重组人凝血因子Ⅶ的表达、激活与功能鉴定  

Expression, Activation and Functional Analysis of Human Recombinant Factor Ⅶ

作　　者：李倩卉 刘旭 王志军 路中枢 高雪寒 薛成峰 张子义 李齐 LI Qianhui;LIU Xu;WANG Zhijun;LU Zhongshu;GAO Xuehan;XUE Chengfeng;ZHANG Ziyi;LI Qi(Beijing Neoletix Biological Technology,Beijing 100176,China)

机构地区：[1]庄亚(北京)生物科技有限公司,北京100176

出　　处：《生物化工》2023年第5期62-68,共7页Biological Chemical Engineering

摘　　要：目的:建立重组人凝血因子Ⅶ(rhFⅦ)中国仓鼠卵巢(CHO)细胞表达系统,并从生物化学与凝血活性等方面对活化的重组人凝血因子Ⅶ(rhFⅦa)进行分析。方法:利用公司专有的CHO细胞表达系统构建rhFⅦ表达载体并筛选稳定高产细胞株。通过经典的离子交换技术纯化并活化rhFⅦ。通过SDS-PAGE、Western Blot对rhFⅦa进行生物化学分析,用生物膜干涉技术(BLI)验证其与组织因子(TF)的结合,利用对发色底物酶切、活化部分凝血活酶时间(APTT)/凝血酶原时间(PT)、血栓弹力仪(TEG)对其凝血活性进行了深入的分析并与成熟产品NovoSeven进行了比较。结果:rhFⅦ在CHO细胞流加培养中得到了成功表达,其表达量可达50 mg/L。成功激活的rhFⅦa显示出与NovoSeven高度相似的生物化学与凝血活性。结论:利用CHO系统成功表达并纯化获得活化的rhFⅦ,其在生化与凝血活性等方面与市售产品一致,为实现rhFⅦa国产化奠定了坚实的基础。Objective:To establish the expression system of recombinant human factorⅦ(rhFⅦ)Chinese Hamster Ovary(CHO)cells,and to present the biochemical and functional characteristics of activated recombinant human factorⅦ(rhFⅦa)from the aspects of biochemistry and coagulation activity.Methods:To manufacture rhFⅦa,the full-length rhFⅦexpression vector is constructed,which is used to generated CHO cell system stably expressing high yield of rhFⅦ.Human rhFⅦis purified by classical Anion-exchange technique and then fully activated.The biochemical analysis of rhFⅦa is determined by SDS-PAGE and Western Blot.Its binding activity to tissue factor(TF)is determined by bio-layer interferometry(BLI).The clotting activity of rhFⅦa is assessed by chromogenic assay,activated partial thromboplastin time(APTT)/prothrombin time(PT)and thrombelastograph coagulation analyzer(TEG),compared with Novoseven.Results:rhFⅦis successfully expressed in fed-batch culture of CHO cells,and the expression of rhFⅦa exceeds 50 mg/L.The fully activated rhFⅦa exhibite biochemical and functional characteristics similar to NovoSeven.Conclusion:The innovative CHO expression system and purification techniques to produce rhFⅦa yields an effective rhFⅦa with high expression level.It is comparable with commercially available products in terms of biochemical property and coagulation activity,laying a solid foundation for domestic manufacture of rhFⅦa.

关 键 词：人重组凝血因子Ⅶ 活化的人重组凝血因子Ⅶ CHO细胞 组织因子 甲型血友病 

分 类 号：R554.1[医药卫生—血液循环系统疾病]