CN115998855B

CN115998855B - A two-unit subunit vaccine for feline infectious rhinotracheitis and feline panleukopenia, and its preparation method and application

Info

Publication number: CN115998855B
Application number: CN202211403538.8A
Authority: CN
Inventors: 康斌; 李雪峰; 张金龙; 武玉梅; 董鹏; 赵炳武; 朱国坡; 王家福
Original assignee: Jinhe Uben Biological Products Co ltd
Current assignee: Jinhe Uben Biological Products Co ltd
Priority date: 2022-11-10
Filing date: 2022-11-10
Publication date: 2025-05-30
Anticipated expiration: 2042-11-10
Also published as: CN115998855A

Abstract

The present invention belongs to the technical field of veterinary biological products, and specifically relates to a bivalent subunit vaccine for feline infectious rhinotracheitis and feline panleukopenia. The present invention innovatively realizes a method for simultaneously producing two viral subunit vaccines in a single expression and purification process. The purification process is simple, and the enzymes used in production can be recycled and reused, thereby reducing production costs. The bivalent subunit vaccine includes a dimer form of feline herpesvirus type 1 gC protein and a VLPs form of feline parvovirus VP2 protein in a molar ratio of 1:1, which improves the purity of the vaccine antigen, has stronger immunogenicity, and lowers the cost.

Description

Bivalent subunit vaccine for cat infectious rhinotracheitis and cat leukopenia, and preparation method and application thereof

Technical Field

The invention belongs to the technical field of biological products for animals, and particularly relates to a bivalent subunit vaccine for cat infectious rhinotracheitis and cat leukopenia, and a preparation method and application thereof.

Background

Infectious rhinotracheitis in cats, also known as cat's nose, is a highly contagious disease characterized by acute upper respiratory symptoms. The pathogen of cat rhino is cat herpesvirus type1 (Felid herpesvirus, FHV-1), FHV-1 belongs to herpesviridae, alpha herpesviridae subfamily, varicella virus genus. The herpesvirus type1 is mainly composed of a core, a capsid, an interlayer and a cyst membrane. The virus capsid is a regular icosahedron with stereo symmetry and has a hexagonal appearance. The FHV-1 genome encodes a variety of proteins, 17 virus-specific proteins and 3 glycoproteins with immunogenicity have been reported. Wherein gB is a major immunogenic protein, expressed in mammalian cells, causing cell fusion and polynuclear formation, which are essential for viral replication, gD plays a major role in firm adsorption of viruses to cells, and is a major neutralizing antibody in body immunization. gC is an important envelope glycoprotein that, by binding to heparan sulfate receptors on the surface of host cells, effects adhesion of FHV-1 to the cells. gC also has immunosuppressive functions, and is capable of binding complement and inhibiting complement-mediated cell killing. In addition, gC is also an important immunogenic protein of FHV-1, capable of inducing cellular immunity and neutralizing antibodies.

Feline panleukopenia, also known as feline pandemic fever, feline infectious enteritis, is an acute, high-contact infectious disease caused by feline parvovirus (Feline panleukopenia virus, FPV). FPV is a single-stranded DNA virus without envelope, belonging to the genus parvoviridae, parvovirus. The FPV genome contains hairpin structures at both ends, and the middle part encodes two proteins, the nonstructural proteins (NS 1, NS 2) and the structural proteins (VP 1, VP 2). The X-ray crystal diffraction technology determination of the FPV capsid protein molecular structure shows that the virus particle is in icosahedral symmetry, and 60 VP1 and VP2 protein molecules form the virus particle capsid protein, wherein 5-6 VP1 molecules and 54-56 VP2 molecules are contained. VP2 protein is the major component of the FPV capsid, and is exposed on the surface of the capsid protein, and VP2 can self-assemble virus-like particles in the absence of VP 1. VP2 is used as a main immunoprotection antigen protein of FPV, can induce the organism to generate neutralizing antibodies, and is the first choice for researching FPV subunit vaccine.

At present, no FHV-1 vaccine is independently developed in China, and the cat nose is mainly prevented and controlled by inoculating an imported vaccine. Although related researches on cat leukopenia vaccines or bivalent and triple vaccines compounded with other cat disease vaccines exist in the prior art, most of bivalent and multi-bivalent vaccines related to the existing cat disease are mixed after being expressed and quantified respectively, so that uniformity of product yield cannot be ensured, for example, bivalent vaccines or bivalent subunit vaccines often have more and less two virus proteins, and equimolar ratio mixing of the two proteins cannot be realized. In addition, the existing bivalent vaccine or bivalent subunit vaccine for preventing and treating diseases related to cat diseases also has the defect that two proteins are used for simultaneously infecting insect cells, so that the infection efficiency of the viruses expressing the proteins is different, and the protein yield is uncontrollable. High frequency recombination also occurs when cells are infected with multiple baculoviruses, resulting in defective virus production that fails to express the desired polyprotein product.

Disclosure of Invention

The invention aims to provide a bigeminal subunit vaccine for cat infectious rhinotracheitis and cat leukopenia, a preparation method and application thereof, the method innovatively realizes the method that two virus subunit vaccines can be produced simultaneously in one expression and purification process, and the purification filler and enzyme for enzyme digestion can be reused, so that the purification process is simple and the production cost is low. The molar ratio of the dimer type feline herpesvirus 1 gC protein to the VLPs type feline parvovirus VP2 protein in the bivalent subunit vaccine is 1:1, the vaccine antigen purity is high, the immunogenicity is stronger, and the cost is lower.

The invention provides a bivalent subunit vaccine for cat infectious rhinotracheitis and cat leukopenia, which comprises SF9-gC-VP2;

The gene encoding SF9-gC-VP2 is a recombinant SF9-gC-VP2 gene, wherein the recombinant SF9-gC-VP2 gene comprises a nucleotide sequence encoding a silkworm immunoglobulin signal peptide, an FHV-1gC gene, a cat IgG FC sequence, a TEV cleavage motif and a FPVVP gene.

Preferably, the amino acid sequence of SF9-gC-VP2 is shown in SEQ ID NO. 7.

Preferably, the nucleotide sequence of the recombinant SF9-gC-VP2 gene is shown as SEQ ID NO. 6.

