茶树Mn-SOD基因在大肠杆菌中的高效表达

High efficient expression of msod gene from tea plants in Escherichia coli

聚合酶链式反应(PCR)扩增茶树嫩叶锰超氧化物歧化酶基因,并与原核表达载体pET22b(+)连接,构建重组质粒pET/msod,将该质粒转化至大肠杆菌Escherichia coliBL21(DE3),获得转基因工程菌BL21-pET/msod。在1 mmol.L-1异丙基硫代-β-半乳糖苷(IPTG)诱导下,重组蛋白得到高效表达,酶活性可达2×105U.L-1。SDS-PAGE检测表达蛋白分子量为25 kD,与通过核苷酸推测的分子量一致。

The msod gene from the young leaves of tea plants was amplified by PCR, cloned and joined the expressing vector pET22b （ ＋ ） so as to construct the recombinant plasmid pET/msod. The pET/msod was transformed into Escherichia coli BI21 to obtain the recombinant E. coli BL21-pET/msod. The recombinant Mn-SOD was expressed under the induction of 1 mmol·L^-1 IPTG,and the activity of the recombinant protein was about 2 ×10^5 U· L^-1. A molecular mass of the expressed protein was 25 kD determined by SDS-PAGE, according with the prediction by the msod nucleic sequence. The msod gene was successfully expressed in E. coli, to which would be referred for the applied research.