人抗凝血酶Ⅲ(AT-Ⅲ)在巴斯德毕赤酵母中表达与纯化  

Expression and purification of recombinant human antithrombin Ⅲ(AT-Ⅲ) in Pichia pastoris

作　　者：于群[1] 贺敏[1] 宋丽雅[1] 卜凤荣[1] 王全立[1] 

机构地区：[1]军事医学科学院野战输血研究所,北京100850

出　　处：《军事医学科学院院刊》2004年第4期350-353,共4页Bulletin of the Academy of Military Medical Sciences

摘　　要：目的 :在巴斯德毕赤酵母中进行表达及纯化AT Ⅲ ,以对其功能进行深入研究。方法 :将携带AT Ⅲ基因的表达载体电转染GS115细胞 ,用G4 18筛选抗性克隆 ,SDS PAGE与Western印迹检测及鉴定表达产物 ;采用生化法测定AT Ⅲ的表达量 ;利用凝血酶空斑法测定表达产物活性 ,经过Heparin hyperD柱纯化。结果 :Western印迹分析发现转移至膜上的表达产物能与抗AT Ⅲ单克隆抗体结合 ,证明成功地表达了AT Ⅲ ,纯度 >95 %。活性测定表明表达产物具有天然AT Ⅲ的生物活性。结论 :在巴斯德毕赤酵母中成功地表达与纯化AT Ⅲ ,得到同天然蛋白相似的比活性人AT Ⅲ。Objectives: To express and purify human antithrombin Ⅲ in yeast Pichia pastoris and to evaluate its functions. Methods: The eukaryotic expression vector incorporated with AT-Ⅲ gene was transformed into P.pastoris GS115 by electroporation. The transformants were selected by growth on G418 and detected for AT-Ⅲ expression by SDS-PAGE and Western blot.The AT-Ⅲ secretion levels were estimated by biochemical methods, AT-Ⅲ biological activity was assayed by a functional assay respectively.The purified protein was obtained by chromatography: Heparin-hyperD. Results: The Western blot analysis showed that the recombinant products could bind to McAb. The recombinant AT-Ⅲ had natural AT-Ⅲ biological activity. Conclusion: The recombinant human antithrombin Ⅲ is successfully expressed and purified in P.pastoris.

关 键 词：抗凝血酶Ⅲ 毕赤酵母 基因表达 

分 类 号：R346[医药卫生—基础医学]