人AQP5真核表达质粒的构建及其在胃癌细胞中的表达

Construction of eukaryotic expression plasmid for human AQP5 and its expression in gastric cell line AGS

目的构建人pcDNA3.1-AQP5/myc-His真核表达质粒,观察水通道蛋白5(AQP5)基因在胃癌AGS细胞中的稳定表达。方法采用基因重组技术将人AQP5cDNA插入真核表达载体pcDNA3.1/myc-His,构建人pcDNA3.1-AQP5/myc-His真核表达质粒。脂质体介导空载体pcDNA3.1/myc-His和表达质粒pcDNA3.1-AQP5/myc-His分别转染人胃癌AGS细胞株,G418筛选,挑取阳性克隆,扩大培养,逆转录-聚合酶链反应(RT-PCR)和蛋白质印迹法(Western Blot)分别检测AQP5mRNA和蛋白表达。结果双酶切和基因测序结果均证实人pcDNA3.1-AQP5/myc-His真核表达质粒构建成功。AQP5转染组与空白对照组、空载体组比较,AQP5mRNA表达显著增加(P<0.05),AQP5蛋白表达显著上调(P<0.05)。结论成功构建人AQP5真核表达质粒并获得稳定表达AQP5基因的胃癌AGS细胞系,为进一步研究AQP5蛋白对胃癌恶性表型的影响奠定实验基础。

Objective To construct and identify human pcDNA3. 1-AQP5/myc-His eukaryotic expression plasmid and to observe the expression of AQP5 in gastric cancer cell line AGS. Methods Human AQP5 cDNA was cloned into eukaryotic expression vector of pcDNA3. 1/myc-His to construct pcDNA3. 1-AQP5/myc-His plasmid. The recombined eukaryotic expression plasmid pcDNA3.1-AQP5/myc-His and empty vector plasmid pcDNA3.1/myc-His were transfected into gastric cancer cell line AGS by Lipofectamine 2000,respectively. The positive colonies were selected by G418. The mRNA and protein expression of AQP5 were assessed by RT-PCR and Western blot, respectively. Results Eukaryotic expression plasmid pcDNA3. 1-AQP5/myc-His was successfully constructed and confirmed by restriction endonuclease digestion and sequencing. And AQP5 mRNA and protein expression were significantly higher in AQP5 transfected group than those of either control or empty vector groups（P〈0.05）. Conclusion Gastric cancer cell line AGS with stable expression of AQP5 is established, and it can be used for further study on the effects of AQP5 protein on gastric malignant phenotypic.