摘要
目的:针对PP2A内源性抑制剂SET基因的不同部位,构建靶向SET的shRNA真核表达质粒,鉴定并筛选出最佳抑制效率的表达质粒.方法:针对SET基因的不同部位设计4对短发卡RNA(shRNA)的寡核苷酸片段,克隆到真核表达载体pGPU6中,构建靶向SET的shRNA真核表达质粒pGPU6/GFP/Neo-SET-shRNA-1、2、3、4.利用脂质体转染人胃癌细胞株BGC-823,Western blot法检测并筛选最佳抑制效率的shRNA表达质粒.结果:4个靶向SET的shRNA真核表达质粒pGPU6/GFP/Neo-SET-shRNA-1、2、3、4经限制性酶切和测序证实基因已正确插入,荧光显示转染效率可达到80%以上.Western blot法证实pGPU6/GFP/Neo-SET-shRNA-3明显降低BGC-823细胞内SET蛋白的表达.pGPU6/GFP/Neo-SET-shRNA-3对SET蛋白表达的抑制效应在72h达到最强.结论:成功构建靶向SET的shRNA真核表达质粒pGPU6/GFP/Neo-SET-shRNA-1、2、3、4,并筛选出最佳抑制效率的表达质粒,为今后研究SET在胃癌中的作用机理奠定了基础.
AIM：To construct eukaryotic vectors expressing short hairpin RNAs（shRNAs） targeting the set gene which encodes an endogenous inhibitor of PP2A.METHODS：Four oligonucleotides targeting the SET gene were synthesized and cloned into the eukaryotic expression plasmid pGPU6.The resulting four recombinant plasmids,pGPU6/GFP/Neo-SET-shRNA-1,2,3 and 4,were introduced into BGC-823 cells by lipofectamine-mediated transfection.The gene silencing eff iciency was measured by Western blot RESULTS：DNA sequencing and enzyme digestion analysis confirmed the identity of the four recombinant shRNA expression vectors.Immunofluorescsence demonstrated that transfection efficiency was above 80%.Transfection of pGPU6/GFP/Neo-SET-shRNA-3 significantly knocked down the expression of SET protein as revealed by Western blot.The silencing effect of pGPU6/GFP/Neo-SET-shRNA-3 on the expression of SET protein was most remarkable at 72 h after transfection.CONCLUSION：Eukaryotic vectors expressing shRNAs targeting the set gene have been constructed successfully and can be used to study the role of SET in gastric cancer.
出处
《世界华人消化杂志》
CAS
北大核心
2011年第24期2521-2526,共6页
World Chinese Journal of Digestology
基金
国家自然科学基金资助项目
No.30870981
No.81001082
No.81000884~~
