重组水蛭素Ⅲ的中试工艺研究

Large-Scale Preparation of Recombinant HirudinⅢ

从大肠杆菌中大规模纯化重组水蛭素Ⅲ,进行重组水蛭素Ⅲ中试发酵工艺和纯化中试工艺研究,连续大量纯化3批,并对纯化终产品进行鉴定。通过菌种活化,一级、二级种子液制备,30L发酵罐补料-批式发酵水蛭素产量达到6000ATU/ml,发酵液经还原性SDS-PAGE分析表明在14000u处有单一条带,为重组水蛭素Ⅲ二聚体。经大孔树脂层析、DEAE-纤维素层析、制备型反相高效液相层析等3步纯化后重组水蛭素Ⅲ的比活达8270ATU/mg。经分析型反相高效液相色谱分析,纯度达95%以上,总收率达到39%以上。通过实验建立了稳定的发酵和纯化工艺流程。

To isolate and purify recombinant hirudinⅢ, which is secreted into the culture, the largescale fermentation and purification method were set up for r-Hirudin Ⅲ, which was expressed in E. coli system. In the three batches of fermentation, the average harvest amount of hirudinⅢ was as high as 6 000ATU/ml. A combined technique of low-temperature centrifugation, macroporous resin adsorption, DEAE-cellulose ion-exchange chromatography and RP-HPLC were developed to isolate and purify the recombinant Hirudin Ⅲ. By analysis of SDS-PAGE, electrophoresis purity of the product was determined to be 95%. By analytical RP-HPLC, its antithrombin activity was 8 270 ATU/mg. The purity and the recovery reached 95% and 39.06%, respectively.