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1. 发明授权

US08435759B2 Plasmid-encoded neurotoxin genes in Clostridium botulinum serotype A subtypes 有权
标题翻译：肉毒毒素肉毒杆菌血清型A亚型中的质粒编码的神经毒素基因
公开(公告)号：US08435759B2
公开(公告)日：2013-05-07
申请号：US12664800
申请日：2008-06-27
申请人： Eric A. Johnson , Kristin M. Marshall , Sabine Pellett , Marite Bradshaw
发明人： Eric A. Johnson , Kristin M. Marshall , Sabine Pellett , Marite Bradshaw
IPC分类号： C12P21/00 , C12N15/74
CPC分类号： C12N9/52 , C07K14/33 , C12N15/74 , C12Y304/24069
摘要： The present invention provides a novel isolated plasmid, wherein the plasmid is a native plasmid found in unique C. botulinum type A strains and encode either BoNT/A3 or BoNT/A4 and BoNT/B. The present invention also provides a method of obtaining a plasmid-encoded botulinum neurotoxin and botulinum neurotoxin complex comprising the step of isolating a plasmid encoding the cntA/A or cntA/B neurotoxin gene and genes encoding protein components of the toxin complex from a C. botulinum type A strain. The inventors performed comparative analyzes of representative BoNT/A subtype strains by pulsed-field gel electrophoresis (PFGE) and Southern hybridizations with probes specific for the BoNT/A and B genes, cntA/A and cntA/B. Unexpectedly, the inventors determined that the genes encoding BoNT/A3 in the A3 strain, and BoNT/A4 and BoNT/B in the A4 strain, are on plasmids.
摘要翻译：本发明提供了一种新的分离质粒，其中质粒是在独特的A型肉毒杆菌中发现的天然质粒，并且编码BoNT / A3或BoNT / A4和BoNT / B。本发明还提供了获得质粒编码的肉毒杆菌神经毒素和肉毒杆菌神经毒素复合物的方法，包括分离编码cntA / A或cntA / B神经毒素基因的质粒和编码来自C的毒素复合物的蛋白质组分的基因的步骤。 A型肉毒杆菌。本发明人通过脉冲场凝胶电泳（PFGE）和针对BoNT / A和B基因cntA / A和cntA / B特异性探针的Southern杂交进行了代表性的BoNT / A亚型菌株的比较分析。出人意料的是，本发明人确定了A3菌株中编码BoNT / A3的基因以及A4菌株中的BoNT / A4和BoNT / B在质粒上。

2. 发明授权

US10253074B2 Nontoxigenic Clostridium botulinum strains and uses thereof 有权
公开(公告)号：US10253074B2
公开(公告)日：2019-04-09
申请号：US12550692
申请日：2009-08-31
申请人： Eric A. Johnson , Marite Bradshaw , Kristin M. Marshall
发明人： Eric A. Johnson , Marite Bradshaw , Kristin M. Marshall
IPC分类号： C12N15/10 , C07K14/33 , C12N15/09 , C12N15/87
摘要： A mutant strain of the bacterium Clostridium botulinum having an inactivated botulinal neurotoxin gene is disclosed. The mutant strain contains an artificially created and inserted modified intron vector between nucleotides 580 and 581 of the sense strand of the gene. The mutant strain can be used in microbiological challenge testing of foods and food processing methods.

3. 发明申请

US20100112149A1 Nontoxigenic Clostridium Botulinum Strains and Uses Thereof 审中-公开
标题翻译：无毒性肉毒梭菌菌株及其用途
公开(公告)号：US20100112149A1
公开(公告)日：2010-05-06
申请号：US12550692
申请日：2009-08-31
申请人： Eric A. Johnson , Marite Bradshaw , Kristin M. Marshall
发明人： Eric A. Johnson , Marite Bradshaw , Kristin M. Marshall
IPC分类号： G01N33/02 , G01N33/04 , G01N33/12 , G01N33/03 , C12N1/21
CPC分类号： C07K14/33 , C12N15/09
摘要： A mutant strain of the bacterium Clostridium botulinum having an inactivated botulinal neurotoxin gene is disclosed. The mutant strain contains an artificially created and inserted modified intron vector between nucleotides 580 and 581 of the sense strand of the gene. The mutant strain can be used in microbiological challenge testing of foods and food processing methods.
摘要翻译：公开了具有失活的肉毒杆菌神经毒素基因的肉毒梭菌的突变菌株。突变菌株在基因的有义链的核苷酸580和581之间含有人工产生和插入的修饰的内含子载体。突变菌株可用于食品和食品加工方法的微生物攻毒检测。

