CN1372567A - 禾草花粉过敏原的分离和提纯方法 - Google Patents
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Abstract
本发明涉及从禾草花粉中将1、2、3、10和13型这5种过敏原快速而有效地分离及提纯的方法。所述禾草花粉的提纯基于疏水作用色谱法、凝胶过滤和阳离子交换色谱法的新型组合。该新型方法获得的蛋白质有助于改进花粉过敏的诊断并可用于花粉原疾病治疗的药物制剂。
Description
本发明涉及从禾草花粉中快速而有效地分离和提纯1、2、3、10和13型这5种过敏原的方法。用来提纯过敏原的天然原料是甜草类,例如梯牧草(Phleum pratense)的花粉。其提纯基于疏水作用色谱法、凝胶过滤和阳离子交换色谱法的新型组合。如此获得的蛋白质可用于改进花粉过敏的诊断并可用于花粉过敏疾病治疗的药物制剂。
1型过敏具有世界范围的重要性。工业化国家人口中高达20%的人受到诸如过敏性鼻炎、结膜炎或支气管哮喘的困扰,这都是由存在于空气中的各种过敏原(空气过敏原)所致,这些过敏原是由各种各样的来源释放的,例如植物、螨虫、猫或狗。这些1型过敏患者中,高达40%又表现出在禾草花粉过敏原情况下的特异IgE反应(Freidhoff等人,1986,J Allergy Clin Immunol 78,1190~201)。
诱发1型过敏的物质是蛋白质、糖蛋白或多肽。经黏膜摄入之后,这些过敏原便与过敏者的肥大细胞表面上键合的IgE分子起反应。如果两个或更多个IgE分子通过一个过敏原彼此连接起来,则导致由效应细胞分泌出递质(例如组胺、前列腺素)和细胞因子,并从而产生相应的临床症状。
根据具有抗特定过敏原的IgE抗体的过敏患者的相对频率,可划分为主要和次要过敏原。在梯牧草的情况下,Phl p1(Petersen等人,1993,J Allergy Clin.Immunol.92,789~796)、Phl p 5(Matthiesen和Lwenstein,1991,Clin.Exp.Allergy,21,297~307;Petersen等人,1992)、Phl p 6(Petersen等人,1995,Int.Arch.AllergyImmunol,108,49~54)以及Phl p 2/3(Dolecek等人,1993)迄今已被表征为主要过敏原;而Phl p 4(Lwenstein,1978,Prog.Allergy25,1~62)和来自黑麦草(Lolium perenne)的10及11型(Ansari等人,1987,J.Allergy Clin.Immunol.80,229~235)则作为次要过敏原。另外,最近又公开了另一种高分子量主要过敏原,被命名为Phlp 13。1和13型过敏原是被糖基化的。
联系到本发明,1、2、3、10和13型过敏特别重要。天然过敏原的现行提纯方法基于在每种情况下个别蛋白质的分离。借助采用特异抗体的亲和色谱法,目前已提纯出,例如来自黑麦草(Boutin等人,1996,Int.Arch.Allergy Immunol.112,218~225)和梯牧草(Grobe等人,1999,Eur.J.Biochem.263,33~40)的1型过敏原。然而该方法的能力有限,并且是采用极端pH值条件实施的,因此不能保证获得天然构象。其它方法是基于色谱步骤的各种多级组合顺序。此时可获得每种情况的个别过敏原,例如10型(Ansari等人,1987,J.Allergy Clin.Immunol.80,229~235)或3型(Ansari等人,1989,Biochemistry,28,8665~8670)。其它过敏原则被丢失或者不能用这类方法制备纯净形式。
DNA序列数据是现成的,尤其是关于Phl p 1的(Laffer等人,1994,J.Allergy Clin.Immunol.94,1190~98;Petersen等人,1995,J.Allergy Clin.Immunol.95(5),987~994)、Phl p 5的(Vrtala等人,1993,J.Immunol.151(9),4773~4781)、Phl p 6的(Petersen等人,1995,Int.Arch.Allergy Immunol.108(1),55~59)以及Phl p2的(Dolecek等人,1993,FEBS 335(3),299~304)。借助cDNA序列,就可能生产出在诊断和治疗中使用的重组过敏原(Scheiner和Kraft,1995,Allergy 50,384~391)。
