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目的优化鸡胚细胞(chick embryo cells,CECs)培养狂犬病病毒(rabies virus,RV)的工艺。方法以M199为基础培养基,向其分别加入人血白蛋白、胎牛血清及HEPES缓冲液,并均加入碳酸氢钠,配制培养基M1、M2和M3。将驯化获得的CTNCEC25株按1∶5 000的比例接种于CECs悬液中,在不同培养条件下,通过单因素试验初步确定培养基配方、培养温度、接种MOI及培养时间4大工艺参数范围,再以正交试验确定上述条件的最佳组合。结果单因素试验初步确定的工艺参数:采用M2或M3,培养温度为33~36℃,MOI=0.01~0.001 FFU/细胞,培养时间为72~120 h,可获得较高的病毒滴度(7.0 lg FFU/ml以上)。正交试验确定的最佳工艺条件:采用M3,培养温度为33℃,MOI=0.01 FFU/细胞,培养时间为120 h,该工艺参数获得病毒滴度可达7.5 lg FFU/ml。结论本实验优化了CECs培养RV的工艺,CTNCEC25株的病毒滴度由约6.0 lg FFU/ml提高至7.0 lg FFU/ml以上,为疫苗生产获得理想抗原量奠定了基础。
Objective To optimize the process of culturing rabies virus (RV) in chick embryo cells (CECs). Methods M199-based medium was added to each of human serum albumin, fetal bovine serum and HEPES buffer, and sodium bicarbonate were added to prepare medium M1, M2 and M3. The domesticated CTNCEC25 strains were inoculated into CECs suspensions at a ratio of 1: 5000, and under the different culture conditions, the formulation parameters of the culture medium, culture temperature, MOI inoculation and culture time were determined by single factor experiments. Orthogonal test to determine the optimal combination of the above conditions. Results The results of single-factor experiment showed that the optimal technological parameters were M2 or M3, the culture temperature was 33-36 ℃, the MOI was 0.01-0.001 FFU / cell and the culture time was 72-120 h, the higher virus titer (7.0 lg FFU / ml or more). Orthogonal test to determine the optimum process conditions: the use of M3, culture temperature of 33 ℃, MOI = 0.01 FFU / cell culture time was 120 h, the process parameters obtained virus titer up to 7.5 lg FFU / ml. Conclusion This experiment optimized the process of culturing RV in CECs. The titer of CTNCEC25 strain increased from about 6.0 lg FFU / ml to 7.0 lg FFU / ml, which lays the foundation for obtaining the ideal antigen quantity for vaccine production.