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    • 2. 发明申请
    • METHOD FOR MASS PRODUCTION OF FACTOR VII/VIIA
    • 大量生产因子VII / VIIA的方法
    • WO2012057527A2
    • 2012-05-03
    • PCT/KR2011/008040
    • 2011-10-26
    • HANMI HOLDINGS CO., LTD.KIM, Chang HwanHONG, Sung KapLEE, Byung SunHONG, Sung HwanKWON, Se Chang
    • KIM, Chang HwanHONG, Sung KapLEE, Byung SunHONG, Sung HwanKWON, Se Chang
    • C12N15/85C12N15/12C07K14/745
    • C12N9/6437C07K14/745C12N15/85C12N2830/85
    • Disclosed is a method for the mass production of human coagulation factor VII. The method comprises a) constructing an expression vector carrying i) a dihydrofolate reductase promoter devoid of one or more CCGCC repeat sequences from the GC-rich region thereof and a dihydrofolate reductase (DHFR) gene operably linked thereto and ii) a cytomegalovirus (CMV) promoter and a human coagulation factor VII gene operably linked thereto; b) transfecting the expression vector of step a) into an animal cell line; c) culturing the transfected animal cell line of step b) in the presence of a dihydrofolate reductase inhibitor to select cells which express human coagulation factor VII with high efficiency; and d) adding sodium butyrate to the selected animal cells of step c), and a cell line for mass production of human coagulation factor VII. Employing a vector carrying a DHFR promoter devoid of GC-rich repeating sequences, the method can produce human coagulation factor VII at high efficiency on a large scale, which contributes to the treatment of hemophilia.
    • 公开了用于大量生产人凝血因子VII的方法。 该方法包括a)构建表达载体,其携带i)不含来自其富GC区的一个或多个CCGCC重复序列的二氢叶酸还原酶启动子和与其可操作连接的二氢叶酸还原酶(DHFR)基因,和ii)巨细胞病毒(CMV) 启动子和与之可操作连接的人凝血因子VII基因; b)将步骤a)的表达载体转染到动物细胞系中; c)在二氢叶酸还原酶抑制剂存在下培养步骤b)的转染的动物细胞系以选择高效表达人凝血因子Ⅶ的细胞; 和d)向步骤c)的所选动物细胞中添加丁酸钠,以及用于大量生产人凝血因子VII的细胞系。 使用携带不含富含GC的重复序列的DHFR启动子的载体,该方法可以大规模高效率地产生人凝血因子VII,这有助于治疗血友病。