大连蛇岛蝮蛇类凝血酶在大肠杆菌中的表达与纯化  

Expression and Purification of a Snake Venom Thrombin-like Enzyme,Gloshedobin,in Escherichia coli

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

机构地区：[1]大连理工大学环境与生命学院生物科学与工程系,大连116023

出　　处：《中国生物工程杂志》2004年第9期44-48,共5页China Biotechnology

基　　金：国家"8 63"计划资助项目 ( 2 0 0 3AA2Z3 5 2 0)

摘　　要：将编码大连蛇岛蝮蛇类凝血酶 (Gloshedobin)的基因克隆于表达载体pET 32a( + )中 ,以融合蛋白形式在大肠杆菌中获得表达。在 2 5℃下经 1mmol LIPTG诱导 6h ,SDS PAGE和蛋白质印迹分析表明 ,部分融合蛋白以可溶形式存在于大肠杆菌的细胞质中。针对金属螯合亲和层析分离某些含His 标签重组蛋白质时专一性不高 ,并且存在配基泄漏的缺陷 ,设计合成了以抗重组类凝血酶的鸡卵黄免疫球蛋白为配基的免疫亲和层析柱。通过疏水色谱OctylSepharoseFF ,IgY免疫亲和层析以及强阴离子交换色谱SourceQ等三步柱色谱分离纯化获得比活力为 45 4 7U mg的重组蛇毒类凝血酶 ,活力回收率为 34 8%。蛋白质印迹分析和纤维蛋白原凝结活性分析表明 。The mature gene of gloshedobin,a snake venom thrombin-like enzyme from the snake,Gloydius shedaoensis,was cloned into expression vector pET-32a(+)and expressed as a fusion protein trx-his-TLE in E.coli.After induction by 1mmol/L IPTG for 5h at 25℃,both SDS-PAGE analysis and western blotting assay showed partial expressed products were found in soluble fractions in the cytoplasm of E.coli.Egg yolk antibody (IgY) of gloshedobin was synthesized as immuno-affinity ligand and coupled to the gel,Sepharose 4B-CNBr activated to prepare immuno-affinity adsorbents.The purification of the soluble fraction was thus performed by a protocol involving Octyl Sepharose FF,IgY-immunoaffinity chromatography and Source Q,resulting in a protein of 454.7U/mg specific activity and 34.8% activity yeild.Both immunological and enzymatic activity of the purified enzyme were observed by western-blotting analysis and fibrinogen-clotting assay,respectively.;

关 键 词：蝮蛇 类凝血酶 大肠杆菌 融合蛋白 蛋白质印迹 配基 ET 蛇类 蛇岛 重组蛋白质 

分 类 号：Q959[生物学—动物学] Q78