A bivalent subunit vaccine against feline infectious rhinotracheitis and feline panleukopenia, its preparation method and application

Abstract

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

Description

Technical Field

[0001] The present invention belongs to the technical field of veterinary biological products, and particularly relates to a bivalent subunit vaccine against feline infectious rhinotracheitis and feline panleukopenia, a preparation method thereof and an application thereof. Background Art

[0002] Feline infectious rhinotracheitis, also known as feline rhinotracheitis, is a highly contagious disease characterized by acute upper respiratory symptoms. The pathogen of feline rhinotracheitis is feline herpesvirus 1 (FHV-1). FHV-1 belongs to the family Herpesviridae, subfamily Alphaherpesvirinae, and genus Varicellovirus. Herpesvirus 1 mainly consists of a core, a capsid, a tegument and an envelope. The viral capsid is an icosahedron with cubic symmetry and has a hexagonal appearance. The FHV-1 genome encodes multiple proteins, and 17 virus-specific proteins and 3 immunogenic glycoproteins have been reported. Among them, gB is the main immunogenic protein, which is expressed in mammalian cells, causes cell fusion and multinucleate formation, and is essential for virus replication; gD plays a major role in the firm adsorption of the virus to cells and is the main neutralizing antibody in the body's immunity. gC is an important envelope glycoprotein that binds to the heparan sulfate receptor on the surface of host cells to achieve the adhesion of FHV-1 to cells. gC also has an immunosuppressive function and can bind to complement to inhibit complement-mediated cell killing. In addition, gC is also an important immunogenic protein of FHV-1 and can induce cellular immunity and neutralizing antibodies.

[0003] Feline panleukopenia, also known as feline distemper and feline infectious enteritis, is an acute and highly contagious disease caused by feline parvovirus (FPV). FPV is a non-enveloped single-stranded DNA virus belonging to the family Parvoviridae and genus Parvovirus. The FPV genome contains hairpin structures at both ends and encodes two proteins in the middle part: non-structural proteins (NS1, NS2) and structural proteins (VP1, VP2). Through the determination of the molecular structure of the FPV capsid protein by X-ray crystallography, it is found that the virus particles are icosahedrally symmetric, and 60 VP1 and VP2 protein molecules form the capsid protein of the virus particles, including 5-6 VP1 molecules and 54-56 VP2 molecules. The VP2 protein is the main component of the FPV capsid, exposed on the surface of the capsid protein. In the absence of VP1, VP2 can self-assemble into virus-like particles. As the main immunoprotective antigen protein of FPV, VP2 can induce the body to produce neutralizing antibodies and is the preferred target for studying FPV subunit vaccines.

[0004] At present, there is no self-developed FHV-1 vaccine in China, and the prevention and treatment of feline rhinotracheitis mainly rely on inoculating imported vaccines. Although there are relevant studies on feline panleukopenia vaccines or combined bivalent and trivalent vaccines combined with other feline disease vaccines in the existing technology, most of the existing bivalent and multivalent vaccines related to feline diseases are mixed after separately expressing and quantifying, which cannot guarantee the uniformity of product yield. For example, in bivalent vaccines or bivalent subunit vaccines, there is often a situation where one of the two viral proteins is more and the other is less, and it is impossible to achieve an equimolar ratio mixture of the two proteins. In addition, the existing bivalent vaccines or bivalent subunit vaccines for preventing and treating feline disease-related diseases also have the problem that two viruses using two proteins infect insect cells at the same time, resulting in different infection efficiencies of the viruses expressing each protein and uncontrollable protein yields. When cells are infected by multiple baculoviruses, high-frequency recombination will also occur, resulting in the production of defective viruses and the inability to express the expected multi-protein products. Summary of the Invention

[0005] The purpose of the present invention is to provide a bivalent subunit vaccine for feline infectious rhinotracheitis and feline panleukopenia, its preparation method and application. This method innovatively realizes a method that can simultaneously produce two viral subunit vaccines in one expression and purification process, and both the purification packing material and the enzyme used for enzymatic cleavage can be reused. The purification process is simple and the production cost is low. In the bivalent subunit vaccine, the molar ratio of the feline herpesvirus 1 gC protein in dimer form to the feline parvovirus VP2 protein in VLPs form is 1:1. The vaccine antigen has high purity, stronger immunogenicity and lower cost.

