CN1205223C - 具有减弱lgE反应活性的昆虫毒性过敏原及其生产方法 - Google Patents
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Abstract
本发明涉及重组昆虫毒性过敏原和产生它们的特异方法。所说的过敏原按照它们是否以与天然一致或不同的折叠(构象)来产生而变化。具有不会在天然状态下出现的折叠的该蛋白具有减弱的IgE反应活性或过敏原性，并因此可在过敏的免疫治疗方面用作治疗试剂。
Description
本发明涉及重组昆虫毒性过敏原和其生产方法，此处所说的过敏原可根据所用的生产方法的不同通过与天然一致的或与天然相反的折叠(构象)来加以区分。
与天然分子一致的折叠形式的应用为对过敏者(尤其是昆虫毒性过敏者)进行单过敏原区分诊断(胞外或胞内)。
与天然相反的折叠形式可作为具有低副作用的特异性免疫治疗的治疗剂使用。因此，这些重组折叠突变体比天然产物有着更安全的治疗效果。该方法以可以在药物生产所必需的条件下(GMP)进行生物技术生产的方法进行设计。
昆虫叮咬过敏主要是由黄蜂和蜜蜂引起的，并可能导致严重的系统性症状或甚至是潜在的致命性过敏性反应(Müller，U.R.，昆虫叮咬过敏，Gustav Fischer Verlag；1990)。引发1型过敏的物质是昆虫毒液的蛋白、糖蛋白或多肽。在毒液被注入之后，这些过敏原与结合在敏感人群柱状细胞表面的IgE分子反应。如果结合有FcεR1的该类型IgE分子通过过敏原彼此交联，就会导致效应细胞释放出介质(例如组胺、白三烯)和细胞素，并因此出现相应的临床症状。
除了该蜂毒肽以外，透明质酸酶和磷脂酶A2也是蜜蜂蜂毒的致敏要素(Habermann，E.，科学，1972，177：314-322)。在黄蜂中，主要的酶活性过敏原同样也是透明质酸酶(其与蜜蜂蜂毒的透明质酸酶非常相似)(Hoffmann，D.R.，过敏临床免疫学杂志，1986，78：337-343)和磷脂酶A1。最重要的黄蜂毒性主过敏原是抗原5，从中没有检测到酶活性(King等，生物化学，1978，17：5156-5174)。所有所说的过敏原都已经用分子生物学术语进行了鉴定并且相应的cDNA也已被克隆(Fang等，PNAS，1988，895-899；Soldatova等，FEBS，1993，145-149；Kuchler等，欧洲生物化学杂志，1989，184：249-254)。用这些cDNA序列，有可能生产出用于过敏诊断和治疗的重组过敏原(Scheiner和Kraft，过敏，1995，50：384-391)。
联系到本发明，主过敏原抗原5尤其重要，因为本发明使用该分子作为实施例。它是一个非糖基化的蛋白，大小约25kDa。其主要序列包含8个半胱氨酸残基，表明其有四个二硫键(Hoffman，D.R.，过敏临床免疫学杂志，1993，92：707-716)。昆虫毒性过敏有效治疗的一个传统方法是特异性免疫治疗或脱敏作用(Müller，U.R.，昆虫叮咬过敏，Gustav FischerVerlag；1990)。在此，天然过敏原提取物以逐渐增加的剂量对病人进行皮下注射。然而，该方法需要承担过敏反应或甚至过敏性休克的危险。因为会有强烈的反应，尤其是在昆虫叮咬脱敏作用时，所以当前只对住院病人使用这种处理方法。
通过重组方法生产的过敏原会尤其适合对昆虫叮咬过敏的治疗。由通过重组方法产生的高纯度过敏原制成的特定鸡尾酒(优选与患者的具体过敏方式相匹配)可以代替天然过敏原来源的提取物(Scheiner和Kraft，1995)。IgE表位被特异性删除而不损伤对治疗至关重要的T-细胞表位，这样的变异重组过敏原给人们提供了这样一个现实的前景，即用这种类型的重组过敏原可能会产生更安全的脱敏作用(Schramm等，免疫学杂志，1999，162：2406-2414)。
从在大肠杆菌中进行的异源表达我们知道，大部分真核蛋白并不采用’天然’的构象或仅有一小部分是这样。这些错误折叠的结果常常是这些蛋白的不溶。这尤其可在含半胱氨酸的蛋白中看到(Kuchler等，欧洲生物化学杂志，1989，184：249-254)。已有关于抗原5的报道，具体地说，就是在细菌中的表达产生了不具有天然构象的不溶性聚集物(Monsalve等，蛋白质表达纯化，1999，16(3)：410-416)。这种类型的不溶性聚集物既不能用于诊断也不能用于治疗。
