Chimeric virus-like particle for feline infectious rhinoconjunctivitis, feline infectious rhinotracheitis and feline panleucopenia, triple vaccine and preparation method and application thereof

Abstract

The invention provides chimeric virus-like particles for feline infectious rhinoconjunctivitis, feline infectious rhinotracheitis and feline panleucopenia, a triple vaccine and a preparation method and application of the chimeric virus-like particles and the triple vaccine, and belongs to the technical field of biological products. The invention provides chimeric virus-like particles assembled by feline parvovirus-like particles and feline herpesvirus type I gD protein and chimeric virus-like particles assembled by feline calicivirus virus-like particles and feline herpesvirus type I gB protein. The invention also provides a triple vaccine which is prepared from the chimeric virus-like particle and an adjuvant. Experiments prove that the chimeric virus-like particle triple vaccine has good safety and antigen compatibility, no local or whole-body adverse reaction occurs after animals are immunized, and a body can be induced to generate good humoral immune and cellular immune responses. Therefore, the chimeric virus-like particle triple vaccine has the advantages of good immunogenicity, high safety, low production cost and the like.

Description

Technical Field

[0001] The present invention belongs to the technical field of biological products, and specifically relates to a chimeric virus-like particle for feline infectious rhinoconjunctivitis, feline infectious rhinotracheitis, and feline panleukopenia, a triple vaccine, and a preparation method and application thereof. Background Art

[0002] Feline viral infectious diseases are a key issue for global pet health and wildlife conservation, and the establishment of a prevention and control system faces significant challenges. Three core diseases caused by feline parvovirus (FPV), feline calicivirus (FCV), and feline herpesvirus-1 (FHV-1)—panleukopenia, infectious rhinoconjunctivitis, and viral rhinotracheitis—have become core biosecurity risks to the health of pet cat populations due to their high contagiousness, wide transmission routes, and persistent infection. They also pose a potential threat to the stability of endangered wild cat populations and public health. While current inactivated vaccine technology is widely used in the prevention and control of feline infectious diseases, its biosecurity risks and immune limitations require urgent attention. In contrast, virus-like particle (VLP) vaccines demonstrate groundbreaking technological advantages. They utilize a recombinant expression system to produce non-replicating particles formed by the self-assembly of viral structural proteins, completely avoiding the risks of handling live viruses. The production process complies with Biosafety Level 2 (BSL-2) standards, significantly reducing the probability of pathogen leakage. The native conformational epitopes presented on the VLP surface can simultaneously activate both humoral and cellular immunity, and enable serological differentiation between vaccination and wild-type virus infection, providing powerful technical support for establishing precise pathogen clearance and purification strategies. Summary of the Invention

[0003] In view of this, the object of the present invention is to provide a chimeric virus-like particle for feline infectious rhinoconjunctivitis, feline infectious rhinotracheitis, and feline panleukopenia. The VP2 protein of FPV and the VP1 protein of FCV are respectively connected and combined with the gD and gB proteins of FHV to form a chimeric virus-like particle, which has good immunogenicity, can induce the body to produce a large amount of specific antibodies, and can be used as a candidate vaccine strain.

[0004] The invention provides a chimeric virus-like particle for feline infectious rhinoconjunctivitis, feline infectious rhinotracheitis and feline panleukopenia, which comprises parvovirus VP2 protein, feline calicivirus VP1 protein, feline herpesvirus type I gD protein and feline herpesvirus type I gB protein.

[0005] Preferably, the chimeric virus-like particle is assembled from two proteins;

[0006] The proteins include VP2 protein of feline parvovirus and gD protein of feline herpesvirus type I, VP1 protein of feline calicivirus and gB protein of feline herpesvirus type I.

[0007] Preferably, the chimeric virus-like particle comprises a first chimeric virus-like particle and a second chimeric virus-like particle;

[0008] The first chimeric virus-like particle is a virus-like particle assembled from the VP2 protein of feline parvovirus and the gD protein of feline herpesvirus type I;

[0009] The second chimeric virus-like particle is obtained by combining virus-like particles assembled from the VP1 protein of feline calicivirus and the gB protein of feline herpesvirus type I.

[0010] Preferably, the mass ratio of the first chimeric virus-like particle to the second chimeric virus-like particle is (0.8-1.2): (0.8-1.2).

[0011] Preferably, in the first chimeric virus-like particle and the second chimeric virus-like particle, the chimeric manner is connected through a spy catcher or a spy tag.

[0012] The present invention provides a method for preparing the chimeric virus-like particle, comprising the following steps:

[0013] The feline parvovirus VP2 protein and the feline calicivirus VP1 protein, each of which was modified with a spy catcher or spy tag at the end, were first recombinantly expressed. The resulting VP2 recombinant protein and VP1 recombinant protein were assembled in vitro to obtain two tagged virus-like particles, FPV-VLPs and FCV-VLPs.

[0014] performing a second recombinant expression on the feline herpesvirus type I gB protein and gD protein modified with another molecule of the spy catcher and spy tag, respectively, to obtain gB recombinant protein and gD recombinant protein;

[0015] The virus-like particles with the tag are respectively connected with one of the gB recombinant protein and the gD recombinant protein to obtain two chimeric virus-like particles.