[0006] The present invention provides a bivalent subunit vaccine for feline infectious rhinotracheitis and feline panleukopenia, and the bivalent subunit vaccine includes SF9-gC-VP2;

[0007] The gene encoding the SF9-gC-VP2 is a recombinant SF9-gC-VP2 gene, and the recombinant SF9-gC-VP2 gene includes a nucleotide sequence encoding a Bombyx mori immunoglobulin signal peptide, an FHV-1 gC gene, a feline IgG FC sequence, a TEV enzyme cleavage motif and an FPV VP2 gene.

[0008] Preferably, the amino acid sequence of the SF9-gC-VP2 is as shown in SEQ ID NO.7.

[0009] Preferably, the nucleotide sequence of the recombinant SF9-gC-VP2 gene is as shown in SEQ ID NO.6.

[0010] Preferably, the nucleotide sequence encoding the signal peptide of silkworm immunoglobulin is as shown in SEQ ID NO.1, the nucleotide sequence of the FHV-1gC gene is as shown in SEQ ID NO.2, the nucleotide sequence of feline IgG FC is as shown in SEQ ID NO.3, the nucleotide sequence of the TEV protease cleavage motif is as shown in SEQ ID NO.4, and the nucleotide sequence of the FPV VP2 gene is as shown in SEQ ID NO.5.

[0011] The present invention also provides a method for preparing a bivalent subunit vaccine against feline infectious rhinotracheitis and feline panleukopenia, comprising the following steps: infecting cells with the recombinant baculovirus comprising the recombinant SF9-gC-VP2 gene described in the above technical solution or the recombinant SF9-gC-VP2 gene described in the above technical solution to obtain a supernatant of the cell infection;

[0012] Purifying the supernatant of the cell infection to obtain a purified target protein;

[0013] Digesting the purified target protein to obtain a digested protein;

[0014] Self-assembling the digested protein into virus-like particles and then mixing with an adjuvant to obtain the bivalent subunit vaccine.

[0015] Preferably, the protease used for digesting the purified target protein is TEV protease.

[0016] Preferably, the expression vector for preparing the recombinant baculovirus comprises the pFastBac1 vector.

[0017] Preferably, the cells include insect cells.

[0018] Preferably, the insect cells include SF9 cells or High Five cells.

[0019] Beneficial effects:

[0020] The present invention provides a bivalent subunit vaccine against feline infectious rhinotracheitis and feline panleukopenia. The bivalent subunit vaccine comprises SF9-gC-VP2, and the SF9-gC-VP2 comprises a truncated FHV-1gC gene sequence, with the transmembrane region and intracellular region at the N-terminus and C-terminus deleted; meanwhile, an FC tag is added to enable the feline herpesvirus gC protein to be expressed in a dimer form, extend the half-life of the protein, promote antigen presentation, and facilitate downstream purification;

[0021] Meanwhile, by adding a TEV protease cleavage motif to the SF9-H-VP2, the feline parvovirus VP2 protein generated by TEV protease cleavage is closest to the natural virus sequence, with only one additional glycine at the N-terminus, which does not affect the formation of VLPs.

[0022] Furthermore, the bivalent subunit vaccine of the present invention includes the feline herpesvirus type 1 gC protein in dimer form and the feline parvovirus VP2 protein in VLPs form. Through a single expression and purification process, a subunit vaccine containing two viruses is obtained, ensuring that the molar ratio of the feline herpesvirus type 1 gC protein in dimer form to the feline parvovirus VP2 protein in VLPs form in the bivalent subunit vaccine is 1:1, with stronger immunogenicity and lower cost.