为了在真核表达系统，例如酵母或昆虫细胞中进行研究，经常会制备这种在大肠杆菌中不溶的蛋白(Monsalve等，蛋白质表达纯化，1999，16(3)：410-416；Soldatova等，过敏临床免疫学杂志，1998，101：691-698)。然而，真核表达系统的不利之处在于其特别有可能产生高糖基化(Grobe等，欧洲生物化学杂志，1999，263：33-40)、蛋白水解降解过程和相对少的产量(Glover和Hames(主编)，1995，表达系统，IRL出版社，牛津-纽约-东京)。因此这种类型的蛋白通常不适于医药诊断和治疗意义上的变态反应用途。
本发明所述方法的具天然构象的产物可优选用于过敏症(尤其是昆虫叮咬过敏)的胞外和胞内诊断。该与天然一致的折叠形式可以以已建立的方法用于IgE抗体的检测。
另一方面，本发明所产生的突变体(其特点为根本没有或仅部分具有IgE反应活性的构象)可在特异性免疫治疗制剂中作为低变应原组分来进行使用。按本发明，上、下文中所用的术语“低变应原的”是指，由于减弱的IgE反应所引起的减弱至没有，优选从5％到95％，尤其是从20％到85％的减弱过敏原性(与天然过敏原相比)。
本发明是一种方法，用此方法可在细菌(大肠杆菌)中产生重组过敏原。第一个纯化步骤是对不溶性蛋白聚集物进行相当可观的富集。然后在不添加还原剂的情况下对这些聚集物进行变性。根据接下来的透析条件的不同，可以得到不同的折叠形式。至关重要的一点是，这些分子是单体的且是可溶的。第一个可溶的折叠突变体具有可与天然过敏原相比的IgE反应活性并可因此用于诊断目的。各种类型的产品可通过用含半胱氨酸的溶液进行透析得到。
另一个替代的可溶性折叠突变体在结构上与天然过敏原不同，并且可由IgE反应活性的减弱或缺乏来加以区别。为此，这种类型的突变体适于推动改良的免疫治疗。按本发明所述，用酸性缓冲液(优选pH在3.5和6.5之间，具体在4.0和5.5之间)透析可得到这种类型的低变应原产品。
因此本发明也涉及具有减弱的IgE反应活性或过敏原性特征的重组昆虫过敏原。按本发明所述，与天然过敏原相比这些蛋白的过敏原性可减少95％。
具体来说，本发明涉及相应的重组黄蜂昆虫过敏原，尤其是来自常见黄胡蜂(Vespula vulgaris)和德国黄蜂(Vespula germanica)的重组黄蜂昆虫过敏原。
本发明涉及分离基本上纯的重组昆虫毒性过敏原的方法，其特征为该过敏原蛋白在细菌细胞内以不溶的“包涵体”形式产生，所说的不溶性聚集体被变性，并且该变性产物通过透析转化成可溶的、不同折叠构象的单体过敏原并得以分离。所说的变性反应优选使用不添加还原剂的氯化胍盐(guanidinium chloride)进行。
具体来说，本发明涉及分离具有减弱过敏原性或IgE反应活性的重组昆虫毒性过敏原的方法，其中透析使用酸性缓冲液，优选pH在4.5和5.0之间的醋酸钠缓冲液，进行。
然而，本发明也涉及分离具有正常过敏原性或IgE反应活性的重组昆虫毒性过敏原的方法，其中透析使用含半胱氨酸的溶液进行。
本发明也涉及可由上述或下述相应方法获得的重组黄蜂毒性过敏原。
本发明又涉及含有具有减弱或消除IgE反应活性的相应重组过敏原和相应辅剂和赋形剂的药物制剂。
最后，本发明涉及可由上述或下述相应方法获得的昆虫毒性过敏原在胞内和胞外昆虫叮咬过敏诊断上的用途。
本方法详述如下：
作为实施例，Vespula vulgaris黄蜂毒性过敏原抗原5(Ves v5)和德国黄蜂黄蜂毒性过敏原抗原5(Ves g5)被克隆至表达载体pSE420并被转入K12细菌菌株M15 pREP4。图1为该方法的流程图。
该重组过敏原使用该菌株的预培养物接种表达培养液来进行生产。表达以IPTG诱导，在LB培养基中以有限供氧(90rpm/min)在三角瓶中37℃进行。表达5小时以后，离心(5000xg，10分钟，20℃)收获该细菌。将此细胞重悬于缓冲液(50mM tris/HCl，25％(w/v)蔗糖，pH8.0)中，添加溶菌酶(10μg/g湿重)进行细菌消化。然后添加同样体积的去污剂溶液(0.2M NaCl，1％(w/v)DOC，1％(w/v)诺乃洗涤剂P40)。随后，该消化液以超声波处理(置冰上3分钟，130瓦，脉冲0.5秒)。既然该表达产物基本上是不溶性聚集体的形式(包涵体)，因此可由其高密度通过离心(3000xg)将它们从大部分剩余的组份(细胞壁片段、核糖体等)中分离出来。用含去污剂的溶液(1％Triton X-100)通过三个连续的洗涤步骤进行进一步的纯化。随后，添加变性缓冲液(6M氯化胍盐，20mMtris/HCl，pH8.0)并于室温振荡2小时对纯化的包涵体进行消化。
为了分离IgE反应活性的折叠形式，该变性批次被转入透析管(消化限度12-14kDa)并于室温对100倍体积的半胱氨酸溶液(5mM半胱氨酸)搅动透析12小时。然后再对蒸馏水透析以除去半胱氨酸。
