs: 12 to 15.

[0051] According to an embodiment of the present invention, the third nucleic acid fragment has a nucleotide sequence at positions 2344 to 4299 of SEQ ID NOs: 12 to 15.

[0052] According to an embodiment of the present invention, the third nucleic acid fragment has a nucleotide sequence that is at least 51% homologous to any one of SEQ ID NOs: 12-15.

[0053] According to an embodiment of the present invention, the third nucleic acid fragment has the nucleotide sequence shown in SEQ ID NOs: 12-15.

[0054] According to an embodiment of the present invention, the pharmaceutical preparation further comprises a fourth nucleic acid fragment encoding a signal peptide sequence of MHC I (major histocompatibility complex I) or a sequence having similar functions to the signal peptide of MHC I. According to an embodiment of the present invention, the addition of the MHC I signal peptide to the N-terminus of the antigen sequence enables ribosomes to attach to the endoplasmic reticulum membrane, thereby directing protein transport within the cell.

[0055] It should be noted that the sequence described in this application that has similar functions to the signal peptide of MHCⅠ can also enable ribosomes to attach to the endoplasmic reticulum membrane and guide the transport of proteins in the cell.

[0056] According to an embodiment of the present invention, the signal peptide sequence of MHC I does not contain a transmembrane region.

[0057] According to an embodiment of the present invention, the signal peptide sequence of MHC I has the amino acid sequence shown in SEQ ID NO:16.

[0058] According to an embodiment of the present invention, the fourth nucleic acid fragment has the nucleotide sequence shown in SEQ ID NOs: 17-22.

[0059] According to an embodiment of the present invention, the fourth nucleic acid fragment is disposed at the 5' end of the nucleic acid fragment or the 5' end of the nucleic acid molecule.

[0060] It should be noted that the fourth nucleic acid fragment is disposed upstream of the nucleic acid fragment or the nucleic acid molecule, and optionally, is located at the 5' end of the nucleic acid fragment or the 5' end of the nucleic acid molecule.

[0061] According to an embodiment of the present invention, the pharmaceutical preparation further comprises a fifth nucleic acid fragment encoding a MITD (MHC class I molecule transport signal or major histocompatibility complex class I molecule transport signal) sequence or a sequence having similar functions to MITD. 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.

[0062] It should be noted that, in this application, the sequence having similar functions to MITD can also stimulate CD4 + T cells proliferate, inducing the production of cytokines, and causing an immune response in animals.

[0063] According to an embodiment of the present invention, the MITD sequence has the amino acid sequence shown in SEQ ID NO: 23.

[0064] According to an embodiment of the present invention, the fifth nucleic acid fragment has the nucleotide sequence shown in SEQ ID NO: 24-32.

[0065] According to an embodiment of the present invention, the fifth nucleic acid fragment is disposed at the 3' end of the nucleic acid fragment or the 3' end of the nucleic acid molecule.

[0066] It should be noted that the fifth nucleic acid fragment is disposed downstream of the nucleic acid fragment or the nucleic acid molecule, optionally, at the 3' end of the nucleic acid fragment or the 3' end of the nucleic acid molecule.

[0067] According to an embodiment of the present invention, the nucleic acid fragment or nucleic acid molecule has the nucleotide sequence shown in Table 4.

[0068] According to an embodiment of the present invention, the pharmaceutical preparation further includes a drug carrier, and the drug carrier includes at least one of liposomes, exosomes, polymer carriers, viral vectors, and nanoparticles.

[0069] It should be noted that the drug carrier refers to a carrier that does not cause obvious irritation to the test animals and does not eliminate the biological activity and properties of the drug preparation.

[0070] In a second aspect of the present invention, the present invention provides a method for preparing the pharmaceutical preparation according to claim 1. According to an embodiment of the present invention, the method comprises: mixing the first nucleic acid fragment and the second nucleic acid fragment in a predetermined ratio to obtain the pharmaceutical preparation;

[0071] 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 first nucleic acid fragment and the second nucleic acid fragment are circular RNA.

[0072] According to an embodiment of the present invention, the method can be used to prepare a pharmaceutical preparation for preventing or treating diseases related to feline infectious peritonitis virus.

[0073] According to an embodiment 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. Alternatively, the mass ratio of the first nucleic acid fragment to the second 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.

[0074] According to an embodiment of the present invention, the mass ratio of the first nucleic acid fragment to the second nucleic acid fragment is 1:1.

[0075] According to an embodiment of the present invention, the mixing process further includes a third nucleic acid segment encoding the S, S_ec or SII protein of feline infectious peritonitis virus.

[0076] 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. Alternatively, the mass ratio of the first nucleic acid fragment to the second 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.

[0077] 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.

[0078] In a third aspect, 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 segment encoding the M protein of feline infectious peritonitis virus; a second nucleic acid segment encoding the N protein of feline infectious peritonitis virus; the first nucleic acid segment and the second nucleic acid segment are connected; and the nucleic acid molecule is a circular RNA.

[0079] According to an embodiment of the present invention, nucleic acid molecules expressing the M and N proteins of feline infectious peritonitis virus can stimulate the somatic cell-mediated immune response of an animal.

