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1. 发明公开

KR1020150026024A 인간 혈액응고 7인자 유도체의 대량 생산 방법 审中-实审
标题翻译：大量生产因子VII模拟的方法
公开(公告)号：KR1020150026024A
公开(公告)日：2015-03-11
申请号：KR1020130104308
申请日：2013-08-30
申请人： 한미약품 주식회사
发明人： 강희철 , 김진영 , 이병선 , 김현욱 , 최인영 , 권세창
IPC分类号： C12N15/85 , C12N15/12 , C12P21/02
CPC分类号： C12N9/6437 , C07K14/745 , C12N9/003 , C12N15/85 , C12P21/02 , C12Y105/01003 , C12Y304/21021
摘要： The present invention relates to a method of mass-producing human blood coagulation factor VII. More specifically, the present invention relates to a mass-production method of human blood coagulation factor VII derivatives, and a cell line for mass-producing human blood coagulation factor VII derivatives, wherein the mass-production method of human blood coagulation factor VII derivatives comprises a) a step of manufacturing an expression vector including i) a base sequence of a dehydrofolate reductase promoter with at least one CCGCCC removed from a GC-rich region, and a base sequence operationally connected thereto and coding the dehydrofolate reductase (DHFR); and ii) a base sequence of early gene promoters of cytomegalosvirus (CMV), and a base sequence operationally connected thereto and coding the human blood coagulation factor VII derivatives; b) a step of transforming animal cell lines with the expression vector of step a); c) a step of cultivating the transformed animal cell lines at step b) under existence of dehydrofolate reductase inhibitors and selecting cell lines expressing human blood coagulation factor VII derivatives at a high efficiency; and d) a step of adding at least one selected from the group consisting of sodium butylate, vitamin K and a culture medium additive to the selected animal cell lines of step c) and cultivating. The present invention can be usefully applied to manufacture of a hemophilia medicine as the human blood coagulation factor VII derivatives can be expressed at a high efficiency and a large quantity by using a vector with GC-rich repeated sequences deleted at a DHFR promotor site.
摘要翻译：本发明涉及一种批量生产人凝血因子VII的方法。更具体地，本发明涉及人凝血因子VII衍生物的批量生产方法和用于大规模生产人凝血因子VII衍生物的细胞系，其中人凝血因子VII衍生物的批量生产方法包括 a）制备表达载体的步骤，所述步骤包括：i）从富含GC的区域除去至少一个CCGCCC的脱氢叶酸还原酶启动子的碱基序列和与其操作连接并编码脱氢叶酸还原酶（DHFR）的碱基序列; 和ii）巨细胞病毒（CMV）的早期基因启动子的碱基序列和与其可操作地连接并编码人血液凝固因子VII衍生物的碱基序列; b）用步骤a）的表达载体转化动物细胞系的步骤; c）在脱氢叶酸还原酶抑制剂的存在下在步骤b）中培养转化的动物细胞系的步骤，并且以高效率选择表达人凝血因子VII衍生物的细胞系; 和d）向步骤c）的所选择的动物细胞系中添加选自丁酸钠，维生素K和培养基添加剂中的至少一种的步骤，并进行培养。本发明可有效地应用于制造血友病药物，因为人血液凝固因子VII衍生物可以通过使用在DHFR启动子位点缺失GC富集重复序列的载体以高效率和大量表达。

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