Quadruple mRNA vaccine for preventing cat infectious diseases and application thereof

Abstract

The invention discloses a quadruple mRNA vaccine for preventing feline infectious diseases and application thereof, the quadruple mRNA vaccine can simultaneously prevent feline rabies, feline calicivirus disease, feline panleucopenia and feline rhinotracheitis through one-time immunization, and experiments show that the vaccine is relatively safe and strong in efficacy, the injection cost is reduced, and the vaccine is suitable for clinical application. The immune procedure is optimized, and the method has great significance in preventing cat infectious diseases.

Description

Technical Field

[0001] This invention belongs to the field of suitable viral vaccines, specifically relating to a quadrivalent mRNA vaccine for the prevention of feline infectious diseases and its application. Background Technology

[0002] Feline rabies is a severe and fatal viral infection in felines caused by the rabies virus (RV). Symptoms after infection include fever, barking, hydrophobia, and other neurological symptoms, ultimately leading to death. The G protein of the rabies virus is an important surface protein with strong immunogenicity. As one of the virus's main antigens, the G protein effectively stimulates the immune system to produce antibodies against viral invasion and spread. Simultaneously, the G protein is a key structural protein for viral invasion of host cells, affecting the virus's infectivity. By selecting the G antigen as a vaccine target, it is possible to induce antibody production in the host and enhance protection against the rabies virus. Currently, there are no reports on feline rabies mRNA vaccines.

[0003] Feline calicivirus disease (FCV) is a common infectious disease in felines caused by feline calicivirus (FCV). FCV is an RNA virus that is primarily transmitted through droplets, direct contact, or contaminated food and water. Cats infected with FCV mainly exhibit upper respiratory tract infection symptoms, including sneezing, runny nose, and conjunctivitis. Some severely infected cats may also develop oral ulcers, eye inflammation, lethargy, and loss of appetite. In some cases, FCV infection can lead to severe oral inflammatory syndrome, threatening the cat's life. The VP1 protein plays a crucial role in the life cycle of FCV and is one of the virus's main structural proteins. The VP1 protein is key to viral replication, immune response, and vaccine development, possessing a highly conserved sequence and unique structure. The VP1 protein can trigger an immune response, clear the virus, and stimulate the production of antiviral factors. Currently, FCV vaccine development focuses primarily on the VP1 protein.

[0004] Feline panleukopenia, also known as feline distemper, is caused by feline parvovirus (FPV). This virus belongs to the family Feline Parvoviridae. FPV is a highly contagious DNA virus, primarily transmitted through contact with infected animals, feces, urine, or infected environments. VP1 and VP2 are the main structural proteins of the FPV virus, playing crucial roles in viral infection and replication. VP2 protein is not only a major structural protein but also plays a vital role in viral immunogenicity, effectively stimulating the body's immune response. Furthermore, VP2 protein has the ability to assemble virus-like particles, making it an important immunogenic protein. Current FPV vaccine development primarily focuses on the VP2 protein.

[0005] Feline rhinotracheitis is an infectious upper respiratory tract infection in cats caused by feline herpesviruses (FHV). FHV is an enveloped, double-stranded DNA virus with a genome composed of UL and US regions. The US region contains genes such as gG, gD, gI, and gE, while the UL region contains genes such as gC, gH, and gB. These genes encode a variety of proteins. Currently, seven glycoproteins—gB, gC, gD, gE, gG, gH, and gI—have been identified, playing crucial roles in viral recognition, invasion, infection, intercellular transmission, and release of infection. Among these, gB and gD proteins are the most important proteins currently being developed for FHV vaccines.

[0006] Feline triple vaccine and rabies vaccine are two different vaccines. Currently, there is research on inactivated triple vaccines for feline calicivirus disease, feline panleukopenia, and feline rhinotracheitis. The feline triple vaccine is mainly used to prevent feline panleukopenia virus, herpesvirus, and calicivirus, while the rabies vaccine is used to prevent rabies in dogs and felines. Therefore, most feline triple vaccines do not contain rabies. Rabies vaccines should not be administered simultaneously with feline triple vaccines because if an allergic reaction occurs, it is difficult to distinguish which vaccine caused it.

[0007] Due to the poor immunogenicity, allergic reactions, and safety issues associated with inactivated vaccines, mRNA vaccines are gaining increasing popularity due to their strong immunogenicity, high efficacy, and relative safety. Although some research is beginning to explore the development of mRNA vaccines, a quadrivalent mRNA vaccine has not yet been developed.

[0008] Developing a quadrivalent mRNA vaccine that can reduce vaccine injection costs, optimize immunization procedures, and enhance vaccine efficacy would be of great significance for preventing feline infectious diseases. Summary of the Invention

[0009] The purpose of this invention is to provide a quadrivalent mRNA vaccine for the prevention of feline infectious diseases and its application. It can prevent feline rabies, feline calicivirus disease, feline panleukopenia, and feline rhinotracheitis simultaneously through a single immunization. Experiments have shown that the vaccine is relatively safe, highly effective, reduces injection costs, and optimizes the immunization program, which is of great significance for the prevention of feline infectious diseases.

[0010] To achieve the above objectives, a specific embodiment of the present invention provides the following technical solution:

[0011] In one aspect, the present invention provides a nucleic acid combination comprising:

[0012] 1) The first nucleic acid molecule, which includes a first nucleotide sequence encoding the rabies virus G protein or an immunogenic fragment thereof;

[0013] 2) A second nucleic acid molecule, which includes a second nucleotide sequence encoding the feline calicivirus VP1 protein or an immunogenic fragment thereof;

[0014] 3) A third nucleic acid molecule, which includes a third nucleotide sequence encoding the feline parvovirus VP2 protein or an immunogenic fragment thereof;

[0015] 4) A fourth nucleic acid molecule comprising a fourth nucleotide sequence encoding the feline herpesvirus gB protein or an immunogenic fragment thereof; and / or

[0016] 5) A fifth nucleic acid molecule comprising a fifth nucleotide sequence encoding a feline herpesvirus gD protein or an immunogenic fragment thereof. In one or more embodiments of the present invention, the amino acid sequence of the G protein or the immunogenic fragment thereof has at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% homology with SEQ ID NO:1.

[0017] The amino acid sequence of the VP1 protein or its immunogenic fragment has at least 85%, 90%, 95%, 96%, 97%, 98% or 99% homology with SEQ ID NO:2;

[0018] The amino acid sequence of the VP2 protein or its immunogenic fragment has at least 85%, 90%, 95%, 96%, 97%, 98% or 99% homology with SEQ ID NO:3;

[0019] The amino acid sequence of the gB protein or its immunogenic fragment has at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% homology with SEQ ID NO:4; or

[0020] The amino acid sequence of the gD protein or its immunogenic fragment has at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% homology with SEQ ID NO:5.

[0021] In one or more embodiments of the present invention, the sequence of the first nucleotide has at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% homology with SEQ ID NO: 6 or 7;

[0022] The sequence of the second nucleotide has at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% homology with SEQ ID NO:8 or 9;

[0023] The sequence of the third nucleotide has at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% homology with SEQ ID NO:10 or 11;

[0024] The sequence of the fourth nucleotide has at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% homology with SEQ ID NO:12 or 13;

[0025] The sequence of the fifth nucleotide has at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% homology with SEQ ID NO:14 or 15, wherein SEQ ID NO:14 is the DNA sequence of the gD protein with the 3' end nucleotides 991 to 1122 deleted.

[0026] In one or more embodiments of the present invention, the 5' end of each nucleotide sequence in the nucleic acid combination is connected to a nucleotide sequence encoding a signal peptide.

[0027] In one or more embodiments of the present invention, the signal peptide is tPA, IL-2, IL-6 or a neuropeptide, preferably VIP, CGRP or NPY.

[0028] In one or more embodiments of the present invention, the 5' end of the nucleotide sequence encoding the signal peptide is connected to a 5' UTR sequence; preferably, each nucleic acid molecule in the nucleic acid combination is provided with a FLAG tag.

[0029] In one or more embodiments of the present invention, each nucleotide sequence in the nucleic acid combination is further connected to a 3' UTR sequence at its 3' end.

[0030] In one or more embodiments of the present invention, the nucleic acid is DNA or RNA.

[0031] In another aspect, the present invention provides a carrier combination comprising a first carrier constructed from a first nucleic acid molecule in the above-described nucleic acid combination, a second carrier constructed from a second nucleic acid molecule, a third carrier constructed from a third nucleic acid molecule, a fourth carrier constructed from a fourth nucleic acid molecule, and a fifth carrier constructed from a fifth nucleic acid molecule.

[0032] In another aspect, the present invention provides a cell assembly comprising cells transformed from the above-described carrier assembly.

[0033] In another aspect, the present invention provides a composition for preventing feline infectious diseases, comprising the above-described nucleic acid combination or mRNA transcribed from the above-described vector combination.

[0034] In one or more embodiments of the present invention, the composition is an mRNA vaccine, wherein the mRNA vaccine is a quadrivalent mRNA vaccine for the prevention of feline infectious diseases, the quadrivalent mRNA vaccine comprising mRNA expressing rabies virus G protein or an immunogenic fragment thereof, mRNA expressing feline calicivirus VP1 protein or an immunogenic fragment thereof, mRNA expressing feline parvovirus VP2 protein or an immunogenic fragment thereof, mRNA expressing feline herpesvirus gB protein or an immunogenic fragment thereof, and mRNA expressing feline herpesvirus gD protein or an immunogenic fragment thereof.

[0035] In one or more embodiments of the present invention, the mRNA sequence expressing the rabies virus G protein or its immunogenic fragment has at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% homology with SEQ ID NO:7.

[0036] The mRNA sequence expressing feline calicivirus VP1 protein or its immunogenic fragment has at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% homology with SEQ ID NO:9.

[0037] The mRNA sequence expressing feline parvovirus VP2 protein or its immunogenic fragment has at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% homology with SEQ ID NO:11.

[0038] The mRNA sequence expressing feline herpesvirus gB protein or its immunogenic fragment has at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% homology with SEQ ID NO:13.

[0039] The mRNA sequence expressing feline herpesvirus gD protein or its immunogenic fragment has at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% homology with SEQ ID NO:15.

[0040] In one or more embodiments of the present invention, the quadrivalent mRNA vaccine comprises the following components in parts by weight: 2-4 parts of mRNA expressing rabies virus G protein or its immunogenic fragment, 2-4 parts of mRNA expressing feline calicivirus VP1 protein or its immunogenic fragment, 2-4 parts of mRNA expressing feline parvovirus VP2 protein or its immunogenic fragment, 1-2 parts of mRNA expressing feline herpesvirus gB protein or its immunogenic fragment, and 1-2 parts of mRNA expressing feline herpesvirus gD protein or its immunogenic fragment.

