靶向ICP4的小干扰RNA对HSV-1病毒复制能力的影响  

siRNA Targeting ICP4 Attenuates HSV-1 Replication

作　　者：刘玉涛[1] 宋波[1] 王亚仑[1] 许予明[1] 韩志强[1] 赵莘瑜[1] 贾丽洁[1] 

机构地区：[1]郑州大学第一附属医院,郑州450052

出　　处：《病毒学报》2010年第3期163-169,共7页Chinese Journal of Virology

基　　金：河南省科技厅(2008-2010)河南省基础与前沿技术研究计划项目(082300450270);河南省科技厅(2008-2010)河南省基础与前沿技术研究计划项目(082300450610);河南省教育厅自然科学研究计划项目(2009A320045)

摘　　要：HSV-1是一种嗜神经病毒,能引起一系列神经系统严重症状,然而目前抗HSV-1药物易反弹、不能完全清除潜伏的病毒。ICP4对HSV复制、转录起主要调节作用,决定着溶细胞型感染或潜伏状态的平衡点。为了探寻新的抗病毒策略,本课题以HSV-1ICP4基因为靶点,设计合成2对siRNA,并构建重组真核慢病毒表达质粒pL-KO-puror-hU6-siRNA,通过脂质体转染和嘌呤霉素筛选建立靶向ICP4的4个siRNA单克隆细胞系,Real-timePCR法检测细胞系中ICP4的mRNA表达水平,TCID50法检测siRNA对HSV-1病毒复制能力的影响。结果显示靶向siRNA能有效抑制单克隆细胞系中的ICP4表达,并且抑制ICP4的表达后HSV-1病毒复制能力明显减弱,表明靶向ICP4的siRNA对HSV-1复制有明显抑制作用,且多位点siRNA联合干扰对病毒复制有协同抑制效果,有望应用于生物抗病毒药物的制备。HSV-1,a neurotropic virus,always leads to severe nervous symptoms. It is hard to completely eradicate the latent viruses after conventional therapy so that recurrence is inevitable. ICP is a key regulator for HSV replication and transcription that determines the cytolytic infection or latent state. In search of new anti-virus strategy targeting HSV-1ICP4,two pairs of siRNA were designed,and a recombinant eukaryotic lentiviral expression plasmid pLKO-puror-hU6-siRNA was constructed. Vero cells were transfected with the designed siRNAs by Lipofectamine 2000 and four stable monoclonal cell lines were established by puromycin screening method. The ICP4 expression at mRNA level was detected with real-time PCR,and the HSV-1 replication was measured with TCID50 assay. SiRNA was shown as an effective way to inhibit the expression of ICP4 in monoclonal cell lines. Meanwhile,HSV-1 replication was significantly inhibited when ICP4 was shut down by siRNA. We conclude that siRNA targeting ICP4 attenuates HSV-1replication. Further more,multi-site siRNAs show stronger inhibitory effect on viral replication,which may be an effective and feasible approach for biological anti-viral drugs.

关 键 词：单纯疱疹病毒1型(HSV-1) ICP4 RNA干扰 抗病毒 

分 类 号：R373.9[医药卫生—病原生物学]