摘要
目的在CHO细胞中表达并纯化人凝血因子Ⅸ(human coagulation factorⅨ,hFⅨ)。方法将高表达hFⅨ的CHO细胞株在摇瓶中用无血清培养基进行批次培养和补料批次培养,收获补料批次培养上清,通过阴离子交换层析进行纯化,测定纯化产物的蛋白浓度及hFⅨ活性,并进行SDS-PAGE分析。结果批次和补料批次培养表达的hFⅨ活性分别高达0.62和14.45 IU/mL。纯化后的hFⅨ比活性高达157.19 IU/mg。SDS-PAGE分析表明重组CHO工程细胞株能正确表达hFⅨ。结论成功在CHO细胞中表达并纯化了高比活性的重组hFⅨ蛋白,为进一步研制重组FⅨ药物奠定了基础。
Objective To express human coagulation factorⅨ(hFⅨ)in CHO cells and purify the expressed product.Methods CHO cell strain for high expression of hFⅨwas subjected to batch and fed-batch cultures with serum-free medium in a shake flask,and the supernatant was harvested and purified by anion exchange chromatography.The purified protein was determined for concentration and hFⅨactivity,and analyzed by SDS-PAGE.Results The activities of hFⅨin batch and fed-batch cultures were as high as 0.62 and 14.45 IU/mL respectively.The purified hFⅨ reached a specific activity of 157.19 IU/mg.SDS-PAGE showed that hFⅨ was expressed in recombinant CHO cell lines correctly.Conclusion Recombinant hFⅨwith high specific activity was successfully expressed in CHO cells and purified,which laid a foundation of further development of recombinant FⅨdrugs.
作者
李德款
张航
聂艳桃
李成
梁洪
寇鹏
费保进
LI De-kuan;ZHANG Hang;NIE Yan-tao;LI Cheng;LIANG Hong;KOU Peng;FEI Bao-jin(Research Management Department,Chengdu Rongsheng Pharmaceuticals Co.Ltd.,Chengdu 610041,Sichuan Province,China.)
出处
《中国生物制品学杂志》
CAS
CSCD
2019年第11期1243-1246,共4页
Chinese Journal of Biologicals
关键词
人凝血因子Ⅸ
中国仓鼠卵巢细胞
阴离子交换层析
Human coagulation factorⅨ(hFⅨ)
Chinese hamster ovary(CHO)cells
Anion exchange chromatography