[0080] It should be noted that in the present application, the M and N proteins of the wild-type FIPV virus can also be adaptively modified as needed to reduce the toxicity of the FIPV virus without affecting its three-dimensional structure and retaining its immunogenicity to prepare a new type of FIPV virus vaccine. According to the sequence alignment results (Tables 1 and 2), the M protein has an amino acid 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 one of SEQ ID NOs: 4 to 7, the N protein has an amino acid 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 one of SEQ ID NOs: 8 to 11. The FIPV virus vaccine is not particularly limited, as long as the binding region of the modified FIPV virus M and N proteins can be produced in the organism, and it has immunogenicity and can stimulate the organism to produce a corresponding immune response. Moreover, the pharmaceutical preparation 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.

[0081] According to an embodiment of the present invention, the nucleic acid molecule may further include at least one of the following additional technical features:

[0082] According to an embodiment of the present invention, the nucleic acid molecule further includes a third nucleic acid segment, which encodes at least one of the S, S_ec and SII proteins of feline infectious peritonitis virus.

[0083] It should be noted that, according to the sequence alignment results (Tables 1 and 2), the S protein has an amino acid sequence that is at least 45% homologous to SEQ ID NO: 3, and the nucleic acid fragment encoding the S protein has a nucleotide sequence that is at least 51% homologous to any one of SEQ ID NOs: 12 to 15. The S_ec protein has an amino acid sequence that is at least 43% homologous to amino acids 1 to 1374 of SEQ ID NO: 3, and the nucleic acid fragment encoding the S_ec protein has a nucleotide sequence that is at least 51% homologous to nucleotides 1 to 4122 of any one of SEQ ID NOs: 12 to 15. The SII protein has an amino acid sequence that is at least 62% homologous to amino acids 782 to 1433 of SEQ ID NO: 3, and the nucleic acid fragment encoding the SII protein has a nucleotide sequence that is at least 57% homologous to nucleotides 2344 to 4299 of any one of SEQ ID NOs: 12 to 15. According to an embodiment of the present invention, the first nucleic acid fragment, the second nucleic acid fragment and the third nucleic acid fragment are connected.

[0084] According to an embodiment 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 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 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;

[0085] Specifically, the connection mode of the M, N, S, S_ec or SII proteins of the feline infectious peritonitis virus (FIPV) can be arbitrarily combined.

[0086] According to an embodiment of the present invention, the M protein has an amino acid sequence that is at least 89% homologous to the amino acid sequence shown in SEQ ID NO: 1.

[0087] According to an embodiment of the present invention, the M protein has an amino acid sequence with at least 89% homology to SEQ ID NO: 1, and the 90th amino acid is Y, the 102nd is V, the 120th is I, the 144th is A, and the 180th is L.

[0088] According to an embodiment of the present invention, the M protein has the amino acid sequence shown in SEQ ID NO: 1.

[0089] According to an embodiment of the present invention, the N protein has an amino acid sequence that is at least 91% homologous to the amino acid sequence shown in SEQ ID NO:2.

[0090] According to an embodiment of the present invention, the N protein has the amino acid sequence shown in SEQ ID NO: 2.

[0091] According to an embodiment of the present invention, the S protein has an amino acid sequence that is at least 45% homologous to the amino acid sequence shown in SEQ ID NO:3.

[0092] According to an embodiment of the present invention, the S protein has an amino acid sequence with at least 45% homology to SEQ ID NO: 3, and the 515th amino acid is V, the 577th amino acid is Q, the 1385th amino acid is V, the 1386th amino acid is V, the 1397th amino acid is F, and the 1415th amino acid is I.

[0093] According to an embodiment of the present invention, the S protein has the amino acid sequence shown in SEQ ID NO:3.

[0094] According to an embodiment of the present invention, the S_ec protein has an amino acid sequence that is at least 43% homologous to amino acids 1 to 1374 of the amino acid sequence shown in SEQ ID NO:3.

[0095] According to an embodiment of the present invention, the S_ec protein has an amino acid sequence with at least 43% homology between amino acids 1 to 1374 and SEQ ID NO: 3, and the amino acid at position 515 is V and the amino acid at position 577 is Q.

[0096] According to an embodiment of the present invention, the S_ec protein has the amino acid sequence at positions 1 to 1374 of SEQ ID NO: 3.

[0097] According to an embodiment of the present invention, the SII protein has an amino acid sequence that is at least 62% homologous to amino acids 782 to 1433 of the amino acid sequence shown in SEQ ID NO: 3.

[0098] According to an embodiment of the present invention, the SII protein has an amino acid sequence with at least 62% homology between amino acids 782 to 1433 and SEQ ID NO: 3, and 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.

[0099] According to an embodiment of the present invention, the SII protein has the amino acid sequence at positions 782 to 1433 of SEQ ID NO: 3.

[0100] According to an embodiment of the present invention, the first nucleic acid fragment has a nucleotide sequence that is at least 67% homologous to any one of SEQ ID NOs: 4 to 7.

[0101] According to an embodiment of the present invention, the first nucleic acid fragment has a nucleotide sequence shown in SEQ ID NOs: 4 to 7.

