内皮细胞抑制素酵母工程菌的高密度发酵及产物纯化和活性分析

High Density Culture of P. pastoris GS115(pPIC9K-EDN)Habouring Endostatin Gene and Purification and Bioactivity Analysis of Product

用 30升发酵罐研究了内皮细胞抑制素 (EDN)酵母工程菌P .pastorisGS115 (pPIC9K EDN)的高密度发酵工艺 ,摸索出发酵培养基 ,pH ,诱导时间等对菌体生长和基因表达的影响 .根据所确定的最适条件进行发酵 ,通过补料和甲醇诱导 4 8h后 ,工程菌GS115 (pPIC9K EDN)生物量的A60 0 值达到 2 5 0 ,分泌量为 15 0mg L .发酵液经StreamlineSP ,SepharoseSPFF离子交换柱和Sepharose HeparinHiTrap亲和柱纯化 ,产物纯度达 97%以上 ,回收率为 6 0 % .Western印迹结果表明 ,酵母工程菌GS115 (pPIC9K EDN)表达的重组人内皮细胞抑制素具有天然EDN的免疫原性 .MTT法和抑癌实验结果显示 :纯化产物能抑制人脐静脉内皮细胞的增殖并能抑制移植于小鼠的黑色素瘤的生长 ,其平均抑瘤率是 94 2 % .

The high cell density of P. pastoris GS115(pPIC9K EDN ) strain harboring human endostatin (EDN) was cultured in a 30 liter fermentor and the effect of media, pH and induction time on cell growth and gene expression were determined. By fed batch fermentation and methanol induction for 48 hours,the biomass A 600 of GS115(pPIC9K EDN ) reached 250, the secreted EDN was 150 mg/L. The fermentation product was purified to 97% by Streamline SP and Sepharose SP F F cation exchanger column and Sepharose\|Heparin Hi Trap affinity chromatography. The total recovery was 60%. Western blot analysis demonstrated that the purified product reacted with anti mEDN antibody. The purified product specifically inhibited the proliferation of human umbilical vein endothelial cells(ECV304) by 68.9% at 2 μg/ml. The purified endostatin also suppressed in vivo growth of B16 murine melanoma by 94 2% at a dose of 10 mg/(kg·d).