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    Gene encoding biologically active human interleukin 1
    3.
    发明授权
    Gene encoding biologically active human interleukin 1 失效
    基因编码生物活性人白细胞介素1

    公开(公告)号：US5122459A

    公开(公告)日：1992-06-16

    申请号：US443399

    申请日：1989-11-30

    申请人： Paul J. Conlon, III ,  David j. Cosman ,  Kenneth H. Grabstein ,  Thomas P. Hopp ,  Shirley R. Kronheim ,  Alf D. Larsen ,  Carl J. March ,  Virginia L. Price ,  Douglas P. Cerretti

    发明人： Paul J. Conlon, III ,  David j. Cosman ,  Kenneth H. Grabstein ,  Thomas P. Hopp ,  Shirley R. Kronheim ,  Alf D. Larsen ,  Carl J. March ,  Virginia L. Price ,  Douglas P. Cerretti

    IPC分类号： C07K14/545 ,  C12N15/25 ,  C12N15/81

    CPC分类号： C07K14/545 ,  C12N15/81 ,  Y10S435/942

    摘要： Double-stranded cDNA is prepared from polyadenylated RNA extracted from activated human peripheral blood adherent mononuclear cells. The cDNA is inserted within a plasmid vector and then the recombinant plasmid employed to transform an appropriate host. Transformed hosts are identified and grouped into pools. Plasmid DNA prepared from these pools is hybridized with a labeled, synthetic oligonucleotide probe corresponding to a portion of the amino acid sequence of the interleukin 1 protein. Pools of host cells that provide a positive signal to the probe are identified, plated out and then employed in direct bacterial colony hybridization with the same probe, thereby to isolate the particular positive colony. Plasmid DNA is prepared from this colony and characterized by restriction enzyme mapping and sequencing by chain-termination method. The coding region for the IL-1 gene is inserted into a shuttle vector for amplification of the vector followed by expression of functional IL-1.

    摘要翻译： 双链cDNA由从活化的人外周血粘附单核细胞提取的聚腺苷酸化RNA制备。 将cDNA插入质粒载体中，然后将重组质粒用于转化合适的宿主。 已识别转化的主机并将其分组到池中。 从这些池制备的质粒DNA与对应于白介素1蛋白的氨基酸序列的一部分的标记的合成的寡核苷酸探针杂交。 确定提供探针的正信号的宿主细胞池，电镀，然后用于与相同探针的直接细菌菌落杂交，从而分离出特定的阳性菌落。 从该菌落制备质粒DNA，并通过限制酶作图和通过链终止法进行测序。 将用于IL-1基因的编码区插入穿梭载体中用于扩增载体，随后表达功能性IL-1。