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CN116240179B - Preparation method of vaccine against strong strain of feline calicivirus disease virus - Google Patents

Preparation method of vaccine against strong strain of feline calicivirus disease virus Download PDF

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CN116240179B
CN116240179B CN202310216788.9A CN202310216788A CN116240179B CN 116240179 B CN116240179 B CN 116240179B CN 202310216788 A CN202310216788 A CN 202310216788A CN 116240179 B CN116240179 B CN 116240179B
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feline calicivirus
fcv
virus
shh202015
preparation
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CN116240179A (en
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刘健
白艺兰
赵洪进
王建
桂亚萍
杨显超
夏炉明
齐新永
陈伟锋
葛菲菲
朱晓英
杨德全
李鑫
唐聪圣
张玉杰
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Shanghai Animal Epidemic Prevention And Control Center (shanghai Veterinary Drug Feed Testing Institute And Shanghai Animal Husbandry Technology Promotion Center)
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Shanghai Animal Epidemic Prevention And Control Center (shanghai Veterinary Drug Feed Testing Institute And Shanghai Animal Husbandry Technology Promotion Center)
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Abstract

The invention provides a preparation method of an anti-feline calicivirus virulent strain immune vaccine, which comprises the following steps of culturing F81 cells, culturing feline calicivirus virulent strain-SHH 202015 virus, preparing immunogens, measuring vaccine immunity and neutralizing antibodies, wherein the neutralizing antibodies generated by the prepared feline calicivirus virulent strain-SHH 202015 immunogen can completely neutralize the feline calicivirus virulent strain, the titer reaches 1280, and meanwhile, the titer reaches 640, so that the feline calicivirus virulent strain-SHH 202015 immunogen can prevent infection of the feline calicivirus virulent strain and the common feline calicivirus pathogen, and provides an important technical means for prevention and control of the feline calicivirus disease.

Description

Preparation method of anti-feline calicivirus virus virulent strain immunity vaccine
Technical Field
The invention belongs to the technical field of vaccine preparation, and particularly relates to a preparation method of an anti-feline calicivirus virulent strain-SHH 202015 immune vaccine.
Background
The incidence of feline calicivirus is high in feline populations, with the primary clinical symptoms being canker sores and upper respiratory symptoms, and atypical symptoms such as lameness, abortion, chronic gastroenteritis, urinary tract infections, etc. also present. Because of the characteristic that the pathogenic cat calicivirus (FCV) of the cat calicivirus disease is easy to mutate, a cat calicivirus virulent strain (VS-FCV) capable of causing the systemic symptoms of the cat appears worldwide, the VS-FCV strain is manifested as common clinical symptoms such as upper respiratory diseases, and the like, and the infected cat also has acute lethal systemic diseases such as skin edema, fever, ulcerative dermatitis, anorexia, jaundice, and the like, and the death rate is as high as 50%.
At present, no domestic feline calicivirus vaccine exists, only one feline triple vaccine (feline calicivirus, feline herpesvirus type 1 and feline parvovirus) exists in the imported vaccine, wherein the nucleotide homology of the feline calicivirus strain and the epidemic strain is only 73.2%, the amino acid homology is only 82.7%, and multiple times of clinical report that the feline immunized cat triple vaccine still has the feline calicivirus disease, so that the early vaccine strain can not provide enough protection, and the life safety of cats such as pet cats is seriously endangered. In order to prevent and control infection and epidemic of the feline calicivirus, a vaccine should be developed by selecting a VS-FCV epidemic strain, so that on one hand, the infection of the VS-FCV can be prevented, the death rate of cats can be reduced, and on the other hand, the immune effect of the feline calicivirus can be improved, and a technical means is provided for prevention and control of the feline calicivirus.
Therefore, it is necessary to provide a new preparation method of an immune vaccine against a virulent strain of feline calicivirus virus to solve the above technical problems.
Disclosure of Invention
Aiming at the defects in the prior art, the invention aims to provide a preparation method of an anti-feline calicivirus virulent strain immune vaccine. Isolation and identification of feline calicivirus from clinical multiple cases of feline calicivirus infection, 1 strain of VS-FCV, designated as VS-FCV-SHH202015, was identified by cell culture, sequencing, electron microscopy, sequence alignment, amino acid comparison characteristic of VS-FCV, animal pathogenicity test, and was based on the strain VS-FCV-SHH 202015.
To achieve the above object, the solution of the present invention is:
a method for preparing an immunity vaccine against a virulent strain of feline calicivirus virus, comprising the steps of:
S1, culturing F81 cells;
s2, culturing the VS-FCV-SHH202015 virus;
S3, preparing an immunogen;
S4, determination of vaccine immunity and neutralizing antibodies.
