应用载体介导的RNAi技术抑制HCMV的UL49基因表达

为了研究RNA干涉抑制HCMV UL49基因的作用,以pLXSN(U6启动子)为模板通过两步PCR的方法扩增含U6启动子的siRNA表达片段,并通过TA克隆将siRNA表达片段克隆到pMD18-T载体构建成siRNA表达质粒,同时以人巨细胞病毒AD169病毒株基因组为模板PCR扩增UL49基因,将其克隆到pEGFP-N1构建融合质粒pEGFP-UL49。通过脂质体介导将siRNA表达质粒和pEGFP-UL49质粒共转染人宫颈癌细胞系HeLa,在荧光显微镜下观察RNA干涉结果。通过这种方法得到具有介导RNA干涉的siRNA片段,为UL49基因沉默研究提供技术基础。

Inhibition of HCMV UL49 Gene Expression by Vector-based RNAi

To study the inhibition of HCMV ULA9 expression by RNAi, siRNA expression plasmid was constructed by TA cloning technique. The siRNA expression cassettes were amplified from the pLXSN (U6 promoter) by two-step PCR. And the recombinant ULA9-EGFP plasmid (pEGFP-UL49) was constructed by cloning the UL49 gene into pEGFP-N1. siRNA expression plasmid and pEGFP-UL49 were cotransfected into human HeLa cells. The effects of RNAi were detected by green fluorescence signals. The results demonstrate that siRNA techniques can be used as an U149 gene silence approach for inhibiting gene expression of HCMV.