[0016] Preferably, the first recombinant expression comprises expression using an Escherichia coli prokaryotic expression system; the second recombinant expression comprises expression using a baculovirus-insect cell expression system;

[0017] The VP2 recombinant protein is obtained by recombinant expression in Escherichia coli of a recombinant vector containing a codon-optimized modified VP2 protein encoding gene; the nucleotide sequence of the codon-optimized modified VP2 protein encoding gene is shown in SEQ ID NO: 5;

[0018] The VP1 recombinant protein is obtained by recombinant expression in Escherichia coli of a recombinant vector containing a codon-optimized modified VP1 protein encoding gene; the nucleotide sequence of the codon-optimized modified VP1 protein encoding gene is shown in SEQ ID NO: 6;

[0019] The gB recombinant protein is obtained by recombinantly expressing a recombinant vector containing a codon-optimized modified gB protein encoding gene in insect cells; the nucleotide sequence of the codon-optimized modified gB protein encoding gene is shown in SEQ ID NO: 7;

[0020] The gD recombinant protein is obtained by recombinantly expressing a recombinant vector containing a codon-optimized modified gD protein encoding gene in insect cells; the nucleotide sequence of the codon-optimized modified gD protein encoding gene is shown in SEQ ID NO:8.

[0021] Preferably, the molar ratio of virus-like particles assembled from the VP2 recombinant protein to the gD recombinant protein is 1:(1-2);

[0022] The molar ratio of virus-like particles assembled by the VP1 recombinant protein to the B recombinant protein is 1:(1-2).

[0023] The present invention provides a chimeric virus-like particle triple vaccine for feline infectious rhinoconjunctivitis, feline infectious rhinotracheitis and feline panleukopenia, comprising the chimeric virus-like particles or the chimeric virus-like particles prepared by the preparation method and an adjuvant.

[0024] The present invention provides the use of the chimeric virus-like particles or the chimeric virus-like particles prepared by the preparation method in preparing vaccines or diagnostic reagents for preventing and controlling feline infectious rhinoconjunctivitis, feline infectious rhinotracheitis and feline panleukopenia.

[0025] The present invention provides a chimeric virus-like particle for feline infectious rhinoconjunctivitis, feline infectious rhinotracheitis, and feline panleukopenia, comprising feline parvovirus VP2 protein, feline calicivirus VP1 protein, feline herpesvirus type I gD protein, and feline herpesvirus type I gB protein. Experiments have shown that the prepared chimeric virus-like particles have high immunogenicity after being used as immunogens to immunize animals. After the first immunization, the body can be induced to produce high-titer specific antibodies. As the immunization time increases, the antibody level also shows an upward trend; after a booster immunization 14 days later, the antibody level reaches a peak one week after the booster immunization and then remains at a high level. In addition, experiments have shown that the chimeric virus-like particle triple vaccine has good safety and antigenic compatibility. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1The SDS-PAGE results of the purification of FPV-SC-VP2 protein (A) and FCV-SC-VP1 protein (B). Note: 1-7: elution of target protein with 500mM imidazole concentration of Buffer B; 8-9: elution with 20mM and 45mM imidazole concentrations of Buffer B, respectively; 10: flow-through; 11: precipitate;

[0027] Figure 2 The following are the results of protease cleavage identification of FPV-SC-VP2 and FCV-SC-VP1, where A is the SDS-PAGE result of FPV-SC-VP2 protease cleavage; B is the WB result of FPV-SC-VP2 protease cleavage; C and D are the SDS-PAGE results of FCV-SC-VP1 protease cleavage; E is the WB result of FCV-SC-VP1 protease cleavage;

[0028] Figure 3 The structural characterization results of FPV-SC-VLPs and FCV-SC-VLPs, where A is the particle size of FPV-SC-VLPs virus-like particles, B is the transmission electron microscopy analysis structure of FPV-SC-VLPs virus-like particles; C is the particle size result of FPV-SC-VLPs virus-like particles, and D is the transmission electron microscopy analysis structure of FCV-SC-VLPs virus-like particles;

[0029] Figure 4 PCR identification results of recombinant bacmid expressing FHV gB and gD genes;

[0030] Figure 5 The SDS-PAGE results of the purification of FHV gB protein (A) and gD protein (B), where 1-5, 6-10: elution of the target protein with 500mM imidazole concentration of Buffer B; 3-5: elution with 5, 20, and 45mM imidazole concentration of Buffer B; 1: flow-through; 2: precipitate;

[0031] Figure 6 These are the immunoblotting results of FHV recombinant proteins, where A is the result of gB recombinant protein and B is the result of gD recombinant protein;

[0032] Figure 7 The SDS-PAGE and WB identification results of FCV-gB-VLPs (A, B) and FPV-gD-VLPs (C, D) chimeric proteins, where 1-3: FCV-gB binding at a molar ratio of 1:1, 1:2, and 1:3, respectively; 1-5: FPV-gD binding at a molar ratio of 1:1, 1:2, 1:3, 1:4, and 1:5, respectively;

[0033] Figure 8Characterization results of FPV-gD-VLPs and FCV-gB-VLPs, where A is the particle size result detected by dynamic light scattering (DLS) and B is the morphology result of VLPs detected by transmission electron microscopy;

[0034] Figure 9 The results of the specific antibody level test of mice immunized with the triple vaccine are shown in Figure 1, where A is the FCV-specific antibody test result, B is the FPV-specific antibody test result, C is the FHV-gB-specific antibody test result, and D is the FHV-gD-specific antibody test result.

