Feline panleukopenia, feline infectious rhinoconjunctivitis and feline infectious rhinotracheitis triple inactivated vaccine and its preparation method and application

Abstract

The invention discloses a kind of cat panleukopenia, feline infectious rhinoconjunctivitis and feline infectious rhinotracheitis triple inactivated vaccine and preparation method and application, screening isolates the high immunogenicity, high virus titer, genetically stable feline infectious rhinoconjunctivitis virus CC3 strain and feline infectious rhinotracheitis virus CP2 strain popular in my country, more suitable for domestic popular trend, the new isolate can be used as vaccine strain after passage, the triple inactivated vaccine prepared has good safety performance, and antigen compatibility is better, and any local and systemic adverse reactions caused by the vaccine do not occur after immunizing animals, and safety is good. High concentration of cytokine secretion can be induced simultaneously, including Th1 type cytokines (TNF α, IFN γ) and Th2 type cytokines (IL 6), immunogenicity is high, and immune duration is long, improves immune effect, ensures the stability of vaccine, fills domestic gap.

Description

Technical Field

[0001] The present invention relates to the technical field of biological products, and in particular to a triple inactivated vaccine for feline panleukopenia, feline infectious rhinoconjunctivitis and feline infectious rhinotracheitis, as well as a preparation method and application thereof. Background Art

[0002] Feline parvovirus (FP), also known as feline panleukopenia, feline distemper, or feline infectious enteritis, is an acute, highly contagious viral disease caused by the feline parvovirus (FPV). It is a serious viral disease affecting cats, characterized by sudden onset of high fever, vomiting, diarrhea, dehydration, and a sharp decrease in circulating white blood cells. FPV primarily infects a variety of species, including felines and mustelids, with particularly high morbidity and mortality rates in young animals under six months of age. The incidence rate is as high as 83.5% in kittens under one year of age, and the disease can occur in entire litters. FPV is the most serious infectious disease in cats and poses a serious threat to their lives.

[0003] Feline infectious rhinoconjunctivitis virus (FCV), also known as feline calicivirus, is a member of the Caliciviridae family and the genus Calicivirus. FCV infects a wide range of animals, including cats and rare wild animals such as tigers, lions, and cheetahs. All felines are susceptible to FCV, with kittens being the most susceptible. Key clinical symptoms include rhinitis, conjunctivitis, acute oral ulcers, pneumonia, chronic gastritis, and lameness. The disease has a high morbidity rate, with a lower mortality rate in adult cats and a higher mortality rate in kittens, reaching over 40%. Current research indicates that FCV has only one serotype, which varies greatly in virulence, antigenicity, and heritability. This high level of antigenic variation makes it difficult to achieve broad protection against FCV in cats.

[0004] Feline infectious rhinotracheitis (FIR) is an infectious disease caused by feline herpesvirus type 1 (FHV-1), primarily characterized by upper respiratory symptoms. FHV-1 infection manifests as upper respiratory tract inflammation, oral ulcers, keratoconjunctivitis, and chronic sinusitis. It can also damage the trigeminal and optic nerves, and in severe cases, can lead to death in young cats. FHV-1 has been reported in countries and regions including the United States, Japan, Germany, and China. In addition to domestic cats, it can also infect wild felines such as tigers and cheetahs, posing a serious threat to pet health and wildlife conservation.

[0005] In order to protect cats from the threat of infectious diseases and maintain their physical and mental health, vaccination is the key. For the three infectious diseases mentioned above, the vaccines currently available on the market are all imported vaccines, mainly cat triple vaccines, including FPV, FCV, and FHV. However, virus mutation is the main reason for the failure of current vaccine immunity. In particular, FCV varies greatly in virulence, antigenicity, and heredity. The foreign vaccines currently on the market belong to different branches of the evolutionary tree of the domestic prevalent strains, and have low homology, and can no longer provide good protection. At the same time, foreign FHV vaccines cannot provide good protection against the prevalent strains in my country. Therefore, it is necessary to establish a safe and effective triple vaccine for the prevalent strains in my country to fill the domestic gap. Summary of the Invention

[0006] Aiming at the technical problems that the existing FPV, FCV and FHV triple vaccines do not conform to the domestic popular trend and cannot achieve a good immune effect, the present invention proposes a new feline panleukopenia, feline infectious rhinoconjunctivitis and feline infectious rhinotracheitis triple inactivated vaccine and its preparation method and application.

[0007] One of the purposes of the present invention is to provide a triple inactivated vaccine for feline panleukopenia, feline infectious rhinoconjunctivitis and feline infectious rhinotracheitis, wherein the antigens of the triple inactivated vaccine include inactivated feline panleukopenia virus VP2 protein, inactivated feline infectious rhinoconjunctivitis virus antigen and inactivated feline infectious rhinotracheitis virus antigen; wherein the feline infectious rhinoconjunctivitis virus antigen is prepared from the feline infectious rhinoconjunctivitis virus CC3 strain, and the feline infectious rhinoconjunctivitis virus CC3 strain is deposited in the General Microbiology Center of the China Culture Collection of Microorganisms, with an address of No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, with a deposit number of CGMCC No. 45218, and is classified as feline infectious rhinoconjunctivitis virus. The deposit date is July 1, 2022. The virus belongs to the same branch as the domestic prevalent strain and has a high homology. The feline infectious rhinotracheitis virus antigen is prepared from the feline infectious rhinotracheitis virus CP2 strain. The feline infectious rhinotracheitis virus CP2 strain is deposited in the General Microbiology Center of the China Culture Collection Administration, located at No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing. The deposit number is CGMCC No. 45219, and the classification name is feline infectious rhinotracheitis virus. The deposit date is July 1, 2022.

[0008] Furthermore, the hemagglutinin titer of the feline panleukopenia virus VP2 protein stock solution is 1:4096. The virus content of the feline infectious rhinoconjunctivitis virus stock solution is 10 9.50 TCID 50 / ml. The virus content of feline infectious rhinotracheitis virus stock solution is 10 8.75 TCID 50 / ml.

[0009] Furthermore, the gene sequence of feline panleukopenia virus VP2 protein is shown in SEQ ID No.1.

[0010] Furthermore, the inactivated feline panleukopenia virus VP2 protein, the inactivated feline infectious rhinoconjunctivitis virus antigen and the inactivated feline infectious rhinotracheitis virus antigen are mixed into the vaccine in a ratio of 1:2:2 to the volume of the virus stock solution.

[0011] Furthermore, the inactivator used was 0.1 mol / L BEI.

[0012] Furthermore, the triple inactivated vaccine also includes Gel 02 PR adjuvant, and the mixed virus liquid of inactivated feline panleukopenia virus VP2 protein, inactivated feline infectious rhinoconjunctivitis virus antigen and inactivated feline infectious rhinotracheitis virus antigen is mixed with Gel 02 PR adjuvant in a volume ratio of 7:1.

