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    • 13. 发明授权
    • Method for identifying genes encoding secreted or membrane-associated
proteins
    • 用于鉴定编码分泌或膜相关蛋白的基因的方法
    • US5952171A
    • 1999-09-14
    • US752307
    • 1996-11-19
    • Sean Anthony McCarthyDavid Paul GearingDouglas Adam Levinson
    • Sean Anthony McCarthyDavid Paul GearingDouglas Adam Levinson
    • C12N15/10C12N15/85C12Q1/68
    • C12Q1/6811C12N15/1034C12N15/1051C12N15/85C12N2800/60C12N2830/38
    • The invention features a method for identifying a cDNA nucleic acid encoding a mammalian protein having a signal sequence, which method includes the following steps: (a) providing library of mammalian cDNA; (b) ligating the library of mammalian cDNA to DNA encoding alkaline phosphatase lacking both a signal sequence and a membrane anchor sequence to form ligated DNA; (c) transforming bacterial cells with the ligated DNA to create a bacterial cell clone library; (d) isolating DNA comprising the mammalian cDNA from at least one clone in the bacterial cell clone library; (e) separately transfecting DNA isolated from clones in step (d) into mammalian cells which do not express alkaline phosphatase to create a mammalian cell clone library wherein each clone in the mammalian cell clone library corresponds to a clone in the bacterial cell clone library; (f) identifying a clone in the mammalian cell clone library which express alkaline phosphatase; (g) identifying the clone in the bacterial cell clone library corresponding to the clone in the mammalian cell clone library identified in step (f); and (h) isolating and sequencing a portion of the mammalian cDNA present in the bacterial cell library clone identified in step (g) to identify a mammalian cDNA encoding a mammalian protein having a signal sequence.
    • 本发明的特征在于鉴定编码具有信号序列的哺乳动物蛋白质的cDNA核酸的方法,该方法包括以下步骤:(a)提供哺乳动物cDNA文库; (b)将哺乳动物cDNA文库与编码缺乏信号序列和膜锚定序列的碱性磷酸酶的DNA连接以形成连接的DNA; (c)用连接的DNA转化细菌细胞以产生细菌细胞克隆文库; (d)从细菌细胞克隆文库中的至少一个克隆分离包含哺乳动物cDNA的DNA; (e)将步骤(d)中从克隆分离的DNA分别转染到不表达碱性磷酸酶的哺乳动物细胞中以产生哺乳动物细胞克隆文库,其中哺乳动物细胞克隆文库中的每个克隆对应于细菌细胞克隆文库中的克隆; (f)鉴定表达碱性磷酸酶的哺乳动物细胞克隆文库中的克隆; (g)鉴定对应于步骤(f)中鉴定的哺乳动物细胞克隆文库中克隆的细菌细胞克隆文库中的克隆; 和(h)分离和测序存在于步骤(g)中鉴定的细菌细胞库克隆中的哺乳动物cDNA的一部分,以鉴定编码具有信号序列的哺乳动物蛋白质的哺乳动物cDNA。