三位点GSK3β shRNA真核表达质粒的构建及鉴定  

Construction of Eukaryotic Vector Expressing Three shRNAs of GSK3β Gene

作　　者：宋晓红[1,2] 翁丹卉[1] 邢辉[3] 卢运萍[1] 马丁[1] 王世宣[1] 

机构地区：[1]华中科技大学同济医学院附属同济医院妇产科,武汉430030 [2]首都医科大学附属北京佑安医院妇产科 [3]华中科技大学同济医学院附属襄樊中心医院妇产科

出　　处：《肿瘤防治研究》2010年第5期495-499,共5页Cancer Research on Prevention and Treatment

基　　金：国家自然科学基金资助项目(30571950);海外青年学者合作研究基金资助项目(30528012);国家973重点基础科学基金资助项目(2002CB513107)

摘　　要：目的构建三位点干扰GSK3β基因的高效shRNA表达质粒载体。方法用DNA重组技术将针对人GSK3β基因不同部位所设计的3对shRNA序列克隆到高效RNAi真核表达质粒载体pshRNA-U3中,构建shRNA表达载体pshRNA-U3/GSK3β。脂质体介导转染人卵巢癌细胞株OV2008,流式细胞仪检测转染效率。运用实时定量PCR和Westernblot分别检测RNA水平和蛋白水平GSK3β的干扰效果。运用流式细胞仪检测紫杉醇诱导的凋亡率。结果经测序证实成功构建携带3个GSK3βshRNA的表达载体pshRNA-U3/GSK3β。RT-PCR和Westernblotting均证实重组质粒pshRNA-U3/GSK3β转染OV2008后可明显降低细胞内GSK3βmRNA丰度及GSK3β蛋白表达。FACS显示,干扰GSK3β可抵抗紫杉醇诱导的凋亡。结论成功构建了针对GSK3β的三位点shRNA表达载体pshRNA-U3/GSK3β,并进行了初步的功能效应验证,为进一步研究GSK3β蛋白分子的生物学功能及应用奠定了基础。Objective To explore the feasibility of selective inhibiting GSK3β expression using GSK3β short hairpin RNA（shRNA）interference.Methods Three 19bp reverse repeated motifs targeted to GSK3β gene were synthesized and cloned into eukaryotic expression plasmid pshRNA-U3 containing three U6 shRNA promoter and termination signal of RNA polymerase.The recombinant plasmids pshRNA-U3/GSK3β and pshRNA-U3/con were transfected into OV2008 cells by lipofectamine reagent,respectively.The alteration of GSK3β expression was examined by real time-PCR and Western blot.Results It was verified by partial nucleotide sequencing that the constructed eukaryotic vector expressing three shRNA of GSK3β was correct.OV2008 cells transfected by pshRNA-U3/GSK3β dramatically down-regulated the expression of GSK3β mRNA and protein compared with untransfected and control cells.Conclusion The shRNA can efficiently suppress expression of GSK3β in OV2008 cells.The results of the study lay the foundation for further studying on biological functions and potential application of GSK3β.

关 键 词：卵巢癌 SHRNA GSK3Β 

分 类 号：R737.31[医药卫生—肿瘤]