siRNA对单纯疱疹病毒2型ICP4基因抑制作用的研究

摘要

Objective To explore the inhibition effect of RNA interference on the IcP4 expression and DNA replication of herpes simplex virus type 2(HSV2).Methods Four pairs of siRNA targeted to HSV2 ICP4 gene and negative control siRNA were synthetized by chemieal method.named as siRNA-1,siRNA-2,siRNA-3,siRNA-4 and siRNA-N respeeticely.HSV2 HG52 was used to attack Vero cell after transfection overnight.Vero cell and supernatant were collected at 1d,2d,3d,4d and 5d after virus attacking.Flurogenic quantitative reverse transcription polymerase chain reaction(FQ-RT-PCR)was used to detect the expression of HSV2 ICP4 mRNA,flurogenic quantitative polymerase chain reaction (FG-PCR) was used to detect the expression of HSV2 DNA and Western-Blot was used to detect the expression of HSV2 ICP4 protein.Results All the four pairs of siRNA could significantly inhibit the expression of HSV2 ICP4 mRNA and protein,especially siRNA-2.The above siRNAs could significantly decrease HSV2 DNA copy number,too.Conclusion siRNAs targeted to HSV2 ICP4 gene could significantly inhibit expression of HSV2 ICP4 mRNA and protein,and decrease HSV2 DNA copy number,suggesting that siRNA can inhibit HSV2 DNA replication through silencing ICP4 gene.%目的 探讨RNA干扰对单纯疱疹病毒2型(HSV2)ICP4基因表达和HSV2 DNA复制的抑制作用.方法 化学合成靶向作用于HSV2 ICP4的四对siRNA和阴性对照siRNA,分别命名为siRNA-1,siRNA-2,siRNA-3,siRNA-4及siRNA-N,转染Vero细胞,过夜后用HSV2 HG52标准毒株感染上述Veto细胞,1、2、3、4d和5d收集Vero细胞和上清液,荧光定量RT-PCR检测ICP4 mRNA的表达水平,荧光定量PCR检测HSV2 DNA拷贝数,Western-Blot检测ICP4蛋白表达水平.结果 与阴性对照相比,四对siRNA对HSV2 ICP4 mRNA和蛋白均有不同程度的抑制作用,以siRNA-2抑制作用最强,并且均可显著降低HSV2 DNA拷贝数.结论 靶向作用于HSV2 ICP4的siRNA可显著抑制ICP4mRNA及蛋白表达和HSV2 DNA拷贝数,提示siRNA可通过抑制HSV2 ICP4基因表达而抑制HSV2的复制.