CN101351224A - 垂枝桦花粉主要变应原的低变应原性变体 - Google Patents
垂枝桦花粉主要变应原的低变应原性变体 Download PDFInfo
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Abstract
本发明提供垂枝桦植物花粉的Bet v 1主要变应原的低变应原性变体，及其预防或治疗变应性疾病的用途。
Description
本发明提供Bet v 1蛋白质的低变应原性序列变体、编码其的核酸分子、包含其的药物组合物以及它们在预防和治疗由来自垂枝桦(Betulaverrucosa)物种的植物花粉导致的变应性疾病中的用途。
背景技术
变态反应由免疫系统功能异常导致，所述免疫系统通过产生IgE-类抗体与花粉、螨、上皮和某些食物中含有的无害蛋白质反应。
最近的数据表明西方国家中超过10％的人口患有该疾病，其症状可随时间恶化，导致例如哮喘或对其他变应原致敏，由此造成选择适当的治疗方法更加困难。
与药理学疗法不同，特定的脱敏免疫疗法是变应性疾病的唯一病原疗法，能够顺利地控制这些疾病所基于的免疫参数。
脱敏免疫疗法包括施用增加剂量的获自导致疾病的物质的标准提取物(疫苗)(1)。这样，患者对该物质的一类免疫耐受逐步降低，随后变应性症状消失。
但是，事实上引起严重副作用的风险(2)限制特定脱敏免疫疗法在治疗变应性疾病中的应用，虽然使用缓释疫苗或使用非注射途径施用的疫苗使该风险显著降低。
近年来大部分注意力已集中在开发有效、安全的疫苗上。具体地，开发包含诱变处理的重组蛋白(即能够有利地影响疾病的自然进程而不导致不希望的副作用的低变应原性变体(3))的疫苗已成为主要的目标。
分类学上称为山毛榉目(Fagales)的植物(桦树、桤木、榛树、橡树、角树)的花粉是温带地区导致变应性鼻炎和哮喘的最主要原因之一。桦树花粉的两种主要变应原，Bet v 1(以登录号X15877保存在GenBank的cDNA)和Bet v 2(登录号M65179)，是分子量分别为17和14kD的蛋白质(4，5)。大约95％对桦树花粉有变态反应的患者产生抗Bet v 1的IgE抗体，60％的这些患者表现出对单独Bet v 1的反应性(6)。
Bet v 1以大于10种同种型天然存在，表现出84.4％至99.4％的序列同一性(7)。该变应原属于“致病相关蛋白质”家族，即植物在应答环境或病理胁迫时产生的遍在蛋白，推测其功能和类固醇转运相关(8，9)。与来自山毛榉目其他植物的花粉中组1变应原的高度序列同源性解释了为什么具有抗Bet v 1的IgE的患者在属于相同分类目的不同植物的传粉季节出现变应性症状(10)。桦树花粉的变态反应常伴随由摄入新鲜水果(例如樱桃、苹果、梨)或蔬菜(例如芹菜和胡萝卜)引发的副反应。原因在于这类食物包含特征在于和Bet v 1高度序列和结构同源性的蛋白质，其被桦树主要变应原产生的特定IgE识别(11)。用Bet v 1变应原的免疫疗法在治疗桦树花粉变态反应，对山毛榉目其他植物的花粉症，以及对包含与Bet v 1交叉反应的变应原的食物的变态反应中有效(12)。
脱敏免疫疗法有益效果的一个相关因素是诱导抗致敏变应原的IgG抗体。这类(保护)抗体能够抑制IgE与抗原结合，特别是抑制与Bet v 1结合，改变该分子的三维构像(13，14)。包含具有较少变应原性和未改变免疫原性的重组蛋白的疫苗的开发将改善变应性疾病的治疗。
发明内容
现已发现通过取代或缺失Bet v 1变应原蛋白质序列中的一个或多个氨基酸残基使得该变应原对IgE抗体的反应性降低。
第一方面，本发明提供低变应原性蛋白质，其是Bet v 1变应原的序列变体，特征在于：
1)与野生型Bet v 1变应原(SEQ ID NO：1)相比，表现出与IgE降低的反应性；
2)具有以下氨基酸序列：
a)与SEQ ID NO：1至少87％相同，优选至少94％相同，进一步优先至少97％相同；
b)与SEQ ID NO：1的序列比对表现出SEQ ID NO：1第54、115和/或123位氨基酸残基中至少一个取代或缺失，其中SEQ ID NO：1的第54、115和/或123位氨基酸残基为Lys残基。
本发明的Bet v 1变应原的变体在所示位置具有1、2或3个Lys-取代和/或缺失，分别称为单、双或三取代和/或缺失。相对缺失变体优选取代变体，特别是那些在所示位点至少一个Lys残基被中性或极性氨基酸取代的变体。更优选所述中性或极性氨基酸选自Ala、Thr、Gly、Pro、Leu、Ile、Ser、Phe，进一步优选选自Ala、Thr和Ser。