[0035] Figure 10 These are the results of spleen lymphocyte proliferation test and cytokine detection, where A is the result of spleen lymphocyte proliferation test, B is the result of IL-4 detection, and C is the result of IFN-γ detection. DETAILED DESCRIPTION

[0036] The invention provides a chimeric virus-like particle for feline infectious rhinoconjunctivitis, feline infectious rhinotracheitis and feline panleukopenia, which comprises feline parvovirus VP2 protein, feline calicivirus VP1 protein, feline herpesvirus type I gD protein and feline herpesvirus type I gB protein.

[0037] In an embodiment of the present invention, the chimeric virus-like particles are preferably assembled from two proteins. The proteins include two selected from feline parvovirus VP2 protein, feline calicivirus VP1 protein, feline herpesvirus type I gD protein, and feline herpesvirus type I gB protein. The chimeric virus-like particles preferably include virus-like particles assembled from feline parvovirus VP2 protein and feline herpesvirus type I gD protein, as well as chimeric virus-like particles assembled from feline calicivirus VP1 protein and feline herpesvirus type I gB protein; or include virus-like particles assembled from feline calicivirus VP1 protein and feline herpesvirus type I gD protein, as well as chimeric virus-like particles assembled from feline parvovirus VP2 protein and feline herpesvirus type I gB protein. The amino acid sequence of the feline calicivirus VP1 protein is shown in SEQ ID NO: 21. The amino acid sequence of the feline parvovirus VP2 protein is shown in SEQ ID NO: 22. The amino acid sequence of the feline herpesvirus type I gD protein is shown in SEQ ID NO: 23. The amino acid sequence of the feline herpesvirus type I gB protein is shown in SEQ ID NO: 24. In the chimeric virus-like particle, the chimeric method is preferably connected by a spy catcher or a spy tag. The present invention does not impose any special restrictions on the type of modified molecules of the feline calicivirus VP1 protein or the feline parvovirus VP2 protein, and any modified spy catcher or spy tag can be used. The mass ratio of the first chimeric virus-like particle and the second chimeric virus-like particle is preferably (0.8-1.2): (0.8-1.2), and can be 1:1.

[0038] The present invention provides a method for preparing the chimeric virus-like particle, comprising the following steps:

[0039] Feline parvovirus VP2 protein and feline calicivirus VP1 protein, each terminally modified with a molecule selected from the group consisting of a spy catcher and a spy tag, were first recombinantly expressed to obtain recombinant VP2 and VP1 proteins, which were then assembled in vitro into tagged FPV-VLPs and FCV-VLPs, respectively.

[0040] performing a second recombinant expression on the feline herpesvirus type I gB protein and gD protein modified with another molecule of the spy catcher and spy tag, respectively, to obtain gB recombinant protein and gD recombinant protein;

[0041] By utilizing the ligation of the spy catcher and the spy tag, two chimeric virus-like particles, FPV-gD-VLPs and FCV-gB-VLPs, were obtained respectively.

[0042] The present invention performs first recombinant expression on feline parvovirus VP2 protein and feline calicivirus VP1 protein, each of which is modified with a molecule of a spy catcher and a spy tag at the end, to obtain VP2 recombinant protein and VP1 recombinant protein.

[0043] In the present invention, the nucleotide sequence of the gene encoding the feline calicivirus VP1 protein is shown in SEQ ID NO: 1. The nucleotide sequence of the gene encoding the feline parvovirus VP2 protein is shown in SEQ ID NO: 2. The nucleotide sequence of the gene encoding the feline herpesvirus type I gD protein is shown in SEQ ID NO: 3. The nucleotide sequence of the gene encoding the feline herpesvirus type I gB protein is shown in SEQ ID NO: 4.

[0044] In the present invention, the first recombinant expression preferably includes expression using a prokaryotic expression system. The N-termini of the feline parvovirus VP2 protein and the feline calicivirus VP1 protein are respectively modified with histidine tags to facilitate purification and extraction of the recombinant proteins. The C-termini of the feline parvovirus VP2 protein and the feline calicivirus VP1 protein are modified with spy catchers, which is conducive to chimerization with other antigenic proteins. The N-termini of the feline herpesvirus type I gB protein and gD protein are respectively modified with histidine tags to facilitate purification and extraction of the recombinant proteins. The C-termini of the feline herpesvirus type I gB protein and gD protein are modified with spy catchers to facilitate chimerization with other antigenic proteins.