[0041] In one or more embodiments of the present invention, the quadrivalent mRNA vaccine comprises the following components in parts by weight: 2 parts of mRNA expressing rabies virus G protein or its immunogenic fragment, 2 parts of mRNA expressing feline calicivirus VP1 protein or its immunogenic fragment, 2 parts of mRNA expressing feline parvovirus VP2 protein or its immunogenic fragment, 1 part of mRNA expressing feline herpesvirus gB protein or its immunogenic fragment, and 1 part of mRNA expressing feline herpesvirus gD protein or its immunogenic fragment.

[0042] In one or more embodiments of the present invention, liposomes are also included, wherein the mRNA in the quadrivalent mRNA vaccine is encapsulated in liposome nanoparticles.

[0043] In one or more embodiments of the present invention, the particle size of the liposome nanoparticles is 50-200 nm.

[0044] In one or more embodiments of the present invention, the liposome nanoparticles are selected from at least one of cationic lipids, distearate phosphatidylcholine (DSPC), cholesterol, and DMG-2000.

[0045] In one or more embodiments of the present invention, the liposome nanoparticles are composed of the following components in parts by weight: 30-80 parts of cationic lipids, 5-15 parts of distearate phosphatidylcholine (DSPC), 24-52 parts of cholesterol, and 1-2 parts of DMG-2000.

[0046] Preferably, the liposome nanoparticles are composed of the following components in parts by weight: 50 parts cationic lipids, 10 parts distearate phosphatidylcholine (DSPC), 38 parts cholesterol, and 1.5 parts DMG-2000.

[0047] In another aspect, the present invention provides the use of the above-mentioned nucleic acid combination, carrier combination, or composition in the preparation of a medicament for the prevention of feline rhinotracheitis, feline calicivirus disease, feline panleukopenia, and feline rabies.

[0048] Compared with existing technologies, the present invention provides a quadrivalent mRNA vaccine for the prevention of feline infectious diseases and its application. Through sequence optimization, a quadrivalent mRNA vaccine containing RV G antigen, FHV gB antigen, and FHV gD antigen was designed. Through sequence optimization, it was unexpectedly discovered that compared with the RV G antigen (without a transmembrane region), FHV gB antigen (without a transmembrane region), and FHV gD antigen used in existing technologies… The mRNA obtained from the sequence of the gD antigen (with a transmembrane region) has a higher titer and expression level after optimization, exhibiting strong immunogenicity. It can simultaneously prevent feline rabies, feline calicivirus disease, feline panleukopenia, and feline rhinotracheitis through a mixed vaccine, overcoming the problems of separate immunizations, complex procedures, and high injection costs in existing technologies for preventing these diseases. Furthermore, experiments have shown that the quadrivalent mRNA vaccine of this invention has a higher immunogenicity than inactivated vaccines, and is relatively safer and more potent, reducing injection costs and optimizing the immunization procedure. This is of great significance for the prevention of feline infectious diseases such as feline rabies, feline calicivirus disease, feline panleukopenia, and feline rhinotracheitis. Attached Figure Description

[0049] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments recorded in the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0050] Figure 1 This is an agarose gel electrophoresis image of the in vitro transcribed mRNA in Example 2 of the present invention, wherein: Marker: RNAmarker 6000; 1 is the mRNA of RV G antigen; 2 is the mRNA of FHV gB antigen; 3 is the mRNA of FCV VP1 antigen; 4 is the mRNA of FPVVP2 antigen; 5 is the mRNA of FHV gD antigen;

[0051] Figure 2This is a Western blotting image for verifying protein expression after in vitro transcription of mRNA in Example 3 of the present invention. In the image, Marker represents the protein marker; 1 is the mRNA of RV G antigen; 2 is the mRNA of FCV VP1 antigen; 3 is the mRNA of FPV VP2 antigen; 4 is the mRNA of FHV gB antigen; 5 is the mRNA of FHV gD antigen; 6 is the mixed mRNA; the right side indicates the bands representing the proteins expressed by each mRNA after mixed immunization.

[0052] Figure 3 The images show the results of mRNA transfection and protein expression of RV G antigen, FHV gB antigen, and FHV gD antigen with and without transmembrane regions in Example 3 of the present invention. In the images, Marker is a protein marker; A is the result of mRNA transfection and protein expression of FHV gB antigen, B is the result of mRNA transfection and protein expression of FHV gD antigen, C is the result of mRNA transfection and protein expression of RV G antigen, 1 represents the protein expressed by mRNA of each antigen without transmembrane regions, and 2 represents the protein expressed by mRNA of each antigen without transmembrane regions.

[0053] Figure 4 The figure shows the titer results of mice immunized with mRNA containing RV G antigen, FHV gB antigen and FHV gD antigen with and without transmembrane regions in Example 3 of the present invention. In this figure, A is the titer result of mice immunized with mRNA containing FHV gB antigen, B is the titer result of mice immunized with mRNA containing FHV gD antigen, and C is the titer result of mice immunized with mRNA containing RV G antigen.

[0054] Figures 5A-5E This is a particle size analysis diagram of LNP-mRNA after packaging in Example 4 of the present invention, wherein... Figure 5A LNP-mRNA vaccine for RV G antigen; Figure 5B LNP-mRNA vaccine based on FCV VP1 antigen; Figure 5C LNP-mRNA vaccine based on FPV VP2 antigen; Figure 5D LNP-mRNA vaccine based on FHV gB antigen; Figure 5E It is an FHV gD antigen LNP-mRNA vaccine;

[0055] Figure 6 This is a flowchart of the animal experiment in Embodiment 5 of the present invention;

[0056] Figure 7The above are statistical charts of IgG antibodies in mice immunized with the quadrivalent mRNA vaccine in Example 5 of this invention. A represents the statistical chart of neutralizing antibodies in RV serum; B represents the statistical chart of IgG antibodies in FCV serum; C represents the statistical chart of IgG antibodies in FPV serum; D represents the statistical chart of IgG antibodies in FHV gD serum; and E represents the statistical chart of IgG antibodies in FHV gB serum.

[0057] Figure 8 The results are pathological sections of mice immunized with an overdose of the quadrivalent mRNA vaccine in Example 6 of this invention. Detailed Implementation

[0058] To enable those skilled in the art to better understand the technical solutions of this invention, the technical solutions of the embodiments of this invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of this invention, and not all embodiments. Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without creative effort should fall within the scope of protection of this invention.

[0059] Unless otherwise specified, all reagents and materials used in this article are available from conventional sources.

[0060] Unless otherwise specified, "RV G antigen" in this article refers to "rabies virus G protein or its immunogenic fragment".

[0061] Unless otherwise specified, "FCV VP1 antigen" in this article refers to "feline calicivirus VP1 protein or its immunogenic fragment".

[0062] Unless otherwise specified, "FPV VP2 antigen" in this article refers to "feline parvovirus VP2 protein or its immunogenic fragment".

[0063] Unless otherwise specified, "FHV gB antigen" in this article refers to "feline herpesvirus gB protein or its immunogenic fragment".

[0064] Unless otherwise specified, "FHV gD antigen" in this article refers to "feline herpesvirus gD protein or its immunogenic fragment".

[0065] Unless otherwise specified, "mRNA-LNP" in this article refers to "LNP-mRNA vaccine".

[0066] Example 1. Obtaining the antigen expression vector for mRNA vaccines

[0067] 1. Optimization design of sequences

[0068] Currently, vaccines for feline rabies, feline calicivirus disease, feline panleukopenia, and feline rhinotracheitis are being developed. Some viral antigens typically use sequences like the one shown below, for example...

[0069] The amino acid sequence of the RV G antigen (without a transmembrane region) is as follows:

[0070] MVPQALLLVPLLGFSLCFGKFPIYTIPDTLGPWSPIDIHHLSCPNNLVVEDEGCTNLSGFSYMELKVGHISAIKVNGFTCTGVVTEAETYTNFVGYVTTTFKRKHFRPTPDACR AAYNWKMAGDTRYEESLHSPYPDYHWLRTVKTTKESLVIISPSVANLDPYDNSLHSRVFPSGKCSGITVPSVYCSTNHDYTVWMPEILRLGTSCDIFTNSRGKRASKGSKTCGFV DERGLYKSLKGACKLKLCGVPGLRLMDGTWVAMQTSNETKWCPPGQLVNLHDLHSDEIEHLVVEELVKKREECLDALESIITTKSVSFRRLSHLRKLVPGFGKAYTIFNKTLMEA EAHYKSVRTWNEIIPSKGCLRVGGRCHPHVNGVFFNGIILGPDGHVLIPEMQSSLLQQHIELLESSVIPLMHPLADPFTVFKDGDEIEDFVEVHLPDVHEQVSGVDLGLPN(SEQ ID NO:21)

[0071] The amino acid sequence of the FHV gB antigen (without a transmembrane region) is as follows:

[0072] MSTRGDLGKRRRGSRWQGHSGYFRQRCFFPSLLGIAATGSRHGNGSSGLTRLARYVSFIWIVLFLVGPRPVEGQSGSTSEQPRRTVATPEVGGTPPKPTTDPTDMSDMREALRASQIEANGPSTFYMCPPPSGSTVVRLEPPRACPDYKLGKNFTEGIAVIFKENIAPYKFKANIYYKNIIMTTVWSGSSYAVTTNRYTDRVPVKVQEITDLIDRRGMCLSKADYVRNNYQFTAFDRDEDPRELPLKPSKFNTPESRGWHTTNETYTKIGAAGFHHSGTSVNCIVEEVDARSVYPYDSFAISTGDVIHMSPFFGLRDGAHVEHTSYSSDRFQQIEGYYPIDLDTRLQLGAPVSRNFLETPHVTVAWNWTPKSGRVCTLAKWREIDEMLRDEYQGSYRFTAKTISATFISNTSQFEINRIRLGDCATKEAAEAIDRIYKSKYSKTHIQTGTLETYLARGGFLIAFRPMISNELAKLYINELARSNRTVDLSALLNPSGETVQRTRRSVPSNQHHRSRRSTIEGGIETVNNASLLKTTSSVEFAMLQFAYDYIQAHVNEMLSRIATAWCTLQNREHVLWTETLKLNPGGVVSMALERRVSARLLGDAVAVTQCVNISSGHVYIQNSMRVTGSSTTCYSRPLVSFRALNDSEYIEGQLGENNELLVERKLIEPCTVNNKRYFKFGADYVYFEDYAYVRKVPLSEIELISAYVDLNLTLLEDREFLPLEVYTRAELEDTGLLDYSEIQRRNQLHALKFYDIDSIVRVDNNLVIMRGMANFFQGLGDVGAGFGKVVLGAASAVISTVSGVSSFLNNPF(SEQ ID NO:22)

[0073] The amino acid sequence of the FHV gD antigen (with transmembrane region) is as follows:

[0074] MDAMKRGLCCVLLLCGAVFVSASMMTRLHFWWCGIFAVLKYLVCTSSLTTTPKTTTVYVKGFNIPPLRYNYTQARIVPKIPQAMDPKITAEVRYVTSMDSCGMVALISEPDIDATIRTIQLSQKKTYNATISWFKVTQGCEYPMFLMDMRLCDPKREFGICALRSPSYWLEPLTKYMFLTDDELGLIMMAPAQFNQGQYR RVITIDGSMFYTDFMVQLSPTPCWFAKPDRYEEILHEWCRNVKTIGLDGARDYHYYWVPYNPQPHHKAVLLYWYRTTHGREPPVRFQEAIRYDRPAIPSGSEDSKRSNDSRGESSGPNWIDIENYTPKNNVPIIISDDDVPTAPPKGMNNQSVVIPAIVLSCLIIALILGVIYYILRVKRSRSTAYQQLPIIHTTHHP(SEQ ID NO:23)

[0075] However, through experimental research, we found that the sequences of these viral antibodies may have problems with expression levels and immunogenicity. Therefore, we attempted to optimize the sequences of the above-mentioned RV G antigen, FHV gB antigen, and FHV gD antigen, and used the optimized sequences to develop a quadrivalent mRNA. Furthermore, through codon optimization, we obtained a quadrivalent mRNA vaccine that can simultaneously prevent feline rabies, feline calicivirus disease, feline panleukopenia, and feline rhinotracheitis with a single immunization.

