Method for identification and isolation of DNA encoding a desired protein
    9.
    发明授权
    Method for identification and isolation of DNA encoding a desired protein 失效
    用于鉴定和分离编码所需蛋白质的DNA的方法

    公开(公告)号:US4675285A

    公开(公告)日:1987-06-23

    申请号:US652316

    申请日:1984-09-19

    摘要: A method for identifying and isolating clones containing DNA coding for a desired protein is described. DNA prepared from a cell that expresses the desired protein is inserted into an isolation expression vector having means for replication (as a means of producing DNA) and a suitable promoter for expression of said DNA in a predetermined mammalian host cell as well as means for replication in a bacterial cell. The transient expression vector is then inserted into a bacterial cell for replication of the DNA. Pools of DNA, prepared from a predetermined number of bacterial clones so that the nucleic acids (DNA and RNA) is substantially free of other bacterial contaminants are transfected or microinjected into mammalian host cells and conditioned medium from growing such cells is tested for the presence of the desired protein. Positive pools are selected and the clones used to make the pool are screened to identify and isolate the clone containing the desired DNA.

    摘要翻译: 描述了鉴定和分离含有编码所需蛋白质的DNA的克隆的方法。 将从表达所需蛋白质的细胞制备的DNA插入具有复制方式的分离表达载体(作为生产DNA的手段)和用于在预定哺乳动物宿主细胞中表达所述DNA的合适启动子以及复制手段 在细菌细胞中。 然后将瞬时表达载体插入用于DNA复制的细菌细胞中。 从预定数量的细菌克隆制备DNA,使得核酸(DNA和RNA)基本上不含其它细菌污染物的DNA转染或显微注射到哺乳动物宿主细胞中,并测试来自培养这样的细胞的条件培养基的存在 所需蛋白质。 选择阳性池,筛选用于制备池的克隆以鉴定并分离含有所需DNA的克隆。