重组 CARDs TX 融合蛋白的表达纯化与复性

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 Wetern 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 CARDs TX 的生物学特性奠定了基础。

Expression,purification and renaturation of recombinant CARDs TX fusion protein

Objective： 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.