[go: up one dir, main page]

WO2025189435A1 - Method for using multi-epitope antigen to construct rna vaccine for fipv - Google Patents

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

Info

Publication number
WO2025189435A1
WO2025189435A1 PCT/CN2024/081756 CN2024081756W WO2025189435A1 WO 2025189435 A1 WO2025189435 A1 WO 2025189435A1 CN 2024081756 W CN2024081756 W CN 2024081756W WO 2025189435 A1 WO2025189435 A1 WO 2025189435A1
Authority
WO
WIPO (PCT)
Prior art keywords
nucleic acid
seq
optionally
protein
acid fragment
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
PCT/CN2024/081756
Other languages
French (fr)
Chinese (zh)
Inventor
刘强
李翠翠
廖微曦
黄慧雅
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Beijing Syngentech Co Ltd
Original Assignee
Beijing Syngentech Co Ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Beijing Syngentech Co Ltd filed Critical Beijing Syngentech Co Ltd
Priority to PCT/CN2024/081756 priority Critical patent/WO2025189435A1/en
Publication of WO2025189435A1 publication Critical patent/WO2025189435A1/en
Pending legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
    • A61K39/215—Coronaviridae, e.g. avian infectious bronchitis virus
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/39—Medicinal preparations containing antigens or antibodies characterised by the immunostimulating additives, e.g. chemical adjuvants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/04—Immunostimulants
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • C07K14/08—RNA viruses
    • C07K14/165—Coronaviridae, e.g. avian infectious bronchitis virus
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
    • C12N15/88—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation using microencapsulation, e.g. using amphiphile liposome vesicle
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/10—Cells modified by introduction of foreign genetic material