[0045] In the present invention, the VP2 recombinant protein is obtained by recombinant expression in Escherichia coli using a recombinant vector containing a codon-optimized modified VP2 protein encoding gene; the nucleotide sequence of the codon-optimized modified VP2 protein (FPV-SC-VP2) encoding gene is shown in SEQ ID NO: 5. The VP1 recombinant protein is obtained by recombinant expression in Escherichia coli using a recombinant vector containing a codon-optimized modified VP1 protein encoding gene; the nucleotide sequence of the codon-optimized modified VP1 protein (FCV-SC-VP1) encoding gene is shown in SEQ ID NO: 6. The backbone vector of the recombinant vector is preferably a pSMA expression vector.

[0046] The invention recombinantly expresses feline herpesvirus type I gB protein and gD protein respectively modified with a spy catcher and another molecule of a spy tag to obtain gB recombinant protein and gD recombinant protein.

[0047] In the present invention, the second recombinant expression is preferably expressed in insect cells. The gB recombinant protein is obtained by recombinant expression in Escherichia coli using a recombinant vector containing a codon-optimized modified gB protein encoding gene; the nucleotide sequence of the codon-optimized modified gB protein (FHV-ST-gB) encoding gene is shown in SEQ ID NO: 7. The gD recombinant protein is obtained by recombinant expression in Escherichia coli using a recombinant vector containing a codon-optimized modified gD protein encoding gene; the nucleotide sequence of the codon-optimized modified gD protein (FHV-ST-gD) encoding gene is shown in SEQ ID NO: 8. The backbone of the recombinant vector is preferably a pFastBac-Dual vector. The present invention has no particular restrictions on the recombinant expression of the recombinant vector in insect cells; conventional transformation methods, recombinant culture methods, and recombinant protein purification methods can be used, such as Cellfectin transfection reagent transformation method, IPTG induction culture, and nickel affinity chromatography purification method. After obtaining the purified recombinant protein, the recombinant protein is enzymatically hydrolyzed with SUMO enzyme to remove the histidine tag.

[0048] After recombinant expression, the present invention assembles the obtained VP2 recombinant protein into virus-like particles in vitro and then chimerizes it with one of the gB recombinant protein and the gD recombinant protein; the VP1 recombinant protein is assembled into virus-like particles in vitro and then chimerized with the other of the gB recombinant protein and the gD recombinant protein to obtain two chimeric virus-like particles.

[0049] In the present invention, the chimerization is preferably performed in an assembly buffer, which is preferably 35 mmol / l KH2PO4, 28 mmol / l K2HPO4·3H2O, 50 mmol / l NaCl, and has a pH value of 7.0 to 7.5.

[0050] In the present invention, the assembled first and second chimeric virus-like particles were analyzed by particle size measurement and transmission electron microscopy, showing that the first chimeric virus-like particles (FPV-gD-VLPs) had a particle size of 25 to 30 nm, and the second chimeric virus-like particles (FCV-gB-VLPs) had a particle size of 30 to 40 nm. Transmission electron microscopy observed virus-like particles with the morphology of natural virus particles.

[0051] In the present invention, the molar ratio of the virus-like particles formed by the VP2 recombinant protein to the gB recombinant protein or the gD recombinant protein is preferably 1:(1-2). The molar ratio of the virus-like particles formed by the VP1 recombinant protein to the gB recombinant protein or the gD recombinant protein is preferably 1:(1-2). Optimization experiments were conducted using different molar ratios (1:1, 1:2, 1:3, 1:4, and 1:5), and the results showed that higher binding efficiencies were achieved at molar ratios of 1:2 and 1:1, respectively.

[0052] The present invention provides a chimeric virus-like particle triple vaccine for feline infectious rhinoconjunctivitis, feline infectious rhinotracheitis and feline panleukopenia, comprising the chimeric virus-like particles or the chimeric virus-like particles prepared by the preparation method and an adjuvant.

[0053] In the present invention, the adjuvant is preferably ISA201 oil adjuvant. In the vaccine, the final concentration of the chimeric virus-like particles is preferably 20-30 μg / ml, and may be 25 μg / ml. The present invention does not particularly limit the preparation method of the vaccine; methods for preparing chimeric virus-like particle vaccines known in the art can be employed.

[0054] The present invention provides the use of the chimeric virus-like particles or the chimeric virus-like particles prepared by the preparation method in preparing vaccines or diagnostic reagents for preventing and controlling feline infectious rhinoconjunctivitis, feline infectious rhinotracheitis, and feline panleukopenia.

[0055] In the present invention, the vaccine is preferably administered by intramuscular injection. The intramuscular injection includes a primary immunization and a booster immunization, and the interval between the primary and booster immunizations is preferably 2 weeks. The dose of the primary or booster immunization is 25 μg / time, respectively, based on the injection dose of the chimeric virus-like particles.

[0056] In this study, indirect ELISA was used to measure specific antibody levels following vaccine immunization. Results showed that after the initial immunization, serum levels of specific antibodies were high, with an increasing trend over time. Following a booster immunization 14 days later, antibody levels peaked one week after the booster immunization and remained high thereafter.

