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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
【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.
- 【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