抑制HBV复制的小干扰RNA的重组腺病毒构建与鉴定  

Construction and identification of a recombinant adenovirus which can inhibit HBV replication by expressing siRNA

作　　者：金晗英[1] 陈智[1] 羊正纲[1] 潘修成[1] 

机构地区：[1]浙江大学医学院附属第一医院传染病研究所,浙江杭州310003

出　　处：《浙江医学》2009年第8期1064-1066,1078,共4页Zhejiang Medical Journal

基　　金：国家自然科学基金资助项目（30371270）;浙江省科技厅重大项目（2003C13015）.

摘　　要：目的构建可同时表达绿荧光蛋白（GFP）和针对HBVS区基因的小干扰RNA（siRNA）的重组腺病毒，并研究该siRNA在体外对HBV的抑制作用。方法采用PCR技术自质粒pEGFP-C1中扩增含CMV启动子的GFP表达框，自质粒pAVU6＋4sh579中扩增含U6启动子的针对HBV S 区579-597位基因的siRNA表达框，分别将两个表达框克隆至穿梭载体质粒pShuttle内，与质粒pADEasy-1在大肠杆菌BJ5183内进行同源重组，将阳性重组体DNA转染至人胚肾细胞株HEK293细胞中进行包装、扩增，并在HepG2.2.15细胞中初步观察其对HBV复制的抑制疗效。结果构建了可同时表达GFP和siRNA的重组腺病毒，其可在HEK293细胞中进行包装、扩增，并在HepG2.2.15细胞中对HBV-DNA、HBsAg和HBeAg的表达具有抑制作用。结论成功构建了可同时表达GFP和针对HBV的siRNA的重组腺病毒，并在体外实验中证实了其对HBV复制具有抑制作用。Objective To construct a recombinant adenovirus capable of expressing both GFP and siRNA targeting HBs gene and to explore the effect of siRNA on HBV replication in vitro. Methods GFP expressing cassette containing CMV promoter was amplified from plasmid pEGFP-C1 by polymerase chain reaction （PCR） technique, siRNA expressing cassette containing U6 promoter was obtained from plasmid pAVU6＋4sh579 by PCR too. Both fragments were cloned into shuttle vector pShuttle in turn then the shuttle plasmid was recombined with plasmid pADEasy-1 in BJ5183 cells. The recombinant adenovirus was transfected into HEK293 cells to acquire primary adenovirus stock which was amplified by infecting HEK293 cells. The inhibition effect of siRNA on HBV replication was observed in cultured HepG2.2.15 cells. Results A recombinant adenovirus expressing GFP and siRNA was acquired. The recombinant adenovirus can be packaged and amplified in HEK293 cells. Furthermore,an inhibition effect of the recombinant adenovirus on HBV DNA,HBsAg and HBeAg expression in HepG2.2.15 cells was observed. Conclusion A recombinant adenovirus expressing GFP and siRNA was successfully constructed. An inhibition effect on HBV replication in vitro was also proved.

关 键 词：HBV 腺病毒载体 RNA干扰 

分 类 号：R512.62[医药卫生—内科学] R734.2[医药卫生—临床医学]