重组pEGFP-C1-Smad4表达载体的构建及在SGC7901细胞中的表达

Construction of recombinant pEGFP-C1-Smad4 and its expression in human gastric cancer SGC7901 cell lines

目的:构建pEGFP-C1-Smad4表达载体,并观察其在人胃癌SGC7901细胞中的表达。方法:应用基因重组技术,构建增强绿色荧光蛋白与Smad4的融合表达载体,经限制性内切酶酶切鉴定和PCR分析,用基因转染技术将其转入人胃癌SGC7901细胞,荧光显微镜、Western blot检测其表达。结果:酶切鉴定和PCR分析证实重组质粒中插入的目的基因片段及载体DNA大小、方向和插入位点均正确,在转染的人胃癌SGC7901细胞中观察到绿色荧光蛋白的表达,Western blot检测到Smad4在蛋白水平的表达。结论:成功构建真核表达质粒pEGFP-C1-Smad4,并在胃癌SGC7901细胞瞬时表达成功,为进一步研究Smad4在胃癌发生中的作用奠定基础。

Objective In order to make basis for gene therapy of gastric cancer, pEGFP-C1-Smad4 expression vector was constructed and we observed its expression in human gastric cancer SGC7901 cell lines. Methods Recombinant pEGFP-C1-Smad4 expression vector was constructed by techniques of gene recombination, screening and identified by restriction enzyme digestion and PCR. Then the recombinant was transfeeted into human gastric cancer cell line SGC7901 by techniques of gene transfection and detected expression by fluoroscopy and Western blot. Results Identification of pEGFP-C1-Smad4 by enzyme digestion and PCR showed that the length, inserted location and direction of the target gene inserted into the recombinant was correct and the expression of EGFP-C1 in transfected human gastric cancer SGC7901 cell was observed. The protein expression of Smad4 was detectable by Western blot. Conclusion The eukaryotic expression plasmid pEGFP-C1-Smad4 has been successfully constructed and it can be expressed transiently in SGC7901 cells, which would play a foundation for the further studies on the role of Smad4 in gastric carcinogenesis.