Cell-adapted strain of h3n2 subtype swine influenza virus and inactivated vaccine prepared and application thereof
A swine flu virus, H3N2 technology, applied in the direction of antisense single-stranded RNA virus, virus, vaccine, etc., can solve the problems of loose virus and high production cost, and achieve the effect of low production cost, high safety and good immune protection
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Embodiment 1H3
[0038] Isolation and Identification of Example 1 H3N2 Subtype Swine Influenza Virus Strains
[0039] 1. Chicken embryo and disease material
[0040] SPF chicken embryos were purchased from Beijing Meria Weitong Experimental Animal Technology Co., Ltd.; disease materials were collected from nasal swabs and lung tissues suspected of swine flu from a pig farm in Heilongjiang. Grind the disease material with a grinder, freeze and thaw repeatedly 3 times, mix it with PBS (0.1mol / L, pH 7.2) at 1:5 (V / V), centrifuge at 3000r / min for 15min, take the supernatant, and pass through 0.22 After sterilizing with a μm filter membrane, store at -70°C for later use.
[0041] 2. Virus isolation
[0042] The above supernatant was inoculated into 5 10-day-old SPF chicken embryos through the allantoic cavity, 0.2ml / embryo, cultured in a 35°C incubator (humidity kept at 60-65%), and the embryos were checked twice a day, within 24 hours. The dead chicken embryos were discarded, and the dead chick...
Embodiment 2
[0071] Embodiment 2 uses bioreactor to cultivate the production technology of swine influenza H3N2 subtype virus
[0072] 1) Screening and recovery of cells
[0073] When MDCK cells are cultured and passaged, the serum content in the culture medium is gradually reduced, and finally the cells that can adapt to the culture in the serum-free medium are obtained, and the cell seed batch for the serum-free culture is established. The above-mentioned cells were cultured in full suspension in shake flasks, and screened by single cell clones to finally obtain a MDCK cell line adaptable to full suspension culture (the MDCK cell line was selected by Harbin Pharmaceutical Group Biological Vaccine Co., Ltd. for sale).
[0074] After the frozen MDCK cells were taken out from the liquid nitrogen, they were immediately melted in a 37°C water bath, transferred to a 75ml cell flask with a pipette, added an appropriate amount of DMEM culture medium (containing 6% fetal bovine serum), and kept ...
Embodiment 3
[0085] Preparation of Example 3 Swine Influenza H3N2 Subtype Inactivated Vaccine and Evaluation of Its Safety and Immunity
[0086] 1) Preparation of virus liquid for seedling production
[0087] The virus liquid prepared in Example 2 was used as the virus liquid for preparing vaccines.
[0088] 2) Preparation of virus inactivator vaccine
[0089] Add the above harvested virus liquid to BEI with a final concentration of 0.2% (m / v, g / mL), and after inactivation at 30°C for 36 hours, add thiosulfuric acid with a final concentration of 0.2% (m / v, g / mL) sodium. Mix the H3N2 subtype swine influenza virus cell-adapted strain SIV-H3N2-HLJ strain virus liquid that has passed the inactivation test according to the ratio of Montanide ISA 15A VG adjuvant = 9:1 (v / v), and add the aqueous phase first Slowly stir in the emulsification tank, then slowly add the oil phase adjuvant, stir at 800r / min for 30 minutes after the addition, and then stand still for 30 minutes to prepare the swine ...
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