人组织型纤溶酶原激活剂原核表达载体的构建及在大肠杆菌中的初步表达  

Construction of the Prokaryotic Expression Vector of Human Tissue-Type Plasminogen Activator and Its Preliminary Expression in Escherichia coli

作　　者：杨震[1] 杨可[1] 侯曼美[2] 景丽丽[1] 张睿[1] 张丽[1] 雷寒[1] 何通川[3] 周建中[1] 

机构地区：[1]重庆医科大学附属第一医院心血管内科,重庆400016 [2]西南大学现代生物医药研究所,重庆400715 [3]美国芝加哥大学分子肿瘤实验室,芝加哥60637

出　　处：《西南大学学报（自然科学版）》2012年第4期71-77,共7页Journal of Southwest University(Natural Science Edition)

基　　金：重庆市自然科学基金资助项目(CSTC2009BB5406)

摘　　要：目的:构建人组织型纤溶酶原激活剂(tissue-type plasminogen activator,tPA)原核表达载体,检测其在大肠杆菌Escherichia coli中的初步表达.方法:以含有人tPA基因的质粒为模板,设计引物扩增出含有RGDS-tPA基因并构建到原核表达载体pET28a中,测序证实为正确的rtPA序列,并将pET28a-tPA转化至菌株E.coli BL21(DE3).用IPTG诱导rtPA在E.coli中表达,并通过聚丙烯酰胺凝胶电泳(SDS-PAGE)进行验证.结果:琼脂糖凝胶电泳获得目的基因片段tPA的条带约在1 700bp处,鉴定为阳性重组体;聚丙烯酰胺凝胶电泳鉴定获得目的蛋白片段rtPA的条带约53KDa,为非活性的包涵体.结论:人组织型纤溶酶原激活剂原核表达载体构建成功,可在大肠杆菌中有效表达.Objective. The construction of the prokaryotic expression vector of human tissue-type plasmino- gen activator （tPA） andidentification of its expression in Escherichia coli. Methods. The human RGDS- tPA gene was amplified from the recombinant plasmid and ligated to the prokaryotic plasmid pET28a. The recombinant PET28a-tPA was identified by sequencing and transformed into E. coli BL21（DE3）. The ex- pression of RGDS-rtPA in E. coli was induced by IPTG and detected by SDS-PAGE. Results. A fragment of 1700bp was amplified from the recombinant plasmid, which was identified as tPA. A 53KDa protein was obtained by induced expression of rtPA and further identified as a non-active inclusion body. Conclu- sion. The prokaryotic expression vector of human tissue-type plasminogen activator was successfully con- structed and was effectively expressed in E. coli.

关 键 词：组织型纤溶酶原激活剂 原核表达载体 转化 表达 

分 类 号：Q814[生物学—生物工程]