在优选实施方案中，具有SEQ ID NO：1的低变应原性蛋白质在第54、115和123位具有至少一个所述Lys残基取代或缺失。
本发明具有1或3个取代的典型低变应原性蛋白质如SEQ ID NO：2、SEQ ID NO：3、SEQ ID NO：4和SEQ ID NO：5所示。
相对于野生型对应物(counterpart)，本发明的Bet v 1变应原取代和/或缺失变体表现出与垂枝桦花粉-变应性患者血清的IgE反应性降低至少25％，优选至少50％，更优选至少80％。
来自变应性患者血清库的蛋白质SEQ ID NO：2-5的IgE反应性由ELISA测定检测(图1)。相对于野生型Bet v 1变应原(SEQ ID NO：1)，当(SEQID NO：2)、(SEQ ID NO：3)、(SEQ ID NO：4)和(SEQ ID NO：5)蛋白质与来自桦树花粉变应性患者的血清库的多种稀释物(1∶2至1∶8)孵育时，观察到IgE反应性平均降低56％(SEQ ID NO：2)、34％(SEQ ID NO：3)、28％(SEQID NO：4)和80％(SEQ ID NO：5)。
这些结果由ELISA抑制试验证实，该试验可以评测来自不同蛋白质的同源表位的反应性。当血清用相同的蛋白质预处理过，野生型Bet v 1(SEQID NO：1)与来自血清库的IgE的结合100％抑制，而当血清与相同量的三取代变体(SEQ ID NO：5)预孵育过，仅观察到40％的抑制(图2)。这些结果明确表明SEQ ID NO：1第54、115和123位的氨基酸取代减少IgE对Bet v 1变应原的识别。
此外，SEQ ID NO：2-5蛋白质对来自垂枝桦花粉阳性血清库的IgE的反应性通过Western印记测定。同时在这种情况下，与野生型Bet v 1(SEQ IDNO：1)相比，在分析的血清中观察到IgE反应性降低，SEQ ID NO：2降低88％，SEQ ID NO：3降低67％，SEQ ID NO：4降低47％以及SEQ ID NO：5降低100％(图3)。
另外，Balb/c小鼠免疫接种试验证明野生型Bet v 1变应原和低变应原性蛋白质SEQ ID NO：5均能诱导IgG-特异性免疫应答(图4)。具体地，抗SEQ ID NO：5抗体能够识别野生型-对应物SEQ ID NO：1(图5)，表明第54、115和/或123位Lys-残基的取代并不导致蛋白质免疫原性的重大改变，特别是并不改变其IgG表位。相反，不相关抗原免疫的小鼠血清中存在的抗体不能够识别野生型Bet v 1和SEQ ID NO：5。
另一方面，本发明提供Bet v 1片段相应的免疫活性肽，所述免疫活性肽包含15至35，更优选15至20个氨基酸残基，并具有至少一种上述取代和/或缺失。本文所用术语“免疫活性肽”意指能够引发不依赖IgE的免疫应答的肽。
使用本领域技术人员已知的方法和技术，通过诱变Bet v 1 cDNA序列(SEQ ID NO：6)可容易地制备本发明的取代和/或缺失变体。
编码SEQ ID NO：2-5单和三取代变体的cDNA序列分别为SEQ IDNO：7-10所示。
另一方面，本发明提供编码本文公开的低变应原性Bet v 1蛋白质的核酸分子、衍生自其的肽、包含该核酸分子的表达载体，其中所述核酸分子与真核细胞或原核细胞中控制所述蛋白质或肽表达的遗传元件(例如转录启动子、增强子、信号及前导序列，或转录调控中涉及的其他序列)功能性连接。载体的例子包括质粒、病毒和噬菌体，然而遗传工程中通常使用的任何其他载体也可以使用。
本发明还包含用上述载体转化或转染的原核或真核宿主细胞。原核细胞例如大肠杆菌(Escherichia coli)或枯草杆菌(Bacillus subtilis)，或真核细胞例如酿酒酵母(Saccharomyces cerevisiae)通常用作克隆或eDNA表达载体。
另外，本发明的低变应原性变体可作为融合蛋白制备。
由于它们降低的IgE反应性，本发明的Bet v 1变体可方便地用于制备药物组合物(例如片剂和胶囊剂)，所述药物组合物用于预防或治疗垂枝桦花粉的变应性个体。
因此本发明的另一方面在于药物组合物，其包含有效量的本文提供的低变应原性Bet v 1变体，该变体任选和垂枝桦的其他变应原，和/或药学可接受载体和赋形剂组合。在本发明的优选实施方案中，药物组合物以疫苗形式用于预防或治疗包括支气管哮喘、变应性鼻炎、变应性皮炎和变应性结膜炎的变应性疾病。免疫接种的理论和操作是本领域技术人员已知的(15，16)。
下列实施例进一步阐明本发明。除非另行指出，实施例中所用方法描述于Sambrook，Fritsch ET Maniatis“Molecular cloning.A laboratorymanual”第二版，第1-2-3卷，CSH Lab出版，1989。