[0102] According to an embodiment of the present invention, the second nucleic acid fragment has a nucleotide sequence that is at least 70% homologous to any one of SEQ ID NOs: 8 to 11.

[0103] According to an embodiment of the present invention, the second nucleic acid fragment has a nucleotide sequence shown in SEQ ID NOs: 8 to 11.

[0104] According to an embodiment of the present invention, the third nucleic acid fragment has a nucleotide sequence that is at least 51% homologous to nucleotides 1 to 4122 of any one of SEQ ID NOs: 12 to 15.

[0105] According to an embodiment of the present invention, the third nucleic acid fragment has a nucleotide sequence at positions 1 to 4122 of SEQ ID NOs: 12 to 15.

[0106] According to an embodiment of the present invention, the third nucleic acid fragment has a nucleotide sequence that is at least 57% homologous to nucleotides 2344 to 4299 of any one of SEQ ID NOs: 12 to 15.

[0107] According to an embodiment of the present invention, the third nucleic acid fragment has a nucleotide sequence at positions 2344 to 4299 of SEQ ID NOs: 12 to 15.

[0108] According to an embodiment of the present invention, the third nucleic acid fragment has a nucleotide sequence that is at least 51% homologous to any one of SEQ ID NOs: 12-15.

[0109] According to an embodiment of the present invention, the third nucleic acid fragment has the nucleotide sequence shown in SEQ ID NOs: 12-15.

[0110] According to an embodiment of the present invention, the pharmaceutical preparation further comprises a fourth nucleic acid fragment encoding a signal peptide sequence of MHC I (major histocompatibility complex I) or a sequence having similar functions to the signal peptide of MHC I. According to an embodiment of the present invention, the addition of the MHC I signal peptide to the N-terminus of the antigen sequence enables ribosomes to attach to the endoplasmic reticulum membrane, thereby directing protein transport within the cell.

[0111] It should be noted that the sequence described in this application that has similar functions to the signal peptide of MHCⅠ can also enable ribosomes to attach to the endoplasmic reticulum membrane and guide the transport of proteins in the cell.

[0112] According to an embodiment of the present invention, the signal peptide sequence of MHC I does not contain a transmembrane region.

[0113] According to an embodiment of the present invention, the signal peptide sequence of MHC I has the amino acid sequence shown in SEQ ID NO:16.

[0114] According to an embodiment of the present invention, the fourth nucleic acid fragment has the nucleotide sequence shown in SEQ ID NOs: 17-22.

[0115] According to an embodiment of the present invention, the fourth nucleic acid fragment is disposed at the 5' end of the nucleic acid fragment or the 5' end of the nucleic acid molecule.

[0116] It should be noted that the fourth nucleic acid fragment is disposed upstream of the nucleic acid fragment or the nucleic acid molecule, and optionally, is located at the 5' end of the nucleic acid fragment or the 5' end of the nucleic acid molecule.

[0117] According to an embodiment of the present invention, the pharmaceutical preparation further comprises a fifth nucleic acid fragment encoding a MITD (MHC class I molecule transport signal or major histocompatibility complex class I molecule transport signal) sequence or a sequence having similar functions to MITD. 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.

[0118] It should be noted that, in this application, the sequence having similar functions to MITD can also stimulate CD4 + T cells proliferate, inducing the production of cytokines, and causing an immune response in animals.

[0119] According to an embodiment of the present invention, the MITD sequence has the amino acid sequence shown in SEQ ID NO: 23.

[0120] According to an embodiment of the present invention, the fifth nucleic acid fragment has the nucleotide sequence shown in SEQ ID NO: 24-32.

[0121] According to an embodiment of the present invention, the fifth nucleic acid fragment is disposed at the 3' end of the nucleic acid fragment or the 3' end of the nucleic acid molecule.

[0122] It should be noted that the fifth nucleic acid fragment is disposed downstream of the nucleic acid fragment or the nucleic acid molecule, optionally, at the 3' end of the nucleic acid fragment or the 3' end of the nucleic acid molecule.

[0123] According to an embodiment of the present invention, the nucleic acid fragment or nucleic acid molecule has the nucleotide sequence shown in Table 4.

[0124] In a fourth 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 third 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.

[0125] According to an embodiment of the present invention, the above-mentioned expression vector may further include at least one of the following additional technical features:

[0126] According to an embodiment of the present invention, the expression vector is a non-viral vector.

[0127] In a fifth 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 third aspect of the present invention. The recombinant virus containing the nucleic acid molecule described in the third aspect can be propagated in large quantities and plays an important role in vaccine development.

[0128] In a sixth aspect, the present invention provides a liposome. According to embodiments 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 and third aspects 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.

[0129] In its seventh aspect, the present invention provides a vaccine. According to embodiments of the present invention, the vaccine comprises the pharmaceutical preparation of the first aspect of the present invention, the nucleic acid molecule of the third aspect, the expression vector of the fourth aspect, the recombinant virus of the fifth aspect, or the liposome of the sixth 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.

[0130] According to an embodiment of the present invention, the above vaccine may further include at least one of the following additional technical features:

[0131] According to an embodiment of the present invention, the vaccine further comprises an adjuvant.