Definitions

  • the present invention relates to the field of biotechnology, and specifically, to a method for constructing an RNA vaccine against the multi-epitope antigens of feline FIPV. More specifically, it relates to an isolated nucleic acid molecule, an expression vector, a recombinant virus, a liposome, a vaccine, a recombinant cell, and a method and use of constructing a feline infectious peritonitis virus vaccine.
  • Feline coronavirus belongs to the coronavirus family.
  • Viruses in the Coronaviridae family are characterized by relatively large, round, enveloped, positive-strand RNA viruses with genomes ranging from 27 to 32 kb. They encode replication polymerases, four structural proteins (S, M, N, and E), and several nonstructural proteins.
  • S protein spike protein
  • the S 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.
  • the S protein is a key antigen in many coronavirus vaccines.
  • the M protein (membrane protein) is involved in the formation and localization of coronavirus particles. It spans the viral envelope and interacts with other proteins to form the structure of the virion.
  • the N protein (nucleocapsid protein) encapsulates the viral RNA genome and is involved in viral gene replication and transcription. It also stimulates the host immune system to respond to the virus.
  • E protein (Envelope protein) is a protein on the coronavirus envelope that can interact with M protein to form the structure of virus particles. It is also involved in the infection and assembly process of the virus.
  • the N-terminus of the N protein has a conserved amino acid site that binds to nucleic acids, which is relatively conserved among coronaviruses and has multiple T cell or B cell epitopes; the C-terminus of the coronavirus's N protein can form a special secondary structure; and studies have reported that the area where T cell or B cell epitopes are concentrated mainly exists in the NSP12 non-structural protein; the two B cell epitopes HR2_4 and HR2_11 are derived from the SII region of FIPV.
  • Feline coronaviruses 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.
  • FIPV FIPV-induced granulomatous lesions in various tissues and organs, including the lungs, liver, spleen, omentum and brain. Infection of macrophages and monocytes is considered to be the key to the pathogenic mechanism. At the end of FIPV infection, a large decrease in T cells in peripheral and lymphoid tissues can be observed, and hypergammaglobulinemia is often present, indicating the presence of severe virus-induced immune disorders. Humoral immunity does not seem to have a protective effect and may lead to "early death syndrome". When S antibodies are present in sub-neutralizing titers, they can enhance the infection of target cells by binding to Fc receptors.
  • the present invention provides an isolated nucleic acid molecule.
  • the isolated nucleic acid molecule comprises at least one of a first nucleic acid fragment, a second nucleic acid fragment, a third nucleic acid fragment, a fourth nucleic acid fragment, and a fifth nucleic acid fragment; wherein the first nucleic acid fragment is derived from the N-terminus (NTD) of the N protein of the QS strain of feline infectious peritonitis virus; the second nucleic acid fragment is derived from the C-terminus (CTD) of the N protein of the QS strain of feline infectious peritonitis virus; the third nucleic acid fragment encodes the NSP12 protein of the QS strain of feline infectious peritonitis virus; the fourth nucleic acid fragment is derived from the epitope HR2_4 of the S protein of the 79-1146 strain of feline infectious peritonitis virus; and the fifth nucleic acid fragment is derived
  • the NTD, CTD, NSP12, HR2_4 or HR2_11 protein sequences of the wild-type FIPV virus can also be adaptively modified as needed to improve antigen expression and reduce the toxicity of the FIPV virus without affecting its three-dimensional structure and retaining its immunogenicity, so as to prepare a new type of FIPV virus vaccine.
  • the NTD protein has an amino acid sequence that is at least 91% homologous to SEQ ID NO: 1, and the NTD protein nucleic acid fragment has a nucleotide sequence that is at least 71% identical to SEQ ID NO: 16-19;
  • the CTD protein has an amino acid fragment that is at least 92% homologous to SEQ ID NO: 2
  • the CTD protein nucleic acid fragment has a nucleotide sequence that is at least 72% identical to SEQ ID NO: 20-23;
  • the NSP12 protein has an amino acid fragment that is at least 97% homologous to SEQ ID NO: 3
  • the CTD protein nucleic acid fragment has a nucleotide sequence that is at least 72% identical to SEQ ID NO: 20-23;
  • the NSP12 protein has an amino acid fragment that is at least 97% homologous to SEQ ID NO: 3.
  • the NSP12 protein nucleic acid fragment has a nucleotide sequence that is at least 71% identical to SEQ ID NOs: 24-27;
  • the HR2_4 protein has an amino acid segment that is at least 50% homologous to SEQ ID NO: 4, and the HR2_4 protein nucleic acid segment has a nucleotide sequence that is at least 50% identical to SEQ ID NOs: 28-31;
  • the HR2_11 protein has an amino acid segment that is at least 95% homologous to SEQ ID NO: 5, and the HR2_11 protein nucleic acid segment has a nucleotide sequence that is at least 73% identical to SEQ ID NOs: 32-35.
  • FIPV virus vaccines are not particularly limited, as long as they can produce the modified FIPV virus NTD, CTD, NSP12, HR2_4, and/or HR2_11 protein receptor binding region in an organism, are immunogenic, and can stimulate the organism to produce a corresponding immune response.
  • the isolated nucleic acid molecule can be used to stimulate an immune response in all animals that can be infected with feline infectious peritonitis virus, including but not limited to cats.
  • linker is a flexible or rigid amino acid chain that acts as a link between two fusion proteins, such as 3 ⁇ flag, EAAAK, GGGS, AAY, GPGPG, (GGGGS)n, etc.
  • linker sequences in experiments can be applied to the embodiments of this application.
  • the NTD, CTD, NSP12, HR2_4, and HR2_11 are connected via a linker, but the connection method is not particularly limited.
  • the nucleic acid molecules can be connected in the form of NTD-linker-CTD-linker-NSP12-linker-HR2_4-linker-HR2_11, or NTD-linker-NSP12-linker-CTD-linker-HR2_4-linker-HR2_11, etc.
  • the isolated nucleic acid molecule may further include at least one of the following technical features:
  • the nucleic acid fragments are connected or not connected.
  • the nucleic acid fragments are connected by a linker.
  • the first to fifth nucleic acid fragments are connected by a linker as a nucleic acid molecule, which can stimulate an immune response mediated by animal somatic cells.
  • the nucleic acid fragments are not connected.
  • any of the first to fifth nucleic acid fragments can stimulate an animal's somatic cell-mediated immune response. For example, immunizing a test animal with the first nucleic acid fragment alone can stimulate an animal's somatic cell-mediated immune response.
  • the first to fifth nucleic acid fragments can be freely combined to stimulate an immune response mediated by animal somatic cells.
  • the first nucleic acid fragment and the second nucleic acid fragment are connected to immunize the test animal, which can also stimulate an immune response mediated by animal somatic cells.
  • the combination method can be set based on actual experimental needs. In some examples of the present application, any combination method can achieve the effect of stimulating an immune response mediated by animal somatic cells.
  • any one of the nucleic acid fragments comprises at least 15 amino acids.
  • the inventors have found through extensive experimental verification that 15 amino acids from any nucleic acid fragment can stimulate an animal's somatic cell-mediated immune response.
  • the 15 amino acids in any nucleic acid fragment can also be connected or not connected.
  • Experimental verification shows that the effect of stimulating the immune response mediated by animal somatic cells can be achieved under the conditions of connection or not.
  • the NTD protein has an amino acid sequence that is at least 91% homologous to SEQ ID NO:1.
  • the NTD protein has the amino acid sequence shown in SEQ ID NO:1.
  • the CTD protein has an amino acid sequence that is at least 92% homologous to SEQ ID NO:2.
  • the CTD protein has the amino acid sequence shown in SEQ ID NO:2.
  • the NSP12 protein has an amino acid sequence that is at least 97% homologous to SEQ ID NO:3.
  • the NSP12 protein has the amino acid sequence shown in SEQ ID NO:3.
  • the HR2_4 protein has an amino acid sequence that is at least 50% homologous to SEQ ID NO:4.
  • the HR2_4 protein has the amino acid sequence shown in SEQ ID NO:4.
  • the HR2_11 protein has an amino acid sequence that is at least 95% homologous to SEQ ID NO:5.
  • the HR2_11 protein has the amino acid sequence shown in SEQ ID NO:5.
  • the first nucleic acid fragment has a nucleotide sequence that is at least 71% identical to any one of SEQ ID NOs: 16 to 19.
  • the first nucleic acid fragment has a nucleotide sequence shown in SEQ ID NO: 16 to 19.
  • the second nucleic acid fragment has a nucleotide sequence that is at least 72% identical to any one of SEQ ID NOs: 20 to 23.
  • the second nucleic acid fragment has a nucleotide sequence shown in SEQ ID NOs: 20 to 23.
  • the third nucleic acid fragment has a nucleotide sequence that is at least 71% identical to any one of SEQ ID NO: 24 to 27.
  • the third nucleic acid fragment has a nucleotide sequence shown in SEQ ID NO: 24 to 27.
  • the fourth nucleic acid fragment has a nucleotide sequence that is at least 50% identical to any one of SEQ ID NO: 28 to 31.
  • the fourth nucleic acid fragment has a nucleotide sequence shown in SEQ ID NO: 28 to 31.
  • the fifth nucleic acid fragment has a nucleotide sequence that is at least 73% identical to any one of SEQ ID NO: 32 to 35.
  • the fifth nucleic acid fragment has a nucleotide sequence shown in SEQ ID NO: 32 to 35.
  • the homology of the amino acid sequence refers to the similarity between two amino acid sequences; the identity of the nucleotide sequence refers to the similarity between two nucleotide sequences.
  • the nucleic acid molecule further includes a sixth nucleic acid segment encoding a signal peptide sequence (MHC-I sp) of MHC-I (major histocompatibility complex I).
  • MHC-I sp signal peptide sequence of MHC-I (major histocompatibility complex I).
  • the purpose of adding the MHC-I signal peptide to the N-terminus of the antigen sequence is to enable ribosomes to attach to the endoplasmic reticulum membrane and guide protein transport within the cell.
  • the signal peptide sequence of MHC-I does not contain a transmembrane region.
  • the signal peptide sequence of MHC-I has an amino acid sequence as shown in SEQ ID NO:6.
  • the sixth nucleic acid fragment has a nucleotide sequence shown in SEQ ID NO: 36 to 38.
  • the sixth nucleic acid fragment is arranged at the 5' end of the nucleic acid molecule.
  • the nucleic acid further comprises a seventh nucleic acid segment encoding a MITD (major histocompatibility complex class I molecule transport signal) sequence.
  • adding the MITD sequence to the C-terminus of the nucleic acid molecule can stimulate CD4 + T cell proliferation and induce the production of more cytokines.
  • the MITD sequence comprises a transmembrane region.
  • the MITD sequence has the amino acid sequence shown in SEQ ID NO:7.
  • the seventh nucleic acid fragment has a nucleotide sequence shown in SEQ ID NO: 39 ⁇ 41.
  • the seventh nucleic acid fragment is arranged at the 3' end of the nucleic acid molecule.
  • the eighth nucleic acid fragment encodes an HBHA (heparin-binding hemagglutinin protein of Mycobacterium tuberculosis) adjuvant sequence.
  • HBHA heparin-binding hemagglutinin protein of Mycobacterium tuberculosis
  • HBHA has a strong immunostimulatory effect, can induce the maturation of DC cells, further planning CD4 + and CD8 + T cells, secreting IFN- ⁇ , and inducing T cell-mediated cytotoxicity.
  • the HBHA sequence has the amino acid sequence shown in SEQ ID NO:8.
  • the eighth nucleic acid fragment has a nucleotide sequence shown in SEQ ID NO: 42 to 44.
  • the ninth nucleic acid fragment encodes a PADRE (Pan HLA-DR reactive epitope) sequence; according to an embodiment of the present invention, PADRE belongs to a "universal" 13-amino acid pan-HLA DR peptide epitope used to activate CD4+T cells.
  • PADRE Pan HLA-DR reactive epitope
  • the PADRE sequence has the amino acid sequence shown in SEQ ID NO:9.
  • the ninth nucleic acid fragment has a nucleotide sequence shown in SEQ ID NO: 45 to 47.
  • the nucleic acid molecule further comprises a rigid or flexible linker sequence.
  • the connecting sequence has an amino acid sequence shown in SEQ ID NO: 11 to 15.
  • the connecting sequence has a nucleotide sequence shown in SEQ ID NO: 51 to 70.
  • the nucleic acid molecule is linear.
  • the present invention provides an expression vector.
  • the expression vector carries the nucleic acid molecule described in the first aspect of the present invention.
  • the expression vector can be expressed in cells, bacteria, yeast, or feline organisms.
  • the above-mentioned expression vector further includes at least one of the following technical features:
  • the expression vector is a non-pathogenic viral vector.
  • the non-pathogenic virus is selected from at least one of a retrovirus, a lentivirus, an adenovirus and an adeno-associated virus.
  • the present invention provides a recombinant virus.
  • the recombinant virus carries the nucleic acid molecule described in the first aspect of the present invention.
  • the recombinant virus containing the nucleic acid molecule described in the first aspect can be stably propagated in large quantities.
  • the present invention provides a liposome.
  • the liposome comprises a liposome carrier and a nucleic acid fragment, wherein the nucleic acid fragment is as defined in the first aspect of the present invention.
  • the liposome containing the liposome carrier and the nucleic acid fragment plays an important role in improving nucleic acid stability, cellular uptake, reducing toxic side effects, and improving delivery efficiency.
  • the present invention provides a vaccine.
  • the vaccine comprises the nucleic acid molecule described in the first aspect, the expression vector described in the second aspect, the recombinant virus described in the third aspect, or the liposome described in the fourth aspect.
  • the aforementioned vaccines can efficiently activate cell-mediated immune responses in animals.
  • the vaccine contains only proteins capable of activating cellular immune responses, thus avoiding toxic side effects and providing enhanced safety.
  • the above vaccine may further include at least one of the following additional technical features:
  • the vaccine includes at least one selected from RNA vaccine, DNA vaccine, protein recombinant vaccine, inactivated vaccine, attenuated vaccine, and viral vector vaccine.
  • the vaccine is an RNA vaccine.
  • the vaccine further comprises an adjuvant.
  • the adjuvant includes at least one of a TLR agonist and Mn 2+ .
  • the TLR agonist includes at least one of HBHA, CpG, R837, MPLA and derivatives thereof.
  • the present invention provides a recombinant cell.
  • the recombinant cell carries the nucleic acid molecule described in the first aspect of the present invention, the expression vector described in the second aspect of the present invention, or the recombinant virus described in the third aspect of the present invention.
  • the recombinant cell is used to package a virus carrying the nucleic acid molecule for use in preparing a nucleic acid vaccine to stimulate an immune response in the body.
  • the present invention provides a method for constructing a feline infectious peritonitis virus vaccine.
  • the method comprises introducing the nucleic acid molecule described in the first aspect of the present invention, the expression vector described in the second aspect, or the recombinant virus described in the third aspect into a recipient cell.
  • the method according to embodiments of the present invention can package a virus carrying the nucleic acid molecule for use in preparing a nucleic acid vaccine. This method for constructing an infectious peritonitis virus vaccine is safe, simple, and highly effective.
  • the above method further includes at least one of the following technical features:
  • the method prior to introduction into the recipient cells, the method further includes encapsulating the nucleic acid, expression vector, or recombinant virus with an encapsulation vector.
  • encapsulating the nucleic acid, expression vector, or recombinant virus with an encapsulation vector can protect the vaccine components from external environmental interference that affects their potency.
  • the encapsulation vector can also reduce the contact of the vaccine components with the external environment, thereby reducing the risk of contamination of the vaccine components and improving the safety of the vaccine.
  • some encapsulation vectors also have the effect of enhancing the infectivity of the vaccine components, thereby enabling them to more effectively stimulate an immune response.
  • the encapsulation carrier is selected from at least one of liposomes, polymer carriers, viral carriers, and nanoparticles.
  • the carrier is a nanoparticle.
  • the recipient cell is a CRFK cell, HEK293FT, HEK293T or BHK cell.
  • the recipient cells are CRFK cells.
  • the present invention provides a use of the nucleic acid molecule described in the first aspect, the expression vector described in the second aspect, the recombinant virus described in the third aspect, the liposome described in the fourth aspect, or the recombinant cell described in the sixth aspect in the preparation of a drug or vaccine.
  • the drug or vaccine is used to prevent or treat diseases associated with feline infectious peritonitis virus infection.
  • the drug or vaccine prepared based on the aforementioned nucleic acid molecule, expression vector, recombinant virus, or recombinant cell has high safety and can activate an animal cell-mediated immune response in a short period of time.
  • the present invention proposes a method for preventing or treating feline infectious peritonitis virus infection.
  • the method comprises: administering the nucleic acid molecule described in the first aspect of the present invention, the expression vector described in the second aspect, the recombinant virus described in the third aspect, the liposome described in the fourth aspect, the vaccine described in the fifth aspect, or the recombinant cell described in the sixth aspect to the test animal.
  • 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.
  • the aforementioned treatment methods have good immune effects on various epidemic strains of FIPV.
  • 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.
  • the effective amount of the nucleic acid molecule, expression vector, recombinant virus, liposome, vaccine or recombinant cell of the present invention may vary depending on the mode of administration and the severity of FIPV infection in the test animal.
  • the selection of the preferred effective amount can be determined by a person of ordinary skill in the art based on various factors (e.g., through clinical trials).
  • Various factors include, but are not limited to: pharmacokinetic parameters of the active ingredient, such as bioavailability, metabolism, half-life, etc.; the severity of FIPV infection in the test animal, the weight of the test animal, the immune status of the test animal, the route of administration, etc. For example, depending on the urgency of the treatment situation, several divided doses may be administered daily, or the dose may be reduced proportionally.
  • the above method may further include at least one of the following technical features:
  • the test animal is selected from cats.
  • the present invention proposes a use of the nucleic acid molecule described in the first aspect, the expression vector described in the second aspect, the recombinant virus described in the third aspect, the liposome described in the fourth aspect, the vaccine described in the fifth aspect or the recombinant cell described in the sixth aspect in preventing or treating feline infectious peritonitis virus infection.
  • administering an effective dose of nucleic acid molecules, expression vectors, recombinant viruses, liposomes, vaccines or recombinant cells to a test animal infected with FIPV can significantly improve the various physiological indicators and survival rates of the test animals.
  • the aforementioned treatment methods have good immune effects on various epidemic strains of FIPV.
  • FIG1 is a result of detecting the expression of target mRNA encapsulated by LNP according to Example 1 of the present invention.
  • FIG2 shows the change in survival rate after immunization with the target mRNA packaged in LNP according to Example 2 of the present invention.
  • FIG3 is a median statistical result of ELISPOT detection stimulated by a multi-epitope antigen polypeptide library after immunization with the target mRNA packaged by LNP according to Example 3 of the present invention.
  • FIG4 is a statistical result of ELISPOT detection of multi-epitope antigen polypeptide stimulation after immunization with LNP-encapsulated target mRNA according to Example 3 of the present invention.
  • FIG5 shows the change in survival rate after immunization with the target mRNA packaged in LNP according to Example 4 of the present invention.
  • 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.
  • a feature defined as “first” or “second” may explicitly or implicitly include at least one such feature.
  • “plurality” means at least two, such as two, three, etc., unless otherwise specifically defined.
  • non-pathogenic viral vector refers to a class of viruses that can express target antigens and are non-pathogenic, and are usually used to prepare vaccines. These vectors are genetically modified and the gene sequence of the target antigen is added, which is then expressed and replicated by the viral vector.
  • non-pathogenic viral vectors as vaccine vectors are that they can induce the immune system to produce a strong immune response, thereby stimulating the body's immune response to the target antigen; non-pathogenic viral vectors are usually designed to be unable to replicate, so they will not reproduce in the body and will not cause disease; compared with other traditional vaccine preparation methods, vaccines prepared with non-pathogenic viral vectors are more convenient and safer in production and storage, and have better stability and purity.
  • the RNA vaccine for preventing feline infectious peritonitis described in the present invention is prepared by constructing a vector encoding multiple epitope antigens of the FIPV virus, and then preparing an RNA vaccine for preventing the FIPV virus through lipid nanoparticles (LNP).
  • the vaccine can produce a strong immune response after immunizing cats.
  • the vaccine described in the present invention uses the N-terminus, C-terminus, NSP12 protein sequence of the N protein of the FIPV virus and the HR2-4 and HR2-11 sequences of the S protein as the main components of the constructed nucleic acid molecule.
  • the RNA vaccine expressed by the nucleic acid molecule has the advantages of simple preparation process, high safety, no toxic side effects, and industrial production; sufficient protection effect can be achieved using a very small dose, and it is superior to existing treatment methods in terms of safety and effectiveness.
  • sequences of the products corresponding to the names in Table 5 or Table 6 are formed by connecting the corresponding sequences in "SEQ ID NO:" in the 5' to 3' direction.
  • sequence of MHC-I sp-HBHA-L-PADRE-L-NTD-L-CTD-L-NSP12-L-HR2_4-L-HR2_11-L-MITD is composed of the MHC-I sp sequence shown in SEQ ID NO:38, the HBHA sequence shown in SEQ ID NO:44, the L (linker) sequence shown in SEQ ID NO:53, the PADRE sequence shown in SEQ ID NO:47, the L (linker) sequence shown in SEQ ID NO:56, the NTD sequence shown in SEQ ID NO:19, the L (linker) sequence shown in SEQ ID NO:59, the CTD sequence shown in SEQ ID NO:23, the L (linker) sequence shown in SEQ ID NO:62, the N
  • the 3’ end of the sequence shown in SEQ ID NO:38 is linked to the 5’ end of the sequence shown in SEQ ID NO:44
  • the 3’ end of the gene sequence shown in SEQ ID NO:44 is linked to the 5’ end of the sequence shown in SEQ ID NO:53
  • the 3’ end of the gene sequence shown in SEQ ID NO:53 is linked to the 5’ end of the sequence shown in SEQ ID NO:47
  • the 3’ end of the gene sequence shown in SEQ ID NO:47 is linked to the 5’ end of the sequence shown in SEQ ID NO:56
  • the 3’ end of the sequence shown in SEQ ID NO:56 is linked to the 5’ end of the sequence shown in SEQ ID NO:19
  • the 3’ end of the gene sequence shown in SEQ ID NO:19 is linked to the 5’ end of the sequence shown in SEQ ID NO:59
  • the 3’ end of the gene sequence shown in SEQ ID NO:59 is linked to the 5’ end of the sequence shown in SEQ ID NO:23
  • the 3’ end of the gene sequence shown in SEQ ID NO: 31 is connected to the 5’ end of the sequence shown in SEQ ID NO: 67
  • the 3’ end of the sequence shown in SEQ ID NO: 67 is connected to the 5’ end of the sequence shown in SEQ ID NO: 35
  • the 3’ end of the gene sequence shown in SEQ ID NO: 35 is connected to the 5’ end of the sequence shown in SEQ ID NO: 70
  • the 3’ end of the gene sequence shown in SEQ ID NO: 70 is connected to the 5’ end of the sequence shown in SEQ ID NO: 41.
  • the linker is not particularly limited, and the above-mentioned linker can be selected, or other flexible peptide or rigid peptide amino acid sequences can be selected for connection according to experimental requirements.
  • the mRNA sequence is synthesized in vitro and then encapsulated using lipid nanoparticles (LNP) and expressed in CRFK (cat kidney) cells and cat DC (dendritic cells).
  • LNP lipid nanoparticles
  • CRFK cat kidney cells
  • cat DC dendritic cells
  • RNA sequences were synthesized in vitro and expressed in CRFK (cat kidney) cells, and the sequences with the highest expression levels were selected as the final target sequences for vaccine preparation.
  • the specific steps are as follows:
  • Codon optimization of multi-epitope antigen sequences was performed using the cat universal codon library and the cat mesenteric lymph node codon library, with three optimizations for each antigen;
  • lipid nanoparticles are prepared. The specific steps are as follows:
  • 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 ⁇ m filter membrane.
  • the effectiveness of the vaccine was evaluated by assessing changes in physiological indicators such as body temperature, body weight, and survival rate of the test animals after mRNA vaccine immunization.
  • the LNP preparation method was the same as in Example 1.
  • D1 was constructed between the cat-derived 5'HBB-UTR (SEQ ID NO: 71) and 3'HBA-UTR (SEQ ID NO: 72), transcribed into mRNA in vitro, and packaged with LNP.
  • SEQ ID NO: 71 cat-derived 5'HBB-UTR
  • SEQ ID NO: 72 3'HBA-UTR
  • the sequence description and composition are shown in Tables 4 and 5.
  • 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:
  • the screened test animals were immunized with the target mRNA encapsulated in LNPs.
  • the immunization procedure is as follows:
  • the first vaccination was performed on D0
  • the second vaccination was performed on D21
  • the virus was challenged on D28 after the second vaccination;
  • the challenge virus strain was QS-1146; 5 kittens/group.
  • LNP-encapsulated target mRNA was transfected into CRFK cells, harvested 24 hours later, and immunoblotting was performed to detect protein expression. The results, as shown in Figure 2, demonstrated normal expression. Following challenge with LNP-encapsulated target mRNA in each group, all five kittens in the PBS group developed fever and weight loss, and autopsies revealed typical feline infectious peritonitis. Physiological indicators of the kittens in the group immunized with multi-epitope antigen mRNA significantly improved, with a significant increase in survival rate, as shown in Figure 2. Only one kitten died after 21 days, and the survival rate remained at 80% by day 30.
  • each antigen epitope was evaluated by detecting the stimulation response of the PBMC of the test animals to each epitope after immunization with the multi-epitope antigen mRNA vaccine.
  • the LNP preparation method and the test animal screening method were the same as those in Examples 1 and 2.
  • D1 was constructed between the cat-derived 5'HBB-UTR (SEQ ID NO: 71) and 3'HBA-UTR (SEQ ID NO: 72), transcribed into mRNA in vitro, and packaged with LNP.
  • SEQ ID NO: 71 cat-derived 5'HBB-UTR
  • SEQ ID NO: 72 3'HBA-UTR
  • the sequence description and composition are shown in Tables 4 and 5.
  • the antigen epitope polypeptide library is synthesized, and the specific implementation steps are as follows:
  • Each epitope peptide sequence was window-cut with a window size of 15 amino acids and a step size of 7 amino acids, and each window peptide and the full-length peptide were synthesized (Sino Biological).
  • PBMC of the test animal is extracted after immunization, stimulated with each epitope, and the response is detected.
  • the specific implementation steps are as follows:
  • PBMCs were extracted from the serum of the test animals and added to activated IFN- ⁇ coated plates. Negative stimulators PBS and positive stimulators PMA were added and incubated with each epitope peptide for 16-24 hours. The cells were counted and statistically analyzed using an ELISPOT detection kit (Dakoway).
  • PBMCs were extracted from the test animals and stimulated with each epitope peptide library.
  • the stimulation response to the entire epitope peptide library was tested, and the results are shown in Figure 3.
  • HR2-4 and HR2-11 are shorter in length and have a correspondingly smaller peptide library size. However, they still produced detectable responses after stimulation with the PBMCs of the immunized test animals.
  • the PBMCs of the test animals immunized with the multi-epitope antigen mRNA vaccine showed significant responses to stimulation with each epitope peptide library compared to the PBS immunization control group.
  • Using the entire peptide library to stimulate the PBMCs of the immunized test animals can obtain a response that is even stronger than when stimulated with each epitope peptide library.
  • PBMCs of the test animals were extracted and stimulated with a single peptide from each antigen epitope peptide library.
  • the stimulation response to each antigen epitope peptide was detected, and the overall distribution of the stimulation response of the peptides in each antigen epitope peptide library was statistically analyzed. The results are shown in Figure 4.
  • the overall distribution of the stimulation response of the peptides in each antigen epitope peptide library showed a lower distribution of the stimulation response of the peptides in the HR2-4 and HR2-11 peptide libraries, while the distribution of the stimulation response of the peptides in the NTD, CTD, and NSP12 peptide libraries was higher. However, they all had stronger responses than the PBS immunized control group. Stimulating the PBMCs of the immunized test animals with the full-length peptides can obtain a significantly stronger response than that of the peptides in each antigen peptide library.
  • Example 4 Validation of the potency of multi-epitope antigen mRNA vaccines against different epidemic strains
  • This example evaluated the effectiveness of a multi-epitope antigen mRNA vaccine against different circulating FIPV strains.
  • the LNP preparation method, test animal screening method, immunization schedule, and evaluation method were the same as in Examples 1 and 2.
  • the challenge strains were HF1902 and SH2211.
  • D1 was constructed between the cat-derived 5'HBB-UTR (SEQ ID NO: 71) and 3'HBA-UTR (SEQ ID NO: 72), transcribed into mRNA in vitro, and packaged with LNP.
  • SEQ ID NO: 71 cat-derived 5'HBB-UTR
  • SEQ ID NO: 72 3'HBA-UTR
  • the sequence description and composition are shown in Tables 4 and 5.
  • the multi-epitope antigen mRNA vaccine can effectively improve the physiological indicators and survival rate of the test animals after challenge with different FIPV epidemic strains.
  • the survival rate is shown in Figure 5.
  • the survival rate of the control group dropped below 50% around 20 days after challenge with the two epidemic strains, while the survival rate of the experimental group remained at 100% until 30 days after challenge.
  • 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.
  • the schematic representations of the above terms do not necessarily refer to the same embodiment or example.
  • the specific features, structures, materials or characteristics described can be combined in any one or more embodiments or examples in a suitable manner.
  • 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.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Engineering & Computer Science (AREA)
  • Organic Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Biomedical Technology (AREA)
  • Wood Science & Technology (AREA)
  • Virology (AREA)
  • Biotechnology (AREA)
  • Zoology (AREA)
  • Microbiology (AREA)
  • General Engineering & Computer Science (AREA)
  • Medicinal Chemistry (AREA)
  • Immunology (AREA)
  • Molecular Biology (AREA)
  • Biochemistry (AREA)
  • Public Health (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Veterinary Medicine (AREA)
  • Animal Behavior & Ethology (AREA)
  • Communicable Diseases (AREA)
  • General Chemical & Material Sciences (AREA)
  • Biophysics (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Physics & Mathematics (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Plant Pathology (AREA)
  • Mycology (AREA)
  • Epidemiology (AREA)
  • Cell Biology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Oncology (AREA)
  • Pulmonology (AREA)
  • Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)

Abstract

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

Description

针对猫FIPV的多表位抗原构建RNA疫苗的方法Method for constructing RNA vaccine targeting multi-epitope antigens of feline FIPV 技术领域Technical Field

本发明涉及生物技术领域，具体地，涉及针对猫FIPV的多表位抗原构建RNA疫苗的方法，更具体地，涉及一种分离的核酸分子、表达载体、重组病毒、脂质体、疫苗、重组细胞、构建猫传染性腹膜炎病毒疫苗的方法及用途。The present invention relates to the field of biotechnology, and specifically, to a method for constructing an RNA vaccine against the multi-epitope antigens of feline FIPV. More specifically, it relates to an isolated nucleic acid molecule, an expression vector, a recombinant virus, a liposome, a vaccine, a recombinant cell, and a method and use of constructing a feline infectious peritonitis virus vaccine.