In step S1, F81 cells are cultured by adding the recovered F81 cells into a fetal bovine serum cell culture solution with the volume percentage concentration of 10%, culturing in a CO 2 incubator with the volume percentage concentration of 5% at 37 ℃, digesting the cell suspension with pancreatin with the mass-volume ratio of 0.1% after growing into a monolayer, and diluting the F81 cell suspension with the cell culture solution with the volume percentage concentration of 10% until the cell density reaches 1X 10 6/mL for later use.
Further, in the step S2, the culture process of the VS-FCV-SHH202015 virus comprises the steps of adding a virus solution of the VS-FCV-SHH202015 into the F81 cell suspension, carrying out virus culture, and freezing and thawing the virus culture solution for three times for standby when 80% of F81 cells have cytopathic effect.
Further, in step S2, the sequence of the VS-FCV-SHH202015 virus is shown as SEQ ID NO. 1.
Further, in step S3, the preparation process of the immunogen comprises S31, virus inactivation, namely, determining TCID 50 of VS-FCV-SHH202015 virus liquid, diluting the VS-FCV-SHH202015 virus liquid to 1X 10 6 TCID 50/mL by using DMEM, adding the diluted VS-FCV-SHH202015 virus liquid into formaldehyde with the final mass volume percentage concentration of 0.2%, and inactivating the virus 28 h at 37 ℃;
S32, inactivating and verifying, namely mixing the inactivated virus liquid with an equal volume F81 cell suspension (1X 10 6/mL), adding the mixture into a 6-hole plate, culturing the mixture in a CO 2 incubator with the volume percentage concentration of 5% at the temperature of 2 mL/hole and 37 ℃, and observing cytopathy;
S33, preparing the immunogen, namely adding the aluminum gel adjuvant after the inactivation verification is qualified, and mixing the mixture according to the volume ratio of the antigen to the aluminum gel adjuvant of 1:1 for use.
Further, in step S4, the determination of vaccine immunization and neutralizing antibodies comprises the steps of:
S41, immunization with vaccine, namely injecting the immunogen into clean New Zealand rabbits subcutaneously at multiple points, injecting the immunogen into the clean New Zealand rabbits twice, and injecting the immunogen into the clean New Zealand rabbits at intervals of 14 d each time, wherein the immunogen is injected into each point by 0.5 mL, and simultaneously comparing the three different cat vaccines;
S42, detecting neutralizing antibody, namely collecting blood and separating serum after 14:26 of the last injection, and detecting neutralizing antibody by using VS-FCV-SHH202015 virus and common FCV virus to ensure that the titer of the neutralizing antibody is more than 320.
By adopting the scheme, the invention has the beneficial effects that:
The neutralizing antibody produced by the VS-FCV-SHH202015 immunogen prepared by the invention can completely neutralize the VS-FCV virus, the titer reaches 1280, the titer reaches 640, the antibody produced by the cat triple vaccine can not completely neutralize the VS-FCV-SHH202015 virus and the common FCV virus, the titer is only 40 and 160 respectively, the titer is unstable, and the duration is shorter. Therefore, the VS-FCV-SHH202015 immunogen can prevent the infection of the VS-FCV virus and the common FCV virus, and provides an important technical means for preventing and controlling the feline calicivirus disease.
Drawings
FIG. 1 is a graph of one-step growth of the VS-FCV-SHH202015 of the present invention.
FIG. 2 is a graph showing cytopathic effects of the strain VS-FCV-SHH202015 of the present invention after F81 cells were seeded (wherein seed 1 is F81 cells of a3 rd generation cell culture of VS-FCV-SHH 202015; 2 is a negative control).
FIG. 3 is a diagram of a VS-FCV-SHH202015 virion according to the present invention.
FIG. 4 is a phylogenetic tree of VP1 gene of VS-FCV-SHH202015 strain of the present invention.
FIG. 5 is a graph showing the neutralizing antibody titer reduction for the VS-FCV-SHH202015 strain of the present invention.
FIG. 6 is a graph showing the neutralizing antibody titer reduction against the FCV-SHH2001 strain of the present invention.
Wherein, the preservation number of the feline calicivirus VS-FCV-SHH202015 is CGMCC No.45209, the preservation date is 2022, 6 and 16 days, and the feline calicivirus is preserved in the China general microbiological culture Collection center with the address of North Star Xiya No.1, 3 in the Chaiyang area of Beijing city.
Description of the embodiments
The invention provides a preparation method of an anti-feline calicivirus virulent strain immune vaccine.
Referring to fig. 1, fig. 2, fig. 3, fig. 4 and fig. 5 in combination, the preparation method of the anti-feline calicivirus virulent strain immune vaccine of the present invention comprises:
Cells and viruses F81 cells were purchased from commercial North and urban Innovative Biotechnology Co., ltd, and the feline calicivirus virulent strain (VS-FCV-SHH 202015) was isolated and stored in the laboratory under the accession number (CGMCC No. 45209) of feline calicivirus virulent strain VS-FCV-SHH 202015.
