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。 OBJECTIVE: To establish a method for cloning and purifying rotavirus by limiting dilution method, and to screen out a recombinant rotavirus LD9 with strong infectivity and stable genetic characteristics. Methods: Gradient dilutions of LD9 virus were used to infect 96-well plate cells covered with monolayer Vero cells with 5 dilutions (10-4 ~ 10-8) of virus, cultured for 6 days and frozen and thawed at -20 ℃. The cultures were passaged The cells were cultured in 24-well plates for 6 days. The cytopathic effect (CPE) was observed under a microscope. The cells were frozen and thawed at -20 ° C. In the same way, the clones were purified three times in succession, and the purified virus with stable virus titer and stable genetic characteristics was screened and amplified step by step from T25 and T75 to 2,4,10-layer cell factories. Results: Stable and highly infectious LD9 vaccine candidates suitable for vaccine production were screened out. Conclusion: A screening method for cloning and purification of rotavirus was established.