[0132] According to an embodiment of the present invention, the adjuvant includes TLR agonist, Mn 2+ At least one of.

[0133] According to an embodiment of the present invention, the TLR agonist comprises at least one of CpG, R837, MPLA and derivatives thereof.

[0134] In an eighth aspect, the present invention provides a recombinant cell. According to an embodiment of the present invention, the recombinant cell carries a nucleic acid fragment, the nucleic acid molecule described in the third aspect of the present invention, the expression vector described in the fourth aspect of the present invention, or the recombinant virus described in the fifth aspect of the present invention;

[0135] The nucleic acid fragments include 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. The first nucleic acid fragment, the second nucleic acid fragment, and the third nucleic acid fragment may be connected or unconnected. The nucleic acid molecule is a circular RNA. According to an embodiment 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 or expressing the M, N, S, S_ec, or SII proteins of the FIPV virus to stimulate an animal to produce a stronger immune response.

[0136] In a ninth aspect, the present invention provides a method for constructing a feline infectious peritonitis virus vaccine. According to an embodiment of the present invention, the method comprises introducing a nucleic acid fragment, a nucleic acid molecule according to the third aspect of the present invention, an expression vector according to the fourth aspect of the present invention, or a recombinant virus according to the fifth aspect of the present invention into a recipient cell;

[0137] Among them, 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; the first nucleic acid fragment, the second nucleic acid fragment and the third nucleic acid fragment are connected or not connected; the nucleic acid molecule is a circular RNA.

[0138] The method according to the embodiment of the present invention can package the virus carrying the nucleic acid molecule for use in preparing a nucleic acid vaccine. The method for constructing an infectious peritonitis virus vaccine is safe, simple, and efficient.

[0139] According to an embodiment of the present invention, the method for constructing a feline infectious peritonitis virus vaccine may further include at least one of the following additional technical features:

[0140] According to an embodiment of the present invention, the method further comprises encapsulating the nucleic acid, expression vector or recombinant virus using an encapsulation vector before introducing the nucleic acid, expression vector or recombinant virus into the recipient cell.

[0141] According to an embodiment of the present invention, the encapsulation carrier is selected from at least one of liposomes, exosomes, polymer carriers, viral vectors, and nanoparticles.

[0142] According to an embodiment of the present invention, the encapsulation carrier is a nanoparticle. Selecting nanoparticles to encapsulate RNA can protect RNA from degradation and bind to cell membranes to facilitate RNA delivery into cells.

[0143] According to an embodiment of the present invention, the recipient cell is a CRFK cell, HEK293FT, HEK293T, BHK cell or insect cell.

[0144] According to an embodiment of the present invention, the recipient cells are CRFK cells. According to an embodiment of the present invention, CRFK cells do not have an immune rejection reaction in a test animal.

[0145] In the tenth aspect of the present invention, the present invention provides a use of the pharmaceutical preparation 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 drug or vaccine. According to an embodiment of the present invention, the drug or vaccine is used to prevent or treat diseases related to feline infectious peritonitis virus infection. According to an embodiment of the present invention, the drug or vaccine prepared based on the aforementioned nucleic acid molecules, expression vectors, recombinant viruses, or recombinant cells has high safety and can activate animal cell-mediated immune responses in a short period of time.

[0146] In the eleventh 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 to the test animal the pharmaceutical preparation of 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, the liposome described in the sixth aspect, the vaccine described in the seventh aspect, or the recombinant cell described in the eighth aspect. 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.

[0147] 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.

[0148] The effective amount of the pharmaceutical preparation, 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.

[0149] According to an embodiment of the present invention, the above method may further include at least one of the following technical features:

[0150] According to an embodiment of the present invention, the test animal is selected from cats.

[0151] In the twelfth aspect of the present invention, the present invention proposes a use of the pharmaceutical preparation of 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 preventing or treating feline infectious peritonitis virus infection. 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 a good immune effect on various epidemic strains of FIPV.

[0152] 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

[0153] 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:

[0154] FIG1 is a result of detecting the expression of target circular RNA encapsulated by LNP according to Example 2 of the present invention.

[0155] FIG2 shows the change in survival rate after immunization with the target circular RNA encapsulated by LNP according to Example 2 of the present invention.

[0156] FIG3 is the expression detection result of the target circular RNA encapsulated by LNP according to Example 3 of the present invention.

[0157] FIG4 shows the change in survival rate after immunization with the target circular RNA encapsulated by LNP according to Example 3 of the present invention.

[0158] Figure 5 shows the changes in survival rate after immunization with the target circular RNA encapsulated by LNP according to Example 4 of the present invention.

[0159] Figure 6 shows the changes in survival rate after immunization with the target circular RNA encapsulated by LNP according to Example 5 of the present invention. DETAILED DESCRIPTION

[0160] 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.

