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

Abstract

The invention provides a method for preparing an anti-feline calicivirus virus strong strain immune vaccine, and the preparation method comprises the following steps: culturing F81 cells; culturing feline calicivirus strong strain-SHH202015 virus; preparing immunogens; vaccine immunization and neutralizing antibody determination; the neutralizing antibodies produced by the feline calicivirus strong strain-SHH202015 immunogen prepared by the invention can completely neutralize the feline calicivirus strong strain, and the titer reaches 1280, and can also neutralize the common feline calicivirus pathogen feline calicivirus, and the titer reaches 640; therefore, the feline calicivirus strong strain-SHH202015 immunogen can prevent infection by the feline calicivirus strong strain and the common feline calicivirus pathogen feline calicivirus, and provides an important technical means for the prevention and control of feline calicivirus disease.

Description

Technical Field

[0001] The invention belongs to the technical field of vaccine preparation, and specifically relates to a method for preparing an immune vaccine against a strong strain of feline calicivirus disease virus-SHH202015. Background Art

[0002] Feline calicivirus disease has a high incidence rate in cats, with the main clinical symptoms being oral ulcers and upper respiratory tract symptoms, and atypical symptoms such as lameness, abortion, chronic gastroenteritis, urinary tract infection, etc. may also occur. Due to the easy mutation of feline calicivirus (FCV), the pathogen of feline calicivirus disease, a strong strain of feline calicivirus (VS-FCV) that can cause systemic symptoms in cats has appeared worldwide. In addition to conventional clinical symptoms such as upper respiratory tract diseases, VS-FCV strains also cause acute and fatal systemic diseases such as skin edema, fever, ulcerative dermatitis, anorexia, jaundice, etc. in infected cats, with a mortality rate of up to 50%.

[0003] There is currently no domestic feline calicivirus vaccine, and there is only one imported feline triple vaccine (feline calicivirus, feline herpesvirus type 1, and feline parvovirus), in which the feline calicivirus strain has only 73.2% nucleotide homology with the epidemic strain, and only 82.7% amino acid homology. There have been many clinical reports that cats immunized with the feline triple vaccine still develop feline calicivirus disease. Therefore, the early vaccine strains can no longer provide sufficient protection, which seriously endangers the life safety of pet cats and other felines. In order to prevent and control the infection and spread of feline calicivirus, the VS-FCV epidemic strain should be selected to develop a vaccine. On the one hand, it can prevent VS-FCV infection and reduce the mortality rate of cats. On the other hand, it can improve the immune effect of feline calicivirus and provide technical means for the prevention and control of feline calicivirus disease.

[0004] Therefore, it is necessary to provide a new method for preparing a vaccine against the virulent strain of feline calicivirus to solve the above technical problems. Summary of the invention

[0005] The present invention aims to provide a method for preparing a strong strain immune vaccine against feline calicivirus disease virus in order to address the deficiencies in the prior art. Feline calicivirus was isolated and identified from multiple clinical cases of feline calicivirus infection, and a strain of VS-FCV was identified through cell culture, sequencing, electron microscopy, sequence alignment, VS-FCV characteristic amino acid comparison, and animal pathogenicity test, named VS-FCV-SHH202015, and based on the VS-FCV-SHH202015 strain.

[0006] To achieve the above object, the solution of the present invention is:

[0007] A method for preparing an anti-feline calicivirus virus strong strain immune vaccine comprises the following steps:

[0008] S1: culture F81 cells;

[0009] S2: culture VS-FCV-SHH202015 virus;

[0010] S3: Preparation of immunogen;

[0011] S4: Vaccine immunity and determination of neutralizing antibodies.

[0012] Further, in step S1, the culture process of F81 cells is as follows: the revived F81 cells are added to a cell culture medium with a volume percentage concentration of 10% fetal bovine serum, cultured at 37°C in an incubator containing a volume percentage concentration of 5% CO2, and digested with 0.1% pancreatic enzyme after growing into a monolayer, and the F81 cell suspension is diluted with a cell culture medium with a 10% serum concentration to a cell density of 1×10 6 pcs / mL for later use.

