重组人组织因子的表达纯化及复性研究  

Expression,Purification and Renaturation of Recombinant Human Tissue Factor

作　　者：徐艺菲[1,2] 李佳萌[1] 齐永[1] 陈红霞[1] 潘英[1] 李素芹[1] 李素梅[1,3] 陈晨[1,2] 王新国[1,2] 张玉彬[2] 李越希[1] XU Yi-fei LI Jia-meng QI Yong CHEN Hong-xia PAN Ying LI Su-qin LI Su-mei CHEN Chen WANG Xin-guo ZHANG Yu-bin LI Yue-xi(Huadong Research Institute of Medicine and Biotechniques, Nanjing 210002, China China Pharmaceutical University, Nanjing 210009, China Nanjing Medical University, Nanjing 210005, China)

机构地区：[1]南京军区军事医学研究所,江苏南京210002 [2]中国药科大学生命科学与技术学院,江苏南京210009 [3]南京医科大学基础医学院,江苏南京210005

出　　处：《药物生物技术》2016年第6期477-480,共4页Pharmaceutical Biotechnology

摘　　要：利用基因工程技术表达重组人组织因子(Human tissue factor,h TF),并进行纯化及复性研究。构建插入目的基因的p ET28a(+)-h TF重组质粒,转化到大肠杆菌中,获得重组菌,用异丙基-β-D-硫代半乳糖苷(Isopropylthio-β-D-galactoside,IPTG)诱导其表达,菌体经超声裂解、离心收集包涵体,用8 mol/L尿素溶解包涵体、Ni-NTA纯化,采用透析复性和柱上复性两种方式获得复性的重组人HTF。柱上复性和透析复性均获得具有生物活性的重组人h TF,但柱上复性的复性率为72%,明显高于透析复性的复性率16%。成功构建高表达人重组h TF的工程菌,通过柱上复性获得高纯度具有生物活性的重组人h TF蛋白,为h TF的研究奠定了基础。To express, purify and refold the recombinant human tissue factor （hTF）, the human hTF gene was inserted into plasmid pET28a（ ＋ ） for construction of the recombinant pET28a（ ＋ ）-hTF. The engineered bacteria expressing human hTF was constructed by transforming the recombinant plasmid into E. coll. BL21 （ DE3 ）. The recombinant human hTF was expressed by inducing the engineered bacteria with isopropyl -β-D- thiogalactoside （IPTG） , and was purified by Ni-NTA immobilized metal affinity chromatography. The purified hTF was renatured by Ni-NTA affinity chromatography or dialysis. The refolding rates of the purified hTF were 72% and 16% for affinity chromatography or dialysis respectively, and the renatured hTF has good bioactivity. The recombinant human tissue factor with bioactivity was expressed, purified and renatured successfully,laid the foundation for the study of hTF.

关 键 词：hTF 表达 纯化 复性 

分 类 号：Q784[生物学—分子生物学]