[0023] The present invention also provides a preparation method for a bivalent subunit vaccine against feline infectious rhinotracheitis and feline panleukopenia. In the preparation method, a commercial protein A packing material is used to purify the protein, and both the purification packing material and the enzyme used for cleavage can be reused. The purification process is simple and does not require complex processes such as protein renaturation and ion exchange chromatography. At the same time, the protease used in the preparation process can be recovered and reused for the next production, saving costs. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for use in the embodiments.

[0025] Figure 1 Results of ELISA for detecting FHV-1 antibodies;

[0026] Figure 2 Results of ELISA for detecting FPV antibodies;

[0027] Figure 3 Map of the pFastBac1 vector. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0028] The present invention provides a bivalent subunit vaccine against feline infectious rhinotracheitis and feline panleukopenia. The bivalent subunit vaccine includes SF9-gC-VP2; the gene encoding the SF9-gC-VP2 is the recombinant SF9-gC-VP2 gene, and the recombinant SF9-gC-VP2 gene includes a nucleotide sequence encoding a Bombyx mori immunoglobulin signal peptide, an FHV-1 gC gene, a feline IgG FC sequence, a TEV protease cleavage motif, and an FPV VP2 gene.

[0029] In the present invention, the nucleotide sequence of the recombinant SF9-gC-VP2 gene is preferably as shown in SEQ ID NO.6.

[0030]

[0031] The present invention also provides a method for preparing a bivalent subunit vaccine against feline infectious rhinotracheitis and feline panleukopenia, comprising the following steps: infecting cells with a recombinant baculovirus comprising the recombinant SF9-gC-VP2 gene described in the above technical solution to obtain a supernatant of the cell infection; purifying the supernatant of the cell infection to obtain a purified target protein; self-assembling the purified target protein into virus-like particles and then mixing it with an adjuvant to obtain the bivalent subunit vaccine.

[0032] Before infecting cells with the recombinant baculovirus comprising the recombinant SF9-gC-VP2 gene, the present invention preferably further comprises preparing the recombinant baculovirus.

[0033] The expression vector for preparing the recombinant baculovirus in the present invention comprises the pFastBac1 vector. The present invention has no special limitation on the specific process of preparing the recombinant expression vector, and the conventional preparation method steps in the art can be adopted.

[0034] After obtaining the recombinant expression vector, the present invention infects cells with the recombinant baculovirus comprising the recombinant SF9-gC-VP2 gene to obtain a supernatant of the cell infection.

[0035] In the present invention, when infecting with the recombinant baculovirus comprising the recombinant SF9-gC-VP2 gene, the infected cells preferably include insect cells, more preferably SF9 cells or High Five cells, and even more preferably SF9 cells. The present invention has no special limitation on the steps of infecting the cells, and the conventional steps in the art can be adopted.

[0036] After obtaining the supernatant of the cell infection, the present invention purifies the supernatant of the cell infection to obtain a purified target protein. The present invention preferably purifies the supernatant of the cell infection by affinity chromatography, and the filler used is preferably ProteinA filler. The present invention has no special limitation on the source of the filler, and the conventional commercial fillers in the art can be used. The present invention has no special limitation on the purification process, and the conventional purification steps in the art can be adopted.

[0037] After obtaining the purified target protein, the present invention self-assembles the purified target protein into virus-like particles (VLPs). The protease preferably used when the present invention self-assembles the purified target protein into virus-like particles is TEV protease.

[0038] After completing the self-assembly of the virus-like particles (VLPs), the present invention preferably removes and recovers TEV protease to obtain an antigen solution containing the dimer of feline herpesvirus type 1 gC protein and feline parvovirus VLPs. The recovered TEV protease can be directly used in the enzymatic digestion process for the next production.