为获得具有减弱IgE反应活性的构象，第一次要对20mM醋酸钠缓冲液(pH5.0)透析。然后再对蒸馏水进行透析。在去除以后，通过离心，该水溶的过敏原从沉淀的聚集物中分离出来。该上清含有所要的可溶性重组过敏原。也可以使用其它的缓冲能力从3.5到6.5，优选从4.0到5.5的酸性缓冲液代替醋酸钠缓冲液。这种类型的缓冲系统的例子在文献中有充分的描述。
用两种方法产生的沉淀下来的重组过敏原可按同样的方案进行再变性及处理。这可明显地增加产量。
在透析步骤之后，产物纯度约为95％。此基本的昆虫毒性过敏原进一步的纯化步骤是用例如Source S(Pharmacia，Freiburg，德国)的阳离子交换层析(缓冲液pH7.2)和凝胶过滤。除了除去极少的高分子量和低分子量的杂质以外，凝胶过滤也用来进行脱盐。
基于下面的特性对产品的质量进行控制，其以抗原5概括于下表：
n-抗原＝天然抗原
| 特性 | 具有天然IgE反应活性的折叠 | 具有减弱IgE反应活性的折叠 |
| SDS-PAGE(非还原条件)中的表观分子量 | 25kDa | 26-27kDa |
| Source S的洗脱盐浓度 | 320mM NaCl | 400mM NaCl |
| 蛋白酶V8剪切 | 15kDa片段+肽 | 肽＜10kDa |
| 抗原5特异性的单克隆抗体 | 可用8E3，1E11检测 | 仅可用8E3检测 |
| 与过敏反应患者血清的IgE反应频率 | ＞95％ | ＜10％ |
| 致敏能力 | 与n-抗原5相似 | ＞10x小于nAg5 |
本发明所述的方法适于所有类型的昆虫毒性过敏原。所使用的纯化技术和重组克隆及表达技术是本领域技术人员所知的和可用的，并且可用已知的相似方法替换之。
Claims (8)
1.分离基本上纯的过敏原性或IgE反应活性减弱的重组黄蜂毒性过敏原抗原5的方法，其特征在于：
-在细菌细胞内以不溶的包涵体形式产生所述过敏原蛋白，
-在不加入还原剂的情况下，将所说的不溶性聚集体变性，并且
-通过在酸性缓冲液中透析将该变性产物转化成可溶的、不同折叠构象的单体过敏原并得以分离。
2.权利要求1所述的方法，其特征在于该变性反应使用氯化胍盐进行。
3.权利要求1或2所述的方法，其特征在于在透析步骤中使用pH在3.5和6.5之间的缓冲液。
4.权利要求1到3中任何一项所述的方法，其特征在于使用黄胡蜂属(Vespula)的种和Paravespula种的过敏原。
5.权利要求4所述的方法，其特征在于所述黄胡蜂属的种为常见黄胡蜂(Vespula vulgaris)或德国黄蜂。
6.可通过权利要求1-5中任何一项所述方法获得的重组黄蜂毒性过敏原抗原5。
7.含有权利要求6所述的重组过敏原和相应的辅剂及赋形剂的药物制剂。
8.可通过权利要求1-3中任何一项所述方法获得的黄蜂毒性过敏原抗原5在制备用于对黄蜂毒性过敏原过敏的患者进行特异性免疫治疗或脱敏的药物中的用途。
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| DE19957904A DE19957904A1 (de) | 1999-12-01 | 1999-12-01 | Insektengift-Allergene mit verminderter IgE-Reaktivität und Verfahren zu ihrer Herstellung |
| DE19957904.0 | 1999-12-01 |
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| DE19957904A1 (de) † | 1999-12-01 | 2001-06-07 | Merck Patent Gmbh | Insektengift-Allergene mit verminderter IgE-Reaktivität und Verfahren zu ihrer Herstellung |
| JPWO2011033588A1 (ja) * | 2009-09-18 | 2013-02-07 | 株式会社アドバンテスト | 試験装置および試験方法 |
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| JP2838800B2 (ja) * | 1989-09-02 | 1998-12-16 | 株式会社林原生物化学研究所 | 減感作剤 |
| DE4139000A1 (de) * | 1991-11-27 | 1993-06-03 | Boehringer Mannheim Gmbh | Verfahren zur gentechnologischen herstellung von biologisch aktivem ss-ngf |
