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1. 发明申请

US20050054038A1 High level constitutive production of anthrax protective antigen 失效
标题翻译：高水平组成型生产炭疽保护性抗原
公开(公告)号：US20050054038A1
公开(公告)日：2005-03-10
申请号：US10494680
申请日：2001-12-07
申请人： Rakesh Bhatnagar , Waheed Mohsin , Vibha Chauhan
发明人： Rakesh Bhatnagar , Waheed Mohsin , Vibha Chauhan
IPC分类号： C12P21/02 , A61K39/07 , A61P31/04 , C07K20060101 , C07K14/32 , C12N1/21 , C12P21/00 , C12R1/07 , A23J1/00 , C07K1/00 , C07K14/00 , C12P21/06 , C07K16/00 , C07K17/00
CPC分类号： C07K14/32
摘要： The present invention relates to a process for preparing anthrax protective antigen protein from E. coli using fed batch culture. This process creates a constitutively expressing system for rapid, efficient, cost-effective and high-level production of anthrax PA from E. coli. The steps of the process involves, transforming E. coli DH5α cells with the recombinant constitutive expression plasmid containing the PA gene to obtain recombinant DH5α cells and testing the PA expression by lysis of said cells followed by denaturing gel electrophoresis and Western Blotting technique using PA antibodies. This is followed by fermentation and harvesting of the high cell density cells. The said cells are solubilized using 6-8 Molar Urea and separated by centrifugation. The urea denatured PA is isolated from said supernatant and purified and thereafter eluted.
摘要翻译：本发明涉及使用补料分批培养从大肠杆菌制备炭疽保护性抗原蛋白的方法。该过程创建了一种组成型表达系统，用于从大肠杆菌中快速，有效，成本有效和高水平地生产炭疽芽孢杆菌。该方法的步骤包括用包含PA基因的重组组成型表达质粒转化大肠杆菌DH5α细胞以获得重组DH5α细胞，并通过裂解所述细胞测试PA表达，然后通过变性凝胶电泳和使用PA抗体的Western Blotting技术。随后发酵和收获高细胞密度的细胞。所述细胞用6-8摩尔尿素溶解并通过离心分离。尿素变性PA从所述上清液中分离并纯化，然后洗脱。

2. 发明申请

US20050063986A1 Process for the preparation of non-toxic anthrax vaccine 失效
标题翻译：无毒性炭疽疫苗的制备工艺
公开(公告)号：US20050063986A1
公开(公告)日：2005-03-24
申请号：US10497673
申请日：2002-03-20
申请人： Rakesh Bhatnagar , Pankaj Gupta , Smriti Batra , Vibha Chauhan , Aparna Singh , Nidhi Ahuja , Praveen Kumar
发明人： Rakesh Bhatnagar , Pankaj Gupta , Smriti Batra , Vibha Chauhan , Aparna Singh , Nidhi Ahuja , Praveen Kumar
IPC分类号： C12N15/09 , A61K38/00 , A61K39/00 , A61K39/07 , A61P31/04 , A61P37/02 , C07K2/00 , C07K14/32 , C07K19/00 , C12N1/21 , C12N15/31 , C12N15/70 , C12P21/02 , C12Q1/68 , A61K39/02 , C07H21/04 , C12N15/74
CPC分类号： C07K14/32 , A61K39/00
摘要： Anthrax toxin, comprising of protective antigen (PA), lethal factor (LF) and edema factor (EF) is a major virulent factor of B. anthracis. Protective antigen, PA is the main component of all the vaccines against anthrax. The protective efficacy of PA is greatly increased if small quantities of LF of EF are incorporated into the vaccines. An ideal vaccine against anthrax should contain PA, LF and EF together, but this combination would be toxic. Therefore, the biologically inactive mutant preparations of PA, LF and EF may be used together for better immunoprotection. The present invention describes the method for generation of recombinant vaccine against anthrax, comprising of non-toxic, mutant anthrax toxin proteins. The procedure involves site-directed mutagenesis of the native genes of the toxin proteins, the expression and purification of the mutant proteins and finally characterization of these proteins.
摘要翻译：包含保护性抗原（PA），致死因子（LF）和水肿因子（EF）的炭疽毒素是炭疽杆菌的主要毒性因子。保护性抗原，PA是所有针对炭疽疫苗的主要成分。如果将小量的EF融入疫苗中，则PA的保护作用大大增加。针对炭疽的理想疫苗应包含PA，LF和EF，但这种组合是有毒的。因此，PA，LF和EF的生物活性突变体制剂可以一起用于更好的免疫保护。本发明描述了用于产生针对炭疽的重组疫苗的方法，其包含无毒的突变体炭疽毒素蛋白质。该方法涉及毒素蛋白质的天然基因的定点突变，突变蛋白的表达和纯化以及这些蛋白质的最终表征。

