人组织型纤溶酶原激活剂-水蛭素融合基因的构建及其在毕赤酵母中的表达

构建并表达兼有溶栓和抗凝活性、减少出血副作用的人组织型纤溶酶原激活剂(t-PA)和水蛭素(HV2)的融合蛋白。通过提取总RNA和RT-PCR获得t-PA基因,与HV2基因通过活化凝血因子X(Fxa)识别序列（IEGR）的对应碱基序列连接构成融合蛋白基因，融合蛋白基因经pGEM-T、pIC9克隆至表达载体pIC9K上，电转导入毕赤酵母（Pichia pastoris）GS115。转化子摇瓶内甲醇诱导表达。纤维蛋白平板溶圈法和纤维蛋白凝块法分别检测溶栓和抗凝活性。琼脂糖凝胶电泳结果显示克隆的t-PA基因片段大小为1700bp，序列测定结果表明其35位氨基酸由文献报道的精氨酸突变为色氨酸。限制性酶切和PCR鉴定结果均表明融合蛋白基因已克隆入表达载体和宿主菌。甲醇利用实验、G418抗性筛选获得多拷贝甲醇利用快型克隆。甲醇诱导表达产物具有纤溶活性并可被抗t-PA抗体抑制。完整融合蛋白无抗凝活性，但以Fxa裂解后可释放抗凝活性。同时，融合蛋白以单链和双链两种形式存在。融合蛋白在血栓部位特有的Fxa作用下靶向释放抗凝活性，具有溶栓抗凝双功能，有望降低临床出血副作用。

Construction and Expression of a Fusion Protein Made of Tissue-type Plasminogen Activator and Hirudin in Pichia pastoris

To combine the fibrinolytic with anticoagulant activities for therapy of thrombotic deseases, a fusion protein made of tissue-type plasminogen activator (t-PA) and hirudin was constructed and expressed in chia pastoris. To improve thrombolytic properties of t-PA and reduce bleeding side effect of hirudin, FXa-recognition sequence was introduced between t-PA and hirudin molecules.The anticoagulant activity of hirudin can be target-released through cleavage of FXa at thrombus site. t-PA gene and hirudin gene with FXa-recognition sequence at its 5'-terminal were obtained by RT-PCR and PCR respectively. The fusion protein gene was cloned into plasmid pIC9K and electroporated into the genome of Pichia pastoris GS115. The expression of fusion protein was induced by methanol in shaking flask and secreted into the culture medium. Two forms of the fusion protein, single-chain and double-chain linked by a disulfide bond (due to the cleveage of t-PA at Arg275-Ile276), were obtained. The intact fusion protein retained the fibrinolytic activity but lacked any anticoagulant activity. After cleavage by FXa, the fusion protein liberated intact free hirudin to exert its anticoagulant activity. So, the fusion protein is a bifunctional molecule having good prospect to develop into a new targeted therapeutic agent with reduced bleeding side effect for thrombotic diseases.