Viral complete Gene sequence the complete gene sequence Genbank (accession number: OQ 296623) of VS-FCV-SHH202015 (7 th generation).
Experimental animals, clean grade New Zealand rabbits, female, 3 months old, 6, body weight 2-2.5 kg, purchased from Shanghai Proteus Biotechnology Co.
The main reagents and instruments are fetal bovine serum from sigma company, DMEM medium from GIBCO company, pancreatin from GIBCO company, alumina gel adjuvant from Chuangzhi Biotechnology Co., ltd, and cell culture from CORNING company.
F81 cell culture, namely recovering F81 cells, culturing in a culture solution containing 10% of fetal bovine serum in a culture box with 5% CO 2 at 37 ℃, digesting the culture solution with 0.1% of pancreatin by mass and volume ratio after growing into a monolayer, and diluting F81 cell suspension with the cell culture solution with 10% of serum concentration until the cell density reaches 1X 10 6 cells/mL for later use.
The optimal virus collection time in the virus culture stage is that 10mL cell suspension is added into a 25 cm 2 cell culture flask, and is cultured in a 5% CO 2 incubator at 37 ℃ for 24h, and 1mL of VS-FCV-SHH202015 virus solution is added after the cells grow into a monolayer. Sampling every 12h hours, detecting virus titer at intervals of 0 hour, 12 hours, 24 hours, 36 hours, 48 hours, 60 hours and 72 hours, drawing a virus one-step growth curve, and determining the optimal virus collection time. The harvested virus solution was centrifuged at 2,000Xg to remove cell debris, and the supernatant was sub-packaged and frozen at-80 ℃. The virus growth kinetics curves show (see FIG. 1) that under 37℃culture conditions, the VS-FCV-SHH202015 had virus titers of 0, 12, 24, 36, 48, 60 and 72h of 1×104、2.65×106、2.01 ×107、4.52 ×108、5.21 ×108、3.78 ×108、 and 4.14X10 8TCID50/mL respectively, and thus the optimal virus recovery time was 48 h.
Virus TCID 50 assay by adding cell suspension to 96 well plates, 50 μl of each well, serial 10-fold dilution of virus with DMEM (10 -1-10-11), sequentially adding to 96 well plates, incubating at 50 μl/well, 37 ℃ in 5% CO 2 incubator, observing cytopathic effect day by day, stopping observation after 4-5d, recording dilution and number of wells at which cytopathic effect finally occurs, and calculating TCID 50 according to Reed-Muench method.
The preparation of the immunogen comprises the steps of adding 5.21 multiplied by 10 8TCID50/mL of virus liquid into formaldehyde with the final concentration of 0.2%, inactivating the virus 28 h at 37 ℃, and preparing the immunogen with an aluminum gel adjuvant according to the ratio of antigen to aluminum gel adjuvant=1:1 after the virus TCID 50 is detected and verified to be qualified.
The experimental rabbits are divided into 3 groups, wherein 2 rabbits are respectively an immune VS-FCV-SHH202015 vaccine group, an immune cat triple vaccine group and an immune DMEM control group. Vaccine immunization the immunogen was subcutaneously injected in clean New Zealand rabbits at multiple points, 2 times per rabbit, at intervals of 14 d each time, 4 points each time, 0.5 mL immunogen each time. Neutralizing antibody assay serum samples were collected at 0,7,14,21,28,35,42 and 49 days and tested with the VS-FCV-SHH202015 strain and FCV-SHH2001 strain, respectively.
Serum neutralizing antibody detection, the values of TCID 50 of the VS-FCV-SHH202015 strain and FCV-SHH2001 strain were determined according to the method of detecting the virus TCID 50. Serum neutralizing antibodies were detected by the fixed virus-diluted serum method by diluting both strains to 200 TCID 50, 20-fold dilution of serum followed by 30 min treatment in a 56℃water bath, followed by 2-fold serial dilutions (2 -1-2-11). 100. Mu.L of serum and 100. Mu.L of virus were mixed and then perceived to be 1.5 h at 37℃with viral control, serum toxicity control, cell blank control and negative and positive serum control. 50 mu L of the perceived mixed solution and the equivalent cell suspension are simultaneously added into a 96-well plate, the mixture is incubated in a 37 ℃ and 5% CO 2 incubator, cytopathy is observed day by day, the observation is stopped after 4-5 d, the serum dilution of the cytopathy finally occurs is recorded, and the maximum serum dilutions capable of neutralizing the two strains of viruses are respectively recorded and determined.
And (3) data analysis, namely respectively drawing neutralizing antibody titer reduction graphs of the two strains of viruses.