背景技术Background Art

猫冠状病毒FCoV属于冠状病毒。冠状病毒科病毒特征都是比较大、圆形、有囊膜的正链RNA病毒，基因组在27～32kb之间，编码复制多聚酶、4种结构蛋白(S蛋白、M蛋白、N蛋白、E蛋白)与几种非结构蛋白。其中，S蛋白(spike protein)是冠状病毒的重要结构蛋白之一。它负责构成这些病毒的表面突起，并且也是这些病毒进行感染的关键因素之一。S蛋白可以结合宿主细胞上的受体，从而进入宿主细胞内部进行感染。除此之外，S蛋白还是许多冠状病毒疫苗的重要抗原。M蛋白(Membrane protein)则参与了冠状病毒的颗粒形态的形成和定位。它是一种跨过病毒囊膜的蛋白，可以与其他蛋白相互作用以形成病毒粒子的结构。N蛋白(Nucleocapsid protein)则包裹着病毒的RNA基因组，并且参与到病毒基因复制和转录过程中。同时，N蛋白还能够诱导宿主免疫系统对病毒进行应答。E蛋白(Envelope protein)则是冠状病毒囊膜上的蛋白，可以与M蛋白相互作用以形成病毒粒子的结构，同时还参与到病毒的感染和装配过程中。Feline coronavirus (FCoV) belongs to the coronavirus family. Viruses in the Coronaviridae family are characterized by relatively large, round, enveloped, positive-strand RNA viruses with genomes ranging from 27 to 32 kb. They encode replication polymerases, four structural proteins (S, M, N, and E), and several nonstructural proteins. The S protein (spike protein) is a key structural protein of coronaviruses. It forms the surface protrusions of these viruses and is a key factor in their infection. The S protein binds to receptors on host cells, allowing them to enter and infect. Furthermore, the S protein is a key antigen in many coronavirus vaccines. The M protein (membrane protein) is involved in the formation and localization of coronavirus particles. It spans the viral envelope and interacts with other proteins to form the structure of the virion. The N protein (nucleocapsid protein) encapsulates the viral RNA genome and is involved in viral gene replication and transcription. It also stimulates the host immune system to respond to the virus. E protein (Envelope protein) is a protein on the coronavirus envelope that can interact with M protein to form the structure of virus particles. It is also involved in the infection and assembly process of the virus.

根据新冠病毒的相关研究发现N蛋白的N端具有保守的与核酸结合的氨基酸位点，在冠状病毒中比较保守，且有多个T细胞或B细胞表位；冠状病毒的N蛋白的C端可以形成特殊的二级结构；而研究报道T细胞或B细胞表位集中的区域主要存在于NSP12非结构蛋白中；HR2_4与HR2_11两个B细胞表位来源于FIPV的SII区域。According to relevant research on the new coronavirus, it was found that the N-terminus of the N protein has a conserved amino acid site that binds to nucleic acids, which is relatively conserved among coronaviruses and has multiple T cell or B cell epitopes; the C-terminus of the coronavirus's N protein can form a special secondary structure; and studies have reported that the area where T cell or B cell epitopes are concentrated mainly exists in the NSP12 non-structural protein; the two B cell epitopes HR2_4 and HR2_11 are derived from the SII region of FIPV.

猫冠状病毒(FCoV)根据生物型/致病力可分为：猫肠道冠状病毒(FECV)和猫传染性腹膜炎病毒(FIPV)。FECV传播非常广泛，感染肠上皮细胞，几乎无症状或只有轻微腹泻。FIPV主要感染猫单核细胞和巨噬细胞。从基因组序列很难区分FECV与FIPV，虽然一些研究表明，FIPV可以通过刺突蛋白氨基酸突变与FECV区分开来，但这些突变后来被发现与组织嗜性更相关。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.

FIPV典型特征为各种组织和器官发生脓性肉芽肿性病变，包括肺、肝、脾、网膜和脑等。巨噬细胞和单核细胞的感染被认为是致病机制的关键。在FIPV感染的终末期可观察到，外周和淋巴组织的T细胞大量减少，并经常出现高γ球蛋白血症，这表明存在严重的病毒诱导的免疫失调。体液免疫似乎不具有保护作用，可能导致“早期死亡综合征”。当S抗体以亚中和滴度存在时，可通过与Fc受体结合增强靶细胞的感染。研究人员曾多次尝试开发FIPV疫苗，但大多都失败了，失败主要原因是存在抗体依赖性的增强(ADE)感染的现象，导致抗体无法发挥有效的保护作用。目前研究人员尝试通过细胞介导的免疫(Cell-Mediated Immunity，CMI)来实现控制FIPV的感染和清除，但仍未取得很好的保护效果。The typical characteristics of FIPV are purulent granulomatous lesions in various tissues and organs, including the lungs, liver, spleen, omentum and brain. Infection of macrophages and monocytes is considered to be the key to the pathogenic mechanism. At the end of FIPV infection, a large decrease in T cells in peripheral and lymphoid tissues can be observed, and hypergammaglobulinemia is often present, indicating the presence of severe virus-induced immune disorders. Humoral immunity does not seem to have a protective effect and may lead to "early death syndrome". When S antibodies are present in sub-neutralizing titers, they can enhance the infection of target cells by binding to Fc receptors. Researchers have tried many times to develop FIPV vaccines, but most of them have failed. The main reason for the failure is the phenomenon of antibody-dependent enhancement (ADE) infection, which makes the antibodies unable to play an effective protective role. Currently, researchers are trying to control the infection and clearance of FIPV through cell-mediated immunity (CMI), but have not yet achieved good protection.

因此，本领域亟需研发一种针对FIPV的疫苗。Therefore, there is an urgent need in this field to develop a vaccine against FIPV.

发明内容Summary of the Invention

本申请是发明人基于对以下问题和事实的发现而提出的：This application is filed by the inventor based on the following findings:

针对目前FIPV疫苗研发长期得不到新突破，导致FIPV感染几乎都以猫的死亡而终结。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.

为此，在本发明的第一方面，本发明提出了一种分离的核酸分子。根据本发明的实施例，所述分离的核酸分子包括第一核酸片段、第二核酸片段、第三核酸片段、第四核酸片段和第五核酸片段中的至少之一；其中，所述第一核酸片段来源于猫传染性腹膜炎病毒QS毒株的N蛋白的N端(NTD)；所述第二核酸片段来源于猫传染性腹膜炎病毒QS毒株的N蛋白的C端(CTD)；所述第三核酸片段编码猫传染性腹膜炎病毒QS毒株的NSP12蛋白；所述第四核酸片段来源于猫传染性腹膜炎病毒79-1146毒株S蛋白的表位HR2_4；所述第五核酸片段来源于猫传染性腹膜炎病毒79-1146毒株S蛋白的表位HR2_11；所述核酸分子为RNA。根据本发明的实施例，表达猫传染性腹膜炎病毒的核酸分子可以刺激动物体细胞介导的免疫反应。 To this end, in a first aspect, the present invention provides an isolated nucleic acid molecule. According to an embodiment of the present invention, the isolated nucleic acid molecule comprises at least one of a first nucleic acid fragment, a second nucleic acid fragment, a third nucleic acid fragment, a fourth nucleic acid fragment, and a fifth nucleic acid fragment; wherein the first nucleic acid fragment is derived from the N-terminus (NTD) of the N protein of the QS strain of feline infectious peritonitis virus; the second nucleic acid fragment is derived from the C-terminus (CTD) of the N protein of the QS strain of feline infectious peritonitis virus; the third nucleic acid fragment encodes the NSP12 protein of the QS strain of feline infectious peritonitis virus; the fourth nucleic acid fragment is derived from the epitope HR2_4 of the S protein of the 79-1146 strain of feline infectious peritonitis virus; and the fifth nucleic acid fragment is derived from the epitope HR2_11 of the S protein of the 79-1146 strain of feline infectious peritonitis virus; and the nucleic acid molecule is RNA. According to an embodiment of the present invention, the nucleic acid molecule expressing feline infectious peritonitis virus can stimulate an animal's somatic cell-mediated immune response.

需要说明的是，在本申请中，也可根据需要对野生型FIPV病毒的NTD、CTD、NSP12、HR2_4或HR2_11蛋白序列进行适应性改造，用以提高抗原表达，降低FIPV病毒的毒性，同时不影响其三维结构，保留其免疫原性，制备新型的FIPV病毒疫苗。根据序列比对结果(表1、2)，所述NTD蛋白具有与SEQ ID NO:1至少91％同源性的氨基酸序列，所述NTD蛋白核酸片段具有与SEQ ID NO:16～19至少71％同一性的核苷酸序列；所述CTD蛋白具有与SEQ ID NO:2至少92％同源性的氨基酸片段，所述CTD蛋白核酸片段具有与SEQ ID NO:20～23至少72％同一性的核苷酸序列；所述NSP12蛋白具有与SEQ ID NO:3至少97％同源性的氨基酸片段，所述NSP12蛋白核酸片段具有与SEQ ID NO:24～27至少71％同一性的核苷酸序列；所述HR2_4蛋白具有与SEQ ID NO:4至少50％同源性的氨基酸片段，所述HR2_4蛋白核酸片段具有与SEQ ID NO:28～31至少50％同一性的核苷酸序列；所述HR2_11蛋白具有与SEQ ID NO:5至少95％同源性的氨基酸片段，所述HR2_11蛋白核酸片段具有与SEQ ID NO:32～35至少73％同一性的核苷酸序列。FIPV病毒疫苗不受特别限制，只要在生物体内能够产生改造后的FIPV病毒NTD、CTD、NSP12、HR2_4和/或HR2_11蛋白的受体结合区，并且具有免疫原性，可以刺激生物体产生相应免疫反应即可。而且，所述分离的核酸分子可用于刺激能够感染猫传染性腹膜炎病毒的所有动物的免疫反应，包括但不仅限于猫。It should be noted that in the present application, the NTD, CTD, NSP12, HR2_4 or HR2_11 protein sequences of the wild-type FIPV virus can also be adaptively modified as needed to improve antigen expression and reduce the toxicity of the FIPV virus without affecting its three-dimensional structure and retaining its immunogenicity, so as to prepare a new type of FIPV virus vaccine. According to the sequence alignment results (Tables 1 and 2), the NTD protein has an amino acid sequence that is at least 91% homologous to SEQ ID NO: 1, and the NTD protein nucleic acid fragment has a nucleotide sequence that is at least 71% identical to SEQ ID NO: 16-19; the CTD protein has an amino acid fragment that is at least 92% homologous to SEQ ID NO: 2, and the CTD protein nucleic acid fragment has a nucleotide sequence that is at least 72% identical to SEQ ID NO: 20-23; the NSP12 protein has an amino acid fragment that is at least 97% homologous to SEQ ID NO: 3, and the CTD protein nucleic acid fragment has a nucleotide sequence that is at least 72% identical to SEQ ID NO: 20-23; the NSP12 protein has an amino acid fragment that is at least 97% homologous to SEQ ID NO: 3. The NSP12 protein nucleic acid fragment has a nucleotide sequence that is at least 71% identical to SEQ ID NOs: 24-27; the HR2_4 protein has an amino acid segment that is at least 50% homologous to SEQ ID NO: 4, and the HR2_4 protein nucleic acid segment has a nucleotide sequence that is at least 50% identical to SEQ ID NOs: 28-31; the HR2_11 protein has an amino acid segment that is at least 95% homologous to SEQ ID NO: 5, and the HR2_11 protein nucleic acid segment has a nucleotide sequence that is at least 73% identical to SEQ ID NOs: 32-35. FIPV virus vaccines are not particularly limited, as long as they can produce the modified FIPV virus NTD, CTD, NSP12, HR2_4, and/or HR2_11 protein receptor binding region in an organism, are immunogenic, and can stimulate the organism to produce a corresponding immune response. Furthermore, the isolated nucleic acid molecule can be used to stimulate an immune response in all animals that can be infected with feline infectious peritonitis virus, including but not limited to cats.

需要说明的是，所述linker(连接肽)为两个融合蛋白间起连接作用的柔性或刚性氨基酸链，如3×flag、EAAAK、GGGS、AAY、GPGPG、(GGGGS)n等。在本申请中，实验中常用linker序列均可适用本申请实施例。It should be noted that the linker (connecting peptide) is a flexible or rigid amino acid chain that acts as a link between two fusion proteins, such as 3×flag, EAAAK, GGGS, AAY, GPGPG, (GGGGS)n, etc. In this application, commonly used linker sequences in experiments can be applied to the embodiments of this application.

需要说明的是，在本申请中，所述NTD、CTD、NSP12、HR2_4和HR2_11通过linker相连，但相连方式并不受特别限制。示例性地，所述核酸分子的相连方式可以是NTD-linker-CTD-linker-NSP12-linker-HR2_4-linker-HR2_11，也可以是NTD-linker-NSP12-linker-CTD-linker-HR2_4-linker-HR2_11等。It should be noted that in this application, the NTD, CTD, NSP12, HR2_4, and HR2_11 are connected via a linker, but the connection method is not particularly limited. For example, the nucleic acid molecules can be connected in the form of NTD-linker-CTD-linker-NSP12-linker-HR2_4-linker-HR2_11, or NTD-linker-NSP12-linker-CTD-linker-HR2_4-linker-HR2_11, etc.

表1：多亚株蛋白序列同源性(％)
Table 1: Protein sequence homology among multiple substrains (%)

表2：多亚株核酸序列同一性(％)


注：-表示无。Table 2: Nucleic acid sequence identity among multiple substrains (%)


Note: - means none.