[0076] The specific sequence of the antigen used in this quadrivalent mRNA vaccine is as follows:

[0077] The DNA sequence of the RV G antigen is shown in SEQ ID NO: 6:

[0078]

[0079] The amino acid sequence of the G antigen of RV is shown in SEQ ID NO: 1:

[0080] MVPQALLLVPLLGFSLCFGKFPIYTIPDTLGPWSPIDIHHLSCPNNLVVEDEGCTNLSGFSYMELKVGHISAIKVNGFTCTGVVTEAETYTNFVGYVTTTFKRKHFRPTPDACRAAYNWKMAGDTRYEESLHSPYPDYHWLRTVKTTKESLVIISPSVANLDPYDNSLHSRVFPSGKCSGITVPSVYCSTNHDYTVWMPEILRLGTSCDIFTNSRGKRASKGSKTCGFVDERGLYKSLKGACKLKLCGVPGLRLMDGTWVAMQTSNETKWCPPGQLVNLHDLHSDEIEHLVVEELVKKREECLDALESIITTKSVSFRRLSHLRKLVPGFGKAYTIFNKTLMEAEAHYKSVRTWNEIIPSKGCLRVGGRCHPHVNGVFFNGIILGPDGHVLIPEMQSSLLQQHIELLESSVIPLMHPLADPFTVFKDGDEIEDFVEVHLPDVHEQVSGVDLGLPNWGEYVLLSAGTLIALMLIIFLITCCKRVDRPESTQRSLRGTGRNVSVTSQSGKFIPSWESYKSGGETGLDYKDDDDK

[0081] The mRNA sequence of the G antigen of RV is shown in SEQ ID NO: 7:

[0082] GAAAUAAGAGAGAAAAGAAGAGUAAGAAGAAAUAUAAGAGCCACCGCUAGCCUCGAGGCCA

[0083] CCAUGGACGCCAUGAAGAGGGGGCUGUGCUGCGUGCUGCUGCUGUGCGGAGCCGUGUUCGUGAG

[0084] CGCCUCCAUGGUGCCCCAGGCCCUGCUGCUGGUGCCCCUGCUGGGAUUCUCCCUGUGCUUCGGCA

[0085] AGUUCCCCAUCUACACCAUCCCCGACACCCUGGGCCCCUGGAGCCCUAUCGACAUCCACCACCUG

[0086] UCCUGCCCCAAUAACCUGGUGGUGGAAGACGAGGGCUGCACCAACCUGAGCGGCUUCUCCUACAU

[0087] GGAACUGAAAGUGGGCCACAUCUCCGCCAUCAAAGUGAACGGCUUCACCUGCACCGGCGUGGUGA

[0088] CCGAAGCCGAAACAUACACAAACUUCGUGGGCUACGUGACCACCACCUUCAAGAGAAAACACUUC

[0089] AGACCCACCCCCGAUGCCUGCCGGGCCGCUUAUAACUGGAAGAUGGCCGGAGACACCAGAUACGA

[0090] GGAGAGCCUGCACAGCCCCUACCCCGACUACCACUGGCUGAGAACCGUGAAGACCACCAAGGAAA

[0091] GCCUGGUGAUCAUCUCUCCCUCCGUGGCCAACCUGGACCCCUACGACAACAGCCUGCACUCCAGA

[0092] GUGUUCCCCUCCGGCAAGUGCUCCGGCAUCACCGUGCCCAGCGUGUACUGUUCCACUAACCACGA

[0093] CUACACCGUGUGGAUGCCCGAGAUCCUGAGACUGGGCACAUCCUGCGACAUCUUUACCAACAGCA

[0094] GAGGCAAGAGGGCUAGCAAAGGGUCUAAAACCUGCGGCUUCGUGGACGAGAGAGGCCUGUACAA

[0095] GAGCCUGAAGGGCGCCUGCAAACUGAAGCUGUGUGGCGUGCCUGGCCUGAGACUGAUGGACGGA

[0096] ACCUGGGUGGGCUAUGCAGACAAGCAAUGAGACGAAGUGGUGUCCUCCCGGACAGCUGGUACC

[0097] UGCAUGACCUGCACUCAGACGAGAUUGAGCACCUGGUGGUGGAGGAGCUGGUGAAGAAGGGGA

[0098] GGAGUGCCUGGACGCCCUGGAGAGCAUCAUCACAACCAAGAGCGUGAGUUUCAGAAGCCUGAGC

[0099] CACCUGAGAAAGCUGGUGCCAGGCUUCGGCAAGGCCUACACCAUCUUUCAACAAGACACUGAUGGA

[0100] GGCCGAGGCCCACUACAAGAGCGUGAGAACAUGGAACGAGAUCAUCCCCAGCAAAGGCUGCC

[0101] GCGUGGCGGACGGUGCCACCCUCAUGUGAACGGGGUGUUCUUCAACGGCAUCAUCCUGGGACC

[0102] GACGGCCACGUGCUGAUCCCCGAAAUGCAGAGCAGCCUGCUGCAGCAGCACAUUGAGCUGCUGGA

[0103] GUCCUCCGUGUAUCCCCCUGAUGCAUCCCCUGGCCGACCCAUCACCGGUGUUCAAGGACGGCGAUG

[0104] AAAUUGAAGACUUCGUGGAAGUGCACCUGCCCCGACGUGCACGAGCAGGUGAGCGGCGUGGAUCU

[0105] GGGCCUGCCUAACUGGGGCGAAUAUUGGCUGCUGAGUGCCCGGCACACUGAUUGCCCUGAUGCUG

[0106] AUAUUUUUUCUGAUACCUGUUGUAAGAGAGUGGACCGGCCAGAGAGUACCCAGAGAUCCCUGA

[0107] GAGGGACAGGCAGGAACGUGAGCGUGACCAGCCAGAGCGGGAAGUUCAUCCCCAGCUGGGAGAG

[0108] CUACAAGAGCGGGGGCGAGACAGGACUGGAUUACAAGGAUGACGACGAUAAGUAAUAAGGUACC

[0109] GAUAUCUGAUAAUAGGCUGGAGCCUCGGUGGCCAUGCUUCUUGCCCCUUGGGCCUCCCCCCAGCC

[0110] CCUCCUCCCCUUCCUGCACCCGUACCCCCGUGGUCUUUGAAUAAAGUCUGAAAAAAAAAAAAAAA

[0111] AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA

[0112] AAAAAAAAAAAAAAAAAAAAAAAAAAA

[0113] The DNA sequence of the VP1 antigen of FCV is shown in SEQ ID NO: 8:

[0114] ATGTGTTCCACCTGTGCAGACGTGCTGAAATACTACAACTGGGACCCACACTTCAAGATGGTGAT

[0115] AAATCCAAACCAGTTTCTCTCCGTGGGATTCTGCGACAATCCACTGATGTGCTGCTACCCTGAACTGT

[0116] TGCCAGAATTGGGAACGGTGTGGGACTGTGACCAGTCACCACTGCAGATTTATCTCGAAAGCATACTT

[0117] GGCGACGACGAATGGTCCAGCACACACGAGGCCATTGATCCAGTGGTGCCACCAATGCACTGGTCCG

[0118] AAATGGGCAAGATCTTTCAGCCACACCCAGGCATTCTGATGCACCACTTGATCAGCCAAGTGGCCAA

[0119] AGGTTGGGACCCAAACCTGCCAAACTTTAGGCTAGAGGCAGACGGTGATGGCTCCATAACAACTCCA

[0120] GAACAGGGCACTGCAGTCGGAGGCGTGATTGCCGAACCATCCGCTCAGATGAGCACTGCTGCAGACA

[0121] TGGCCTCCGGAAAGTCCGTGGACAGCGAATGGGAGGCCTTCTTCAGCTTTCACACCTCCGTGAACTG

[0122] GTCCACCTCCGAGACTCAAGGCAAGATACTGTTCAAACAGAACCTCTCTCCATTGCTGAATCCATATC

[0123] TGAGCCACTTGAGCAAACTCTACGTCGCTTGGTCCGGAAGCATCGACGTGAGGTTCTCCATTTCCGGC

[0124] TCCGGCGTCTACGGCGGTAAACTGGCCGCAATTGTCGTGCCACCAGGCATAGAGCCAGTGCAGTCCA

[0125] CCAGCATGCTCCAGTATCCACACGTGCTCTTTGATGCCAGGCAAGTCGAGCCAGTGATCTTCTCCGTT

[0126] CCAGACCTGAGATCCACTCTCTACCACCTGATGTCCGACGTTGACACCACCTCTCTGGTGATCATGAT

[0127] CTACAACGACCTCATCAACCCGTACGCCTCCGAAACCAACAGCTCCGGCTGCATTGTCACAGTCGAA

[0128] ACCAAACCGGGACCAGACTTTAAGTTCCACTTACTGAAACCACCTGGCTCAATGCTCACTCACGGCT

[0129] CAGTTCCATCCGACCTGATACCTAAATCCAGTAGCCTGTGGATCGGAAACAGGCACTGGACCGACATC

[0130] GTGGACTTCGTCATAAGGCCATTTGTGTTCCAGGCTAACAGACACTTCGACTTCAACCAGGAGACTGC

[0131] CGGATGGTCCACACCAAGGTTCAGGCCAATTACCATCAACATCAGCCAGTCCAAAGGCGAAAGGCTC

[0132] GGAATTGGAGTCGCTACTGACTACATCGTGCCAGGCATACCAGACGGATGGCCGGACACCACCATACC

[0133] AGAGAAACTCATACCAGCCGGAAAGTACGCCAATTACCTCCGGAAAACAACAACGACTTGGAAAGCGCC

[0134] AAAGAGTACGACGAGGCAGATGTGATTCGGAACAACCAACTTTAAGAGCATGTACATCTGCGGAA

[0135] GCCTGCAGAGGGCATGGGGGAGACAAAGAGATCTCCAACACCGGCTTTATCACTACAGCCACCGTGGT

[0136] GGGAAACGCCTTGGTGCCATCTAACACCATTGACCAGACCAAGATTGCCGTGTTCCAAGACAACCAC

[0137] GTCAACACCGAGGTGCAGACCAGCGACGTGACACTGGCACTGTTGGGATACACCGGAATCGGAGAA

[0138] GAGGCTATCGGAGTGGACAGAGAAAGTGGTCAGAATCAGCGTGTTACCAGAAACTGGTGCCAGA

[0139] GGTGGAAACCACCCAATCTTCTCAAGAACAAGATGAAACTCGCTACGTCATCAGGGAGATCGACG

[0140] TGTTCAACAGCCAGATTCTCCACACCAGCAGGCAGCTCTCTCTGAATAACTACCTATTGCCACCAGAC

[0141] AGCTTTGCCGTCTACAGAATCATCGACGCCAACGGAAGCTGGTTTGACATCGGAATTGACAGCGACG

[0142] GATTCAGCTTTGTCGGAGTCTCCAACATTTCCAAACTAGAGTTTCCGCTGACCGCATCTTACATGGGA

[0143] ATCCAGCTGGCAAAGATCAGGCTGGCATCCAATATCCGCTCATCCATGACCAAACTCGACTACAAAGA

[0144] CGACGACGACAAGTAA

[0145] The amino acid sequence of the VP1 antigen of FCV is shown in SEQ ID NO: 2:

[0146] MCSTCADVLKYYNWDPHFKMVINPNQFLSVGFCDNPLMCCYPELLPELGTVWDCDQSPLQIYLESILGDDEWSSTHEAIDPVVPPMHWSEMGKIFQPHPGILMHHLISQVAKGWDPNLPNFRLEADGDGSITTPEQGTAVGGVIAEPSAQMSTAADMASGKSVDSEWEAFFSFHTSVNWSTSETQGKILFKQNLSPLLNPYLSHLSKLYVAWSGSIDVRFSISGSGVYGGKLAAIVVPPGIEPVQSTSMLQYPHVLFDARQVEPVIFSVPDLRSTLYHLMSDVDTTSLVIMIYNDLINPYASETNSSGCIVTVETKPGPDFKFHLLKPPGSMLTHGSVPSDLIPKSSSLWIGNRHWTDIVDFVIRPFVFQANRHFDFNQETAGWSTPRFRPITINISQSKGERLGIGVATDYIVPGIPDGWPDTTIPEKLIPAGKYAITSGNNNDIGSAKEYDEADVIRNNTNFKSMYICGSLQRAWGDKEISNTGFITTATVVGNALVPSNTIDQTKIAVFQDNHVNTEVQTSDVTLALLGYTGIGEEAIGVDREKVVRISVLPETGARGGNHPIFYKNKMKLGYVIREIDVFNSQILHTSRQLSLNNYLLPPDSFAVYRIIDANGSWFDIGIDSDGFSFVGVSNISKLEFPLTASYMGIQLAKIRLASNIRSSMTKLDYKDDDDK

[0147] The mRNA sequence of the VP1 antigen of FCV is shown in SEQ ID NO: 9:

[0148] GAAAUAAGAGAGAAAAGAAGAGUAAGAAGAAAUAUAAGAGCCACCGCUAGCCUCGAGGCCA

[0149] CCAUGGACGCCAUGAAGAGGGGGCUGUGCUGCGUGCUGCUGCUGUGCGGAGCCGUGUUCGUGAG

[0150] CGCCUCCAUGUGUUCCACCUGUGCAGACGUGCUGAAAAUACUACAACUGGGACCCACACUUCAAGA

[0151] UGGUGAUAAAUCCAAACCAGUUUCUCUCCGUGGGAUUCUGCGACAAUCCACUGAUGUGCUGCUA

[0152] CCCUGAACUGUUGCCAGAAUUGGGAACGGUUGGGACUGUGGACGACCAGUCACCACUGGCAGAUUUAU

[0153] CUCGAAAGCAUACUUGGCGACGACGAAUGGUCCAGCACACACGAGGCCAUUGAUCCAGUGGUGCC

[0154] ACCAAUGCACUGGUCCGAAAUGGGCAAGAUCUUUCAGCCACACCCAGGCAUUCUGAGCACCACU

[0155] UGAUCAGCCAAGUGGCCAAAGGUUGGACCCAAACCUGCCAAACUUUAGGCUAGCAGACGG

[0156] UGAUGGCUCCAUAAACACUCCAGAACAGGGCACUGCAGUCGGGAGGCGUGAUUGCCGAACCAUCCG

[0157] CUCAGAUGAGCACUGCUGCAGACAUGGCCUCCGGAAAGUCCGUGGACAGCGAAUGGGAGGCCUU

[0158] CUUCAGCUUUCACACCUCCGUGAACUGGUCCACCUCCGAGACUCAAGGCAAGAUACUGUUCAAAC

[0159] AGAACCUCUCUCCAUUGCUGAAUCCAUAUCUGAGCCACUUGAGCAAACUCUACGUCGCUUGGUCC

[0160] GGAAGCAUCGACGUGAGGUUCUCCCAUUUCCGGCUCCGGCGUCUACGGCGGUAAACUGGCCGCAAU

[0161] UGUCGUGCCACCAGGCAUAGAGCCAGUGCAGUCCACCAGCAUGCUCCAGUAUCCACACGUGCUCU

[0162] UUGAUGCCAGGCAAGUCGAGCCAGUGAUCUUCUCCGUUCCAGACCUGAGAUCCACUCUCUACCAC

[0163] CUGAUGUCCGACGUUGACACCACCUCUCUGGUGAUCAUGAUCUACAACGACCUCAUCAACCCGUA

[0164] CGCCUCCGAAACCAACAGCUCCGGCUGCAUUGUCACAGUCGAAACCAAACCGGGACCAGACUUUA

[0165] AGUUCCACUUACUGAAACCACCUGGCUCAAUGCUCACUCACGGCUCAGUUCCAUCCGACCUGAUA

[0166] CCUAAAUCCAGUAGCCUGUGGAUCGGAAACAGGCACUGGACCGACAUCGUGGACUUCGUCAUAA

[0167] GGCCAUUUGUGUUCCAGGCUAACAGACACUUCGACUUCAACCAGGAGACUGCCGGAUGGUCCACA

[0168] CCAAGGUUCAGGCCAAUUACCAUCAACAUCAGCCAGUCCAAAGGCGAAAGGCUCGGAAUUGGAG

[0169] UCGCUACUGACUACAUCGUGCCAGGCAUACCAGACGGAUGGCCGGACACCACCAUACCAGAGAAA

[0170] CUCAUACCAGCCGGAAAGUACGCAAUUACCUCCGGAAACAACAACGACAUUGGAAGCGCCAAAGA

[0171] GUACGACGAGGCAGAUGUGAUUCGGAACAACACCAACUUUAAGAGCAUGUACAUCUGCGGAAGC

[0172] CUGCAGAGGGCAUGGGGAGACAAAGAGAUCUCCAACACCGGCUUUAUCACUACAGCCACCGUGG

[0173] UGGGAAACGCCUUGGUGCCAUCUAACACCAUUGACCAGACCAAGAUUGCCGUGUUCCAAGACAAC

[0174] CACGUCAACACCGAGGUGCAGACCAGCGACGUGACACUGGCACUGUUGGGAUACACCGGAAUCGG

[0175] AGAAGAGGCUAUCGGAGUGGACAGAGAGAAAGUGGUCAGAAUCAGCGUGUUACCAGAAACUGGU

[0176] GCCAGAGGUGGAAACCACCCAAUCUUCUACAAGAACAAGAUGAAACUCGGCUACGUCAUCAGGG

[0177] AGAUCGACGUGUUCAACAGCCAGAUUCUCCACACCAGCAGGCAGCUCUCUCUGAAUAACUACCUA

[0178] UUGCCACCAGACAGCUUUGCCGUCUACAGAAUCAUCGACGCCAACGGAAGCUGGUUUGACAUCGG

[0179] AAUUGACAGCGACGGAUUCAGCUUUGUCGGAGUCUCCAACAUUUCCAAACUAGAGUUUCCGCUG

[0180] ACCGCAUCUUACAUGGGAAUCCAGCUGGCAAAGAUCAGGCUGGCAUCCAAUAUCCGCUCAUCCAU

[0181] GACCAAACUCGACUACAAAGACGACGACGACAAGUAAUAAGGUACCGAUAUCUGAUAAUAGGCU

[0182] GGAGCCUCGGUGGCCAUGCUUCUUGCCCCUUGGGCCUCCCCCCAGCCCCUCCUCCCCUUCCUGCAC

[0183] CCGUACCCCCGUGGUCUUUGAAUAAAGUCUGAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA

[0184] AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA

[0185] AAAAAAAAA

[0186] The DNA sequence of the VP2 antigen of FPV is shown in SEQ ID NO: 10:

[0187]

[0188] The amino acid sequence of the VP2 antigen of FPV is shown in SEQ ID NO: 3:

[0189] MSDGAVQPDGGQPAVRNERATGSGNGSGGGGGGGSGGVGISTGTFNNQTEFKFLENGWVEITANSSRLVHLNMPESENYKRVVVNNMDKTAVKGNMALDDTHVQIVTPWSLVDANAWGVWFNPGDWQLIVNTMSELHLVSFEQEIFNVVLKTVSESATQPPTKVYNNDLTASLMVALDSNNTMPFTPAAMRSETLGFYPWKPTIPTPWRYYFQWDRRLIPSHTGTSGTPTNVYHGTDPDDVQFYTIENSVPVHLLRTGDEFATGTFFFDCKPCRLTHTWQTNRALGLPPFLNSLPQSEGATNFGDIGVQQDKRRGVTQMGNTDYITEATIMRPAEVGYSAPYYSFEASTQGPFKTPIAAGRGGAQTDENQAADGDPRYAFGRQHGQKTTTTGETPERFTYIAHQDTGRYPEGDWIQNINFNLPVTNDNVLLPTDPIGGKTGINYTNIFNTYGPLTALNNVPPVYPNGQIWDKEFDTDLKPRLHVNAPFVCQNNCPGQLFVKVAPNLTNEYDPDASANMSRIVTYSDFWWKGKLVFKAKLRASHTWNPIQQMSINVDNQFNYVPNNIGAMKIVYEKSQLAPRKLYDYKDDDDK

[0190] The mRNA sequence of the VP2 antigen of FPV is shown in SEQ ID NO: 11:

