人粒细胞-巨噬细胞集落刺激因子的克隆及在毕赤酵母中的表达  

Cloning and Expression in Pichia pastoris of Human Granulocyte-macrophage Colony Stimulating Factor

作　　者：李俏俏[1] 王清路[1] 张玉军[1] 张锐[2] 徐寿增[1] 

机构地区：[1]山东万杰医学院,淄博255213 [2]中国农业科学院生物技术研究所,北京100081

出　　处：《中国生物工程杂志》2010年第1期35-40,共6页China Biotechnology

基　　金：山东省教育厅科研项目(J06L77)资助项目

摘　　要：采用加长引物5'端的方法克隆了hGM-CSF的编码基因,克隆过程中对该基因的局部做了密码子优化。然后克隆进毕赤酵母分泌型表达载体pPIC9K,电击转化毕赤酵母。PCR、SDS-PAGE与Western blotting证实hGM-CSF已整合进酵母基因组,摇瓶水平粗蛋白表达量达389mg/L,动物实验证实蛋白产物活性正常,SDS-PAGE与N-糖苷酶F去糖基化分析发现hGM-CSF被糖基化。Code gene being cloned from the human genome by the primer-5＇-extension,as the same time that code had been optimized partly to gene in the cloned procession.It was integrated into the gene secreted Pichia pastoris expression vector pPIC9K and translated yeast Pichia pastoris.PCR,SDS-PAGE and Western blotting confirmed hGM-CSF gene was integrated into the yeast genome and expression was successful.The expressed hGM-CSF was secreted into medium at the level of 389mg/L.Experiments in vivo showed that activity of hGM-CSF was normal.SDS-PAGE electrophoresis revealed that the hGM-CSF expression had occurred in glycosylation,N-glycosidase F to glycosylation also confirmed this point.

关 键 词：粒细胞-巨噬细胞集落刺激因子 毕赤酵母 引物5'端加长法 糖基化 

分 类 号：Q78[生物学—分子生物学]