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目的构建靶向肠道病毒71型(EV71)VP1基因的siRNA表达质粒,并进行鉴定。方法构建靶向EV71VP1基因的siRNA表达质粒pSVSi1、pSVSi2和pSVSi3;脂质体法转染A549细胞株,筛选阳性克隆,通过荧光定量PCR和Western blot,分别在mRNA水平和蛋白水平鉴定EV71VP1基因的表达。结果质粒pSVSi1、pSVSi2和pSVSi3经测序证明构建正确。pSVSi1对A549细胞VP1基因的抑制效果最好,在mRNA和蛋白水平上的抑制率分别为87%和89%,pSVSi2和pSVSi3在mR-NA水平上的抑制率分别为60%和70%。结论已成功构建了靶向EV71VP1基因的siRNA表达质粒,并筛选出一种对VP1基因有显著抑制作用的siRNA,为手足口病的siRNA基因治疗奠定了基础。
Objective To construct siRNA expression vector targeting VP1 gene of enterovirus 71 (EV71) and to identify it. Methods The siRNA expression plasmids pSVSi1, pSVSi2 and pSVSi3 targeting EV71VP1 gene were constructed and transfected into A549 cell line by lipofectamine 2000. The positive clones were screened by fluorescence quantitative PCR and Western blot to identify the expression of EV71VP1 gene at mRNA and protein levels . Results The plasmids pSVSi1, pSVSi2 and pSVSi3 were verified by sequencing. pSVSi1 had the best inhibitory effect on VP1 gene in A549 cells with 87% and 89% inhibition rates at mRNA and protein levels respectively. The inhibition rates of pSVSi2 and pSVSi3 at mR-NA levels were 60% and 70%, respectively. Conclusion siRNA expression vector targeting EV71VP1 gene was successfully constructed and a siRNA targeting VP1 gene was screened out, which laid the foundation for the siRNA gene therapy of HFMD.