论文部分内容阅读
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 design and construct β-cate-nin shRNA eukaryotic expression plasmid and transfect gastric cancer cell line AGS. Methods: A template oligonucleotide with short hairpin was designed by targeting β-catenin and annealed to form a complementary double-stranded structure. The eukaryotic expression vector pGPU6-β-cate-nin-1,2 was constructed. Human gastric cancer cell line AGS was transfected with liposome, and the interference effect was detected by RT-PCR and Western blotting. Results: The recombinant plasmid pGPU6-β-catenin-1,2 was identified by restriction enzyme digestion and sequencing. After transfection of AGS cells, RT-PCR and Western blotting showed that pGPU6-β-catenin-2 significantly down-regulated β-catenin gene transcription and protein expression. The stable passage cell line was successfully screened, and the expression rate of green fluorescent protein was 94%. Conclusion: The eukaryotic expression plasmid pGPU6-β-catenin-2 of β-catenin gene was successfully constructed and stably transfected into gastric cancer cell line AGS, which provided a theoretical reference for further study on the role of β-catenin in gastrointestinal cancer.