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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 screen siRNAs targeting the HSV-1 gene of HSV-1 gene silencing HS12 in vitro and in vivo. Methods: The recombinant plasmid pEGFP-N1-US12 was constructed and three pairs of siRNA targeting US12 gene were designed and synthesized. The recombinant plasmid was co-transfected with pEGFP-N1-US12 plasmid into Vero cells. Flow cytometry was used to screen pEGFP-N1 -US12 fusion protein. Real-time fluorescence quantitative PCR was used to detect the inhibitory effect of siRNA on the expression of US12 mRNA in cells. The plaque reduction test was used to evaluate the inhibitory effect of siRNA on the proliferation of HSV-1. Results: The siR-NAS121, siRNAS122 and siRNAS123.3, which were highly effective in inhibiting the pEGFP-N1-US12 fusion protein, were screened by co-transfection. The expression of US12 mRNA in infected cells was significantly reduced by siRNA. However, no plaque reduction test was found 3 had a significant inhibitory effect on the proliferation of HSV-1 by siRNA. CONCLUSIONS: The pEGFP-N1-US12 fusion expression plasmid was successfully constructed and 3 pairs of siRNAs that efficiently inhibit the expression of US12 gene were screened. However, there was no significant effect on the virus proliferation in vitro, indicating that the function of immediate early protein ICP47 expressed by US12 may be related to that of HSV-1 No direct correlation with proliferation in vitro.