蛇毒类凝血酶基因在大肠杆菌中的融合表达及纯化  

Fusion Expression and Purification of Gloshedobin, a Thrombin-Like Enzyme from Gloydius Shedaoensis in E. coli

作　　者：李民[1] 杨青[1] 包永明[1] 雷旭宇[1] 许建强[1] 安利佳[1] 

机构地区：[1]大连理工大学生物工程系,辽宁大连116024

出　　处：《无锡轻工大学学报（食品与生物技术）》2003年第2期22-25,共4页Journal of Wuxi University of Light Industry

基　　金：辽宁省科技基金项目(No.99305001)资助课题.

摘　　要：将大连蛇岛蝮蛇毒类凝血酶(Gloshedobin)基因克隆到载体pET32-a(+)上,在T7lac启动子下以N端融合硫氧还蛋白的形式在大肠杆菌BL21(DE3)中进行表达,诱导条件为:30℃诱导6 h,IPTG浓度为1 mmol/L.利用固定化金属离子(Ni2+)亲和层析分别对胞内可溶物和变性条件下的包涵体进行纯化.通过SDS-PAGE检测融合蛋白的纯度,采用点杂交和纤维蛋白凝结活性检测分析,显示纯化产物具有较高生物活性.The gene of snake venom thrombin-like enzyme from Gloydius shedaoensis, Gloshedobin, was cloned into expression vector pET32-a( + ) . The enzyme fused with thioredoxin as its N-terminal part was expressed in E . coli BL21(DE3) under the control of T7 lac promoter at 30 ℃ with 1 mmol/ L IPTG for 6 hours. Recombinant proteins which distributed among soluble and insoluble cellular frac-tions were purified by Immobilized Metai Affinity Chromatography(IMAC) individually. SDS-PAGE was used to detect the purity of target protein. A higher bioactivity of purified products was shown by the determination of dot-blotting assay and fibrinogen-clotting analysis.

关 键 词：类凝血酶 固定化金属亲和层折 融合表达 大肠杆菌 

分 类 号：Q78[生物学—分子生物学]