hTFF3基因真核表达载体构建及表达鉴定  

Construction of EGFP-hTFF3 Vector and Identification of Its Eukaryotic Expression

作　　者：郑倩倩[1] 郭文东[1] 朱亚勤[1] 

机构地区：[1]中国医科大学基础医学院细胞生物学教研室,教育部医学细胞生物学重点实验室,沈阳110001

出　　处：《中国医科大学学报》2011年第10期877-880,共4页Journal of China Medical University

基　　金：辽宁省自然科学基金资助项目(20092123);辽宁省教育厅高校科研计划(2009S108)

摘　　要：目的克隆人肠三叶因子基因hTFF3,构建重组真核表达载体pEGFP-C1-TFF3,并在真核细胞中表达。方法应用逆转录聚合酶链式反应(RT-PCR)技术,从真核细胞中扩增得到人TFF3的全长序列,克隆至表达增强型绿色荧光蛋白载体中,经酶切、PCR鉴定后测序证实克隆成功。将重组载体转至真核细胞,荧光显微镜观察下转染效率,同时采用RT-PCR、Western blot技术检测外源基因TFF3的表达。结果成功将hTFF3基因克隆到真核表达载体中,酶切片段与预期大小一致(200 bp)。在荧光显微镜下观察转染后的细胞可见明显的绿色荧光,RT-PCR及Western blot结果表明:转染后的细胞中TFF3在转录和翻译水平的表达均有明显提高。结论成功构建了真核表达载体pEGFP-C1-TFF3,并在真核细胞中显著表达,为进一步研究TFF3因子的生物学功能奠定了基础。Objective To construct the eukaryotic expression vector pEGFP-C1-TFF3. Methods The full-length TFF3 gene sequence was amplified fwm Cos7 cells by reverse transcription polymerase chain reaction （RT-PCR）. The fragment was inserted into Green Fluorescent protein （GFP） expression vector digested by Bgl Ⅱ and EcoR Ⅰ enzymes, the same with PCR product digestion. The recombinant was transfected into Cos7 cells and the exogenous expression of TFF3 gene was detected by RT-PCR and Western blot. Results The pEGFP- C1-TFF3 eukaryotic expression vector was successfully constructed and the inserted sequence was 200 bp in accordance with our expectation. The vivid green fluorescence was observed in transfected cells under a fluorescent microscope. RT-PCR and Western blot showed the predominant elevation of TFF3 expression in transfected cells at both transcription and translation levels. Conclusion The pEGFP-C1-TFF3 eukaryotic expression vector was successfully constructed and TFF3 was significantly expressed in eukaryocyte,which provides the foundation for the biologic function study of TFF3.

关 键 词：肠三叶因子 克隆 表达载体 

分 类 号：Q291[生物学—细胞生物学]