Results analysis As shown in FIGS. 5 and 6, antibody monitoring was continued until day 49 post-immunization, and the antibodies produced by the VS-FCV-SHH202015 vaccine group were able to neutralize the VS-FCV-SHH202015 virus, and were able to achieve an antibody titer of 1280, and also able to neutralize the FCV-SHH2001 virus, and an antibody titer of 640. Antibodies generated by the cat triple vaccine can not completely neutralize the VS-FCV-SHH202015 virus and the common FCV virus, the titer is only 40 and 160 respectively, and the titer is unstable and the duration is short.
In conclusion, the VS-FCV-SHH202015 vaccine induces a body to generate a neutralizing antibody with higher titer on the 28 th day after one immunization (2 times of immunization are performed), the VS-FCV-SHH202015 vaccine antibody can neutralize the FCV virulent strain and can also neutralize the FCV classical strain well, and meanwhile, the antibody generated by the cat triple vaccine can not completely neutralize the FCV virulent strain and has poor neutralizing capability on the FCV classical strain. Therefore, the invention is helpful for preventing and controlling the feline calicivirus, becomes an important technical means for preventing and controlling the FCV, solves the problem of poor effect of the current imported vaccine, and fills the blank that no domestic FCV vaccine is produced in China.
The complete gene sequence (SEQ ID NO. 1) (accession number: OQ 296623) of the VS-FCV-SHH202015 strain ：GTAAAAGAAATTTGAGACAATGTCTCAAACTCTGAGCTTCGTGCTTAAAACTCACAATGTCAAGAAAGACTTTGTGCACTCCGTCAAGTTAACACTTGCTCGGAGGCGCGATCTTCAGTATTTCTATAACAGGCTCTCTCACACAATTCGTGCTGAGGCCTGTCCCTCTTGTGCTAGTTATGACGTTTGTCCTAACTGCACCTCTGGTAATATCCCTGATGACGGGTCGTCGATAAATTCGATCCCATCTTGGGAAGACATTACCAAAACCTCTACTTACTCTCTCCTGTTATCTGAGGATACGACCGATGAACTTTGCCCTGATGACTTGGCCAACATCGCATCCCACATCCGCAAGGCATTATCGACTCAGTCTCACCCCGCTAACAATGATATGTGCAAAGAACAGCTCACATCATTGTTGGTTGTGGCTGAAGCGATGCTGCCCCAGCGATCACGGTCCACTATCCCTCTTCATCAACAACACCAGGCAGCTCGCTTGGAATGGAGAGAGAAGTTCTTCTCTAAACCAATTGACTTCCTCCTGGAAAGACTTGGGCTGTCAAAGGATATCCTTCAGACTACCGCGATTTGGAAAATTCTTTTGGAAAAGGCCTGCTATTGCAAATCCTATGGGGAACAATGGTACACCACCGCAAGAACAAAATTACGTGAAATCAAATGTTTTGAAGGAAACACCCTTAAACCACTGGTTGGAGCTTTCATTGATGGCCTTCGATTCATGACTGTTGACAACCCAAATCCCATTGGCTTTCTTCCAAAATTAATTGGGTTGATTAAACCTCTCAACTTGGCTATGATAATTGACAACCATGAAAATACAATGTCGGGATGGATAGTTACAATAACAGCCATTATGGAGTTGTATAATATAACTGAATGTACCATTGATATCATAACTTCATTGATAACTGGGTTTTATGACAAACTTGCTAAGGCTACTCGATTCTATAGCCAGGTAAAGAGCTTATTTACTGGCTTTAGATCTGAGGATGTTTCTAATTCATTCTGGTACATGGCAGCTGCAGTCTTGTGTTACTTGATTACTGGGCTTCTCCCTAACAACGGAAGGTTTTCAAAAATTAAGGCTTGCCTGTCAGGTGCATCTACATTGGTCTCTGGTATTATTGCAACCCAAAAGTTAGCTGCTATGTTTGCAACGTGGAATTCGGAAACTATCGTTAATGAGCTATCTGCTAGGACAGTTGCCCTCTCTGAGTTGAACAACCCAACCACTACCTCTGACACGGATTCCGTGGAAAAATTATTAGAATTGGCTAAGATCTTGCATGAAGAGATAAAAGTACACACATTAAACCCAATAATGCAATCATACAACCCGATTCTCAGGAATTTGATGTCCACGCTTGATGGCGTTATAACATCATGTAACAAAAGGAAGGCAATTGCCAAAAAGAGGCCTGTCCCAGTTTGCTATATCCTCACCGGTCCTCCTGGTTGTGGGAAAACTACAGCTGCCCTGGCATTGGCAAAGAAGTTGTCTGAACAAGAGCCATCAGTTATTAACCTTGATGTGGATCATCATGATACTTACACCGGCAATGAGGTTTGCATTATTGATGAATTTGACTCATCTGACAAAGTTGATTTTGCAAATTTTGTTATTGGGATGGTAAATTCGGCCCCTATGGTCCTAAACTGTGATATGCTTGAGAACAAGGGTAAACTCTTTACTTCTAAATATATCATTATGACCTCTAACTCTGAAACTCCTGTCAAACCAGCTTCTAAGCGTGCTGGCGCATTCTACCGAAGGGTAACAATCATAGATGTAACAAACCCTTTGGTGGAGTCGCACAAGCGTGCCAGACCTGGTACCACTGTACCTCGTAGTTGCTACAAGAAAAACTTTTCCCACTTGTCCCTGGCAAAACGGGGAGCTGAGTGTTGGTGCAAGGAATATGTCTTGGACCCAAAGGGACTTCAACACCAAAGCATTAAAGCCCCTCCTCCTAGTTTCTTGAATATTGACTCTCTTGCACAAACCATGAAACAAGATTTCACCCTTAAAAACATGGCTTTTGATGCTGAGGAAGGATGTAGTGAACACCGTTATGGTTTTGTCTGTCAAAGAGATGAGGTTGAAACAGTGCGCAGGCTACTCAATGCCATAAGAGTCAGGCTCAATGCCACCTTCACAGTTTGTGTGGGATCTGAAGCCTCAACCAACTCTGTAGGGTGTACGGCACACGTACTGACACCTGAGGAGCCATTCAATGGTAAGAGGTTTGTGGTCTCGCGCTGCAATGAGGCATCCCTAGCTGCACTAGAAGGCAACTGTGTCCAAACTGCATTGGGCATATGCATGTCCGACAAGGATCTTACCCATTTGTGCCACTTTATAAAAGGGAAGATTGTCAATGATAGTGTTAGGTTGGATGAACTACCCGCCAATCAACATGTGGTAACTGTTAATTCAGTGTTTGATCTGGCCTGGGCTCTTCGCCGACACCTATCATTAGCAGGACAGTTTCAAGCCATCAGAGCCGCATATGATGTGCTTACTGTCCCTGACAAAATCCCAGCTATGTTGCGGCATTGGATGGATGAGACCTCTTTCTCGGATGAGCATGTTGTGACGCAGTTTGTAACCCCAGGGGGGATAGTTATCCTTGAATCGTGTGGGGGTGCACGCATCTGGGCCATCGGTCACAATGTGATCAGGGCCGGAGGCATCACTGCCACACCAACCGGGGGTTGCGTTAGATTGGTTGGCCTATCCGCGCAAACAATGCCATGGTCTGAAATCTTTAGGGAACTCTTCACCCTGTTAGGGAAAATCTGGTCTAGTGTTAAGGTCTCCACTCTTGTTCTTACTGCCCTCGGGATGTACGCATCAAGATTTAGGCCAAAGTCTGAAGCAAAAGGTAAAACAAAATCCAAAATTGGGCCATACAGGGGTCGCGGGGTAGCTCTAACTGATGATGAATATGATGAATGGAAAGAGCACAATGCAAGTAGAAAACTGGACCTATCAGTGGAGGATTTTCTAATGCTAAGACATCGCGCCGCCCTAGGCGCTGATGATGCAGACGCAGTAAAATTTAGATCTTGGTGGAACTCAAGGTCAAAATTGGCATATGATGATTTTGAGGACGTTACCGTAATTGGAAAAAGTGGTGTCAAACATGAAAGGGTTAGAACAAATGTTATGAGAGCCGTTGATCGTGGTTACGATGTAAGCTTTGCAGAGGAATCTGGTCCTGGTACAAAATTCCACAAGAATGCAATTGGCTCTGTTACTGATGTGTGTGGTGAACACAAAGGATACTGTGTCCATATGGGTCACGGTGTGTACGCATCTGTTGCTCATGTGGTCAAGGGAGACTCCTTCTTCTTGGGTGAGCGGATCTTTGATCTAAAGACAAATGGTGAGTTCTGCTGCTTCAGAAGTACTAAGATTCTCCCAAGTGCAGCTCCTTTCTTTTCTGGCAAACCCACTCGTGATCCATGGGGATCCCCTGTGGCAACGGATTGGAAGCCAAAAGCCTACACCACAACATCGGGAAAAATTGTGGGTTGTTTTGCAACCACATCAACAGAAACTCACCCAGGCGACTGCGGCCTGCCATACATTGATGACAACGGCAGGGTAACTGGATTGCATACTGGATCAGGTGGCCCAAAAACTCCTAGTGCAAAGTTGGTTGTCCCCTACATTCACATTGACATGAAGACAAAATCAGTCACCGCCCAAAAATACGATGTCACCAAGCCAGACATTAGCTATAAGGGATTAATTTGTAAACAATTGGATGAAATTAGGATTATACCAAAAGGAACACGACTTCATGTTTCTCCAGCCCACGTTGATGATTATGAAGAGTGTTCACACCAACCTGCATCCCTAGGTAGTGGTGATCCTCGGTGTCCCAAATCATTAACCGCAATTGTTGTTGATTCCCTTAAACCCTACTGTGATAAGGTTGATGGTCCCCCTCATGATGTTTTGCACCGTGTTCAAAAGATGTTGATAGATCATCTGTCTGGATTTGTTCCCATGAATATCTCCTCTGAAACTTCTATGCTATCTGCGTTCCACAAGCTTAATCATGACACTTCTTGTGGACCCTATTTAGGTGGTAGAAAGAAGGACCATATGACCAATGGTGAACCTGACAAACCCCTTCTGGATCTTTTATCTTCAAAGTGGAAACTGGCTACTCAAGGTATTGCCCTCCCTCATGAGTACACAATTGGACTGAAAGACGAACTCCGGCCCATAGAGAAAGTGCAAGAAGGGAAGAGAAGAATGATCTGGGGATGTGATGTTGGGGTGGCTACTGTTTGTGCAGCTGCATTCAAGGGTGTTAGCGATGCGATCACGGCAAATCACCAATATGGGCCTGTTCAAGTTGGCATAAACATGGACAGCCCTAGTGTTGATGCGTTGTATCAAAGGATCAAAAGTGCTGCTAAGGTATTTGCTGTTGATTACTCTAAGTGGGATTCAACACAATCACCCCGTGTCAGTGCTGCTTCAATTGATATTCTACGATATTTCTCTGATCGATCGCCCATTGTGGATTCTGCGGCCAACACCCTCAAATCCCCCCCAATTGCAATCTTTAATGGGGTTGCTGTGAAGGTGTCATCTGGTCTACCATCTGGAATGCCCCTAACTTCTGTAATCAATTCTTTAAACCATTGCTTGTATGTTGGATGTGCTATCTTGCAGTCATTGGAAGCTAAGAATATCCCTGTCACTTGGAACTTGTTCTCCTCCTTCGATATGATGACTTACGGTGATGATGGTGTCTACATGTTCCCTACTATGTTTGCTAGTGTGAGTGACCAGATATTTGGAAACCTGTCTGCCTATGGCCTCAAACCTACTAGAGTTGACAAGTCTGTTGGAGCAATTGAACCCATTGACCCGGAGACCGTAGTGTTTCTCAAACGAACCATTACAAGGACTCCTAACGGTATAAGAGGATTGCTCGACCGCAGCTCCATACTGCGGCAGTTCTACTATATCAAGGGAGAGAATTCGGATGACTGGAAAACCCCACCCAAAACGATAGACCCAACATCCAGAGGTCAGCAACTATGGAATGCCTGTCTCTATGCTAGTCAGCATGGTGTTGAGTTCTACAATAAGGTTTTAAAATTGGCACAAAAAGCAGTTGAATATGAGGAGCTCCATTTAGATCCCCCAAATTACTCAACAGCTCTTGAACATTACAACAGCCAATTCAATGGTGTGGAGGCGCGGACTGATCAGATCGGAACGAGCCATGCTACCGCCCTTCACTGTGATGTGTTCGAAGTTTGAGCATGTGCTCAACCTGCGCTAACGTGCTAAAGTACTATAATTGGGATCCCCATTTTAGGCTCACAATAAACCCCAATGATTTTCTATCTGTAGGTTTCTGTGATAACCCCCTTATGTGTTGCTACCCTGAACTCCTTCCAGAATTCGGAACTGTATGGGATTGTGATCAACCCCCTCTTCAAATTTATCTGGAGTCTATCCTTGGTGATGATGAATGGTCTTCAACACATGAGGCCATCGATCCTGTTGTTCCCCCAATGCATTGGAGTGAAATGGGAAAGATCTTCCAACCGCACCCTGGAGTTCTTATGCACCATCTCATTGGCCAAGTTGCGAAAGGTTGGGATCCTAATTTACCAAATTTTCGTTTGGAAGCCGGGGATGGCTCCATTACAACGCCTGAGCAGGGGACTGCCGTTGGCGGTGTAATTGCTGAACCTAGCGCCCAAATGTCAACTGCTGCAGATATGGCAACAGGGAAGAGTGTTGATTCTGAATGGGAAGCATTCTTTTCTTTCCATACGAGTGTCAATTGGAGCACTTCTGAAACCCAAGGGAAGATTCTATTCAAACAAAACCTTAGCCCTCTCTTAAACCCTTACCTCTCTCACTTGGCTAAACTGTACGTTGCATGGTCTGGATCTATCGATGTCCGCTTCTCTATCTCAGGCTCTGGTGTGTATGGTGGTAAGCTTGCTGCAATTGTAGTGCCACCAGGGATTGAACCCGTACAAAGCACTTCAATGTTACAGTACCCTCATGTTCTATTTGACGCTCGTCAAGTAGAGCCTGTCATTTTCTCCGTCCCTGACTTAAGAAGCACTCTCTATCACTTAATGTCTGATGTTGACACTACATCTTTAGTCATAATGATTTACAATGACCTTATCAACCCCTATGCTAGTGAAGCAAATTCTTCTGGTTGTATTGTAACTGTTGAAACCAAACCTGGCCCTGATTTCAAATTTCACCTTCTGAAGCCTCCTGGATCAATGCTCACTCATGGATCTGTTCCATCTGACCTGATTCCAAAAAGTTCATCACTGTGGATTGGAAATCGCCATTGGACTGATATTGATGATTTCATCATTCGACCCTTCGTGTTCCAGGCAAATCGTCACTTCGATTTCAACCAAGAAACTGCTGGGTGGAGCACACCACGATTTCGACCAATTACCATAAGTATCAGCCAGCGGGATGGTGCAAAATTGGGAACTGGGATTGCCACTGATTATATCGTACCTGGAATACCTGATGGTTGGCCTGACACGACAATCCCTGAGACACTAACTCCGGCAGGCGACTACGCCATTACCTCAAGAGCTGGCAACGATATAACAACTCCCGCCCAGTACGATACGGCAGATGTGATAGAGAACAACACTAATTTCAAAAGTATGTATATTTGTGGATCATTACAAAGGGCATGGGGTGACAAGAAGATTTCGAATACTGGTTTCATTACCACAGCCACGGTTAGGGACAACCGTCTTGAACCATCCAACACCATCGATCAAACAAAGATTGCCGTGTTTCAAGACAATCATGTTAACAGTGATGTCCAAACATCAGATGTTACACTGGCATTACTTGGATACACAGGAATAGGAGAAGAGGCAATTGGTGCTGATAGGGAGAAGGTTGTGCGGATCAGTGTGTTGCCGGAAACTGGGGCTCGTGGTGGAAATCACCCAATATTCTACAAGAATAAAATGAAACTTGGATATGTAATTAGAGAAATTGATGTGTTTAACTCCCAAATTTTACACACCTCTAGACAGTTATCACTCAATAATTACTTGTTGCCTCCCGATTCCTTTGCAGTTTATAGAATTATTGATGCTAATGGTTCTTGGTTTGACATAGGGATTGATTCAGATGGTTTCTCTTTTGTTGGTGTTTCTAACATAGGTAAATTAGAGTTTCCTCTCACTGCCTCCTACATGGGAATTCAGCTGGCAAAGATTCGGCTTGCCTCTAATATTAGGAGTTCAATGACTAAACTATGAATTCAATCCTTGGTCTGATCGACTCTGTAACCAATACAGTATCTAAGGCGCAGCAAATTGAATTGGACAAAGCTGCCCTTAATCAAAATAGGGACTTGGCTCTTCGACGCATGCAATTGGACAAAAGAGCGTTGGACAATCAAGTTGACCAGTTTAACAAAATTCTTGAGCAAAGGGTACATGGCCCCATCCAGTCGGTCCGCCTAGCGCGTGCCGCTGGTTTTCGGGTTGACCCTTACTCATACACAAATCAAAATTTTTATGAAGATCAATTGAACATAATTAGGAATTGTTACAAGAATTTGTTTAAAATGTGATCATGTATCCCTTCGGGCTGCCGCTCTTGCGCCTAACCCCAGGG.
The foregoing description is only illustrative of the present invention and is not intended to limit the scope of the invention, and all equivalent structures or equivalent processes or direct or indirect application in other related technical fields are included in the scope of the present invention.

