特异性干扰ICP27基因的siRNA对HSV-1病毒复制的影响

摘要

针对单纯疱疹病毒1型(herpes simplex virus 1,HSV-1)的ICP27基因和其他疱疹病毒相关基因的高度保守区设计小干扰RNA (small interfering RNA,siRNA),研究其抑制病毒复制的效果.首先构建相应的小发夹RNA (small hairpin RNA,shRNA),然后通过病毒滴度测定、real-time PCR和细胞致病变效应(cytopathic effect,CPE)检测所设计的siRNA抑制病毒复制的能力.结果显示,所设计的shRNA-2(靶序列起始位置815)和shRNA-3(靶序列起始位置1 367)具有明显地抑制病毒复制的效果.尤其是shRNA-3,抑制病毒复制的效果更明显,在病毒滴度实验中,与阴性对照相比,其抑制倍数为81,同时可以下调ICP27基因的mRNA表达水平.实验结果表明shRNA-3能够显著抑制HSV-1病毒复制的能力,可以作为HSV-1感染性疾病的补充治疗手段,其对应的靶序列可以作为抗HSV-1新的靶标.%Small interfering RNAs (siRNAs) targeting ICP27 gene for herpes simplex virus 1 (HSV-1) and other highly conserved sequences for herpesviruses were designed and detected for their abilities to inhibit herpesvirus replication in vitro.Virus titer assay,real-time PCR and cytopathic effect (CPE) experiment were used to observe their inhibition effect in Vero cells challenged with HSV-1 by three small hairpin RNAs (shRNA-1,shRNA-2 and shRNA-3).The results suggested shRNA-2 (target sequence from 815 position) and shRNA-3 (target sequence from 1 367 position) could depress the replication of HSV-1.Especially,virus replication was obviously inhibited by shRNA-3 in the virus titer assay,and the inhibition ratio was 81 times comparing with Negative group.At the same time,the mRNA expression level of ICP27 gene could be decreased by shRNA-3.It indicated that shRNA-3 could effectively suppress the replication of HSV-1 and be used as a supplementary therapy for HSV-1 infectious diseases.The corresponding target sequence of shRNA-3 could therefore be used as a new target for anti-HSV-1 drugs.