根据本发明的实施例，上述分离的核酸分子还可以包括下列技术特征中的至少之一：According to an embodiment of the present invention, the isolated nucleic acid molecule may further include at least one of the following technical features:

根据本发明的实施例，所述核酸片段相连或不相连。According to an embodiment of the present invention, the nucleic acid fragments are connected or not connected.

根据本发明的实施例，所述核酸片段通过linker相连。在本申请的一些示例中，第一核酸片段～第五核酸片段通过linker相连作为一个核酸分子，能够刺激动物体细胞介导的免疫反应。According to an embodiment of the present invention, the nucleic acid fragments are connected by a linker. In some examples of the present application, the first to fifth nucleic acid fragments are connected by a linker as a nucleic acid molecule, which can stimulate an immune response mediated by animal somatic cells.

在本申请的一些示例中，所述核酸片段不相连。在本申请的一些示例中，上述第一核酸片段～第五核酸片段中任一核酸片段均能够刺激动物体细胞介导的免疫反应。示例性地，单独采用第一核酸片段对受试动物进行免疫，能够刺激动物体细胞介导的免疫反应。In some examples of this application, the nucleic acid fragments are not connected. In some examples of this application, any of the first to fifth nucleic acid fragments can stimulate an animal's somatic cell-mediated immune response. For example, immunizing a test animal with the first nucleic acid fragment alone can stimulate an animal's somatic cell-mediated immune response.

在本申请的另一些示例中，上述第一核酸片段～第五核酸片段可进行自由组合刺激动物体细胞介导的免疫反应。示例性地，采用第一核酸片段和第二核酸片段相连对受试动物进行免疫，同样能够刺激动物体细胞介导的免疫反应。其中，组合方式可基于实际实验需求，进行设定，在本申请的一些示例中，任一组合方式均能实现刺激动物体细胞介导的免疫反应的效果。In other examples of the present application, the first to fifth nucleic acid fragments can be freely combined to stimulate an immune response mediated by animal somatic cells. For example, the first nucleic acid fragment and the second nucleic acid fragment are connected to immunize the test animal, which can also stimulate an immune response mediated by animal somatic cells. Among them, the combination method can be set based on actual experimental needs. In some examples of the present application, any combination method can achieve the effect of stimulating an immune response mediated by animal somatic cells.

根据本发明的实施例，所述核酸片段中的任意一个至少包括15个氨基酸。在本申请的一些示例中，发明人经过大量实验验证发现，在任一核酸片段截取15个氨基酸，即可实现刺激动物体细胞介导的免疫反应的效果。According to an embodiment of the present invention, any one of the nucleic acid fragments comprises at least 15 amino acids. In some examples of this application, the inventors have found through extensive experimental verification that 15 amino acids from any nucleic acid fragment can stimulate an animal's somatic cell-mediated immune response.

在本申请的一些示例中，同样可对任一核酸片段中的15个氨基酸进行相连或不相连处理。经过实验验证，在相连或不相连的条件下，均能够实现刺激动物体细胞介导的免疫反应的效果。In some examples of the present application, the 15 amino acids in any nucleic acid fragment can also be connected or not connected. Experimental verification shows that the effect of stimulating the immune response mediated by animal somatic cells can be achieved under the conditions of connection or not.

根据本发明的实施例，所述NTD蛋白具有与SEQ ID NO:1至少91％同源性的氨基酸序列。According to an embodiment of the present invention, the NTD protein has an amino acid sequence that is at least 91% homologous to SEQ ID NO:1.

根据本发明的实施例，所述NTD蛋白具有SEQ ID NO:1所示的氨基酸序列。According to an embodiment of the present invention, the NTD protein has the amino acid sequence shown in SEQ ID NO:1.

根据本发明的实施例，所述CTD蛋白具有与SEQ ID NO:2至少92％同源性的氨基酸序列。According to an embodiment of the present invention, the CTD protein has an amino acid sequence that is at least 92% homologous to SEQ ID NO:2.

根据本发明的实施例，所述CTD蛋白具有SEQ ID NO:2所示的氨基酸序列。According to an embodiment of the present invention, the CTD protein has the amino acid sequence shown in SEQ ID NO:2.

根据本发明的实施例，所述NSP12蛋白具有与SEQ ID NO:3至少97％同源性的氨基酸序列。According to an embodiment of the present invention, the NSP12 protein has an amino acid sequence that is at least 97% homologous to SEQ ID NO:3.

根据本发明的实施例，所述NSP12蛋白具有SEQ ID NO:3所示的氨基酸序列。According to an embodiment of the present invention, the NSP12 protein has the amino acid sequence shown in SEQ ID NO:3.

根据本发明的实施例，所述HR2_4蛋白具有与SEQ ID NO:4至少50％同源性的氨基酸序列。According to an embodiment of the present invention, the HR2_4 protein has an amino acid sequence that is at least 50% homologous to SEQ ID NO:4.

根据本发明的实施例，所述HR2_4蛋白具有SEQ ID NO:4所示的氨基酸序列。According to an embodiment of the present invention, the HR2_4 protein has the amino acid sequence shown in SEQ ID NO:4.

根据本发明的实施例，所述HR2_11蛋白具有与SEQ ID NO:5至少95％同源性的氨基酸序列。According to an embodiment of the present invention, the HR2_11 protein has an amino acid sequence that is at least 95% homologous to SEQ ID NO:5.

根据本发明的实施例，所述HR2_11蛋白具有SEQ ID NO:5所示的氨基酸序列。According to an embodiment of the present invention, the HR2_11 protein has the amino acid sequence shown in SEQ ID NO:5.

根据本发明的实施例，所述第一核酸片段具有与SEQ ID NO:16～19任一序列至少71％同一性的核苷酸序列。According to an embodiment of the present invention, the first nucleic acid fragment has a nucleotide sequence that is at least 71% identical to any one of SEQ ID NOs: 16 to 19.

根据本发明的实施例，所述第一核酸片段具有SEQ ID NO:16～19所示的核苷酸序列。According to an embodiment of the present invention, the first nucleic acid fragment has a nucleotide sequence shown in SEQ ID NO: 16 to 19.

根据本发明的实施例，所述第二核酸片段具有与SEQ ID NO:20～23任一序列至少72％同一性的核苷酸序列。According to an embodiment of the present invention, the second nucleic acid fragment has a nucleotide sequence that is at least 72% identical to any one of SEQ ID NOs: 20 to 23.

根据本发明的实施例，所述第二核酸片段具有SEQ ID NO:20～23所示的核苷酸序列。 According to an embodiment of the present invention, the second nucleic acid fragment has a nucleotide sequence shown in SEQ ID NOs: 20 to 23.

根据本发明的实施例，所述第三核酸片段具有与SEQ ID NO:24～27任一序列至少71％同一性的核苷酸序列。According to an embodiment of the present invention, the third nucleic acid fragment has a nucleotide sequence that is at least 71% identical to any one of SEQ ID NO: 24 to 27.

根据本发明的实施例，所述第三核酸片段具有SEQ ID NO:24～27所示的核苷酸序列。According to an embodiment of the present invention, the third nucleic acid fragment has a nucleotide sequence shown in SEQ ID NO: 24 to 27.

根据本发明的实施例，所述第四核酸片段具有与SEQ ID NO:28～31任一序列至少50％同一性的核苷酸序列。According to an embodiment of the present invention, the fourth nucleic acid fragment has a nucleotide sequence that is at least 50% identical to any one of SEQ ID NO: 28 to 31.

根据本发明的实施例，所述第四核酸片段具有SEQ ID NO:28～31所示的核苷酸序列。According to an embodiment of the present invention, the fourth nucleic acid fragment has a nucleotide sequence shown in SEQ ID NO: 28 to 31.

根据本发明的实施例，所述第五核酸片段具有与SEQ ID NO:32～35任一序列至少73％同一性的核苷酸序列。According to an embodiment of the present invention, the fifth nucleic acid fragment has a nucleotide sequence that is at least 73% identical to any one of SEQ ID NO: 32 to 35.

根据本发明的实施例，所述第五核酸片段具有SEQ ID NO:32～35所示的核苷酸序列。According to an embodiment of the present invention, the fifth nucleic acid fragment has a nucleotide sequence shown in SEQ ID NO: 32 to 35.

需要说明的是，在本申请中，所述氨基酸序列的同源性是指两个氨基酸序列之间的相似度；所述核苷酸序列的同一性是指两个核苷酸序列之间的相似度。It should be noted that, in the present application, the homology of the amino acid sequence refers to the similarity between two amino acid sequences; the identity of the nucleotide sequence refers to the similarity between two nucleotide sequences.

根据本发明的实施例，所述核酸分子进一步包括第六核酸片段，所述第六核酸片段编码MHC-Ⅰ(主要组织相容性复合体I)的信号肽序列(MHC-I sp)。根据本发明的实施例，在抗原序列的N端添加MHC-I信号肽的目的是使核糖体附着于内质网膜，指引蛋白在细胞内的运输。According to an embodiment of the present invention, the nucleic acid molecule further includes a sixth nucleic acid segment encoding a signal peptide sequence (MHC-I sp) of MHC-I (major histocompatibility complex I). According to an embodiment of the present invention, the purpose of adding the MHC-I signal peptide to the N-terminus of the antigen sequence is to enable ribosomes to attach to the endoplasmic reticulum membrane and guide protein transport within the cell.

根据本发明的实施例，所述MHC-Ⅰ的信号肽序列不包含跨膜区。According to an embodiment of the present invention, the signal peptide sequence of MHC-I does not contain a transmembrane region.

根据本发明的实施例，所述MHC-Ⅰ的信号肽序列具有如SEQ ID NO:6所示的氨基酸序列。According to an embodiment of the present invention, the signal peptide sequence of MHC-I has an amino acid sequence as shown in SEQ ID NO:6.

根据本发明的实施例，所述第六核酸片段具有SEQ ID NO:36～38所示的核苷酸序列。According to an embodiment of the present invention, the sixth nucleic acid fragment has a nucleotide sequence shown in SEQ ID NO: 36 to 38.

根据本发明的实施例，所述第六核酸片段设置于所述核酸分子的5’端。According to an embodiment of the present invention, the sixth nucleic acid fragment is arranged at the 5' end of the nucleic acid molecule.

根据本发明的实施例，所述进一步包括第七核酸片段，所述第七核酸片段编码MITD(主要组织相容性复合体I类分子转运信号)序列。根据本发明的实施例，在所述核酸分子的C端添加MITD序列可刺激CD4+T细胞增殖，诱导产生更多的细胞因子。According to an embodiment of the present invention, the nucleic acid further comprises a seventh nucleic acid segment encoding a MITD (major histocompatibility complex class I molecule transport signal) sequence. According to an embodiment of the present invention, adding the MITD sequence to the C-terminus of the nucleic acid molecule can stimulate CD4 + T cell proliferation and induce the production of more cytokines.

根据本发明的实施例，所述MITD序列包含跨膜区。According to an embodiment of the present invention, the MITD sequence comprises a transmembrane region.

根据本发明的实施例，所述MITD序列具有SEQ ID NO:7所示的氨基酸序列。According to an embodiment of the present invention, the MITD sequence has the amino acid sequence shown in SEQ ID NO:7.

根据本发明的实施例，所述第七核酸片段具有SEQ ID NO:39～41所示的核苷酸序列。According to an embodiment of the present invention, the seventh nucleic acid fragment has a nucleotide sequence shown in SEQ ID NO: 39~41.

根据本发明的实施例，所述第七核酸片段设置于所述核酸分子的3’端。According to an embodiment of the present invention, the seventh nucleic acid fragment is arranged at the 3' end of the nucleic acid molecule.

根据本发明的实施例，所述第八核酸片段编码HBHA(结核分枝杆菌的肝素结合血凝素蛋白)佐剂序列。根据本发明的实施例，HBHA具有较强的免疫刺激作用，可以诱导DC细胞的成熟，进而计划CD4+和CD8+T细胞，分泌IFN-γ，并诱导T细胞介导的细胞毒性。According to an embodiment of the present invention, the eighth nucleic acid fragment encodes an HBHA (heparin-binding hemagglutinin protein of Mycobacterium tuberculosis) adjuvant sequence. According to an embodiment of the present invention, HBHA has a strong immunostimulatory effect, can induce the maturation of DC cells, further planning CD4 + and CD8 + T cells, secreting IFN-γ, and inducing T cell-mediated cytotoxicity.

根据本发明的实施例，所述HBHA序列具有SEQ ID NO:8所示的氨基酸序列。According to an embodiment of the present invention, the HBHA sequence has the amino acid sequence shown in SEQ ID NO:8.

根据本发明的实施例，所述第八核酸片段具有SEQ ID NO:42～44所示的核苷酸序列。According to an embodiment of the present invention, the eighth nucleic acid fragment has a nucleotide sequence shown in SEQ ID NO: 42 to 44.

根据本发明的实施例，所述第九核酸片段编码PADRE(Pan HLA-DR reactive epitope)序列；根据本发明的实施例，PADRE属于“通用”的13个氨基酸的泛HLA DR肽表位，用于激活CD4+T细胞。According to an embodiment of the present invention, the ninth nucleic acid fragment encodes a PADRE (Pan HLA-DR reactive epitope) sequence; according to an embodiment of the present invention, PADRE belongs to a "universal" 13-amino acid pan-HLA DR peptide epitope used to activate CD4+T cells.

根据本发明的实施例，所述PADRE序列具有SEQ ID NO:9所示的氨基酸序列。According to an embodiment of the present invention, the PADRE sequence has the amino acid sequence shown in SEQ ID NO:9.

根据本发明的实施例，所述第九核酸片段具有SEQ ID NO:45～47所示的核苷酸序列。According to an embodiment of the present invention, the ninth nucleic acid fragment has a nucleotide sequence shown in SEQ ID NO: 45 to 47.

根据本发明的实施例，所述核酸分子进一步包括刚性或柔性的连接序列。According to an embodiment of the present invention, the nucleic acid molecule further comprises a rigid or flexible linker sequence.

根据本发明的实施例，所述连接序列具有SEQ ID NO:11～15所示的氨基酸序列。According to an embodiment of the present invention, the connecting sequence has an amino acid sequence shown in SEQ ID NO: 11 to 15.

根据本发明的实施例，所述连接序列具有SEQ ID NO:51～70所示的核苷酸序列。According to an embodiment of the present invention, the connecting sequence has a nucleotide sequence shown in SEQ ID NO: 51 to 70.

根据本发明的实施例，所述核酸分子为线性。According to an embodiment of the present invention, the nucleic acid molecule is linear.

在本发明的第二方面，本发明提出了一种表达载体。根据本发明的实施例，所述表达载体携带本发明第一方面所述的核酸分子。根据本发明的实施例，所述表达载体可以在细胞、细菌、酵母或猫科动物生物体内表达。In a second aspect, the present invention provides an expression vector. According to an embodiment of the present invention, the expression vector carries the nucleic acid molecule described in the first aspect of the present invention. According to an embodiment of the present invention, the expression vector can be expressed in cells, bacteria, yeast, or feline organisms.

根据本发明的实施例，上述表达载体还包括下列技术特征的至少之一：According to an embodiment of the present invention, the above-mentioned expression vector further includes at least one of the following technical features:

根据本发明的实施例，所述表达载体是非致病性病毒载体。According to an embodiment of the present invention, the expression vector is a non-pathogenic viral vector.

