作　　者： ; ;

机构地区： 北京大学生命科学学院蛋白质工程及植物基因工程国家重点实验室

出　　处： 《北京大学学报（自然科学版）》 2005年第4期506-513,共8页

摘　　要： 人工合成山蛭素基因后,利用基因重组的方法将山蛭素的基因克隆到pPicZαA载体,经过线性化,电转至毕赤酵母GS115中表达。使用浓度高达1500μgmL的zeocin筛选得到高拷贝插入的GS115H菌株,经过优化摇瓶表达条件表达量达到100mgL。摇瓶培养物经离心取上清,分别使用3kD和10kD的超滤膜超滤,阳离子交换层析和凝胶过滤层析等纯化步骤之后,得到纯度高于95%的目的蛋白,产出量为40mgL,回收率为40%。通过以chromozymTH为底物的凝血酶酰胺水解实验证实了其体外生物学活性:其抑制凝血酶常数为(2.46±0.13)×10-13molL。 The gene of haemadin is synthesized and cloned into vector of pPicZαA. The recombinant plasmid is then linearized with Sac I and transformed into pichia pastoris GS115. GS115 strain with high copy inserts is obtained by 1500 mg/L zeocin selection, which, in the optimized condition, express as high as 100 mg/L target protein in flask fermentation. The culture supernatant is collected by centrifuge and then purificated by combination of ultrafiltration, cation exchange chromatography and gel filtration chromatography. Haemadin with above 95% purity is obtained. The yield per liter culture of purified haemadin is 40 mg and overoll recovery yield is 40%. Amidolytic assay of thrombin approved that the recombinant haemadin does have the antithrombin activity. Its inhibition constant (Ki) is (2.46 ± 0.13) × 10-13 mol/L.

关 键 词： 山蛭素 凝血酶抑制剂 毕赤酵母表达 抗凝剂

领　　域： [生物学]