CN116240179A - Preparation method of anti-feline calicivirus virus virulent strain immunity vaccine - Google Patents

Preparation method of anti-feline calicivirus virus virulent strain immunity vaccine Download PDF

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CN116240179A
CN116240179A CN202310216788.9A CN202310216788A CN116240179A CN 116240179 A CN116240179 A CN 116240179A CN 202310216788 A CN202310216788 A CN 202310216788A CN 116240179 A CN116240179 A CN 116240179A
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feline calicivirus
virulent strain
shh
immunogen
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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: culturing F81 cells; culturing a feline calicivirus virulent strain-SHH 202015 virus; preparing an immunogen; determination of vaccine immunization and neutralizing antibodies; the neutralizing antibody generated by the feline calicivirus virulent strain-SHH 202015 immunogen can completely neutralize the feline calicivirus virulent strain, the titer reaches 1280, and meanwhile, the neutralizing antibody can neutralize the pathogenic feline calicivirus of common feline calicivirus disease, and the titer reaches 640; therefore, the feline calicivirus virulent strain-SHH 202015 immunogen can prevent the infection of the feline calicivirus virulent strain and the common feline calicivirus disease pathogenic feline calicivirus, and provides an important technical means for the 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: vaccine immunization and determination of neutralizing antibodies.
Further, in step S1, the culture process of F81 cells is: adding recovered F81 cells into 10% by volume of fetal bovine serum cell culture solution, and adding 5% by volume of CO at 37deg.C 2 Culturing in incubator, digesting with pancreatin with mass-volume ratio of 0.1%, diluting F81 cell suspension with 10% serum concentration cell culture solution to cell density of 1×10 6 And (3) spare the sample at the time of one/mL.
Further, in step S2, the culture process of the VS-FCV-SHH202015 virus is as follows: and (3) adding a virus solution of VS-FCV-SHH202015 into the F81 cell suspension, culturing the virus, and freezing and thawing the virus culture solution for three times when 80% of F81 cells have cytopathy.
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 is as follows: s31, virus inactivation: determination of TCID of VS-FCV-SHH202015 virus liquid 50 Diluting the VS-FCV-SHH202015 virus solution to 1X 10 with DMEM 6 TCID(s) 50 Adding 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, inactivation verification: the inactivated virus solution was mixed with an equal volume of F81 cell suspension (1X 10) 6 individual/mL), was added to a 6-well plate, 2 mL/well, 37 ℃, at a concentration of 5% CO by volume 2 Culturing in an incubator while observing cytopathy;
s33, preparing an immunogen: and after the inactivation verification is qualified, adding the aluminum gel adjuvant, and mixing 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, vaccine immunization: the immunogen is subcutaneously injected into clean New Zealand rabbits at multiple points, each rabbit is injected twice, each time at intervals of 14 and d, 4 points are injected each time, each point is injected with 0.5 and mL immunogen, and meanwhile, the comparison is carried out by using a cat triple vaccine;
s42, determination of neutralizing antibody: after the last injection, 14 d, blood is collected, serum is separated, and the neutralizing antibody is detected by using the VS-FCV-SHH202015 virus and the common FCV virus, so that the neutralizing antibody titer is ensured to be more than 320.
By adopting the scheme, the invention has the beneficial effects that:
the neutralizing antibody generated by the VS-FCV-SHH202015 immunogen can completely neutralize the VS-FCV virus, the titer reaches 1280, and the titer reaches 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. 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 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 a 3 rd generation cell culture of VS-FCV-SHH 202015; 2 is a negative control).
FIG. 3 is a graph of the VS-FCV-SHH202015 virion of 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 against 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 Silu No.1, 3 in the Chaoyang 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 North Nakagaku Biotechnology Co., ltd. In commercial city, and the feline calicivirus virulent strain (VS-FCV-SHH 202015) was isolated from the laboratory and stored under the accession number (CGMCC No. 45209) of feline calicivirus virulent strain VS-FCV-SHH 202015.
Viral whole 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.
Main reagents and instruments: fetal bovine serum was purchased from sigma company; DMEM medium, pancreatin from GIBCO company; aluminum gel adjuvants were purchased from developing intelligent biotechnology limited company; cell cultures were purchased from CORNING corporation.
