CN1436245A - 从大肠杆菌中提取的脂多糖 - Google Patents
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Abstract
本发明涉及由大肠杆菌DSM 6601提取的脂多糖。这种LPS与大肠杆菌提取的目前已知的LPS特别在核心的磷酰化的糖组分和O－链的聚合度方面不同。脂质A在结构和生物学上相应于大肠杆菌的常规类型。所述的LPS不只适用于鉴定带有它的大肠杆菌菌株，还在保持其免疫调制作用的同时赋予其降低了的致病性。
Description
本发明涉及从大肠杆菌中提取的新的脂多糖。
所谓的内毒素是指细菌的结构成分，其不同于外毒素，不是由活细菌排泄出的，而是特别在自体消化作用后释放出来的。所谓的典型的内毒素涉及热稳定的脂多糖，下面称为LPS，从革兰氏阴性细菌的外细胞膜中提取。LPS由所谓的脂质A，核低聚糖以及特定的O-链构成，其中脂质A是LPS的毒素作用的主要贡献成分。
内毒素刺激宏观生物中免疫系统的介质，如白介素1，简称IL-1和肿瘤-坏死因子，简称TNFα的产生。
对肠细菌的内毒素，特别是大肠杆菌内毒素的组成，已经进行了一系列的研究，其中已经确定，S/R突变株一般只含有其O-链的一个重复单元(参见图1)。出发点是，在这些情况下编码O-链的聚合酶的基因有缺陷，因此只将一个重复单元转移到核低聚糖。与LPS结构相似，但是为其它结构的类型，经常也在人体病原菌，如奈瑟氏菌，弧菌，弯曲杆菌，螺杆菌等中发现。这些细菌拥有一种LPS，使它们可能通过一种特殊的分子模拟，此外像哺乳动物的糖蛋白和糖脂质的唾液酸和含有唾液酸的低聚糖的存在，避开宿主的免疫防护。大肠杆菌DSM 6601(分类号为大肠杆菌(E.Coli)NISSLE 1917 SK22/1，于1991年7月11日保藏于德意志微生物保藏中心，该保藏单位的地址为德国(D-38124)不伦瑞克马歇尔奥德路1b号)确定为O6-血清型。这种结构由P.E.Jansson等在Carbohydr.Res.131(1984)277-283中研究并发表。这种结构相应于图2中描述的结构式。
也有不同的研究组研究了大肠杆菌的脂质A，其中确定，脂质A的结构在一般情况下以六酰基形式存在，对于大肠杆菌的所有血清型是一致的(图3)。此六酰基化合物的结构在1984年由Th.Rietschel等的脂多糖的脂质A组分的结构和构象(Structure and conformationof the lipid A component of lipopolysaccharides)；内毒素手册(Handbook of Endotoxins)(Proctor，R.编辑)，第一卷，内毒素化学(E.Th.Rietschel编写)，Elsevier，阿姆斯特丹(1984)，187-220页中公开，相应于图3中的描述。
特定的O-链和脂质A通过核低聚糖互相连接。目前已知大肠杆菌有五种不同的核低聚糖，这方面参阅O.Holst等的脂多糖的核心区的化学结构，在：细菌的内毒素的脂多糖(Bacterial EndotoxicLipopolysacehariides)，第一卷，Morrison D.C.和Ryan，J.L.(编辑)，Boca Raton，FL，美国(1992)第135-170页(参见图4)。
在大肠杆菌菌株DSM 6601的LPS的研究方面已经确定，脂质A在其组成上对应于通常对大肠杆菌描述的脂质A的六酰基形式。
关于IL-1和TNFα的释放方面的研究在人体的单核细胞中确认，脂质A具有相同的活性，因此很有可能对应于大肠杆菌的脂质A的已知结构(参见图5，图6)。这种假设通过化学分析证实。
有关大肠杆菌DSM 6601的特定O-抗原的结构令人惊奇的事实是，明显地链中总是只存在唯一一个“重复单元”(参见图1)，由此可以得出的结论是，菌株DSM 6601涉及S/R突变株，这对于一个人体分离物是极其特殊的。但是，这种“重复单元”的结构，如血清学分析表明，对应于大肠杆菌O6的O-链的基本模式。
