H3N2 subtype swine influenza virus cell adapted strain, inactivated vaccine prepared from H3N2 subtype swine influenza virus cell adapted strain, and applications of H3N2 subtype swine influenza virus cell adapted strain
A swine flu virus, H3N2 technology, applied in the direction of antisense single-stranded RNA virus, vaccine, virus, etc., can solve the problems of loose virus and high production cost, and achieve the generation of induced specific antibodies, good safety and immune effect good effect
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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] Example 2 The production process of culturing swine influenza H3N2 subtype virus with bioreactor
[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 cells that can adapt to serum-free medium culture are obtained, and a cell seed batch for serum-free culture is established. The above cells were subjected to full suspension adaptation culture in a shaker flask, and screened through single cell clones to finally obtain an MDCK cell line that could be adapted to full suspension culture (the MDCK cell line was selected and bred by Harbin Pharmaceutical Group Biological Vaccine Co., Ltd. for sale abroad).
[0074] After the cryopreserved MDCK cells were taken out from the liquid nitrogen, they were immediately thawed in a 37°C water bath, transferred to a 75ml cell flask with a pipette, and an appropriate amount of DMEM medium (containing 6% fetal bovine seru...
Embodiment 3
[0085] Example 3 Preparation of swine influenza H3N2 subtype inactivated vaccine and evaluation of its safety and immune efficacy
[0086] 1) Preparation of virus liquid for seedling production
[0087] The virus liquid prepared in Example 2 was used as the virus liquid for vaccine preparation.
[0088] 2) Preparation of virus inactivator vaccine
[0089] The virus solution harvested above was added with BEI with a final concentration of 0.2% (m / v, g / mL), inactivated at 30°C for 36 hours, and then added with thiosulfuric acid with a final concentration of 0.2% (m / v, g / mL). sodium. Mix the virus liquid of H3N2 subtype swine influenza virus cell-adapted strain SIV-H3N2-HLJ strain with qualified inactivation test according to the ratio of Montanide ISA 15A VG adjuvant=9:1 (v / v), first add the water phase Slowly stir in the emulsification tank, then slowly add the oil phase adjuvant, stir at 800 r / min for 30 minutes after adding, and then stand still for 30 minutes to prepare t...
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