短双链RNA对柯萨奇B组3型病毒体外感染的抑制作用研究  

Short interfering RNA-mediated inhibition of Coxsakievirus B3 infection in vitro

作　　者：韩继生[1] 肖宗慧[1] 姚海兰[1] 任红雁[1] 刘哲伟[1] 

机构地区：[1]首都儿科研究所分子免疫室,北京100020

出　　处：《中华实验和临床病毒学杂志》2007年第2期150-152,共3页Chinese Journal of Experimental and Clinical Virology

基　　金：国家自然科学基金资助项目（30371343）;北京市自然科学基金资助项目（5042005）

摘　　要：目的利用RNA干扰技术，在细胞、蛋白和基因水平上观察短双链RNA对柯萨奇病毒体外感染Hela细胞的增殖抑制作用、对病毒RNA复制和蛋白合成的影响。方法应用病毒致细胞病变作用保护实验、空斑形成减少实验、Western Blot、RT-PCR等方法，在体外检测其抗CVB3病毒作用。结果设计、合成的8条SiRNA中，针对基因组2B、VP4、2A、3C区的SiRNA-2、SiRNA-3、SiRNA-6、SiRNA-7具有不同程度的抑制病毒作用。其中，病毒基因组2B的SiRN．2作用效果最好，对Hela细胞CVB3感染72h后致细胞病变和空斑形成的抑制率为95％，抑制病毒蛋白的合成，病毒基因的复制水平也显著降低，在病毒0．1、0．01、0．001、0．0001 MOI感染水平上对CVB3致细胞病变作用的抑制率分别为30％、70％、88％和99％（48h）。结论针对CVB3基因组2B的SiRNA-2具有较强的抑制柯萨奇B3型病毒感染的作用。Objective To evaluate feasibility of inhibiting Coxsackievirus B3 （CVB3） infection at cellular, protein and gene levels by using small interfering RNA （siRNA）. Methods Antiviral activities of siRNAs were evaluated by observing cytopathic effect （CPE）, using plaque reduction Western blotting assays and RT-PCR. Results Eight siRNAs were synthesized, among them, SiRNA-2, SiRNA-3, SiRNA-6 and SiRNA-7 which were targeted against sequences located in 2B, VP4, 2A and 3C section of CVB3 genome, were designed to have different effect of inhibiting CVB3 infection in vitro. SiRNA-2 showed the best protective effect, 95 % inhibition of CVB3 cytopathic effect and plaque forming effect was observed at 0.0001 MOI, viral protein synthesis and replication were inhibited. SiRNA-2 showed 30% inhibition of virus at 0. 1 MOI, 70% inhibition at 0.01 MOI, 88%inhibition at 0.001 MOI, and 99% inhibition at 0.0001 MOI 48 hours after CVB3 infection. Conclusion SiRNA could effectively inhibit CVB3 infection in vitro, siRNA-2, which is targeted against sequence in 2B section of CVB3 genome, seemed to be the best one among those synthesized in this study.

关 键 词：RNA 肠道病毒B型 人 RNA病毒感染 

分 类 号：R686[医药卫生—骨科学]