[0057] The present invention adopts a multi-platform synergistic strategy: FPV-VLPs and FCV-VLPs are prepared separately through a prokaryotic expression system, and the FHV-1 glycoprotein gB / gD is obtained using a eukaryotic expression system. The gB and gD epitopes are site-specifically coupled to FPV-VLPs and FCV-VLPs respectively through Spy Catcher / Spy Tag molecular covalent linkage technology, successfully constructing two structurally stable chimeric virus-like particles. Transmission electron microscopy (TEM) and dynamic light scattering (DLS) techniques confirmed that the chimeric virus-like particles prepared by the present invention have a uniform particle size distribution and a typical icosahedral symmetrical hollow structure. The above-mentioned antigens are combined with the ISA201 adjuvant system to prepare a triple vaccine. Evaluation in a Balb / c mouse model showed that after 28 days of immunization, the multi-vaccine can simultaneously induce the body to produce good humoral and cellular immune responses.

[0058] The triple virus-like particle vaccine prepared by this invention utilizes a "live virus-free" production process, which not only reduces the risk of zoonotic transmission but also induces a potent immune response that blocks the virus's transmission within pet cats, effectively alleviating the accompanying pathogenic contamination pressure in the human habitat. This "green vaccine" technology provides an innovative solution for building a biosafety control system that integrates humans, pets, and the ecosystem.

[0059] The following is a detailed description of the feline infectious rhinoconjunctivitis, feline infectious rhinotracheitis, feline panleukopenia chimeric virus-like particles, triple vaccine, and preparation method and application thereof provided by the present invention in conjunction with the examples, but they should not be construed as limiting the scope of protection of the present invention.

[0060] The specific primer sequences designed in the examples of the present invention are shown in Table 1. The primers were all synthesized by Xi'an Qingke Biotechnology Co., Ltd.

[0061] Table 1 Specific primer sequences

[0062]

[0063]

[0064] Example 1

[0065] Recombinant expression method of FPV and FCV immune antigens

[0066] 1. Construction of recombinant vectors expressing FPV and FCV immune antigens

[0067] 1.1 Sequence design and synthesis

[0068] Using the genomes of FPV and FCV strains as reference sequences, His tags were added to the N-termini of the FPV-VP2 protein and the FCV-VP1 protein, respectively, and a Spy catcher tag was added to their C-termini. The modified sequences were codon-optimized and synthesized, and the codon-optimized FPV-SC-VP2 gene sequences (SEQ ID NO: 5) and FCV-SC-VP1 (SEQ ID NO: 6) were respectively inserted into the pSMA expression vector containing a His tag and Sumo (see CN101914501A or CN106479986A) between the HindIII and BamHI sites to form the pSMA-His-Sumo-FPV-SC-VP2 and pSMA-His-Sumo-FCV-SC-VP1 recombinant vectors. The construction of the recombinant vectors was entrusted to Nanjing GenScript Biotech Co., Ltd.

[0069] 2. Recombinant Plasmid Transformation

[0070] The pSMA-His-Sumo-FPV-SC-VP2 and pSMA-His-Sumo-FCV-SC-VP1 recombinant vectors were transformed into BL21 competent cells, respectively, and spread onto LB plates containing 20 μg / ml ampicillin. The cells were cultured at 37°C overnight, and single colonies were picked and inoculated into liquid LB medium for expansion and identification by PCR (primers see SEQ ID NO: 9 to SEQ ID NO: 12). The correctly identified strains were named FPV-SC-VP2 and FCV-SC-VP1 and stored at -80°C for future use.

[0071] 3 Expression and purification of recombinant plasmids FPV-SC-VP2 and FCV-SC-VP1 in Escherichia coli

[0072] Inoculate the stored FCV-SC-VP1 and FPV-SC-VP2 strains at a 1% inoculum ratio into 5 ml of LB medium containing 20 μg / ml ampicillin. Incubate at 37°C for 12 hours, then transfer to 500 ml of liquid LB medium. When the OD value reaches 0.6-0.8, add 0.2 mM IPTG and continue incubation at 16°C for 14-16 hours. Harvest the cells and disrupt them by ultrasonication. After centrifugation, collect the supernatant, purify them by nickel affinity chromatography, and analyze them by SDS-PAGE.

[0073] See the results Figure 1 The results showed that FPV-SC-VP2 protein and FCV-SC-VP1 protein were successfully recombinantly expressed in recombinant E. coli.

[0074] Example 2

[0075] Assembly of FCV-SC-VLPs and FPV-SC-VLPs

[0076] 1. The FPV-SC-VP2 and FCV-SC-VP1 recombinant proteins correctly identified in Example 1 were added to 10 kDa dialysis tubing at a ratio of 1:40 (v:v) with 100 μg / ml SUMO enzyme. The dialysis tubing was placed in assembly buffer and subjected to enzymatic digestion and assembly at 4°C for 48 h. The assembly buffer components are as follows: FPV-SC-VP2 buffer (20 mM Tris, 0.1 M NaCl, pH adjusted to 8.0), FCV-SC-VP1 buffer (20 mM Tris, 0.3 M NaCl, pH adjusted to 7.0). The magnetic stirrer was operated at 300 rpm. Identification was performed by SDS-PAGE and Western blot.

