人血管抑制素在酿酒酵母中的分泌表达和活性鉴定

将酵母交配因子 (MF)α1信号肽编码序列和人血管抑制素 (hAGN)cDNA融合序列插入穿梭载体pYADE4 ,构建得到分泌型重组表达质粒pYADEMA18.转化酿酒酵母JG110 7后 ,用 2 %乙醇和2 %甘油联合诱导表达 .ELISA分析表明 ,在诱导 14h～ 30h期间 ,hAGN获得了表达并分泌至细胞外 .发酵上清液经 75 %饱和度硫酸铵沉淀、CM 5 2纤维素离子交换层析和Superdex 75凝胶过滤层析纯化 .SDS PAGE分析显示 ,表达产物重组人血管抑制素 (rhAGN)相对分子质量约 5 0kD ,电泳纯度达到 94 %.生物活性分析证明 ,rhAGN在 0 0 1mg L～ 3 0mg L浓度范围内能够抑制人真皮内皮细胞株HDMEC增殖 ,抑制作用随剂量的增加而增强 .

Secretive Expression, Purification and Bioactivity Assay of Human Angiostatin in S. cerevisiae

The human angiostatin cDNA and yeast mating factor α1(MFα1) signal sequence were cloned into an E. coli/S.cerevisiae shuttle vector pYADE4 resulting in recombinant expression plasmid pYADEMA18 which was then transformed into S. cerevisiae JG1107. By combined induction with 2% ethanol and 2% glycerol, ELISA analysis indicated that human angiostatin(hAGN) was expressed and secreted into the culture medium. After precipitation with 75% saturation ammonium sulfate and purification with CM-52 cellulose and Superdex-75 chromatography, SDS-PAGE analysis showed that the recombinant hAGN (rhAGN) was about 50 kD, and its purity achieved 94%. Among the concentration range of 0.01 mg/L～3.0 mg/L, rhAGN was capable of inhibiting the proliferation of human dermal endothelial cell (HDMEC)line in a dose-dependent manner.