CN1194093C - 细菌质粒 - Google Patents
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- CN1194093C CN1194093C CNB988044471A CN98804447A CN1194093C CN 1194093 C CN1194093 C CN 1194093C CN B988044471 A CNB988044471 A CN B988044471A CN 98804447 A CN98804447 A CN 98804447A CN 1194093 C CN1194093 C CN 1194093C
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Abstract
本发明涉及带有图1或图4所示核苷酸序列的质粒和DNA序列以及它们的用途。
Description
本发明涉及细菌质粒。
质粒是染色体外小的可独立复制的DNA分子，大多为环状，几乎存在于所有细菌和部分真核细胞以及线粒体中。质粒的大小在约1.5-300kb之间。
通常，细菌质粒为环状、共价、闭合以及超螺旋的。它们常常携带针对抗生素或重金属的抗性基因、用于代谢非典型底物的基因或者一系列种属特异性特征的基因，如代谢特性或毒力因子。一些质粒可被从一个细胞转移至另一个细胞。因为根据目前的观点，细菌的致病性也部分由其质粒的性质决定，因此日益需要澄清质粒DNA的性质。
已知肠杆菌科细菌家族中有14个主要变种合6个其它变种，其可形成不同的特性。典型的例子是埃希氏菌属(Escherichia)、沙门氏菌属(Salmonella)和克雷伯氏菌属(Klebsiella)。大肠杆菌(E.coli)是细菌遗传学的经典对象。只有发现并表征了大肠杆菌不同的毒力因子，才能发现该种属菌株在人和动物病原学上不同表现的普遍令人满意的解释。其中有的菌株为极端性的，从无毒力到高度毒力，如最近传播的称为“EHEC”的变种的例子。已经有一系列肠道外以及肠道大肠杆菌菌株的毒力因子被描述，其中部分已被很好地表征。对致病性大肠杆菌菌株，已发现血清型变种O6:K5毒力因子例如溶血蛋白和P伞毛粘附等，这些曾认为不出现在该血清型变种的非致病性代表菌中。
通常毒力基因在肠杆菌属的大质粒(约60kb)上可见。也有带小的所谓隐蔽性质粒的肠杆菌属，其功能至今无法方便的测定。
因为已知大肠杆菌的毒力因子也至少部分存在于质粒基因上，因此需要针对肠杆菌属特别是大肠杆菌中质粒的鉴定和表征的进一步研究，以改善例如肠杆菌属感染后疾病的诊断和治疗的可能性。质粒或其细菌性载体或相应的合成DNA可用于治疗或预防疾病，也可用于在微生物分析或诊断中以及营养生理学或微生态制剂方面的用途。此外，特别是在大肠杆菌中有的质粒已知为遗传工程中的表达载体，因此也需要增进对这类质粒性质的了解。
根据上述目的进行了针对大肠杆菌菌株DSM 6601的分子遗传学研究。在DNA序列分析的数据库程序帮助下，研究了获自该菌株的DNA序列，并与已知的其它细菌DNA序列比较。
菌株DSM6601含有两个大小分别为3177bp和5552bp的小质粒，分别表述为pMUT1和pMUT2。在本发明的一个具体实施方案中，涉及带有如图1所示DNA的序列的质粒。
小质粒pMUT1的DNA以线性片段形式被亚克隆至载体pUC18，用HindIII限制性酶切后测定了DNA序列。由图1可见。在本发明的一个具体实施方案中，涉及带有如图1所示核苷酸序列的DNA序列。所得DNA序列通过与GenEMBL数据库进行同源性比较进行检查；比较结果示于图2。
此次比较中，HindHI插入位点固定为位置1。质粒pMUT1的DNA位置200至800bp处与其它肠杆菌属质粒的不同复制起始位置(复制起点)有明显的同源性，特别是同质粒NTP1、NTP16和CloDF13。在位置950bp开始有一个570bp长的质粒NTP16同源区，其最初从鼠伤寒沙门氏菌(Salmonella typhimurium)中分离出来。此同源区含有mobA基因，该基因是质粒NTP16移动以及转录起点(oriT)。此外，从位置1790至1920bp发现与基因parA和cer明显同源的区域，其对于质粒分子的稳定性、连续转录和细胞分裂过程中的质粒分配十分重要。对于剩余DNA区域未鉴定出明显同源区。
此外，将质粒pMUT1的DNA序列转录为氨基酸序列并分析开放阅读框架的存在。研究了6种不同阅读框架的可能性。总共发现5个大小为143、62、56、49和48个氨基酸的开放阅读框架。分析的图示结果示于图3。
类似地，将较大质粒pMUT2用限制性酶SphI线性化后亚克隆至载体pUC18，随后全部测序。DNA序列示于图4。在GeneEMBL数据库程序帮助下研究用此法获得的DNA序列与已知DNA序列的同源性。结果图示于图5。质粒pMUT2的DNA与大肠杆菌不同ColE1质粒的复制区在位置890至1660bp明显同源。在3800至4950bp区也发现一个与ColE质粒的明显同源区。在此处认为其为ColE1质粒移动区的同源区。发现位置3770至4980bp区与溶血巴斯德氏菌(Pasteurella haemolytica)菌株A1同源。在该质粒上有在巴斯德氏菌属中编码抗微生物抗性蛋白质的基因。然而，由于同源区横跨属间区，所以可能也含有质粒移动必须的序列。
鉴定了与其它肠杆菌属质粒明显同源的两个区。由此发现它含有质粒移动必需的复制区域起点和移动区。pMUT2的剩余DNA部分中未鉴定出明显同源区。
同样，随后将质粒pMUT2的DNA序列转化为氨基酸序列，并分析开放阅读框架的存在。类似地，结果图示于图3。发现了带有327、318、264、76和63个氨基酸的5个开放阅读框架。
不仅质粒pMUT1和pMUT2此前未知，其组合也未在任何大肠杆菌菌株或其它肠杆菌属中发现。