[0013] The second object of the present invention is to provide a method for preparing the above-mentioned triple inactivated vaccine for feline panleukopenia, feline infectious rhinoconjunctivitis and feline infectious rhinotracheitis, including a method for preparing feline panleukopenia virus VP2 protein, feline infectious rhinoconjunctivitis virus antigen and feline infectious rhinotracheitis virus antigen and a method for preparing the vaccine.

[0014] The third object of the present invention is to provide the use of the above-mentioned triple inactivated vaccine for feline panleukopenia, feline infectious rhinoconjunctivitis and feline infectious rhinotracheitis in the preparation of medicaments for feline panleukopenia, feline infectious rhinoconjunctivitis and feline infectious rhinotracheitis.

[0015] Compared with the prior art, the present invention has the following beneficial effects:

[0016] The present invention screens and isolates the feline infectious rhinoconjunctivitis virus CC3 strain and the feline infectious rhinotracheitis virus CP2 strain, which are popular in my country, highly immunogenic, have high viral titers and are genetically stable. The strains are more suitable for the domestic epidemic trend. The new isolates can be used as vaccine strains after passage. The prepared triple inactivated vaccine has good safety performance and good antigen compatibility. After immunizing animals, no local or systemic adverse reactions caused by the vaccine occur, and the vaccine has good safety. At the same time, the vaccine can induce the secretion of high concentrations of cytokines, including Th1 type cytokines (TNF-α, IFN-γ) and Th2 type cytokines (IL-6). The vaccine has high immunogenicity and a long immune duration, further improving the immune effect and ensuring the stability of the vaccine. Especially in terms of the protective efficacy of the feline infectious rhinoconjunctivitis virus, the effect is particularly outstanding, filling a domestic gap. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] In order to more clearly illustrate the technical solutions of the embodiments of the present application, the following briefly introduces the drawings required for use in the embodiments. Obviously, the drawings described below are only some embodiments recorded in the present invention. For ordinary technicians in this field, other drawings can also be obtained based on these drawings.

[0018] Figure 1 This is the result of immunofluorescence staining to identify VP2 expression provided in Example 1 of the present invention. In the figure, A is Sf9 cells infected with the recombinant baculovirus FPV-VP2 strain; B is normal Sf9 cells.

[0019] Figure 2 The identification results of the feline panleukopenia virus VP2 structural protein provided in Example 1 of the present invention are shown in Figure A. A is an SDS-PAGE image (1: cell control; 2: cell culture; 3: supernatant after centrifugation of the cell culture, 4: supernatant after centrifugation of the cell culture with NaHCO3 treatment and centrifugation; 5: precipitate after centrifugation of the cell culture with NaHCO3 treatment and centrifugation; M: pre-stained marker); B is a Western blotting image (1: cell culture; 2: cell control; M: pre-stained marker).

[0020] Figure 3 This is the electron microscope negative staining observation of virus-like particles provided in Example 1 of the present invention.

[0021] Figure 4 This is the genetic evolution analysis of the FCV CC3 strain provided in Example 2 of the present invention.

[0022] Figure 5 The results of FCV isolation and culture provided in Example 2 of the present invention, wherein A: normal F81 cells; B: FCV-infected pathological F81 cells.

[0023] Figure 6 This is the FCV PCR identification result provided in Example 2 of the present invention, wherein 1: CC1; 2: CC2; 3: CC3; 4: negative control; 5: positive control; M: Marker 2000.

[0024] Figure 7 This is the PCR identification result of the exogenous virus FHV provided in Example 2 of the present invention, wherein 1: CC1; 2: CC2; 3: CC3; 4: positive control; 5: negative control; M: Marker 2000.

[0025] Figure 8 This is the indirect immunofluorescence result provided in Example 2 of the present invention, wherein A: CC1; B: CC2; C: CC3; D: normal F81 cells.

[0026] Figure 9 This is the FCV electron microscopy observation result provided by Example 2 of the present invention, wherein A: CC1; B: CC2; C: CC3.

[0027] Figure 10 This is the genetic evolution analysis of the gB gene of the FHV CP2 strain provided in Example 3 of the present invention.

[0028] Figure 11 The cytopathic effect results provided in Example 3 of the present invention, in which A: CP2 cell cytopathic effect; B: normal cell control.

[0029] Figure 12 The indirect immunofluorescence results provided in Example 3 of the present invention, in which A: P3 virus solution infected F81 cells; B: normal F81 cells; virus-infected F81 cells showed high-intensity green fluorescence ( Figure 12 A), while cells in the control group showed no visible fluorescence ( Figure 12 Middle B).

[0030] Figure 13 This is the FHV electron microscopy observation result provided by Example 3 of the present invention.

[0031] Figure 14 This is a comparison of the cytokine secretion level results provided in Example 10 of the present invention. DETAILED DESCRIPTION

[0032] In order to make those skilled in the art better understand the technical scheme of the present invention, the present invention will be further described in detail below in conjunction with Examples and accompanying drawings. Obviously, the described embodiments are only a part of embodiments of the present invention, rather than all embodiments. Based on the embodiments in the present invention, all other embodiments obtained by those of ordinary skill in the art without making creative work premise belong to the scope of protection of the present invention. The experimental method in the following examples, unless otherwise specified, is conventional method. The test materials used in the following examples, unless otherwise specified, are purchased from conventional biochemical reagent stores.

[0033] Example 1 Construction and identification of recombinant baculovirus FPV-VP2 strain

[0034] The present invention inserts two optimized feline panleukopenia virus VP2 genes (the gene sequence of the feline panleukopenia virus VP2 gene is shown in SEQ ID No. 1) into the transfer vector pFastBacDual, transforms competent cells containing the baculovirus genome, and obtains the Bacmid-dVP2 recombinant plasmid. After transfection into Sf9 cells, the recombinant baculovirus AcMNPV-VP2 expressing the feline panleukopenia virus capsid protein VP2 is successfully rescued and named the recombinant baculovirus FPV-VP2 strain. The test results show that the viral content of the recombinant baculovirus FPV-VP2 strain is 4.4×10 7 IFU / ml or more; the results of indirect immunofluorescence and Western blotting showed that the constructed recombinant baculovirus successfully expressed specific VP2 protein (such as Figure 1 and Figure 2 ); The results of electron microscopy and hemagglutination tests showed that the protein expressed in this experiment could be assembled into virus-like particles (such as Figure 3 ), the hemagglutinin titer of the harvested recombinant baculovirus cell culture can reach 1:2 14 The strain's morphology and hemagglutination properties are consistent with those of natural feline panleukopenia virus, and animal testing has shown good immunogenicity. When the strain's hemagglutinin titer is no less than 1:1024, the protection rate is no less than 80%. To ensure effective immunity, the hemagglutinin titer used in the immunogenicity of the strain is set at 1:2048.