对过敏进行有效治疗的经典做法是特异性免疫疗法或减敏疗法(Fiebig,1995,Allergo J.4(6),336~339;Bousquet等人,1998,J.Allergy Clin Immunol.102(4),558~562)。在这些方法中,患者接受剂量递增的天然过敏原提取物的皮下注射。然而,该方法会带来过敏反应或甚至过敏性休克的危险。为了最大限度减少此种危险,可采用类变应原形式的创新制剂。它们是比未处理提取物明显减少IgE反应性但T-细胞反应性相同的化学改性过敏原提取物(Fiebig,1995,Allergo J.4(7),377~382)。
若有高度提纯的过敏原,可更大程度地达到疗效的优化。来自天然过敏原的规定混合物能够代替上述提取物,因为后者,除了含有各种各样过敏原之外还含有相对大量的免疫原性但非变应性的伴生蛋白质,该蛋白质并非该特异性免疫疗法所必须的。使用过敏原混合物还使得制备对应于患者过敏谱的患者-特异过敏原混合物成为可能。用高纯度天然过敏原达到安全减敏目的的现实前景是由改性过敏原提供的,其中IgE抗原决定簇因二级和三级结构的不可逆改性而被破坏,但不会损害疗效所必需的T-细胞抗原决定簇。
本发明也可有利地用于过敏性疾病,特别是花粉热的体外和体内诊断。为此,在用来检测IgE抗体的成熟方法中,使用各类提纯的过敏原。
本发明涉及一种生化提纯方法,它通过高效三级提纯从水性短期花粉提取物中分离4种主要过敏原和1种次要过敏原。所使用的天然原料是禾本科花粉,例如,梯牧草(Phleum pratense)、黑麦草(Lolium perenne)、鸭茅(Dactylis glomerata)、草地早熟禾(Poa pratensis)、绊根草(Cynodon dactylon)、绒毛草(Holcuslanatus)等。图1表示出从禾草花粉提取物中提纯所提到的5种过敏原的流程。过敏原名称Phl p 1~Phl p 13对应于本文中其它地方使用的过敏原1~13的名称。
因此,本发明涉及一种分离基本纯净的1、2、3、10和13型禾草过敏原的方法,其中制备一种禾本科花粉的水性提取物,然后可溶性组分接受疏水作用色谱法处理、凝胶过滤步骤,以及如果需要的话,阳离子交换色谱法处理。
按照本发明,也可实施一类色谱术的多个步骤,然而一般而言,该方法非常有效,以致每种情况下一个分离步骤就足够了。
该方法尤其适合从物种:梯牧草(Phleum pratense)、黑麦草(Lolium perenne)、鸭茅(Dactylis glomerata)、牛尾草(Festucapratensis)、绒毛草(Holcus lanatus)、草地早熟禾(Poa pratensis)、黑麦(Secale cereale)的花粉中提取所述过敏原。
在优选的实施方案中,利用tris/HCl缓冲水溶液来进行提取。然而,按照本发明也可使用其它已知的缓冲水溶液。
为提纯所述过敏原,采用提取物的可溶性组分。为此,提取物在18,000~30,000×g下离心3~8min、优选5min,然后取上清液以做进一步提纯。此外也可通过其它方法,例如用过滤,分离掉不溶解组分。
第一色谱提纯步骤采用疏水作用色谱法,例如用Sepharose实施。在该步骤中,大量杂质被固定在载体上,而所需要的过敏原则处于流过色谱柱的级分中。也可使用相应的其它载体材料。
因此,本发明涉及一种相应的方法,其中1、2、3、10和13型禾草过敏原借助于疏水作用色谱法与其它组分分离开来。
在随后的提纯步骤中,禾草过敏原被分成三个级分,其中1型和13型各代表一个级分,而2、3和10型则代表第3级分。因此,本发明特别是涉及这样一种方法,其中1和13型过敏原在随后的凝胶过滤步骤中作为单独的级分得到,并与2、3和10型过敏原分离开来。
然后,按照本发明,后者可以利用随后的色谱步骤通过阳离子交换剂彼此分离开来。因此,本发明涉及这样一种方法,其中在凝胶过滤步骤后获得的2、3和10型过敏原借助随后的阳离子交换色谱法彼此分离开来。