[0013] Furthermore, in step S2, the culture process of VS-FCV-SHH202015 virus is as follows: adding VS-FCV-SHH202015 virus solution to F81 cell suspension for virus culture, and when 80% of F81 cells show cytopathic effects, freezing and thawing the virus culture solution three times for later use.

[0014] Furthermore, in step S2, the sequence of the VS-FCV-SHH202015 virus is shown in SEQ ID NO.1.

[0015] Further, in step S3, the preparation process of the immunogen is as follows: S31, virus inactivation: determining the TCID of the VS-FCV-SHH202015 virus solution 50 , dilute the VS-FCV-SHH202015 virus solution to 1×10 6 TCID 50 / mL, add the diluted VS-FCV-SHH202015 virus solution into formaldehyde with a final mass volume percentage concentration of 0.2%, and inactivate the virus at 37°C for 28 h;

[0016] S32, Inactivation verification: The inactivated virus solution was mixed with an equal volume of F81 cell suspension (1×10 6 / mL) were mixed and added to a 6-well plate, 2 mL / well, and cultured at 37°C in a CO2 incubator with a volume percentage concentration of 5%, while observing the cell lesions;

[0017] 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.

[0018] Further, in step S4, the determination of vaccine immunity and neutralizing antibodies includes the following steps:

[0019] S41. Vaccine immunization: The immunogen was injected subcutaneously at multiple points in clean New Zealand rabbits. Each rabbit was injected twice, with an interval of 14 days. Each injection was at 4 points, and 0.5 mL of the immunogen was injected at each point. At the same time, a cat triple vaccine was used for comparison.

[0020] S42. Neutralizing antibody determination: 14 days after the last injection, blood was collected, serum was separated, and neutralizing antibody detection was performed using VS-FCV-SHH202015 virus and common FCV virus to ensure that the neutralizing antibody titer was greater than 320.

[0021] Due to the adoption of the above scheme, the beneficial effects of the present invention are:

[0022] The neutralizing antibodies produced by the VS-FCV-SHH202015 immunogen prepared by the present invention can completely neutralize the VS-FCV virus, and the titer reaches 1280, and can also neutralize the common FCV virus, and the titer reaches 640; the antibodies produced by the cat triple vaccine cannot completely neutralize the VS-FCV-SHH202015 virus and the common FCV virus, and the titers are 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 provide an important technical means for the prevention and control of feline calicivirus disease. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 This is a one-step growth curve diagram of VS-FCV-SHH202015 of the present invention.

[0024] Figure 2 This is a diagram of the cytopathic effect after the VS-FCV-SHH202015 strain of the present invention was inoculated with F81 cells (where inoculation 1 is F81 cells of the third generation cell culture of VS-FCV-SHH202015; 2 is a negative control).

[0025] Figure 3 This is a picture of the VS-FCV-SHH202015 virus particles of the present invention.

[0026] Figure 4 This is the evolutionary tree of the VP1 gene of the VS-FCV-SHH202015 strain of the present invention.

[0027] Figure 5 This is a graph showing the reduction in neutralizing antibody titer of the VS-FCV-SHH202015 strain of the present invention.

[0028] Figure 6This is a graph showing the reduction in neutralizing antibody titer of the FCV-SHH2001 strain of the present invention.

[0029] Among them, the deposit number of feline calicivirus VS-FCV-SHH202015 is CGMCC No.45209, the deposit date is June 16, 2022, and it is deposited in the General Microbiology Center of China National Microbiological Culture Collection, located at No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing. Implementation

[0030] The invention provides a method for preparing an immune vaccine against a strong strain of feline calicivirus disease virus.

[0031] Please refer to Figure 1 , Figure 2 , Figure 3 , Figure 4 and Figure 5 , the preparation method of the anti-feline calicivirus virus strong strain immune vaccine of the present invention:

[0032] Cells and viruses: F81 cells were purchased from Shangcheng Beina Chuanglian Biotechnology Co., Ltd. The feline calicivirus virulent strain (VS-FCV-SHH202015) was isolated and preserved by our laboratory. The deposit number of the feline calicivirus virulent strain VS-FCV-SHH202015 is (CGMCC No. 45209).