尽管菌株DSM 6601的核心区相应于已知的R1结构。然而，结构的特殊性在于，分析确定了每个LPS分子具有8个磷酸酯残基，其中脂质A一般分摊到只有2个磷酸酯残基。此外还发现了非化学计量含量的焦磷酸乙醇胺。
因此概括地确定，菌株DSM 6601的LPS与目前已知的由大肠杆菌提取的LPS特别在核心的磷酰化的糖组分方面和在O-链的聚合度方面区别很大。脂质A在结构和生物学上相应于对于大肠杆菌常规的类型，这强调了这种物质在生物活性方面的作用。所述的LPS不只是适用于鉴别带有它的大肠杆菌菌株，还在保持其免疫调制作用的情况下赋予其降低了的致病性。O-链是β-糖苷而不是α-糖苷连接的事实，对于大肠杆菌可以用S/R突变株DSM 6601的例子首次明确地表明。
大肠杆菌DSM 6601的脂多糖(LPS)涉及一种新型的平滑-粗糙(S/R)结构，其一方面由目前已知的部分结构组成(O-特定链，核低聚糖和脂质A)，另一方面以这里存在的复杂的形式表征为第一次且完整的结构(参见图7)。仅由血清型O6的一个唯一的重复单元构成的O-特定链以β-糖苷连接到核低聚糖上，因此不同于O-链内部(α-糖苷)的连接。核低聚糖具有R1结构，一种通过用R1特异性抗体进行血清学研究证实的化学判断。脂质A组分具有大肠杆菌脂质A特征性的某种化学结构。
大肠杆菌菌株DSM 6601的LPS具有一个奇特的均一性。仅仅在磷酸酯取代基方面存在非均一性(PP和P-Etn对P和P)可以加以确定，其首次以这种形式被描述。借助复杂的NMR分析可以明确地确定在核低聚糖R1核低聚糖中的P-Etn-取代基在第二个庚糖(HepII)的2-位。
菌株DSM 6601的LPS的完整的结构在图7中描述。
下面借助实施例对本发明进一步阐述。
实施例1
LPS的制备
LPS由洗涤并干燥的细菌生物质在改进的苯酚/水萃取工艺后获得；就此方面参阅O.Westphal等，细菌的脂多糖，用苯酚/水萃取和这种工艺的进一步应用，Meth.Carbohydr.Chem.，第V卷(1965)第83-91页。
47g事先用蒸馏水洗涤两次的冻干的细菌按照Westphal和Jann相应的改进的规程提取。这种改进在于，随后进行的对含水提取物的酶处理(DNA酶，RNA酶，蛋白质酶K)，其用于除去可能出现的杂质蛋白质和DNA/RNA组分。为此室温下向这种水相中加入各20mgRNA酶(核糖核酸酶A，牛胰，Sigma)和20mg DNA酶(DNA酶1，牛胰，II级，Sigma)，混合物在室温下搅拌30h。然后，加入20mg蛋白质酶K(Tritirachium album，Boehringer，Mannheim)，再搅拌12h。然后，悬浮液在4℃下对15L蒸馏水渗析24小时三次，再冻干。酶处理的提取物重新吸收在蒸馏水中，以致于最后浓度达到50mg/mL。这种悬浮液在冷状态下超速离心分离(155，000×g，4℃，4h)三次。沉淀物(LPS)用150mL蒸馏水吸收，再相对于水渗析三天一次，然后冻干(产量LPS：1.45g，3.1％m/m)。
实施例2
来自大肠杆菌菌株DSM 6601的LPS的分析
己糖胺(HexN)(这里指葡糖胺+半乳糖胺，GlcN+GalN)按照改进的Morgan-Elson试验来测定(Strominger，J.L.，Park，J.T.Thompson，R.E.J.Biol.Chem.234，3263-3268(1959))，但是也可选择利用HPLC(PICO-TAG，Waters)来测定。不同于Morgan-Elson试验，这种分析方法不仅可以分开测定和量化GlcN和GalN，而且还可以平行查出经常在LPS中出现的GlcN磷酸酯，2-乙醇胺(Etn)和2-乙醇胺磷酸酯(Etn-P)的存在。气液色谱(GC)利用Varian 3700GC或Hewlett Packard(HP 5890系列II)色谱通过毛细管柱(熔融硅胶SPB-5，30m，Supelco)进行。联合的气液色谱/质谱(GC-MS)利用装有HP-1毛细管柱(30m，Hewlett Packard)的质谱仪(HP Modell5989)完成。GC或GC-MS分析用于测定糖醇乙酸酯形式的中性糖(Glc，Gal，Hep，Man)(Sawardeker，J.S.