[0035] Example 2 Isolation and Identification of Feline Infectious Rhinoconjunctivitis Virus CC3 Strain

[0036] The present invention processes 10 eye and nasal swabs collected from cats suspected of having feline infectious rhinoconjunctivitis in Changchun City, Jilin Province, and then isolates and cultures them on F81 cells. After sterility testing, mycoplasma testing, exogenous virus testing, PCR testing, virus isolation and culture, virus content determination, virus morphology testing, specificity testing, and pathogenicity testing, a strain of feline infectious rhinoconjunctivitis virus is finally screened out. The results of genetic evolution analysis show that the isolated strain is on the same branch as the main domestic isolated strains (most foreign isolated strains (including vaccine strain 255 and classic strain F9) are in another branch), and are consistent with the domestic prevalent strains, and are named feline infectious rhinoconjunctivitis virus CC3 strain. By sequence analysis of the ORF2 gene of the P1 and P10 generations of viruses, it was found that the ORF2 gene of the CC3 strain had no difference (such as Figure 4 ), while CC1 and CC2 strains have amino acid mutations. The viral contents of CC3 strain P1 to P3 generations are 10 8.00 TCID 50 / ml, 10 9.23 TCID 50 / ml, 109.33 TCID 50 / ml, inoculation of F81 cells can cause cell shrinkage, shedding and the appearance of grape-like cell lesions (such as Figure 5 ) and can be specifically neutralized by feline infectious rhinoconjunctivitis virus positive serum; the harvested P3 generation virus liquid was subjected to PCR identification using FCV identification primers, and the results were as follows Figure 6 As shown, the PCR amplification product was 618 bp; at the same time, the exogenous virus FHV was detected, and the results were all negative (such as Figure 7 ); Indirect immunofluorescence showed that the virus could be recognized by specific antibodies (such as Figure 8 ), transmission electron microscopy can reveal spherical, non-enveloped virus particles of about 35 nm in size (e.g. Figure 9 ). The test cats were inoculated with feline infectious rhinoconjunctivitis virus CC3 strain by nasal drops. Two days after inoculation, symptoms of varying degrees appeared, including increased discharge from the corners of the eyes, ulcers on the tongue, depression, and other typical symptoms of feline infectious rhinoconjunctivitis, and even death. This indicates that feline infectious rhinoconjunctivitis virus CC3 strain is a highly pathogenic and virulent strain of feline infectious rhinoconjunctivitis virus. This strain is deposited at the General Microbiology Center of the China Culture Collection Administration, located at No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing. The deposit number is CGMCC No. 45218, and the classification name is feline infectious rhinoconjunctivitis virus. The deposit date is July 1, 2022.

[0037] The feline infectious rhinoconjunctivitis virus CC3 strain was propagated on F81 cells for 8 generations, and the P2, P3, P5 and P8 generations of viruses were collected to conduct a comprehensive identification of the virus content and virulence of the virus liquid. The test results showed that the strain was not contaminated by bacteria, molds, mycoplasmas, or exogenous viruses. The P2, P3, P5 and P8 generations of feline infectious rhinoconjunctivitis virus CC3 strain could be neutralized by feline infectious rhinoconjunctivitis virus positive serum, showing the specificity of feline infectious rhinoconjunctivitis virus. The virus content was 10 9.23 TCID 50 / ml, 10 9.33 TCID 50 / ml, 10 9.50 TCID 50 / ml, 10 9.23 TCID 50 / ml, and 5 / 5 cats developed disease after intranasal challenge. Therefore, the P2 generation of the CC3 strain of feline infectious rhinoconjunctivitis virus was used as the primary virus, and generations P3 to P5 were used as potent test viruses. The P5 generation virus solution was stored at -70°C for 42 months, and its viral content showed no significant change, giving it a shelf life of 36 months.

[0038] The P5 generation of feline infectious rhinoconjunctivitis virus CC3 strain was inoculated into F81 cells and passaged continuously to the 19th generation. The P6, P7, P10, P13, P16, and P19 generations were fully identified and tested. The results showed that the P6, P7, P10, P13, P16, and P19 generations were free of bacteria, molds, mycoplasmas, and exogenous viruses. The virus strains were specific and had good immunogenicity. The virus titers of the virus strains were not less than 10 9.50 TCID 50 The virus strain was tested for shelf life, and the results showed that the virus strain was stored below -70°C for 30 months, and the production virus strain was stored below -20°C for 9 months, with no significant change in virus content (the virus content change value was not higher than 10 0.50 TCID 50 / ml).

[0039] Example 3 Isolation and Identification of Feline Infectious Rhinotracheitis Virus CP2 Strain

[0040] The present invention collected nasal and ocular swabs from 18 cats suspected of having feline infectious rhinotracheitis at an animal hospital in Changchun, Jilin Province, processed them, isolated and cultured them on F81 cells, and plaque purified them. Sterility tests, mycoplasma tests, exogenous virus tests, PCR tests, virus content determination, virus morphology tests, specificity tests, and pathogenicity tests were performed on them. Finally, a feline infectious rhinotracheitis virus CP2 strain was successfully obtained. Genetic analysis results showed that the nucleotide and amino acid homology with the reference strain published in NCBI were very high, and they were in the same branch (such as Figure 10 ), named as feline infectious rhinotracheitis virus CP2 strain. The virus content of P1 to P3 generations was 10 6.00 TCID 50 / ml, 10 7.23 TCID 50 / ml, 10 7.33 TCID 50 / ml, inoculation of F81 cells may cause obvious cytopathic effects (such as Figure 11 ), the cells showed aggregated rounding, shrinkage, netting, and beading, and could be specifically neutralized by feline infectious rhinotracheitis virus positive serum; indirect immunofluorescence showed that the virus could be recognized by specific antibodies (such as Figure 12 ), transmission electron microscopy showed round, enveloped, herpes-like virus particles (such as Figure 13). When cats were inoculated with feline infectious rhinotracheitis virus CP2 strain by nasal drops, symptoms of varying degrees appeared two days after inoculation, including increased eye discharge covering the upper and lower eyelids; increased nasal discharge blocking the nostrils and nasal cavity; sneezing and difficulty breathing, and other typical symptoms of feline infectious rhinotracheitis, and even death. This indicates that the feline infectious rhinotracheitis virus CP2 strain is a highly pathogenic and virulent strain. The strain is deposited at the General Microbiology Center of the China Culture Collection Administration, located at No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing. The deposit number is CGMCC No. 45219, and the classification name is feline infectious rhinotracheitis virus. The deposit date is July 1, 2022.

