人IL-24基因原核表达载体的构建及蛋白的表达纯化

The Construction of Prokaryotic Expression Vector for Human Gene IL-24 and Expression and Purification of Its Protein

构建人白细胞介素24(IL-24)原核表达载体并利用ELP-Intein系统表达纯化可溶性的IL-24蛋白。通过PCR扩增不含信号肽的人IL-24基因,将IL-24基因插入p ET-ELP-Intein质粒构建重组表达载体p ET-ELP-Intein-IL-24。将重组质粒转化至E.coliBLR(DE3),20℃下经IPTG诱导表达。利用ELP蛋白在不同温度下发生相变的特点和Intein蛋白的自切割反应纯化可溶性IL-24蛋白,将纯化得到的IL-24蛋白进行Western blot鉴定。用Annexin V-FITC/PI细胞凋亡检测试剂盒检测IL-24蛋白的生物学活性。成功构建了重组表达载体p ET-ELP-Intein-IL-24,第一次通过原核表达方法表达并纯化出了可溶性的IL-24蛋白。Western blot检测显示目的蛋白能与IL-24抗体特异性结合,表明纯化出的蛋白确实为IL-24蛋白。细胞凋亡检测实验证明IL-24蛋白能显著地诱导hep G2细胞发生凋亡。

This work aims to construct a prokaryotic expression vector for human gene IL-24, and express and purify soluble IL-24 protein via ELP-Intein system. The human gene IL-24 without signal peptide was amplified by PCR and cloned into vector p ET-ELP-Intein, and the recombinant expression vector p ET-ELP-Intein-IL-24 was constructed. The recombinant plasmid was transformed to Escherichia coli BLR（DE3）, and the expression was induced at 20℃ by IPTG. The soluble IL-24 protein was purified based on the transformation of ELP protein at different temperatures and the self-cleavage reaction of Intein protein. The purified protein was identified by Western blot. The bioactivity of IL-24 protein was measured by Annexin V-FITC/PI apoptosis detection kit. The recombinant expression vector p ET-ELP-Intein-IL-24 was successfully constructed, and the soluble IL-24 protein was expressed for the first time by prokaryotic vector and purified. Western blot analysis showed the target protein specifically bound with IL-24 antibody, which proved that the purified protein was indeed IL-24 protein. Apoptosis detection experiment confirmed that IL-24 protein significantly induced the apoptosis of hep G2 cells.