Preferably, the nucleotide sequence of the encoded silkworm immunoglobulin signal peptide is shown as SEQ ID NO.1, the nucleotide sequence of the FHV-1gC gene is shown as SEQ ID NO.2, the nucleotide sequence of the cat IgG FC is shown as SEQ ID NO.3, the nucleotide sequence of the TEV cleavage motif is shown as SEQ ID NO.4, and the nucleotide sequence of the FPVVP gene is shown as SEQ ID NO. 5.

The invention also provides a preparation method of the bigeminal subunit vaccine for cat infectious rhinotracheitis and cat leukopenia, which comprises the following steps of infecting cells with recombinant SF9-gC-VP2 gene or recombinant baculovirus of recombinant SF9-gC-VP2 gene according to the technical scheme to obtain cell infectious matter supernatant;

Purifying the cell infecting supernatant to obtain a purified target protein;

Performing enzyme digestion on the purified target protein to obtain enzyme-digested protein;

And (3) carrying out virus-like particle self-assembly on the enzyme-cleaved protein, and then mixing with an adjuvant to obtain the bivalent subunit vaccine.

Preferably, the protease used in cleavage of the purified target protein is TEV protease.

Preferably, the expression vector used to prepare the recombinant baculovirus comprises a pFastBac1 vector.

Preferably, the cells comprise insect cells.

Preferably, the insect cells comprise SF9 cells or High Five cells.

The beneficial effects are that:

The invention provides a bivalent subunit vaccine for cat infectious rhinotracheitis and cat leukopenia, which comprises SF9-gC-VP2, wherein the SF9-gC-VP2 comprises a truncated FHV-1gC gene sequence, and a transmembrane region and an intracellular region at the N end and the C end are deleted;

Meanwhile, through adding a TEV protease cleavage motif into the SF9-H-VP2, the cat parvovirus VP2 protein generated by TEV protease cleavage is closest to a natural virus sequence, and only one glycine is added at the N end, so that the formation of VLPs is not affected.

Furthermore, the bivalent subunit vaccine comprises the dimeric form of the feline herpesvirus 1 type gC protein and the VLPs form of the feline parvovirus VP2 protein, and the subunit vaccine comprising the two viruses is obtained through one-time expression and purification process, so that the molar ratio of the dimeric form of the feline herpesvirus 1 type gC protein to the VLPs form of the feline parvovirus VP2 protein in the bivalent subunit vaccine is 1:1, the immunogenicity is stronger, and the cost is lower.

The invention also provides a preparation method of the cat infectious rhinotracheitis and cat panleukopenia bigeminal subunit vaccine, wherein commercial protein A filler is adopted to purify protein in the preparation method, the purified filler and enzyme for enzyme digestion can be repeatedly used, the purification process is simple, and complex processes such as protein renaturation, ion exchange chromatography and the like are not needed. Meanwhile, protease adopted in the preparation process can be recycled and reused for next production, so that the cost is saved.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings that are required to be used in the embodiments will be briefly described below.

FIG. 1 shows the results of ELISA detection of FHV-1 antibody;

FIG. 2 shows the results of ELISA detection of FPV antibodies;

FIG. 3 is a pFastBac1 vector map.

Detailed Description

The invention provides a bivalent subunit vaccine for cat infectious rhinotracheitis and cat leukopenia, which comprises SF9-gC-VP2, wherein a gene encoding the SF9-gC-VP2 is a recombinant SF9-gC-VP2 gene, and the recombinant SF9-gC-VP2 gene comprises a nucleotide sequence encoding a silkworm immunoglobulin signal peptide, a FHV-1gC gene, a cat IgG FC sequence, a TEV enzyme cutting motif and FPVVP genes.

In the present invention, the nucleotide sequence of the recombinant SF9-gC-VP2 gene is preferably shown as SEQ ID NO. 6.

