【摘要】 构建重组人血清白蛋白粒细胞集落刺激因子(HSA-hG-CSF)表达载体,用毕赤酵母表达该重组蛋白。PCR扩增出人血清白蛋白基因(HSA)和粒细胞集落刺激因子基因(hG-CSF),GGGGS作为小肽接头,采用重叠PCR的方法将HSA和hG-CSF拼接起来,与质粒载体pPIC9K连接,转化大肠杆菌感受态细胞DH-5α。抽提质粒,用SalI酶切重组质粒,电转化法导入毕赤酵母SMD1168中,通过表型筛选和诱导表达实验得到蛋白表达工程菌。Western-blotting分析表明融合蛋白具有粒细胞集落刺激因子免疫原性。NFS-60细胞测活实验分析表明体外活性达到约4.0×107IU/mg。更多还原

【Abstract】 Human serum albumin and granulocyte colony-stimulating factor(HSA-hG-CSF)were recombined and expressed in Pichia pastoris.DNA of HSA and hG-CSF was amplified by PCR with GGGGS as connector.Overlap PCR was used to combine HSA with hG-CSF by and ligate with pPIC9K,then transform into competent cell of Escherichia coli named DH-5α.Extracted plasmid was digested by SalI.The recombinated vector was transformed into Pichia pastoris SMD1168 by electroporation.By phenotype selection and inducing assay,the expression engineering strain was obtained.Western blotting analyses showed the expression productions had immunogenicity of hG-CSF.It indicated that the activity of expression productions was 4.0 107IU/mg by NFS-60 cell examines.更多还原