应用干扰RNA体外抑制严重急性呼吸综合征冠状病毒N基因的表达  

The function of Th and dendritic cells in patients with different hepatitis B virus genotypes

作　　者：陶鹏 [1] 张君 [1] 张秉强 [1] 王超 [2] Tong-Chuan He  黄爱龙 [1] 

机构地区：[1]重庆医科大学病毒性肝炎研究所,400016 [2]四川泸州医学院附属医院感染科 [3]Molecular Oncology Laboratory, The University of Chicago Medical Center, Chicago, IL 60637, USA

出　　处：《中华传染病杂志》2005年第3期166-169,共4页Chinese Journal of Infectious Diseases

基　　金：国家杰出青年基金资助(30228026);国家"十五"863计划资助(2001AA217121)

摘　　要：目的构建绿色荧光蛋白(GFP)与严重急性呼吸综合征(SARS)冠状病毒N蛋白的融合表达载体pEGFP-C1-N以及针对SARS冠状病毒N基因的小发卡型shRNA表达载体pshRNAN,观察shRNA对SARS冠状病毒N基因复制和表达的影响。方法将构建成功的pEGFPC1N与pshRNAN共转染293细胞,于转染后24、48、72h观察GFP表达的强弱,采用Western Blot分别检测GFP与N蛋白的表达,逆转录PCR检测N基因的mRNA。结果psh-RNA-N作用组绿色荧光强度明显弱于未干扰组,Western Blot和逆转录PCR结果也证实pshRNA-N对N基因和N蛋白的表达有明显抑制作用,而无关序列的shRNA无此作用。结论针对SARS冠状病毒N基因的shRNA具有显著和特异的抑制N蛋白表达的作用。Objective To develop a RNAi approach that specifically targets the N gene sequence of SARS-CoV by synthesizing short hairpin RNA （shRNA） in vivo, and to assess the inhibitory effect of this shRNA on SARS-CoV-N expression. Methods The eukaryotic expression plasmid pEGFP-C1- N, which contains SARS-CoV-N, were cotransfected into 293 cells with either the RNAi plasmid pshRNA-N or unrelated control plasmid pshRNA-HBV. At 24, 48, 72 hours post transfection （p. t. ）,the Green Fluorescent Proteins （GFP） were observed by fluorescence microscope. The levels of SARS-CoV-N RNA were determined by RT-PCR. The expression of GFP and protein N were determined by Western blot. Results Successfully constructed expressing shRNA vector pshRNA-N which targeted the N gene of SARS-CoV. The introduction of RNAi plasmid was shown to efficiently and specifically inhibit the synthesis of protein N. RT-PCR showed that the RNAs of N gene were distinctly reduced when the pEGFP-C1-N was cotransfected with pshRNA-N,whereas the control vector did not exhibit inhibitory effect on the N gene transcription. Conclusions Our results demonstrate that the short hairpin RNA targeting SARS-CoV-N exerts robust inhibition on SARS-CoV-N expression, suggesting that RNAi-based anti-SARS-CoV strategy may represent a potential approach to the management of SARS.

关 键 词：RNA干扰 SARS病毒 基因疗法 基因表达 严重急性呼吸综合征(SARS) SARS冠状病毒 shRNA N基因 体外抑制 干扰 

分 类 号：R346[医药卫生—基础医学]