F81 cell culture: resuscitates F81 cells with 10% fetal bovine serum in culture medium at 37deg.C and 5% CO 2 Culturing in incubator, digesting with pancreatin with mass-volume ratio of 0.1%, diluting F81 cell suspension with 10% serum concentration cell culture solution to cell density of 1×10 6 And (3) spare the sample at the time of one/mL.
Optimal virus collection time in virus culture stage: 10 mL cell suspension was added to 25 cm 2 In a cell culture flask, at 37℃and 5% CO 2 Culturing 24h in an incubator, and adding 1mL of VS-FCV-SHH202015 virus solution after cells grow into a monolayer. Samples are taken every 12 hours h, virus titer is detected at intervals of 0 hours, 12 hours, 24 hours, 36 hours, 48 hours, 60 hours and 72 hours, a virus one-step growth curve is drawn, and the optimal virus collection time is determined. Removing cell debris by centrifugation of 2 000Xg of the harvested virus solutionCleaning, packaging, and freezing at-80deg.C. The kinetics of viral growth curve shows (see FIG. 1) that VS-FCV-SHH202015 has a viral titer of 1X 10 at 0, 12, 24, 36, 48, 60 and 72h, respectively, under culture conditions at 37 ℃ 4 、2.65×10 6 、2.01 ×10 7 、4.52 ×10 8 、5.21 ×10 8 、3.78 ×10 8 And 4.14X10 8 TCID 50 The optimal toxin-receiving time is 48 h.
Virus TCID 50 And (3) detection: cell suspensions were added to 96-well plates, 50 μl per well, and virus was serially diluted 10-fold in DMEM (10 -1 -10 -11 ) Then sequentially added to a 96-well plate at 50. Mu.L/well, 37℃and 5% CO 2 Culturing in an incubator, observing cytopathy day by day, stopping observing after 4-5d, recording dilution and hole number of cytopathy, and calculating TCID according to Reed-Muench method 50 。
Immunogen preparation: the titer was 5.21×10 8 TCID 50 Adding formaldehyde with final concentration of 0.2% into/mL virus solution, inactivating virus 28 h at 37deg.C, passing through virus TCID 50 After the detection and verification are qualified, the immunogen is prepared with the aluminum gel adjuvant according to the ratio of antigen to aluminum gel adjuvant=1 to 1.
Grouping and immunizing experimental rabbits: the experimental rabbits were randomly divided into 3 groups of 2 animals each: immunization of the VS-FCV-SHH202015 vaccine group, immunization of the cat triple vaccine group, and immunization of the DMEM control group. Vaccine immunization: the immunogen was subcutaneously injected in multiple spots into clean grade New Zealand rabbits, 2 times per rabbit, 14 d intervals, 4 spots per injection, 0.5 mL immunogen per spot. Neutralizing antibody assay: serum samples were collected at days 0,7, 14, 21, 28, 35, 42 and 49 and tested for neutralizing antibodies using VS-FCV-SHH202015 strain and FCV-SHH2001 strain, respectively.
Serum neutralizing antibody detection: according to the virus TCID 50 Detection method for measuring TCID of VS-FCV-SHH202015 strain and FCV-SHH2001 strain 50 Is a value of (2). Serum neutralizing antibodies were detected using a fixed virus-diluted serum method: two strains of virus were diluted to 200 TCID 50 The serum was diluted 20-fold and then treated in a 56℃water bath for 30 min, followed by 2-fold serial dilution (2 -1 -2 -11 ). 100. Mu.L of serum and 100. Mu.L of virus were mixed and then perceived as 1.5. 1.5 h to 37℃with virus control, serum toxicity control, cell blank control and yin-yang serum control. mu.L of the perceived mixture and an equal amount of the cell suspension were simultaneously added to a 96-well plate at 37℃with 5% CO 2 Incubation in incubator, observation of cytopathic effect was stopped after 4-5d days, and the dilutions of serum that eventually developed cytopathic effect were recorded, and the maximum dilutions of serum that could neutralize both strains were recorded separately.
Data analysis: neutralizing antibody titers of the two strains were plotted separately.
Analysis of results: as shown in fig. 5 and 6, antibody monitoring was continued until day 49 post-immunization, and the VS-FCV-SHH202015 vaccine group produced antibodies that were able to neutralize VS-FCV-SHH202015 virus, had an antibody titer of 1280, and also were able to neutralize FCV-SHH2001 virus, and had 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 summary, the VS-FCV-SHH202015 vaccine induces the organism to generate a neutralizing antibody with higher titer on the 28 th day after one immunization (2 times of immunization are performed), and the VS-FCV-SHH202015 vaccine antibody can neutralize the FCV virulent strain and can also neutralize the FCV classical strain well; meanwhile, antibodies generated by the cat triple vaccine can not completely neutralize the FCV virulent strain, and the neutralizing capacity of the cat triple vaccine on the FCV classical strain is poor. 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 is: 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 (6)