[0191] GAAAUAAGAGAGAAAAGAAGAGUAAGAAGAAAUAUAAGAGCCACCGCUAGCCUCGAGGCCA

[0192] CCAUGGACGCCAUGAAGAGGGGGCUGUGCUGCGUGCUGCUGCUGUGCGGAGCCGUGUUCGUGAG

[0193] CGCCUCCAUGUCUGAUGGAGCCGUCCAGCCUGAUGGUGGACAGCCAGCCGUCAGAAACGAAAGAG

[0194] CAACCGGAUCUGGAAACGGAAGCGGAGGCGGAGGUGGAGGCGGCUCCGGCGGAGUCGGCAUCUC

[0195] CACCGGCACCUUUAACAACCAGACCGAAAUUUAAGUUCUUGGAAAACGGAUGGGUCGAGAUCACC

[0196] GCCAACUCCAGCAGUUGGACACCUGAACAUGCCAGAUCAGAGAACUACGGGUCGUGG

[0197] UGAACAACAUGGACAAGACCGCAGUGAAGGCAACAUGGCACUGGACGACACUCACGUGCAGAU

[0198] UGUGACUCCGUGGAGCCUAGUGGACGCCAACGCAUGGGGUGUGUGGUUUAAUCCAGGCGACUGG

[0199] CAGUUGAUGUCAACACCAUGUCCGAGUUACACUUAGUGAGCCUUUGAGAGGAAUAUUCAACG

[0200] UCGUGCUCAAGACAGUGAGCGAGAGCCGUACACAGCCACCAACGAAAGUGUAUAACAACGACCU

[0201] GACCGCCAGCCUAAUGGUGGCUCUGGACUCCAACAACACCAUGCCAUUCACACCAGCCGCAAUGA

[0202] GGUCCGAGACUCUGGGAUUCUAUCCGUGGAAACCAACCAUACCUACCCCUUGGAGGUACUACUUC

[0203] CAGUGGGACAGACGGCUGAUUCCAUCUCACACAGGCACCUCCGGAACACCUACUAACGUGUACCA

[0204] CGGUACAGAUCCAGACGACGUGCAGUUCUACACCAUCGAGAACUCCGUGCCAGUGCACUUGCUCA

[0205] GAACCGGAGACGAAUUUGCCACCGGAACCUUCUUCUUUGACUGCAAACCUUGCAGAUUAACGCAC

[0206] ACCUGGCAGACCAACAGAGCACUCGGAUUGCCACCAAUUUCUGAGACAGUCUGCCCACAAAGCGAGGG

[0207] AGCAACCAACUUCGGCGACAUUGGAGUGCAACAAGACAAACGAAGGGUGUGACUCAGAUGGGA

[0208] AACACCGACUACAUCACCGAGGCUACCAUCAUGAGGCCUGCAGAAGUGGGAUACUCCGCUCCAUA

[0209] CUACAGCUUCGAGGCCAGCACACAGGGACCAUUCAAGACGCCAAUUGCAGCCGGAAGAGGCGGAG

[0210] CACAGACUGACGAGAACCAGGCAGCAGACGGCGACCCCAAGAUACGCCUUCGGGCAGACAGCACGGA

[0211] CAGAAAAACAACCACUACCGGCGAAAACACGAAACGCUUCACCUACAUCGCACAACCAAGACACCGG

[0212] AAGAUAUCCAGAGGGAGAGACUGGAUACAGAACAUCAACUUCAACCAUCCAGUGAGACCAACGACAAC

[0213] GUCUUGCUUCCAACCGAUCCAAUCGGCGGAAAGACUGGAAUCAACUACACCAACAUCUUAACAC

[0214] CUACGGACCACUGAGCGACAUUAAACAACGUUCCACCAGUCUAUCCAAACGGACAAAUCUGGGACA

[0215] AAGAGUUGACCCGACCUGAAACCUAGACUGCACGUGAGAAUGCACCAUUCGUCUCAGAACAA

[0216] CUGCCCUGGACAGCUGUUCGUCAAAGUGGCUCCAAACCUGACCAACGAGUACGAUCCAGACGCCU

[0217] CAGCAAACAUGUCCAGAAUUGUGACUUACAGCGACUUCUGGUGGAAGGGCAAACUGGUGUUCAA

[0218] GGCCAAGCUGCGAGCAAGCCACACCUGGAAUCCAAUCCAGCAGAUGUCCAUCAACGUGGACAACC

[0219] AGUUCAACUACGUGCCAAACAACAUCGGCGCAAUGAAGAUUGUGUACGAGAAAUCACAGCUGGC

[0220] ACCACGCAAAUUGUACGACUACAAAGACGACGACGACAAAUAAGGUACCGAUAUCUGAUAAUAG

[0221] GCUGGAGCCUCGGUGGCCAUGCUUCUUGCCCCUUGGGCCUCCCCCCAGCCCCUCCUCCCCUUCCU

[0222] GCACCCGUACCCCCGUGGUCUUUGAAUAAAGUCUGAAAAAAAAAAAAAAAAAAAAAAAAAAAAA

[0223] AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA

[0224] AAAAAAAAAAAAAA

[0225] The DNA sequence of the gD antigen of FHV is shown in SEQ ID NO: 14:

[0226] ATGATGACACGATTGCACTTCTGGTGGTGCGGAATATTTGCCGTGCTGAAATACCTGGTCTGCACC

[0227] TCCAGTCTGACTACCACGCCAAAGACGACCACCGTGTACGTGAAAGGCTTCAACATTCCACCACTGA

[0228] GGTACAATTACACGCAGGCCAGAATCGTGCCAAAGATTCCACAGGCAATGGACCCAAAGATTACAGC

[0229] TGAGGTCAGATACGTGACTAGCATGGACAGCTGCGGAATGGTGGCTCTGATCTCCGAGCCAGACATC

[0230] GACGCCACCATCCGCACCATTCAGCTAAGCCAGAAAAAAACCTATAACGCCACCATTTCTTGGTTCAA

[0231] GGTCACACAGGGATGTGAATACCCTATGTTTCTCATGGACATGAGGCTCTGTGATCCAAAGAGGGAGT

[0232] TTGGCATCTGCGCACTGAGATCTCCGTCTTACTGGTTGGAGCCACTCACCAAATACATGTTTCTGACG

[0233] GACGACGAACTCGGCCTGATTATGATGGCACCAGCACAGTTCAACCAGGGACAGTACAGAAGGGTGA

[0234] TCACCATTGACGGCAGCATGTTCTACACCGACTTTATGGTGCAGCTGAGTCCAACACCATGCTGGTTT

[0235] GCCAAACCTGACAGATACGAAGAGATTCTCCACGAATGGTGCAGAAACGTCAAGACCATCGGACTGG

[0236] ACGGAGCCAGAGACTACCACTACTACTGGGTGCCATACAATCCACAGCCACACCACAAGGCAGTTCT

[0237] GCTCTACTGGTACAGAACACACGGAAGAGAACCACCAGTGAGATTCCAAGAAGCAATCAGATACGAC

[0238] AGACCAGCAATACCATCCGGCTCCGAAGATTCCAAACGGAGCAACGACTCCAGAGGCGAAAGCTCC

[0239] GGACCAAACTGGATAGACATCGAGAACTACACGCCTAAGAACAACGTGCCAATCATCATCTCCGACG

[0240] ACGACGTGCCAACCGCACCACCAAAGGGAATGAACAACCAGTCAGTGGTCGATTATAAAGACGATGA

[0241] TGATAAATAA

[0242] The amino acid sequence of the gD antigen of FHV is as shown in SEQ ID NO: 5:

[0243] MMTRLHFWWCGIFAVLKYLVCTSSLTTTPKTTTVYVKGFNIPPLRYNYTQARIVPKIPQAMDPKITAE

[0244] VRYVTSMDSCGMVALISEPDIDATIRTIQLSQKKTYNATISWFKVTQGCEYPMFLMDMRLCDPKREFGICA

[0245] LRSPSYWLEPLTKYMFLTDDELGLIMMAPAQFNQGQYRRVITIDGSMFYTDFMVQLSPTPCWFAKPDRYE

[0246] EILHEWCRNVKTIGLDGARDYHYYWVPYNPQPHHKAVLLYWYRTHGREPPVRFQEAIRYDRPAIPSGSE

[0247] DSKRSNDSRGESSGPNWIDIENYTPKNNVPIIISDDDVPTAPPKGMNNQSVVDYKDDDDK

[0248] The mRNA sequence of the gD antigen of FHV is as shown in SEQ ID NO: 15:

[0249] GAAAUAAGAGAGAAAAGAAGAGUAAGAAGAAAUAUAAGAGCCACCGCUAGCCUCGAGGCCA

[0250] CCAUGGACGCCAUGAAGAGGGGGCUGUGCUGCGUGCUGCUGCUGUGCGGAGCCGUGUUCGUGAG

[0251] CGCCUCCAUGAUGAUGACACGAUUGCACUUCUGGUGGUGCGGAAUAUUUGCCGUGCUGAAAUAC

[0252] CUGGUCUGCACCUCCAGUCUGACUACCACGCCAAAGACGACCACCGUGUACGUGAAAGGCUUCAA

[0253] CAUUCCACCACUGAGGUACAAUUACACGCAGGCCAGAAUCGUGCCAAAGAUUCCACAGGCAAUGG

[0254] ACCCAAAGAUUACAGCUGAGGUCAGAUACGUGACUAGCAUGGACAGCUGCGGAAUGGUGGCUCU

[0255] GAUCUCCGAGCCAGACAUCGACGCCACCAUCCGCACCAUUCAGCUAAGCCAGAAAAAAACCUAUA

[0256] ACGCCACCAUUUCUUGGUUCAAGGUCACACAGGGAUGUGAAUACCCUAUGUUUCUCAUGGACAU

[0257] GAGGCUCUGUGAUCCAAAGAGGGAGUUUGGCAUCUGCGCACUGAGAUCUCCGUCUUACUGGUUG

[0258] GAGCCACUCACCAAAUACAUGUUUCUGACGGACGACGAACUCGGCCUGAUUAUGAUGGCACCAGC

[0259] ACAGUUCAACCAGGGACAGUACAGAAGGGUGAUCACCAUUGACGGCAGCAUGUUCUACACCGAC

[0260] UUUAUGGUGCAGCUGAGUCCAACACCAUGCUGGUUUGCCAAACCUGACAGAUACGAAGAGAUUC

[0261] UCCACGAAUGGUGCAGAAACGUCAAGACCAUCGGACUGGACGGAGCCAGAGACUACCACUACUAC

[0262] UGGGUGCCAUACAAUCCACAGCCACACCACAAGGCAGUUCUGCUCUACUGGUACAGAACACACGG

[0263] AAGAGAACCACCAGUGAGAUUCCAAGAAGCAAUCAGAUACGACAGACCAGCAAUACCAUCCGGC

[0264] UCCGAAGAUUCCAAACGGAGCAACGACUCCAGAGGCGAAAGCUCCGGACCAAACUGGAUAGACA

[0265] UCGAGAACUACACGCCUAAGAACAACGUGCCAAUCAUCAUCUCCGACGACGACGUGCCAACCGCA

[0266] CCACCAAAGGGAAUGAACAACCAGUCAGUGGUCGAUUAUAAAGACGAUGAUGAUAAAUAAUAAG

[0267] GUACCGAUAUCUGAUAAUAGGCUGGAGCCUCGGUGGCCAUGCUUCUUGCCCCUUGGGCCUCCCCC

[0268] CAGCCCCUCCUCCCCUUCCUGCACCCGUACCCCCGUGGUCUUUGAAUAAAGUCUGAAAAAAAAAA

[0269] AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA

[0270] AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA

[0271] The DNA sequence of the gB antigen of FHV is shown in SEQ ID NO: 12:

