东方粘虫中肠V-ATP酶A亚基突变体TSCA的原核表达及复性纯化

根据得到的东方粘虫Mythimna separate(Walker)]中肠V-ATPase A亚基(VHA-A)基因(VATPA),预测其三维结构,构建定点突变并复性。利用Swiss-Model在线预测VHA-A三维结构,确定突变位点,通过重折叠PCR(Overlap-PCR)技术构建突变基因并将其整合到pET-22b(+)载体上,得到重组质粒pET-22b(+)-TSCA,转入大肠杆菌BL21中表达,最后通过亲和层析纯化包涵体蛋白质并进行透析复性。结果表明,成功构建突变基因和融合表达载体,融合蛋白质以包涵体形式表达,并得到大量纯化的可溶性蛋白。

Prokaryotic Expression, Refolding and Purification of V-ATPase Subunit A Mutant TSCA in the Midgut of Mythimna separate

Based on V-ATPase A subunit gene(VATPA) in the midgut of Mythimna separate, three-dimensional structure was predicted, site-directed mutant was built and recombinant protein was obtained after renaturing.The protein structure of VHA-A was analyzed, three-dimensional structure was predicted by Swiss-Model online,and the mutation site was designed.The mutant gene was constructed with overlap PCR, and inserted into pET-22b(+) vector. The recombinant plasmid pET-22b(+)-TSCA was transformed into E.coli BL21(DE3) for expression. After purified by Ni-NTA column, the inclusion body was renatured. The results showed the recombinant plasmid was successfully built, the fusion proteins were expressed as inclusion bodies, and the soluble proteins were successfully obtained in large scale.The study will facilitate the investigation of structure and function of V-ATPase subunit A and creation of environmental friendly pesticides.