3. 发明授权

US07329513B2 High level constitutive production of anthrax protective antigen 失效
标题翻译：高水平组成型生产炭疽保护性抗原
公开(公告)号：US07329513B2
公开(公告)日：2008-02-12
申请号：US10494680
申请日：2001-12-07
申请人： Rakesh Bhatnagar , Waheed Sayed Mohsin , Vibha Chauhan
发明人： Rakesh Bhatnagar , Waheed Sayed Mohsin , Vibha Chauhan
IPC分类号： C12P21/04
CPC分类号： C07K14/32
摘要： The present invention relates to a process for preparing anthrax protective antigen protein from E. coli using fed batch culture. This process creates a constitutively expressing system for rapid, efficient, cost-effective and high-level production of anthrax PA from E. coli. The steps of the process involves, transforming E. coli DH5α cells with the recombinant constitutive expression plasmid containing the PA gene to obtain recombinant DH5α cells and testing the PA expression by lysis of said cells followed by denaturing gel electrophoresis and Western Blotting technique using PA antibodies. This is followed by fermentation and harvesting of the high cell density cells. The said cells are solubilized using 6-8 Molar Urea and separated by centrifugation. The urea denatured PA is isolated from said supernatant and purified and thereafter eluted.
摘要翻译：本发明涉及使用补料分批培养从大肠杆菌制备炭疽保护性抗原蛋白的方法。该过程创建了一种组成型表达系统，用于从大肠杆菌中快速，有效，成本有效和高水平地生产炭疽芽孢杆菌。该方法的步骤包括用包含PA基因的重组组成型表达质粒转化大肠杆菌DH5α细胞以获得重组DH5α细胞，并通过裂解所述细胞测试PA表达，然后通过变性凝胶电泳和使用PA抗体的Western Blotting技术。随后发酵和收获高细胞密度的细胞。所述细胞用6-8摩尔尿素溶解并通过离心分离。尿素变性PA从所述上清液中分离并纯化，然后洗脱。

4. 发明授权

US07888490B2 Process for the preparation of non-toxic anthrax vaccine 失效
标题翻译：无毒性炭疽疫苗的制备工艺
公开(公告)号：US07888490B2
公开(公告)日：2011-02-15
申请号：US10497673
申请日：2002-03-20
申请人： Rakesh Bhatnagar , Pankaj Gupta , Smriti Batra , Vibha Chauhan , Aparna Singh , Nidhi Ahuja , Praveen Kumar
发明人： Rakesh Bhatnagar , Pankaj Gupta , Smriti Batra , Vibha Chauhan , Aparna Singh , Nidhi Ahuja , Praveen Kumar
IPC分类号： C07H21/04 , C07H21/02 , A61K39/07
CPC分类号： C07K14/32 , A61K39/00
摘要： Anthrax toxin, comprising of protective antigen (PA), lethal factor (LF) and edema factor (EF) is a major virulent factor of B. anthracis. Protective antigen, PA is the main component of all the vaccines against anthrax. The protective efficacy of PA is greatly increased if small quantities of LF of EF are incorporated into the vaccines. An ideal vaccine against anthrax should contain PA, LF and EF together, but this combination would be toxic. Therefore, the biologically inactive mutant preparations of PA, LF and EF may be used together for better immunoprotection. The present invention describes the method for generation of recombinant vaccine against anthrax, comprising of non-toxic, mutant anthrax toxin proteins. The procedure involves site-directed mutagenesis of the native genes of the toxin proteins, the expression and purification of the mutant proteins and finally characterization of these proteins.
摘要翻译：包含保护性抗原（PA），致死因子（LF）和水肿因子（EF）的炭疽毒素是炭疽杆菌的主要毒性因子。保护性抗原，PA是所有针对炭疽疫苗的主要成分。如果将小量的EF融入疫苗中，则PA的保护作用大大增加。针对炭疽的理想疫苗应包含PA，LF和EF，但这种组合是有毒的。因此，PA，LF和EF的生物活性突变体制剂可以一起用于更好的免疫保护。本发明描述了用于产生针对炭疽的重组疫苗的方法，其包含无毒的突变体炭疽毒素蛋白质。该方法涉及毒素蛋白质的天然基因的定点突变，突变蛋白的表达和纯化以及这些蛋白质的最终表征。

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