[0033] Virus complete genome sequence: The complete genome sequence of VS-FCV-SHH202015 (generation 7) Genbank (accession number: OQ296623).

[0034] Experimental animals: 6 female clean-grade New Zealand rabbits, 3 months old, weighing 2-2.5 kg, purchased from Shanghai Proton Biotechnology Co., Ltd.

[0035] Main reagents and instruments: Fetal bovine serum was purchased from Sigma; DMEM culture medium and trypsin were purchased from GIBCO; aluminum gel adjuvant was purchased from Chuangzhi Biotechnology Co., Ltd.; cell culture was purchased from CORNING.

[0036] F81 cell culture: F81 cells were revived and cultured in a 37°C, 5% CO2 incubator with a culture medium containing 10% fetal bovine serum. After growing into a monolayer, they were digested with 0.1% pancreatic enzyme by mass volume ratio. The F81 cell suspension was diluted with a cell culture medium with a 10% serum concentration to a cell density of 1×10 6 pcs / mL for later use.

[0037] Optimal virus collection time during virus culture: Add 10 mL of cell suspension to a 25 cm 2In a cell culture flask, culture in a 37°C, 5% CO2 incubator for 24 h. When the cells grow into a monolayer, add 1 mL of VS-FCV-SHH202015 virus solution. Samples were taken every 12 h, and the virus titer was tested at 0h, 12h, 24h, 36h, 48h, 60h and 72h. The virus one-step growth curve was drawn to determine the optimal virus collection time. The harvested virus solution was centrifuged at 2 000 × g to remove cell debris, and the supernatant was packaged and frozen at -80°C. The virus growth kinetic curve shows (see Figure 1 ), under 37°C culture conditions, the virus titers of VS-FCV-SHH202015 at 0, 12, 24, 36, 48, 60 and 72 h were 1×10 4 , 2.65×10 6 , 2.01 ×10 7 , 4.52 × 10 8 , 5.21 ×10 8 , 3.78 × 10 8 , and 4.14×10 8 TCID 50 / mL, so the optimal collection time is 48 h.

[0038] Virus TCID 50 Detection: The cell suspension was added to a 96-well plate, 50 μL per well, and the virus was serially diluted 10-fold in DMEM (10 -1 -10 -11 ) were added to 96-well plates in sequence, 50 μL / well, and cultured in a 37°C, 5% CO2 incubator. The cytopathic effect was observed daily. The observation was stopped after 4-5 days, and the final dilution and number of wells with cytopathic effect were recorded. TCID was calculated according to the Reed-Muench method. 50 .

[0039] Immunogen preparation: The titer is 5.21×10 8 TCID 50 / mL virus solution was added with formaldehyde at a final concentration of 0.2%, and the virus was inactivated at 37℃ for 28 h. 50 After the test is verified to be qualified, the immunogen is prepared with aluminum gel adjuvant in the ratio of antigen: aluminum gel adjuvant = 1:1.

[0040] Rabbit grouping and immunization: The rabbits were randomly divided into 3 groups, with 2 rabbits in each group, namely: VS-FCV-SHH202015 vaccine group, cat triple vaccine group, and DMEM control group. Vaccine immunization: The immunogen was subcutaneously injected into clean New Zealand rabbits at multiple points. Each rabbit was injected twice, with an interval of 14 days, 4 points each time, and 0.5 mL of immunogen was injected at each point. Neutralizing antibody determination: Serum samples were collected on days 0, 7, 14, 21, 28, 35, 42, and 49, and neutralizing antibodies were detected using VS-FCV-SHH202015 strain and FCV-SHH2001 strain, respectively.

[0041] Serum neutralizing antibody detection: according to virus TCID 50 The detection method was to measure the TCID of VS-FCV-SHH202015 strain and FCV-SHH2001 strain respectively. 50 The fixed virus-diluted serum method was used to detect serum neutralizing antibodies: the two viruses were diluted to 200 TCID 50 After 20-fold dilution, the serum was treated in a 56°C water bath for 30 min and then serially diluted 2-fold (2 -1 -2 -11 ). 100 μL serum and 100 μL virus were mixed and incubated at 37℃ for 1.5 h. Virus control, serum toxicity control, cell blank control and positive and negative serum control were set up at the same time. 50 μL of the infective mixture and an equal amount of cell suspension were added to a 96-well plate at the same time. They were cultured in a 37℃, 5% CO2 incubator. The cytopathic effect was observed daily. The observation was stopped after 4-5 days. The serum dilution that finally caused cytopathic effect was recorded, and the maximum serum dilution that could neutralize the two viruses was recorded respectively.

