重组HSA-hG-CSF融合蛋白在毕赤酵母中的表达

为了延长G-CSF半衰期，我们利用甲醇酵母表达重组人血清白蛋白融合的集落细胞刺激因子（rHSA-G-CSF）。用PCR方法从人胎肝cDNA文库扩增出HSA cDNA序列，hG-CSFcDNA序列从大肠表达载体中酶切获取。将HSA和hG-CSF两片段连接后，克隆到酵母分泌型表达载体pGENYK中，酶切线性化后原生质体转化导入酵母细胞进行整合。工程菌经发酵灌培养表达，层析法分离纯化融合蛋白。纯化的融合蛋白经Western 印迹分析表明具有HSA和G-CSF的免役原性，体外生物学活性分析表明，同縻尔数的融合表达产物的活性为E.coli表达G-CSF单体的活性的50%以上。体内动物实验研究表明，经HSA融合的G-CSF的半衰期为G-CSF单体的15-20倍。甲醇酵母表达的融合HSA的G-CSF具有比G-CSF更长的半衰期，有良好的临床应用前景。

Expression of the Recombinant HSA-G-CSF in Pichia pastoris

The complete gene of HSA-G-CSF was obtained by follow protocol: the HSA gene was obtained from the library of cDNA by PCR, and the gene of G-CSF was obtained from vector we constructed before. After ligation of the two genes, the complete gene was cloned to recombinant plasmid of pGENYD and then the linearized vector was transfected into Pichia pastoris. The target protein secreted by the recombinant Pichia pastoris was induced by methanol. The target protein showed single band in the SDS-PAGE. Western Blot showed the HSA-G-CSF combining activity both to HSA and G-CSF. The in intro activity showed that the activity of HSA-G-CSF is more than half of the G-CSF by the same molar ratio. And the animal study indicated the HSA-G-CSF have 15-20 multiple half-life in vivo than G-CSF.