[0077] Western-blot identification results are shown in Figure 2The results showed that the sizes of SC-VP2 and SC-VP1 bands after enzyme digestion were consistent with the expected target band sizes.

[0078] 2. Determination of Virus-like Particle Size and Transmission Electron Microscopy Observation

[0079] 500 μL of each assembled FCV-SC-VLPs and FPV-SC-VLPs sample was taken and the sample size was measured using a nanoparticle size analyzer (DLS). 10 μL of each sample was added to a 200-mesh copper grid and adsorbed at room temperature for 1 minute. The remaining liquid on the copper grid was then blotted with filter paper. The grid was then washed with 10 μL of PBS for 1 minute and then blotted with filter paper. The grid was then stained with 10 μL of 3% phosphotungstic acid for 30 seconds. The morphology of the FCV-SC-VLPs and FPV-SC-VLPs was observed using transmission electron microscopy (TEM).

[0080] The results are as follows Figure 3 As shown, the results of DLS and TEM showed that the particle size of the obtained FPV-SC-VLPs was 25-30 nm, and the particle size of FCV-SC-VLPs was 30-40 nm. The morphology and particle size of the two VLPs were similar to those of their natural virus particles.

[0081] Example 3

[0082] Construction of recombinant expression vectors pFastBac-gB and pFastBac-gD for feline herpesvirus gB and gD proteins

[0083] Based on the published gB and gD sequences of the FHV virus strain, a SpyTag tag was added to the N-terminus of the protein sequence, and a His tag was added to the C-terminus. After codon modification, the modified protein coding sequence was synthesized by GenScript and constructed into the pFastBac-Dual vector to obtain the recombinant vectors pFastBac-ST-gB and pFastBac-ST-gD.

[0084] 1. Construction of recombinant bacmid

[0085] Correctly identified recombinant plasmids, pFastBac-ST-gB and pFastBac-ST-gD, were transformed into competent DH10 Bac cells carrying the baculovirus shuttle vector. After 48 hours of incubation at 37°C on LB solid medium supplemented with 50 μg / ml kanamycin, 7 μg / ml gentamicin, 10 μg / ml tetracycline, 100 μg / ml X-gal, and 40 μg / ml IPTG, blue-white colonies were screened. White colonies were picked and inoculated into 5 ml of LB liquid medium supplemented with 50 μg / ml kanamycin, 7 μg / ml gentamicin, and 10 μg / ml tetracycline and incubated at 37°C for 48 hours. Identification was performed using the upstream primer of the M13 universal primer and the downstream primers of gB / gD. According to the instructions for the pFastBac-Dual recombinant vector, the amplified bands were 4976 bp / 3437 bp, respectively. The identified bacmids were named rBcmid-ST-gB and rBcmid-ST-gD, respectively.

[0086] 2. Preparation of Recombinant Baculovirus

[0087] Recombinant bacmids rBcmid-ST-gB and rBcmid-ST-gD were transfected into sf9 insect cells using Cellfectin transfection reagent and cultured in a 27°C incubator for 72 hours. When cells showed obvious pathological changes, the culture medium was collected and centrifuged to obtain the supernatant, which was the P1 strain and stored at -80°C until further use. The P1 strain was amplified twice after infection of sf9 insect cells to obtain the P3 strain, which was then identified by PCR.

[0088] See the results Figure 4 The results showed that a band of the expected size was obtained.

[0089] 3. Purification of recombinant ST-gB and ST-gD proteins

[0090] The pH of the collected ST-gB and ST-gD supernatants was adjusted to 8.5. The filtered supernatants were added to equilibrated nickel columns and allowed to bind at 4°C for 2 hours to elute the target protein. Wash with Buffer A containing 5mM, 20mM, and 45mM imidazole, respectively. Elution was performed five times with Buffer B containing 500mM imidazole (500mM / L NaCl, 20mM / L Tris-HCl, 5% glycerol, 500mM / L imidazole, adjusted to pH=8.5), using 2ml per column. The eluted samples were subjected to SDS-PAGE electrophoresis.

[0091] See the results Figure 5 The results showed that after purification, recombinant ST-gB (molecular weight 100kDa) and ST-gD proteins (molecular weight 43kDa) with the expected molecular weight were obtained.

[0092] 4. Western-blot analysis of recombinant ST-gB and ST-gD proteins

[0093] Recombinant ST-gB and ST-gD proteins were subjected to 10% SDS-PAGE and electrotransferred to polyvinylidene difluoride hybridization membrane (PVDF) using the wet transfer method. The membrane was blocked with blocking buffer (TBST, 50 g / L skim milk powder, pH 7.0) at 37°C for 2 h. HRP-labeled mouse anti-His IgG antibody (1:5000) was then incubated at 37°C for 1 h, followed by thorough washing with PBST. Luminescent substrate was added for 3 min in the dark, and the membrane was exposed to light.

