靶向肠道病毒71型VP1基因的siRNA表达质粒的构建  

Construction of Expression Vector of siRNA Targeting to VP1 gene of Enterovirus 71

作　　者：吕玉春[1,2] 陈全[1,2] 严莎[1,2] 候艳[1,2] 

机构地区：[1]重庆医科大学基础医学院免疫学教研室,重庆400016 [2]重庆医科大学分子医学与肿瘤研究中心,重庆400016

出　　处：《中国生物制品学杂志》2010年第6期573-576,共4页Chinese Journal of Biologicals

基　　金：高等学校博士学科点专项科研基金(20070631006)

摘　　要：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 and identify the expression vector of siRNA targeting the VP1 gene of enterovirus 71（EV71）.Methods The expression vectors pSVSi1,pSVSi2 and pSVSi3 of siRNA targeting EV71 VP1 gene were constructed and transfected to A549 cells in mediation of liposome.The positive clones were screened,in which the expression of EV71 VP1 gene at mRNA and protein levels were identified by fluorescent quantitative PCR and Western blot respectively.Results Sequencing proved that recombinant plasmids pSVSi1,pSVSi2 and pSVSi3 were constructed correctly.Of the three recombinant plasmids,pSVSi1 was more effective than the other two in inhibiting the expression of VP1 gene in A549 cells,with inhibitory rates at mRNA and protein levels of 87% and 89% respectively.However,the inhibitory rates of pSVSi2 and pSVSi3 to the expression of VP1 gene at mRNA level were 60% and 70% respectively.Conclusion The expression vector of siRNA targeting the VP1 gene of EV71 was successfully constructed,and a siRNA inhibiting VP1 gene significantly was screened,which laid a foundation of siRNA gene therapy of hand-footmouth disease.

关 键 词：肠道病毒A型 人 VP1基因 RNA干扰 RNA 小分子干扰 

分 类 号：R512.5[医药卫生—内科学]