[0161] 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. Beneficial effects

[0162] The RNA vaccine for preventing feline infectious peritonitis described in the present invention is prepared by constructing a vector encoding at least one of the M, N and S, S_ec or SII proteins of the FIPV virus, and then preparing the RNA vaccine for preventing the FIPV virus through lipid nanoparticles (LNP). The antibodies stimulated by the vaccine can prevent the recognition of the FCoV serotype II virus and the host cells, and avoid inducing the body to produce antibody-dependent enhancement (ADE). After immunizing the cat with the vaccine, it can produce a strong immune response and produce neutralizing antibodies with protective efficacy. The vaccine described in the present invention uses RNA containing the amino acid sequences of M, N and S, S_ec or SII of the FIPV virus as the main component, and has the advantages of simple preparation process, high safety, no toxic side effects, and industrial production; it can achieve sufficient protection effect using a very small dose, and is superior to existing treatment methods in terms of safety and effectiveness.

[0163] The sequences involved in this application are shown in Table 3.

[0164] Table 3:

[0165] Table 4: Single antigen sequence composition in the examples of the present invention Note: sp represents signal peptide.

[0166] Table 5: Composition of multiple antigen sequences in the examples of the present invention

[0167] It should be noted that the sequences of the products corresponding to the names in Tables 4 and 5 are formed by connecting the corresponding sequences in SEQ ID NO: in the 5' to 3' direction. For example, in Table 4, the sequence of sp-Flag-SII6-MITD is formed by connecting the signal peptide sequence shown in SEQ ID NO: 22, the gene sequence shown in SEQ ID NO: 15, and the MITD sequence shown in SEQ ID NO: 32, wherein the 3' end of the signal peptide sequence shown in SEQ ID NO: 22 is connected to the 5' end of the gene sequence shown in SEQ ID NO: 15, and the 3' end of the gene sequence shown in SEQ ID NO: 15 is connected to the 5' end of the MITD sequence shown in SEQ ID NO: 32.

[0168] 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.

[0169] Example 1: Vaccine potency verification

[0170] The embodiments of the present invention evaluate the effectiveness of the vaccine by evaluating the changes in physiological indicators such as body temperature, body weight and survival rate of the test animals after immunization with the circular RNA vaccine and challenge.

[0171] According to an embodiment of the present invention, sp-HA-M6-MITD, sp-His-N2, and sp-SII2-MITD were constructed downstream of CVB3 IRES (SEQ ID NO: 44), transcribed in vitro, and circularized into circular RNAs. The sequences are shown in Table 4.

[0172] According to an embodiment of the present invention, lipid nanoparticles (LNP) are prepared. The specific steps are as follows:

[0173] 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.

[0174] 2) Dilute the target circular RNA 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 a circular RNA vaccine liposome solution, immediately place it in 30 volumes of PBS, concentrate using a 15ml ultrafiltration tube with a cutoff of 100KDa, and centrifuge at 3000rpm for 20min. Finally, use 600mM sucrose solution (prepared in PBS, filtered with a 0.22μm filter membrane) to preserve the equal volume dilution. Store the sample at -20°C for later use;

[0175] 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 testing. The specific experimental steps are as follows:

[0176] 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.

[0177] 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.

[0178] According to an embodiment of the present invention, the screened test animals were immunized using LNP-encapsulated target circular RNA. The immunization procedure is as follows:

[0179] 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.

[0180] The target circular RNA encapsulated by LNP was transfected into CRFK cells, collected after 24 hours, and immunoblotting was performed to detect protein expression. The results are shown in Figure 1, and all were expressed normally. After immunization with the target circular RNA encapsulated by LNP in each group, the five kittens in the PBS group developed symptoms of fever and weight loss. The autopsy revealed typical feline infectious peritonitis. The physiological indicators of the kittens in the immunization group expressing circular RNA of M, N, and SII antigens and their combination improved significantly, and the kittens in the M+N+SII group were all normal. The survival rate is shown in Figure 2. Immunization with circular RNA expressing M, N, and SII antigens can improve the survival rate after the virus attack. The combination of multiple antigens can further improve the survival rate after the virus attack. The survival rate of the M+N+SII group reached 100%.

[0181] The above results show that circular RNA expressing M, N, SII antigens and their combinations have a good immune effect on FIPV as a vaccine, significantly improving various physiological indicators and survival rates, and the M+N+SII combination has the best effect.

[0182] Example 2: Potency Verification of Multi-Antigen Circular RNA Vaccines with Different Linking Methods

[0183] The present invention evaluates the effectiveness of multi-antigen circular RNA vaccines with different linkage methods. The LNP preparation and expression verification methods, test animal screening methods, immunization procedures, and evaluation methods are the same as those in Example 1.

[0184] According to an embodiment 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 downstream of CVB3 IRES (SEQ ID NO: 44), transcribed in vitro, and circularized into circular RNA. The sequences are shown in Tables 4 and 5.

[0185] The expression verification results of the target circular RNA after LNP encapsulation are shown in Figure 3, showing normal expression. After immunization, the multi-antigen circular RNA vaccine with different linkage methods effectively improved the physiological indicators and survival rate of the test animals after challenge. The survival rate is shown in Figure 4, and there was no significant difference between the different linkage methods.

[0186] The above results show that whether it is a separated single-antigen circular RNA vaccine as a pharmaceutical composition or a multi-antigen circular RNA vaccine connected by different connecting peptides, it can show good immune effect against FIPV.