[0094] See the results Figure 6 The results showed that recombinant ST-gB (molecular weight 100kDa) and ST-gD proteins (molecular weight 43kDa) with the expected molecular weight were obtained.

[0095] Example 4

[0096] Preparation method of FCV-gB-VLPs and FPV-gD-VLPs

[0097] The coupling technology between Spy catcher (SC) and Spy tag (ST) can spontaneously form an irreversible isopeptide bond, but the binding efficiency between the two is related to the molar concentration ratio. In this experiment, FCV-SC-VLPs and ST-gB and FPV-SC-VLPs and ST-gD were combined overnight at 4°C in a buffer solution (35mmol / l KH2PO4, 28mmol / l K2HPO4·3H2O, 50mmol / l NaCl, pH 7.0-7.5) at different molar concentration ratios (FCV-SC-VP1:ST-gD are 1:1, 1:2, 1:3, 1:4 and 1:5; similarly, FPV-SC-VP2:ST-gB are 1:1, 1:2 and 1:3), trying to ensure that the residual amount of VP2 and VP1 is minimal. After the binding is completed, the binding efficiency of the two proteins is verified by SDS-PAGE and Western blot. The results are shown in Figure 7 , the optimal binding molar ratios were determined to be 1:2 and 1:1 respectively.

[0098] Then the particle size and transmission electron microscopy were tested. Figure 8 The particle size of FPV-SC-VLPs is 25-30 nm, and that of FCV-SC-VLPs is 30-40 nm. Both VLPs are similar in morphology and size to their native viral particles.

[0099] Example 5

[0100] Preparation method of chimeric virus-like particle triple vaccine for feline infectious rhinoconjunctivitis, feline infectious rhinotracheitis and feline panleukopenia

[0101] ISA201 oil was selected as an adjuvant, and the FCV-gB-VLPs and FPV-gD-VLPs prepared in Example 4 were mixed in a volume ratio of 1:1 to obtain a triple vaccine with a final concentration of 25 μg / ml for FCV-gB-VLPs and a final concentration of 25 μg / ml for FPV-gD-VLPs.

[0102] Example 6

[0103] Validation of the immunogenicity of the triple vaccine

[0104] Female BALB / c mice aged 5-6 weeks were randomly divided into five groups of 5 mice each: the first group received FCV-VLPs, the second group received FPV-VLPs, the third group received FHV-VLPs, the fourth group received FPV-gD + FCV-gB VLPs-ISA201 oil adjuvant, and the fifth group received a PBS control group. The experimental groups received 25 μg of the corresponding VLPs intramuscularly, while the control group received 200 μL of PBS intramuscularly. Fourteen days after the initial immunization, each immunized group received a booster immunization with the same dose as the initial immunization. Blood was collected from the eye sockets weekly after immunization. The collected blood was incubated at 37°C for 1 hour, then at 4°C for 3 hours, and centrifuged twice at 3000 rpm for 15 minutes. After four weeks of continuous blood collection, serum was isolated and tested for specific antibodies.

[0105] The specific IgG antibody levels of each immune group were detected using the indirect ELISA method:

[0106] FPV, FCV-VP1, FHV-gB, and FHV-gD were used as antigens to coat ELISA plates, respectively. HRP-labeled mouse anti-IgG antibody was used as the secondary antibody. The serum of healthy non-immunized mice was used as a negative control. The titer of antibodies produced by mice immunized with the triple virus-like particle vaccine was detected by indirect ELISA. The specific steps are as follows:

[0107] (1) Antigen coating: Dilute the prepared antigen to 0.5 μg / mL using the coating solution. Add 100 μL of the diluted antigen solution to each well of the 96-well plate. Cover the plate with the lid and shake it slightly manually to evenly distribute the antigen. Incubate in a refrigerator at 4°C overnight.

[0108] (2) Discard the liquid in the ELISA plate, wash 5 times with PBST, 200 μL per well, and pat dry. Add 100 μL of 1% BSA to each well of the 96-well plate, seal the plate, and incubate at 37°C for 1 hour.

[0109] (3) The mouse serum samples were serially diluted at a ratio of 1:4 to ensure that the volume in each well was 50 μL, and then incubated at 37°C for 1 h.

[0110] (4) Discard the liquid in the ELISA plate, wash 5 times with PBST, 200 μL per well, and pat dry. Dilute HRP-labeled mouse anti-IgG antibody at a ratio of 1:8000, add 50 μL to each well of the 96-well plate, and incubate at 37°C for 1 h.

[0111] (5) Repeat step 2, add 50 mL of TMB substrate solution to each well, and develop color at 37°C in the dark for 10 min.

[0112] (6) Add 50 μL of stop solution to each well to terminate the reaction and read the OD value using a microplate reader. 450nm value, record the result.

[0113] Test results are shown in Figure 9 Data from each group were collated and plotted using GraphPad Prism 9 software. The results showed that after the initial immunization, mice produced high levels of specific antibodies in their serum. These levels increased with increasing immunization duration. Following a 14-day booster immunization, antibody levels peaked one week after the booster immunization and remained high thereafter.