4. 发明申请

US20110171734A1 CONJUGATIVE PLASMIDS AND METHODS OF USE THEREOF 审中-公开
标题翻译：结合剂及其使用方法
公开(公告)号：US20110171734A1
公开(公告)日：2011-07-14
申请号：US12905592
申请日：2010-10-15
申请人： Eric A. Johnson , Kristin M. Marshall , Marite Bradshaw
发明人： Eric A. Johnson , Kristin M. Marshall , Marite Bradshaw
IPC分类号： C12N15/74 , C12N15/63
CPC分类号： C12N15/74
摘要： The present invention provides novel C. botulinum conjugatively transmissible plasmids and methods of use thereof. Specifically, described herein are novel, conjugatively transmissible clostridial plasmids which are capable of being transferred among and between clostridial species. The novel plasmids of the present invention therefore permits the delivery of heterologous clostridial genes into a clostridial host, such as C. botulinum, and the expression of genes of interest in that host, including clostridial toxins and the nontoxigenic components of the toxin complex, toxin fragments, or antigenic portions thereof, in a way both that ensures abundant expression and facilitates purification. Furthermore, toxins with altered structures, chimeric, hybrid toxins, and other toxin derivatives valuable in medicine could be synthesized in this system.
摘要翻译：本发明提供了新型肉毒杆菌共轭传递质粒及其使用方法。具体地，本文描述的是能够在梭菌之间和之间转移的新颖的，共轭传播的梭菌质粒。因此，本发明的新型质粒允许将异源梭菌基因递送至梭菌宿主，例如肉毒杆菌，以及该宿主中感兴趣的基因的表达，包括梭菌毒素和毒素复合物的无毒成分，毒素片段或其抗原部分，以确保丰富的表达并促进纯化的方式。此外，在该系统中可以合成具有改变的结构的毒素，嵌合型，杂合毒素和在医学中有价值的其它毒素衍生物。

5. 发明申请

US20090017495A1 ESCHERICHIA COLI-DERIVED VACCINE AND THERAPY AGAINST BOTULISM 有权
标题翻译： ESCHERICHIA派生的疫苗和治疗效果
公开(公告)号：US20090017495A1
公开(公告)日：2009-01-15
申请号：US12040542
申请日：2008-02-29
申请人： Michael Baldwin , Eric A. Johnson , Joseph T. Barbieri , Marite Bradshaw , William H. Tepp , Christina L. Pier
发明人： Michael Baldwin , Eric A. Johnson , Joseph T. Barbieri , Marite Bradshaw , William H. Tepp , Christina L. Pier
IPC分类号： C12N15/09 , C07K14/33
CPC分类号： C12N9/52 , A61K39/0258 , A61K39/08 , A61K2039/523 , C12Y304/24069 , Y02A50/469 , Y02A50/474
摘要： A method of producing botulinum toxin C-terminal receptor binding domain (HCR) is disclosed. The one embodiment, the method comprises the steps of (a) preparing E. coli transformed with an expression vector comprising DNA encoding HCR protein, (b) inducing expression of the HCR protein at a reduced temperature in a culture media, and (c) purifying the HCR protein via extraction, wherein the extraction comprises a clarification by centrifugation and a filtration, wherein the purified HCR protein is at least 10 mg/L of culture medium.
摘要翻译：公开了产生肉毒杆菌毒素C末端受体结合结构域（HCR）的方法。该方法包括以下步骤：（a）用包含编码HCR蛋白的DNA的表达载体转化大肠杆菌，（b）在培养基中降低温度诱导HCR蛋白的表达，和（c）通过提取纯化HCR蛋白质，其中提取物通过离心和过滤进行澄清，其中纯化的HCR蛋白质为至少10mg / L的培养基。

