摘要
目的:设计并构建β-连环蛋白(β-cate-nin)shRNA真核表达质粒,并转染胃癌细胞株AGS。方法:以β-catenin为靶基因设计具有短发夹结构的模板寡核苷酸,退火形成互补双链结构,构建shRNA真核表达载体pGPU6-β-cate-nin-1,2。脂质体转染人胃癌AGS细胞株,RT-PCR、蛋白质印迹法检测干扰效果。结果:重组质粒pGPU6-β-catenin-1,2经酶切和测序鉴定。转染AGS细胞后,RT-PCR和蛋白质印迹法显示,pGPU6-β-catenin-2明显下调β-catenin基因转录和蛋白表达。成功筛选稳定传代细胞株,绿色荧光蛋白表达率94%。结论:成功构建了β-catenin基因shRNA真核表达质粒pGPU6-β-catenin-2,稳定转染胃癌细胞株AGS,为进一步研究β-catenin在胃肠道恶性肿瘤中的作用提供了理论参考。
OBJECTIVE： To construct β-catenin shRNA eukaryotic expression vectors which are transfected into AGS in order to silence β catenin gene. METHODS： The shRNA oligonucleotides targeting for β-catenin gene were synthesized and cloned into pGPU6, pGPU6-β-catenin 1,2 were identifi cated by sequencing and transfected into AGS by lipofectamine reagent. The gene silencing effects were determined by RT PCR and Western blot. RESULTS： shRNA expression vectors were measured hy restricted endonuclease analysis and partial nucleotide sequencing, pGPU6-β-catenin-2 slowed down the expression of β-catenin evidently. Steady cell line was selected successfully, and expression rate of GFP was 94%. CONCLUSIONS： shRNA of β-catenin recombinant were established successfully by RNAi technique and transfected in to AGS cells. It supports further investigation of the role of β-catenin in gastric cancer.
出处
《中华肿瘤防治杂志》
CAS
2009年第15期1151-1154,共4页
Chinese Journal of Cancer Prevention and Treatment