[0114] The proliferation of T lymphocytes in the spleen of mice was measured by MTS method. The results showed that the spleen lymphocytes in the immunized group were activated. Compared with the PBS group, both the single vaccine and the chimeric virus-like particle triple vaccine groups increased the level of spleen lymphocyte proliferation (P<0.05). The FCV proliferation index was 2.6, the FPV proliferation index was 3.6, and the chimeric triple vaccine proliferation index was 3.2. The cytokine detection results showed that IL-4 and IFN-γ in the immunized group increased significantly. The above results indicate that the chimeric virus-like particle triple vaccine can induce the body to produce a good cellular immune response ( Figure 10 ).

[0115] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. A chimeric virus-like particle for feline infectious rhinoconjunctivitis, feline infectious rhinotracheitis, or feline panleukopenia, characterized in that: Contains feline parvovirus VP2 protein, feline calicivirus VP1 protein, feline herpesvirus type 1 gD protein and feline herpesvirus type 1 gB protein.

2. The chimeric virus-like particle according to claim 1, characterized in that The chimeric virus-like particles are prepared from two proteins; The proteins include VP2 protein of feline parvovirus and gD protein of feline herpesvirus type I or VP1 protein of feline calicivirus and gB protein of feline herpesvirus type I.

3. The chimeric virus-like particle according to claim 2, characterized in that The chimeric virus-like particle comprises a first chimeric virus-like particle and a second chimeric virus-like particle; The first chimeric virus-like particle is a virus-like particle assembled from feline parvovirus VP2 protein and feline herpesvirus type I gD protein; The second chimeric virus-like particle is obtained by chimerizing the virus-like particle assembled by the feline calicivirus VP1 protein and the feline herpesvirus type I gB protein.

4. The chimeric virus-like particle according to claim 3, characterized in that The mass ratio of the first chimeric virus-like particle to the second chimeric virus-like particle is (0.8-1.2): (0.8-1.2).

5. The chimeric virus-like particle according to claim 3, characterized in that In the first chimeric virus-like particle or the second chimeric virus-like particle, the chimeric manner is connected via a spy catcher or a spy tag.

6. A method for preparing the chimeric virus-like particle according to any one of claims 1 to 5, characterized in that: The following steps are involved: The feline parvovirus VP2 protein and the feline calicivirus VP1 protein, each of which is terminally modified with one of the spy catcher and spy tags, are first recombinantly expressed, and the resulting VP2 recombinant protein and VP1 recombinant protein are assembled in vitro to obtain two tagged virus-like particles; performing a second recombinant expression on the feline herpesvirus type I gB protein and gD protein modified with another molecule of the spy catcher and spy tag, respectively, to obtain gB recombinant protein and gD recombinant protein; The virus-like particles with the tag are respectively connected with one of the gB recombinant protein and the gD recombinant protein to obtain two chimeric virus-like particles.

7. The preparation method according to claim 6, characterized in that: The first recombinant expression comprises expression using a prokaryotic expression system; the second recombinant expression comprises expression using a baculovirus-insect cell expression system; The VP2 recombinant protein is obtained by recombinant expression in Escherichia coli of a recombinant vector containing a codon-optimized modified VP2 protein encoding gene; the nucleotide sequence of the codon-optimized modified VP2 protein encoding gene is shown in SEQ ID NO: 5; The VP1 recombinant protein is obtained by recombinant expression in Escherichia coli of a recombinant vector containing a codon-optimized modified VP1 protein encoding gene; the nucleotide sequence of the codon-optimized modified VP1 protein encoding gene is shown in SEQ ID NO: 6; The gB recombinant protein is obtained by recombinant expression in insect cells of a recombinant vector containing a codon-optimized modified gB protein encoding gene; the nucleotide sequence of the codon-optimized modified gB protein encoding gene is shown in SEQ ID NO: 7; The gD recombinant protein is obtained by recombinantly expressing a recombinant vector containing a codon-optimized modified gD protein encoding gene in insect cells; the nucleotide sequence of the codon-optimized modified gD protein encoding gene is shown in SEQ ID NO:

8.

8. The preparation method according to claim 7, characterized in that: The molar ratio of the virus-like particles assembled by the VP2 recombinant protein to the gD recombinant protein is 1:(1-2); The molar ratio of the virus-like particles assembled by the VP1 recombinant protein to the gB recombinant protein is 1:(1-2).

9. A chimeric virus-like particle triple vaccine for feline infectious rhinoconjunctivitis, feline infectious rhinotracheitis, and feline panleukopenia, characterized in that: The invention comprises the chimeric virus-like particle according to any one of claims 1 to 5 or the chimeric virus-like particle prepared by the preparation method according to any one of claims 6 to 8 and an adjuvant.

10. Use of the chimeric virus-like particle according to any one of claims 1 to 5 or the chimeric virus-like particle prepared by the preparation method according to any one of claims 6 to 8 in the preparation of vaccines or diagnostic reagents for preventing and controlling feline infectious rhinoconjunctivitis, feline infectious rhinotracheitis and feline panleukopenia.

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