针对S基因的siRNA或shRNA表达框对HepG2.2.15细胞中HBV基因表达和病毒复制的抑制作用比较  

Inhibition of HBV gene expression and replication in HepG2.2.15 cells by siRNA or shRNA expressing cassettes targeting HBV S gene

作　　者：温伟红[1] 刘家云[1] 王凯[2] 薛茜[1] 孟艳玲[1] 赵晶[2] 贾林涛[2] 薛采芳[3] 王成济[2] 杨安钢[1] 

机构地区：[1]肿瘤生物学国家重点实验室,第四军医大学免疫学教研室,西安710032 [2]肿瘤生物学国家重点实验室,生物化学与分子生物学教研室 [3]肿瘤生物学国家重点实验室,病原生物学教研室

出　　处：《中华微生物学和免疫学杂志》2007年第4期353-357,共5页Chinese Journal of Microbiology and Immunology

基　　金：国家自然科学基金资助（30400379,30500433）;国家重点基础研究发展规划（973）基金资助（2004CB518805）;长江学者和创新团队发展计划资助（IRT0459）

摘　　要：目的 化学合成针对乙型肝炎病毒（HBV）S基因的siRNA，同时制备针对相同区段的shRNA表达框，并比较二者对HepG2．2．15细胞中HBV基因表达和病毒复制的抑制作用。方法 化学合成针对HBVS基因的带有FITC标记的siRNA，设计合成带有FTrc标记的引物，PCR扩增含有RNA聚合酶Ⅲ启动子H1序列和shRNA编码序列的表达框，并对扩增产物进行纯化，将等摩尔数siRNA和shRNA表达框分别用脂质体转染稳定表达HBV的HepG2．2．15细胞，转染1d后流式细胞仪检测转染效率，转染3d后RT-PCR检测靶基因mRNA的水平，用SDS-PAGE、Westem blot及间接免疫荧光染色检测HBsAg的表达。收集转染前和转染后1、3、5和7d的细胞培养上清，检测HBsAg水平，同时用荧光定量PCR方法检测HBV　DNA的含量。结果成功制备了针对HBVS基因的shRNA表达框，将其与等摩尔数siRNA分别转染HepG2．2．15细胞后，RT-PCR证实细胞中HBV　mRNA水平降低，SDS-PAGE、Westem blot及间接免疫荧光染色检测到HBsAg的表达受到抑制，细胞培养上清中HBsAg和HBV DNA含量下降。与siRNA相比，shRNA表达框对HBV的抑制作用虽然起效较慢，但持续时间更长。结论shRNA表达框和siRNA均可明显抑制HBV的转录和表达，与siRNA相比，shRNA表达框能够更持久地抑制靶基因的表达。Objective To synthesize siRNA and to construct shRNA expressing cassettes targeting hepatitis B virus S gene and to evaluate their inhibitory effect in HepG2.2.15 ceils. Methods FITC-siRNA targeting HBV S gene was synthesized;corresponding shRNA expressing cassettes containing RNA polymerase H1 was amplified. The synthesized FITC-siRNA and the purified shRNA expressing cassettes targeting the same sequence of HBV were respectively transiently transfected into HepG2.2.15 cells. Transfection efficiency was determined by flow cytometry at day 2, HBV mRNA level was detected by RT-PCR and HBsAg expression was examined by SDS- PAGE, Western blot and immunotluorescent staining on day 3 after transfection. Medium HBsAg and HBV DNA level were analyzed on 0, 1, 3, 5 and 7 days after transfecfion. Results shRNA expressing cassettes was successfully constructed. HBV mRNA and HBsAg expression in HepG2.2.15 cells was significantly decreased. Secretion of HBsAg and HBV particles was also suppressed. Compared with siRNA, although the inhibitory effect induced by shRNA expressing cassettes occured a little late, it maintained a longer time. Conclusion Both shRNA expressing cassettes and siRNAs can effectively down-regulate the transcription and expression of HBV. Compared with siRNA, shRNA expressing cassettes showed a more persistent inhibitory effect.

关 键 词：乙型肝炎病毒 RNA干扰 shRNA表达框 SIRNA 

分 类 号：R686[医药卫生—骨科学]