The recombinant SF9-gC-VP2 gene comprises a nucleotide sequence for encoding a silkworm immunoglobulin signal peptide, an FHV-1gC gene, a cat IgG FC sequence, a TEV enzyme cutting motif and an FPV VP2 gene. the nucleotide sequence of the encoded silkworm immunoglobulin signal peptide preferably comprises 60 bases, particularly shown as SEQ ID NO.1 and particularly 5'-ATGGCATTTAAGAGTATCGCAGTGCTAAGTGCCTGCATCATTGTGGGCTCCGCTCTGCCT-3', the FHV-1gC gene preferably comprises 1413 bases, the nucleotide sequence of the encoded silkworm immunoglobulin signal peptide is shown as SEQ ID NO.2, particularly 5'-GAGAATTCGGACAATTCTACCGCTGAGATGTTAAGTTCAACCTCGATGAGCGCAACAACCCCCATAAGTCAACCAACTTCACCTTTTACGACTCCGACTCGGCGGTCGACGAATATTGCTACGTCTTCATCCACAACCCAGGCTTCCCAACCAACCAGTACCCTGACAACACTCACACGTTCGTCAACAACTATCGCCACATCCCCGAGCACTACGCAGGCCGCGACATTTATTGGATCAAGCACAGACTCCAACACTACTCTCCTGAAGACGACGAAAAAGCCAAAAAGAAAGAAGAATAAAAATAATGGAGCGCGTTTCAAACTTGATTGCGGCTACAAGGGCGTGATCTATAGGCCGTACTTCTCACCCCTCCAATTGAACTGTACGCTACCTACTGAGCCCCACATTACTAATCCGATTGATTTTGAGATCTGGTTCAAACCAAGAACTCGTTTCGGAGATTTCTTGGGGGACAAGGAAGACTTCGTGGGCAACCATACCCGCACCTCTATATTGCTTTTTAGTTCTAGAAACGGTAGTGTAAACAGTATGGATCTAGGAGATGCGACGTTGGGTATTCTGCAAAGTCGGATACCAGATTATACTTTATACAACATCCCCATACAACACACAGAGGCCATGTCGCTAGGGATCAAGTCAGTCGAAAGCGCTACTTCTGGCGTCTACACATGGCGCGTCTATGGGGGCGATGGACTGAACAAGACTGTTCTGGGGCAGGTGAACGTGTCAGTTGTAGCATATCATCCTCCCTCCGTGAATCTTACACCCCGAGCTTCCCTATTCAACAAAACGTTTGAAGCGGTATGCGCAGTAGCAAATTATTTTCCACCCCGCAGTACAAAATTGACGTGGTATCTTGATGGAAAACCTATAGAACGGCAGTATATATCTGATACCGCAAGCGTCTGGATCGACGGGCTAATAACACGATCGTCCGTACTCGCCATCCCAACGACCGAGACAGACTCGGAGAAGCCCGATATACGATGCGATCTAGAATGGCACGAATCTCCTGTATCGTACAAGCGCTTCACCAAATCTGTTGCCCCGGACGTTTACTATCCTCCGACGGTTTCGGTTACTTTTGCGGATACCAGGGCAATTTGTGACGTAAAATGTGTCCCGAGGGACGGTATTAGCTTGATGTGGAAAATCGGGAACTACCATTTACCGAAGGCCATGTCTGCTGACATTTTAATCACCGGACCCTGCATAGAACGACCGGGCCTTGTTAATATCCAGTCAATGTGCGACATTTCCGAGACGGACGGTCCAGTGAGCTACACCTGTCAAACGATAGGTTACCCTCCTATTTTACCAGGTTTTTATGATACTCAGGTCTACGACGCGTCCCCTGAAATTGTCAGCGAGAGTATGCTCGTTAGCGTG-3';, the encoded protein of the FHV-1gC gene preferably is a truncated FHV-1gC gene sequence, and N-terminal and C-terminal transmembrane regions are deleted, the intracellular region may be expressed in dimer form. The cat IgG FC of the invention preferably comprises 687 bases, the nucleotide sequence of which is shown as SEQ ID NO.3, and particularly CCCAAGCCTTGTGACTGTCCTAAGTGTCCTCCCCCTGAGATGCTGGGCGGACCTTCCATCTTCATCTTTCCCCCCAAGCCCAAGGACACCCTGTCCATCAGCAGGACACCTGAAGTGACCTGCCTGGTCGTGGATCTGGGACCTGACGATTCCGACGTGCAGATTACCTGGTTCGTGGACAATACCCAAGTGTACACCGCAAAGACCTCTCCTAGAGAAGAGCAGTTTAACAGTACTTACAGAGTGGTGTCCGTGCTCCCGATCCTGCACCAGGACTGGCTGAAAGGCAAAGAGTTCAAGTGCAAGGTGAACAGCAAGAGCCTGCCGAGCCCTATCGAGAGAACCATTTCCAAGGCCAAGGGACAGCCACACGAGCCTCAGGTGTATGTACTGCCCCCGGCCCAGGAGGAGCTCAGCCGGAACAAGGTCTCCGTCACATGTCTGATCAAGAGCTTCCACCCTCCCGACATCGCTGTGGAGTGGGAGATCACCGGACAGCCTGAACCAGAGAACAACTATCGGACCACCCCCCCTCAGCTGGACTCCGATGGCACGTACTTCGTGTACTCCAAGCTGTCTGTGGATCGATCGCACTGGCAGCGGGGCAACACGTACACCTGTTCCGTGTCTCACGAGGCCCTGCACTCCCACCACACCCAAAAGTCTCTGACCCAGTCCCCTGGCAAG-3';, which is a tag sequence, can promote the truncated FHV-1gC protein to form a dimer, prolong the half-life of the protein and promote antigen presentation. The TEV enzyme cutting motif is preferably 21 bases, the nucleotide sequence of which is shown as SEQ ID NO.4, specifically 5'-GAGAACCTGTACTTCCAGGGC-3', and by adding the TEV enzyme cutting motif into the recombinant SF9-gC-VP2 gene, the TEV protease can be adopted to carry out enzyme cutting on the cat parvovirus VP2 protein, and the cat parvovirus VP2 protein obtained by enzyme cutting is closest to a natural virus sequence, and only one glycine is added at the N end, so that the formation of VLPs is not influenced. The FPV VP2 gene of the invention preferably