[0041] The feline infectious rhinotracheitis virus CP2 strain was propagated on F81 cells for 8 generations, and the P2, P3, P5 and P8 generations of viruses were collected to conduct a comprehensive identification of the virus content and virulence of the virus liquid. The test results showed that the strain was not contaminated by bacteria, molds, mycoplasmas, or exogenous viruses. The P2, P3, P5 and P8 generations of feline infectious rhinotracheitis virus CP2 strain could be neutralized by feline infectious rhinotracheitis virus positive serum, showing specificity for feline infectious rhinotracheitis virus. The virus titers were 10 7.23 TCID 50 / ml, 10 7.33 TCID 50 / ml, 10 7.50 TCID 50 / ml, 10 7.77 TCID 50 / ml, and 5 / 5 cats developed disease after intranasal challenge. Therefore, the P2 generation of the feline infectious rhinotracheitis virus CP2 strain was used as the primary virus, and generations P3 to P5 as the test strains. The P5 generation virus solution was stored at -70°C for 42 months, and its viral content showed no significant change, giving it a shelf life of 36 months.

[0042] The P5 generation of feline infectious rhinotracheitis virus CP2 strain was inoculated into F81 cells and passaged continuously to the 19th generation. The P6, P7, P10, P13, P16, and P19 generations were fully identified and tested. The results showed that the P6, P7, P10, P13, P16, and P19 generations were free of bacteria, molds, mycoplasmas, and exogenous viruses. The virus strains were specific and had good immunogenicity. The virus titers of the virus strains were not less than 10 8.75 TCID 50 The virus strain was tested for shelf life, and the results showed that the virus strain was stored below -70°C for 30 months, and the production virus strain was stored below -20°C for 9 months, with no significant change in virus content (the virus content change value was not higher than 10 0.50 TCID 50 / ml).

[0043] Example 4 Preparation of Inactivated Feline Panleukopenia Virus VP2 Protein

[0044] Construction of S101, recombinant baculovirus FPV-VP2 strain

[0045] By inserting two optimized feline panleukopenia virus VP2 genes (the gene sequence of the feline panleukopenia virus VP2 gene is shown in SEQ ID No. 1) into the transfer vector pFastBacDual, the Bacmid-dVP2 recombinant plasmid was obtained after transformation into competent cells containing the baculovirus genome. After transfection into Sf9 cells, the recombinant baculovirus AcMNPV-VP2 expressing the feline panleukopenia virus capsid protein VP2 was successfully rescued and named the recombinant baculovirus FPV-VP2 strain.

[0046] S102, culture of recombinant baculovirus FPV-VP2 strain

[0047] Inoculate Sf9 cells with the virus at an MOI of 0.1 to 1.0, and harvest the cell cultures 96 to 120 hours later;

[0048] S103, Feline Panleukopenia Virus VP2 Protein Harvesting

[0049] The cell culture of the recombinant baculovirus FPV-VP2 strain was harvested, and the cells were enriched using a 750KD ultrafiltration hollow fiber column. The cells were lysed using an equal volume of 25mmol / L NaHCO3 in the original solution for 1 hour at room temperature. Cell debris was removed using a 0.65μm microfiltration hollow fiber filter column. The lysed VP2 protein solution was harvested after sterilization and filtration through a 0.22μm filter cartridge. The hemagglutinin titer of the VP2 protein solution was not less than 1:2. 13 ;

[0050] S104, Feline Panleukopenia Virus VP2 Protein Inactivation

[0051] The feline panleukopenia virus VP2 protein stock solution was diluted to a hemagglutinin titer of 1:4096, and BEI was added to a final concentration of 0.001 mol / L and inactivated at 30°C for 44 hours. After inactivation, sodium thiosulfate was added to a final concentration of 0.001 mol / L to neutralize BEI.

[0052] S105, Feline Panleukopenia Virus VP2 Protein Purification

[0053] Use a 100KDa membrane package to replace the inactivated solution of feline panleukopenia virus VP2 protein with buffer A of Tris 20mmol / L, pH 8.0, and NaCl 0.05mol / L, and then perform Q column ion exchange chromatography. Use buffer B and buffer A of Tris 20mmol / L, pH 8.0, and NaCl 1.0mol / L for linear elution, collect elution peak 1, use a 100KDa membrane package for 10-50 times ultrafiltration concentration, load 6FF molecular sieves with 4% column bed volume, elute with 10mmol / L, pH 7.0 PBS as the mobile phase, and collect peak 1 target peak to obtain.

[0054] Example 5 Preparation of inactivated feline infectious rhinoconjunctivitis virus antigen

[0055] S201, Feline Infectious Rhinoconjunctivitis Virus CC3 Strain Culture

[0056] Inoculate F81 cells with the CC3 strain of feline infectious rhinoconjunctivitis virus at an MOI of 0.01 to 0.1 and culture for 24 to 48 hours. Observe the pathological changes daily. Harvest the cell culture when the cells are completely detached. After clarification, harvest the virus solution. The virus content should be no less than 10 9.50 TCID 50 / ml;

[0057] S202, Feline Infectious Rhinoconjunctivitis Virus Concentrate

[0058] After the virus liquid was harvested, cell debris was removed using a 20 μm hollow fiber filter. The virus liquid after cell debris removal was concentrated 10-fold using a 10KD ultrafiltration membrane to obtain the feline infectious rhinoconjunctivitis virus stock solution.

[0059] S203, Feline Infectious Rhinoconjunctivitis Virus Inactivated

[0060] Dilute the feline infectious rhinoconjunctivitis virus stock solution to a virus content of 10 9.50 TCID 50 / ml, add BEI with a final concentration of 0.001mol / L, inactivate at 30℃ for 20 hours, and then add sodium thiosulfate with a final concentration of 0.001mol / L to neutralize BEI to obtain the product.

[0061] Example 6 Preparation of Inactivated Feline Infectious Rhinotracheitis Virus Antigen

[0062] S301, Feline Infectious Rhinotracheitis Virus CP2 Strain Culture

[0063] Inoculate F81 cells with feline infectious rhinotracheitis virus CP2 strain at an MOI of 0.1-1 and culture for 24-72 hours. Observe the cytopathic effect daily. Harvest the cell culture when the cytopathic effect reaches 80%. After freeze-thaw clarification, harvest the virus solution. The virus content should be no less than 10 8.75 TCID 50 / ml;

[0064] S302, Feline Infectious Rhinotracheitis Virus Concentration

[0065] After the virus liquid was harvested, the cell debris was removed by a 20 μm hollow fiber filter, and the virus liquid after the cell debris was removed was concentrated 10 times using a 10KD ultrafiltration membrane to obtain the feline infectious rhinotracheitis virus stock solution;

[0066] S303, inactivation

[0067] Dilute the feline infectious rhinotracheitis virus stock solution to a virus content of 10 8.00 TCID 50 / ml, add BEI with a final concentration of 0.001mol / L, inactivate at 30℃ for 44-48 hours, and then add sodium thiosulfate with a final concentration of 0.001mol / L to neutralize BEI to obtain the product.

