内皮细胞抑制素在毕节酵母中的表达与活性分析  

Expression of Human Endostatin in P.pastoris and the Bioactivity of Its Product

作　　者：吴江雪[1] 符武钊[1] 张添元[1] 罗进贤[1] 吴群悦[1] 

机构地区：[1]中山大学基因工程教育部重点实验室生物化学系,广东广州510275

出　　处：《中山大学学报（自然科学版）》2003年第6期83-86,共4页Acta Scientiarum Naturalium Universitatis Sunyatseni

基　　金：广东省重点科技攻关资助项目(2KMO2502G)

摘　　要：采用PCR技术从人胎脑cDNA库中扩增了人内皮细胞抑制素基因。经DNA序列分析后将扩增的基因克隆至酵母载体pPIC9K,获得的重组质粒pPIC9K＿EDN转化毕节酵母GS115,构建表达内皮细胞抑制素的酵母工程菌P.pastorisGS115(pPIC9K＿EDN)。SDS＿PAGE分析结果显示:人内皮细胞抑制素在重组酵母GS115(pPIC9K＿EDN)中获得高效表达。用30L发酵罐构对建的工程菌进行高密度发酵,经甲醇诱导48h,生物量达到250OD,分泌量为150mg L,发酵液经SPStreamline,SPSepharoseFF阳离子交换柱和SepharoseHeparinHiTrap柱纯化,产物纯度达到98%。纯化产物具有免疫活性并能抑制bFGF诱导的鸡胚绒毛尿囊膜血管生成。Human endostatin gene was amplified from human fetal brain cDNA library by PCR technique and cloned into E.coli-yeast vector pPIC9K. The resultant plasmid pPIC9K-EDN was transformed into P.pastoris GS115. SDS-PAGE analysis indicated that endostatin was expressed in yeast engineered strain GS115(pPIC9K-EDN). The high cell density culture of GS115(pPIC9K-EDN) was performed in a 30 liter bioreactor. After 48 h methanol induction, the cell density reaches A_(600)=250 and secretion yield is 150 mg/L. The purity of the expressed product after puritication with SP Streamline, SP Sepharose FF cation exchanger chromatographies and Heparin Sepharose affinity charomatography reached 98%. The purified product possessed immunoactivity and could inhibit CAM angiogenesis induced by bFGF.

关 键 词：人内皮细胞抑制素 基因表达 毕节酵母 高密度发酵 纯化 生物活性分析 肿瘤 

分 类 号：R730.1[医药卫生—肿瘤]