靶向β-catenin的shRNA真核表达质粒的构建及鉴定

Construction of eukaryotic vector expressing shRNA of β-catenin

目的:针对β-连环蛋白(β-catenin)基因的不同部位,构建靶向β-catenin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β-catenin基因的不同部位设计3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β-catenin的shRNA真核表达质粒pGPU6-β-catenin-shRNA-1、2、3。利用脂质体转染人胃癌细胞株AGS,Western blot法检测并筛选最佳抑制效率的shRNA表达质粒。结果:3个靶向β-catenin的shRNA真核表达质粒pGPU6-β-catenin-shRNA-1、2、3经限制性酶切和测序确实基因插入正确,Westernblot法证实pGPU6-β-catenin-shRNA-3明显降低细胞内β-catenin蛋白的表达。结论:成功构建了靶向β-catenin的shRNA真核表达质粒pGPU6-β-catenin-shRNA-1、2、3,并筛选出最佳抑制效率的表达质粒,为进一步研究Wnt/β-catenin信号通路在胃癌中的作用奠定了基础。

Objective：To construct short hairpin RNA（shRNA） eukaryotie expression vectors selectively silencing β-catenin gene. Methods：Three oligonucleotides targeting of β-catenin gene were synthesized and cloned into eukaryotie expression plasmids pGPU6. Three combinant plasmids pGPU6-β-catenin-shRNA-1,2,3 were transfeeted into AGS cells by lipofectamine reagent. The gene silencing efficiency was measured by Western blotting. Results：Three shRNA expression vectors were confirmed by restricted endonuelease analysis and partial nucleotide sequencing, pGPU6-β-catenin-shRNA-3 knocked down the expression of β-catenin protein dramatically by Western blot. Conclusion：The eukaryotic vectors expressing shRNA of β-catenin were constructed successfully. The results of this study lay the foundation for further studying on the role of Wnt/β-catenin signaling pathway in gastric cancer.