comprises 1752 bases, the nucleotide sequence of which is shown as SEQ ID NO.5, and particularly 5'-AGTGATGGTGCTGTCCAGCCCGATGGCGGACAACCTGCCGTGCGGAATGAGCGTGCCACCGGCAGCGGCAATGGCTCTGGAGGCGGCGGCGGCGGCGGATCTGGCGGAGTAGGCATCAGCACCGGCACCTTTAACAACCAGACCGAGTTCAAGTTCCTGGAGAACGGCTGGGTGGAAATCACCGCCAACTCCTCCAGGCTGGTGCACCTGAACATGCCTGAATCTGAGAACTACAAGCGGGTGGTGGTGAATAACATGGACAAGACCGCCGTGAAGGGCAACATGGCTCTGGACGACACCCATGTGCAGATCGTGACACCTTGGTCCCTGGTGGACGCCAATGCCTGGGGCGTGTGGTTCAACCCTGGAGATTGGCAGCTAATCGTGAACACCATGTCCGAACTGCATCTGGTCTCCTTCGAGCAAGAAATCTTCAACGTGGTCCTGAAGACCGTGTCTGAGTCCGCTACCCAGCCTCCTACCAAGGTGTACAACAACGATCTGACCGCTTCTCTGATGGTCGCCTTGGACTCCAACAACACCATGCCCTTCACCCCAGCTGCTATGAGATCTGAAACCCTGGGCTTTTACCCTTGGAAGCCTACCATCCCTACTCCTTGGAGATACTACTTTCAGTGGGACAGAACACTGATCCCTAGCCACACAGGGACTTCTGGCACACCGACCAATGTGTACCATGGTACCGATCCTGATGATGTGCAGTTCTACACCATCGAGAATTCTGTGCCTGTTCACCTGCTGAGAACAGGCGACGAGTTCGCCACCGGAACCTTCTTCTTCGACTGCAAGCCCTGCCGGCTGACCCACACCTGGCAGACCAACAGAGCCCTGGGCCTTCCTCCTTTCCTGAATAGCCTGCCTCAGTCCGAAGAAGCGACCAACTTCGGCGACATCGGCGTGCAGCAGGATAAGCGGAGAGGCGTGACCCAGATGGGCAATACGGATTATATCACCGAGGCCACCATCATGCGGCCTGCTGAGGTGGGCTACTCTGCTCCTTACTACTCCTTCGAGGCTTCTACACAAGGCCCTTTCAAAACCCCTATCGCCGCTGGCAGAGGCGGCGCTCAAACAGACGAGAACCAGGCCGCCGACGGCGACCCAAGATACGCCTTCGGCAGACAGCACGGCCAGAAAACCACTACCACAGGCGAGACACCTGAACGCTTTACATACATCGCCCACCAGGACACCGGGAGATACCCTGAAGGCGACTGGATACAGAACATCAACTTCAACCTGCCTGTGACCAACGACAACGTGCTGCTGCCAACCGATCCTATCGGCGGAAAGACCGGCATCAACTACACCAACATCTTCAACACATACGGCCCTCTGACAGCTCTGAACAATGTGCCTCCAGTGTACCCTAACGGCCAGATCTGGGACAAAGAATTCGACACCGACCTGAAGCCTCGGCTGCACGTGAACGCCCCCTTCGTGTGCCAGAACAACTGCCCTGGCCAGCTGTTCGTGAAGGTGGCCCCTAACCTGACCAACGAATACGACCCAGACGCCTCTGCCAACATGTCCCGGATCGTGACCTACAGCGACTTCTGGTGGAAGGGCAAGCTGGTTTTCAAGGCCAAGCTGCGGGCCTCTCACACCTGGAACCCCATCCAGCAGATGTCTATCAACGTGGATAACCAGTTCAACTACGTGCCTAACAACATCGGCGCCATGAAGATCGTGTACGAGAAGTCCCAACTCGCTCCTAGAAAGCTGTATTGA-3'; the protein coded by the FPV VP2 gene of the invention is preferably VP2 protein of cat parvovirus. The two ends of the recombinant SF9-gC-VP2 gene synthesized by the invention contain Hin dIII and EcoRI enzyme cutting sites, the full length is 3951 bases, the nucleotide sequence is shown as SEQ ID NO.6, specifically 5'-GGATCCGCCACCATGGCATTTAAGAGTATCGCAGTGCTAAGTGCCTGCATCATTGTGGGCTCCGCTCTGCCTGAGAATTCGGACAATTCTACCGCTGAGATGTTAAGTTCAACCTCGATGAGCGCAACAACCCCCATAAGTCAACCAACTTCACCTTTTACGACTCCGACTCGGCGGTCGACGAATATTGCTACGTCTTCATCCACAACCCAGGCTTCCCAACCAACCAGTACCCTGACAACACTCACACGTTCGTCAACAACTATCGCCACATCCCCGAGCACTACGCAGGCCGCGACATTTATTGGATCAAGCACAGACTCCAACACTACTCTCCTGAAGACGACGAAAAAGCCAAAAAGAAAGAAGAATAAAAATAATGGAGCGCGTTTCAAACTTGATTGCGGCTACAAGGGCGTGATCTATAGGCCGTACTTCTCACCCCTCCAATTGAACTGTACGCTACCTACTGAGCCCCACATTACTAATCCGATTGATTTTGAGATCTGGTTCAAACCAAGAACTCGTTTCGGAGATTTCTTGGGGGACAAGGAAGACTTCGTGGGCAACCATACCCGCACCTCTATATTGCTTTTTAGTTCTAGAAACGGTAGTGTAAACAGTATGGATCTAGGAGATGCGACGTTGGGTATTCTGCAAAGTCGGATACCAGATTATACTTTATACAACATCCCCATACAACACACAGAGGCCATGTCGCTAGGGATCAAGTCAGTCGAAAGCGCTACTTCTGGCGTCTACACATGGCGCGTCTATGGGGGCGATGGACTGAACAAGACTGTTCTGGGGCAGGTGAACGTGTCAGTTGTAGCATATCATCCTCCCTCCGTGAATCTTACACCCCGAGCTTCCCTATTCAACAAAACGTTTGAAGCGGTATGCGCAGTAGCAAATTATTTTCCACCCCGCAGTACAAAATTGACGTGGTATCTTGATGGAAAACCTATAGAACGGCAGTATATATCTGATACCGCAAGCGTCTGGATCGACGGGCTAATAACACGATCGTCCGTACTCGCCATCCCAACGACCGAGACAGACTCGGAGAAGCCCGATATACGATGCGATCTAGAATGGCACGAATCTCCTGTATCGTACAAGCGCTTCACCAAATCTGTTGCCCCGGACGTTTACTATCCTCCGACGGTTTCGGTTACTTTTGCGGATACCAGGGCAATTTGTGACGTAAAATGTGTCCCGAGGGACGGTATTAGCTTGATGTGGAAAATCGGGAACTACCATTTACCGAAGGCCATGTCTGCTGACATTTTAATCACCGGACCCTGCATAGAACGACCGGGCCTTGTTAATATCCAGTCAATGTGCGACATTTCCGAGACGGACGGTCCAGTGAGCTACACCTGTCAAACGATAGGTTACCCTCCTATTTTACCAGGTTTTTATGATACTCAGGTCTACGACGCGTCCCCTGAAATTGTCAGCGAGAGTATGCTCGTTAGCGTGCCCAAGCCTTGTGACTGTCCTAAGTGTCCTCCCCCTGAGATGCTGGGCGGACCTTCCATCTTCATCTTTCCCCCCAAGCCCAAGGACACCCTGTCCATCAGCAGGACACCTGAAGTGACCTGCCTGGTCGTGGATCTGGGACCTGACGATTCCGACGTGCAGATTACCTGGTTCGTGGACAATACCCAAGTGTACACCGCAAAGACCTCTCCTAGAGAAGAGCAGTTTAACAGTACTTACAGAGTGGTGTCCGTGCTCCCGATCCTGCACCAGGACTGGCTGAAAGGCAAAGAGTTCAAGTGCAAGGTGAACAGCAAGAGCCTGCCGAGCCCTATCGAGAGAACCATTTCCAAGGCCAAGGGACAGCCACACGAGCCTCAGGTGTATGTACTGCCCCCGGCCCAGGAGGAGCTCAGCCGGAACAAGGTCTCCGTCACATGTCTGATCAAGAGCTTCCACCCTCCCGACATCGCTGTGGAGTGGGAGATCACCGGACAGCCTGAACCAGAGAACAACTATCGGACCACCCCCCCTCAGCTGGACTCCGATGGCACGTACTTCGTGTACTCCAAGCTGTCTGTGGATCGATCGCACTGGCAGCGGGGCAACACGTACACCTGTTCCGTGTCTCACGAGGCCCTGCACTCCCACCACACCCAAAAGTCTCTGACCCAGTCCCCTGGCAAGGAGAACCTGTACTTCCAGGGCAGTGATGGTGCTGTCCAGCCCGATGGCGGACAACCTGCCGTGCGGAATGAGCGTGCCACCGGCAGCGGCAATGGCTCTGGAGGCGGCGGCGGCGGCGGATCTGGCGGAGTAGGCATCAGCACCGGCACCTTTAACAACCAGACCGAGTTCAAGTTCCTGGAGAACGGCTGGGTGGAAATCACCGCCAACTCCTCCAGGCTGGTGCACCTGAACATGCCTGAATCTGAGAACTACAAGCGGGTGGTGGTGAATAACATGGACAAGACCGCCGTGAAGGGCAACATGGCTCTGGACGACACCCATGTGCAGATCGTGACACCTTGGTCCCTGGTGGACGCCAATGCCTGGGGCGTGTGGTTCAACCCTGGAGATTGGCAGCTAATCGTGAACACCATGTCCGAACTGCATCTGGTCTCCTTCGAGCAAGAAATCTTCAACGTGGTCCTGAAGACCGTGTCTGAGTCCGCTACCCAGCCTCCTACCAAGGTGTACAACAACGATCTGACCGCTTCTCTGATGGTCGCCTTGGACTCCAACAACACCATGCCCTTCACCCCAGCTGCTATGAGATCTGAAACCCTGGGCTTTTACCCTTGGAAGCCTACCATCCCTACTCCTTGGAGATACTACTTTCAGTGGGACAGAACACTGATCCCTAGCCACACAGGGACTTCTGGCACACCGACCAATGTGTACCATGGTACCGATCCTGATGATGTGCAGTTCTACACCATCGAGAATTCTGTGCCTGTTCACCTGCTGAGAACAGGCGACGAGTTCGCCACCGGAACCTTCTTCTTCGACTGCAAGCCCTGCCGGCTGACCCACACCTGGCAGACCAACAGAGCCCTGGGCCTTCCTCCTTTCCTGAATAGCCTGCCTCAGTCCGAAGAAGCGACCAACTTCGGCGACATCGGCGTGCAGCAGGATAAGCGGAGAGGCGTGACCCAGATGGGCAATACGGATTATATCACCGAGGCCACCATCATGCGGCCTGCTGAGGTGGGCTACTCTGCTCCTTACTACTCCTTCGAGGCTTCTACACAAGGCCCTTTCAAAACCCCTATCGCCGCTGGCAGAGGCGGCGCTCAAACAGACGAGAACCAGGCCGCCGACGGCGACCCAAGATACGCCTTCGGCAGACAGCACGGCCAGAAAACCACTACCACAGGCGAGACACCTGAACGCTTTACATACATCGCCCACCAGGACACCGGGAGATACCCTGAAGGCGACTGGATACAGAACATCAACTTCAACCTGCCTGTGACCAACGACAACGTGCTGCTGCCAACCGATCCTATCGGCGGAAAGACCGGCATCAACTACACCAACATCTTCAACACATACGGCCCTCTGACAGCTCTGAACAATGTGCCTCCAGTGTACCCTAACGGCCAGATCTGGGACAAAGAATTCGACACCGACCTGAAGCCTCGGCTGCACGTGAACGCCCCCTTCGTGTGCCAGAACAACTGCCCTGGCCAGCTGTTCGTGAAGGTGGCCCCTAACCTGACCAACGAATACGACCCAGACGCCTCTGCCAACATGTCCCGGATCGTGACCTACAGCGACTTCTGGTGGAAGGGCAAGCTGGTTTTCAAGGCCAAGCTGCGGGCCTCTCACACCTGGAACCCCATCCAGCAGATGTCTATCAACGTGGATAACCAGTTCAACTACGTGCCTAACAACATCGGCGCCATGAAGATCGTGTACGAGAAGTCCCAACTCGCTCCTAGAAAGCTGTATTGAAAGCTT-3'. the amino acid sequence of SF9-gC-VP2 coded by the recombinant SF9-gC-VP2 gene is shown as SEQ ID NO.7, specifically MAFKSIAVLSACIIVGSALPENSDNSTAEMLSSTSMSATTPISQPTSPFTTPTRRSTNIATSSSTTQASQPTSTLTTLTRSSTTIATSPSTTQAATFIGSSTDSNTTLLKTTKKPKRKKNKNNGARFKLDCGYKGVIYRPYFSPLQLNCTLPTEPHITNPIDFEIWFKPRTRFGDFLGDKEDFVGNHTRTSILLFSSRNGSVNSMDLGDATLGILQSRIPDYTLYNIPIQHTEAMSLGIKSVESATSGVYTWRVYGGDGLNKTVLGQVNVSVVAYHPPSVNLTPRASLFNKTFEAVCAVANYFPPRSTKLTWYLDGKPIERQYISDTASVWIDGLITRSSVLAIPTTETDSEKPDIRCDLEWHESPVSYKRFTKSVAPDVYYPPTVSVTFADTRAICDVKCVPRDGISLMWKIGNYHLPKAMSADILITGPCIERPGLVNIQSMCDISETDGPVSYTCQTIGYPPILPGFYDTQVYDASPEIVSESMLVSVPKPCDCPKCPPPEMLGGPSIFIFPPKPKDTLSISRTPEVTCLVVDLGPDDSDVQITWFVDNTQVYTAKTSPREEQFNSTYRVVSVLPILHQDWLKGKEFKCKVNSKSLPSPIERTISKAKGQPHEPQVYVLPPAQEELSRNKVSVTCLIKSFHPPDIAVEWEITGQPEPENNYRTTPPQLDSDGTYFVYSKLSVDRSHWQRGNTYTCSVSHEALHSHHTQKSLTQSPGKENLYFQGSDGAVQPDGGQPAVRNERATGSGNGSGGGGGGGSGGVGISTGTFNNQTEFKFLENGWVEITANSSRLVHLNMPESENYKRVVVNNMDKTAVKGNMALDDTHVQIVTPWSLVDANAWGVWFNPGDWQLIVNTMSELHLVSFEQEIFNVVLKTVSESATQPPTKVYNNDLTASLMVALDSNNTMPFTPAAMRSETLGFYPWKPTIPTPWRYYFQWDRTLIPSHTGTSGTPTNVYHGTDPDDVQFYTIENSVPVHLLRTGDEFATGTFFFDCKPCRLTHTWQTNRALGLPPFLNSLPQSEEATNFGDIGVQQDKRRGVTQMGNTDYITEATIMRPAEVGYSAPYYSFEASTQGPFKTPIAAGRGGAQTDENQAADGDPRYAFGRQHGQKTTTTGETPERFTYIAHQDTGRYPEGDWIQNINFNLPVTNDNVLLPTDPIGGKTGINYTNIFNTYGPLTALNNVPPVYPNGQIWDKEFDTDLKPRLHVNAPFVCQNNCPGQLFVKVAPNLTNEYDPDASANMSRIVTYSDFWWKGKLVFKAKLRASHTWNPIQQMSINVDNQFNYVPNNIGAMKIVYEKSQLAPRKLY.

