人TIMP-2基因的克隆及其原核表达  

Gene clone and expression of human tissue inhibitor of matrix metalloproteinases2 in Escherichia coli

作　　者：徐国强[1] 王伟[1] 孙达权[2] 黄小琼[1] 陈腾祥[1] 

机构地区：[1]贵州医科大学生理学教研室 [2]贵州医科大学生物化学与分子生物学教研室,贵州贵阳550004

出　　处：《贵州医药》2016年第4期344-347,共4页Guizhou Medical Journal

基　　金：贵州省科学技术基金[黔科合LG字(2012)007];贵州省科学技术基金[黔科合J字(2014)2025]

摘　　要：目的构建人TIMP-2重组表达载体,并在大肠杆菌BL21(DE3)中表达。方法提取人肝癌细胞(Hep G2)总RNA后,经RT-PCR扩增获得目的片段,插入克隆载体pMD18-T;酶切鉴定和测序后将TIMP-2导入原核表达载体pGEX-4T-1中,构建重组质粒pGEX-TIMP-2。将重组质粒转化至大肠杆菌菌株BL21(DE3)中,IPTG诱导融合蛋白表达后用SDS-PAGE电泳检测并用western blot验证(蛋白质印迹法)。结果成功构建原核重组表达载体pGEX-TIMP-2,并在原核宿主BL21中大量表达融合蛋白GST-TIMP-2。结论克隆人TIMP-2基因并在原核生物中大量表达。Objective To construct the recombinant prokaryotic vector of human TIMP-2（Tissue inhibitor of matrix metalloproteinases 2,TIMP-2）,and then express it in E.coli BL21（DE3）.Methods Total RNA was extracted from human hepatoma cell line（Hep G2）,and the TIMP-2cDNA was obtained with RT-PCR.Then,the amplified TIMP-2coding region by PCR technology was cloned into pMD18-T.After identification and sequence,the TIMP-2DNA fragment was inserted into the prokaryotic expression vector of pGEX-4T-1,and the recombinant was transformed into E.coli BL21（DE3）for expression.After induced by IPTG,the recombinant GST-TIMP-2had been expressed and its molecular weight was approximately 48 kD and identified by western blot.Results TIMP-2gene coding region was cloned from Hep G2 cell and the recombinant plasmid of pGEX-TIMP-2 was successfully constructed and expressed fusion protein GST-TIMP-2in BL21 induced by IPTG.Conclusion The pGEX-TIMP-2recombinant vector was successfully constructed.

关 键 词：基质金属蛋白酶抑制因子2 PGEX-4T-1 原核表达 肝癌 

分 类 号：R329.25[医药卫生—人体解剖和组织胚胎学]