【摘要】 为了延长G-CSF半衰期,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HSAcDNA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。工程菌经发酵罐培养表达,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的免疫原性,体外生物学活性分析表明,同等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。更多还原

【Abstract】 To enhance pharmacokinetics of GCSF, a chimeric gene encoding human serum albumin (HSA)human GCSF fusion protein was overexpressed in Pichia pastoris. the HSA sequence was obtained from the library of cDNA by PCR, and the sequence of GCSF was obtained from vector constructed before. After ligation of the two sequences, the complete chimeric gene was cloned to recombinant plasmid of pGENYD and then the linearized vector was transfected into Pichia pastoris. After fermentation in bioreactor , the fusion protein was purified from the culture of the recombinant yeast by a threesteps chromatography methods. Western blot showed the purified HSAGCSF protein combining activity both to HSA and GCSF. The in vitro 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. The enhanced pharmacokinetics of GCSF fused to human serum albumin suggest its promissing application in clinic medicine.更多还原