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    • 1. 发明授权
    • Plasmid shuttle vector between Escherichia coli and Brevibacillus
    • 质粒穿梭载体在大肠杆菌和短杆菌之间
    • US07332331B2
    • 2008-02-19
    • US10467930
    • 2002-02-07
    • Koji Yashiro
    • Koji Yashiro
    • C07H21/04C12N1/20C12N15/00C12P21/06
    • C12N15/77C12N15/70C12N15/74
    • The invention provides a plasmid shuttle vector characterized by being replicable in both of bacteria of the genus Brevibacillus and Escherichia coli, and by comprising a promoter capable of functioning in bacteria of the genus Brevibacillus and a DNA sequence(s) capable of functioning as a selective marker(s) for Escherichia coli and bacteria of the genus Brevibacillus, and a method of transforming bacteria of the genus Brevibacillus using the said plasmid shuttle vector. The plasmid shuttle vector enables the efficient transformation of bacteria of the genus Brevibacillus. Further, since plasmid shuttle vector pNCMO2 of the invention in particular has a gene expression control region which efficiently expresses and secretes an objective gene in bacteria of the genus Brevibacillus but significantly suppresses the expression in Escherichia coli, a recombinant gene expression plasmid can efficiently be constructed using Escherichia coli, and it is suited for protein production in bacteria of the genus Brevibacillus.
    • 本发明提供了一种质粒穿梭载体,其特征在于可以在短杆菌属和大肠杆菌属的细菌中复制,并且包含能够在短杆菌属细菌中起作用的启动子和能够作为选择性的DNA序列 用于大肠杆菌的标记物和Brevibacillus属的细菌,以及使用所述质粒穿梭载体转化短杆菌属细菌的方法。 质粒穿梭载体能够有效转化Brevibacillus属的细菌。 此外,由于本发明的质粒穿梭载体pNCMO2特别具有有效表达并分泌短杆菌属细菌中的目标基因但显着抑制大肠杆菌表达的基因表达控制区,因此能够有效地构建重组基因表达质粒 使用大肠杆菌,适用于短杆菌属细菌中的蛋白质生产。
    • 8. 发明授权
    • Brevibacillus choshinensis and process for prodcuing protein wtih use of the microbe as host
    • 使用微生物作为宿主生产蛋白质的短杆菌(Brevibacillus choshinensis)及其制备方法
    • US07655452B1
    • 2010-02-02
    • US10578781
    • 2004-11-08
    • Hiroshi HanagataTakayuki Nishijyo
    • Hiroshi HanagataTakayuki Nishijyo
    • C12N1/20
    • C12P21/02C12R1/01
    • Brevibacillus choshinensis is characterized in that its extracellular proteolytic activity is extremely low and its protein secretion productivity is excellent, but it is desired that not only the extracellular proteolytic activity of the strain is further reduced but also the intracellular proteolytic activity thereof is further reduced. When Brevibacillus choshinensis is used as a host for the production of protein pharmaceuticals and the like, it is also desired that it does not form spores and is readily sterilized. The above problems have been solved by inactivating the sporulation-associated gene thereof and by cloning and inactivating the extracellular and intracellular protease genes thereof.
    • 特征在于其细胞外蛋白水解活性极低,其蛋白质分泌生产性优异,但是不仅要进一步降低菌株的细胞外蛋白水解活性,而且进一步降低其细胞内的蛋白水解活性。 当将短杆菌属(Clovibacillus choshinensis)用作生产蛋白质药物等的宿主时,也希望它不形成孢子并容易灭菌。 通过灭活其孢子形成相关基因并通过克隆和灭活其细胞外和细胞内的蛋白酶基因来解决上述问题。