薇甘菊几丁质酶基因的原核表达及活性分析

Expression and Activity Analysis of a Chitinase Gene from Mikania micrantha in Escherichia coli

将薇甘菊Mmchi1基因cDNA序列的编码区插入到原核表达载体pET-32a（＋）中,构建融合表达质粒,并在大肠杆菌中进行了融合蛋白6×His-Mmchi1的诱导表达、Western blot和酶活性分析.结果表明,0.1 mmol/LIPTG、25℃诱导4 h,在大肠杆菌Rosetta-gami（DE3）中能获得可溶性的6×His-Mmchi1;Western blot证实表达的6×His-Mmchi1能与抗6×His的单抗发生特异性反应,分子量约为55 kD,与预测的融合蛋白分子量相符;纯化后的6×His-Mmchi1最佳酶活性pH和温度分别为5.5~6.5和35~40℃.为进一步研究融合蛋白6×His-Mmchi1的功能奠定了基础.

The fused expression plasmid was constructed by inserting the coding region of chitinase gene（Mmchi1） from Mikania micrantha H.B.K.into the prokaryotic expression vector pET-32a（＋）.Its induced expression in Escherichia coli cells as fusion protein 6×His-Mmchi1 carried out Western blot and enzyme activity analyses.The results showed that the soluble fusion protein（6×His-Mmchi1） was produced at a high level in E.coli Rosetta-gami（DE3） cells when induced by 0.1 mmol/L IPTG at 25℃ for 4 h.Western blot analysis revealed that the fusion protein（6×His-Mmchi1） could be recognized by anti-6×His monoclonal antibody.The molecular mass of 6×His-Mmchi1 was about 55 kD which was agreed with the predicted molecular mass.The optimal temperature and pH of purified 6×His-Mmchi1 were determined to be 5.5~6.5 and 35~40℃,respectively.This study established the foundation for future researches on the function of the fusion protein（6×His-Mmchi1）.