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

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.