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按照shRNA(small hairpin RNA)设计原则,针对Ⅱ型单纯疱疹病毒(herpes simplex virus type2,HSV-2)的UL27基因序列保守区域筛选设计、合成4条干扰靶序列并构建表达UL27序列特异性siRNA(short interfering RNA)的质粒载体pGPU6/GFP/Neo.通过脂质体介导重组表达载体转染HEK293细胞(human embryonic kidney 293 cell)再接种HSV-2.采用实时荧光定量PCR(real-timefluorescent quantitative PCR)技术检测UL27各组的mRNA转录水平,终点滴定法检测细胞上清液中的病毒滴度,四甲基偶氮唑盐(four methyl thiazolyl tetrazolium,MTT)法测定细胞存活率,Western印迹法检测蛋白表达效果.结果显示,UL27shRNA75组对UL27基因mRNA表达抑制效果最佳,同时能显著抑制感染细胞的CPE(cytopathic effect,CPE),降低上清液中的病毒感染滴度,提高细胞的生存率,抑制UL27基因的蛋白表达.提示本研究构建的pGPU6/GFP/Neo-UL27表达载体能在细胞水平上不同程度地干扰HSV-2 UL27基因表达,抑制HSV-2在HEK293细胞中复制.
According to the design principle of shRNA (small hairpin RNA), four target sequences were designed and synthesized based on the conservative region of UL27 gene of herpes simplex virus type 2 (HSV-2) (short interfering RNA) plasmid pGPU6 / GFP / Neo was transfected into HEK293 cells by liposome-mediated recombination expression vector and then inoculated with HSV-2. Real-time fluorescence quantitative PCR ) Technique was used to detect the transcription level of mRNA in each group of UL27. The titer of virus in the cell supernatant was detected by end-point titration method. The cell viability was measured by MTT method and Western blotting The results showed that UL27shRNA75 group had the best inhibitory effect on the expression of UL27 gene mRNA and the cytopathic effect (CPE) of infected cells was significantly inhibited, the virus titer in supernatant was decreased, and the cell survival rate was increased , Which inhibited the expression of UL27 gene, suggesting that the pGPU6 / GFP / Neo-UL27 expression vector constructed in this study could interfere with HSV-2 UL27 Gene expression, inhibition of HSV-2 replication in HEK293 cells.