人肠三叶因子真核载体的构建与表达  

Construction of a recombinant eukaryotic vector of human intestinal trefoil factor and its expression in 293-T cells

作　　者：卢雅丕[1] 周飞[1] 王琳[1] 张波[1] 董菁[1] 任建林[1] 

机构地区：[1]厦门大学附属中山医院消化内科,福建厦门361004

出　　处：《南方医科大学学报》2008年第9期1630-1633,共4页Journal of Southern Medical University

基　　金：福建省自然科学基金(2006J0380);福建省卫生厅青年科研基金(2004-2-6;2006-1-53)

摘　　要：目的构建人肠三叶因子(hITF/hTFF3)的真核载体并在真核细胞中表达。方法从人结肠粘膜提取总RNA,逆转录多聚酶反应制备总cDNA,PCR扩增hTFF3基因,TA克隆至pGEMT载体中,测序鉴定后将hTFF3基因重组到真核表达载体pCMV5-myc中,转染真核细胞293-T,裂解细胞后分别采用鼠抗人c-myc抗体和鼠抗人TFF3抗体行Western-blot了解hTFF3表达情况。结果从人结肠粘膜成功克隆了hTFF3基因并经测序验证,构建了pCMV5-myc-hTFF3真核表达载体,转染细胞后行western-blot提示hTFF3可在293-T细胞中表达。结论成功构建pCMV5-myc-hTFF3重组真核载体并在293-T细胞中表达hTFF3蛋白,为进一步研究hTFF3的功能、作用机制及相互作用蛋白奠定了基础。Objective To clone human intestinal trefoil factor （hITF/hTFF3） gene into an eukaryotic expression vector for its expression in eukaryotic cells. Methods The total RNA was extracted from normal human colon mucosa, and transcribed into cDNAs using RT-PCR. hTFF3 gene was amplified by PCR and ligated into pGEMT vector by TA cloning method. After sequencing, the hTFF3 gene was transfered into the eukaryotic expression vector pCMV5-myc. The recombinant vector was transfected into 293-T cells, and the expression of the recombinant protein was detected by Western blotting. Results and Conclusion hTFF3 gene was successfully cloned from normal human colon mucosa. The vector pCMV5-myc-hTFF3 was reconstructed, and in 293-T cells transfected with the vector, hTFF3 expression was detected by Western blotting.

关 键 词：人肠三叶因子 基因重组 真核表达载体 

分 类 号：R735[医药卫生—肿瘤]