Abstract: The gene of haemadin is synthesized and cloned into vector of pPicZαA. The recombinant plasmid is then linearized with SacⅠ 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 (K_i) is (2.46±0.13)×10_-13 mol/L.

Key words: thrombin inhibitor, expression in methylotrophic yeast pichia pastoris, anticoagulant

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1500 μg/mL的zeocin筛选得到高拷贝插入的GS115-H菌株，经过优化摇瓶表达条件表达量达到100 mg/L。摇瓶培养物经离心取上清，分别使用3 kD和10 kD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40 mg/L，回收率为40%。通过以chromozym TH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^-13 mol/L。

关键词: 山蛭素, 凝血酶抑制剂, 毕赤酵母表达, 抗凝剂