，Slonerker，J.H.Jeanes，A.Anal.Chem.37，1602-1604(1967))，以及在强烈的甲醇醇解(2MHCl/MeOH，120℃，16h)(Wollenweber，H.W.和Rietschel，E.Th.，脂多糖(脂质A)脂肪酸的分析，J.Microbiol.Meth.11，(1990)195-211)和氯仿提取后进行测定和量化脂肪酸甲酯衍生物形式的脂肪酸。两种GC分析方法都在150℃(恒温3min)开始，借助5℃/min的线性温度梯度升温至320℃。磷酸酯根据Lowry等(Lowry，O.H.，Roberts，N.R.，Leiner，K.Y.，Wu，M.KL.Farr，A.L.，J.Biol.Chem.207，1-17(1954))，2-氧代-3-脱氧-D-甘露辛酮糖酸(Kdo)借助硫代巴比妥酸试验测定(Waravdekar，V.C.& Saslaw，L.D.，J.Biol.Chem.234，1945-1950(1959))。
游离脂质A和核低聚糖的制备和纯化
LPS(258.8mg)悬浮在25mL 0.1M NaOAc/HOAc(pH4.4)中，在100℃进行温和的酸水解1h。然后用25mL氯仿从水解产物中萃取亲油组分(脂质A)三次(产量23.2mg)。有机相中的脂质A借助制备的薄层色谱(PSC)进一步提纯(2mmPSC Kieselgel 60板，E.Merck，Darmstadt)，其用氯仿-甲醇-水100∶75∶15(v/v/v)进行色谱提纯，通过浸入在蒸馏水中展开。用这种方法得到6个级分，其中主要的级分(Rf≈0.4)是纯化的二磷酰化的六酰基脂质A(DPHLA-Ec6601)。提纯的DPHLA-Ec6601(产量2.06mg)溶解在氯仿-甲醇8∶2(v/v)中，在进行MALDI-TOF-MS分析前用离子交换剂(Amberlite IRA 120，H+型)处理。纯化的DPHLA-Ec6601的等分试样(250μg)用于生物学试验。
对氯仿抽提物的含水相进行冻干(产量272mg)，低聚糖借助TSK-柱[3.5×90cm，TSK HW-40(S)，E.Merck]用吡啶-醋酸-水8∶20∶2000，(v/v/v)进一步纯化。各个低聚糖级分(Pool A，B，C和D)借助GC-MS和NMR-谱分析。含有O-链的糖组分(Man，GalNac)和核低聚糖的糖组分(Hep，Kdo)的主要级分(Pool A，#28-41；49.05mg)，进行进一步纯化。其它级分含有单糖，没有详细研究的Kdo的人造物(Artefakte)(酐和内酯)和最终盐。TSK分离的主要级分不仅在GC-MS分析中而且在NMR-分析中都表现出核低聚糖(Kdo，Gal，Hep)和O-链(Man，GalNac)的所有组分，因此进一步处理。
为此首先检测是否分析用的高压-阴离子交换色谱(HPAEC)适合将低聚糖纯化为均相。这里应用带有分析用的CarboPac PA1柱；(4.6mm×250mm)和线性的盐梯度(5min在0值，然后50min内升至0.5M NaOAc)，流速为1mL/min的用于分析复杂糖结构的特殊的HPLC方法(DIONEX体系)。洗脱液借助脉冲电流检测器(PAD)检测还原当量(糖分子)。用这种方法可以得到四种低聚糖级分，其借助半制备的HPAEC，按类似的方法进一步提纯。
半制备的HPAEC借助CarboPac PA1柱；[(9mm×250mm)Dionex体系]用与如在分析用的HPAEC(5min在0值，然后50min内升为0.5M NaOAc)中一样的盐梯度和4mL/min的流速完成。低聚糖(42mg；从TSK-柱得到的pool A)在半制备的HPAEC上的装载是在两个类似的HPAEC进程中进行。洗脱液每分钟收集一个级分，各个级分利用分析用的HPAEC分别进行研究。按照这种方法利用半制备的HPAEC得到两个主要的级分(级分I，保留时间tR～12min和组分II，tR～15min)。两种HPAEC级分必须在MALDI-TOF-MS和NMR分析之前借助G-10柱(2.5×120cm)脱盐(产量：级分I 4.68mg；级分II 4.39mg)。
