毕赤氏酵母/hGM-CSF工程菌的构建及表达分析  

Construction of Recombinant Pichia Pastoris Containing hGM-CSF Gene and Assay of It' s Secretory Expression Proteins

作　　者：杨国峰[1,2] 周鹏[1] 张泰芳[1] 

机构地区：[1]中国热带农业科学院热带作物生物技术国家重点实验室,热带生物技术所,海南海口571 1O1 [2]海南师范学院生物系,海南海口571158

出　　处：《药物生物技术》2003年第6期349-352,共4页Pharmaceutical Biotechnology

摘　　要：为了探索利用毕赤氏酵母系统表达和生产人粒细胞-巨噬细胞集落刺激因子(human granulocyte-macrophage colony-stimulating factor,hGM-CSF)的可行性,使用经改造的hGM-CSF基因构建pGAPZa-A/hGM-CSF分泌型表达载体,转入毕赤氏酵母SMD1168(his 4,pep 4)菌株和GS115(his 4)菌株,利用斑点杂交技术筛选hGM-CSF基因阳性菌株,利用SDS-PAGE和TF-1依赖细胞株/MTT比色法对工程菌株的分泌产物进行表达和生物活性分析,最后筛选得到7个hGM-CSF基因阳性菌株,其中有3个工程菌株的杂蛋白较少且主带蛋白质相对分子质量与经改造的hGM-CSF基因相对分子质量保持一致,这3个工程菌株的分泌蛋白质样品都具有天然hGM-CSF的生物活性,经初步提取,活性单位分别为2.28×105U/ml、2.22×105U/ml和2.46 × 105U/ml。从而证明利用毕赤氏酵母系统表达hGM-CSF是可行的,为hGM-CSF的生产和临床应用打下了基础。To study the feasibility of expression and production of hGM-CSF in recombinant Pichia pastoris yeast, the recombinant pGAPZa-A vector on which the gene encoding human granulocyte-macrophage colony-stimulating factor (hGM-CSF) was cloned to a downstream site of promoter GAP, was transfered into SMD1168 strain (his4, pep4) and GS115 strain (his4) of Pichia pastoris. Recombinant strains were identified by DNA dot blot. SDS-PAGE and MTT methods were used in assaying the secretory expression products. At last, 7 recombinant Pichia pastoris strains containing hGM-CSF gene were identified,3 strains of which were able to express a protein whose relative molecular mass was corresponding to hGM-CSF. Secretory proteins from 3 trains were as activity as natural hGM-CSF, which were 2.28 × 105 U/ml, 2.22 × 105 U/ml and 2.46 × 105 U/ml respectively. These data demonstrated the feasibility of expression and production of recombinant hGM-CSF in Pichia pastoris .

关 键 词：HGM-CSF 毕赤氏酵母 载体构建 表达分析 

分 类 号：Q786[生物学—分子生物学]