ATF-PAI2CD融合蛋白基因在毕赤酵母中克隆、表达和鉴定

Cloning, Expression and Characterization of Human ATF-PAI2CD Fused Gene in Pichia pastoris

为了克隆尿激酶型纤溶酶原激活物 (uPA)的氨基末端片段与纤溶酶原激活剂抑制物 2型(PAI 2 )突变体所构成的融合蛋白基因 ,并在Pichiapastoris中表达 ,应用PCR获得了人ATF PAI2CD融合蛋白基因cDNA(简称ATF PAI2CD) ,将其克隆到酵母表达载体pPIC9K ,获得融合基因表达质粒pZWY ATF PAI2CD .该质粒转化毕赤酵母菌GS115 ,用G4 18 YPD平板筛选高拷贝转化子 ,然后用甲醇诱导表达 .工程菌用摇瓶发酵 ,表达产物ATF PAI2CD占培养液中总蛋白 5 0 %以上 .经硫酸铵沉淀、分子筛和离子交换层析纯化得到的目标表达产物纯度达 95 % .Western印迹检测具有PAI 2与uPA的免疫原性 ,经牛奶板法检测具有纤溶抑制活性 .经流式细胞仪 (FCM )检测 ,能与肿瘤细胞特异性结合 .结果表明 ,ATF PAI2CD融合蛋白成功地在毕赤酵母中表达 ,且具有抑制uPA及与肿瘤细胞表面uPAR特异性结合的双重功能 .提示该融合蛋白可能具有良好的应用前景 .

In order to construct expression vector of ATF-PAI2CD and express ATF-PAI2CD in Pichia pastoris, the amino-terminal fragment (ATF) of human uPA, specifically binding to uPAR, was fused with PAI-2CD, mutants of human plasminogen activator inhibitor 2, through a flexible linker by recombinant DNA techniques. The recombinant plasmid containing ATF-PAI2CD fused gene, pZWY-ATF-PAI2CD, was transferred to the competent cells of yeast strain GS115. After the phenotype selection, the positive clones were induced with methanol to express ATF-PAI2CD. The expression product was purified by the protocols including precipitation by ammonium sulfate, Sephadex G-75 gel filtration, Q-Sepharose ion-exchange chromatography. The PAI activity was measured by chromogenic assay. The fusion protein could inhibit urokinase-type plasminogen activator measured by milk agarose plate assay and be able to bind to the surface of uPAR-bearing cells by flow cytometry. The results show that the fusion protein ATF-PAI2CD may be promising in application.