乙酰化酶基因 RNA 干扰文库的构建及其对 HepG2．2．15细胞的感染  

Establishment of interfering RNA library of acetyltransferase genes and its infection of HepG2.2.15 cells

作　　者：李凤棣 刘柯慧[1] 吴海清[1] 汤伟亮[1] 赵钢德[1] 项晓刚[1] 徐玉敏[1] 谢青[1] 王晖[1] 

机构地区：[1]上海交通大学医学院附属瑞金医院感染科,200025

出　　处：《中华传染病杂志》2014年第11期649-652,共4页Chinese Journal of Infectious Diseases

基　　金：国家自然科学基金资助项目（81070334）;上海市公共卫生优秀学科带头人培养计划资助项目（GWDTR201202）;十二五“艾滋病和病毒性肝炎等重大传染病防治”科技重大专项课题（2012ZX10005004-002）;上海市卫生和计划生育委员会重点课题（20134004）;上海市科委科技支撑项目（1340i902900）

摘　　要：目的：构建人类基因组中16个组蛋白乙酰化酶的短发夹 RNA（shRNA）慢病毒载体文库，为进一步探索表观遗传学基因在 HBV 调控中的作用机制提供有利工具。方法根据 shRNA 引物设计原则，针对每个基因选择8对确保干扰效率的 shRNA 序列（A～H），将引物退火后连接至 shRNA空慢病毒载体上转化，PCR 法验证菌落克隆，确认阳性克隆；对质量合格的质粒再进行单切酶验证。分别将同一基因的4个 shRNA 慢病毒质粒混合，分别包装病毒。293T 细胞转染48 h 和72 h 后收集病毒粗液，感染 HepG2．2．15细胞。感染72 h 后用荧光显微镜观察并评估荧光细胞比例。结果针对16个乙酰化酶基因，构建128个慢病毒载体 RNA 干扰（RNAi）文库，72 h 后慢病毒感染 HepG2．2．15细胞的效率＞80％。结论成功构建16个组蛋白乙酰化酶的 shRNA 慢病毒载体，从而为研究人组蛋白乙酰化酶对 HBV 复制的影响奠定了坚实的基础。Objective To construct lentivirus vectors carrying 16 short hairpin RNA （shRNA） expression cassettes targeting histone acetyltransferases and provide a powerful research approach to explore the mechanism of epigenetic genes in regulating hepatitis B virus （HBV）.Methods Following the rule of short shRNA primer design,eight-pair primers （A ~ H ）for each gene,which had stable interfering efficiency,were designed.The annealed primers were connected to the empty lentiviral vectors of shRNA for transformation.In order to confirm the positive clones,clones were analyzed by real-time polymerase chain reaction （RT-PCR ）.Then, qualified plasmids were verified by enzyme digestion technology.Four shRNA lentivirus plasmids against the same gene were mixed to build lentivirus respectively.After the virus transfected into 293T cells for 48 and 72 hours,supernatants were collected to infect HepG2.2.15 cells.The percentage of fluorescent cells were observed and assessed by microscope 72 hours after infection.Results One hundred and twenty-eight lentiviral vectors of RNA interference （RNAi）library were constructed against 16 histone acetyltransferases and more than 80% of HepG2.2.15 cells were infected with lentivirus 72 hours after infection.Conclusions Sixteen shRNA lentivirus vectors against histone acetyltransferase are successfully constructed.Thus,a solid foundation for the study of the effect of human histone deacetylase on HBV replication is established.

关 键 词：肝炎病毒 乙型 病毒复制 组蛋白酰基转移酶 短发夹 RNA RNA 干扰 

分 类 号：R373.21[医药卫生—病原生物学]