实施例1-Bet v 1变应原编码eDNA的位点特异性诱变
Bet v 1变应原编码eDNA(SEQ ID NO：6)的位点特异性诱变通过原核载体(pBluescript，GenBank登录号X52327)中的cDNA克隆，然后是PCR扩增进行。PCR反应中用作引物的寡核苷酸(表)具有合适的碱基取代。对于每个诱变，使用结合到DNA链相应区域的互补寡核苷酸(17)。扩增后，限制酶DpnI催化的酶促消化选择性降解未改变的原模板。然后用诱变的分子转化大肠杆菌细胞。根据桑格(法)对获自单细菌菌落的克隆进行测序，以确定cDNA中正确的碱基修饰以及不存在非特异性突变。
表.位点特异性诱变中用作引物的寡核苷酸序列。
突变的碱基为黑体。
| 寡核苷酸 | 序列 |
| Bet v 1 54 | cct gga acc att gcg aag atc agc ttt ccc |
| Bet v 1 115 | ggt tcc atc ttg gcg atc aac aac aag tac c |
| Bet v 1 123 | aag tac cat acc gca gga gac cat gag |
实施例2-制备Bet v 1蛋白及其变体
根据标准方案(18，19)在大肠杆菌中克隆并表达野生型(SEQ ID NO.6)和诱变的(SEQ ID NO.7-10)Bet v 1 cDNA，所述诱变的Bet v 1 cDNA侧接编码六个组氨酸的序列。细胞离心收集，重悬于50mM NaH2PO4、300mM NaCl缓冲液(pH 8)中并超声裂解。离心分离重组蛋白。含有不溶蛋白质聚集体的沉淀重悬于100mM NaH2PO4、10mM Tris-HCl、8M尿素(pH 8)(变性缓冲液)中并搅拌60分钟。溶解的重组蛋白从不溶的残渣中离心分离，并通过在变性条件下使用连接有次氮基三乙酸的琼脂糖柱亲和层析纯化，所述次氮基三乙酸与融合到变应原的六-组氨酸部分相互作用的镍离子螯合。纯化的蛋白质通过在0.68％NaCl、0.275％NaHCO3溶液中4℃透析16小时来重折叠。
实施例3-变应性受试者血清的表征
血清收集自具有对垂枝桦花粉有季节性变态反应临床既往症，并具有对垂枝桦变应原有RAST 4+特异反应性的个体，然后集中这些血清。来自非变应性患者的血清库用作阴性对照。
实施例4-Bet v 1变体对血清库中IgE的反应性的ELISA分析
50mM碳酸盐/碳酸氢盐缓冲液(pH 9.6)中相同量的野生型变应原和突变变体(0.5μg)在4℃下温育16小时以吸附到ELISA分析用聚苯乙烯(polystirene)板的孔上。该孔用洗涤溶液(60mM含有0.05％Tween-20的磷酸盐缓冲液，pH 6.5)洗涤，并用稀释溶液(150mM磷酸盐缓冲液中的25％马血清、1mM EDTA、0.05％ Tween 20、0.01％乙基汞硫代水杨酸钠，pH 7.4)封闭。100μl人血清RAST 4+库的连续稀释物(在稀释缓冲液中)的整分试样加入至每个样品，并于25℃温育2小时。洗涤三次后，加入过氧化物酶缀合的抗人-IgE血清(稀释缓冲液中1∶1500)，接着25℃温育1.5小时。洗涤三次后加入100μl TMB试剂(BioFX Laboratories，Owings Mills，MD)并在25℃温育15分钟进行比色反应。加入100μl 1N HCl终止反应并使用酶标仪(microplate reader spectrophotometer)在450nm处读数。
实施例5-ELISA-抑制测定。Bet v 1变体抑制野生型Bet v 1与血清库中IgE的结合
50mM碳酸盐/碳酸氢盐缓冲液(pH 9.6)中的1μg野生型变应原的整分试样在4℃下温育16小时以吸附到ELISA分析用聚苯乙烯板的孔上。该孔用洗涤溶液(60mM含有0.05％Tween-20的磷酸盐缓冲液，pH 6.5)洗涤，并用稀释缓冲液(150mM磷酸盐缓冲液中的25％马血清、1mM EDTA、0.05％ Tween 20、0.01％乙基汞硫代水杨酸钠，pH 7.4)封闭。100μl 1∶3稀释于稀释缓冲液中的RAST 4+人血清库的整分试样与野生型变应原以及诱变变体的连续稀释物在25℃下预温育2小时。获得的溶液置于孔中并于4℃温育16小时。在用0.06M磷酸盐缓冲液(pH 6.5)、Tween-20 0.05％洗涤三次后，加入1∶1500稀释(稀释缓冲液中)的过氧化物酶缀合的抗人-IgE血清，接着25℃温育1.5小时。洗涤三次后加入100μl TMB试剂(BioFXLaboratories，Owings Mills，MD)并在25℃温育15分钟进行比色反应。加入100μl 1N HCl终止反应并使用分光光度仪在450nm处读数。