基体辅助的激光解吸/离子化时间飞行(MALDI-TOF)质谱分析
基体辅助的激光解吸/离子化时间飞行质谱分析(MALDI-TOF-MS)在Bruker-ReflexII飞行时间质谱仪(Bruker-Franzen Analytik，Bremen)中只以线性的组态和负模式，在加速电压为20kV和“延迟的离子萃取”的状态下进行。试样首先以10μg/μL的浓度溶解在氯仿(脂质A)或蒸馏水(低聚糖级分)中，其中2μL的等分试样用2μL于甲醇含有0.5M 2，4，6-三羟基乙酰苯(Aldrich，Steinheim)的基体溶液溶解。这种混合物的等分试样(0.5μL)涂敷在金属杆上，用电吹风机干燥。
NMR谱图分析
一维(1D)1H和31P-NMR-谱图与二维(2D)NMR谱图用Bruker AvanceDRX-600谱图仪(Bruker，Rheinstetten)进行，13C NMR谱图用BrukerAMX-360谱图仪在300K用2H2O进行。每次测量前，样品用氘代水2H2O冻干两次。作为外标使用丙酮(δH2.225ppm，δC31.45ppm)或85％H3PO4(δPOppm)。标准的Bruker软件(XWINNMR 1.3)用于采集NMR数据。用于TOCSY(整体相关谱图)或NOESY(核奥佛好塞增强谱图)的混合时间是100或500ms。
血清学分析
血清学分析作为用三种不同的抗体显现的Western-Blots进行。
1.多克隆抗-O6-抗血清(家兔)用大肠杆菌菌株DSM 6601(血清型O6∶K5∶H1)在汉堡的卫生研究院制备(Bockemühl教授)。
2.多克隆抗-大肠杆菌R1-抗血清(家兔，内部记号：K299/d58)通过用粗糙型突变株的免疫得到，其具有R1-核心(抗-R1)。
3.应用一种单克隆抗体(WN1-222-5，内部记号F167)，其对从最小结构(＞Rd)起的所有的大肠杆菌核低聚糖广泛地交叉反应。
表：
由菌株DSM 6601提取的大肠杆菌LPS的成分分析成分 成分含量
nmol/mg(mol/LPS)a糖 1.分析 2.分析GlcNb 283(1.8) n.b.GalN 139(0.9) n.b.HexNc 591(3.8) 589(2.9)Kdo 248(1.6) 242(1.2)Man 321(2.1) 383(1.9)Gal 474(3.0) 557(2.8)Glc 1069(6.9) 1291(6.4)L，D-Hep 566(3.6) 442(2.2)极性端基P 1188(7.6) 1146(5.7)Etn-P 85(0.5) n.b.脂肪酸12∶0 130(0.8) 162(0.8)14∶0 156(1.0) 201(1.0)14∶0(3-OH) 460(3.0) 504(2.5)16∶0 痕量 痕量a各组分的摩尔比(在括号中)因为O-链中GalNAc和GlcNAc的存在而标化到肉豆蔻酸的值(14∶0)(1.0mol 14∶0/mol LPS)。
bGlcN借助氨基酸分析仪测定。数值由GlcN和GlcN-6P的总和得到。
cHexN根据Morgan-Elson光度测定。
n.b.未确定。
按上述方法得到的LPS-制品与比较的LPS一起进行聚丙烯酰胺凝胶电泳(参见图1)。对于LPS的SDS-PAGE-分析用16％聚丙烯酰胺凝胶进行(英国，Laemmli，噬菌体T4的头部的组装过程中结构蛋白质的断裂，Nature，227，680-685(1970))。LPS-带借助灵敏的碱性银染方法着色(C.M.Tsai和Frasch，C.F.，一种用于检测聚丙烯酰胺凝胶中的脂多糖的敏感的银染方法Anal.Biochem.，119，1982，115-119)。
研究结果在图1中说明。
实施例3
生物活性
a)IL-1-活性
