有限稀释法筛选轮状病毒LD9株纯化方法的建立及应用  

Development and application of limiting dilution method for screening rotavirus LD9

作　　者：寇桂英[1] 余黎[1] 朱传凤[1] 王名强[1] 周旭[2] 

机构地区：[1]兰州生物制品研究所有限责任公司,兰州730046 [2]上海生物制品研究所有限责任公司,上海200052

出　　处：《中国新药杂志》2012年第10期1135-1139,共5页Chinese Journal of New Drugs

基　　金：国家科技支撑计划(2008BAI66B08)

摘　　要：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 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 -s ） of the ressortant rotavirus LD9 were prepared, the virus was inoculated in 96-well plates with mono- layer of Vero cells. The cell culture 6 days postinfeetion 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.

关 键 词：轮状病毒 基因重配株 梯度稀释 筛选 有限稀释法 

分 类 号：R186[医药卫生—流行病学]