体外转录制备的siRNAs对SARS-CoV复制的抑制作用  

Inhibition of SARS-CoVs replication by in vitro-prepared siRNAs

作　　者：石孝民[1] 周高标[1] 毛丽伟[2] 姜曼[2] 黎万玲[2] 何仰东[2] 吴玉章[2] 倪兵[2] 

机构地区：[1]解放军空军总医院泌尿外科,北京100036 [2]第三军医大学全军免疫学研究所,重庆400038

出　　处：《免疫学杂志》2005年第6期438-441,共4页Immunological Journal

基　　金：国家自然科学基金(30400437);国家重点基础研究计划"973"项目SARS预防基础研究基金(2003CB514108)资助

摘　　要：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 effects of RdRp gene-specific siRNAs on SARS-CoV replication in vitro. Methods Four of siRNAs targeting RdRp gene of SARS-CoV were designed and synthesized by in vitro transcription. The siRNAs were transformed into Veto E6 cells by [ipofectamine 2000 reagent one day before the cells were infected with SARS-CoV. The cytopathic effect （CPE） was observed every day postinfection. Titers of vires in cultured cells transformed with various siRNAs were assayed by plaque formation unit （PFU）. Results CPE assay showed that all cells protected by RdRp-specific siRNAs could survive for 4 d after infection. On day 5, 3 of 4 siRNAs-protected Vero E6 cells could survive. In accordance with the result of CPE assay, PFU assay revealed that viruses from culture supernatant of protected cells were much less than that of control cells. Conclusions Our results indicate that the siRNAs targeting RdRp gene of SARS-CoV could effectively and specifically silence the replication and production of the virus, which suggests that RNAi strategy, with appropriate administration method, might be used for inhibiting SARS-CoV replication in vivo.

关 键 词：严重急性呼吸器官综合征 RNA干扰 RNA依赖性RNA聚合酶 小干扰RNA 

分 类 号：R346[医药卫生—基础医学]