端粒重复序列结合因子2真核表达载体的构建及表达

Construction of TRF2 eukaryotic expression vector

目的:构建端粒重复序列结合因子2(TRF2)真核表达载体,观察其在胃癌细胞中的表达。方法:EcoR I,HindⅢ双酶切AdTRF2质粒,胶回收1600bp小片段,连接入pcDNA3.1载体。经双酶切及测序鉴定后转染SGC7901细胞,空载体质粒转染SGC7901细胞为对照。G418选择培养液培养2个月,选取转染组及对照组细胞克隆。①细胞用阿霉素处理6h后Westernblot法检测TRF2表达。②MTT法检测转染对细胞生长增殖速度的影响。结果:成功构建TRF2真核表达载体,转染后SGC7901细胞TRF2表达明显增高,对细胞的生长增殖速度无明显影响(P>0.05)。结论:成功构建TRF2真核表达载体及稳定转染细胞株,为进一步研究TRF2功能奠定了基础。

Aim： To construct TRF2 eukaryotic expression vector and detect its expression in gastric cancer cell. Motbods：The AdTRF2 plasmid was digested by EcoR Ⅰ and Hind Ⅲ. A fragment of 1.6 kb was recovered by agarose electrophoresis and subcloned to the responding site of the eukaryotic expression vector pcDNA3. 1. After confirmation by double enzyme digestion analysis and DNA sequencing,positive recombinant plasmid was transfeeted into SGC7901 cells.Stable cell lines of SGC7901-TRF2 and SGC7901-cont were aequired after 2 months selection.The cell growth veolocity was analysed by MTT.T he expression of TRF2 was detected by Westem blot after cells were treated with ADR for 6 h.Results：The recombinant expression vector pcDNA-TRF2 was suceesfully construeted and the transfected SGC7901 cells showed significantly upregulated TRF2 expression.The Transfection did not influence the growth of cells（p〉0.05）.Conclusion：TRF2 eukaryotic expression vector has been successfully construeted and can stably expressed in gastric cancer cells.which provides a useful model for further study of the biological functions of TRF2.