[0068] Example 7 Study on the ratio and antigen compatibility in triple inactivated vaccine

[0069] In order to study the ratio and antigen compatibility of each active component in the combined vaccine, the present invention uses five inactivated vaccines, namely, feline panleukopenia virus inactivated single vaccine (the purified feline panleukopenia virus liquid is diluted to a hemagglutinin titer of 1:4096, and mixed with aluminum hydroxide adjuvant at a ratio of 7:1), feline infectious rhinoconjunctivitis virus inactivated single vaccine (the inactivated feline infectious rhinoconjunctivitis virus liquid is mixed with aluminum hydroxide adjuvant at a ratio of 7:1), feline infectious rhinotracheitis virus inactivated single vaccine (the inactivated feline infectious rhinotracheitis virus liquid is mixed with aluminum hydroxide adjuvant at a ratio of 7:1), triple inactivated vaccine ( Target animal immunization trials were conducted using purified feline panleukopenia virus (FPV) diluted to a hemagglutinin titer of 1:4096 and mixed with FIV and FIV at a ratio of 1:2:2. The mixed virus solution was then mixed with aluminum hydroxide adjuvant at a ratio of 7:1. Furthermore, a second inactivated triple vaccine (purified FPV) was diluted to a hemagglutinin titer of 1:4096 and mixed with FIV and FIV at a ratio of 1:1:1. The mixed virus solution was then mixed with aluminum hydroxide adjuvant at a ratio of 7:1. Twenty-one days after the booster immunization, blood was collected, serum was isolated, and antibody titer levels were tested.

[0070] The experimental animals were divided into six groups, each consisting of five cats: control, FPV (Fluid Proliferation Virus) alone, FCV (Fertilizer for Hypertension) alone, FHV (Fertilizer for Hypertension) alone, triple vaccine group 1, and triple vaccine group 2. Each group received two immunizations, 21 days apart. The control group received MEM (1 ml per cat) subcutaneously in the neck; the FPV (Fluid Proliferation Virus) alone group received inactivated feline panleukopenia virus vaccine (0.2 ml per cat); the FCV (Fertilizer for Hypertension) alone group received inactivated feline infectious rhinoconjunctivitis virus vaccine (0.4 ml per cat); and the FHV (Fertilizer for Hypertension) alone group received inactivated feline infectious rhinotracheitis virus vaccine (0.4 ml per cat). Triple vaccine groups 1 and 2 received inactivated triple vaccine 1 and 2, respectively, subcutaneously in the neck (1 ml per cat).

[0071] Venous blood was collected from all test cats before immunization and 21 days after booster immunization, and the serum was separated to determine the FPV HI antibody titer, FCV neutralizing antibody titer and FHV neutralizing antibody titer in the serum.

[0072] The research results showed that the antibody levels of the triple inactivated vaccine group were comparable to those of the single vaccine groups, with no significant differences. This indicated that the three viruses in the triple inactivated vaccine did not interfere with each other, and the antigen compatibility was good according to the 1:2:2 ratio. There was no antigen interference, and the immune effects of the three single vaccines were maintained. Moreover, the immune effects of the antigens in the 1:2:2 ratio were better than those in the 1:1:1 ratio.

[0073] Example 8 Screening of adjuvants for triple inactivated vaccine and study of immune effects

[0074] In order to screen suitable adjuvants, the present invention used aluminum hydroxide adjuvant vaccine and MONTANIDE GEL02 PR adjuvant vaccine to conduct a comparative study on immunogenicity.

[0075] The purified inactivated feline panleukopenia virus solution was diluted to a hemagglutinin titer of 1:4096, and mixed evenly with the inactivated feline infectious rhinoconjunctivitis virus solution and the inactivated feline infectious rhinotracheitis virus solution at a ratio of 1:2:2. The mixed virus solution was mixed with aluminum hydroxide adjuvant and MONTANIDE GEL02 PR adjuvant at a ratio of 7:1 to prepare the vaccine.

[0076] The experimental animals were divided into three groups: a control group, an aluminum hydroxide adjuvant vaccine group, and a MONTANIDE GEL 02 PR adjuvant vaccine group. Each group consisted of five cats. The aluminum hydroxide adjuvant vaccine group received a 1.0 ml dose of the aluminum hydroxide adjuvant vaccine subcutaneously in the neck. The MONTANIDE GEL 02 PR adjuvant vaccine group received a 1.0 ml dose of the MONTANIDE GEL 02 PR adjuvant vaccine subcutaneously in the neck. The control group received a 1.0 ml subcutaneous injection of MEM in the neck. Immunizations were performed twice, 21 days apart. Venous blood was collected from all experimental cats before immunization and 21 days after the booster immunization. Serum was isolated and assayed for FPV HI antibody titers, FCV neutralizing antibody titers, and FHV neutralizing antibody titers.

[0077] Results: Twenty-one days after booster immunization, FPV HI antibody titers ranged from 1:4 to 1:8 in the control group, from 1:128 to 1:512 in the aluminum hydroxide adjuvanted vaccine group, and from 1:256 to 1:1024 in the Montanide Gel 02 PR adjuvanted vaccine group. FCV neutralization antibody titers were no higher than 1:4 in the control group, from 1:81 to 1:121 in the aluminum hydroxide adjuvanted vaccine group, and from 1:182 to 1:323 in the Montanide Gel 02 PR adjuvanted vaccine group. FHV neutralization antibody titers were no higher than 1:2 in the control group, from 1:48 to 1:77 in the aluminum hydroxide adjuvanted vaccine group, and from 1:64 to 1:98 in the Montanide Gel 02 PR adjuvanted vaccine group.

[0078] Immunogenicity testing with different adjuvants revealed that the FPV HI antibody titer, FCV neutralizing antibody titer, and FHV neutralizing antibody titer were all lower in the aluminum hydroxide-adjuvanted vaccine group than in the Montanide Gel 02 PR-adjuvanted vaccine group. FPV HI antibody titers in the Montanide Gel 02 PR-adjuvanted vaccine group were significantly different from those in the aluminum hydroxide-adjuvanted vaccine group (P < 0.05). Therefore, Montanide Gel 02 PR was determined to be the adjuvant for the feline triple inactivated vaccine against panleukopenia, infectious rhinoconjunctivitis, and infectious rhinotracheitis.