The invention also provides a preparation method of the bivalent subunit vaccine for cat infectious rhinotracheitis and cat leukopenia, which comprises the following steps of infecting cells with recombinant baculovirus containing the recombinant SF9-gC-VP2 gene in the technical scheme to obtain cell infectious supernatant, purifying the cell infectious supernatant to obtain purified target protein, carrying out virus-like particle self-assembly on the purified target protein, and mixing with an adjuvant to obtain the bivalent subunit vaccine.

The present invention preferably further comprises preparing a recombinant baculovirus comprising said recombinant SF9-gC-VP2 gene before infecting cells with said recombinant baculovirus.

The expression vector used for preparing the recombinant baculovirus comprises a pFastBac1 vector. The specific process for preparing the recombinant expression vector is not particularly limited, and the steps of the conventional preparation method in the field can be adopted.

After the recombinant expression vector is obtained, the recombinant baculovirus comprising the recombinant SF9-gC-VP2 gene is infected with cells to obtain cell infectious matter supernatant.

In the present invention, when a recombinant baculovirus comprising the recombinant SF9-gC-VP2 gene is infected, the infected cell preferably comprises an insect cell, more preferably an SF9 cell or a High Five cell, and still more preferably an SF9 cell. The steps for infecting the cells are not particularly limited in the present invention, and conventional steps in the art may be employed.

After the cell infectious agent supernatant is obtained, the cell infectious agent supernatant is purified to obtain the purified target protein. The present invention preferably uses affinity chromatography to purify the cell infecting supernatant, and the filler used is preferably a protein A filler, and the source of the filler is not particularly limited, and the filler is conventionally commercialized in the art. The purification process is not particularly limited, and conventional purification steps in the art may be employed.

After the purified protein of interest is obtained, the present invention self-assembles the purified protein of interest into virus-like particles (VLPs). The protease used in the present invention for self-assembling the purified target protein into virus-like particles is preferably TEV protease.

After the virus-like particles (VLPs) are self-assembled, the present invention preferably removes and recovers TEV protease to yield an antigenic solution containing feline herpesvirus type 1 gC protein dimer and feline parvovirus VLPs. The recovered TEV protease can be directly used in the enzyme digestion process of the next production.

After the antigen liquid containing the feline herpesvirus gC protein dimer and the feline parvovirus VLPs is obtained, the invention mixes the antigen liquid containing the feline herpesvirus type 1 gC protein dimer and the feline parvovirus VLPs with an adjuvant to obtain the bivalent subunit vaccine. The adjuvant according to the invention preferably comprises ISA 201VG. The concentration of the antigen solution containing the feline herpesvirus type 1 gC protein dimer and feline parvovirus VLPs of the invention is preferably 60 μg/mL. The volume ratio of antigen solution containing feline herpesvirus type 1 gC protein dimer and feline parvovirus VLPs to adjuvant of the invention is preferably 46:54.

The technical solutions provided by the present invention are described in detail below with reference to the drawings and examples for further illustrating the present invention, but they should not be construed as limiting the scope of the present invention.

Example 1

Sequence synthesis

Referring to the feline herpesvirus type 1gC amino acid sequence (yp_ 003331535.1) and feline parvovirus VP2 amino acid sequence (QSG 73888.1), the SF9-gC-VP2 sequence was synthesized by codon optimization. SF9-gC-VP2 sequence is sequentially shown as a silkworm immunoglobulin signal peptide with the size of 60 bases, the nucleotide sequence of which is shown as SEQ ID NO.8, FHV-1gC gene sequence with the size of 1413 bases, the nucleotide sequence of which is shown as SEQ ID NO.9, cat IgG FC sequence with the size of 687 nucleotides, the nucleotide sequence of which is shown as SEQ ID NO.10, TEV cleavage motif with the size of 21 bases, the nucleotide sequence of which is shown as SEQ ID NO.11, FPVVP gene sequence with the size of 1752 bases, the nucleotide sequence of which is shown as SEQ ID NO.12, and the two ends of the synthesized SF9-gC-VP2 sequence containing BamHI and HindIII cleavage sites with the total length of 3951 bases, as shown as SEQ ID NO. 13. The amino acid sequence encoded by the SF9-gC-VP2 sequence is shown as SEQ ID NO. 14.

Example 2

Baculovirus expression vector construction

SF9-gC-VP2 sequence and pFastBac1 (pFastBac 1 vector map is shown in FIG. 3) vector sequence synthesized in practical example 2 are used by BamHI and HindIII enzymes, fragments of the digested SF9-gC-VP2 and pFastBac1 sequences are recovered, and subjected to ligation transformation cloning and sequencing verification to construct an SF9-gC-VP2-pFastBac1 expression vector.

Example 3

Recombinant rod granule preparation

The SF9-gC-VP2-pFastBac1 expression vector prepared in example 2 is transformed into competent cells DH10Bac, cultured at 37 ℃, and then screened and PCR identified by using a blue-white spot screening method to obtain SF9-gC-VP2-Bacmid recombinant rod particles. The bacmid transformation and screening method was operated with reference to the Invitrogen Bac-to-Bac ^TM baculovirus expression system user guide.