抑制百分数计算如下：100x[(A-B)/A]，其中A是无抑制剂时450nm处的吸光度，而B是存在抑制剂时的吸光度。
实施例6-Bet v 1变体与来自血清库的IgE反应性的Western印记分析
如Towbin(20)所述，等量野生型变应原和诱变形式(1.5μg)在聚丙烯酰胺凝胶上电泳分析，随后在硝化纤维素膜上电印记。
该膜首先在含有5％脱脂奶粉的TBST(TBS，0.05％ Tween-20，饱和缓冲液)中孵育1小时，然后与表现4+反应性，1∶3稀释于TBST 2％脱脂奶粉中的对垂枝桦变应性的受试者的血清库孵育过夜。孵育1小时后，用TBST洗涤膜3次。膜结合的抗体与过氧化物酶缀合的抗人IgE血清接触，洗涤数次后，使用鲁米诺(ECL，Amersham)作为过氧化物酶底物，用化学发光检测系统检测。
实施例7-免疫接种Balb/c小鼠的方案
5只雌性Balb/c小鼠(Charles River)的两个组用200μl的乳剂皮下免疫，该乳剂含有100μl完全弗氏佐剂和100μl盐水中的20μg抗原(SEQ ID NO：1和SEQ ID NO：5)。另外三次加强免疫以1周的间隔进行，用不完全佐剂替换完全佐剂。作为对照，5只小鼠施用不相关的抗原。末次免疫后7天，从尾巴获取血样，用于ELISA以检验抗体与每种免疫原性物质的应答。对于SEQ ID NO：5免疫的小鼠，还分析了识别野生型蛋白质的能力。
实施例8-ELISA分析免疫小鼠中的IgG-特异性应答
50mM碳酸盐/碳酸氢盐缓冲液(pH 9.6)中相同量的野生型Bet v 1和SEQ ID NO：5变体(0.25μg)在4℃下温育16小时以吸附到ELISA分析用聚苯乙烯板的孔上。该孔用洗涤溶液(60mM含有0.05％Tween-20的磷酸盐缓冲液，pH 6.5)洗涤，并用稀释溶液(150mM磷酸盐缓冲液中的25％马血清、1mM EDTA、0.05％ Tween 20、0.01％乙基汞硫代水杨酸钠，pH 7.4)封闭。100μl每只小鼠血清的连续稀释物(在稀释缓冲液中)的整分试样置于每孔中，并于25℃温育2小时。
洗涤三次后，在稀释缓冲液中1∶2000稀释过氧化物酶缀合的抗小鼠IgG血清，并加入到小孔中，接着25℃温育1.5小时。洗涤三次后加入100μlTMB试剂(BioFX Laboratories，Owings Mills，MD)并在25℃温育15分钟进行比色反应。用100μl 1N HCl终止反应并使用分光光度仪在450nm处读数。图4和5显示通过分析每组的5只小鼠的血清获得的平均反应性。
附图说明
图1：ELISA分析IgE与Bet v 1变应原和Bet v 1低变应原性变体的反应性；
图2：IgE与Bet v 1变应原结合的抑制；
图3：Western印记分析IgE与Bet v 1变应原及其变体的反应性；
图4：鼠IgG与各免疫原性蛋白质的应答；
图5：SEQ ID NO：5免疫的小鼠中的IgG应答。
参考文献
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2)Toubi E.，Kessel A.，Blant A.，Golan T.D.，(1999) “Follow-up aftersystemic adverse reactions of immunotherapy”. Allergy，54(6)：617-620.
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5)Valenta R.，Duchene M.，Pettenburger K.，Sillaber C.，Valent P.，Bettelheim P.，Breitenbach M.，Rumpold H.，Kraft D.，Scheiner O.(1991).“Identification of profilin as a novel pollen allergen；IgE autoreactivity insensitized individuals.” Science 253：557-560.
6)Batard T.，Didierlaurent A.，Chabre H.等人，(2005).“Characterization of wild-type recombinant Bet v 1a as a candidatevaccine against birch pollen allergy”. Int Arch Allergy Immunol.136：239-249.
