有限稀释法筛选轮状病毒LD9株纯化方法的建立及应用

目的:建立有限稀释法克隆纯化轮状病毒的试验方法,筛选出感染性强、遗传特性稳定的轮状病毒基因重配株LD9。方法:梯度稀释LD9毒种,用5个稀释度(10-4～10-8)的病毒感染铺满单层Vero细胞的96孔板细胞,培养6 d,-20℃冻融,培养物传代至24孔板继续培养6 d,显微镜下观察细胞病变(CPE),-20℃冻融后检测。以相同方法重复克隆纯化3次,筛选获得病毒滴度稳定、具有稳定遗传特性的纯化病毒,由T25、T75到2,4,10层细胞工厂逐级放大培养。结果:筛选出适用于疫苗生产用的遗传特性稳定、感染性强的LD9疫苗候选株。结论:建立了适用于轮状病毒克隆纯化的筛选方法。

Development and application of limiting dilution method for screening rotavirus LD9

Objective: To establish the limiting dilution method for purifying rotavirus;to screen and obtain reassortant rotavirus strain LD9 with strong infectivity and genetic stability.Methods: A serial 10 time dilution(10-4~10-8) of the ressortant rotavirus LD9 were prepared,the virus was inoculated in 96-well plates with monolayer of Vero cells.The cell culture 6 days postinfection was frozen at-20 ℃ and then thawed.The subpassage was cultured in 24-well plate for 6 days,and CPE of cells was observed under a microscope.Then the culture was examined after freezing-thawing at-20 ℃ and room temperature.The purification process was repeated three times to screen and obtain the highly-infectious reassortant rotavirus with genetic stability.The subpassage cultures were then enlarged into T25,T75,2-Layer,4-Layer,10-layer cell factories gradually.Results: We had screened and obtained the highly-infectious reassortant rotavirus with genetic stability.Conclusion: The limiting dilution approach of purifying rotavirus has been established.