[0039] After obtaining the antigen solution containing the dimer of feline herpesvirus gC protein and feline parvovirus VLPs, the present invention mixes the antigen solution containing the dimer of feline herpesvirus type 1 gC protein and feline parvovirus VLPs with an adjuvant to obtain the bivalent subunit vaccine. The adjuvant of the present invention preferably includes ISA 201VG. The concentration of the antigen solution containing the dimer of feline herpesvirus type 1 gC protein and feline parvovirus VLPs in the present invention is preferably 60 μg / mL. The volume ratio of the antigen solution containing the dimer of feline herpesvirus type 1 gC protein and feline parvovirus VLPs to the adjuvant in the present invention is preferably 46:54.

[0040] To further illustrate the present invention, the technical solutions provided by the present invention will be described in detail below in conjunction with the drawings and embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0041] Example 1

[0042] Sequence synthesis

[0043] Referring to the amino acid sequence of feline herpesvirus type 1 gC (YP_003331535.1) and the amino acid sequence of feline parvovirus VP2 (QSG73888.1), after codon optimization, the SF9-gC-VP2 sequence was synthesized. The SF9-gC-VP2 sequence is as follows: the signal peptide of Bombyx mori immunoglobulin, with a size of 60 bases, and its nucleotide sequence is shown in SEQ ID NO.8; the FHV-1 gC gene sequence, with a size of 1413 bases, and its nucleotide sequence is shown in SEQ ID NO.9; the feline IgG FC sequence, with a size of 687 nucleotides, and its nucleotide sequence is shown in SEQ ID NO.10; the TEV protease cleavage motif, with a size of 21 bases, and its nucleotide sequence is shown in SEQID NO.11; the FPV VP2 gene sequence, with a size of 1752 bases, and its nucleotide sequence is shown in SEQ ID NO.12; both ends of the synthesized SF9-gC-VP2 sequence contain BamHI and HindIII cleavage sites, and the full length is 3951 bases, as shown in SEQID NO.13. The amino acid sequence encoded by the SF9-gC-VP2 sequence is shown in SEQ ID NO.14.

[0044] Example 2

[0045] Construction of baculovirus expression vector

[0046] Use BamHI and HindIII to digest the SF9-gC-VP2 sequence synthesized in Example 2 and pFastBac1 (the pFastBac1 vector map is as Figure 3The carrier sequence as shown, recover the digested SF9-gC-VP2 and pFastBac1 sequence fragments, and perform ligation transformation cloning and sequencing verification to construct the SF9-gC-VP2-pFastBac1 expression vector.

[0047] Example 3

[0048] Preparation of recombinant bacmid

[0049] Transform the SF9-gC-VP2-pFastBac1 expression vector prepared in Example 2 into competent cells DH10Bac. After culturing at 37 °C, use the blue-white screening method for screening and perform PCR identification to obtain the SF9-gC-VP2-Bacmid recombinant bacmid. The methods for bacmid transformation and screening refer to the Bac-to-Bac TM Baculovirus Expression System User Guide for operation.

[0050] Example 4

[0051] Harvest of recombinant baculovirus

[0052] Transfect the SF9-gC-VP2-Bacmid recombinant bacmid obtained in Example 3 into SF9 cells in the logarithmic growth phase using a transfection reagent After culturing for 72 h, harvest the P1 generation of recombinant baculovirus SF9-gC-VP2-rBV. Continuously passage the harvested P1 generation of recombinant baculovirus on SF9 cells until the P3 generation. Centrifuge the P3 generation of virus, and the supernatant is the virus solution. Determine the virus titer of the P3 generation by the plaque method. Infect High Five cells with the P3 generation of virus at an inoculation amount of 1 MOI. After culturing for 96 h, the cell infection supernatant contains the target protein.