6. 发明授权

US06444209B1 Hybrid botulinal neurotoxins 有权
标题翻译：混合肉毒杆菌神经毒素
公开(公告)号：US06444209B1
公开(公告)日：2002-09-03
申请号：US09706047
申请日：2000-11-03
申请人： Eric A. Johnson , Michael C. Goodnough , Marite Bradshaw , William H. Tepp
发明人： Eric A. Johnson , Michael C. Goodnough , Marite Bradshaw , William H. Tepp
IPC分类号： A61K39385
CPC分类号： C07K14/33 , A61K39/00 , A61K47/6415 , Y10S435/842 , Y10S530/825
摘要： A hybrid botulinal neurotoxin is disclosed. In one embodiment, the neurotoxin comprises a combination of a botulinal neurotoxin heavy chain and light chain, wherein the light chain and heavy chain are not of the same serotype and wherein the heavy and light chains are linked by a homobifunctional sulfydryl linker. A method for creating hybrid neurotoxins comprised of different functional domains is also disclosed.
摘要翻译：公开了一种混合肉毒杆菌神经毒素。在一个实施方案中，神经毒素包括肉毒杆菌神经毒素重链和轻链的组合，其中轻链和重链不是相同的血清型，并且其中重链和轻链通过同双功能巯基接头连接。还公开了由不同功能域组成的混合神经毒素的方法。

7. 发明授权

US07820411B2 Escherichia coli-derived vaccine and therapy against botulism 有权
标题翻译：大肠杆菌来源的疫苗和针对肉毒中毒的治疗
公开(公告)号：US07820411B2
公开(公告)日：2010-10-26
申请号：US12040542
申请日：2008-02-29
申请人： Michael Baldwin , Marite Bradshaw , William H. Tepp , Eric A. Johnson , Joseph T. Barbieri , Christina L. Pier
发明人： Michael Baldwin , Marite Bradshaw , William H. Tepp , Eric A. Johnson , Joseph T. Barbieri , Christina L. Pier
IPC分类号： C12P21/02
CPC分类号： C12N9/52 , A61K39/0258 , A61K39/08 , A61K2039/523 , C12Y304/24069 , Y02A50/469 , Y02A50/474
摘要： A method of producing botulinum toxin C-terminal receptor binding domain (HCR) is disclosed. The one embodiment, the method comprises the steps of (a) preparing E. coli transformed with an expression vector comprising DNA encoding HCR protein, (b) inducing expression of the HCR protein at a reduced temperature in a culture media, and (c) purifying the HCR protein via extraction, wherein the extraction comprises a clarification by centrifugation and a filtration, wherein the purified HCR protein is at least 10 mg/L of culture medium.
摘要翻译：公开了产生肉毒杆菌毒素C末端受体结合结构域（HCR）的方法。该方法包括以下步骤：（a）用包含编码HCR蛋白的DNA的表达载体转化大肠杆菌，（b）在培养基中降低温度诱导HCR蛋白的表达，和（c）通过提取纯化HCR蛋白质，其中提取物通过离心和过滤进行澄清，其中纯化的HCR蛋白质为至少10mg / L的培养基。

8. 发明申请

US20090182123A1 Method of Preparing Botulinum Neurotoxin Type E Light Chain 有权
标题翻译：肉毒杆菌神经毒素E型轻链的制备方法
公开(公告)号：US20090182123A1
公开(公告)日：2009-07-16
申请号：US12273157
申请日：2008-11-18
申请人： Eric A. Johnson , Marite Bradshaw , Michael Baldwin , Joseph T. Barbieri
发明人： Eric A. Johnson , Marite Bradshaw , Michael Baldwin , Joseph T. Barbieri
IPC分类号： C07K14/33
CPC分类号： C07K14/33
摘要： The present invention provides a preparation of botulinum toxin light chain type A or E, wherein the preparation is both catalytically active and soluble. Preferably, the preparation consists essentially of amino acid residues 1 through 425 of the botulinum toxin light chain type A. A method of screening inhibitors is also provided, wherein the method comprises exposing a test inhibitor to the preparation of botulinum toxin light chain type A and evaluating the biological activity of the preparation. In another embodiment, a method of providing a catalytically active, soluble preparation of botulinum toxin light chain, type A is provided, wherein the method comprises obtaining an expression vector comprising a DNA sequence encoding amino acid residues 1-425 and expressing a polypeptide.
摘要翻译：本发明提供了A型或E型轻链型肉毒杆菌毒素的制备方法，其中所述制剂既具有催化活性，又具有可溶性。优选地，制剂基本上由A型肉毒毒素轻链的氨基酸残基1至425组成。还提供了筛选抑制剂的方法，其中所述方法包括将测试抑制剂暴露于A型轻链A型肉毒杆菌毒素的制备中，评估制剂的生物活性。在另一个实施方案中，提供了提供A型肉毒毒素轻链的催化活性可溶性制剂的方法，其中所述方法包括获得包含编码氨基酸残基1-425的DNA序列并表达多肽的表达载体。

