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目的体外研究针对SARSCoVRNA依赖性RNA聚合酶(RNAdependentRNApolymerase,RdRp)的小干扰RNA(SmallinterferenceRNA,siRNA)对SARSCoV感染的抑制效应。方法利用Ambion公司在线设计工具,设计出4条针对RdRp基因的siRNAs,并在体外利用试剂盒进行转录合成。在SARSCoV感染前1d,将这4条体外转录的siRNAs用Lipofectamine2000试剂转染入VeroE6细胞中。感染后,每天观察感染细胞的细胞病变效应(Cytopathiceffect,CPE)。第5天,用空斑形成实验(Plaqueformationunit,PFU)检测感染细胞培养上清液中SARSCoV滴度。结果CPE结果显示,在感染后的前4d,各siRNAs均能有效地保护细胞免受感染;在第5天,4条siRNAs中有3条仍能有效保护细胞不被感染。PFU实验结果显示,在第5天,所有siRNAs转染的细胞培养上清液中,SARSCoV的滴度均显著低于阳性对照(P<0.001)。结论实验结果表明针对SARSCoV的RdRp基因的siRNAs能有效而特异地抑制该病毒在哺乳动物细胞中的复制和繁殖,提示如果应用合适的给药途径,RNAi策略可以用于体内抑制SARSCoV的感染。
Objective To investigate the inhibitory effect of small interfering RNA (siRNA) targeting SARSCoV RNA dependent RNA polymerase (RdRp) on SARSCoV infection in vitro. Methods Four siRNAs targeting RdRp gene were designed using Ambion’s online design tool and synthesized in vitro using a kit. One day before SARSCoV infection, the four in vitro transcribed siRNAs were transfected into VeroE6 cells using Lipofectamine 2000 reagent. After infection, infected cells were observed daily for cytopathic effect (CPE). On day 5, the SARSCoV titers in infected cell culture supernatants were examined using Plaque formation unit (PFU). Results CPE results showed that all siRNAs effectively protected cells from infection 4 days before infection. On day 5, 3 of the 4 siRNAs still effectively protected cells from infection. The results of PFU showed that on day 5, the titers of SARSCoV in all siRNAs transfected cell culture supernatants were significantly lower than those in the positive controls (P <0.001). Conclusion The experimental results show that the siRNA targeting RdRp gene of SARSCoV can effectively and specifically inhibit the replication and multiplication of the virus in mammalian cells, suggesting that the RNAi strategy can be used to inhibit SARSCoV infection in vivo if the proper route of administration is used.