人组织因子在毕赤酵母中的表达和纯化

 目的 制备具有凝血活性的重组人组织因子（recombinant tissue factor, r-TF） 用于构建 PT 试剂盒。 方法 将人组织因子胞外区编码基因与酵母表达载体重组，构建酵母表达质粒 pPIC9K-TF，利用电穿孔法转化宿主菌 Pichia pastoris GS115，转化子经 G418 抗性筛选后，获得高表达克隆。工程菌在摇瓶中经甲醇诱导，表达 r-TF，表达产物经纯化后，鉴定生物活性。 结果 SDS-PAGE 证实表达产物的分子量为 37000-45000,Western-Blot证明其为人组织因子衍生物，纯化后，r-TF 经脂化后，具有促凝血活性，为研制凝血活酶时间测定试剂盒及研究 TF 的构效关系创造条件。 结论 用毕赤酵母高效表达具有活性的rTF，表达量达到182mg/L。纯化工艺简便易行，蛋白回收率高，纯化后的rTF可用于组装PT测定试剂盒。

Expression and purification of the recombinant human tissue factor in Pichia pastoris

Objective To obtain recombinant human extracellular tissue factor(r-TF) which can be used to set up PT kit. Methods Expression plasmid,pPIC9K-TF,was constructed by inserting the sequence encoding human extracellular tissue factor into yeast expression vector pPIC9K and transformed into Pichia pastoris GS115 with electroporation.Having been selected by G418,transformants containing TF cDNA were induced by methanol for the expression of rTF,purificated and anlaysis activity. Results SDS-PAGE showed that the molecular weight of the expression product was about 37 000-45 000.Western-blotting indicated that it was human extracellular tissue factor.After phorspholipids treatment,purified rTF was able to initiate blood coagulation. Conclusions rTF gene is expressed in Pichia pastoris and the active products are secreted into the medium with concentration up to 182 mg/L.The recombinant protein is purified with a simple process and high rate of protein recovery,the purified protein can be used in PT test.