内皮细胞抑制素在毕节酵母中的表达与活性分析

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pPICgK，获得的重组质粒pPIC9K-EDN转化毕节酵母GSll5，构建表达内皮细胞抑制素的酵母工程菌P.pastoris GSll5(pPIC9K-EDN)。SDS-PAGE分析结果显示：人内皮细胞抑制素在重组酵母GSll5(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SP Streamline，SP Sepharose FF阳离子交换柱和SephamseHeparin Hi Trap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。

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

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.