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6d,- 20℃冻融,培养物传代至24孔板继续培养6d,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 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.
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