1. 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 a feline calicivirus virulent strain-SHH 202015 virus;
s3: preparing an immunogen;
s4: vaccine immunization and neutralizing antibody assay.
2. The method according to claim 1, wherein in step S1, the F81 cells are cultured as follows: adding recovered F81 cells into 10% by volume of fetal bovine serum cell culture solution, and adding 5% by volume of CO at 37deg.C 2 Culturing in incubator, digesting with pancreatin with mass-volume ratio of 0.1%, diluting F81 cell suspension with 10% serum concentration cell culture solution to cell density of 1×10 6 And (3) spare the sample at the time of one/mL.
3. The method according to claim 1, wherein in step S2, the culture process of the feline calicivirus virulent strain-SHH 202015 virus is as follows: and (3) adding a virus solution of a virulent strain of feline calicivirus-SHH 202015 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 cytopathy.
4. The preparation method according to claim 1, wherein in step S2, the sequence of the feline calicivirus virulent strain-SHH 202015 virus is shown in SEQ ID No. 1.
5. The method according to claim 1, wherein in step S3, the immunogen is prepared by: s31, virus inactivation: determination of TCID of feline calicivirus virulent strain-SHH 202015 Virus liquid 50 Dilute the feline calicivirus virulent strain SHH202015 virus solution to 1X 10 with DMEM 6 TCID(s) 50 Adding diluted feline calicivirus virulent strain-SHH 202015 virus solution into formaldehyde with the final mass volume percentage concentration of 0.2%, and inactivating the virus 28 h at 37 ℃;
s32, inactivation verification: mixing the inactivated virus liquid with F81 cell suspension with equal volume, adding into a pore plate, 2 mL/pore, and adding CO with concentration of 5% by volume at 37deg.C 2 Culturing in an incubator while observing cytopathy;
s33, preparing an immunogen: and after the inactivation verification is qualified, adding the aluminum gel adjuvant, and mixing according to the volume ratio of the antigen to the aluminum gel adjuvant of 1:1 for use.
6. The method of claim 1, wherein in step S4, the vaccine immunization and neutralizing antibody determination comprises the steps of:
s41, vaccine immunization: the immunogen is subcutaneously injected into clean New Zealand rabbits at multiple points, each rabbit is injected twice, each time at intervals of 14 and d, 4 points are injected each time, each point is injected with 0.5 and mL immunogen, and meanwhile, the comparison is carried out by using a cat triple vaccine;
s42, determination of neutralizing antibody: after the last injection, 14 to d, collecting blood, separating serum, and detecting a neutralizing antibody by using the feline calicivirus virulent strain-SHH 202015 virus and the pathogenic feline calicivirus of common feline calicivirus disease, wherein the neutralizing antibody titer is ensured to be more than 320.
CN202310216788.9A 2023-03-08 2023-03-08 Preparation method of anti-feline calicivirus virus virulent strain immunity vaccine Pending CN116240179A (en)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN116875560A (en) * 2023-06-16 2023-10-13 衡阳师范学院 Callicarpa virus strain and application thereof

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN116875560A (en) * 2023-06-16 2023-10-13 衡阳师范学院 Callicarpa virus strain and application thereof

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