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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

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【作者】 徐艺菲李佳萌齐永陈红霞潘英李素芹李素梅陈晨王新国张玉彬李越希

【Author】 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;China Pharmaceutical University;Nanjing Medical University;

【机构】 南京军区军事医学研究所中国药科大学生命科学与技术学院南京医科大学基础医学院

【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。

【Abstract】 To express,purify and refold the recombinant human tissue factor(h TF),the human h TF gene was inserted into plasmid p ET28a(+) for construction of the recombinant p ET28a(+)-h TF. The engineered bacteria expressing human h TF was constructed by transforming the recombinant plasmid into E. coli. BL21(DE3). The recombinant human h TF 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 h TF was renatured by Ni-NTA affinity chromatography or dialysis. The refolding rates of the purified h TF were 72%and 16% for affinity chromatography or dialysis respectively,and the renatured h TF has good bioactivity. The recombinant human tissue factor with bioactivity was expressed,purified and renatured successfully,laid the foundation for the study of h TF.

【关键词】 hTF表达纯化复性
【Key words】 HTF geneExpression productsPurificationRenaturation
  • 【文献出处】 药物生物技术 ,Pharmaceutical Biotechnology , 编辑部邮箱 ,2016年06期
  • 【分类号】Q78
  • 【被引频次】1
  • 【下载频次】128
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