Claims (5)

1.一种抗猫杯状病毒病病毒强毒株免疫疫苗的制备方法，其特征在于，其包括以下步骤：1. A method for preparing an anti-feline calicivirus virus virulent strain immune vaccine, characterized in that it comprises the following steps: S1：培养F81细胞；S1: culture F81 cells; S2：培养猫杯状病毒VS-FCV-SHH202015；S2: culture of feline calicivirus VS-FCV-SHH202015; S3：制备免疫原；S3: Preparation of immunogen; S4：疫苗免疫和中和抗体测定；S4: Vaccine immunity and neutralizing antibody assay; 步骤S2中，所述猫杯状病毒VS-FCV-SHH202015已于2022年6月16日保藏在中国科学微生物研究所，保藏编号为CGMCC No.45209；In step S2, the feline calicivirus VS-FCV-SHH202015 was deposited at the Institute of Microbiology, Chinese Academy of Sciences on June 16, 2022, with a deposit number of CGMCC No.45209; 步骤S3中，所述免疫原的制备过程为：S31、病毒灭活：测定猫杯状病毒VS-FCV-SHH202015病毒液的TCID50，用DMEM将猫杯状病毒VS-FCV-SHH202015病毒液稀释至1×106个TCID50/mL，将稀释好的猫杯状病毒VS-FCV-SHH202015病毒液加入终质量体积百分比浓度为0.2%的甲醛中，37℃灭活病毒28 h；In step S3, the preparation process of the immunogen is as follows: S31, virus inactivation: determine the TCID 50 of the feline calicivirus VS-FCV-SHH202015 virus solution, dilute the feline calicivirus VS-FCV-SHH202015 virus solution to 1×10 6 TCID 50 /mL with DMEM, add the diluted feline calicivirus VS-FCV-SHH202015 virus solution to formaldehyde with a final mass volume percentage concentration of 0.2%, and inactivate the virus at 37°C for 28 h; S32、灭活验证：将灭活后的病毒液与等体积F81细胞悬液进行混合，加至孔板中，2 mL/孔，37℃，在体积百分比浓度为5%的CO2培养箱培养，同时观察细胞病变；S32. Inactivation verification: Mix the inactivated virus solution with an equal volume of F81 cell suspension and add to the well plate, 2 mL/well, incubate at 37°C in a CO2 incubator with a volume percentage concentration of 5%, and observe the cytopathic effect; S33、制备免疫原：灭活验证合格后，加入铝胶佐剂，按抗原﹕铝胶佐剂体积比为1:1的比例混合后使用。S33. Preparation of immunogens: After inactivation verification, add aluminum gel adjuvant and mix in a volume ratio of 1:1 between antigen and aluminum gel adjuvant before use. 2.根据权利要求1所述的制备方法，其特征在于，步骤S1中，所述F81细胞的培养过程为：将复苏的F81细胞加入体积百分比浓度为10%的胎牛血清细胞培养液中，于37℃，含有体积百分比浓度为5%的CO2培养箱中培养，待长成单层后用质量体积比为0.1%的胰酶进行消化，F81细胞悬液用10%血清浓度的细胞培养液稀释至细胞密度达1×106个/mL时备用。2. The preparation method according to claim 1 is characterized in that in step S1, the culturing process of the F81 cells is: adding the revived F81 cells to a fetal bovine serum cell culture medium with a volume percentage concentration of 10%, culturing at 37°C in an incubator containing a volume percentage concentration of 5% CO2 , digesting them with 0.1% pancreatic enzyme after growing into a monolayer, and diluting the F81 cell suspension with a cell culture medium with a 10% serum concentration to a cell density of 1× 106 cells/mL for standby use. 3.根据权利要求1所述的制备方法，其特征在于，步骤S2中，所述猫杯状病毒VS-FCV-SHH202015的培养过程为：在F81细胞悬液中加入猫杯状病毒VS-FCV-SHH202015的病毒液，进行病毒培养，待80%的 F81细胞出现细胞病变时将病毒培养液冻融三次备用。3. The preparation method according to claim 1, characterized in that, in step S2, the culture process of the feline calicivirus VS-FCV-SHH202015 is: adding the virus liquid of the feline calicivirus VS-FCV-SHH202015 to the F81 cell suspension, carrying out virus culture, and freezing and thawing the virus culture liquid three times for standby use when 80% of the F81 cells have cytopathic effects. 4.根据权利要求1所述的制备方法，其特征在于，步骤S2中，所述猫杯状病毒VS-FCV-SHH202015的序列如SEQ ID NO.1所示。4. preparation method according to claim 1, is characterized in that, in step S2, the sequence of described feline calicivirus VS-FCV-SHH202015 is shown in SEQ ID NO.1. 5.根据权利要求1所述的制备方法，其特征在于，步骤S4中，所述疫苗免疫和中和抗体的测定包括以下步骤：5. The preparation method according to claim 1, characterized in that in step S4, the determination of vaccine immunity and neutralizing antibodies comprises the following steps: S41、疫苗免疫：免疫原皮下多点注射清洁级新西兰兔，每只兔子注射两次，每次间隔14d，每次注射4个点，每个点注射0.5 mL免疫原，同时用猫三联疫苗作比较；S41. Vaccine immunization: The immunogen was injected subcutaneously at multiple points into clean New Zealand rabbits. Each rabbit was injected twice, with an interval of 14 days between each injection. Each injection was at 4 points, and 0.5 mL of the immunogen was injected at each point. A cat triple vaccine was used for comparison. S42、中和抗体测定：最后一次注射后14 d，采集血液，分离血清，用猫杯状病毒VS-FCV-SHH202015和普通猫杯状病毒病病原猫杯状病毒进行中和抗体检测，保证中和抗体效价大于320。S42. Neutralizing antibody assay: 14 days after the last injection, collect blood, separate serum, and use feline calicivirus VS-FCV-SHH202015 and feline calicivirus, the pathogen of common feline calicivirus disease, for neutralizing antibody testing to ensure that the neutralizing antibody titer is greater than 320.
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