质粒的发现使菌株DSM6601存在用于代谢和医药和/或营养性生理或微生态制剂方面用途的可能性。本发明的一个具体实施方案中，涉及质粒或DNA序列在微生物分析和/或诊断中的用途，和在制备医药和/或营养性生理或微生态制剂中的用途。
质粒的研究使肠杆菌属特别是埃希氏菌属的测定和分析更为精确。而且，这些质粒使它们作为可靠的表达载体用于遗传工程。本发明的一个具体实施方案中，涉及质粒或DNA序列作为表达载体的用途。
现对本发明进一步以实施例的方式解释。
实施例1
质粒分离
根据Birnboim等人(Birnboim，A.C.和Doly，J.(1979)核酸研究(Nucl.Acids Res.)7：1513-1523，一种用于筛选重组质粒DNA的快速碱提取方法)的方法进行质粒DNA的分离。
用一个细菌菌落接种3毫升LB培养基，37℃下搅拌培养过夜。在Eppendorf管中离心培养液，用吸尖去除培养基沉淀。细胞沉淀用100微升溶液I(50mM葡萄糖；10mM EDTA，pH8；25mM Tris-HCl，pH8)重悬。室温下温育5分钟后加入200微升溶液II(0.2N NaOH；1％SDS)，混合至澄清，将Eppendorf管置于冰上5分钟。然后向其中加入150微升溶液III(3M乙酸钠，pH4.8)，短暂搅拌直至染色体DNA出现絮状沉淀，将沉淀再次置于冰上5分钟。沉淀染色体DNA和细胞残片于离心管中离心5分钟被沉淀，带有质粒DNA的上清液被移至新管中。为纯化质粒DNA，加入50微升苯酚和150微升氯仿/异丙醇(24∶1)，短暂搅拌后离心2分钟。水相用吸尖移至新管中。用2倍体积冰冷的乙醇沉淀质粒DNA，离心10分钟。沉淀用70％乙醇洗涤，在Speedvac中干燥。质粒DNA在20微升双蒸水中重悬，保存于-20℃。
实施例2
DNA测序
根据F.Sanger等人(Sanger，F.，Nicklen，S.，和Coulson，A.R.(1977)，美国国家科学院院(Proc.Natl.Acad.Sci.USA)74：5463-54-67，用链终止抑制剂进行DNA测序)的方法进行DNA测序。
用Pharmacia LKB公司的T7测序试剂盒进行DNA测序。
变性步骤中，8微升(1.5至2微克)质粒DNA与2微升2N NaOH混合，短暂离心后室温下温育10分钟。用3微升3M乙酸钠，pH4.8，并用7微升双蒸水和60微升无水乙醇在-70℃下15分钟沉淀DNA。沉淀的DNA离心10分钟，用70％乙醇洗涤并干燥。
退火反应中，变性的DNA重悬于10微升双蒸水中，于2微升退火液和2微升引物(40ng)混合。沉淀在37℃下温育20分钟，以使引物与模板DNA结合。在室温下冷却反应物10分钟，然后立即用于标记反应或冻存于-20℃。对于标记反应，用吸尖将3微升标记混合物、1微升[α-P32]dATP和2微升T7聚合酶(T7聚合酶用酶稀释液以1∶5稀释)加入退火反应沉淀中，短暂混合后室温下温育5分钟。同时，已经制备好的用于末端终止反应的测序混合物(每管中带有2.5微升“G”、“A”、“T”和“C”混合物)在37℃预热。标记反应完成后，取其中4微升加入四种测序反应混合物的各一种，用吸尖短暂混合。末端终止反应在37℃下温育5分钟。分别加入5微升终止溶液结束末端终止反应。然后将沉淀转移至95℃的温育器中变性2分钟，再置于冰上。按“G”、“A”、“T”、“C”的顺序将2.5微升每种反应混合物加入测序胶[25.2g尿素、22ml双蒸水、6ml 10×TBE、10ml聚丙烯酰胺(40％)、2ml过硫酸铵(16mg/ml)、60微升TEMED]。在40瓦和1500伏条件下电泳4.5小时。
Claims (4)
1.带有如图1所示DNA的序列的质粒。
2.带有如图1所示核苷酸序列的DNA序列。
3.根据权利要求1的质粒或权利要求2的DNA序列在微生物分析和/或诊断中的用途。
4.根据权利要求1的质粒或权利要求2的DNA序列作为表达载体的用途。
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| DE19713543A DE19713543B4 (de) | 1997-04-02 | 1997-04-02 | Bakterielle Plasmide |
| DE19713543.9 | 1997-04-02 |
Related Child Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CNB2005100059434A Division CN100429313C (zh) | 1997-04-02 | 1998-04-01 | 细菌质粒 |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN1253590A CN1253590A (zh) | 2000-05-17 |
| CN1194093C true CN1194093C (zh) | 2005-03-23 |
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| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CNB988044471A Expired - Fee Related CN1194093C (zh) | 1997-04-02 | 1998-04-01 | 细菌质粒 |
| CNB2005100059434A Expired - Fee Related CN100429313C (zh) | 1997-04-02 | 1998-04-01 | 细菌质粒 |
Family Applications After (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CNB2005100059434A Expired - Fee Related CN100429313C (zh) | 1997-04-02 | 1998-04-01 | 细菌质粒 |