[0053] Example 5

[0054] Protein purification

[0055] Collect the cell infection supernatant in Example 4, centrifuge at 8000 g at 4 °C for 30 minutes, take the supernatant, and filter it through a 0.8 μm filter membrane. Equilibrate the Protein A column material with 5 - 10 column volumes of PBS. Repeat loading the treated cell supernatant onto the column 3 times, wash the column material with 10 column volumes of PBST, wash the column material with 2 column volumes of PBS, elute the protein with 2 column volumes of 0.1 M glycine (pH 3.0), collect the eluate, and add 1 M Tris (pH 9.0) to neutralize to pH 7.5 to obtain the purified protein.

[0056] Example 6

[0057] Digestion of protein and self-assembly of VLPs

[0058] According to the instructions of the BCA protein quantification detection kit (purchased from Sangon Biotech, Shanghai), the concentration of the purified protein was determined, and the total mass of the protein was calculated. 1 U of TEV protease (His-tag, purchased from Beyotime Biotechnology) was added per 8 μg of protein, and 10× digestion buffer (500 mM NaH 2 PO 4 , 150 mM NaCl, 10 mM EDTA, 10 mM DTT, 1% Tween-20, pH 8.0) was added according to volume, and digestion was carried out at 4 °C for 12 - 16 hours. After the digestion was completed, the digested protein was loaded into a dialysis bag (3500 Da), and the dialysis solution was 50 mM NaH 2 PO 4 , 500 mM NaCl, pH 8.0, and dialysis was carried out for 12 - 16 hours, with the solution changed 2 - 3 times during the period. After the first dialysis was completed, it was left to stand at 4 °C for 8 - 12 hours to complete protein refolding. The dialysis solution was changed to PBS buffer, and dialysis was carried out for 12 - 16 hours, with the solution changed 2 - 3 times during the period.

[0059] Example 7

[0060] Removal and recovery of TEV protease

[0061] A nickel column filled with packing material was taken, and the nickel column was equilibrated with PBS at 2 - 5 times the column volume. The liquid after dialysis in Example 6 was loaded onto the column repeatedly 3 times, and the flow-through was the antigen solution containing the dimer of feline herpesvirus 1 gC protein and feline parvovirus VLPs. The nickel column was eluted with PBS buffer containing 200 mM imidazole, and the eluate was collected, which was the recovered TEV protease. After the enzyme activity was measured, it could be directly used for the digestion process in the next production.

[0062] Example 8

[0063] Vaccine preparation

[0064] The antigen solution in Example 7 was diluted with PBS buffer to a final concentration of 60 μg / mL and mixed and emulsified with ISA201 VG adjuvant according to a volume ratio of 46:54, which was the feline infectious rhinotracheitis and feline panleukopenia bivalent subunit vaccine.

[0065] Example 9

[0066] Application of the feline infectious rhinotracheitis and feline panleukopenia bivalent subunit vaccine

[0067] 1. ELISA method for detecting FHV-1 and FPV antibodies

[0068] Equilibrate the Protein A column with PBS buffer. Repeat the loading of the antigen solution containing the dimer of feline herpesvirus type 1 gC protein and feline parvovirus VLPs described in Example 7 three times. The flow-through fraction is the feline parvovirus VP2 protein. Wash the column material with 10-fold PBST, then with 2-fold PBS, and elute the protein with 2-column-volume of 0.1 M glycine (pH 3.0). Collect the eluate and neutralize it to pH 7.5 by adding 1 M Tris (pH 9.0), which is the feline herpesvirus type 1 gC protein. Detect the feline herpesvirus type 1 gC protein and feline parvovirus VP2 protein respectively according to the following steps:

[0069] Quantify the concentration by BCA method and dilute it to 0.2 μg / mL with PBS buffer for coating the ELISA plate, 100 μL per well, and coat at 4°C for 16 h. After coating, discard the liquid in the wells, add 300 μL of PBST washing solution to each well and rinse once. Add 200 μL of freshly prepared blocking solution (5% skim milk, PBS) to each well and block at 37°C for 2 h. After blocking, discard the liquid in the wells, add 300 μL of PBST washing solution to each well and rinse once, then pat dry on the absorbent filter paper. Dilute the test serum 100-fold with PBS buffer, add it to the antigen-coated plate, and incubate at 37°C for 1 h. Discard the liquid in the wells, add 300 μL of washing solution to each well and rinse three times. Dilute the HRP-labeled goat anti-cat IgG Fab secondary antibody 10,000-fold with PBS containing 5% skim milk, add 100 μl to each well, and incubate at 37°C for 1 h. Discard the liquid in the wells, add 300 μL of PBST washing solution to each well and rinse three times. Add TMB to the antigen-coated plate, 100 μL per well, and develop color at room temperature in the dark for 10 min. Add 50 μL of stop solution to each well and read the absorbance at 450 nm on the microplate reader.

[0070] Criteria for judging positive FHV-1 antibody: P / N ≥ 2.1, S / P value ≥ 0.25; Criteria for judging positive FPV antibody: P / N ≥ 2.1, S / P value ≥ 0.4.

[0071] 2. Immunization experiment

[0072] Select 7 Chinese rural cats aged 6 - 8 weeks (negative for FHV-1 and FPV antibodies), randomly divide them into 2 groups, with 5 cats in the vaccine immunization group and 2 cats in the blank control group. The vaccine immunization group is immunized with the feline infectious rhinotracheitis and feline panleukopenia bivalent subunit vaccine, and the blank control group is immunized with PBS. Inject 1 mL subcutaneously into the neck each time, and boost immunize once 21 days after the first immunization. Collect serum before immunization and 21 days after the second immunization.

[0073] Refer to the ELISA method in step 1 to detect FHV-1 antibody, and the results are as Figure 1, Numbers 1 to 5 are the vaccine immunization groups, and numbers 6 and 7 are the blank groups. The results showed that the S / P values of FHV-1 antibodies in the pre-immunization and blank groups were both less than 0.25, all negative, and the S / P values of FHV-1 antibodies were around 1.5 21 days after the second immunization. ELISA was used to detect FPV antibodies, and the results are as Figure 2 , Numbers 1 to 5 are the vaccine immunization groups, and numbers 6 and 7 are the blank groups. The results showed that the S / P values of FHV-1 antibodies in the pre-immunization and blank groups were both less than 0.4, all negative, and the lowest S / P value of FHV-1 antibodies was 1.367 and the highest could reach 2.133 21 days after the second immunization. The detection results of FHV-1 and FPV antibodies indicate that the feline infectious rhinotracheitis and feline panleukopenia bivalent subunit vaccine has good immunogenicity.

[0074] 3. Neutralizing antibody experiment

[0075] Feline herpesvirus type 1 (FVRm strain) and feline parvovirus (Philips-Roxane strain) were both purchased from ATCC, USA. CRFK cells (feline kidney cells) were purchased from the Cell Bank of the Chinese Academy of Sciences.

[0076] One day before the experiment, CRFK cells were digested with trypsin, resuspended with MEM containing 10% FBS, and seeded into a 96-well cell culture plate. The cell seeding density was 2×10 5 cells / mL, 0.1 mL was inoculated into each well, and it was placed in an incubator at 37°C with 5% CO 2 for culture. The serum samples 21 days after the second immunization were diluted with MEM cell culture medium containing 2% FBS, and the dilution factors were 2 1 , 2 2 , 2 3 , 2 4 , 2 5 , 2 6 , 2 7 , 2 8 and mixed evenly. The FHV-1 virus solution was diluted to 200 TCID 50 / 0.1 ml with MEM cell culture medium containing 2% FBS according to the measured virus titer. Take 100 μL of the serum dilution, mix it evenly with an equal volume of FHV-1 virus (200 TCID 50 ), and the final virus dose is 100 TCID 50 / 0.1 ml, and it was placed in an incubator at 37°C with 5% CO 2Cultivate in an incubator for 90 min; Take the CRFK cells in a 96-well culture plate cultured for 24 h, discard the growth medium, transfer the mixture of the virus and serum neutralized for 90 min to the corresponding wells of the 96-well cell culture plate, 0.1 mL / well. When transferring the serum / virus mixture with different dilutions to the cell plate, change the pipette tip in time. Make 4 replicates for each dilution, and at the same time set up normal negative control cells without neutralized virus; Incubate at 37 °C, 5% CO 2 Incubate under the conditions of. Inoculate the serum-FHV-1 virus mixture with different dilutions and CRFK cells in an incubator for 72 h, and observe the cytopathic effect. If cytopathic effect occurs, it indicates that FHV-1 is not neutralized by the neutralizing antibodies produced in the serum; If the cell growth state is good, the same as the control group result, it indicates that FHV-1 is neutralized by the neutralizing antibodies produced in the serum, and no cytopathic effect will occur in the cells. The maximum dilution at which no CPE appears in CRFK cells is the neutralizing antibody titer of this serum sample, and the neutralizing antibody titer < 1:4 is negative. The operation of the FPV neutralizing antibody experiment is the same as above. The results of the FHV-1 and FPV neutralizing antibody experiments are shown in Table 1.

