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本文采用分子克隆技术成功地构建了反义核酸表达载体pDOR-Bcl-2 cDNA.以脂质体介导法将重组反义核酸表达载体转染受体细胞SGC7901,并经C418筛选,从2×10~5细胞中筛选出大约150个抗性克隆,转导率大于1‰,随机挑选2个克隆继续筛选与扩增培养,获得了1株稳定的抗性细胞,从而建立了Bcl-2表达阻断的胃癌细胞株,我们命名为7901anBcl-2 cells.通过Southern印迹法、Northern印迹法、Western印迹法检测显示,转导株与非转导株均有Bcl-2 cDNA的表达,但转导株Bcl-2 mRNA及蛋白的表达明显低于非转导株.说明在胃癌细胞中Bcl-2基因处于高表达状态,通过真核表达载体介导的转染,反义Bcl-2可成功地导入胃癌细胞,并有效地阻断Bcl-2表达.
In this study, the antisense nucleic acid expression vector pDOR-Bcl-2 cDNA was successfully constructed by molecular cloning. The recombinant antisense nucleic acid expression vector was transfected into recipient cell SGC7901 by liposome-mediated method and screened by C418 from 2×. About 150 resistant clones were screened out in 10~5 cells. The transduction rate was greater than 1‰. Two clones were randomly selected to continue screening and expansion. A stable resistant cell was obtained, and Bcl-2 expression was established. Blocked gastric cancer cell lines, we named 7901anBcl-2 cells. Southern blotting, Northern blotting, and Western blotting showed that Bcl-2 cDNA was expressed in both transduced and non-transduced strains, but transduction was observed. The expression of Bcl-2 mRNA and protein in the strain was significantly lower than that in the non-transduced strain. It was demonstrated that the Bcl-2 gene was highly expressed in gastric cancer cells, and the antisense Bcl-2 was successfully transfected by the eukaryotic expression vector. Introduce gastric cancer cells and effectively block Bcl-2 expression.