[0079] Example 9 Safety Test of Triple Inactivated Vaccine

[0080] Purified inactivated feline panleukopenia virus (FPV) was diluted to a hemagglutinin titer of 1:4096 and mixed with inactivated feline infectious rhinoconjunctivitis virus (FIN) and inactivated feline infectious rhinotracheitis virus (FIN) at a ratio of 1:2:2. The mixed virus solution was then mixed with MONTANIDE GEL02 PR adjuvant at a ratio of 7:1 to prepare the vaccine. Vaccines were administered using a single dose, repeated doses, and a single superdose. The test animals included the youngest target animal (8-week-old kittens), healthy kittens (8-12 weeks of age), and healthy adult cats (12-36 months of age). Cats in the single-dose safety trial underwent repeated vaccinations (second and third vaccinations). Cats were grouped as in the single-dose safety trial and immunized two more times, 14 days apart. Patients were observed for 14 consecutive days, focusing on clinical symptoms and body temperature. At the end of the observation period, the injection site was carefully inspected for abnormalities. Cats with abnormalities were dissected for safety evaluation using anatomical and histopathological methods.

[0081] The results of a single-dose vaccination safety test showed that a single-dose vaccination of the triple inactivated vaccine had no effect on the spirit, drinking / appetite, defecation, etc. of the test cats. No vomiting or allergic reactions occurred, and there was no inflammatory reaction in the injected part. The body temperature of all test animals was within the normal range (38.5-39.5℃). There was no significant difference in the body temperature of the vaccine group at different time points after vaccination compared with the body temperature before vaccination (P>0.05).

[0082] The results of the single-dose repeated safety test showed that single-dose repeated vaccination had no effect on the spirit, drinking / appetite, defecation, etc. of the test cats. No vomiting or allergic reactions occurred, and there was no inflammatory reaction in the injection part. The body temperature of all test animals was within the normal range (38.5-39.5℃). There was no significant difference in the body temperature of the vaccine group at different time points after vaccination compared with the body temperature before vaccination (P>0.05).

[0083] The results of a single overdose safety test showed that a single overdose vaccination had no effect on the spirit, drinking / appetite, defecation, etc. of the test cats. No vomiting or allergic reactions occurred, and there was no inflammatory reaction in the injected part. The body temperature of all test animals was within the normal range (38.5-39.5℃). There was no significant difference in the body temperature of the vaccine group at different time points after vaccination compared with the body temperature before vaccination (P>0.05).

[0084] In summary, the triple inactivated vaccine for panleukopenia, infectious rhinoconjunctivitis, and infectious rhinotracheitis was administered using a single dose, repeated single doses, and a single superdose inoculation in the target animals (8-week-old kittens), healthy kittens (8-12 weeks of age), and healthy adult cats (12-36 months of age). No adverse reactions were observed throughout the entire trial. Dissections of the test animals in the single superdose group revealed no abnormalities at the injection site anatomy or tissue sections, demonstrating the safety of the triple inactivated vaccine.

[0085] Example 10 Cytokine Test of Triple Inactivated Vaccine in Mice

[0086] The test vaccine consisted of purified inactivated feline panleukopenia virus diluted to a hemagglutinin titer of 1:4096. The mixture was then mixed with inactivated feline infectious rhinoconjunctivitis virus and inactivated feline infectious rhinotracheitis virus at a ratio of 1:2:2. The mixed virus solution was then mixed with MONTANIDE GEL02 PR adjuvant at a ratio of 7:1 to prepare the test veterinary drug. The control veterinary drug was a triple inactivated feline rhinotracheitis, calicivirus, and panleukopenia vaccine produced by Boehringer Ingelheim Animal Health (USA).

[0087] Animal grouping: 24 mice were divided into 2 groups, with 6 mice in each group.

[0088] Immunization: Immunize the test animal drug (immune group) and the control animal drug (control group) subcutaneously at a dose of 0.1 ml / mouse. Three weeks after the first immunization, perform a booster immunization using the same route and dose as the first immunization.

[0089] Blood collection: Collect blood from the ocular vein 1 week after the first immunization, 1 week after the booster immunization, and 5 weeks after the booster immunization. Separate the serum. The serum should not be less than 100ul, and 50ul / tube should be dispensed into small centrifuge tubes and stored at -80℃. Freezing and thawing are strictly prohibited.

[0090] Tests: After sample collection, nine mouse cytokine tests were performed, including IFN-γ, IL-10, IL-12p70, IL-1β, IL-2, IL-4, IL-5, IL-6, and TNF-α / mouse.

[0091] The data of 9 cytokines were collected one week after the first vaccination (W1), five weeks after the first vaccination (W5, two weeks after the booster vaccination), and eight weeks after the first vaccination (W8, five weeks after the booster vaccination). The results showed that one week after the first vaccination, some cytokines (IFN-γ, IL-2, IL-5, and IL-6) in the immunization group were different from those in the control group. The cytokines that showed differences one week after the first vaccination were plotted as bar graphs, as shown in Figure 2. Figure 14As shown, the immunized group induced high levels of cytokine secretion, including Th1 cytokines (IL-2, IFN-γ) and Th2 cytokines (IL-5, IL-6). The functional regulation of Th cell subsets and the cytokine environment contribute to enhanced protective immunity. Th1 cells produce cytotoxic T cells and complement-fixing antibodies, while Th2 cells produce a range of cytokines that promote antibody production. Vaccine-induced cytokines act synergistically. If the antigen binding to the B cell receptor does not interact synergistically with the membrane molecules and cytokines expressed by Th cells, an effective immune response cannot be induced. Cytokines are immunomodulatory polypeptides primarily secreted by activated immune cells. IL-2 is a pleiotropic cytokine that primarily promotes lymphocyte growth, proliferation, and differentiation, playing a vital role in the body's immune response and resistance to viral infection. IFN-γ has antiviral, immunomodulatory, and cell proliferation-regulating effects. IL-5 promotes the differentiation of antigen-stimulated B cells into antibody-producing cells, primarily targeting B cells entering the late proliferation phase and increasing IL-2R expression on activated B cells. IL-6 regulates immune responses, acute phase reactions, and hematopoiesis, and plays an important role in the body's anti-infective immune response. The immunized group secreted significantly higher levels of IFN-γ, IL-2, IL-5, and IL-6 than the control group, indicating that the tested veterinary drug can stimulate balanced Th1 and Th2 cell immunity and has a higher immune protection effect.

[0092] Example 11 Study on the immune efficacy and duration of immunity of triple inactivated vaccine

[0093] The test vaccine consisted of purified inactivated feline panleukopenia virus diluted to a hemagglutinin titer of 1:4096. The mixture was then mixed with inactivated feline infectious rhinoconjunctivitis virus and inactivated feline infectious rhinotracheitis virus at a ratio of 1:2:2. The mixed virus solution was then mixed with MONTANIDE GEL02 PR adjuvant at a ratio of 7:1 to prepare the test veterinary drug. The control veterinary drug was a triple inactivated feline rhinotracheitis, calicivirus, and panleukopenia vaccine produced by Boehringer Ingelheim Animal Health (USA).

