干扰EV71病毒2Apro基因的siRNA具有潜在的抗病毒作用

目的: 探讨以肠病毒71(entrovirus 71, EV71) 2A蛋白酶(2Apro)基因为靶序列合成的siRNA是否具有潜在的抗病毒作用。方法: 用Lipofectamine 2000脂质体分别将40、60、80 nmol/L FITC标记的BLOCK iT Fluorescent Oligo转染RD细胞，通过流式细胞仪和倒置荧光显微镜检测siRNA的转染效率。将化学合成的3个siRNA 2Apro (siRNA 69、294、319)和阴性对照的乱序si RNA(siRNA SCS)转染RD细胞，siRNA 2Apro为实验组，siRNA SCS为阴性对照组，另设模拟转染组，仅用脂质体处理；用EV71分别感染各组RD细胞，观察细胞形态学改变，荧光定量PCR和蛋白质印迹检测病毒RNA和VP1蛋白。结果： 当siRNA浓度为60 nmol/L时转染效率最高，达87%。细胞形态学结果显示，与对照组比较，实验组细胞只发生了轻微的病变。荧光定量PCR和蛋白质印迹结果显示，siRNA 2Apro组的病毒RNA和VP1蛋白明显低于对照组(P<0.01)。结论： 以EV71 2Apro基因为靶序列化学合成的siRNA能有效地抑制病毒的复制。

siRNA against the 2Apro genomic region of EV71 exerts potential antiviral effects

Objective: To explore whether siRNA targeted the 2A protease genomic (2Apro) region of enterovirus 71 (EV71) possesses potential antiviral effects. Methods: RD cells were transfected with 40, 60 and 80 nmol/L BLOCK iT Fluorescent Oligo labeled with FITC, transfection efficiency of siRNA was detected by flow cytometry and fluorescence microscopy. RD cells were transfected with three siRNA 2Apro (siRNA 69, 294, 319), the experimental group, and siRNA SCS,the negative group; the MOCK group only dealed with Lipofectamine 2000. Morphological changes of RD cells in each group were observed under the microscopy, viral RNA and protein were tested by quantitative RT PCR and western blotting, respectively. Results: The maximal transfection efficiency occurred when the siRNA concentration was at 60 nmol/L, the transfection efficiency was up to 87%. RD cells of siRNA 2Apro group showed slight cytopathic effect and a higher cell survival rate compared to control groups, through the observation of the morphological changes of the cells infected with EV71. The results of quantitative RT PCR and western blotting revealed that the levels of viral RNA and protein of siRNA 2Apro group were significantly lower than the control group (P<0.01). Conclusion: siRNAs targeted the 2Apro genomic region of EV71 exerts potential antiviral effects.