hMTERF3基因真核表达载体的构建与鉴定  

Construction and Identification of Eukaryotic Expression Vector of Human MTERF3

作　　者：孙美涛 梅雯 杨泽芳 丁小倩 张若鹏 张晓娟 熊伟 Sun Meitao;Mei Wen;Yang Zefang;Ding Xiaoqian;Zhang Ruopeng;Zhang Xiaojuan;Xiong Wei(Pre-clinical College, Dali University, Dali, Yunnan 671000, China;Department of Reproductive Medicine, The First Affiliated Hospital of Dali University, Dali, Yunnan 671000, China;Department of Respiratory Medicine, Dali Teaching Hospital of Dali University, Dali, Yunnan 671000, China)

机构地区：[1]大理大学基础医学院,云南大理671000 [2]大理大学第一附属医院生殖科,云南大理671000 [3]大理大学大理教学医院呼吸内科,云南大理671000

出　　处：《大理大学学报》2017年第8期18-22,共5页Journal of Dali University

基　　金：国家自然科学基金资助项目(31601155;81560458);云南省教育厅科学研究基金资助项目(2014Z126);大理大学大学生创新创业计划资助项目(CXCY-X-2016-18);大理大学大学生科研基金资助项目(KYSX201609)

摘　　要：目的:构建和鉴定带3×Flag标签的人线粒体转录终止因子3基因(hMTERF3)真核表达载体。方法:根据NCBI数据库中hMTERF3基因序列设计引物,采用聚合酶链式反应从pGEM-hMTERF3重组克隆载体中扩增出hMTERF3基因开放阅读框序列,经HindⅢ和Bam HⅠ双酶切后,连接入真核表达载体p3×FLAG-CMV-14的多克隆位点中,构建重组质粒p3×FLAGCMV-hMTERF3,分别用菌落PCR、限制性核酸内切酶分析和DNA序列分析3种方法鉴定重组子。结果:重组质粒p3×FLAGCMV-hMTERF3经双酶切鉴定和菌落PCR鉴定均获得大小为1 254 bp的目的条带,DNA序列分析和Blast序列比对结果表明该序列与Gen Bank中hMTERF3基因序列的同源性达到100%,插入基因的大小和方向均正确。结论:成功构建了hMTERF3基因的真核表达载体,为进一步研究hMTERF3基因在恶性肿瘤发生发展中的作用奠定了基础。Objective:To construct and identify the eukaryotic expression vector of FLAG-tagged human MTERF3gene.Methods:The human MTERF3gene was amplified by Polymerase Chain Reaction(PCR).The recombinant products of the p3×FLAG-CMVhMTERF3were gained by T4DNA ligase connecting MTERF3gene fragment and eukaryotic expression vector p3×FLAG-CMV-14with restriction enzymes HindⅢand BamHⅠ.The recombinant pasmid p3×FLAG-CMV-hMTERF3were identified by colony PCR,double restriction enzyme digest and DNA sequencing.Results:A specific band of1254bp was detected from recombinant plasmidp3×FLAG-CMV-hMTERF3by digestion of HindⅢand BamHⅠ.The sequencing and identification showed that homology betweenthis sequence and the human MTERF3gene sequence from GenBank was100%,and the size and the direction of the inserted genewere right.Conclusion:The eukaryotic expression vector of human MTERF3was constructed successfully,which may lay thefoundation for a further study of the interaction mechanism between behavior of MTERF3in tumor cells and its development.

关 键 词：人线粒体转录终止因子3 真核表达载体 鉴定 

分 类 号：Q78[生物学—分子生物学]