根据本发明的实施例，所述非致病性病毒选自反转录病毒、慢病毒、腺病毒和腺病毒相关病毒中的至少之一。 According to an embodiment of the present invention, the non-pathogenic virus is selected from at least one of a retrovirus, a lentivirus, an adenovirus and an adeno-associated virus.

在本发明的第三方面，本发明提出了一种重组病毒。根据本发明的实施例，所述重组病毒携带本发明第一方面所述的核酸分子。所述包含第一方面所述核酸分子的重组病毒可以稳定大量繁殖。In a third aspect, the present invention provides a recombinant virus. According to an embodiment of the present invention, the recombinant virus carries the nucleic acid molecule described in the first aspect of the present invention. The recombinant virus containing the nucleic acid molecule described in the first aspect can be stably propagated in large quantities.

在本发明的第四方面，本发明提出了一种脂质体。根据本发明的实施例，所述脂质体包括脂质体载体以及核酸片段，所述核酸片段如本发明第一方面所限定的。所述含有脂质体载体以及核酸片段的脂质体在提高核酸稳定性、细胞摄取率、减少毒副作用、提高递送效率等方面具有重要作用。In a fourth aspect, the present invention provides a liposome. According to an embodiment of the present invention, the liposome comprises a liposome carrier and a nucleic acid fragment, wherein the nucleic acid fragment is as defined in the first aspect of the present invention. The liposome containing the liposome carrier and the nucleic acid fragment plays an important role in improving nucleic acid stability, cellular uptake, reducing toxic side effects, and improving delivery efficiency.

在本发明的第五方面，本发明提出了一种疫苗。根据本发明的实施例，所述疫苗包括本发明第一方面所述的核酸分子、第二方面所述的表达载体、第三方面所述的重组病毒或第四方面所述的脂质体。根据本发明的实施例，采用前述疫苗可以高效激活动物细胞介导的免疫反应。此外，疫苗中仅包含能够激活细胞免疫反应的蛋白，避免产生毒副作用，具有更高的安全性。In its fifth aspect, the present invention provides a vaccine. According to embodiments of the present invention, the vaccine comprises the nucleic acid molecule described in the first aspect, the expression vector described in the second aspect, the recombinant virus described in the third aspect, or the liposome described in the fourth aspect. According to embodiments of the present invention, the aforementioned vaccines can efficiently activate cell-mediated immune responses in animals. Furthermore, the vaccine contains only proteins capable of activating cellular immune responses, thus avoiding toxic side effects and providing enhanced safety.

根据本发明的实施例，上述疫苗还可以包括下列附加技术特征中的至少之一：According to an embodiment of the present invention, the above vaccine may further include at least one of the following additional technical features:

根据本发明的实施例，所述疫苗包括选自RNA疫苗、DNA疫苗、蛋白重组疫苗、灭活疫苗、减活疫苗、病毒载体疫苗的至少之一。According to an embodiment of the present invention, the vaccine includes at least one selected from RNA vaccine, DNA vaccine, protein recombinant vaccine, inactivated vaccine, attenuated vaccine, and viral vector vaccine.

根据本发明的实施例，所述疫苗为RNA疫苗。According to an embodiment of the present invention, the vaccine is an RNA vaccine.

根据本发明的实施例，所述疫苗进一步包括佐剂。According to an embodiment of the present invention, the vaccine further comprises an adjuvant.

根据本发明的实施例，所述佐剂包括TLR激动剂、Mn2+的至少之一。According to an embodiment of the present invention, the adjuvant includes at least one of a TLR agonist and Mn 2+ .

根据本发明的实施例，所述TLR激动剂包括HBHA、CpG，R837，MPLA和其衍生物的至少之一。According to an embodiment of the present invention, the TLR agonist includes at least one of HBHA, CpG, R837, MPLA and derivatives thereof.

在本发明的第六方面，本发明提出了一种重组细胞。根据本发明的实施例，所述重组细胞携带本发明第一方面所述的核酸分子、第二方面所述的表达载体或本发明第三方面所述的重组病毒。根据本发明的实施例，所述重组细胞用于包装携带所述核酸分子的病毒，以用于制备核酸疫苗，以刺激机体产生免疫反应。In a sixth aspect, the present invention provides a recombinant cell. According to embodiments of the present invention, the recombinant cell carries the nucleic acid molecule described in the first aspect of the present invention, the expression vector described in the second aspect of the present invention, or the recombinant virus described in the third aspect of the present invention. According to embodiments of the present invention, the recombinant cell is used to package a virus carrying the nucleic acid molecule for use in preparing a nucleic acid vaccine to stimulate an immune response in the body.

在本发明的第七方面，本发明提出了一种构建猫传染性腹膜炎病毒疫苗的方法。根据本发明的实施例，所述方法包括讲本发明第一方面所述的核酸分子、第二方面所述的表达载体或第三方面所述的重组病毒导入受体细胞。根据本发明实施例的方法可包装携带所述核酸分子的病毒，以用于制备核酸疫苗。所述构建传染性腹膜炎病毒疫苗的方法安全、简便、高效。In its seventh aspect, the present invention provides a method for constructing a feline infectious peritonitis virus vaccine. According to embodiments of the present invention, the method comprises introducing the nucleic acid molecule described in the first aspect of the present invention, the expression vector described in the second aspect, or the recombinant virus described in the third aspect into a recipient cell. The method according to embodiments of the present invention can package a virus carrying the nucleic acid molecule for use in preparing a nucleic acid vaccine. This method for constructing an infectious peritonitis virus vaccine is safe, simple, and highly effective.

根据本发明的实施例，上述方法还包括下列技术特征的至少之一：According to an embodiment of the present invention, the above method further includes at least one of the following technical features:

根据本发明的实施例，所述导入受体细胞前，进一步包括使用包裹载体对所述核酸、表达载体或重组病毒进行包裹处理。根据本发明的实施例，使用包裹载体对所述核酸、表达载体或重组病毒进行包裹处理可以保护疫苗成分免受外界环境干扰影响其效价，而且包裹载体可以减少疫苗成分与外界环境的接触，从而减少疫苗成分受到污染的风险，提高疫苗的安全性。此外，部分包裹载体还具有增强疫苗成分的感染力的作用，从而使其能够更有效地激发免疫反应。According to an embodiment of the present invention, prior to introduction into the recipient cells, the method further includes encapsulating the nucleic acid, expression vector, or recombinant virus with an encapsulation vector. According to an embodiment of the present invention, encapsulating the nucleic acid, expression vector, or recombinant virus with an encapsulation vector can protect the vaccine components from external environmental interference that affects their potency. The encapsulation vector can also reduce the contact of the vaccine components with the external environment, thereby reducing the risk of contamination of the vaccine components and improving the safety of the vaccine. In addition, some encapsulation vectors also have the effect of enhancing the infectivity of the vaccine components, thereby enabling them to more effectively stimulate an immune response.

根据本发明的实施例，所述包裹载体选自脂质体、聚合物载体、病毒载体、纳米颗粒中的至少之一。According to an embodiment of the present invention, the encapsulation carrier is selected from at least one of liposomes, polymer carriers, viral carriers, and nanoparticles.

根据本发明的具体实施例，所述包括载体为纳米颗粒。According to a specific embodiment of the present invention, the carrier is a nanoparticle.

根据本发明的实施例，所述受体细胞为CRFK细胞、HEK293FT、HEK293T或BHK细胞。According to an embodiment of the present invention, the recipient cell is a CRFK cell, HEK293FT, HEK293T or BHK cell.

根据本发明的实施例，所述受体细胞为CRFK细胞。According to an embodiment of the present invention, the recipient cells are CRFK cells.

在本发明的第八方面，本发明提出了一种本发明第一方面所述核酸分子、第二方面所述表达载体、第三方面所述重组病毒、第四方面所述脂质体或第六方面所述重组细胞在制备药物或疫苗中的用途。根据本发明的实施例，所述药物或疫苗用于预防或治疗猫传染性腹膜炎病毒感染相关疾病。根据本发明的实施例，基于前述核酸分子、表达载体、重组病毒或重组细胞制备的药物或疫苗具有高安全性，可短时间内激活动物细胞介导的免疫反应。In an eighth aspect, the present invention provides a use of the nucleic acid molecule described in the first aspect, the expression vector described in the second aspect, the recombinant virus described in the third aspect, the liposome described in the fourth aspect, or the recombinant cell described in the sixth aspect in the preparation of a drug or vaccine. According to an embodiment of the present invention, the drug or vaccine is used to prevent or treat diseases associated with feline infectious peritonitis virus infection. According to an embodiment of the present invention, the drug or vaccine prepared based on the aforementioned nucleic acid molecule, expression vector, recombinant virus, or recombinant cell has high safety and can activate an animal cell-mediated immune response in a short period of time.

在本发明的第九方面，本发明提出了一种预防或治疗猫传染性腹膜炎病毒感染疾病的方法。根据本发明的实施例，该方法包括：向受试动物施用本发明第一方面所述的核酸分子、第二方面所述的表达载体、第三方面所述的重组病毒、第四方面所述的脂质体、第五方面所述的疫苗或第六方面所述的重组细胞。根据本发明的实施例，向受到FIPV感染的受试动物施用有效剂量的药物制剂、核酸分子、表达载体、重组病毒、脂质体、疫苗或重组细胞，能够显著改善受试动物的各项生理指标与生存率。此外，前述治疗方法对FIPV的各流行株均有良好的免疫效果。In the ninth aspect of the present invention, the present invention proposes a method for preventing or treating feline infectious peritonitis virus infection. According to an embodiment of the present invention, the method comprises: administering the nucleic acid molecule described in the first aspect of the present invention, the expression vector described in the second aspect, the recombinant virus described in the third aspect, the liposome described in the fourth aspect, the vaccine described in the fifth aspect, or the recombinant cell described in the sixth aspect to the test animal. According to an embodiment of the present invention, administering an effective dose of a pharmaceutical preparation, nucleic acid molecule, expression vector, recombinant virus, liposome, vaccine or recombinant cell to a test animal infected with FIPV can significantly improve the various physiological indicators and survival rate of the test animal. In addition, the aforementioned treatment methods have good immune effects on various epidemic strains of FIPV.

在本文中，术语“有效剂量”是指可对受试动物产生功能或活性的且可被受试动物所接受的量。 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.

本发明所述的核酸分子、表达载体、重组病毒、脂质体、疫苗或重组细胞的有效量可随给药的模式和受试动物感染FIPV的严重程度等而变化。优选的有效量的选择可以由本领域普通技术人员根据各种因素来确定(例如通过临床试验)。各种因素包括但不限于：所述的活性成分的药代动力学参数例如生物利用率、代谢、半衰期等；受试动物感染FIPV的严重程度、受试动物的体重、受试动物的免疫状况、给药的途径等。例如，由治疗状况的迫切要求，可每天给予若干次分开的剂量，或将剂量按比例地减少。The effective amount of the nucleic acid molecule, expression vector, recombinant virus, liposome, vaccine or recombinant cell of the present invention may vary depending on the mode of administration and the severity of FIPV infection in the test animal. The selection of the preferred effective amount can be determined by a person of ordinary skill in the art based on various factors (e.g., through clinical trials). Various factors include, but are not limited to: pharmacokinetic parameters of the active ingredient, such as bioavailability, metabolism, half-life, etc.; the severity of FIPV infection in the test animal, the weight of the test animal, the immune status of the test animal, the route of administration, etc. For example, depending on the urgency of the treatment situation, several divided doses may be administered daily, or the dose may be reduced proportionally.

根据本发明的实施例，上述方法还可以包括下列技术特征的至少之一：According to an embodiment of the present invention, the above method may further include at least one of the following technical features:

根据本发明的实施例，所述受试动物选自猫。According to an embodiment of the present invention, the test animal is selected from cats.

在本发明的第十二方面，本发明提出了一种第第一方面所述的核酸分子、第二方面所述的表达载体、第三方面所述的重组病毒、第四方面所述的脂质体、第五方面所述的疫苗或第六方面所述的重组细胞在预防或治疗猫传染性腹膜炎病毒感染疾病中的用途。根据本发明的实施例，向受到FIPV感染的受试动物施用有效剂量的核酸分子、表达载体、重组病毒、脂质体、疫苗或重组细胞，能够显著改善受试动物的各项生理指标与生存率。此外，前述治疗方法对FIPV的各流行株均有良好的免疫效果。In the twelfth aspect of the present invention, the present invention proposes a use of the nucleic acid molecule described in the first aspect, the expression vector described in the second aspect, the recombinant virus described in the third aspect, the liposome described in the fourth aspect, the vaccine described in the fifth aspect or the recombinant cell described in the sixth aspect in preventing or treating feline infectious peritonitis virus infection. According to an embodiment of the present invention, administering an effective dose of nucleic acid molecules, expression vectors, recombinant viruses, liposomes, vaccines or recombinant cells to a test animal infected with FIPV can significantly improve the various physiological indicators and survival rates of the test animals. In addition, the aforementioned treatment methods have good immune effects on various epidemic strains of FIPV.

本发明的附加方面和优点将在下面的描述中部分给出，部分将从下面的描述中变得明显，或通过本发明的实践了解到。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.

附图说明BRIEF DESCRIPTION OF THE DRAWINGS

本发明的上述和/或附加的方面和优点从结合下面附图对实施例的描述中将变得明显和容易理解，其中：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:

图1是根据本发明实施例1所述LNP包裹的目标mRNA表达检测结果。FIG1 is a result of detecting the expression of target mRNA encapsulated by LNP according to Example 1 of the present invention.

图2是根据本发明实施例2所述LNP包裹的目标mRNA免疫后攻毒的生存率变化。FIG2 shows the change in survival rate after immunization with the target mRNA packaged in LNP according to Example 2 of the present invention.

图3是根据本发明实施例3所述LNP包裹的目标mRNA免疫后多表位抗原多肽库刺激的ELISPOT检测中位数统计结果。FIG3 is a median statistical result of ELISPOT detection stimulated by a multi-epitope antigen polypeptide library after immunization with the target mRNA packaged by LNP according to Example 3 of the present invention.

图4是根据本发明实施例3所述LNP包裹的目标mRNA免疫后多表位抗原多肽刺激的ELISPOT检测统计结果。FIG4 is a statistical result of ELISPOT detection of multi-epitope antigen polypeptide stimulation after immunization with LNP-encapsulated target mRNA according to Example 3 of the present invention.

图5是根据本发明实施例4所述LNP包裹的目标mRNA免疫后攻毒的生存率变化。FIG5 shows the change in survival rate after immunization with the target mRNA packaged in LNP according to Example 4 of the present invention.

具体实施方式DETAILED DESCRIPTION

下面详细描述本发明的实施例，所述实施例的示例在附图中示出。下面通过参考附图描述的实施例是示例性的，旨在用于解释本发明，而不能理解为对本发明的限制。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.

此外，术语“第一”、“第二”仅用于描述目的，而不能理解为指示或暗示相对重要性或者隐含指明所指示的技术特征的数量。由此，限定有“第一”、“第二”的特征可以明示或者隐含地包括至少一个该特征。在本发明的描述中，“多个”的含义是至少两个，例如两个，三个等，除非另有明确具体的限定。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.

