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外部引导序列(EGS)技术(The EGS-based technology)是一种新型的基因沉默技术,能诱导内源性的核酶P(RNase P)对靶mRNA进行有效切割.以人类巨细胞病毒(HCMV)UL49基因mRNA片段为靶序列,基于前人实验基础上设计出更为精简与高效的改进型EGS(miniEGS),DNA片段长度仅为12bp.构建稳定表达HCMVUL49的细胞系,通过应用荧光定量PCR及Western印迹分别鉴定miniEGS对内源性UL49的抑制效率.结果显示,miniEGS能在HeLa细胞中能达到很高的转染效率(97.9%),并且在转染稳定表达UL49的HeLa细胞系后,发现UL49基因的mRNA与蛋白表达水平都出现明显下降(50%).研究表明,改进型的EGS序列不仅能有效抑制目的基因的表达,同时因其序列设计的精简性与高效性,可更好地应用到以后的抗病毒研究中.
The EGS-based technology is a novel gene silencing technology that induces the efficient cleavage of target mRNA by the endogenous RNase P. Human cytomegalovirus (HCMV ) UL49 gene mRNA fragment as target sequence based on previous experiments to design a more streamlined and efficient modified EGS (miniEGS), the DNA fragment length of only 12bp.Construct stable expression of HCMVUL49 cell line, by using quantitative PCR And Western blot were used to identify the inhibitory efficiency of miniEGS on endogenous UL49.The results showed that miniEGS could achieve high transfection efficiency (97.9%) in HeLa cells, and after transfection with HeLa cells stably expressing UL49, The results showed that the expression of UL49 gene was significantly decreased (50%). The results showed that the improved EGS sequence can not only effectively inhibit the expression of the target gene, but also better because of the streamlined and efficient sequence design Applied to future anti-virus research.