干扰素诱导的跨膜蛋白-1基因的扩增、克隆及蛋白表达  

PCR amplification, cloning and protein expression of interferon-inducible transmembrane protein-1 gene

作　　者：刘宇虎[1] 钟东[2] 柳娟[1] 张振书[2] 陈村龙[2] 武金宝[2] 肖冰[2] 郭文英[1] 

机构地区：[1]东莞市人民医院消化科,广东东莞523018 [2]南方医科大学南方医院消化病研究所,广东广州510515

出　　处：《第一军医大学学报》2005年第10期1221-1224,共4页Journal of First Military Medical University

基　　金：国家自然科学基金(30171053);广东省医学科学技术研究基金(B2005089);东莞市科技计划项目(B200501)~~

摘　　要：目的研究干扰素诱导的跨膜蛋白-1(interferon-inducibletransmembraneprotein-1,IFITMP-1)基因的扩增、克隆及蛋白表达。方法以含IFITMP-1基因的cDNA为模板,用Pfu酶做PCR扩增,用EcoRⅠ和HindⅢ双酶切,将目的基因克隆到pUCm-T质粒测序。进一步克隆到pET-Trx蛋白表达载体质粒,优化蛋白表达条件。结果含有IFITMP-1基因的PCR产物约1000bp。重组pUCm-T质粒经EcoRⅠ、HindⅢ双酶切,有相应大小的cDNA片段插入,测序结果显示其序列正确,证实目的基因的扩增、克隆成功,并成功克隆进pET-Trx蛋白表达载体,经IPTG诱导,在BL21(DE3)plysS中有融合蛋白表达。结论成功扩增克隆IFITMP-1基因,并成功表达该基因编码的蛋白,为进一步研究IFITMP-1基因在大肠癌中的作用奠定了基础。Objective To study the PCR amplification, cloning and protein expression of interferon-inducible transmembrane protein-1 （IFITMP-1） gene. Methods With the cDNA fragment containing IFITMP-1 gene as template, IFITMP-1 gene was amplified using Pfu enzyme by means of PCR. After EcoR I and Hind Ⅲ digestion, the target gene fragment was linked to pUCm-T plasmid and sequenced. The IFITMP-1 gene was cloned into pET-Trx protein expression plasmid, and the condition for protein expression was optimized. Results The length of the PCR product of IFITMP-1 gene-containing cDNA fragment was about 1000 bp. The IFITMP-1 gene was successfully inserted into pUCm-T plasmid with correct sequence and cloning of the IFITMP-1 gene into the pET-Trx protein expression plasmid was achieved. Expression of the fusion protein ofpUCm-T plasmid and IFITMP-1 gene was detected after IPTG induction. Conclusion Successful amplification and cloning of the IFITMP-1 gene and its protein expression may facilitate further study of the role of IFITMP-1 gene in colorectal cancer.

关 键 词：干扰素 跨膜蛋白-1基因 大肠肿瘤 PCR 克隆 蛋白表达 

分 类 号：R346[医药卫生—基础医学]