VHS特异性siRNA对单纯疱疹病毒-1复制的影响

The impact of VHS specific siRNA on Herpes simplex virus typeⅠreplication

目的:探讨在RNAi技术条件下,干扰VHS合成对HSV-1复制的影响及对宿主细胞Vero的保护作用。方法:根据siRNA设计原则,用T7RNA聚合酶体外合成3种针对VHS(UL41)mRNA的siRNA,用lipofectamine 2000将siRNA转染到Vero细胞中后感染HSV-1,MTT法检测了细胞12h到72h的细胞活力,并测定了各时间点的病毒滴度值,绘制了子病毒一步生长曲线。结果:子代病毒一步生长曲线和MTT活力测底结果表明,转染的3对实验组siRNA都出现了病毒滴度明显降低,细胞病变相对空白组延迟的现象。结论:体外合成VHS基因特异性siRNA,能够有效的抑制病毒的复制,同时保护宿主细胞,这为siRNA治疗HSV-1的感染提供的初步的理论依据。

Objective To study the role of VHS protein during HSV-1 replication by RNAi. Methods Three siRNAs were transcripted in vitro by T7RNA polymerase. The Vero cells were transfected with siRNAs followed by HSV-1 infection. Then we detected the activity of cell by MTF method and monitored virus titer at 12 to 72 h after infection using single step growth curve assay, and used RT-PCR method to detect the expression of UL41 mRNA after R2 transfeetion. Results One step growth carves of HSV-1 and cell activity curves indicated that groups with UIAl-specific siRNAs decreased significantly in virus titer and have higher cell activity than control. The RT-PCR result indicated that UL41-specific siRNA R2 inhibited the expression of UIA1 gene considerably. Conclusion SiRNA transcripted in vitro by T7RNA polymerase can be used to inhibit viral replication, and the results provided a preliminary theory for treating HSV-1 by siRNA.