[0042] Data analysis: Draw the neutralizing antibody titer reduction graphs for the two virus strains respectively.

[0043] Result analysis: Figure 5 and Figure 6 As shown, antibody monitoring continued until the 49th day after immunization, and the antibodies produced by the VS-FCV-SHH202015 vaccine group were able to neutralize the VS-FCV-SHH202015 virus, with an antibody titer of 1280, and could also neutralize the FCV-SHH2001 virus, with an antibody titer of 640. The antibodies produced by the cat triple vaccine could not completely neutralize the VS-FCV-SHH202015 virus and the common FCV virus, with titers of only 40 and 160, respectively, and the titers were unstable and of short duration.

[0044] In summary, the VS-FCV-SHH202015 vaccine of the present invention induced the body to produce a high titer of neutralizing antibodies on the 28th day after one immunization (a total of 2 immunizations were performed). The VS-FCV-SHH202015 vaccine antibodies can neutralize FCV strong strains and can also neutralize FCV classic strains well; at the same time, the antibodies produced by the cat triple vaccine cannot completely neutralize FCV strong strains, and the neutralization ability of FCV classic strains is also poor. Therefore, the present invention will contribute to the prevention and control of feline calicivirus, and become an important technical means for FCV prevention and control, which not only solves the problem of poor effect of current imported vaccines, but also fills the gap of no domestically produced FCV vaccines in China.

[0045]

[0046] The above descriptions are merely embodiments of the present invention and are not intended to limit the patent scope of the present invention. Any equivalent structure or equivalent process transformation made using the contents of the present invention specification and drawings, or directly or indirectly applied in other related technical fields, are also included in the patent protection scope of the present invention.

Claims

1. A method for preparing an anti-feline calicivirus virus strong strain immune vaccine, characterized in that: It includes the following steps: S1: culture F81 cells; S2: culture of feline calicivirus VS-FCV-SHH202015; S3: Preparation of immunogen; S4: Vaccine immunization and neutralizing antibody assay; In step S2, the feline calicivirus VS-FCV-SHH202015 was deposited in the Institute of Microbiology, Chinese Academy of Sciences on June 16, 2022, with the deposit number CGMCC No.45209; In step S3, the preparation process of the immunogen is as follows: S31, virus inactivation: determination of the TCID of the feline calicivirus VS-FCV-SHH202015 virus solution 50 Dilute the feline calicivirus VS-FCV-SHH202015 virus solution to 1×10 6 TCID 50 / mL, add the diluted feline calicivirus VS-FCV-SHH202015 virus solution into formaldehyde with a final mass volume percentage concentration of 0.2%, and inactivate the virus at 37°C for 28 h; 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. 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. The preparation method according to claim 1, characterized in that: In step S1, the culturing process of the F81 cells is as follows: the revived F81 cells are added to a cell culture medium with a volume percentage concentration of 10% fetal bovine serum, cultured at 37°C in an incubator containing a volume percentage concentration of 5% CO2, and digested with 0.1% pancreatic enzyme after growing into a monolayer. The F81 cell suspension is diluted with a cell culture medium with a 10% serum concentration to a cell density of 1×10 6 pcs / mL for later use.

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 as follows: adding the virus solution of the feline calicivirus VS-FCV-SHH202015 to the F81 cell suspension for virus culture, and when 80% of the F81 cells show cytopathic effects, freezing and thawing the virus culture solution three times for standby use.

4. The preparation method according to claim 1, characterized in that: In step S2, the sequence of the feline calicivirus VS-FCV-SHH202015 is shown in SEQ ID NO.

1.

5. The preparation method according to claim 1, characterized in that: In step S4, the vaccine immunity and neutralizing antibody determination comprises the following steps: 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. 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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