7)Swoboda L.，Jilek A.，Ferreira F.，Engel E.，Hoffmann-Sommergruber K.，Scheiner O.，Kraft D.，Breiteneder H.，Pittenauer E.，Schmid E.，Vicente O.，Heberle-Bors E.，Ahorn H.，Breitenbach M.(1995).“Isoforms of Bet v 1，the major birch pollenallergen，analyzed by liquid chromatography，mass spectrometry，andcDNA cloning”. JBC.270(6)：2607-2613.
8)Swoboda I.，Scheiner O.，Heberle-Bors E.，Vicente O.，(1992).“cDNA cloning and characterisation of three genes in the Bet v 1 genefamily that encode pathogenesis-related proteins”. Plant Cell Environ. 18：865-874.
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10)Niederberger V.，Pauli G.，Gronlund H.等人(1998).“Recombinant birch pollen allergens(rBet v 1 and rBetv 2)contain most ofthe IgE epitopes present in birch，alder，hornbeam，hazel，and oak pollen：aquantitative IgE inhibition study with sera from different populations”.JAllergy Clin Immunol. 102：579-591.
11)Neudecker P，Lehmann K，Nerkamp J，Haase T，Wangorsch A，Fotisch K，Hoffmann S，Rosch P，Vieths S，Scheurer S.(2003).“Mutationalepitope analysis of Pru av 1 and Api g 1，the major allergens of cherry(Prunus avium) and celery(Apium graveolens)：correlating IgE reactivitywith three-dimensional structure”.Biochem J.376(Pt 1)：97-107.
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序列表
<110>洛法玛有限公司
<120>垂枝桦花粉主要变应原的低变应原性变体
<130>7683Meur
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<170>PatentIn版本3.3
<210>1
<211>159
<212>PRT
<213>未知
<220>
<223>垂枝桦花粉的野生型Bet v 1
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Gly Val Phe Asn Tyr Glu Thr Glu Thr Thr Ser Val Ile Pro Ala Ala
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Arg Leu Phe Lys Ala Phe Ile Leu Asp Gly Asp Asn Leu Phe Pro Lys
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Val Ala Pro Gln Ala Ile Ser Ser Val Glu Asn Ile Glu Gly Asn Gly
35 40 45
Gly Pro Gly Thr Ile Lys Lys Ile Ser Phe Pro Glu Gly Phe Pro Phe
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Lys Tyr Val Lys Asp Arg Val Asp Glu Val Asp His Thr Asn Phe Lys
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Tyr Asn Tyr Ser Val Ile Glu Gly Gly Pro Ile Gly Asp Thr Leu Glu
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Lys Ile Ser Asn Glu Ile Lys Ile Val Ala Thr Pro Asp Gly Gly Ser
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Ile Leu Lys Ile Asn Asn Lys Tyr His Thr Lys Gly Asp His Glu Val
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Lys Ala Glu Gln Ile Lys Ala Ser Lys Glu Met Gly Glu Thr Leu Leu
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Arg Ala Val Glu Ser Tyr Leu Leu Ala His Ser Asp Ala Tyr Asn
145 150 155
<210>2
<211>159
<212>PRT
<213>来知
<220>
<223>Bet v 1突变体
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Gly Val Phe Asn Tyr Glu Thr Glu Thr Thr Ser Val Ile Pro Ala Ala
1 5 10 15
Arg Leu Phe Lys Ala Phe Ile Leu Asp Gly Asp Asn Leu Phe Pro Lys
20 25 30
Val Ala Pro Gln Ala Ile Ser Ser Val Glu Asn Ile Glu Gly Asn Gly
35 40 45
Gly Pro Gly Thr Ile Ala Lys Ile Ser Phe Pro Glu Gly Phe Pro Phe
50 55 60
Lys Tyr Val Lys Asp Arg Val Asp Glu Val Asp His Thr Asn Phe Lys
65 70 75 80
Tyr Asn Tyr Ser Val Ile Glu Gly Gly Pro Ile Gly Asp Thr Leu Glu
85 90 95
