人NANOGP8基因原核表达及融合蛋白纯化

Prokaryotic expression of human NANOGP8 gene and its fusion protein purification

目的构建人NANOGP8基因原核表达载体,并纯化其融合蛋白。方法从plvxNANOGP8重组质粒中扩增出NANOGP8基因,插入pGEX-4T-1载体中构建pGEX-4T-1-NANOGP8重组载体,并转化大肠杆菌BL21,优化其表达条件,分别以可溶及包涵体形式得到了融合蛋白并进行纯化,采用Western blot检测其抗原性。结果成功构建了重组表达载体pGEX-4T-1-NANOGP8,经37℃诱导6h获得主要以包涵体形式存在的融合蛋白;而经16℃过夜诱导可获得可溶性融合蛋白,并分别成功对两种形式融合蛋白进行纯化且经Western blot检测显示其抗原性良好。结论成功获得NANOGP8纯化蛋白,可用于进一步研究NANOGP8基因在肿瘤中的作用。

Objective To construct the prokaryotic expression vector of human NANOGP8 gene and purify its fusion protein.Methods The DNA fragment of human NANOGP8 was amplified from recombinant plasmid plvx-NANOGP8 and then cloned into pGEX-4T-1 to construct a recombinant vector pGEX-4T-1-NANOGP8,which was transformed into E.coli BL21.After the expression condition was optimized,NANOGP8 fusion protein in the form of solubility or inclusion body was obtained and purified,the antigenicity of which was then detected by Western blot.Results The recombinant expression vector pGEX-4T-1-NANOGP8 was successfully constructed.The fusion protein in the form of inclusion body or solubility induced for 6 h at 37℃ or overnight at 16℃ was obtained and successfully purified.Western blot showed that the antigenicity of two forms of fusion protein was satisfactory.Conclusion NANOGP8 purified protein is successfully achieved,which may be used in studying the role of NANOGP8 gene in tumor.