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    Gene encoding biologically active human interleukin 1
    9.
    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。

    Purification of the IL-2 receptor
    10.
    发明授权
    Purification of the IL-2 receptor 失效
    IL-2受体的纯化

    公开(公告)号：US5317087A

    公开(公告)日：1994-05-31

    申请号：US630331

    申请日：1990-10-22

    申请人： Douglas P. Cerretti ,  ; David J. Cosman ,  Steven K. Dower ,  Carl J. March ,  David L. Urdal ,  Alf D. Larsen

    发明人： Douglas P. Cerretti ,  ; David J. Cosman ,  Steven K. Dower ,  Carl J. March ,  David L. Urdal ,  Alf D. Larsen

    IPC分类号： C07K14/715 ,  C07K16/28 ,  C07K13/00 ,  C07K15/00

    CPC分类号： C07K14/7155 ,  C07K16/2866

    摘要： Interleukin-2 receptor derived from normal and malignant cells has been purified by use of various techniques including affinity chromatography in conjunction with a monoclonal antibody directed to the receptor. The purification process also includes reversed phased high performance liquid chromatography. By these techniques, interleukin-2 receptor has been purified to homogeneity. The high purification of the interleukin-2 receptor has made possible the sequencing of the amino acid residues at the N-terminal of this protein molecule. Double-stranded cDNA is prepared from polyadenylated RNA extracted from cell lines or other sources known to produce IL-2 receptor. 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 purified IL-2 receptor. The cDNA clone isolated with the probe is characterized by restriction enzyme mapping and sequenced by chain-termination method. The particular DNA clone that actually contains the gene coding for the functional IL-2 receptor is identified by expressing the clones in COS-7 monkey kidney cells and assaying for the expressed IL-2 receptor by its ability to bind IL-2 or a monoclonal antibody directed against the IL-2 receptor.

    摘要翻译： 来自正常和恶性细胞的白细胞介素-2受体已经通过使用各种技术进行纯化，包括亲和层析与针对受体的单克隆抗体。 纯化方法还包括反相高效液相色谱法。 通过这些技术，白细胞介素-2受体已经被纯化到均一性。 白细胞介素-2受体的高度纯化使得可以对该蛋白质分子的N-末端的氨基酸残基进行测序。 从从已知产生IL-2受体的细胞系或其他来源提取的聚腺苷酸化的RNA制备双链cDNA。 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。 从这些库制备的质粒DNA与对应于纯化的IL-2受体的氨基酸序列的一部分的标记的合成寡核苷酸探针杂交。 用探针分离的cDNA克隆的特征在于限制酶作图并通过链终止法进行测序。 通过在COS-7猴肾细胞中表达克隆并通过其结合IL-2或单克隆抗体的能力测定表达的IL-2受体来鉴定实际含有编码功能性IL-2受体的基因的特定DNA克隆 针对IL-2受体的抗体。