Lys Ile Ser Asn Glu Ile Lys Ile Val Ala Thr Pro Asp Gly Gly Ser
100 105 110
Ile Leu Lys Ile Asn Asn Lys Tyr His Thr Lys Gly Asp His Glu Val
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Lys Ala Glu Gln Ile Lys Ala Ser Lys Glu Met Gly Glu Thr Leu Leu
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Arg Ala Val Glu Ser Tyr Leu Leu Ala His Ser Asp Ala Tyr Asn
145 150 155
<210>3
<211>159
<212>PRT
<213>未知
<220>
<223>Bet v 1突变体
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Gly Val Phe Asn Tyr Glu Thr Glu Thr Thr Ser Val Ile Pro Ala Ala
1 5 10 15
Arg Leu Phe Lys Ala Phe Ile Leu Asp Gly Asp Asn Leu Phe Pro Lys
20 25 30
Val Ala Pro Gln Ala Ile Ser Ser Val Glu Asn Ile Glu Gly Asn Gly
35 40 45
Gly Pro Gly Thr Ile Lys Lys Ile Ser Phe Pro Glu Gly Phe Pro Phe
50 55 60
Lys Tyr Val Lys Asp Arg Val Asp Glu Val Asp His Thr Asn Phe Lys
65 70 75 80
Tyr Asn Tyr Ser Val Ile Glu Gly Gly Pro Ile Gly Asp Thr Leu Glu
85 90 95
Lys Ile Ser Asn Glu Ile Lys Ile Val Ala Thr Pro Asp Gly Gly Ser
100 105 110
Ile Leu Ala Ile Asn Asn Lys Tyr His Thr Lys Gly Asp His Glu Val
115 120 125
Lys Ala Glu Gln Ile Lys Ala Ser Lys Glu Met Gly Glu Thr Leu Leu
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Arg Ala Val Glu Ser Tyr Leu Leu Ala His Ser Asp Ala Tyr Asn
145 150 155
<210>4
<211>159
<212>PRT
<213>未知
<220>
<223>Bet v 1突变体
<400>4
Gly Val Phe Asn Tyr Glu Thr Glu Thr Thr Ser Val Ile Pro Ala Ala
1 5 10 15
Arg Leu Phe Lys Ala Phe Ile Leu Asp Gly Asp Asn Leu Phe Pro Lys
20 25 30
Val Ala Pro Gln Ala Ile Ser Ser Val Glu Asn Ile Glu Gly Asn Gly
35 40 45
Gly Pro Gly Thr Ile Lys Lys Ile Ser Phe Pro Glu Gly Phe Pro Phe
50 55 60
Lys Tyr Val Lys Asp Arg Val Asp Glu Val Asp His Thr Asn Phe Lys
65 70 75 80
Tyr Asn Tyr Ser Val Ile Glu Gly Gly Pro Ile Gly Asp Thr Leu Glu
85 90 95
Lys Ile Ser Asn Glu Ile Lys Ile Val Ala Thr Pro Asp Gly Gly Ser
100 105 110
Ile Leu Lys Ile Asn Asn Lys Tyr His Thr Ala Gly Asp His Glu Val
115 120 125
Lys Ala Glu Gln Ile Lys Ala Ser Lys Glu Met Gly Glu Thr Leu Leu
130 135 140
Arg Ala Val Glu Ser Tyr Leu Leu Ala His Ser Asp Ala Tyr Asn
145 150 155
<210>5
<211>159
<212>PRT
<213>未知
<220>
<223>Bet v 1突变体
<400>5
Gly Val Phe Asn Tyr Glu Thr Glu Thr Thr Ser Val Ile Pro Ala Ala
1 5 10 15
Arg Leu Phe Lys Ala Phe Ile Leu Asp Gly Asp Asn Leu Phe Pro Lys
20 25 30
Val Ala Pro Gln Ala Ile Ser Ser Val Glu Asn Ile Glu Gly Asn Gly
35 40 45
Gly Pro Gly Thr Ile Ala Lys Ile Ser Phe Pro Glu Gly Phe Pro Phe
50 55 60
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85 90 95
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<210>6
<211>480
<212>DNA
<213>未知
<220>
<223>野生型Bet v 1编码序列
<400>6
ggtgttttca attacgaaac tgagaccacc tctgttatcc cagcagctcg actgttcaag 60
gcctttatcc ttgatggcga taatctcttt ccaaaggttg caccccaagc cattagcagt 120
gttgaaaaca ttgaaggaaa tggagggcct ggaaccatta agaagatcag ctttcccgaa 180
ggcttccctt tcaagtacgt gaaggacaga gttgatgagg tggaccacac aaacttcaaa 240
tacaattaca gcgtgatcga gggcggtccc ataggcgaca cattggagaa gatctccaac 300
gagataaaga tagtggcaac ccctgatgga ggttccatct tgaagatcaa caacaagtac 360
cataccaaag gagaccatga ggtgaaggca gagcagatta aggcaagtaa agaaatggga 420
gagacacttt tgagggccgt tgagagctac ctcttggcac actccgatgc ctacaactaa 480
