Japanese encephalitis vaccine prepared by human embryonic lung fibroblasts and preparation method thereof
A technology of fibroblasts and Japanese encephalitis, applied in the field of bioengineering, can solve the problems of inability to use human embryonic lung fibroblasts, and achieve the effects of quality control, safety assurance, and pollution avoidance
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Embodiment 1
[0040] Use revived human embryonic lung fibroblast WI-38, the cell nutrient solution is MEM culture solution, which contains 8-10% calf serum and 24-32U / ml gentamicin, adjust the pH to 7.0-7.2, and then Cultured in a 15L spinner bottle at 37°C to form a dense monolayer and then amplified according to the seeding ratio of 1:8. When the expansion reached the production batch, after 6 days, the cells grew into a dense monolayer. Discard the cell nutrient solution and use pH7.0 Earle's solution of -7.4 washes the cell surface, inoculates the P3 virus seed of the virulent strain of Japanese encephalitis virus in human embryonic lung fibroblasts, and uses the third generation working virus seed (P3V) of the P3 strain on WI-38 cells. 3 ), virus seed concentration MOI 0.001, cell maintenance medium is MEM solution containing 0.2% (w / w) human albumin, its pH is 7.4-7.6, cultured at 33°C, after 24h, replace with fresh cell maintenance medium and continue to cultivate for 48- After 72 ho...
Embodiment 2
[0042] Use resuscitated human embryonic lung fibroblasts MRC-5, the cell concentration is 10 6 cells / ml, cultured in a 50L bioreactor with 5g / L Cytodex 2, working volume 40L, rotating speed 40-45 rpm, dissolved oxygen 25-50%. The cell nutrient solution uses MEM culture solution containing 8%-10% calf serum and 24-32U / ml gentamycin, pH 7.0-7.2, and the culture temperature is 37°C. Observe the growth of the cells at any time, adjust the pH of the culture solution according to the automatic replenishment of the reactor according to the growth of the cells, and continue culturing for 6 days until the cell density reaches 10 7 In the case of cells / ml, after washing twice with Earle's solution, add MEM solution without calf serum and containing 0.2% (w / w) human albumin, pH7.4-7.6. Then inoculate human embryonic lung fibroblast JE virus attenuated strain SA14-14-2 virus seed, use the third generation working virus seed of SA14-14-2 strain passed on MRC-5 cells, virus seed concentrat...
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