siRNA对SARS冠状病毒复制的抑制作用

为探讨siRNA在哺乳动物细胞中对SARS冠状病毒复制的抑制作用,针对BJ0 1株SARS冠状病毒复制酶基因(Pol)和刺突蛋白基因(S) ,设计4个siRNA ,并构建相应的siRNA表达载体及克隆细胞系.利用间接免疫荧光法及实时定量反转录PCR法,检测所设计的siRNA对SARS冠状病毒复制的抑制作用.结果表明,针对Pol基因的siRNA(psOe)在Vero细胞中可阻断BJ0 1株SARS病毒RNA的复制及其蛋白的表达.该结果为深入阐明SARS冠状病毒的致病机理及探讨SARS病毒防治新途径奠定了基础.

Interference of SARS-CoV Replication by Small Interfering RNAs

To investigate interference of SARS-CoV replication in mammalian cells by small interfering RNAs(siRNAs),siRNA expression plasmids targeting the polymerase gene and the S gene of SARS-CoV were constructed.Vero cells were stably transfected with these plasmids.Clonal cell lines,psOa,psOe,psSk and psNe were selected by growth in hygromycin B-containing media and characterized further by PCR analysis to determine the organization of plasmid DNA in the clonal cell lines.Cell lines in which the plasmid DNA was correctly integrated into the genome were challenged with SARS-CoV BJ01 strain at a multiplicity of infection (MOI) of 0.05.Cytopathic effect (CPE) of cells infected by SARS-CoV was observed every day.At 3rd day,postchallenged cells which was no CPE were fixed in the slides and immunofluorescence assays (IFA) were performed.One of the clonal cell lines,psOe were resistant to SARS-CoV infection.In addition,SARS-CoV RNA failed to accumulate in psOe by real-time quantitive PCR analysis.Observations have showed that siRNA (psOe) targeting polymerase gene could inhibit SARS-CoV RNA replication and protein expression in Vero cells.These results suggested a feasibility of using RNAi to elucidate pathogenesis of SARS-CoV and that RNAi might represent a new approach for the treatment of SARS infection.