<210>7
<211>480
<212>DNA
<213>未知
<220>
<223>Bet v 1-突变体编码序列
<400>7
ggtgttttca attacgaaac tgagaccacc tctgttatcc cagcagctcg actgttcaag 60
gcctttatcc ttgatggcga taatctcttt ccaaaggttg caccccaagc cattagcagt 120
gttgaaaaca ttgaaggaaa tggagggcct ggaaccattg cgaagatcag ctttcccgaa 180
ggcttccctt tcaagtacgt gaaggacaga gttgatgagg tggaccacac aaacttcaaa 240
tacaattaca gcgtgatcga gggcggtccc ataggcgaca cattggagaa gatctccaac 300
gagataaaga tagtggcaac ccctgatgga ggttccatct tgaagatcaa caacaagtac 360
cataccaaag gagaccatga ggtgaaggca gagcagatta aggcaagtaa agaaatggga 420
gagacacttt tgagggccgt tgagagctac ctcttggcac actccgatgc ctacaactaa 480
<210>8
<211>480
<212>DNA
<213>未知
<220>
<223>Bet v 1-突变体编码序列
<400>8
ggtgttttca attacgaaac tgagaccacc tctgttatcc cagcagctcg actgttcaag 60
gcctttatcc ttgatggcga taatctcttt ccaaaggttg caccccaagc cattagcagt 120
gttgaaaaca ttgaaggaaa tggagggcct ggaaccatta agaagatcag ctttcccgaa 180
ggcttccctt tcaagtacgt gaaggacaga gttgatgagg tggaccacac aaacttcaaa 240
tacaattaca gcgtgatcga gggcggtccc ataggcgaca cattggagaa gatctccaac 300
gagataaaga tagtggcaac ccctgatgga ggttccatct tggcgatcaa caacaagtac 360
cataccaaag gagaccatga ggtgaaggca gagcagatta aggcaagtaa agaaatggga 420
gagacacttt tgagggccgt tgagagctac ctcttggcac actccgatgc ctacaactaa 480
<210>9
<211>480
<212>DNA
<213>未知
<220>
<223>Bet v 1-突变体编码序列
<400>9
ggtgttttca attacgaaac tgagaccacc tctgttatcc cagcagctcg actgttcaag 60
gcctttatcc ttgatggcga taatctcttt ccaaaggttg caccccaagc cattagcagt 120
gttgaaaaca ttgaaggaaa tggagggcct ggaaccatta agaagatcag ctttcccgaa 180
ggcttccctt tcaagtacgt gaaggacaga gttgatgagg tggaccacac aaacttcaaa 240
tacaattaca gcgtgatcga gggcggtccc ataggcgaca cattggagaa gatctccaac 300
gagataaaga tagtggcaac ccctgatgga ggttccatct tgaagatcaa caacaagtac 360
cataccgcag gagaccatga ggtgaaggca gagcagatta aggcaagtaa agaaatggga 420
gagacacttt tgagggccgt tgagagctac ctcttggcac actccgatgc ctacaactaa 480
<210>10
<211>480
<212>DNA
<213>未知
<220>
<223>Bet v 1突变体-编码序列
<400>10
ggtgttttca attacgaaac tgagaccacc tctgttatcc cagcagctcg actgttcaag 60
gcctttatcc ttgatggcga taatctcttt ccaaaggttg caccccaagc cattagcagt 120
gttgaaaaca ttgaaggaaa tggagggcct ggaaccattg cgaagatcag ctttcccgaa 180
ggcttccctt tcaagtacgt gaaggacaga gttgatgagg tggaccacac aaacttcaaa 240
tacaattaca gcgtgatcga gggcggtccc ataggcgaca cattggagaa gatctccaac 300
gagataaaga tagtggcaac ccctgatgga ggttccatct tggcgatcaa caacaagtac 360
cataccgcag gagaccatga ggtgaaggca gagcagatta aggcaagtaa agaaatggga 420
gagacacttt tgagggccgt tgagagctac ctcttggcac actccgatgc ctacaactaa 480
Claims (15)
1.蛋白质，其是垂枝桦花粉主要变应原(Bet v 1)的低变应原性变体，并且特征在于：
a)与野生型Bet v 1(SEQ ID NO：1)相比，表现出与IgE降低的反应性；
b)具有以下氨基酸序列：
i)与SEQ ID NO：1具有至少87％同一性，优选至少94％同一性，更优选至少97％同一性；
ii)与SEQ ID NO：1的序列比对表现出SEQ ID NO：1第54、115和/或123位氨基酸对应位置上的至少一个Lys残基取代或缺失。
2.根据权利要求1的蛋白质，其中所述Lys残基被中性或极性氨基酸取代。
3.根据权利要求2的蛋白质，其中所述中性或极性氨基酸选自Ala、Thr、Gly、Pro、Leu、Ile、Phe、Ser。
4.根据权利要求3的蛋白质，其中所述氨基酸是Ala、Ser或Thr。
5.根据权利要求1的蛋白质，其选自SEQ ID NO：2、SEQ ID NO：3、SEQ ID NO：4、SEQ ID NO：5。
6.权利要求1-5的蛋白质的免疫活性肽片段，其包含15至35个氨基酸并具有如上定义的至少一个Lys取代和/或缺失。
7.根据权利要求6的肽，其含有15至20个氨基酸。
8.核酸分子，其编码权利要求1-5的蛋白质或权利要求6-7的肽。
9.根据权利要求8的核酸分子，其选自SEQ ID NO：7、SEQ ID NO：8、SEQ ID NO：9、SEQ ID NO：10。