[0077] Table 1 Results of FHV-1 and FPV neutralizing antibody experiments

[0078]

[0079] It can be concluded from Table 1 that the neutralizing antibody titer of FHV-1 is not less than 1:32, and the neutralizing antibody titer of FPV is not less than 1:64. High levels of neutralizing antibodies are produced in the sera 21 days after the second immunization, indicating that the feline infectious rhinotracheitis and feline panleukopenia bivalent subunit vaccine can provide immune protection for immunized cats.

[0080] It can be concluded from the above examples that the feline infectious rhinotracheitis and feline panleukopenia bivalent subunit vaccine of the present invention has good immunogenicity and can prevent the harm of feline herpesvirus type 1 and feline parvovirus.

[0081] The above are only the preferred embodiments of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and modifications can be made, and these improvements and modifications should also be regarded as the protection scope of the present invention.

Claims

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

6. The recombinant SF9-gC-VP2 gene comprises a nucleotide sequence encoding a Bombyx mori immunoglobulin signal peptide, an FHV-1 gC gene, a feline IgG FC sequence, a TEV protease cleavage motif, and an FPV VP2 gene; the bivalent subunit vaccine comprises the dimeric form of feline herpesvirus type 1 gC protein and the VLPs form of feline parvovirus VP2 protein, and the molar ratio of the dimeric form of feline herpesvirus type 1 gC protein to the VLPs form of feline parvovirus VP2 protein is 1:1; the preparation method of the bivalent subunit vaccine comprises the following steps: infecting cells with the recombinant baculovirus of the recombinant SF9-gC-VP2 gene to obtain the supernatant of the cell infection; purifying the supernatant of the cell infection to obtain the purified target protein; digesting the purified target protein with TEV protease to obtain the digested protein; self-assembling the digested protein into virus-like particles and then mixing with an adjuvant to obtain the bivalent subunit vaccine.

2. The bivalent subunit vaccine according to claim 1, characterized in that, the nucleotide sequence encoding the Bombyx mori immunoglobulin signal peptide is as shown in SEQ ID NO.1, the nucleotide sequence of the FHV-1 gC gene is as shown in SEQ ID NO.2, the nucleotide sequence of the feline IgG FC is as shown in SEQ ID NO.3, the nucleotide sequence of the TEV protease cleavage motif is as shown in SEQ ID NO.4, and the nucleotide sequence of the FPV VP2 gene is as shown in SEQ ID NO.

5.

3. The bivalent subunit vaccine according to claim 1, characterized in that, the expression vector for preparing the recombinant baculovirus comprises the pFastBac1 vector.

4. The bivalent subunit vaccine according to claim 1, characterized in that, the cells comprise insect cells.

5. The bivalent subunit vaccine according to claim 1, characterized in that, the insect cells comprise SF9 cells or High Five cells.

Images