摘要
目的 研究重组AT Ⅲ基因在巴斯德毕赤酵母菌中进行表达。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 ,用SDS PAGE及WesternBlot检测表达产物的纯度及特异性 ;用生化法检测AT Ⅲ的表达量 ;用凝血酶空斑法检测表达产物活性。结果 表达产物能与抗AT Ⅲ单克隆抗体结合 ,并具有天然AT Ⅲ的生物活性。结论 已在毕赤酵母菌中成功地表达了AT Ⅲ。
Objective To express human antithrombin Ⅲ(AT-Ⅲ) in P.pastoris.Methods Transfect the eukaryotic expression vector incorporated with AT-Ⅲ gene into P.pastoris GS115 by electroporation, select the recombinants by growth in G418 medium and identify the expressed protein by SDS-PAGE and Western blot.Determine the expression level and activity of AT-Ⅲ by biochemical method and thrombin plaque formation test respectively.Results The expressed protein bound to McAb against AT-Ⅲ and showed biological activity of natural AT-Ⅲ.Conclusion AT-Ⅲ was successfully expressed in P.pastoris.
出处
《中国生物制品学杂志》
CAS
CSCD
2004年第5期277-279,共3页
Chinese Journal of Biologicals
