摘要
为了鉴定抑制麻疹病毒体外复制的靶向Rab9 GTPase基因效应性小干扰RNA(siRNA),根据siRNA设计原则和Rab9 GTPase基因的mRNA序列,设计并化学合成8对靶向Rab9 GTPase基因的siRNAs和1对阴性对照siRNA,经脂质体法转染Vero-E6细胞株,转染10 h后感染麻疹病毒Edmonston株。通过逆转录聚合酶链反应(RT-PCR)检测转染后细胞内Rab9 GTPase mRNA水平;通过标准蚀斑试验检测麻疹病毒滴度。同对照组相比,8对靶向Rab9 GTPase基因siRNAs中的2对(Rab9-4和Rab9-7),以时间和剂量依赖性的方式显著地抑制细胞内Rab9 GTPase mRNA表达和麻疹病毒的复制(抑制率高达90%以上),其他的siRNAs对细胞内Rab9 GTPase mRNA表达和麻疹病毒的复制的抑制性效应则低于50%。结果表明,Rab9-4和Rab9-7是体外抑制麻疹病毒复制的最有效的siRNAs,这些siRNAs靶序列能被用来深入研究RNA干扰治疗麻疹病毒感染的可能性。
In order to identify effective small interference RNA （siRNA） that could inhibit measles virus （MV） replication in vitro, eight pairs of siRNAs target oriented Rab9 GTPase gene and one pair of negative control siRNA were designed and synthesized chemically according to siRNA design rule and Rab9 GTPase gene mRNA sequence. The siRNAs were transfected into Vero-E6 cells strain via liposome, and then infected the cells by MV Edmonston strain ten hours after. The level of Rab9 GTPasc mRNA was assayed through reverse transcription PCR （ RT-PCR）, and the titer of MV was detected by standard plaque assay. As compared with negative control, two pairs of the eight pairs target oriented Rab9 GTPase gene siRNAs, Rab9-4 and Rab9-7 obviously inhibited Rab9 GTPase mRNA in the host cells to express and to replicate MV （ the inhibitory rate was as high as above 90% ） in the way of time and dosage dependence, while the expression of and the replication of MV the other siRNAs in the host cells on the inhibitory effect were lower than 50%. The results indicated that Rab9-4 and Rab9-7 were the most effective siRNAs in vitro to inhibit the replication of MV; and these siRNAs target sequence could be used for further study on the possibility of the treatment of MV infection by the RNA interference.
出处
《微生物学杂志》
CAS
CSCD
2009年第4期44-48,共5页
Journal of Microbiology
基金
辽宁省教育厅资助项目(2008758)
