针对2B区域的短双链RNA抑制柯萨奇B3病毒感染HeLa细胞的特性研究  

Study on Inhibition of Coxsackievirus B3 Infection in HeLa Cell by Short Interfering RNA Targeting 2B Protein

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

机构地区：[1]首都儿科研究所,北京100020

出　　处：《病毒学报》2007年第4期276-281,共6页Chinese Journal of Virology

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

摘　　要：为了研究短双链RNA(Small interfering RNA,siRNA)对柯萨奇B组3型病毒(CVB3)复制的影响及其作用特性,合成针对CVB3基因组2B区的siRNA-2B,脂质体法转染HeLa细胞后感染CVB3病毒,观测转染效率及存留时间、毒性作用、病毒致细胞病变效应、病毒滴度、病毒RNA含量、siRNA-2B对重组基因的特异性降解及培养上清有限稀释后再感染情况。结果发现siRNA-2B能高效转染入HeLa细胞并存留长达48h,高剂量的siRNA-2B对培养细胞无明显毒性,siRNA-2B能特异性针对2B区有效地降解病毒RNA,能明显抑制病毒RNA的复制。随着转染浓度的增加,siRNA-2B的抗病毒作用逐渐增强。siRNA-2B还能明显降低CVB3的再感染能力。这些结果提示,针对基因组2B区的siRNA-2B可以明显抑制CVB3基因复制,有效控制病毒再感染,并具有高效性、特异性和量效关系等特点。为siRNA可能成为预防和治疗CVB3感染的新途径奠定基础。To study the inhibitory effect and function characteristics of small interfering RNA （siRNA） on cosxackievirus B3（CVB3） infection by RNA interference technique, siRNA-2B against the viral 2B region was synthesized and transfected into HeLa cell, which was then infected with CVB3. The efficiency of siRNA transfection was examined by FCM, the cell toxicity of siRNA-2B by MTT, and the antiviral ability of siRNA-2B by cytopathic effect （CPE）, plaque reduction assay and RT-PCR. The results showed that siRNA-2B could be transfected efficiently into HeLa cell and lasted at least 48h. High concentration of siRNA-2B didn＇t show any sign of toxicity to cells, siRNA-2B exhibited a significant protective effect on cell viability by specific inhibition of viral replication. It showed a close relationship between the concentrations of siRNA-2B and the antiviral effects, siRNA-2B led to dramatical reduction of viral titers in supernatant of cell culture and weakened the reinfection ability of the virus. These data proposed that siRNA-2B, targeting 2B protein, can effectively inhibit CVB3 infection in HeLa cell and exhibits its transfection efficiency, viral inhibition specificity and adose-dependant manner, suggesting its potential role in prevention and treatement of CVB3 infection.

关 键 词：RNAI SIRNA 柯萨奇B3病毒 抗病毒效应 

分 类 号：R373.23[医药卫生—病原生物学]