所述过敏原,鉴于本身是已知的,故可通过其已知的不同物理、化学、生物学或免疫学性质加以鉴别,特别是利用等电聚焦、紫外吸收测定、SDS-PAGE电泳以及特异性抗体。这些方法和技术是已知的并普遍描述过。
按照本发明获得的过敏原的得率,以最初使用的禾草花粉总蛋白为基准介于0.5~1.5%。
本发明也可用于改进体内和体外诊断,作为分辨过敏原组分的患者-特异性过敏谱鉴别的一部分。因此,本发明涉及一种利用按权利要求1~6获得的过敏原来实现花粉过敏体内和体外诊断的方法。
本发明还可用于制备禾草花粉过敏特异性免疫疗法用的改良制剂,这是通过分离掉那些虽是致免疫的、但与该疗法无关的提取物组分来实现的。此外还可以通过提纯后过敏原的化学反应来制取类变应原制剂。因此,本发明还涉及一种药物制剂,它包含一种或多种按照本发明方法制取的过敏原以及,如果需要的话,相应的助剂和赋形剂。
该方法详细内容如下:
来自梯牧草花粉的天然过敏原按三步法(见图1)进行提纯。将花粉用tris/HCl缓冲溶液(20mM tris/HCl,1mM EDTA,pH8.0)进行30min水相提取之后,通过离心分离提取物,优选20,000×g进行5min。该tris/HCl缓冲溶液(20mM tris/HCl,1mM EDTA,pH8.0)的上清液用1M硫酸铵处理,随后接受疏水作用色谱法处理(高效苯基-琼脂糖凝胶(Phenyl-Sepharose High Performance),Pharmacia出品)。在典型的色谱柱中装填50~100ml载体材料,并在约5ml/min的流速下进行操作。流过色谱柱的级分包含五种过敏原类别的蛋白质:1型(30~35kDa)、2型(11kDa)、3型(12kDa)、10型(13kDa)和13型(55~60kDa)。这之后是减少其体积,优选用超滤或冷冻干燥。
随后在第二步中,根据其不同的分子量,采用Superdex75制剂级(Pharmacia出品)或者适合此目的的类似已知载体材料通过凝胶过滤将13和1型过敏原从低分子量过敏原中分离出来。洗脱介质优选是50mM的碳酸氢铵。在约5ml/min的流速下操作色谱柱。获得3个级分,分别包含过敏原1和13(各一个单独级分)以及2、3和10(在一个级分中)。
一同洗脱到凝胶过滤的第3级分中的低分子量2、3和10型过敏原,用阳离子交换色谱法彼此分开。为此,冻干的样品被溶解在水性缓冲液中,优选20mM的磷酸盐缓冲液,pH7.2,然后加入到与此种缓冲液(例如,Source S)处于平衡的阳离子交换柱中。流过交换柱的级分包含酸性过敏原2。被结合的过敏原3和10在约20倍于柱体积的洗脱过程中依靠0~500mM NaCl盐梯度先后被洗脱下来。由此,各类低分子量过敏原被分离为其各自的单独过敏原。其它阳离子交换剂材料也可用于本发明中。
鉴于本发明方法的特征在于特定的色谱步骤顺序以及色谱介质的选择,因此凭借所提供的本发明方法,本发明提供一种提取多个高纯度天然禾草过敏原的高级生产方法,它不费事或不费时并且在技术上可行。由于所用提纯方法对蛋白质作用非常温和,因此它们的构象和抗原性得以保留。这是过敏性疾病成功诊断的先决条件。
Claims (8)
1.一种用来分离基本纯净的1、2、3、10和13型禾草过敏原的方法,其特征在于,制备一种禾本科花粉的水性提取物,然后此可溶性组分接受疏水作用色谱法处理、凝胶过滤步骤以及,如果需要的话,阳离子交换色谱法处理。
2.权利要求1的方法,其特征在于,使用物种:梯牧草、黑麦草、鸭茅、牛尾草、绒毛草、草地早熟禾、黑麦的花粉来进行提取。
3.权利要求1或2的方法,其特征在于,提取是利用tris/HCl缓冲水溶液进行的。
4.权利要求1~3之一的方法,其特征在于,在第一步中,1、2、3、10和13型禾草过敏原利用疏水作用色谱法从其它组分中分离出来。
5.权利要求4的方法,其特征在于,1和13型过敏原通过随后的凝胶过滤步骤作为单独的级分得到,并从2、3和10型过敏原中分离出来。
6.权利要求5的方法,其特征在于,凝胶过滤步骤后获得的2、3和10型过敏原通过随后的阳离子交换色谱法彼此分离开来。
7.一种采用按照权利要求1~6获得的过敏原对花粉过敏进行体内和体外诊断的方法。
8.一种药物制剂,它包含按照权利要求1~6之一获得的一种或多种过敏原以及相应的助剂和赋形剂。
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