[0272] ATGTCTACACGCGGAGATTTGGGAAAGCGAAGAAGAGGTTCCAGATGGCAAGGTCACAGTGGAT

[0273] ACTTCCGACAAAGGTGCTTCTTTCCGTCTCTTCTGGGAATTGCAGCAACTGGAAGTAGACACGGAAA

[0274] CGGAAGTAGTGGTCTGACAAGGTTGGCAAGGTACGTGAGCTTCATCTGGATCGTGCTGTTCTTGGTGG

[0275] GACCAAGACCAGTAGAAGGTCAGTCTGGAAGCACTTCTGAGCAACCAAGACGAACTGTAGCAACTC

[0276] CAGAAGTAGGTGGAACACCACCAAAGCCAACCACTGATCCAACTGACATGTCCGACATGAGAGAAG

[0277] CACTGAGAGCATCTCAGATCGAAGCAAACGGACCATCTACCTTCTACATGTGTCCACCACCATCTGGA

[0278] AGTACTGTGGTAAGACTGGAACCACCAAGAGCATGTCCAGACTACAAGCTGGGAAAGAACTTCACC

[0279] GAAGGTATCGCAGTGATCTTCAAAGAGAACATCGCACCATACAAGTTCAAGGCCAACATCTACTACAA

[0280] GAACATCATCATGACCACTGTGTGGAGTGGAAGCTCTTACGCAGTGACTACCAACAGGTACACTGAC

[0281] AGAGTACCAGTGAAAGTGCAAGAGATCACTGACCTGATCGATAGACGCGGAATGTGTCTGTCCAAAG

[0282] CAGACTATGTGCGCAACAACTACCAGTTCACAGCATTCGATCGAGATGAAGATCCACGAGAATTGCCA

[0283] CTGAAACCGTCCAAGTTCAACACGCCAGAATCTCGCGGATGGCACACAACCAACGAAACCTACACCA

[0284] AGATCGGAGCAGCAGGCTTTCACCACTCTGGAACCTCTGTGAACTGCATCGTGGAAGAAGTGGATGC

[0285] AAGGTCTGTGTATCCGTACGACTCTTTCGCCATCTCTACTGGAGATGTGATCCACATGAGTCCGTTCTT

[0286] CGGACTGAGAGATGGAGCACATGTGGAACACACCAGCTACAGCTCCGATAGGTTCCAGCAGATTGAG

[0287] GGCTACTATCCGATCGATCTGGACACAAGGTTGCAACTTGGAGCACCAGTAAGTCGCAACTTTCTGGA

[0288] GACTCCACACGTAACTGTGGCTTGGAACTGGACACCAAAGTCTGGAAGAGTGTGTACACTGGCTAAA

[0289] TGGCGAGAGATTGACGAGATGCTGAGAGACGAGTATCAGGGATCTTACCGCTTCACAGCCAAGACCA

[0290] TCAGTGCAACCTTCATCTCCAACACAAGCCAGTTCGAGATCAACCGCATCAGACTGGGAGATTGTGC

[0291] AACCAAAGAAGCAGCAGAAGCAATCGATCGCATCTACAAGTCCAAGTACTCCAAGACACACATCCAG

[0292] ACAGGTACACTGGAAACCTACCTAGCAAGAGGTGGATTCTTGATCGCCTTCAGACCGATGATCTCCAA

[0293] CGAACTGGCAAAGCTGTACATCAACGAACTGGCAAGATCCAACCGAACTGTGGACTTGTCTGCACTA

[0294] CTGAATCCAAGTGGAGAAACCGTACAACGCACAAGAAGGTCTGTACCATCCAATCAGCACCATAGAA

[0295] GCCGAAGATCCACCATCGAAGGTGGAATCGAGACTGTGAACAACGCATCTCTGCTCAAGACCACTTC

[0296] CAGTGTAGAGTTCGCAATGCTGCAGTTTGCCTACGACTACATCCAAGCTCACGTAAACGAGATGCTGT

[0297] CCAGAATCGCAACAGCATGGTGTACCTTGCAGAACAGAGAACACGTACTGTGGACTGAGACTCTGAA

[0298] GCTGAATCCAGGTGGAGTAGTGTCTATGGCACTAGAACGAAGAGTGTCTGCAAGGTTACTGGGAGAT

[0299] GCAGTAGCAGTGACTCAATGCGTGAACATCAGCTCTGGTCATGTGTACATCCAGAACTCCATGAGAGT

[0300] GACTGGATCTTCCACAACCTGCTACAGTAGGCCATTGGTGAGCTTTCGAGCACTAAACGACAGTGAGT

[0301] ACATCGAAGGTCAACTTGGAGAGAACAACGAGCTTCTGGTAGAGAGAAAGCTGATCGAGCCATGTAC

[0302] CGTGAACAACAAGCGCTACTTCAAGTTCGGAGCAGATTACGTGTACTTCGAGGACTATGCCTACGTAC

[0303] GCAAAGTACCACTGAGTGAGATCGAGCTGATCTCTGCATACGTGGATCTGAACCTGACACTGCTTGAA

[0304] GATCGCGAATTTCTGCCACTAGAGGTGTACACCAGAGCTGAACTGGAAGATACTGGCTTACTGGACTA

[0305] CTCCGAGATTCAGCGAAGAAACCAGCTTCATGCTCTGAAGTTCTACGACATCGACTCCATCGTGAGAG

[0306] TGGACAACAACCTGGTGATCATGAGAGGTATGGCCAACTTCTTCCAGGGACTTGGAGATGTTGGAGC

[0307] TGGATTTGGCAAGGTAGTGTTAGGTGCAGCATCTGCAGTGATCTCCACTGTGAGTGGAGTATCCAGCT

[0308] TTCTGAACAATCCGTTTGGAGCATTAGCAGTTGGATTGCTGATTCTGGCTGGAATCGTAGCAGCATTTC

[0309] TGGCATACAGGTACATCAGTCGCTTGAGAGCCAATCCGATGAAGGCACTGTATCCAGTGACTACACGC

[0310] AATCTGAAGCAGACTGCAAAGAGTCCAGCATCTACTGCTGGAGGTGATAGTGATCCAGGTGTAGATG

[0311] ACTTCGACGAAGAGAAGCTGATGCAAGCAAGAGAGATGATCAAGTACATGTCTCTGGTGTCTGCAAT

[0312] GGAACAGCAAGAACACAAGGCCATGAAGAAGAACAAGGGACCAGCAATTCTGACCTCTCATCTGAC

[0313] CAACATGGCACTGAGAAGAAGAGGTCCAAAGTACCAACGACTGAACAACCTGGATTCTGGAGATGAT

[0314] ACCGAGACAAACCTAGTGGACTACAAAGACGATGACGACAAG

[0315] The amino acid sequence of the gB antigen of FHV is shown in SEQ ID NO: 4:

[0316] MSTRGDLGKRRRGSRWQGHSGYFRQRCFFPSLLGIAATGSRHGNGSSGLTRLARYVSFIWIVLFLVG

[0317] PRPVEGQSGSTSEQPRRTVATPEVGGTPPKPTTDPTDMSDMREALRASQIEANGPSTFYMCPPPSGSTVVR

[0318] LEPPRACPDYKLGKNFTEGIAVIFKENIAPYKFKANIYYKNIIMTTVWSGSSYAVTTNRYTDRVPVKVQEIT

[0319] DLIDRRGMCLSKADYVRNNYQFTAFDRDEDPRELPLKPSKFNTPESRGWHTTNETYTKIGAAGFHHSGTS

[0320] VNCIVEEVDARSVYPYDSFAISTGDVIHMSPFFGLRDGAHVEHTSYSSDRFQQIEGYYPIDLDTRLQLGAP

[0321] VSRNFLETPHVTVAWNWTPKSGRVCTLAKWREIDEMLRDEYQGSYRFTAKTISATFISNTSQFEINRIRLG

[0322] DCATKEAAEAIDRIYKSKYSKTHIQTGTLETYLARGGFLIAFRPMISNELAKLYINELARSNRTVDLSALLN

[0323] PSGETVQRTRRSVPSNQHHRSRRSTIEGGIETVNNASLLKTTSSVEFAMLQFAYDYIQAHVNEMLSRIATA <​​​​

[0326] AYVDLNLTLLEDREFLPLEVYTRAELEDTGLLDYSEIQRRNQLHALKFYDIDSIVRVDNNLVIMRGMANFF

[0327] QGLGDVGAGFGKVVLGAASAVISTVSGVSSFLNNPFGALAVGLLILAGIVAAFLAYRYISRLRANPMKALY

[0328] PVTTRNLKQTAKSPASTAGGDSDPGVDDFDEEKLMQAREMIKYMSLVSAMEQQEHKAMKKNKGPAILTS

[0329] HLTNMALRRRGPKYQRLNNLDSGDDTETNLVDYKDDDDK

[0330] The mRNA sequence of the gB antigen of FHV is shown in SEQ ID NO: 13:

[0331]

[0332] 2. Sequence synthesis and plasmid construction

[0333] The sequences of the different antigens designed in section 1 above and the optimized antigens were synthesized, and plasmids were constructed as follows:

[0334] The following gene sequences were synthesized by Shanghai Sangon Biotech Co., Ltd.: from 5' to 3' end, they are T7 promoter (SEQ ID NO: 16), 5'UTR (SEQ ID NO: 17), kozak sequence, tPA signal peptide sequence (SEQ ID NO: 18), DNA sequence of rabies virus G protein (SEQ ID NO: 6), or DNA sequence of feline calicivirus VP1 protein (SEQ ID NO: 8), or DNA sequence of feline parvovirus VP2 protein (SEQ ID NO: 10), or DNA sequence of feline herpesvirus gB protein (SEQ ID NO: 12), or DNA sequence of feline herpesvirus gD protein (SEQ ID NO: 14), 3'UTR (SEQ ID NO: 19), and polyA (SEQ ID NO: 20). A Bsa I restriction site was introduced after the polyA sequence to facilitate plasmid linearization. Using genetic engineering, the above target gene sequences were constructed into the cloning vector pUC57, and plasmids pUC57-RV-G, pUC57-FCV-VP1, pUC57-FPV-VP2, pUC57-FHV-gD, and pUC57-FHV-gB were constructed. These plasmids were then transformed into competent E. coli cells, and large-scale culture of the five bacteria and large-scale extraction of the five plasmids were performed.

