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    • 4. 发明申请
    • PROTEIN EXPRESSION FROM MULTIPLE NUCLEIC ACIDS
    • 来自多种核酸的蛋白质表达
    • WO2009046978A1
    • 2009-04-16
    • PCT/EP2008/008523
    • 2008-10-09
    • F. HOFFMANN-LA ROCHE AGGOEPFERT, UlrichKNOETGEN, HendrikKOPETZKI, ErhardSTERN, Anne
    • GOEPFERT, UlrichKNOETGEN, HendrikKOPETZKI, ErhardSTERN, Anne
    • C12N15/69C12N15/85C07K16/00
    • C12N15/85C07K16/18C07K16/28C07K16/2812C07K16/2854C07K16/2866C07K16/2896C07K2317/14C07K2317/21C07K2317/51C07K2317/515C12P21/00
    • The current invention reports a method for the recombinant production of a secreted heterologous immunoglobulin in a CHO cell comprising the following steps: i) providing a CHO cell, which is adapted to growth in suspension culture, adapted to growth in serum-free medium, mycoplasma free, and virus free, ii) providing a vector comprising a prokaryotic origin of replication, a first nucleic acid conferring resistance to a prokaryotic selection agent, a second nucleic acid encoding the heavy chain of said heterologous immunoglobulin, a third nucleic acid encoding the light chain of said heterologous immunoglobulin, a fourth nucleic acid conferring resistance to a eukaryotic selection agent, iii) transfecting said CHO cell, wherein said transfecting comprises a) transfecting said CHO cell with said vector comprising a fourth nucleic acid conferring resistance to a first eukaryotic selection agent, b) selecting a CHO cell by growth in cultivation medium containing said first eukaryotic selection agent, c) transfecting said selected CHO cell with said vector comprising a fourth nucleic acid conferring resistance to a second eukaryotic selection agent different to said first eukaryotic selection agent, d) selecting a CHO cell by selected growth in cultivation medium containing said first and said second eukaryotic selection agent, iv) cultivating said transfected CHO cell in a medium in the presence of said first and second eukaryotic selection agent, under conditions suitable for the expression of said second, and third nucleic acid, and v) recovering said secreted heterologous immunoglobulin from the cultivation medium.
    • 本发明报告了在CHO细胞中重组产生分泌的异源免疫球蛋白的方法,其包括以下步骤:i)提供适于在悬浮培养物中生长的CHO细胞,适于在无血清培养基中生长,支原体 免费和无病毒,ii)提供包含原核复制起点的载体,赋予原核选择剂抗性的第一核酸,编码所述异源免疫球蛋白的重链的第二核酸,编码光的第三核酸 所述异源免疫球蛋白链,赋予对真核选择剂的抗性的第四核酸,iii)转染所述CHO细胞,其中所述转染包括a)用包含第四核酸的所述载体转染所述CHO细胞,赋予对第一真核选择性的抗性 试剂,b)通过在含有所述第一种真核生物的培养基中生长来选择CHO细胞 c)用所述载体转染所述选择的CHO细胞,所述载体包含赋予与所述第一真核选择试剂不同的第二真核选择剂的抗性的第四核酸,d)通过在含有所述第一和第二真核选择试剂的培养基中选择生长来选择CHO细胞, 所述第二真核选择剂,iv)在所述第一和第二真核选择剂的存在下,在适于表达所述第二和第三核酸的条件下,在培养基中培养所述转染的CHO细胞,和v)回收所述分泌的异源 免疫球蛋白。