[0094] The experimental animals were healthy kittens aged 10 to 12 weeks (FPV HI antibody titer no higher than 1:8, FCV neutralizing antibody titer no higher than 1:4, FHV neutralizing antibody titer no higher than 1:2), and the breed was Chinese rural cat.

[0095] The experimental groups were divided into 9-month challenge group, 12-month challenge group, and 15-month challenge group according to the challenge time. Each group was further divided into FPV group, FCV group, and FHV group according to the challenge virus. Each group was further divided into Group I (test veterinary drug group), Group II (control veterinary drug group), and Group III (non-immune control group).

[0096] The animal immunization test veterinary drug group was immunized with the test veterinary drug prepared by the present invention, with 45 healthy susceptible cats immunized twice with a 21-day interval by subcutaneous injection in the neck. The control veterinary drug group was immunized with the commercially available control veterinary drug, with 45 healthy susceptible cats immunized twice with a 21-day interval by subcutaneous injection in the neck. The non-immunization control group was immunized with MEM, with 1 ml per cat immunized twice with a 21-day interval by subcutaneous injection in the neck.

[0097] Antibody tests were performed before immunization, 7 days, 14 days, and 21 days after the first immunization, and 7 days, 14 days, 21 days, and 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 9 months, 12 months, and 15 months after the booster immunization to detect the FPV HI antibody titer and the neutralizing antibody titer of FCV and FHV in the immunized cats.

[0098] The FPV challenge group was challenged 9 months after booster immunization, 12 months after booster immunization, and 15 months after booster immunization. Each cat was orally administered 1.5 ml of feline panleukopenia virus JL-3 strain (hemagglutinin titer was 1:64), and the cats were isolated and kept for observation for 14 days. The incidence and protection status of each group were recorded.

[0099] The FCV challenge group was challenged with FCV 9 months after booster immunization, the FCV challenge group was challenged with FCV 12 months after booster immunization, and the FCV challenge group was challenged with FCV CC3 strain (virus content of 10 8.50 TCID 50 / ml) 0.5ml, isolated and raised, observed for 14 days, and the incidence and protection status of each group were recorded.

[0100] The FHV challenge group 9 months after booster immunization, the FHV challenge group 12 months after booster immunization, and the FHV challenge group 15 months after booster immunization were challenged with FHV CP2 strain (virus content 10 5.00 TCID 50 / ml)1ml, isolated and raised, observed for 14 days, and the incidence and protection status of each group were recorded.

[0101] In the FPV group, clinical symptoms of the animals were observed before challenge and on days 2, 4, 6, 8, 10, 12, and 14 after challenge. Changes in white blood cell counts were measured in each group and compared across the same groups at different time points. Fecal FPV was detected using FPV antigen test strips before challenge and on days 1 to 14 after challenge. In the FIR and FIR groups, body temperature was measured daily after challenge, and clinical symptoms, such as eyes, nose, and mental state, were observed daily. Body weight was measured before challenge and on days 7 and 14 after challenge. Weight on day 7 after challenge was compared with that before challenge and on day 14 after challenge. Eye and nasal swabs were collected before challenge and on days 2 to 14 after challenge for viral detection by PCR.

[0102] Results showed that in both the experimental and control groups, antibody levels began to rise 14 days after the initial vaccination, increased significantly 7 days after the booster vaccination, and peaked 21 days after the booster vaccination. Antibody levels began to decline 2 months after the booster vaccination, and by 15 months after the booster vaccination, antibody levels had significantly decreased. Antibody levels in the control group and the experimental group showed no significant difference 15 months after the booster vaccination (P>0.05), and serum FPV HI antibody titers in all immunized cats were no less than 1:32. All animals in the non-immunized control group excreted the virus by day 4 after challenge; none of the animals in the experimental or control groups excreted the virus. Following challenge, the non-immunized control group experienced 100% morbidity, with some animals dying. There were no cases of morbidity or mortality in either the experimental or control groups, resulting in a protection rate of no less than 80%, meeting the standard for protection against feline panleukopenia and comparable to the duration of immunity of similar commercially available imported products.

[0103] In the feline infectious rhinoconjunctivitis virus (FICV) group, antibody levels began to rise 14 days after the first vaccination in both the experimental and control groups. Antibody levels increased significantly 14 days after the booster vaccination, reaching a peak 21 days after the booster vaccination. Compared to the control group, antibody levels began to decline 1 month after the booster vaccination, with a significant decrease by 15 months after the booster vaccination. In the experimental group, antibody levels began to decline 2 months after the booster vaccination, with a significant decrease by 15 months after the booster vaccination. However, antibody levels in the experimental group and the control group were significantly different 15 months after the booster vaccination (P < 0.05). Serum FCV neutralizing antibody titers in all immunized cats were no less than 1:32. The eye and nasal swabs of the test cats in the non-immune control group and the control veterinary drug group were positive for feline infectious rhinoconjunctivitis virus on the 2nd to 3rd day after the infection, and the shedding of toxins lasted for more than 7 days; the eye and nasal swabs of the test cats in the test veterinary drug group were positive for feline infectious rhinoconjunctivitis virus on the 2nd to 7th day after the infection, and the shedding of toxins lasted for 5 to 7 days for some test cats, and for less than 4 days for some test cats. When the virus was attacked 9 months after immunization, the difference between the test veterinary drug group and the control veterinary drug group and the non-immune control group was extremely significant (P < 0.01); when the virus was attacked 12 months after immunization, the difference between the test veterinary drug group and the control veterinary drug group and the non-immune control group was extremely significant (P < 0.01); when the virus was attacked 15 months after immunization, the difference between the test veterinary drug group and the control veterinary drug group and the non-immune control group was extremely significant (P < 0.01); therefore, the test veterinary drug group was protected 9 months, 12 months, and 15 months after immunization, while the protective efficacy of the control drug group was far inferior to that of the test veterinary drug group, and the protective efficacy was significantly reduced at 15 months after immunization. Therefore, the immune efficacy of the test veterinary drug group was better and the duration of immunity was longer.

[0104] In the FIRTV group, antibody levels in the experimental drug group began to rise 21 days after the first vaccination, and significantly increased 7 days after the booster vaccination, reaching a peak 21 days after the booster vaccination. Antibody levels began to decline 1 month after the booster vaccination, and by 15 months after the booster vaccination, antibody levels had significantly decreased. Antibody levels in the control drug group and the experimental drug group showed no significant difference 15 months after vaccination (P>0.05), and serum FHV neutralizing antibody titers in all immunized cats were no less than 1:8. Eye and nasal swabs from cats in the non-immunized control group tested positive for FIRTV on day 4 after challenge, with virus shedding persisting for more than 7 days. Eye and nasal swabs from cats in the experimental and control drug groups tested positive for FIRTV from day 7 to 10 after challenge, with some cats shedding virus for more than 7 days, while others continued to shed virus for 5 to 7 days. At 9 months after immunization, the differences between the test and control groups and the non-immunized control group were extremely significant (P < 0.01). At 12 months after immunization, the differences between the test and control groups and the non-immunized control group were extremely significant (P < 0.01). At 15 months after immunization, the differences between the test and control groups and the non-immunized control group were extremely significant (P < 0.01). Therefore, at 9, 12, and 15 months after immunization, the test and control groups all provided protection, meeting the standard for protection against feline infectious rhinotracheitis. The duration of immunity is comparable to that of similar imported products.