IL-1-活性借助MNC-增殖试验在培养液的上清液中测定。人体单核细胞(MNC)由志愿捐献者的外周血液分离(8×105MNC/200mL)，转移到玻璃瓶中，同时掺入试验物质。对于体外生物活性的试验，首先用LPS刺激(10ng/mL)细胞。在8个小时的温育时间后研究150mL培养液的上清液的细胞因子的释放。IL-1-活性借助纤维细胞-增殖试验在培养液的上清液中测定。对此需要的成纤维细胞从人体的包皮取得。这种成纤维细胞的增殖通过IL-1提高。通过在概率分析中比较培养液的上清液的剂量作用曲线与标准曲线，测定培养液的上清液中的生物活性。由已知内毒素活性的细菌菌株(弗里德奥沙门氏菌)提取的LPS用作阳性参照(阳性对照)，因此在图5中引用。
b)TNFα-活性
培养液的上清液的TNFα-活性在细胞毒性试验中用TNF敏感的细胞系L929测定。在概率分析中比较培养液的上清液的剂量作用曲线与标准曲线，可以测定TNF活性。这里也使用已知内毒素活性的由弗里德奥沙门氏菌中提取的LPS作为阳性对照。结果在图6中图解说明。
结果表明，关于IL-1和TNFα-释放(Ausschuettung)，可以确定在用作标准和阳性对照的由弗里德奥沙门氏菌中提取的LPS与由菌株DSM 6601中提取的LPS之间没有显著的区别(图5和6，下图)。这也可以由菌株DSM 6601的脂质A与大肠杆菌的高纯化的脂质A几乎具有全等的活性来证明(图5和6，上图)。
Claims (6)
1.具有图7描述的结构的脂多糖(LPS)。
2.权利要求1的LPS，其特征为，每个LPS分子含有8个磷酸酯残基。
3.权利要求1或2的LPS，其特征为，每个LPS分子含有0.5molP-Etn。
4.获得按权利要求1至3的LPS的方法，其特征为，洗涤并干燥的大肠杆菌生物质按已知方式进行苯酚/水提取，这样得到的提取物用RNA酶/DNA酶和蛋白酶K处理。
5.权利要求4的方法，其特征为，使用大肠杆菌菌株DSM 6601。
6.按权利要求1至3的由大肠杆菌菌株DSM 6601提取的脂多糖在微生物学，生物技术，分析，诊断和/或医药方面的应用。
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| US20130066064A1 (en) * | 2010-05-20 | 2013-03-14 | Glaxosmithkline Biologicals S.A, | Novel process |
| CN105137073A (zh) * | 2015-08-06 | 2015-12-09 | 中国兽医药品监察所 | 牛布鲁氏菌胶体金抗体检测试纸条 |
| PL432842A1 (pl) * | 2020-02-10 | 2021-08-16 | Uniwersytet Warszawski | LPS do zastosowania w indukowaniu u osobnika tolerancji na alergeny pokarmowe wywołujące alergie pokarmowe zależne od IgE |
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| DE19844191A1 (de) * | 1998-09-28 | 2000-03-30 | Pharma Zentrale Gmbh | Lipopolysaccharide aus Escherichia coli |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN100395328C (zh) * | 2003-06-26 | 2008-06-18 | 制药中心有限公司 | 大肠杆菌菌株dsm 6601的无质粒的克隆 |
| CN100354428C (zh) * | 2005-01-13 | 2007-12-12 | 丁友玲 | 隧道式干热灭菌设备测试用内毒素指示剂的制备方法 |
| CN116249720A (zh) * | 2020-03-13 | 2023-06-09 | 赫菲斯托斯制药公司 | 解毒脂多糖(lps)，天然无毒lps及其用途 |
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