单纯疱疹病毒1型US12基因siRNA筛选及其对病毒增殖的影响

Screening of highly efficient siRNA for silencing US12 gene of HSV-1

目的:筛选高效沉默单纯疱疹病毒1型(HSV-1)US12基因的siRNA,研究siRNA沉默US12基因后对HSV-1增殖的影响。方法:构建pEGFP-N1-US12融合表达质粒,设计并化学合成3对靶向US12基因的siRNA,与pEGFP-N1-US12融合表达质粒共转染Vero细胞,流式细胞术筛选高效抑制pEGFP-N1-US12融合蛋白的siRNA,实时荧光定量PCR检测siRNA对细胞内US12 mRNA表达水平的抑制效果,空斑减数实验评价siRNA对HSV-1增殖的抑制效果。结果:共转染实验筛选出高效抑制pEGFP-N1-US12融合蛋白的siR-NAS121、siRNAS122及siRNAS123。3对siRNA均能显著降低感染细胞US12 mRNA的表达水平,但空斑减数实验均未发现3对siRNA对HSV-1的增殖有显著抑制效果。结论:成功构建pEGFP-N1-US12融合表达质粒,筛选到高效抑制US12基因表达的3对siRNA,但是对病毒的体外增殖无显著影响,表明US12表达的立即早期蛋白ICP47的功能可能与HSV-1体外增殖无直接相关。

AIM： To explore the influence of siRNA on the reproduction of herpes simplex virus 1（HSV-1） by screening the siRNA that has high efficiency for silencing US12 gene of HSV-1.METHODS： Three siRNAs targeting US12 gene were designed,chemosynthesized and cotransfected into Vero cells with the fusion expression plasmid pEGFP-N1-US12.Fluorescence-activated cell sorter was used to screen inhibitory effect of siRNAs on the production of fusion protein US12-EGFP.The inhibitory effect of siRNAs on the mRNA expression of US12 in infected cells was also detected by real-time PCR.The reproduction of HSV-1 was assayed via plaque reduction experiment.RESULTS： siRNAS121,siRNAS122 and siRNAS123,which effectively inhibited the production of fusion protein US12-EGFP,were screened via cotransfection experiment,and the expression level of intra-cellular US12 mRNA was significantly reduced.However,no significant inhibitory effect on the reproduction of HSV-1 was observed as determined by plaque reduction assay.CONCLUSION： The three siRNAs effectively inhibit the mRNA expression of US12 but has no inhibitory effect on the in vitro production of progeny viruses when US12 gene is silenced,probably because the function of ICP47,the immediate early phase viral protein edcoded by US12 gene,is involved in immune evasion during in vivo infection.