人血管内皮抑制素在毕赤酵母中的表达纯化及活性测定

血管内皮抑制素是胶原ⅩⅧ C-末端的一个片段，是一个有效的血管生成抑制因子。本文克隆了人血管内皮抑制素的基因，并用毕赤酵母进行表达，表达量为50.5 mg/L。发酵液上清经SP Sepharose Fast Flow凝胶一步层析，产物纯度达到98%以上，纯化收率达到95%以上。毕赤酵母分泌表达的人血管内皮抑制素具有免疫活性；能明显抑制鸡胚尿囊膜新生血管的生长；并且能特异性地抑制bFGF刺激的人微血管内皮细的迁移，达到抑制效果为50%时所需的蛋白浓度（IC50）为0.4μg/ml。本研究为应用人血管内皮抑制素治疗肿瘤奠定了初步实验基础。

Expression , Purification of Human Endostatin in Pichia pastoris and the Detection of Its Anti-angiogenic Activity

Endostatin is a potent, endogenous inhibitor of angiogenesis, which corresponds to the C-terminal fragment of collagen ⅩⅧ. The human endostatin gene was amplified from a human fetal liver cDNA library by means of PCR and was cloned into pPIC9K vector. Soluble endostatin was superexpressed in Pichia pastoris (50.5mg/L) . The recombinant protein was purified by cation exchange chromatography with the final yield of more than 95%. Western blotting analysis showed positive immunoreactivity to the expressed product. Recombinant endostatin was able to suppress the angiogenesis on the CAMs. Also, the paper showed that endostatin inhibited specifically of the migration of HMECs stimulated by bFGF, with EC50 being about 0.4 μg/ml.