[0105] The above description is merely illustrative of certain exemplary embodiments of the present invention. It goes without saying that those skilled in the art will be able to modify the described embodiments in various ways without departing from the spirit and scope of the present invention. Therefore, the above drawings and description are illustrative in nature and should not be construed as limiting the scope of protection of the claims.

Claims

1. A triple inactivated vaccine for feline panleukopenia, feline infectious rhinoconjunctivitis and feline infectious rhinotracheitis, characterized in that: The antigens of the triple inactivated vaccine are inactivated feline panleukopenia virus VP2 protein, inactivated feline infectious rhinoconjunctivitis virus antigen and inactivated feline infectious rhinotracheitis virus antigen; wherein, the feline infectious rhinoconjunctivitis virus antigen is prepared from the feline infectious rhinoconjunctivitis virus CC3 strain, which is deposited in the General Microbiology Center of the China Culture Collection of Microorganisms, with an address of No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, with a deposit number of CGMCC No. 45218, and is classified as feline infectious rhinoconjunctivitis virus, with a deposit date of July 1, 2022; the feline infectious rhinotracheitis virus antigen is prepared from the feline infectious rhinotracheitis virus CP2 strain, which is deposited in the General Microbiology Center of the China Culture Collection of Microorganisms, with an address of No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, with a deposit number of CGMCC No.45219, classified as feline infectious rhinotracheitis virus, with a deposit date of July 1, 2022; the gene sequence of the feline panleukopenia virus VP2 protein is shown in SEQ ID No.1; the hemagglutinin titer of the feline panleukopenia virus VP2 protein stock solution is 1:4096; the virus content of the feline infectious rhinotracheitis virus stock solution is 10 9.50 TCID 50 / ml; the virus content of feline infectious rhinotracheitis virus stock solution is 10 8.75 TCID 50 / ml; inactivated feline panleukopenia virus VP2 protein, inactivated feline infectious rhinoconjunctivitis virus antigen and inactivated feline infectious rhinotracheitis virus antigen are prepared in a virus liquid volume ratio of 1:2:2; the inactivator adopts 0.1mol / LBEI; the triple inactivated vaccine also includes Gel 02PR adjuvant, and the inactivated feline panleukopenia virus VP2 protein, inactivated feline infectious rhinoconjunctivitis virus antigen and inactivated feline infectious rhinotracheitis virus antigen mixed virus liquid and Gel 02PR adjuvant are mixed in a volume ratio of 7:

1.

2. The preparation method of the triple inactivated vaccine for cat panleukopenia, feline infectious rhinoconjunctivitis and feline infectious rhinotracheitis according to claim 1, wherein The following steps are involved: S1. Preparation of inactivated feline panleukopenia virus VP2 protein Two optimized feline panleukopenia virus VP2 genes are inserted into a transfer vector pFastBacDual, the gene sequence of which is shown in SEQ ID No.

1. Competent cells containing a baculovirus genome are transformed to obtain a Bacmid-dVP2 recombinant plasmid. After transfection into Sf9 cells, a recombinant baculovirus AcMNPV-VP2 expressing feline panleukopenia virus capsid protein VP2 is successfully rescued and named as a recombinant baculovirus FPV-VP2 strain. The virus is inoculated into Sf9 cells at an MOI of 0.1 to 1.0, and the cell culture is harvested after 96 to 120 hours. The cell culture is enriched using an ultrafiltration hollow fiber column, lysed using NaHCO3, and cell debris is removed using a microfiltration hollow fiber filter column. The lysed VP2 protein solution is then sterile filtered and harvested, and the hemagglutinin titer of the VP2 protein solution is no less than 1:

2. 13 The feline panleukopenia virus VP2 protein stock solution was diluted to a hemagglutinin titer of 1:4096, and then inactivated by adding BEI at a final concentration of 0.001 mol / L at 30°C for 44-48 hours. After inactivation, the BEI was neutralized by adding sodium thiosulfate at a final concentration of 0.001 mol / L. The inactivated VP2 protein was purified and diluted to a hemagglutinin titer of 1:4096 to obtain the inactivated feline panleukopenia virus VP2 protein. S2. Preparation of inactivated feline infectious rhinoconjunctivitis virus antigen Feline infectious rhinoconjunctivitis virus CC3 strain was inoculated into F81 cells at an MOI of 0.01 to 0.1 and cultured for 24 to 48 hours. The pathological changes were observed daily. When the cells were completely detached, the cell culture was harvested and the virus solution was harvested after clarification. The virus content was not less than 10 9.50 TCID 50 / ml, add BEI with a final concentration of 0.001mol / L, inactivate at 30℃ for 20-24 hours, and then add sodium thiosulfate with a final concentration of 0.001mol / L to neutralize BEI to obtain inactivated feline infectious rhinoconjunctivitis virus antigen; S3. Preparation of inactivated feline infectious rhinotracheitis virus antigen Feline infectious rhinotracheitis virus CP2 strain was inoculated into F81 cells at an MOI of 0.01 to 0.1 and cultured for 24 to 48 hours. The pathological changes were observed daily. When the cells were completely detached, the cell culture was harvested and the virus solution was harvested after clarification. The virus content was not less than 10 8.75 TCID 50 / ml, add BEI with a final concentration of 0.001mol / L, inactivate at 30℃ for 40-48 hours, and then add sodium thiosulfate with a final concentration of 0.001mol / L to neutralize BEI to obtain inactivated feline infectious rhinotracheitis virus antigen; S4. Seedling preparation The inactivated feline panleukopenia virus VP2 protein obtained in step S1, the inactivated feline infectious rhinoconjunctivitis virus antigen obtained in step S2, and the inactivated feline infectious rhinotracheitis virus antigen obtained in step S3 are mixed in a virus liquid volume ratio of 1:2:2, and Gel 02PR adjuvant is added. The mixed virus liquid and Gel 02PR adjuvant are mixed in a volume ratio of 7:

1.

3. the application of cat panleukopenia according to claim 1, feline infectious rhinoconjunctivitis and feline infectious rhinotracheitis triple inactivated vaccine in preparation prevention cat panleukopenia, feline infectious rhinoconjunctivitis and feline infectious rhinotracheitis medicine.

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