岸蟹金属硫蛋白在大肠杆菌BL21中的表达

Expression of Carcinus maenas Metallothionein Gene in Escherichia Coli BL21

目的 :在大肠杆菌BL2 1中表达岸蟹金属硫蛋白基因 ,建立金属硫蛋白原核表达的流程。方法 :将重组质粒pET GST R转化至大肠杆菌 ,经PCR扩增和酶切检测 ,得到含重组质粒pET GST R的工程菌。结果 :从IPTG浓度、IPTG诱导时间、金属离子浓度等几个方面摸索诱导金属硫蛋白表达的条件 ,表达出分子量约为 33kD的融合蛋白。结论 :成功制备金属硫蛋白的工程菌菌株。金属硫蛋白对大肠杆菌细胞毒性很大 ,培养基需添加金属离子。

Objective: To express Carcinus maenas Metallothionein gene in Escherichia Coli BL21 and establish the procedure of expressing Metallothionein gene in prokaryote. Methods: Transform the recombined plasmid pET-GST-R into Escherichia Coli BL21 and use PCR and restriction enzyme digestion to confirm the positive plasmid. Results:The expression condition was studied through the concentration of IPTG,the induction time of IPTG and metallic ion concentration. A fusion protein with molecular weight about 33 kD was obtained. Conclusion:The recombinant MT expression vector was successfully constructed. The metallothionein was harmful to Escherichia Coli. It is necessary to add metallic ion into the culture medium.