Example 4

Recombinant baculovirus harvesting

The SF9-gC-VP2-Bacmid recombinant Bacmid obtained in example 3 was used as transfection reagentSF9 cells in the logarithmic growth phase are transfected, and after culturing for 72 hours, the recombinant baculovirus SF9-gC-VP2-rBV of the P1 generation is harvested. The harvested P1 generation recombinant baculovirus is continuously subjected to subculture on SF9 cells until the generation of the P3, the generation P3 virus is centrifuged, the supernatant is used as virus liquid, and the titer of the generation P3 virus is measured by a plaque method. The High Five cells were infected with the P3 virus at an inoculum size of 1 MOI, and after 96h of culture, the cell-infected supernatant contained the protein of interest.

Example 5

Protein purification

The cell-infected supernatant of example 4 was collected, centrifuged at 8000g for 30 minutes at 4℃and filtered through a 0.8 μm filter. 5-10 times of the column volume PBS balances the ProteinA column material, the treated cell supernatant is repeatedly applied to the column for 3 times, 10 times of PBST is used for flushing the column material, 2 times of PBS is used for flushing the column material, 2 times of 0.1M glycine (pH 3.0) of the column volume is used for eluting protein, eluent is collected, and 1M Tris (pH 9.0) is added for neutralizing to pH7.5, thus obtaining purified protein.

Example 6

Self-assembly of cleaved proteins and VLPs

According to the BCA protein quantitative determination kit (purchased from Shanghai Ind), the purified protein concentration was determined, the total protein mass was calculated, 1U of TEV protease (His-tag, purchased from Biyun-Tian) was added per 8. Mu.g of protein, 10 Xof cleavage buffer (500 mM NaH ₂PO₄, 150mM NaCl,10mM EDTA,10mM DTT,1%Tween-20, pH 8.0) was added by volume, and cleavage was performed at 4℃for 12-16 hours. After the enzyme digestion is completed, the enzyme digested proteins are put into a dialysis bag (3500 Da), the dialysis solution is 50mM NaH ₂PO₄, 500mM NaCl, pH8.0, and the dialysis is carried out for 12-16 hours, and the solution is changed for 2-3 times. After the first dialysis is completed, the protein refolding is completed by standing at 4 ℃ for 8-12 hours. Changing the dialysis solution into PBS buffer solution, dialyzing for 12-16 hours, and changing the solution for 2-3 times.

Example 7

Removal and recovery of TEV protease

And (3) taking a nickel column filled with a filler, balancing the nickel column by using PBS (phosphate buffer solution) with the volume which is 2-5 times that of the column, and repeatedly loading the liquid subjected to dialysis in the example 6 for 3 times, wherein the fluid is an antigen liquid containing the feline herpesvirus 1 type gC protein dimer and the feline parvovirus VLPs. Eluting the nickel column by using PBS buffer solution containing 200mM imidazole, and collecting the eluent, namely the recovered TEV protease, and directly using the enzyme activity determination in the enzyme digestion process of the next production.

Example 8

Vaccine preparation

The antigen solution of example 7 is diluted with PBS buffer solution to a final concentration of 60 mug/mL, and mixed and emulsified with ISA201 VG adjuvant according to a volume ratio of 46:54, thus obtaining the combined subunit vaccine for cat infectious rhinotracheitis and cat leukopenia.

Example 9

Use of feline infectious rhinotracheitis and feline panleukopenia bivalent subunit vaccine

1. ELISA method for detecting FHV-1 and FPV antibody

The PBS buffer solution balances the ProteinA column, the antigen solution containing the feline herpesvirus type 1 gC protein dimer and the feline parvovirus VLPs described in the example 7 is repeatedly sampled for 3 times, and the penetrating fluid is the feline parvovirus VP2 protein. Washing the column material by 10 times of PBST, washing the column material by 2 times of PBS, eluting protein by 2 times of 0.1M glycine (pH 3.0), collecting eluent, adding 1M Tris (pH 9.0) to neutralize to pH7.5, and obtaining the feline herpesvirus type 1 gC protein. The feline herpesvirus type 1 gC protein and the feline parvovirus VP2 protein were detected as follows:

The ELISA plates were coated with PBS buffer to a concentration of 0.2. Mu.g/mL, 100. Mu.L/well, and 4℃for 16h. After the coating was completed, the wells were discarded, 300. Mu.L of PBST wash was added to each well, and the wells were rinsed 1 time. Fresh prepared blocking solution (5% skim milk, PBS) was added to each well at 200. Mu.L and blocked for 2h at 37 ℃. After the end of the blocking, the wells were discarded, 300. Mu.L of PBST wash was added to each well, rinsed 1 time, and dried on a bibulous filter paper. The serum to be tested was diluted 100-fold with PBS buffer, added to the antigen-coated plate and incubated at 37℃for 1h. The wells were discarded, 300 μl of wash solution was added to each well, and rinsed 3 times. HRP-labeled goat anti-cat IgG Fab secondary antibody was diluted 10000-fold with 5% skim milk in PBS, 100 μl per well was added and incubated at 37 ℃ for 1h. The wells were discarded, 300. Mu.L of PBST wash was added to each well, and the wells were rinsed 3 times. TMB was added to the antigen coated plate at 100. Mu.L per well and developed at room temperature in the dark for 10min. 50 μl of stop solution was added to each well, and the absorbance at 450nm was read on a microplate reader.

Judging positive standard of FHV-1 antibody, wherein P/N is more than or equal to 2.1, S/P value is more than or equal to 0.25; judging the FPV antibody positive standard: P/N is more than or equal to 2.1, S-the P value is more than or equal to 0.4.

2. Immunoassay test

7 Chinese rural cats (FHV-1 and FPV are negative) with 6-8 weeks of age are selected and randomly divided into 2 groups, 5 vaccine immune groups and 2 blank control groups, and the vaccine immune groups are used for immunizing cat infectious rhinotracheitis and cat panleukopenia bivalent subunit vaccine and the blank control groups are used for immunizing PBS. 1mL of the serum was subcutaneously injected into the neck each time, and the immunization was boosted once 21 days after the first immunization and the serum was collected 21 days before the immunization and after the second immunization.