[0335] The sequences involved in the construction of the above plasmids are as follows:

[0336] The DNA sequence of the 5' untranslated region (5'UTR) is shown in SEQ ID NO: 17:

[0337] GAAATAAGAGAGAAAAGAAGAGTAAGAAGAAATATAAGAGCCACCGCTAGCCTCGAG

[0338] The DNA sequence of the 3' untranslated region (3'UTR) is shown in SEQ ID NO: 19:

[0339] GATATCTGATAATAGGCTGGAGCCTCGGTGGCCATGCTTCTTGCCCCTTGGGCCTCCCCCCAGCCC CTCCTCCCCTTCCTGCACCCGTACCCCCGTGGTCTTTGAATAAAGTCTG

[0340] Polyadenylate (polyA) has the sequence shown in SEQ ID NO: 20, containing 104 bases A:

[0341] AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA

[0342] The DNA sequence of the T7 promoter is shown in SEQ ID NO: 16:

[0343] TAATACGACTCACTATAGG

[0344] The DNA sequence of the signal peptide tPA is shown in SEQ ID NO: 18:

[0345] ATGGACGCCATGAAGAGGGGGCTGTGCTGCGTGCTGCTGCTGTGCGGAGCCGTGTTCGTGAGCGCCTCC

[0346] In addition, the plasmids obtained by following the above method with the sequences of RV G antigen (without transmembrane region) (SEQ ID NO: 21), FHV gB antigen (without transmembrane region) (SEQ ID NO: 22) and FHV gD antigen (with transmembrane region) (SEQ ID NO: 23) were pUC57-RV-G-1, pUC57-FHV-gD-1 and pUC57-FHV-gB-1.

[0347] Example 2. mRNA transcription verification experiment

[0348] The plasmids pUC57-RV-G, pUC57-FCV-VP1, pUC57-FPV-VP2, pUC57-FHV-gD, and pUC57-FHV-gB obtained in Example 1 were digested with Bsa I restriction endonuclease from Novizan Biosciences and linearized at 37°C for 1 hour. DNA templates were recovered using a DNA recovery kit from TransGen. The linearization system is shown in Table 1.

[0349] Table 1. Plasmid linearization reaction system

[0350]

[0351] The plasmid was then used to synthesize mRNA in vitro using the T7 transcription kit from Yisheng Biotechnology, while a capping structure was added to the mRNA using Cap101 from Cangzhou Weikexin Biotechnology. The reaction was carried out at 37°C for 3 hours, with specific reaction conditions as shown in Table 2. After the reaction, 1.5 volumes of lithium chloride solution were added to the system, and the mixture was incubated at -20°C for 1 hour to precipitate the mRNA. Subsequently, the mixture was centrifuged at 10,000 rpm for 10 minutes, the supernatant was removed, and the mRNA was redissolved in DEPC-treated water. The RNA concentration was determined using a Nanodrop device and stored at -80°C. A 1% agarose gel was prepared and electrophoresis was performed to evaluate the quality of the synthesized RNA.

[0352] The results are as follows Figure 1 As shown, from Figure 1 As can be seen, the five in vitro synthesized mRNA bands of plasmids pUC57-RV-G, pUC57-FCV-VP1, pUC57-FPV-VP2, pUC57-FHV-gD, and pUC57-FHV-gB are all clear, single, and consistent with the expected size.

[0353] Table 2 In vitro transcription reaction system

[0354]

[0355] Example 3. Protein expression validation experiment and antigen optimization selection

[0356] The mRNA synthesized in vitro in Example 2 was introduced into HEK-293T cells using TransGen's PEI transfection reagent, and Western blot experiments were performed to examine its expression capacity. First, 293T cells were evenly seeded into 24-well culture plates, and then PEI was used for mRNA transfection, adding 0.8 μg of mRNA to each well, following the manufacturer's instructions. After 48 hours of culture, cell samples were collected and analyzed by SDS-PAGE electrophoresis. After electrophoresis, the proteins were transferred to a PVDF membrane. The membrane was then blocked overnight with 5% skim milk powder and incubated with TransGen's anti-FLAG-tagged monoclonal antibody and TransGen's HRP-labeled goat anti-rabbit IgG secondary antibody. After incubation at room temperature for 1 hour, ECL chemiluminescence staining was performed using Beyotime Biotechnology's ECL chromogenic solution to obtain the Western blot results.

[0357] The results are as follows Figure 2 As shown, from Figure 2As can be seen, compared with the blank control group, the experimental group transfected with five mRNAs (transcribed from plasmids pUC57-RV-G, pUC57-FCV-VP1, pUC57-FPV-VP2, pUC57-FHV-gD, and pUC57-FHV-gB) all showed specific and uniform bands on the Western blot, consistent with the expected protein size. This confirms that the mRNAs can be successfully expressed in cells. Transfecting cells with a mixture of the five mRNAs in equal proportions showed that all five mRNAs were expressed simultaneously, indicating that their expression does not interfere with each other and can be used for mixed immunization.

[0358] Furthermore, following the above method, the three mRNAs transcribed from pUC57-RV-G, pUC57-FHV-gD, and pUC57-FHV-gB in Example 2, as well as the three mRNAs transcribed from plasmids pUC57-RV-G-1, pUC57-FHV-gD-1, and pUC57-FHV-gB-1, were transfected in vitro. The results... Figure 3 As shown in AC, from Figure 3 As can be seen from AC, RV G antigen with and without transmembrane regions, FHV gB antigen, and FHV gD antigen can all express proteins, indicating that the mRNA was successfully constructed.

[0359] The mRNAs of RV G antigen (without a transmembrane region), FHV gB antigen (without a transmembrane region), and FHV gD antigen (with a transmembrane region) constructed in Example 2, as well as the mRNAs of RV G antigen, FHV gB antigen, and FHV gD antigen, were used in mouse immunization experiments according to the materials and methods in Example 4 (15 8-week-old SPF-grade BALB / c mice purchased from Liaoning Changsheng Biotechnology Co., Ltd. were randomly selected). Each mRNA was divided into three groups: a blank control group, an mRNA-1 group, and an mRNA-2 group, with 5 mice in each group. The mRNA-1 group consisted of mRNAs without a transmembrane region, while the mRNA-2 group consisted of mRNAs with a transmembrane region. Each mouse was injected intramuscularly with 10 μg of mRNA. Two weeks after the initial immunization, a second immunization was performed. Two weeks after the second immunization, the titer was measured. The results are as follows: Figure 4 As shown in AC.

[0360] from Figure 4 As can be seen from A, the mRNA of FHV gB antigen with a transmembrane region has a higher titer than the mRNA of FHV gB antigen (without a transmembrane region); from Figure 4 As can be seen from B, the mRNA of FHV gD antigen without a transmembrane region has a higher titer than the mRNA of FHV gD antigen (with a transmembrane region); from Figure 4As can be seen from C, the mRNA of RVG antigen with a transmembrane region has a higher titer than the mRNA of RVG antigen (without a transmembrane region).

[0361] Therefore, based on the commonly used RV G antigen (without transmembrane region) (SEQ ID NO: 21), FHV gB antigen (without transmembrane region) (SEQ ID NO: 22), and FHV gD antigen (with transmembrane region) (SEQ ID NO: 23) in the prior art, after sequence optimization, the sequences of RV G antigen (with transmembrane region) (SEQ ID NO: 1), FHV gB antigen (with transmembrane region) (SEQ ID NO: 4), and FHV gD antigen (without transmembrane region) (SEQ ID NO: 5) were selected and combined with FCV-VP1 antigen (SEQ ID NO: 2) and FPV-VP2 antigen (SEQ ID NO: 3) to prepare a quadrivalent mRNA vaccine. That is, the mRNA transcribed from plasmids pUC57-RV-G, pUC57-FCV-VP1, pUC57-FPV-VP2, pUC57-FHV-gD, and pUC57-FHV-gB is used to prepare a quadrivalent mRNA vaccine.

[0362] Example 4. Preparation of mRNA vaccine

[0363] The mRNA package obtained in Example 2 was prepared as an mRNA vaccine (LNP-mRNA vaccine).

[0364] The specific experimental steps are as follows: First, an alcohol phase was prepared by dissolving lipids (SM102), distearate phosphatidylcholine (DSPC), cholesterol, and DMG-2000 in anhydrous ethanol at a ratio of 50:10:38.5:1.5 (mass ratio). Next, an aqueous phase was prepared using 50 mM citrate buffer (pH 4.0) as the medium for dissolving mRNA. The mRNA was packaged using a microfluidic device with the alcohol and aqueous phases (volume ratio 1:3). Then, it was diluted with RNase-free PBS buffer and concentrated using a 30 kDa ultrafiltration tube. An equal volume of 20% sucrose PBS solution was added to adjust the mRNA concentration to 100 μg / ml, with a sucrose concentration of 10%. Finally, the mixture was filtered through a 0.22 μm filter to obtain the prepared LNP-mRNA vaccine, which was then aliquoted and stored at -20°C.

[0365] The liposome nanoparticles contain at least one of cationic lipids, distearate phosphatidylcholine (DSPC), cholesterol, and DMG-2000.

[0366] Particle size distribution was detected by dynamic light, and the results are as follows: Figures 5A-5E As shown, from Figures 5A-5EIt can be seen that all five LNP-mRNA vaccines prepared exhibit uniform particle size, with a particle size of approximately 120 nm.

[0367] The LNP-mRNA vaccine expressing rabies virus G protein, the LNP-mRNA vaccine expressing feline calicivirus VP1 protein, the LNP-mRNA vaccine expressing feline parvovirus VP2 protein, the LNP-mRNA vaccine expressing feline herpesvirus gB protein, and the LNP-mRNA vaccine expressing feline herpesvirus gD protein were mixed in a mass ratio of 2:2:2:1:1 to obtain the quadrivalent mRNA vaccine for the prevention of feline infectious diseases of the present invention. The mixture was then filtered, sterilized, and packaged.

[0368] Example 5. Mouse Immunological Evaluation Experiment

[0369] The quadrivalent mRNA vaccine prepared in Example 4 was used to immunize BALB / c mice, and its immunization effect was evaluated. Fifteen 8-week-old SPF-grade BALB / c mice purchased from Liaoning Changsheng Biotechnology Co., Ltd. were randomly selected and divided into three groups of five mice each. The mRNA vaccine group received an intramuscular injection of 20 μg of mRNA vaccine. Three weeks after the initial immunization, a booster immunization with the same dose of vaccine was administered. Blood samples were collected from the mice six weeks later. The positive control group adopted a "2+1" immunization strategy: an initial subcutaneous injection of 20 μL of Zoetis' feline triple vaccine (Fel-O-Vax PCT) in the back, followed by a booster immunization with the same dose of feline triple vaccine three weeks later, and then a subcutaneous injection of 20 μL of Invesco's inactivated rabies vaccine (Nobivac Rabies) in the back three weeks later. Blood samples were collected three weeks after the final immunization. The blank control group received an intramuscular injection of 100 μL of PBS every three weeks for a total of three times. Blood samples were collected three weeks after the final immunization (the specific procedure is as follows). Figure 6 (As shown). Serum was separated and relevant antibody tests were performed. The feline herpesvirus gD protein ELISA kit was purchased from Shanghai Hengyuan Biotechnology; the feline herpesvirus gB protein ELISA kit, feline calicivirus antibody detection kit, and feline poxvirus antibody detection kit were all purchased from Shanghai Enzyme-Linked Biotechnology. Serum antibody titers were detected using the ELISA kits. The rabies virus serum neutralization test (fluorescent antibody virus neutralization test FAVN) was performed according to the national standard method (GB / T201117 / 97-T-326). The results showed that after mRNA vaccine immunization, the serum rabies virus neutralizing antibody level was significantly increased compared to the blank control group, and the level was higher than that of the inactivated vaccine group (P<0.05).