[0187] Example 3: Potency Verification of Multi-Antigen Circular RNA Vaccines at Different Ratios

[0188] This example evaluated the effectiveness of multi-antigen circular RNA vaccines at different ratios. The LNP preparation method, test animal screening method, immunization procedure, and evaluation method were the same as in Example 1.

[0189] According to an embodiment of the present invention, sp-HA-M6-MITD, sp-His-N2, and sp-SII2-MITD were constructed downstream of CVB3 IRES (SEQ ID NO: 44), transcribed in vitro, and circularized into circular RNAs. The sequences are shown in Table 4.

[0190] After immunization, different ratios of multi-antigen circular RNA vaccines can effectively improve the physiological indicators and survival rate of the test animals after challenge. The survival rate is shown in Figure 5.

[0191] The above results show that multi-antigen circular RNA vaccines with different ratios can exhibit good immune effects against FIPV.

[0192] Example 4: Validation of the potency of multi-antigen circular RNA vaccines against different epidemic strains

[0193] This example evaluated the effectiveness of a multi-antigen circular RNA vaccine against different circulating FIPV strains. The LNP preparation method, test animal screening method, immunization schedule, and evaluation method were the same as in Example 1. The challenge strains were HF1902 and SH2211.

[0194] According to an embodiment of the present invention, sp-HA-M6-MITD, sp-His-N2, and sp-SII2-MITD were constructed downstream of CVB3 IRES (SEQ ID NO: 44), transcribed in vitro, and circularized into circular RNAs. The sequences are shown in Table 4.

[0195] After immunization, the multi-antigen circular RNA 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 6.

[0196] The above results show that the multi-antigen circular RNA vaccine has a good immune effect on different FIPV strains.

[0197] 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.

[0198] 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. A pharmaceutical preparation, characterized in that include: A nucleic acid fragment, wherein the nucleic acid fragment is a circular RNA, and the nucleic acid fragment comprises a first nucleic acid fragment and a second 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 first nucleic acid fragment is connected to the second nucleic acid fragment or not.

2. The pharmaceutical preparation according to claim 1, characterized in that The nucleic acid fragment further includes a third nucleic acid fragment, which encodes the S, S_ec or SII protein of feline infectious peritonitis virus.

3. The pharmaceutical preparation according to claim 2, characterized in that The first nucleic acid fragment, the second nucleic acid fragment and the third nucleic acid fragment are connected or not connected.

4. The pharmaceutical preparation according to claim 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, preferably 1:

1.

5. The pharmaceutical preparation according to claim 3, 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 preparation according to claims 3 to 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 preparation according to claim 3, characterized in that The first nucleic acid fragment, the second nucleic acid fragment and the third nucleic acid fragment are connected.

8. A method for preparing the pharmaceutical preparation according to any one of claims 1 to 7, characterized in that: include: Mixing the first nucleic acid fragment and the second nucleic acid fragment in a predetermined ratio to obtain the pharmaceutical preparation; 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; The first nucleic acid fragment and the second nucleic acid fragment are circular RNA.

9. The method according to claim 8, 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 claim 9, characterized in that The mass ratio is 1:

1.

11. The method according to claim 9, characterized in that The mixed treatment further includes a third nucleic acid fragment, which encodes the S, S_ec or SII protein of feline infectious peritonitis virus.

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

10.

13. The method according to claim 12, 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.

14. An isolated nucleic acid molecule, characterized in that include: A first nucleic acid segment encoding the M protein of feline infectious peritonitis virus; a second nucleic acid segment encoding the N protein of feline infectious peritonitis virus; The first nucleic acid fragment is linked to the second nucleic acid fragment; The nucleic acid molecule is a circular RNA.

15. The nucleic acid molecule according to claim 14, characterized in that The nucleic acid molecule further includes a third nucleic acid fragment, which encodes the S, S_ec or SII protein of feline infectious peritonitis virus, and the first nucleic acid fragment, the second nucleic acid fragment and the third nucleic acid fragment are connected.

16. The pharmaceutical preparation according to claim 2 or the nucleic acid molecule according to claim 15, characterized in that The first nucleic acid fragment is connected to 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 first nucleic acid fragment is connected to 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 second nucleic acid fragment is connected to the third nucleic acid fragment, and the first nucleic acid fragment is connected to the second nucleic acid fragment and the third nucleic acid fragment. The segments are not connected; 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 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 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 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; or 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 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.

17. The pharmaceutical preparation according to claim 1 or the nucleic acid molecule according to claim 14, characterized in that The M protein has an amino acid sequence that is at least 89% homologous to the amino acid sequence shown in SEQ ID NO:

1.

18. The pharmaceutical preparation or nucleic acid molecule according to claim 17, characterized in that The M protein has an amino acid sequence with at least 89% homology to SEQ ID NO: 1, and the 90th amino acid is Y, the 102nd is V, the 120th is I, the 144th is A, and the 180th is L.

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

1.

20. The pharmaceutical preparation according to claim 1 or the nucleic acid molecule according to claim 14, characterized in that The N protein has an amino acid sequence that is at least 91% homologous to the amino acid sequence shown in SEQ ID NO:

2.

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

2.

22. The pharmaceutical preparation according to claim 2 or the nucleic acid molecule according to claim 15, characterized in that The S protein has an amino acid sequence that is at least 45% homologous to the amino acid sequence shown in SEQ ID NO:

3.