在本申请中，如无特别说明，术语“非致病性病毒载体”是指一类能够表达目标抗原并且不具有致病性的病毒，通常被用来制备疫苗。这些载体经过基因改造后，被加入了目标抗原的基因序列，进而被病毒载体表达和复制。将非致病性病毒载体作为疫苗载体的优势在于：能够诱导免疫系统产生强烈的免疫反应，从而刺激体内对目标抗原的免疫应答；非致病性病毒载体通常都被设计成无法复制，因此不会在体内繁殖，也不会引起疾病；与其他传统的疫苗制备方法相比，非致病性病毒载体制备的疫苗在生产和保存方面更为便捷和安全，且具有更好的稳定性和纯度。In this application, unless otherwise specified, the term "non-pathogenic viral vector" refers to a class of viruses that can express target antigens and are non-pathogenic, and are usually used to prepare vaccines. These vectors are genetically modified and the gene sequence of the target antigen is added, which is then expressed and replicated by the viral vector. The advantages of using non-pathogenic viral vectors as vaccine vectors are that they can induce the immune system to produce a strong immune response, thereby stimulating the body's immune response to the target antigen; non-pathogenic viral vectors are usually designed to be unable to replicate, so they will not reproduce in the body and will not cause disease; compared with other traditional vaccine preparation methods, vaccines prepared with non-pathogenic viral vectors are more convenient and safer in production and storage, and have better stability and purity.

有益效果Beneficial effects

本发明所述用于预防猫传染性腹膜炎的RNA疫苗，通过构建编码FIPV病毒多表位抗原的载体，而后通过脂质纳米颗粒(LNP)制备出用于预防FIPV病毒的RNA疫苗，该疫苗免疫猫后能够产生较强的免疫反应。本发明所述疫苗以含有FIPV病毒的N蛋白的N端、C端、NSP12蛋白序列以及S蛋白HR2-4和HR2-11序列作为的构建核酸分子作为主要成分，所述核酸分子表达的RNA疫苗具有制备工艺简单、安全性高、无毒副作用、可工业化生产等优点；使用极小剂量能达到足够的保护效果，在安全性、有效性方面优于现有的治疗手段。 The RNA vaccine for preventing feline infectious peritonitis described in the present invention is prepared by constructing a vector encoding multiple epitope antigens of the FIPV virus, and then preparing an RNA vaccine for preventing the FIPV virus through lipid nanoparticles (LNP). The vaccine can produce a strong immune response after immunizing cats. The vaccine described in the present invention uses the N-terminus, C-terminus, NSP12 protein sequence of the N protein of the FIPV virus and the HR2-4 and HR2-11 sequences of the S protein as the main components of the constructed nucleic acid molecule. The RNA vaccine expressed by the nucleic acid molecule has the advantages of simple preparation process, high safety, no toxic side effects, and industrial production; sufficient protection effect can be achieved using a very small dose, and it is superior to existing treatment methods in terms of safety and effectiveness.

本申请涉及的序列如表3所示：








The sequences involved in this application are shown in Table 3:








表4：本发明实施例中涉及序列描述
Table 4: Description of sequences involved in the examples of the present invention

表5：本发明实施例中涉及序列组成


注：L表示linker；-表示无。Table 5: Sequence composition involved in the examples of the present invention


Note: L stands for linker; - stands for none.

表6：本发明实施例中涉及序列组成

注：L表示linker；-表示无。Table 6: Sequence composition involved in the examples of the present invention

Note: L stands for linker; - stands for none.

需要说明的是，表5或表6中名称对应产品的序列由“SEQ ID NO:”中对应的序列以5’到3’方向依此连接而成，以表6中d3为例，MHC-I sp-HBHA-L-PADRE-L-NTD-L-CTD-L-NSP12-L-HR2_4-L-HR2_11-L-MITD的序列由SEQ ID NO:38所示的MHC-I sp序列、SEQ ID NO:44所示的HBHA序列、SEQ ID NO:53所示的L(linker)序列、SEQ ID NO:47所示的PADRE序列、SEQ ID NO:56所示的L(linker)序列、SEQ ID NO:19所示的NTD序列、SEQ ID NO:59所示的L(linker)序列、SEQ ID NO:23所示的CTD序列、SEQ ID NO:62所示的L(linker)序列、SEQ ID NO:27所示的NSP12序列、SEQ ID NO:65所示的L(linker)序列、SEQ ID NO:31所示 的HR2_4序列、SEQ ID NO:67所示的L(linker)序列、SEQ ID NO:35所示的HR2_11序列、SEQ ID NO:70所示的L(linker)序列、SEQ ID NO:41所示的MITD序列连接而成。It should be noted that the sequences of the products corresponding to the names in Table 5 or Table 6 are formed by connecting the corresponding sequences in "SEQ ID NO:" in the 5' to 3' direction. Taking d3 in Table 6 as an example, the sequence of MHC-I sp-HBHA-L-PADRE-L-NTD-L-CTD-L-NSP12-L-HR2_4-L-HR2_11-L-MITD is composed of the MHC-I sp sequence shown in SEQ ID NO:38, the HBHA sequence shown in SEQ ID NO:44, the L (linker) sequence shown in SEQ ID NO:53, the PADRE sequence shown in SEQ ID NO:47, the L (linker) sequence shown in SEQ ID NO:56, the NTD sequence shown in SEQ ID NO:19, the L (linker) sequence shown in SEQ ID NO:59, the CTD sequence shown in SEQ ID NO:23, the L (linker) sequence shown in SEQ ID NO:62, the NSP12 sequence shown in SEQ ID NO:27, the L (linker) sequence shown in SEQ ID NO:65, Shown in NO:31 The HR2_4 sequence, the L (linker) sequence shown in SEQ ID NO: 67, the HR2_11 sequence shown in SEQ ID NO: 35, the L (linker) sequence shown in SEQ ID NO: 70, and the MITD sequence shown in SEQ ID NO: 41 are connected.

其中，SEQ ID NO:38所示序列的3’端与SEQ ID NO:44所示序列的5’端相连，所述SEQ ID NO:44所示的基因序列的3’端与SEQ ID NO:53所示的序列的5’端相连，所述SEQ ID NO:53所示的基因序列的3’端与SEQ ID NO:47所示的序列的5’端相连，所述SEQ ID NO:47所示的基因序列的3’端与SEQ ID NO:56所示的序列的5’端相连，所述SEQ ID NO:56所示序列的3’端与SEQ ID NO:19所示序列的5’端相连，所述SEQ ID NO:19所示的基因序列的3’端与SEQ ID NO:59所示的序列的5’端相连，所述SEQ ID NO:59所示的基因序列的3’端与SEQ ID NO:23所示的序列的5’端相连，所述SEQ ID NO:23所示的基因序列的3’端与SEQ ID NO:62所示的序列的5’端相连，所述SEQ ID NO:62所示序列的3’端与SEQ ID NO:27所示序列的5’端相连，所述SEQ ID NO:27所示的基因序列的3’端与SEQ ID NO:65所示的序列的5’端相连，所述SEQ ID NO:65所示的基因序列的3’端与SEQ ID NO:31所示的序列的5’端相连，所述SEQ ID NO:31所示的基因序列的3’端与SEQ ID NO:67所示的序列的5’端相连，所述SEQ ID NO:67所示序列的3’端与SEQ ID NO:35所示序列的5’端相连，所述SEQ ID NO:35所示的基因序列的3’端与SEQ ID NO:70所示的序列的5’端相连，所述SEQ ID NO:70所示的基因序列的3’端与SEQ ID NO:41所示的序列的5’端相连。在本申请中，所述linker不受特别限制，可以选择上述linker，也可根据实验需求选择其他的柔性肽或刚性肽氨基酸序列进行连接。wherein, the 3’ end of the sequence shown in SEQ ID NO:38 is linked to the 5’ end of the sequence shown in SEQ ID NO:44, the 3’ end of the gene sequence shown in SEQ ID NO:44 is linked to the 5’ end of the sequence shown in SEQ ID NO:53, the 3’ end of the gene sequence shown in SEQ ID NO:53 is linked to the 5’ end of the sequence shown in SEQ ID NO:47, the 3’ end of the gene sequence shown in SEQ ID NO:47 is linked to the 5’ end of the sequence shown in SEQ ID NO:56, the 3’ end of the sequence shown in SEQ ID NO:56 is linked to the 5’ end of the sequence shown in SEQ ID NO:19, the 3’ end of the gene sequence shown in SEQ ID NO:19 is linked to the 5’ end of the sequence shown in SEQ ID NO:59, the 3’ end of the gene sequence shown in SEQ ID NO:59 is linked to the 5’ end of the sequence shown in SEQ ID NO:23, and the 3’ end of the gene sequence shown in SEQ ID NO:23 is linked to the 5’ end of the sequence shown in SEQ ID NO: The 3’ end of the gene sequence shown in SEQ ID NO: 27 is connected to the 5’ end of the sequence shown in SEQ ID NO: 65, the 3’ end of the gene sequence shown in SEQ ID NO: 65 is connected to the 5’ end of the sequence shown in SEQ ID NO: 31, and the 5’ end of the gene sequence shown in SEQ ID NO: 62 is connected to the 5’ end of the sequence shown in SEQ ID NO: 27. The 3’ end of the gene sequence shown in SEQ ID NO: 31 is connected to the 5’ end of the sequence shown in SEQ ID NO: 67, the 3’ end of the sequence shown in SEQ ID NO: 67 is connected to the 5’ end of the sequence shown in SEQ ID NO: 35, the 3’ end of the gene sequence shown in SEQ ID NO: 35 is connected to the 5’ end of the sequence shown in SEQ ID NO: 70, and the 3’ end of the gene sequence shown in SEQ ID NO: 70 is connected to the 5’ end of the sequence shown in SEQ ID NO: 41. In the present application, the linker is not particularly limited, and the above-mentioned linker can be selected, or other flexible peptide or rigid peptide amino acid sequences can be selected for connection according to experimental requirements.

下面参考具体实施例，对本发明进行描述，需要说明的是，这些实施例仅仅是描述性的，而不以任何方式限制本发明。实施例中未注明具体技术或条件的，按照本领域内的文献所描述的技术或条件或者按照产品说明书进行。所用试剂或仪器未注明生产厂商者，均为可以通过市购获得的常规产品。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.

实施例1：目标序列选择Example 1: Target sequence selection

根据本发明的实施例，体外合成mRNA序列后使用脂质纳米颗粒(LNP)包裹并在CRFK(猫肾)细胞及猫DC(树突状)细胞中进行表达，选用表达量较高的序列作为最终目标序列，进行疫苗的制备。According to an embodiment of the present invention, the mRNA sequence is synthesized in vitro and then encapsulated using lipid nanoparticles (LNP) and expressed in CRFK (cat kidney) cells and cat DC (dendritic cells). The sequence with the higher expression level is selected as the final target sequence for vaccine preparation.

具体步骤如下：The specific steps are as follows:

根据本发明的实施例，体外合成RNA序列并在CRFK(猫肾)细胞中进行表达，选用表达量较高的序列作为最终目标序列，进行疫苗的制备。具体步骤如下：According to an embodiment of the present invention, RNA sequences were synthesized in vitro and expressed in CRFK (cat kidney) cells, and the sequences with the highest expression levels were selected as the final target sequences for vaccine preparation. The specific steps are as follows:

1)使用猫通用密码子库与猫肠系膜淋巴结密码子库对多表位抗原序列进行密码子优化，每种抗原优化3条；1) Codon optimization of multi-epitope antigen sequences was performed using the cat universal codon library and the cat mesenteric lymph node codon library, with three optimizations for each antigen;

2)序列合成(来源：通用生物)后构建载体，载体带有猫源的5'HBB-UTR(SEQ ID NO:71)、3'HBA-UTR(SEQ ID NO:72)序列及polyA，将目标序列构建至5'UTR与3'UTR之间；2) After sequence synthesis (Source: Universal Bio), construct a vector containing the feline 5'HBB-UTR (SEQ ID NO: 71), 3'HBA-UTR (SEQ ID NO: 72) sequences, and polyA, and insert the target sequence between the 5'UTR and 3'UTR.

3)体外转录成mRNA后，使用LNP包裹，并转染至CRFK细胞及猫的DC细胞，筛选能在两种猫源细胞系表达量较高的序列。3) After in vitro transcription into mRNA, it was packaged using LNPs and transfected into CRFK cells and cat DC cells to screen for sequences that could be expressed at high levels in both cat-derived cell lines.

根据本发明的实施例，制备脂质纳米颗粒(LNP)。具体步骤如下所示：According to an embodiment of the present invention, lipid nanoparticles (LNP) are prepared. The specific steps are as follows:

1)配制脂质溶液：脂质体体系平均分子质量约为620.62，配置12mM脂质溶液：称取SM-102：42.61mg，PEG-DMG：4.52mg，DSPC：9.48mg，Chol：17.86mg，溶于10mL无水乙醇后用0.22μm滤膜过滤；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.

2)使用柠檬酸缓冲液(pH为4)稀释目标mRNA，混合均匀后，使用快速纳米药物制备系统(铭汰)，流速预条件设置为1：3(有机相X体积(含阳离子脂质)：水相Y体积(含核酸)＝1：3)，制备mRNA疫苗脂质体溶液，立即置于30倍体积的PBS中，使用截留量100K的15ml超滤管浓缩，3000rpm 20min。最后使用600mM的蔗糖溶液(PBS配制，0.22μm滤膜过滤)保存等体积稀释。将样品置于-20℃保存备用；2) Dilute the target mRNA with citric acid buffer (pH 4), mix well, and use a rapid nanodrug preparation system (Mingtai) with a flow rate precondition of 1:3 (organic phase X volume (containing cationic lipids): aqueous phase Y volume (containing nucleic acids) = 1:3) to prepare an mRNA vaccine liposome solution. Immediately place it in 30 volumes of PBS and concentrate using a 15 ml ultrafiltration tube with a cutoff of 100K at 3000 rpm for 20 minutes. Finally, use 600 mM sucrose solution (prepared in PBS, filtered with a 0.22 μm membrane) to store the equal volume dilution. Store the sample at -20°C until use.

结果如图1所示，成功筛选出能在两种细胞系高表达的目标序列B1、C1、D1(序列描述及组成见表4、5)。The results are shown in FIG1 , and target sequences B1, C1, and D1 that can be highly expressed in both cell lines were successfully screened (sequence descriptions and compositions are shown in Tables 4 and 5 ).

实施例2：疫苗效价验证 Example 2: Vaccine potency verification

通过评估mRNA疫苗免疫后受试动物攻毒后体温、体重等生理指标及生存率变化，来评估疫苗的有效性。LNP制备方法同实施例1。The effectiveness of the vaccine was evaluated by assessing changes in physiological indicators such as body temperature, body weight, and survival rate of the test animals after mRNA vaccine immunization. The LNP preparation method was the same as in Example 1.

根据本发明的实施例，将D1构建至猫源5'HBB-UTR(SEQ ID NO:71)与3'HBA-UTR(SEQ ID NO:72)之间，体外转录为mRNA，并用LNP包裹，序列描述及组成见表4、5。According to an embodiment of the present invention, D1 was constructed between the cat-derived 5'HBB-UTR (SEQ ID NO: 71) and 3'HBA-UTR (SEQ ID NO: 72), transcribed into mRNA in vitro, and packaged with LNP. The sequence description and composition are shown in Tables 4 and 5.

根据本发明的实施例，通过体格检查和实验室检查筛选符合试验标准的受试动物。体格检查项目包括：体温、体重；筛选项目包括：PCR检测、N与S结合抗体、中和抗体的测试。具体实验步骤如下：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:

1)体格检查：免疫前7天，每天测量小猫的体温与体重，正常体温在38.5℃左右，1周岁的宠物猫体重在3kg左右。1) Physical examination: Measure the kitten's body temperature and weight every day for 7 days before immunization. The normal body temperature is around 38.5℃, and the weight of a one-year-old pet cat is around 3kg.