[0370] Serum IgG antibody test results as follows Figure 7 As shown, from Figure 7It can be seen that the immune efficacy of both vaccine groups was higher than that of the blank control group, and the immune efficacy of FCV, FPV and FHV gB in the mRNA vaccine group was better than that in the inactivated vaccine group.

[0371] Based on the above results, the two-dose combination of the quadrivalent mRNA vaccine is more effective than the "2+1" combination of the inactivated vaccine.

[0372] Example 6. Safety experiment in mice

[0373] The quadrivalent mRNA vaccine prepared in Example 4 was used to immunize BALB / c mice, and its safety was evaluated. Ten 8-week-old SPF-grade BALB / c mice purchased from Liaoning Changsheng Biotechnology Co., Ltd. were randomly selected and divided into a normal control group and a vaccine-immunized group, with five mice in each group. Each group received a single intraperitoneal injection of 50 μg of mRNA vaccine or 100 μL of PBS. Ten days later, mouse tissues were collected for HE staining to observe the pathological changes in the mouse tissues.

[0374] The results are as follows Figure 7 As shown, from Figure 7 It can be seen that a single overdose immunization of mice did not cause damage to the liver, kidneys, or spleen, indicating that the mRNA vaccine has high safety.

[0375] In summary, the present invention relates to a quadrivalent mRNA vaccine for the prevention of feline infectious diseases and its application. Through sequence optimization, a quadrivalent mRNA vaccine containing RV G antigen, FHV gB antigen, and FHV gD antigen was designed. Unexpectedly, through sequence optimization, it was found that compared to the RV G antigen (without a transmembrane region), FHV gB antigen (without a transmembrane region), and FHV gD antigen used in the prior art, this vaccine offers significant advantages. The mRNA obtained from the sequence of the gD antigen (with a transmembrane region) has a higher titer and expression level after optimization, exhibiting strong immunogenicity. It can simultaneously prevent feline rabies, feline calicivirus disease, feline panleukopenia, and feline rhinotracheitis through a mixed vaccine, overcoming the problems of separate immunizations, complex procedures, and high injection costs in existing technologies for preventing these diseases. Furthermore, experiments have shown that the quadrivalent mRNA vaccine of this invention has a higher immunogenicity than inactivated vaccines, and is relatively safer and more potent, reducing injection costs and optimizing the immunization procedure. This is of great significance for the prevention of feline infectious diseases such as feline rabies, feline calicivirus disease, feline panleukopenia, and feline rhinotracheitis.

[0376] It will be apparent to those skilled in the art that the present invention is not limited to the details of the exemplary embodiments described above, and that the invention can be implemented in other specific forms without departing from its spirit or essential characteristics. Therefore, the embodiments should be considered in all respects as exemplary and non-limiting, and the scope of the invention is defined by the appended claims rather than the foregoing description. Thus, all variations falling within the meaning and scope of equivalents of the claims are intended to be included within the present invention. No reference numerals in the claims should be construed as limiting the scope of the claims.

[0377] Furthermore, it should be understood that although this specification describes embodiments, not every embodiment contains only one independent technical solution. This narrative style is merely for clarity. Those skilled in the art should consider the specification as a whole, and the technical solutions in each embodiment can also be appropriately combined to form other embodiments that can be understood by those skilled in the art.

Claims

1. A combination of nucleic acids, comprising: 1) The first nucleic acid molecule, which includes a first nucleotide sequence encoding the rabies virus G protein or an immunogenic fragment thereof; 2) A second nucleic acid molecule, which includes a second nucleotide sequence encoding the feline calicivirus VP1 protein or an immunogenic fragment thereof; 3) A third nucleic acid molecule, which includes a third nucleotide sequence encoding the feline parvovirus VP2 protein or an immunogenic fragment thereof; 4) A fourth nucleic acid molecule comprising a fourth nucleotide sequence encoding the feline herpesvirus gB protein or an immunogenic fragment thereof; and / or 5) The fifth nucleic acid molecule, which includes the fifth nucleotide sequence encoding the feline herpesvirus gD protein or an immunogenic fragment thereof.

2. The nucleic acid combination according to claim 1, characterized in that, The amino acid sequence of the G protein or its immunogenic fragment has at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% homology with SEQ ID NO:1; The amino acid sequence of the VP1 protein or its immunogenic fragment has at least 85%, 90%, 95%, 96%, 97%, 98% or 99% homology with SEQ ID NO:2; The amino acid sequence of the VP2 protein or its immunogenic fragment has at least 85%, 90%, 95%, 96%, 97%, 98% or 99% homology with SEQ ID NO:3; The amino acid sequence of the gB protein or its immunogenic fragment has at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% homology with SEQ ID NO:4; or The amino acid sequence of the gD protein or its immunogenic fragment has at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% homology with SEQ ID NO:

5.

3. The nucleic acid combination according to claim 1 or 2, characterized in that, The sequence of the first nucleotide has at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% homology with SEQ ID NO: 6 or 7; The sequence of the second nucleotide has at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% homology with SEQ ID NO:8 or 9; The sequence of the third nucleotide has at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% homology with SEQ ID NO:10 or 11; The sequence of the fourth nucleotide has at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% homology with SEQ ID NO:12 or 13; The sequence of the fifth nucleotide has at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% homology with SEQ ID NO:14 or 15, wherein SEQ ID NO:14 is the DNA sequence of the gD protein with the 3' end nucleotides 991 to 1122 deleted.

4. The nucleic acid combination according to any one of claims 1 to 3, characterized in that, Each nucleotide sequence in the nucleic acid combination has a nucleotide sequence encoding a signal peptide linked to its 5' end. Preferably, the signal peptide is tPA, IL-2, IL-6, or a neuropeptide, and the neuropeptide is preferably VIP, CGRP, or NPY.

5. The nucleic acid combination according to claim 4, characterized in that, The nucleotide sequence encoding the signal peptide has a 5' UTR sequence attached to its 5' end, or each nucleotide sequence in the nucleic acid combination has a 3' UTR sequence attached to its 3' end; preferably, each nucleic acid molecule in the nucleic acid combination has a FLAG tag.

6. The nucleic acid combination according to any one of claims 1 to 5, characterized in that, The nucleic acid is either DNA or RNA.

7. A vector combination comprising a first vector constructed from a first nucleic acid molecule in any one of the nucleic acid combinations of claims 1 to 6, a second vector constructed from a second nucleic acid molecule, a third vector constructed from a third nucleic acid molecule, a fourth vector constructed from a fourth nucleic acid molecule, and a fifth vector constructed from a fifth nucleic acid molecule.

8. A cell assembly comprising cells transformed from the carrier assembly of claim 7.

9. A composition for preventing feline infectious diseases, comprising mRNA transcribed from the nucleic acid combination of claims 1 to 6 or the vector combination of claim 7.

10. The composition according to claim 9, characterized in that, The composition is an mRNA vaccine, which is a quadrivalent mRNA vaccine for the prevention of feline infectious diseases. The quadrivalent mRNA vaccine comprises mRNA expressing rabies virus G protein or its immunogenic fragment, mRNA expressing feline calicivirus VP1 protein or its immunogenic fragment, mRNA expressing feline parvovirus VP2 protein or its immunogenic fragment, mRNA expressing feline herpesvirus gB protein or its immunogenic fragment, and mRNA expressing feline herpesvirus gD protein or its immunogenic fragment.

11. The composition according to claim 10, characterized in that, The mRNA sequence expressing the rabies virus G protein or its immunogenic fragment has at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% homology with SEQ ID NO:

7. The mRNA sequence expressing feline calicivirus VP1 protein or its immunogenic fragment has at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% homology with SEQ ID NO:

9. The mRNA sequence expressing feline parvovirus VP2 protein or its immunogenic fragment has at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% homology with SEQ ID NO:

11. The mRNA sequence expressing feline herpesvirus gB protein or its immunogenic fragment has at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% homology with SEQ ID NO:

13. The mRNA sequence expressing feline herpesvirus gD protein or its immunogenic fragment has at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% homology with SEQ ID NO:

15.

12. The composition according to claim 11, characterized in that, The quadrivalent mRNA vaccine comprises the following components in parts by weight: 2-4 parts of mRNA expressing rabies virus G protein or its immunogenic fragment, 2-4 parts of mRNA expressing feline calicivirus VP1 protein or its immunogenic fragment, 2-4 parts of mRNA expressing feline parvovirus VP2 protein or its immunogenic fragment, 1-2 parts of mRNA expressing feline herpesvirus gB protein or its immunogenic fragment, and 1-2 parts of mRNA expressing feline herpesvirus gD protein or its immunogenic fragment; Preferably, the quadrivalent mRNA vaccine comprises the following components in parts by weight: 2 parts of mRNA expressing rabies virus G protein or its immunogenic fragment, 2 parts of mRNA expressing feline calicivirus VP1 protein or its immunogenic fragment, 2 parts of mRNA expressing feline parvovirus VP2 protein or its immunogenic fragment, 1 part of mRNA expressing feline herpesvirus gB protein or its immunogenic fragment, and 1 part of mRNA expressing feline herpesvirus gD protein or its immunogenic fragment.

13. The composition according to any one of claims 9 to 12, characterized in that, It also includes liposomes, wherein the mRNA in the quadrivalent mRNA vaccine is encapsulated in liposome nanoparticles, and the particle size of the liposome nanoparticles is preferably 50-200 nm.

14. The composition according to claim 13, characterized in that, The liposome nanoparticles are selected from at least one of cationic lipids, distearate phosphatidylcholine (DSPC), cholesterol, and DMG-2000. Preferably, the liposome nanoparticles are composed of the following components in parts by weight: 30-80 parts cationic lipids, 5-15 parts distearate phosphatidylcholine (DSPC), 24-52 parts cholesterol, and 1-2 parts DMG-2000. More preferably, the liposome nanoparticles are composed of the following components in parts by weight: 50 parts cationic lipids, 10 parts distearate phosphatidylcholine (DSPC), 38 parts cholesterol, and 1.5 parts DMG-2000.

15. The use of the nucleic acid combination according to any one of claims 1 to 6, the carrier combination according to claim 7, or the composition according to any one of claims 9 to 14 in the preparation of a medicament for the prevention of feline infectious diseases, wherein the feline infectious diseases are feline rhinotracheitis, feline calicivirus disease, feline panleukopenia, and feline rabies.

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