人白血病抑制因子基因在巴斯德毕赤酵母中的表达  

EXPRESSION OF HUMAN LEUKEMIA INHIBITORY FACTOR GENE IN PICHIA PASTORIS

作　　者：王全忠[1] 杨林[1] 欧阳菁[1] 龙綮新[1] 王珣章[1] 

机构地区：[1]中山大学生物医药中心

出　　处：《微生物学报》2001年第5期582-586,共5页Acta Microbiologica Sinica

摘　　要：将 575bp的人白血病抑制因子基因克隆到表达载体pPICZαA上 ,构建成重组质粒pPICZαA hLIF。pPICZαA hLIF经SacI酶切使之线性化后转化到巴斯德毕赤酵母细胞X 33中。转化子经Mut表型筛选和PCR分析鉴定后 ,利用甘油增菌和甲醇诱导 ,实现了hLIF基因在毕赤酵母系统中的表达。SDS PAGE检测和Westernblot分析结果表明表达产物的分子量约为58 5kD ,与天然hLIF大小相近 ,并且具有免疫原性。凝胶薄层扫描分析显示 ,重组hLIF约占上清总蛋白的 32 8%。生物活性测定结果表明 。The 575bp artificial synthesized human leukemia inhibitory factor (hLIF) gene was cloned into vector pPICZαΑ and the constructed expression vector pPICZαA\|hLIF was linearized by Sac I and transformed into Pichia pastoris X\|33.After the screening for Mut phenotype and the PCR analysis of the transformants,hLIF gene expressed in Pichia pastoris. SDS\|PAGE and Western blot analysis of the culture supernatants showed that hLIF gene could express as 58.5kD protein with the desired immunogenicity.Density scanning of the SDS\|PAGE gel revealed that the targeted protein accounted for 32.8% of the total protein in the supernatants.The expressed products can inhibit the clone formation of murine's teratoma cells.

关 键 词：人白血病抑制因子 巴斯德毕赤酵母 基因表达 生物活性测定 

分 类 号：Q786[生物学—分子生物学]