2)筛选FIPV阴性猫：通过PCR方法，检测FIPV的7ab基因；ELISA方法，分别以N、S蛋白作为抗原，检测猫血清中的结合抗体；利用假病毒的中和实验，检测FIPV的中和抗体。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.

根据本发明的实施例，使用LNP包裹的目标mRNA对经过筛选的受试动物进行免疫。其中，免疫程序如下所示：According to an embodiment of the present invention, the screened test animals were immunized with the target mRNA encapsulated in LNPs. The immunization procedure is as follows:

D0一免，D21二免，二免后D28攻毒；攻毒毒株为QS-1146；5只小猫/组。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.

将LNP包裹的目标mRNA转染进CRFK细胞，24h后收集，进行免疫印记测试，检测蛋白的表达情况。结果如图2所示，可以正常表达。各组LNP包裹的目标mRNA免疫攻毒后，PBS组5只小猫都先后出现了发烧、体重下降的症状，解剖发现属于典型的猫传腹病变；多表位抗原mRNA的免疫组的小猫生理指标明显好转，生存率如图2所示显著上升，21天后才出现1只死亡，至30天时生存率维持在80％。LNP-encapsulated target mRNA was transfected into CRFK cells, harvested 24 hours later, and immunoblotting was performed to detect protein expression. The results, as shown in Figure 2, demonstrated normal expression. Following challenge with LNP-encapsulated target mRNA in each group, all five kittens in the PBS group developed fever and weight loss, and autopsies revealed typical feline infectious peritonitis. Physiological indicators of the kittens in the group immunized with multi-epitope antigen mRNA significantly improved, with a significant increase in survival rate, as shown in Figure 2. Only one kitten died after 21 days, and the survival rate remained at 80% by day 30.

以上结果表明，表达多表位抗原的mRNA疫苗对于FIPV表现出良好的免疫效果。The above results show that the mRNA vaccine expressing multiple epitope antigens exhibits good immune effect against FIPV.

实施例3：表位有效性验证Example 3: Epitope Validation

通过检测多表位抗原mRNA疫苗免疫后受试动物PBMC对于各表位的刺激响应，来评估各抗原表位的有效性。LNP制备方法、受试动物筛选方法同实施例1、2。The effectiveness of each antigen epitope was evaluated by detecting the stimulation response of the PBMC of the test animals to each epitope after immunization with the multi-epitope antigen mRNA vaccine. The LNP preparation method and the test animal screening method were the same as those in Examples 1 and 2.

根据本发明的实施例，将D1构建至猫源5'HBB-UTR(SEQ ID NO:71)与3'HBA-UTR(SEQ ID NO:72)之间，体外转录为mRNA，并用LNP包裹，序列描述及组成见表4、5。According to an embodiment of the present invention, D1 was constructed between the cat-derived 5'HBB-UTR (SEQ ID NO: 71) and 3'HBA-UTR (SEQ ID NO: 72), transcribed into mRNA in vitro, and packaged with LNP. The sequence description and composition are shown in Tables 4 and 5.

根据本发明的实施例，合成各抗原表位多肽库，具体实施步骤如下：According to an embodiment of the present invention, the antigen epitope polypeptide library is synthesized, and the specific implementation steps are as follows:

将各抗原表位多肽序列以15氨基酸为窗口大小、7氨基酸为步长进行滑窗截取，并合成各窗口多肽与全长多肽(义翘神州)。Each epitope peptide sequence was window-cut with a window size of 15 amino acids and a step size of 7 amino acids, and each window peptide and the full-length peptide were synthesized (Sino Biological).

根据本发明的实施例，免疫后提取受试动物PBMC，进行各表位刺激，并检测响应。具体实施步骤如下：According to an embodiment of the present invention, PBMC of the test animal is extracted after immunization, stimulated with each epitope, and the response is detected. The specific implementation steps are as follows:

从受试动物血清中提取PBMC，加入活化的IFN-γ包被板，并分别加入阴性刺激物PBS、阳性刺激物PMA与各表位多肽孵育16-24小时，使用ELISPOT检测试剂盒(达科为)检测计数并统计分析。PBMCs were extracted from the serum of the test animals and added to activated IFN-γ coated plates. Negative stimulators PBS and positive stimulators PMA were added and incubated with each epitope peptide for 16-24 hours. The cells were counted and statistically analyzed using an ELISPOT detection kit (Dakoway).

分别使用PBS与包裹多表位抗原mRNA疫苗的LNP免疫受试动物后提取受试动物PBMC并分别使用各抗原表位多肽库进行刺激，检测对于各抗原表位多肽库整体的刺激响应，结果如图3所示。HR2-4与HR2-11长度较短，多肽库规模相应较小，免疫受试动物PBMC刺激后仍可产生可检出的响应；而对于多肽库规模更大的抗原表位NTD、CTD与NSP12，相较于PBS免疫对照组，多表位抗原mRNA疫苗免疫的受试动物PBMC对于各抗原表位多肽库刺激均有显著响应；使用整个多肽库对免疫受试动物PBMC进行刺激可以获得进一步强于各抗原表位多肽库刺激时的响应。After immunizing the test animals with PBS and LNPs containing multi-epitope antigen mRNA vaccines, PBMCs were extracted from the test animals and stimulated with each epitope peptide library. The stimulation response to the entire epitope peptide library was tested, and the results are shown in Figure 3. HR2-4 and HR2-11 are shorter in length and have a correspondingly smaller peptide library size. However, they still produced detectable responses after stimulation with the PBMCs of the immunized test animals. For the epitopes NTD, CTD, and NSP12, which have larger peptide libraries, the PBMCs of the test animals immunized with the multi-epitope antigen mRNA vaccine showed significant responses to stimulation with each epitope peptide library compared to the PBS immunization control group. Using the entire peptide library to stimulate the PBMCs of the immunized test animals can obtain a response that is even stronger than when stimulated with each epitope peptide library.

分别使用PBS与包裹多表位抗原mRNA疫苗的LNP免疫受试动物后提取受试动物PBMC并分别使用各抗原表位多肽库中单一多肽进行刺激，检测对于各抗原表位多肽的刺激响应，统计各抗原表位多肽库中多肽的刺激响应的整体分布，结果如图4所示。与使用各抗原表位多肽库整体进行刺激时相一致，对于各抗原表位多肽库中多肽刺激响应的整体分布，HR2-4与HR2-11多肽库中多肽刺激响应分布较低而NTD、CTD与NSP12多肽库中多肽刺激响应分布较高，但较PBS免疫对照组均有较强响应，而使用全长多肽对免疫受试动物PBMC进行刺激可以获得显著强于各抗原多肽库中多肽的响应。After immunizing the test animals with PBS and LNPs encapsulating multi-epitope antigen mRNA vaccines, PBMCs of the test animals were extracted and stimulated with a single peptide from each antigen epitope peptide library. The stimulation response to each antigen epitope peptide was detected, and the overall distribution of the stimulation response of the peptides in each antigen epitope peptide library was statistically analyzed. The results are shown in Figure 4. Consistent with the stimulation using the entire antigen epitope peptide library, the overall distribution of the stimulation response of the peptides in each antigen epitope peptide library showed a lower distribution of the stimulation response of the peptides in the HR2-4 and HR2-11 peptide libraries, while the distribution of the stimulation response of the peptides in the NTD, CTD, and NSP12 peptide libraries was higher. However, they all had stronger responses than the PBS immunized control group. Stimulating the PBMCs of the immunized test animals with the full-length peptides can obtain a significantly stronger response than that of the peptides in each antigen peptide library.

以上结果表明，多表位抗原mRNA疫苗的各抗原表位均能有效激活免疫反应。 The above results show that each antigen epitope of the multi-epitope antigen mRNA vaccine can effectively activate the immune response.

实施例4：不同流行株的多表位抗原mRNA疫苗效价验证Example 4: Validation of the potency of multi-epitope antigen mRNA vaccines against different epidemic strains

本发明实施例评估多表位抗原mRNA疫苗对于不同FIPV流行株的有效性。LNP制备方法、受试动物筛选方法、免疫程序与评估方法同实施例1、2。攻毒毒株为HF1902与SH2211。This example evaluated the effectiveness of a multi-epitope antigen mRNA vaccine against different circulating FIPV strains. The LNP preparation method, test animal screening method, immunization schedule, and evaluation method were the same as in Examples 1 and 2. The challenge strains were HF1902 and SH2211.

根据本发明的实施例，将D1构建至猫源5'HBB-UTR(SEQ ID NO:71)与3'HBA-UTR(SEQ ID NO:72)之间，体外转录为mRNA，并用LNP包裹，序列描述及组成见表4、5。According to an embodiment of the present invention, D1 was constructed between the cat-derived 5'HBB-UTR (SEQ ID NO: 71) and 3'HBA-UTR (SEQ ID NO: 72), transcribed into mRNA in vitro, and packaged with LNP. The sequence description and composition are shown in Tables 4 and 5.

免疫攻毒后，多表位抗原mRNA疫苗对于不同FIPV流行株均能有效改善受试动物攻毒后生理指标与生存率，生存率如图5所示，两种流行株攻毒后20天前后对照组生存率均下降至50％以下，而实验组至攻毒30天后生存率仍维持100％。After immunization, the multi-epitope antigen mRNA vaccine can effectively improve the physiological indicators and survival rate of the test animals after challenge with different FIPV epidemic strains. The survival rate is shown in Figure 5. The survival rate of the control group dropped below 50% around 20 days after challenge with the two epidemic strains, while the survival rate of the experimental group remained at 100% until 30 days after challenge.

以上结果表明，多表位抗原mRNA疫苗对于不同FIPV流行株均表现出良好的免疫效果。The above results show that the multi-epitope antigen mRNA vaccine exhibits good immune effects against different FIPV strains.

在本说明书的描述中，参考术语“一个实施例”、“一些实施例”、“示例”、“具体示例”、或“一些示例”等的描述意指结合该实施例或示例描述的具体特征、结构、材料或者特点包含于本发明的至少一个实施例或示例中。在本说明书中，对上述术语的示意性表述不必须针对的是相同的实施例或示例。而且，描述的具体特征、结构、材料或者特点可以在任一个或多个实施例或示例中以合适的方式结合。此外，在不相互矛盾的情况下，本领域的技术人员可以将本说明书中描述的不同实施例或示例以及不同实施例或示例的特征进行结合和组合。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.

尽管上面已经示出和描述了本发明的实施例，可以理解的是，上述实施例是示例性的，不能理解为对本发明的限制，本领域的普通技术人员在本发明的范围内可以对上述实施例进行变化、修改、置换和变型。 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 (25)