23. The pharmaceutical preparation or nucleic acid molecule according to claim 22, characterized in that The S protein has an amino acid sequence with at least 45% homology to SEQ ID NO: 3, and the 515th amino acid is V, the 577th amino acid is Q, the 1385th amino acid is V, the 1386th amino acid is V, the 1397th amino acid is F, and the 1415th amino acid is I.

24. The pharmaceutical preparation or nucleic acid molecule according to claim 23, characterized in that The S protein has the amino acid sequence shown in SEQ ID NO:

3.

25. The pharmaceutical preparation according to claim 2 or the nucleic acid molecule according to claim 15, characterized in that The S_ec protein has an amino acid sequence that is at least 43% homologous to the amino acids 1 to 1374 of the amino acid sequence shown in SEQ ID NO:

3.

26. The pharmaceutical preparation or nucleic acid molecule according to claim 25, characterized in that The S_ec protein has an amino acid sequence with at least 43% homology between amino acids 1 to 1374 and SEQ ID NO: 3, and the amino acid at position 515 is V and the amino acid at position 577 is Q.

27. The pharmaceutical preparation or nucleic acid molecule according to claim 26, characterized in that The S_ec protein has the amino acid sequence at positions 1 to 1374 of SEQ ID NO:

3.

28. The pharmaceutical preparation according to claim 2 or the nucleic acid molecule according to claim 15, characterized in that The SII protein has an amino acid sequence that is at least 59% homologous to the amino acids at positions 661 to 1433 of the amino acid sequence shown in SEQ ID NO:

3.

29. The pharmaceutical preparation or nucleic acid molecule according to claim 28, characterized in that The SII protein has an amino acid sequence with at least 59% homology between amino acids 661 to 1433 and SEQ ID NO: 3, and 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.

30. The pharmaceutical preparation or nucleic acid molecule according to claim 29, characterized in that The SII protein has the amino acid sequence at positions 661 to 1433 of SEQ ID NO:

3.

31. The pharmaceutical preparation according to claim 1 or the nucleic acid molecule according to claim 14, characterized in that The first nucleic acid fragment has a nucleotide sequence that is at least 67% homologous to any one of SEQ ID NOs: 4-7.

32. The pharmaceutical preparation or nucleic acid molecule according to claim 31, characterized in that The first nucleic acid fragment has a nucleotide sequence shown in SEQ ID NOs: 4-7.

33. The pharmaceutical preparation according to claim 1 or the nucleic acid molecule according to claim 14, characterized in that The second nucleic acid fragment has a nucleotide sequence that is at least 70% homologous to any one of SEQ ID NOs: 8-11.

34. The pharmaceutical preparation or nucleic acid molecule according to claim 33, characterized in that The second nucleic acid fragment has the nucleotide sequence shown in SEQ ID NOs: 8-11.

35. The pharmaceutical preparation according to claim 2 or the nucleic acid molecule according to claim 15, characterized in that The third nucleic acid fragment has a nucleotide sequence that is at least 51% homologous to nucleotides 1 to 4122 of any one of SEQ ID NOs: 12 to 15.

36. The pharmaceutical preparation or nucleic acid molecule according to claim 35, characterized in that The third nucleic acid fragment has the nucleotide sequence at positions 1 to 4122 of SEQ ID NOs: 12 to 15.

37. The pharmaceutical preparation or nucleic acid molecule according to claim 36, characterized in that The third nucleic acid fragment has a nucleotide sequence that is at least 56% homologous to nucleotides 1981 to 4299 of any one of SEQ ID NOs: 12 to 15.

38. The pharmaceutical preparation or nucleic acid molecule according to claim 37, characterized in that Optionally, the third nucleic acid fragment has the nucleotide sequence at positions 1981 to 4299 of SEQ ID NOs: 12 to 15.

39. The pharmaceutical preparation or nucleic acid molecule according to claim 38, characterized in that The third nucleic acid fragment has a nucleotide sequence that is at least 51% homologous to any one of SEQ ID NOs: 12-15.

40. The pharmaceutical preparation or nucleic acid molecule according to claim 39, characterized in that The third nucleic acid fragment has the nucleotide sequence shown in SEQ ID NOs: 12-15.

41. The pharmaceutical preparation according to claim 1 or 2 or the nucleic acid molecule according to claim 14 or 15, characterized in that The invention further comprises a fourth nucleic acid fragment, wherein the fourth nucleic acid fragment encodes a signal peptide sequence of MHC I or a sequence having a similar function to the signal peptide of MHC I.

42. The pharmaceutical preparation or nucleic acid molecule according to claim 41, characterized in that The signal peptide sequence of MHC I does not contain a transmembrane region.

43. The pharmaceutical preparation or nucleic acid molecule according to claim 41, characterized in that The signal peptide sequence of MHC I has an amino acid sequence as shown in SEQ ID NO:

16.

44. The pharmaceutical preparation or nucleic acid molecule according to claim 41, characterized in that The fourth nucleic acid fragment has the nucleotide sequence shown in SEQ ID NOs: 17-22.

