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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。
OBJECTIVE: To express and purify AT Ⅲ in Pichia pastoris to further study its function. Methods: The transfected GS115 cells were transfected with the expression vector of AT Ⅲ gene. The resistant clones were screened by G4 18, and the expressed products were detected by SDS PAGE and Western blot. The expression of AT Ⅲ was determined by biochemical method. The expressed products were assayed for activity and purified by Heparin hyperD column. RESULTS: Western blot analysis revealed that the expressed product transferred to the membrane bound to anti-AT III monoclonal antibody, demonstrating the successful expression of AT III with> 95% purity. The activity assay showed that the expressed product had the biological activity of native AT Ⅲ. CONCLUSION: AT Ⅲ is successfully expressed and purified in Pichia pastoris and the specific activity of human AT Ⅲ is obtained.