摘要
背景:解旋酶-引发酶复合体是Ⅱ型单纯疱疹病毒进行复制的必需基因,UL5基因为Ⅱ型单纯疱疹病毒解旋酶-引发酶复合体的组成单位之一。目的:应用RNA干扰技术分析特异性小干扰RNA对Ⅱ型单纯疱疹病毒UL5基因的干扰作用。方法:以Ⅱ型单纯疱疹病毒UL5基因为靶目标,设计、合成5对特异性小干扰RNA。通过LipofeCtamine 2000脂质体将特异性小干扰RNA转染HEK293细胞。48h后行荧光定量RT-PCR检测UL5基因转录水平,终点滴定法测定干扰后病毒滴度,观察其干扰效果。结果与结论:小干扰RNA成功转染入细胞内。荧光定量RT-PCR结果显示,siRNA722、siRNA2394、siRNA2513和siRNA2627均可不同程度降低靶mRNA表达,病毒滴度检测结果显示siRNA722、siRNA2394、siRNA2513和siRNA2627均可不同程度降低上清液中病毒感染滴度,阴性对照组和siRNA374对病毒感染滴度无影响。针对UL5基因的有效小干扰RNA可以特异性降低Ⅱ型单纯疱疹病毒的复制水平。
BACKGROUND：Helicase-primase complex is the necessary gene for the replication of herpes simplex virus type Ⅱ （HSV-2）, and UL5 gene is one of the composing units of HSV-2 helicase-primase complex. OBJECTIVE：To analyze the interference effect of specific small interfering RNA （siRNA） on HSV-2 UL5 genes using RNA interference technology. METHODS：To target at HSV-2 UL5 gene, we designed and synthesized five pairs of specific siRNA. By means of liposome LipofeCtamine 2000, the specific siRNA would be transfected into the HEK293 cells. After 48 hours, fluorescence quantitative reverse transcription-PCR test was performed to determine the UL5 gene transcription as well as virus titer detection in order to observe the interference effect of siRNA. RESULTS AND CONCLUSION：siRNA would be successfully transfected into the cells. The fluorescence quantitative reverse transcription-PCR test showed that siRNA 722, siRNA 2 394, siRNA 2 513 and siRNA 2 627 could variously reduce the expression of the target mRNA. The virus titer detection showed siRNA 722, siRNA 2 394, siRNA 2 513 and siRNA 2 627 could in various degrees lower the titer of virus infection in the supernatant, negative control group and siRNA 374 had no effect on the titer of virus infection. The siRNA which is effective to UL5 gene could specifically reduce the replication of HSV-2.
出处
《中国组织工程研究》
CAS
CSCD
2012年第37期6931-6936,共6页
Chinese Journal of Tissue Engineering Research
基金
贵州省科学技术基金(黔科合J字[2008]2313号)
贵州省优秀科技教育人才省长专项资金(黔省专合字(2009)122号)资助项目~~
