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1. 发明申请

WO0047750A3 METALLOPROTEASES OF THE NEPRILYSIN FAMILY 审中-公开
标题翻译： NEPRILYSIN家族的金属蛋白质
公开(公告)号：WO0047750A3
公开(公告)日：2000-11-30
申请号：PCT/CA0000147
申请日：2000-02-11
申请人： UNIVERSTIE DE MONTREAL , DESGROSEILLERS LUC , BOILEAU GUY
发明人： DESGROSEILLERS LUC , BOILEAU GUY
IPC分类号： G01N33/53 , C07K16/40 , C12N1/15 , C12N1/19 , C12N1/21 , C12N5/10 , C12N9/48 , C12N9/64 , C12N15/09 , C12N15/57 , C12Q1/68 , G01N33/566 , G01N33/573 , C12N15/62 , C12N15/85
CPC分类号： C12N9/6489 , C07K2319/02
摘要： In this paper, we describe RT-PCR strategies that allowed us to identify and clone members of the NEP-like family. Degenerate oligoncleotide primers corresponding to consensus sequences located on either side of the HEXXH consensus sequence for zincins were designed and used in RT-PCR with mouse and human testis cDNAs. DNA fragments with lengths expected from the sequence of this class of enzympes were obtained. These DNA fragments were cloned and sequenced. Using this PCR strategy and the PCR fragments as probes to screen cDNA libraries, three zincin-like peptidases were identified in addition of known members of the family. The cDNA sequences allowed to derive specific probes for Northern and in situ hybridization, and probe human chromosomes to localize the gene and establish potential links to genetic diseases. Furthermore, these cDNA sequences were used to produce recombinant fusion proteins in Escherichia coli in order to raise specific antibodies. Finally, the cDNA sequences were cloned in mammalian expression vectors and transfected in various mammalian cell lines to produce active recombinant enzymes suitable for testing specific inhibitors.
摘要翻译：在本文中，我们描述了RT-PCR策略，使我们能够识别和克隆NEP样家族的成员。设计了与位于HEXXH共有序列两侧的共有序列相对应的用于锌的简并寡核苷酸引物，并用于小鼠和人睾丸cDNA的RT-PCR。获得了从该类酶的序列预期的长度的DNA片段。克隆并测序这些DNA片段。使用该PCR策略和PCR片段作为筛选cDNA文库的探针，鉴定出已知家族成员的三种类似锌的肽酶。 cDNA序列允许导出用于Northern和原位杂交的特异性探针，并探测人染色体来定位基因并建立与遗传疾病的潜在联系。此外，这些cDNA序列用于在大肠杆菌中产生重组融合蛋白以产生特异性抗体。最后，将cDNA序列克隆到哺乳动物表达载体中并转染到各种哺乳动物细胞系中以产生适于测试特异性抑制剂的活性重组酶。

2. 发明申请

WO0047750B1 METALLOPROTEASES OF THE NEPRILYSIN FAMILY 审中-公开
标题翻译： NEPRILYSIN家族的金属蛋白质
公开(公告)号：WO0047750B1
公开(公告)日：2001-05-25
申请号：PCT/CA0000147
申请日：2000-02-11
申请人： UNIVERSTIE DE MONTREAL , DESGROSEILLERS LUC , BOILEAU GUY
发明人： DESGROSEILLERS LUC , BOILEAU GUY
IPC分类号： G01N33/53 , C07K16/40 , C12N1/15 , C12N1/19 , C12N1/21 , C12N5/10 , C12N9/48 , C12N9/64 , C12N15/09 , C12N15/57 , C12Q1/68 , G01N33/566 , G01N33/573 , C12N15/62 , C12N15/85
CPC分类号： C12N9/6489 , C07K2319/02
摘要： In this paper, we describe RT-PCR strategies that allowed us to identify and clone members of the NEP-like family. Degenerate oligoncleotide primers corresponding to consensus sequences located on either side of the HEXXH consensus sequence for zincins were designed and used in RT-PCR with mouse and human testis cDNAs. DNA fragments with lengths expected from the sequence of this class of enzympes were obtained. These DNA fragments were cloned and sequenced. Using this PCR strategy and the PCR fragments as probes to screen cDNA libraries, three zincin-like peptidases were identified in addition of known members of the family. The cDNA sequences allowed to derive specific probes for Northern and in situ hybridization, and probe human chromosomes to localize the gene and establish potential links to genetic diseases. Furthermore, these cDNA sequences were used to produce recombinant fusion proteins in Escherichia coli in order to raise specific antibodies. Finally, the cDNA sequences were cloned in mammalian expression vectors and transfected in various mammalian cell lines to produce active recombinant enzymes suitable for testing specific inhibitors.
摘要翻译：在本文中，我们描述了RT-PCR策略，使我们能够识别和克隆NEP样家族的成员。设计了与位于HEXXH共有序列两侧的共有序列相对应的用于锌的简并寡核苷酸引物，并用于小鼠和人睾丸cDNA的RT-PCR。获得了从该类酶的序列预期的长度的DNA片段。克隆并测序这些DNA片段。使用该PCR策略和PCR片段作为筛选cDNA文库的探针，鉴定出已知家族成员的三种类似锌的肽酶。 cDNA序列允许导出用于Northern和原位杂交的特异性探针，并探测人染色体来定位基因并建立与遗传疾病的潜在联系。此外，这些cDNA序列用于在大肠杆菌中产生重组融合蛋白以产生特异性抗体。最后，将cDNA序列克隆到哺乳动物表达载体中并转染到各种哺乳动物细胞系中以产生适于测试特异性抑制剂的活性重组酶。

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