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| Country | Link |
|---|---|
| US (1) | US6391631B1 (zh) |
| EP (1) | EP0975774B1 (zh) |
| JP (2) | JP2000514661A (zh) |
| KR (1) | KR100398558B1 (zh) |
| CN (2) | CN1194093C (zh) |
| AR (1) | AR010140A1 (zh) |
| AT (1) | ATE277190T1 (zh) |
| AU (1) | AU739941B2 (zh) |
| BR (1) | BR9808458A (zh) |
| CA (1) | CA2285633C (zh) |
| CO (1) | CO4790114A1 (zh) |
| CZ (2) | CZ294686B6 (zh) |
| DE (2) | DE19713543B4 (zh) |
| DK (1) | DK0975774T3 (zh) |
| DZ (1) | DZ2454A1 (zh) |
| EA (2) | EA003738B1 (zh) |
| EE (1) | EE04944B1 (zh) |
| ES (1) | ES2229492T3 (zh) |
| HK (2) | HK1026233A1 (zh) |
| HU (1) | HU228182B1 (zh) |
| IL (2) | IL132101A0 (zh) |
| NZ (1) | NZ338000A (zh) |
| PE (1) | PE96799A1 (zh) |
| PL (2) | PL192699B1 (zh) |
| PT (1) | PT975774E (zh) |
| SI (1) | SI0975774T1 (zh) |
| SK (2) | SK284928B6 (zh) |
| TR (1) | TR199902434T2 (zh) |
| TW (1) | TW567225B (zh) |
| UA (1) | UA73072C2 (zh) |
| WO (1) | WO1998044134A2 (zh) |
| ZA (1) | ZA982733B (zh) |
Families Citing this family (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| DE19751242C2 (de) * | 1997-11-19 | 2001-02-08 | Pharma Zentrale Gmbh | DNA-Sequenzen aus Fimbriengenen von Escherichia coli Stamm DSM 6601 |
| EP1038035A1 (de) * | 1997-11-19 | 2000-09-27 | Pharma-Zentrale Gmbh | VERFAHREN ZUR IDENTIFIZIERUNG VON $i(ESCHERICHIA COLI) STAMM DSM 6601 |
| DE19915772C2 (de) * | 1998-11-18 | 2001-08-30 | Pharma Zentrale Gmbh | Verfahren zur Identifizierung von Escherichia coli Stamm DSM 6601 |
| DE10155928A1 (de) * | 2001-11-15 | 2003-06-12 | Degussa | recA-negativer und rhaB-negativer Mikroorganismus |
| US7648886B2 (en) * | 2003-01-14 | 2010-01-19 | Globalfoundries Inc. | Shallow trench isolation process |
| DE10328669B4 (de) * | 2003-06-26 | 2005-12-01 | Pharma-Zentrale Gmbh | Plasmidfreier Klon des E. coli Stammes DSM 6601 |
| CN100368549C (zh) * | 2006-03-29 | 2008-02-13 | 北京未名凯拓作物设计中心有限公司 | 一种细菌质粒及其衍生质粒与应用 |
| EP2466678B1 (en) | 2009-08-10 | 2017-11-22 | LG Chem, Ltd. | Lithium secondary battery |
Family Cites Families (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5843882A (en) * | 1995-04-27 | 1998-12-01 | The United States Of America As Represented By The Department Of Health And Human Services | Antiviral proteins and peptides |
| CN1060807C (zh) * | 1995-05-25 | 2001-01-17 | 中国科学院微生物所 | 大肠杆菌中表达外源基因的质粒和牛凝乳酶原的生产方法 |
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