9. 发明授权

US07833524B2 Method of preparing botulinum neurotoxin type E light chain 有权
标题翻译：制备肉毒杆菌神经毒素E型轻链的方法
公开(公告)号：US07833524B2
公开(公告)日：2010-11-16
申请号：US12273157
申请日：2008-11-18
申请人： Eric A. Johnson , Marite Bradshaw , Michael Baldwin , Joseph T. Barbieri
发明人： Eric A. Johnson , Marite Bradshaw , Michael Baldwin , Joseph T. Barbieri
IPC分类号： C12P1/04
CPC分类号： C07K14/33
摘要： The present invention provides a preparation of botulinum toxin light chain type A or E, wherein the preparation is both catalytically active and soluble. Preferably, the preparation consists essentially of amino acid residues 1 through 425 of the botulinum toxin light chain type A. A method of screening inhibitors is also provided, wherein the method comprises exposing a test inhibitor to the preparation of botulinum toxin light chain type A and evaluating the biological activity of the preparation. In another embodiment, a method of providing a catalytically active, soluble preparation of botulinum toxin light chain, type A is provided, wherein the method comprises obtaining an expression vector comprising a DNA sequence encoding amino acid residues 1-425 and expressing a polypeptide.
摘要翻译：本发明提供了A型或E型轻链型肉毒杆菌毒素的制备方法，其中所述制剂既具有催化活性，又具有可溶性。优选地，制剂基本上由A型肉毒毒素轻链的氨基酸残基1至425组成。还提供了筛选抑制剂的方法，其中所述方法包括将测试抑制剂暴露于A型轻链A型肉毒杆菌毒素的制备评估制剂的生物活性。在另一个实施方案中，提供了提供A型肉毒毒素轻链的催化活性可溶性制剂的方法，其中所述方法包括获得包含编码氨基酸残基1-425的DNA序列并表达多肽的表达载体。

10. 发明授权

US5955368A Expression system for clostridium species 失效
标题翻译：梭菌物种的表达系统
公开(公告)号：US5955368A
公开(公告)日：1999-09-21
申请号：US56075
申请日：1998-04-06
申请人： Eric A. Johnson , Marite Bradshaw , Julian I. Rood , Dena Lyras
发明人： Eric A. Johnson , Marite Bradshaw , Julian I. Rood , Dena Lyras
IPC分类号： C07K14/33 , C12N1/21 , C12N15/74 , C12N15/64 , C12N15/70
CPC分类号： C07K14/33 , C12N15/74
摘要： A system is used to express clostridial gene constructions in a clostridial host. A mobilizable transfer plasmid is described which permits the direct transfer of the plasmid, and genes carried on it, from E. coli into Clostridium species. A promoter is described for use in clostridial species. Also, a useful host strain is used which is nontoxigenic and which permits high levels of expression of clostridial genes using the clostridial promoter.
摘要翻译：系统用于在梭菌宿主中表达梭菌基因构建。描述了可移动的转移质粒，其允许质粒及其上携带的基因从大肠杆菌直接转移到梭菌属物种中。描述了用于梭菌物种的启动子。此外，使用无毒的有用的宿主菌株，并且使用梭菌启动子允许梭菌基因的高水平表达。

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