| CA2148713A1 (en) * | 1992-11-12 | 1994-05-26 | Mei-Chang Kuo | Allergenic proteins and peptides from japanese cedar pollen |
| JP3578787B2 (ja) * | 1993-05-14 | 2004-10-20 | ラホヤ インスティチュート フォア アレルギー アンド イムノロジー | 生物学的に活性なポリペプチドの組換え製造方法 |
| US5804201A (en) * | 1996-03-11 | 1998-09-08 | The Rockefeller University | Immunomodulatory peptides of vespid antigen 5 |
| AU714318B2 (en) * | 1996-06-11 | 2000-01-06 | Roche Diagnostics Gmbh | Process for the activation of denatured protein |
| EP1007648A4 (en) * | 1996-12-20 | 2003-01-15 | Lilly Co Eli | ANTI-OBESITY PROTEINS |
| DE19957904A1 (de) * | 1999-12-01 | 2001-06-07 | Merck Patent Gmbh | Insektengift-Allergene mit verminderter IgE-Reaktivität und Verfahren zu ihrer Herstellung |
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| PT1512694E (pt) | 2007-01-31 |
| RU2264462C2 (ru) | 2005-11-20 |
| US8048424B2 (en) | 2011-11-01 |
| PL355166A1 (en) | 2004-04-05 |
| CN1451015A (zh) | 2003-10-22 |
| EP1233984B1 (de) | 2004-11-24 |
| ES2271764T5 (es) | 2011-02-28 |
| ES2271764T3 (es) | 2007-04-16 |
| DE50008782D1 (de) | 2004-12-30 |
| WO2001040266A2 (de) | 2001-06-07 |
| US20070280884A1 (en) | 2007-12-06 |
| ATE339444T1 (de) | 2006-10-15 |
| CA2393975C (en) | 2012-06-19 |
| CA2393975A1 (en) | 2001-06-07 |
| AU2359401A (en) | 2001-06-12 |
| ATE283284T1 (de) | 2004-12-15 |
| EP1233984A2 (de) | 2002-08-28 |
| AU783505B2 (en) | 2005-11-03 |
| DE50013480D1 (de) | 2006-10-26 |
| ES2232518T3 (es) | 2005-06-01 |
| PL201904B1 (pl) | 2009-05-29 |
| EP1512694A1 (de) | 2005-03-09 |
| EP1512694B2 (de) | 2010-11-03 |
| JP5597336B2 (ja) | 2014-10-01 |
| CZ20021874A3 (cs) | 2002-08-14 |
| DE19957904A1 (de) | 2001-06-07 |
| JP2004525601A (ja) | 2004-08-26 |
| JP5265745B2 (ja) | 2013-08-14 |
| HUP0401012A2 (hu) | 2004-08-30 |
| JP2012087119A (ja) | 2012-05-10 |
| US7285397B1 (en) | 2007-10-23 |
| PT1233984E (pt) | 2005-04-29 |
| WO2001040266A3 (de) | 2002-02-28 |
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