45. The pharmaceutical preparation or nucleic acid molecule according to claim 41, characterized in that The fourth nucleic acid fragment is disposed at the 5' end of the nucleic acid fragment or the 5' end of the nucleic acid molecule.

46. ​​The pharmaceutical preparation according to claim 1 or 2 or the nucleic acid molecule according to claim 14 or 15, characterized in that The invention further comprises a fifth nucleic acid fragment, wherein the fifth nucleic acid fragment encodes a MITD sequence or a sequence having a similar function to the MITD sequence.

47. The pharmaceutical preparation or nucleic acid molecule according to claim 46, characterized in that The MITD sequence has the amino acid sequence shown in SEQ ID NO:

23.

48. The pharmaceutical preparation or nucleic acid molecule according to claim 46, characterized in that The fifth nucleic acid fragment has the nucleotide sequence shown in SEQ ID NOs: 24-32.

49. The pharmaceutical preparation or nucleic acid molecule according to claim 46, characterized in that The fifth nucleic acid fragment is disposed at the 3' end of the nucleic acid fragment or the 3' end of the nucleic acid molecule.

50. The pharmaceutical preparation according to claim 1 or the nucleic acid molecule according to claim 14, characterized in that The nucleic acid fragment or nucleic acid molecule has the nucleotide sequence shown in Table 4.

51. The pharmaceutical preparation according to claim 1, characterized in that It further includes a drug carrier, which includes at least one of liposomes, exosomes, polymer carriers, viral vectors, and nanoparticles.

52. An expression vector, characterized in that Carrying the nucleic acid molecule according to any one of claims 14 to 50.

53. The expression vector according to claim 52, characterized in that The expression vector is a non-viral vector.

54. A recombinant virus, characterized in that The recombinant virus carries the nucleic acid molecule according to any one of claims 14 to 50.

55. 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 7 and 15 to 50.

56. A vaccine, characterized in that It comprises the pharmaceutical preparation according to any one of claims 1 to 7 and 15 to 51, the nucleic acid molecule according to any one of claims 14 to 50, the expression vector according to any one of claims 52 to 53, the recombinant virus according to claim 54 or the liposome according to claim 55.

57. The vaccine according to claim 56, characterized in that Further included is an adjuvant.

58. The vaccine according to claim 57, characterized in that The adjuvant includes TLR agonist, Mn 2+ At least one of.

59. The vaccine according to claim 58, characterized in that The TLR agonist includes at least one of CpG, R837, MPLA and derivatives thereof.

60. A recombinant cell, characterized in that Carrying a nucleic acid fragment, a nucleic acid molecule according to any one of claims 14 to 50, an expression vector according to any one of claims 52 to 53, or a recombinant virus according to claim 54; Wherein, the nucleic acid fragment is a circular RNA, and the nucleic acid fragment includes 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 The first nucleic acid fragment, the second nucleic acid fragment and the third nucleic acid fragment are connected or not connected.

61. A method for constructing a feline infectious peritonitis virus vaccine, characterized in that: include: Introducing the nucleic acid fragment, the nucleic acid molecule according to any one of claims 14 to 50, the expression vector according to any one of claims 52 to 53, or the recombinant virus according to claim 54 into a recipient cell; Wherein, the nucleic acid fragment is a circular RNA, and the nucleic acid fragment includes 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; The first nucleic acid fragment, the second nucleic acid fragment and the third nucleic acid fragment are connected or not connected.

62. The method according to claim 61, characterized in that Before introduction into the recipient cell, the method further includes using a packaging vector to package the nucleic acid, expression vector or recombinant virus.

63. The method according to claim 62, characterized in that The encapsulation carrier is selected from at least one of liposomes, exosomes, polymer carriers, viral vectors, and nanoparticles.

64. The method according to claim 63, characterized in that The encapsulating carrier is a nanoparticle.

65. The method according to claim 61, characterized in that The recipient cells are CRFK cells, HEK293FT, HEK293T, BHK cells or insect cells.

66. The method according to claim 65, characterized in that The recipient cells are CRFK cells.

67. Use of the pharmaceutical preparation described in any one of claims 1 to 7, 15 to 51, the nucleic acid molecule described in any one of claims 14 to 50, the expression vector described in any one of claims 52 to 53, the recombinant virus described in claim 54 or the liposome described in claim 55 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.

68. A method for preventing or treating feline infectious peritonitis virus infection, characterized in that: include: Administering the pharmaceutical preparation of any one of claims 1 to 7, 16 to 51, the nucleic acid molecule of any one of claims 14 to 50, the expression vector of any one of claims 52 to 53, the recombinant virus of claim 54, the liposome of claim 55, the vaccine of any one of claims 56 to 59 or the recombinant cell of claim 60 to a test animal.

69. The method according to claim 68, characterized in that The animal is selected from cats.

70. Use of the pharmaceutical preparation of any one of claims 1 to 7, 16 to 51, the nucleic acid molecule of any one of claims 14 to 50, the expression vector of any one of claims 52 to 53, the recombinant virus of claim 54, the liposome of claim 55, the vaccine of any one of claims 56 to 59 or the recombinant cell of claim 60 in preventing or treating feline infectious peritonitis virus infection.

Images