重组双功能水蛭素的发酵、纯化和鉴定

Fermentation, Purification and Identification of Recombinant RGD-Hirudin

构建了重组双功能水蛭素 (Recombinant RGD Hirudin、r RGD Hirudin )cDNA的表达质粒RGD Hirudin pPIC9K ,转化入毕赤酵母中 ,经筛选得到高表达的阳性克隆。种子菌经过 3d发酵培养 ,其培养液上清经超滤浓缩、凝胶过滤层析和离子交换层析后 ,得到纯度大于 97%、比活性为 12 0 0 0ATU mg的r RGD Hirudin ,回收率大于 6 0 % ,发酵产率为 1g L。纯化后的r RGD Hirudin经过还原SDS PAGE ,抗凝血酶活力分析、抗血小板聚集分析、质谱分析及等电聚焦分析等方法鉴定 ,证明该表达产物为水蛭素的衍生物 ,具有抗凝血酶和抗血小板聚集双重功能。

Recombinant RGD-Hirudin ( r-RGD-Hirudin ) has double functions: anti-thrombin activity and anti-platelet aggregation activity. To identify these functions, the expression plasmid, RGD-Hirudin-pPIC9K, was constructed by inserting cDNA of RGD-hirudin in yeast expression vector pPIC9K. The high expression clone was gained after screening. This clone was fermented for 3 days. The r-RGD-hirudin was secreted into the culture. It was ultra-filtrated from culture supernatant, then after gel filtration chromatography and anion exchange chromatography, the purified r-RGD-hirudin was gained. Its purity was larger than 97% and its specific activity was 12 000 ATU/mg. The yield per liter culture of purified r-RGD-hirudin was 1 g and overall recovery yield was more than 75%. The purified r-RGD-hirudin was identified by reductive SDS-PAGE, anti-thrombin activity assay, anti-platelet aggregation assay, LC/MS and isoelectrofocusing assay. It is proved that r-RGD-Hirudin is ramification of wt-Hirudin and it has anti-thrombin activity and anti-platelet aggregation activity .