重组CARDsTX融合蛋白的表达纯化与复性  

Expression, purification and renaturation of recombinant CARDs TX fusion protein

作　　者：牛健[1] 于晓旭[1] 李雪[1] 鲍会静[1] 刘运德[1] 

机构地区：[1]天津医科大学医学检验学院,天津300203

出　　处：《天津医科大学学报》2014年第3期184-187,共4页Journal of Tianjin Medical University

基　　金：天津医科大学重点学科建设与发展规划项目资助(201301)

摘　　要：目的:构建pET28α-CARDs TX重组质粒,诱导CARDs TX融合蛋白表达,并对其进行纯化与复性研究。方法:将CARDs TX基因(MPN 372)克隆入pET28α载体,经8次点突变获得pET28α-CARDs TX重组质粒。转化大肠杆菌BL21,IPTG诱导表达。利用亲和层析技术纯化蛋白并通过SDS-PAGE和Western blot检测CARDs TX的表达和纯化效果。利用尿素梯度复性法和扩大体积透析法对蛋白进行复性研究。结果:酶切和测序结果证明pET28α-CARDs TX重组质粒的DNA序列完全正确,在BL21中CARDs TX融合蛋白可高效表达,并可获得高纯度目的蛋白。利用扩大体积透析法对目的蛋白复性有较好的效果。结论:成功构建出pET28α-CARDs TX重组质粒,且CARDs TX可在大肠杆菌中高效表达,并可获得高纯度的目的蛋白,为深入研究CARDsObjective： To obtain recombinant CARDs TX fusion protein from BL21 cells. Methods： The CARDs TX gene （MPN 372） was cloned into vector pET28α and the integral CARDs TX expression vector was obtained after single gene point-mutations. The constructed recombinant plasmid was transformed into E.coli BL21 for expression under induction of IPTG. The Ni-NTA was used to purify the CARDs TX and the urea was desahed to get the refolding protein. Results： Enzyme digestion and gene sequencing analysis showed that pET28α- CARDs TX recombinant plasmid DNA sequences were completely correct. CARDs TX fusion protein could be expressed efficiently in BL21 and high purity target protein could be obtained. Expanded volume dialysis method applied had significant positive effect on the renaturation of the target protein. Conclusion： To successfully purify the CARDs TX and construct the CARDs TX expression vector that can be efficiently expressed in E. coli.

关 键 词：CARDsTX 质粒构建 点突变 蛋白纯化 蛋白复性 

分 类 号：Q7[生物学—分子生物学]