Referring to ELISA in step 1, FHV-1 antibodies were detected, and the results were shown in FIG. 1, with numbers 1 to 5 being vaccine immunized groups and numbers 6 and 7 being blank groups. The results showed that both pre-immunization and blank FHV-1 antibodies had S/P values less than 0.25, both negative and FHV-1 antibodies had S/P values above and below 1.5 after 21 days of double immunization. The results of ELISA detection of FPV antibodies are shown in FIG. 2, with numbers 1 to 5 for the vaccine immunization groups and numbers 6 and 7 for the blank groups. The results show that the S/P values of the FHV-1 antibody before immunization and the blank group are less than 0.4, are negative, and the S/P value of the FHV-1 antibody after 21 days of secondary immunization is 1.367 at the lowest and can reach 2.133 at the highest. FHV-1 and FPV antibody detection results indicate that the cat infectious rhinotracheitis and cat leukopenia bigeminal vaccine has good immunogenicity.

3. Neutralizing antibody assay

Feline herpes virus type 1 (FVRm strain) and feline parvovirus (Philips-Roxane strain), both purchased from ATCC in the united states. CRFK cells (feline kidney cells) were purchased from the cell bank of the national academy of sciences.

After digesting CRFK cells with pancreatin one day before the test, the cells were resuspended in MEM containing 10% FBS, plated in 96-well cell culture plates at a cell seeding density of 2X 10 ⁵ cells/mL, 0.1mL per well, and incubated at 37℃under 5% CO ₂. Serum samples were diluted with MEM cell culture medium containing 2% fbs for 21 days after the double immunization at a dilution of 2 ¹,2²,2³,2⁴,2⁵,2⁶,2⁷,2⁸ and mixed well. FHV-1 virus solution was diluted to 200TCID ₅₀/0.1 ml with MEM cell culture medium containing 2% FBS according to the determined virus titer. Mixing 100 μl of serum dilution with equal volume of FHV-1 virus (200 TCID ₅₀), culturing in an incubator with final toxicity of 100TCID ₅₀/0.1 mL at 37deg.C and 5% CO ₂ for 90min, collecting 96-well culture plate CRFK cells cultured for 24 hr, discarding growth solution, transferring the mixture of virus and serum neutralized for 90min into corresponding well of 96-well cell culture plate, and transferring 0.1 mL/well of serum/virus mixture with different dilutions to cell plate while changing pipette gun head for 4 times for each dilution, and culturing under 37 deg.C and 5% CO ₂. serum-FHV-1 virus mixtures of different dilutions were inoculated and CRFK cells were cultured in a cell incubator for 72h to observe cytopathic effects. If the cells are diseased, the FHV-1 is not neutralized by neutralizing antibodies generated in serum, and if the cell growth state is good, the result is the same as that of a control group, the FHV-1 is neutralized by neutralizing antibodies generated in serum, and the cells cannot be diseased. The maximum dilution at which CPE did not occur in CRFK cells was the neutralizing antibody titer of this serum sample, and the neutralizing antibody titer <1:4 was negative. FPV neutralizing antibody experiments were performed as described above. The results of FHV-1 and FPV neutralizing antibody experiments are shown in Table 1.

TABLE 1 Experimental results of neutralizing antibodies to FHV-1 and FPV

As can be seen from Table 1, FHV-1 neutralizing antibody titres are not lower than 1:32, FPV neutralizing antibody titres are not lower than 1:64, and serum produced higher levels of neutralizing antibodies 21 days after the second immunization, indicating that the feline infectious rhinotracheitis and feline panleukopenia bivalent subunit vaccine can provide immune protection to an immunized cat.

From the above examples, it can be seen that the bivalent subunit vaccine for cat infectious rhinotracheitis and cat leukopenia according to the present invention has good immunogenicity, and can prevent the damage of feline herpesvirus type 1 and feline parvovirus.

The foregoing is merely a preferred embodiment of the present invention and it should be noted that modifications and adaptations to those skilled in the art may be made without departing from the principles of the present invention, which are intended to be comprehended within the scope of the present invention.

Claims

1. A bivalent subunit vaccine for feline infectious rhinotracheitis and feline panleukopenia, characterized in that the bivalent subunit vaccine comprises SF9-gC-VP2, and the amino acid sequence of the SF9-gC-VP2 is shown in SEQ ID NO.7;

The gene encoding the SF9-gC-VP2 is a recombinant SF9-gC-VP2 gene, the nucleotide sequence of the recombinant SF9-gC-VP2 gene is shown in SEQ ID NO.6, and the recombinant SF9-gC-VP2 gene includes a nucleotide sequence encoding a silkworm immunoglobulin signal peptide, a FHV-1gC gene, a cat IgGFC sequence, a TEV restriction endonuclease motif and a FPVVP2 gene;

The bivalent subunit vaccine comprises a dimerized feline herpesvirus type 1 gC protein and a VLPs-form feline parvovirus VP2 protein, wherein the molar ratio of the dimerized feline herpesvirus type 1 gC protein to the VLPs-form feline parvovirus VP2 protein is 1:1;

The preparation method of the bivalent subunit vaccine comprises the following steps:

Infect cells with the recombinant baculovirus of the recombinant SF9-gC-VP2 gene to obtain a cell infection supernatant;

Purifying the supernatant of the cell infection to obtain a purified target protein;

Digesting the purified target protein with TEV protease to obtain digested protein;

The enzyme-cleaved protein is subjected to virus-like particle self-assembly and then mixed with an adjuvant to obtain the bivalent subunit vaccine.

2. The bivalent subunit vaccine according to claim 1 is characterized in that the nucleotide sequence encoding the silkworm immunoglobulin signal peptide is shown in SEQ ID NO.1, the nucleotide sequence of the FHV-1gC gene is shown in SEQ ID NO.2, the nucleotide sequence of the cat IgGFC is shown in SEQ ID NO.3, the nucleotide sequence of the TEV cleavage motif is shown in SEQ ID NO.4, and the nucleotide sequence of the FPVVP2 gene is shown in SEQ ID NO.5.

3 . The bivalent subunit vaccine according to claim 1 , wherein the expression vector used to prepare the recombinant baculovirus comprises a pFastBac1 vector.

4. The bivalent subunit vaccine according to claim 1, wherein the cells comprise insect cells.

5. The bivalent subunit vaccine according to claim 1, wherein the insect cells include SF9 cells or High Five cells.