一种分离的核酸分子，其特征在于，包括：第一核酸片段、第二核酸片段、第三核酸片段、第四核酸片段和第五核酸片段中的至少之一；An isolated nucleic acid molecule, characterized by comprising: at least one of a first nucleic acid fragment, a second nucleic acid fragment, a third nucleic acid fragment, a fourth nucleic acid fragment, and a fifth nucleic acid fragment; 其中，in, 所述第一核酸片段来源于猫传染性腹膜炎病毒QS毒株的N蛋白的N端(NTD)；The first nucleic acid fragment is derived from the N-terminus (NTD) of the N protein of the QS strain of feline infectious peritonitis virus; 所述第二核酸片段来源于猫传染性腹膜炎病毒QS毒株的N蛋白的C端(CTD)；The second nucleic acid fragment is derived from the C-terminus (CTD) of the N protein of the QS strain of feline infectious peritonitis virus; 所述第三核酸片段编码猫传染性腹膜炎病毒QS毒株的NSP12蛋白；The third nucleic acid segment encodes the NSP12 protein of the QS strain of feline infectious peritonitis virus; 所述第四核酸片段来源于猫传染性腹膜炎病毒79-1146毒株S蛋白的表位HR2_4；The fourth nucleic acid fragment is derived from epitope HR2_4 of the S protein of feline infectious peritonitis virus 79-1146 strain; 所述第五核酸片段来源于猫传染性腹膜炎病毒79-1146毒株S蛋白的表位HR2_11；The fifth nucleic acid fragment is derived from the epitope HR2_11 of the S protein of the feline infectious peritonitis virus 79-1146 strain; 所述核酸分子为RNA。The nucleic acid molecule is RNA. 根据权利要求1所述的核酸分子，其特征在于，所述核酸片段相连或不相连；The nucleic acid molecule according to claim 1, wherein the nucleic acid fragments are connected or not connected; 任选地，所述核酸片段通过linker相连；Optionally, the nucleic acid fragments are connected via a linker; 任选地，所述核酸片段中的任意一个至少包括15个氨基酸；Optionally, any one of the nucleic acid fragments comprises at least 15 amino acids; 任选地，所述NTD蛋白具有与SEQ ID NO:1至少91％同源性的氨基酸序列；Optionally, the NTD protein has an amino acid sequence that is at least 91% homologous to SEQ ID NO: 1; 任选地，所述NTD蛋白具有SEQ ID NO:1所示的氨基酸序列；Optionally, the NTD protein has the amino acid sequence shown in SEQ ID NO: 1; 任选地，所述CTD蛋白具有与SEQ ID NO:2至少92％同源性的氨基酸序列；Optionally, the CTD protein has an amino acid sequence that is at least 92% homologous to SEQ ID NO: 2; 任选地，所述CTD蛋白具有SEQ ID NO:2所示的氨基酸序列；Optionally, the CTD protein has the amino acid sequence shown in SEQ ID NO: 2; 任选地，所述NSP12蛋白具有与SEQ ID NO:3至少97％同源性的氨基酸序列；Optionally, the NSP12 protein has an amino acid sequence that is at least 97% homologous to SEQ ID NO: 3; 任选地，所述NSP12蛋白具有SEQ ID NO:3所示的氨基酸序列；Optionally, the NSP12 protein has the amino acid sequence shown in SEQ ID NO: 3; 任选地，所述HR2_4蛋白具有与SEQ ID NO:4至少50％同源性的氨基酸序列；Optionally, the HR2_4 protein has an amino acid sequence that is at least 50% homologous to SEQ ID NO:4; 任选地，所述HR2_4蛋白具有SEQ ID NO:4所示的氨基酸序列；Optionally, the HR2_4 protein has the amino acid sequence shown in SEQ ID NO: 4; 任选地，所述HR2_11蛋白具有与SEQ ID NO:5至少95％同源性的氨基酸序列；Optionally, the HR2_11 protein has an amino acid sequence that is at least 95% homologous to SEQ ID NO:5; 任选地，所述HR2_11蛋白具有SEQ ID NO:5所示的氨基酸序列；Optionally, the HR2_11 protein has the amino acid sequence shown in SEQ ID NO: 5; 任选地，所述第一核酸片段具有与SEQ ID NO:16～19任一序列至少71％同一性的核苷酸序列；Optionally, the first nucleic acid fragment has a nucleotide sequence that is at least 71% identical to any one of SEQ ID NOs: 16 to 19; 任选地，所述第一核酸片段具有SEQ ID NO:16～19所示的核苷酸序列；Optionally, the first nucleic acid fragment has a nucleotide sequence shown in SEQ ID NO: 16 to 19; 任选地，所述第二核酸片段具有与SEQ ID NO:20～23任一序列至少72％同一性的核苷酸序列；Optionally, the second nucleic acid fragment has a nucleotide sequence that is at least 72% identical to any one of SEQ ID NOs: 20 to 23; 任选地，所述第二核酸片段具有SEQ ID NO:20～23所示的核苷酸序列；Optionally, the second nucleic acid fragment has a nucleotide sequence shown in SEQ ID NO: 20 to 23; 任选地，所述第三核酸片段具有与SEQ ID NO:24～27任一序列至少71％同一性的核苷酸序列；Optionally, the third nucleic acid fragment has a nucleotide sequence that is at least 71% identical to any one of SEQ ID NOs: 24 to 27; 任选地，所述第三核酸片段具有SEQ ID NO:24～27所示的核苷酸序列；Optionally, the third nucleic acid fragment has a nucleotide sequence shown in SEQ ID NO: 24-27; 任选地，所述第四核酸片段具有与SEQ ID NO:28～31任一序列至少50％同一性的核苷酸序列；Optionally, the fourth nucleic acid fragment has a nucleotide sequence that is at least 50% identical to any one of SEQ ID NOs: 28 to 31; 任选地，所述第四核酸片段具有SEQ ID NO:28～31所示的核苷酸序列；Optionally, the fourth nucleic acid fragment has a nucleotide sequence shown in SEQ ID NO: 28 to 31; 任选地，所述第五核酸片段具有与SEQ ID NO:32～35任一序列至少73％同一性的核苷酸序列；Optionally, the fifth nucleic acid fragment has a nucleotide sequence that is at least 73% identical to any one of SEQ ID NOs: 32 to 35; 任选地，所述第五核酸片段具有SEQ ID NO:32～35所示的核苷酸序列。Optionally, the fifth nucleic acid fragment has a nucleotide sequence shown in SEQ ID NO: 32 to 35. 根据权利要求1所述的核酸分子，其特征在于，进一步包括第六核酸片段，所述第六核酸片段编码MHC-Ⅰ的信号肽序列；The nucleic acid molecule according to claim 1, further comprising a sixth nucleic acid segment encoding a signal peptide sequence of MHC-I; 任选地，所述MHC-Ⅰ的信号肽序列不包含跨膜区；Optionally, the signal peptide sequence of MHC-I does not contain a transmembrane region; 任选地，所述MHC-Ⅰ的信号肽序列具有如SEQ ID NO:6所示的氨基酸序列；Optionally, the MHC-I signal peptide sequence has the amino acid sequence shown in SEQ ID NO: 6; 任选地，所述第六核酸片段具有SEQ ID NO:36～38所示的核苷酸序列；Optionally, the sixth nucleic acid fragment has a nucleotide sequence shown in SEQ ID NO: 36-38; 任选地，所述第六核酸片段放置于所述核酸分子的5’端。Optionally, the sixth nucleic acid fragment is placed at the 5' end of the nucleic acid molecule. 根据权利要求1所述的核酸分子，其特征在于，进一步包括第七核酸片段，所述第七核酸片段编码MITD序列；The nucleic acid molecule according to claim 1, further comprising a seventh nucleic acid segment encoding a MITD sequence; 任选地，所述MITD序列包含跨膜区；Optionally, the MITD sequence comprises a transmembrane region; 任选地，所述MITD序列具有SEQ ID NO:7所示的氨基酸序列；Optionally, the MITD sequence has the amino acid sequence shown in SEQ ID NO: 7; 任选地，所述第七核酸片段具有SEQ ID NO:39～41所示的核苷酸序列；Optionally, the seventh nucleic acid fragment has a nucleotide sequence shown in SEQ ID NO: 39-41; 任选地，所述第七核酸片段放置于所述核酸分子的3’端。 Optionally, the seventh nucleic acid fragment is placed at the 3' end of the nucleic acid molecule. 根据权利要求1所述的核酸分子，其特征在于，进一步包括第八核酸片段，所述第八核酸片段编码HBHA佐剂序列；The nucleic acid molecule according to claim 1, further comprising an eighth nucleic acid segment encoding an HBHA adjuvant sequence; 任选地，所述HBHA序列具有SEQ ID NO:8所示的氨基酸序列；Optionally, the HBHA sequence has the amino acid sequence shown in SEQ ID NO: 8; 任选地，所述第八核酸片段具有SEQ ID NO:42～44所示的核苷酸序列。Optionally, the eighth nucleic acid fragment has a nucleotide sequence shown in SEQ ID NO: 42 to 44. 根据权利要求1所述的核酸分子，其特征在于，进一步包括第九核酸片段，所述第九核酸片段编码PADRE序列。The nucleic acid molecule according to claim 1 is characterized in that it further comprises a ninth nucleic acid segment, wherein the ninth nucleic acid segment encodes a PADRE sequence. 根据权利要求6所述的核酸分子，其特征在于，所述PADRE序列具有SEQ ID NO:9所示的氨基酸序列。The nucleic acid molecule according to claim 6 is characterized in that the PADRE sequence has the amino acid sequence shown in SEQ ID NO:9. 根据权利要求6所述的核酸分子，其特征在于，所述第九核酸片段具有SEQ ID NO:45～47所示的核苷酸序列。The nucleic acid molecule according to claim 6 is characterized in that the ninth nucleic acid fragment has a nucleotide sequence shown in SEQ ID NO: 45 to 47. 根据权利要求1所述的核酸分子，其特征在于，所述核酸分子进一步包括刚性或柔性的连接序列；The nucleic acid molecule according to claim 1, characterized in that the nucleic acid molecule further comprises a rigid or flexible linker sequence; 任选地，所述连接序列具有SEQ ID NO:11～15所示的氨基酸序列；Optionally, the linker sequence has an amino acid sequence shown in SEQ ID NO: 11 to 15; 任选地，所述连接序列具有SEQ ID NO:51～70所示的核苷酸序列。Optionally, the connecting sequence has a nucleotide sequence shown in SEQ ID NO: 51 to 70. 根据权利要求1所述的核酸分子，其特征在于，所述核酸分子为线性。The nucleic acid molecule according to claim 1, wherein the nucleic acid molecule is linear. 一种表达载体，其特征在于，携带权利要求1～10任一项所述的核酸分子。An expression vector, characterized in that it carries the nucleic acid molecule according to any one of claims 1 to 10. 根据权利要求11所述的表达载体，其特征在于，所述表达载体是非致病性病毒载体。The expression vector according to claim 11, characterized in that the expression vector is a non-pathogenic viral vector. 根据权利要求12所述的表达载体，其特征在于，所述非致病性病毒选自反转录病毒、慢病毒、腺病毒和腺病毒相关病毒中的至少之一。The expression vector according to claim 12, characterized in that the non-pathogenic virus is selected from at least one of a retrovirus, a lentivirus, an adenovirus, and an adeno-associated virus. 一种重组病毒，所述重组病毒携带权利要求1～10任一项所述的核酸分子。A recombinant virus carrying the nucleic acid molecule according to any one of claims 1 to 10. 一种脂质体，其特征在于，包括脂质体载体以及核酸片段，所述核酸片段如权利要求1～10任一项所限定的。A liposome, characterized in that it comprises a liposome carrier and a nucleic acid fragment, wherein the nucleic acid fragment is as defined in any one of claims 1 to 10. 一种疫苗，其特征在于，包括权利要求1～10任一项所述的核酸分子、权利要求11～13所述的表达载体、权利要求14所述的重组病毒或权利要求15所述的脂质体；A vaccine, characterized in that it comprises the nucleic acid molecule according to any one of claims 1 to 10, the expression vector according to claims 11 to 13, the recombinant virus according to claim 14, or the liposome according to claim 15; 任选地，所述疫苗包括选自RNA疫苗、DNA疫苗、蛋白重组疫苗、灭活疫苗、减活疫苗、病毒载体疫苗的至少之一；Optionally, the vaccine comprises at least one selected from RNA vaccine, DNA vaccine, protein recombinant vaccine, inactivated vaccine, attenuated vaccine, and viral vector vaccine; 优选地，所述疫苗为RNA疫苗。Preferably, the vaccine is an RNA vaccine. 根据权利要求16所述的疫苗，其特征在于，进一步包括佐剂；The vaccine according to claim 16, further comprising an adjuvant; 任选地，所述佐剂包括TLR激动剂、Mn2+的至少之一；Optionally, the adjuvant comprises at least one of a TLR agonist and Mn 2+ ; 任选地，所述TLR激动剂包括HBHA、CpG，R837，MPLA和其衍生物的至少之一。Optionally, the TLR agonist comprises at least one of HBHA, CpG, R837, MPLA and derivatives thereof. 一种重组细胞，其特征在于，携带权利要求1～10任一项所述的核酸分子、权利要求11～13所述的表达载体或权利要求14所述的重组病毒。A recombinant cell, characterized in that it carries the nucleic acid molecule according to any one of claims 1 to 10, the expression vector according to claims 11 to 13, or the recombinant virus according to claim 14. 一种构建猫传染性腹膜炎病毒疫苗的方法，其特征在于，包括：A method for constructing a feline infectious peritonitis virus vaccine, comprising: 将权利要求1～10任一项所述的核酸分子、权利要求11～13所述的表达载体或权利要求14所述的重组病毒导入受体细胞。The nucleic acid molecule according to any one of claims 1 to 10, the expression vector according to claims 11 to 13, or the recombinant virus according to claim 14 is introduced into a recipient cell. 根据权利要求19所述的方法，其特征在于，导入受体细胞前，进一步包括使用包裹载体对所述核酸、表达载体或重组病毒进行包裹处理；The method according to claim 19, characterized in that before introducing into the recipient cell, it further comprises encapsulating the nucleic acid, expression vector or recombinant virus with an encapsulation vector; 任选地，所述包裹载体选自脂质体、外泌体、聚合物载体、病毒载体、纳米颗粒中的至少之一；Optionally, the encapsulation carrier is selected from at least one of liposomes, exosomes, polymer carriers, viral vectors, and nanoparticles; 优选地，所述包裹载体为纳米颗粒。Preferably, the encapsulating carrier is a nanoparticle. 根据权利要求19所述的方法，其特征在于，所述受体细胞为CRFK细胞、HEK293FT、HEK293T或BHK细胞；The method according to claim 19, wherein the recipient cell is a CRFK cell, HEK293FT, HEK293T or BHK cell; 优选地，所述受体细胞为CRFK细胞。Preferably, the recipient cells are CRFK cells. 权利要求1～10任一项所述的核酸分子、权利要求11～13所述的表达载体、权利要求14所述的重组病毒、权利要求15所述的脂质体或权利要求18所述的重组细胞在制备药物或疫苗中的用途，所述药物或疫苗用于预防或治疗猫传染性腹膜炎病毒感染相关疾病。 Use of the nucleic acid molecule according to any one of claims 1 to 10, the expression vector according to claims 11 to 13, the recombinant virus according to claim 14, the liposome according to claim 15, or the recombinant cell according to claim 18 in the preparation of a drug or vaccine, wherein the drug or vaccine is used to prevent or treat diseases related to feline infectious peritonitis virus infection. 一种预防或治疗猫传染性腹膜炎病毒感染疾病的方法，其特征在于，包括：向受试动物施用权利要求1～10任一项所述的核酸分子、权利要求11～13任一项所述的表达载体、权利要求14所述的重组病毒、权利要求15所述的脂质体、权利要求16或17所述的疫苗或权利要求18所述的重组细胞。A method for preventing or treating feline infectious peritonitis virus infection, comprising administering to a test animal the nucleic acid molecule of any one of claims 1 to 10, the expression vector of any one of claims 11 to 13, the recombinant virus of claim 14, the liposome of claim 15, the vaccine of claim 16 or 17, or the recombinant cell of claim 18. 根据权利要求23所述的方法，其特征在于，所述受试动物选自猫。The method according to claim 23, wherein the test animal is selected from cats. 权利要求1～10任一项所述的核酸分子、权利要求11～13任一项所述的表达载体、权利要求14所述的重组病毒、权利要求15所述的脂质体、权利要求16或17所述的疫苗或权利要求18所述的重组细胞在预防或治疗猫传染性腹膜炎病毒感染疾病中的用途。 Use of the nucleic acid molecule according to any one of claims 1 to 10, the expression vector according to any one of claims 11 to 13, the recombinant virus according to claim 14, the liposome according to claim 15, the vaccine according to claim 16 or 17, or the recombinant cell according to claim 18 in preventing or treating feline infectious peritonitis virus infection.
PCT/CN2024/081756 2024-03-14 2024-03-14 Method for using multi-epitope antigen to construct rna vaccine for fipv Pending WO2025189435A1 (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
PCT/CN2024/081756 WO2025189435A1 (en) 2024-03-14 2024-03-14 Method for using multi-epitope antigen to construct rna vaccine for fipv

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
PCT/CN2024/081756 WO2025189435A1 (en) 2024-03-14 2024-03-14 Method for using multi-epitope antigen to construct rna vaccine for fipv

Publications (1)

Publication Number Publication Date
WO2025189435A1 true WO2025189435A1 (en) 2025-09-18

Family

ID=97062581

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2024/081756 Pending WO2025189435A1 (en) 2024-03-14 2024-03-14 Method for using multi-epitope antigen to construct rna vaccine for fipv

Country Status (1)

Country Link
WO (1) WO2025189435A1 (en)

Similar Documents

Publication Publication Date Title
CN115551545A (en) SARS-COV-2 mRNA structure domain vaccine
WO2021185310A1 (en) Mvsv virus vector and virus vector vaccine thereof, and novel coronavirus pneumonia vaccine based on mvsv mediation
US9801927B2 (en) Pharmaceutical compositions comprising a polypeptide comprising at least one CXXC motif and heterologous antigens and uses thereof
CN113151184B (en) Method for cell membrane-based display of coronavirus immunogens to induce neutralizing antibodies
WO2020063370A2 (en) Immune composition, preparation method therefor, and application thereof
CN103747797A (en) Liposome formulation
AU2024205755B2 (en) Treatment of covid-19 and methods therefor
WO2022110099A1 (en) Coronavirus vaccines and uses thereof
CN113666990A (en) T cell vaccine immunogen for inducing broad-spectrum anti-coronavirus and application thereof
CN114213548B (en) Methods for simultaneously inducing immune responses against multiple viruses
US20250000968A1 (en) Sars-cov-2 spike fused to a hepatitis b surface antigen
WO2023051701A1 (en) Mrna, protein and vaccine against sars-cov-2 infection
EP4205762A1 (en) Improved dna vaccine for sars-cov-2
WO2023023940A1 (en) Immunogen for inducing broad-spectrum anti-coronavirus t cell vaccine and use thereof
WO2021254270A1 (en) Method for inducing neutralizing antibody based on cell membrane to display coronavirus immunogen
WO2021214703A1 (en) A vaccine against sars-cov-2 and preparation thereof
WO2024055273A1 (en) Rabies mrna vaccine and preparation and use thereof
WO2025189435A1 (en) Method for using multi-epitope antigen to construct rna vaccine for fipv
WO2025189436A1 (en) Method for constructing circular rna polytope vaccine for feline infectious peritonitis virus (fipv)
WO2024234754A1 (en) Method for constructing circular rna and vaccine against fipv
WO2024234755A1 (en) Development of multi-antigen mrna vaccine against feline fipv
WO2024234756A1 (en) Method for constructing mrna and vaccine targeting m and n antigens of feline fipv
CN118389541A (en) Method for constructing RNA vaccine against cat FIPV multi-epitope antigen
CN118185961A (en) Method for constructing circular RNA vaccine against cat FIPV multi-epitope antigen
He et al. A novel nanoparticle vaccine, based on S1-CTD, elicits robust protective immune responses against porcine deltacoronavirus

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 24928907

Country of ref document: EP

Kind code of ref document: A1