LRP16短发夹RNA慢病毒载体的构建及LRP16稳定抑制细胞的建立  

Construction and Stable Expression of Lentiviral Vector Carrying LRP16 in HeLa Cells

作　　者：王春萌[1] 柏苗苗 伍志强[1] 李小雷[1] 李祥[1] 梅倩[1] 韩庆旺[1] 韩为东[1] 

机构地区：[1]解放军总医院基础医学所分子生物室,北京100853

出　　处：《生物技术通讯》2014年第1期29-32,共4页Letters in Biotechnology

基　　金：国家自然科学基金(31270820;81230061);北京市科技新星计划(2011113)

摘　　要：目的:构建靶向LRP16基因的短发夹RNA(shRNA)慢病毒表达载体,鉴定其在HeLa细胞中对LRP16的抑制效果。方法:构建pWPT-U6-LRP16shRNA-CMV-GFP慢病毒载体,通过病毒感染、细胞筛选、Western印迹等步骤,获得LRP16基因稳定抑制的细胞株。结果:构建了具有LRP16干扰效果的慢病毒载体,感染HeLa细胞后获得了稳定沉默LRP16及对照的细胞株;经克隆筛选,在荧光显微镜下观察到近似100%感染细胞发出绿色荧光;Western印迹证实pWPT-U6-L374-CMV-GFP和pWPT-U6-L668-CMV-GFP均可显著抑制HeLa细胞株中LRP16蛋白的表达,其中pWPT-Gsi-L374-GFP的抑制效果更好。结论:构建了靶向人LRP16基因shRNA慢病毒载体及LRP16稳定抑制的HeLa细胞系。Objective： To construct the recombinant lentiviral vector of short hairpin RNA （shRNA） against LRP16 and verified the stable knockdown efficiency of LRP16 in infected HeLa ceils. Methods： We constructed lentiviral vector carrying LRP16. After PCR verification, virus infecting, cell screening and Western blotting, we got HeLa cell lines stable expressing LRP16 shRNA. Results： Two lentiviral vectors carrying LRPI6 shRNA（L668 and L374） were successfully constructed. LRP16 stable knock-downed HeLa cell lines were establiehed and screened to harvest the pure positive clones which were almost reached 100% infection efficiency. Western blot an alyze confirmed that both L668 and L374 down-regulated the LRP16 expression in HeLa ceils which L374 was more significant. Conclusion： The lentivirus containing shRNA targeting the LRP16 gene has been constructed suc cessfully. HeLa cell lines stable expressing LRP16 shRNA were successfully established with lentiviral system.

关 键 词：LRP16 HELA细胞 RNA干扰 慢病毒载体 

分 类 号：Q78[生物学—分子生物学]