10.载体，其含有权利要求8-9的核酸分子。
11.宿主细胞，其含有权利要求10的载体。
12.药物组合物，其含有有效量的权利要求1-5的蛋白质，或权利要求6-7的肽，以及药学可接受的载体和赋形剂。
13.根据权利要求12的组合物，其为疫苗形式。
14.权利要求1-5的蛋白质或权利要求6-7的肽在制备用于预防或治疗变应性疾病的药物组合物中的用途。
15.根据权利要求14的用途，其用于治疗支气管哮喘、鼻炎、结膜炎和特应性皮炎。
Applications Claiming Priority (3)
| Application Number | Priority Date | Filing Date | Title |
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| ITMI2005A002517 | 2005-12-29 | ||
| IT002517A ITMI20052517A1 (it) | 2005-12-29 | 2005-12-29 | Varianbtio ipoallergeniche dell'allergene maggiore bet v 1 di polline di betula verrucosa |
| PCT/EP2006/012237 WO2007073907A1 (en) | 2005-12-29 | 2006-12-19 | Hypoallergenic variants of the major allergen from betula verrucosa pollen |
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| CN101351224A true CN101351224A (zh) | 2009-01-21 |
| CN101351224B CN101351224B (zh) | 2013-04-03 |
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| EP (2) | EP2172215B1 (zh) |
| JP (1) | JP2009521910A (zh) |
| CN (1) | CN101351224B (zh) |
| AT (1) | ATE457738T1 (zh) |
| AU (1) | AU2006331005B2 (zh) |
| BR (1) | BRPI0620749A2 (zh) |
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| IT (1) | ITMI20052517A1 (zh) |
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| NO (1) | NO20082881L (zh) |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103649315A (zh) * | 2011-04-18 | 2014-03-19 | 国家技术研究中心Vtt | 低过敏原 |
| CN103874509A (zh) * | 2011-08-03 | 2014-06-18 | 洛法玛有限公司 | 来自苹果的主要变应原Mal d 1的低变应原性变体 |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP2028188A1 (en) * | 2007-08-21 | 2009-02-25 | Biomay AG | Hypoallergenic molecules |
| ITMI20071819A1 (it) | 2007-09-19 | 2009-03-20 | Lofarma Spa | Varianti ipoallergeniche dell'allergene maggiore bet v 2 di polline di betula verrucosa |
| FI20115375A0 (fi) | 2011-04-18 | 2011-04-18 | Teknologian Tutkimuskeskus Vtt Oy | Uudet hypoallergeenit |
| WO2018222854A1 (en) | 2017-06-01 | 2018-12-06 | Regeneron Pharmaceuticals, Inc. | Human antibodies to bet v 1 and methods of use thereof |
| BR112022026279A2 (pt) | 2020-07-01 | 2023-02-14 | Regeneron Pharma | Métodos de tratamento de alergia usando anticorpos anti-bet v 1 |
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| AU5411594A (en) * | 1992-10-27 | 1994-05-24 | Biomay Produktions- Und Handelsgesellschaft M.B.H. | Molecule fragments (peptides) of the main allergens contained in the pollen of trees of the (fagales) order |
| DK0885244T4 (da) * | 1996-03-01 | 2008-06-16 | Novartis Ag | Peptidimmunogener til vaccination imod og til behandling af allergi |
| CN1332029C (zh) * | 1998-03-16 | 2007-08-15 | 阿尔克-阿贝洛有限公司 | 突变的重组过敏原 |
| NZ525820A (en) * | 2000-11-16 | 2005-01-28 | Alk Abello As | Mutant allergens |
| US20040043438A1 (en) * | 2002-05-16 | 2004-03-04 | Alk-Abello A/S | Allergen mutants |
| EP1504089A2 (en) * | 2002-05-16 | 2005-02-09 | Alk Abell A/S | Allergen mutants |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103649315A (zh) * | 2011-04-18 | 2014-03-19 | 国家技术研究中心Vtt | 低过敏原 |
| CN103649315B (zh) * | 2011-04-18 | 2016-12-07 | 德森特姆有限公司 | 低过敏原 |
| CN103874509A (zh) * | 2011-08-03 | 2014-06-18 | 洛法玛有限公司 | 来自苹果的主要变应原Mal d 1的低变应原性变体 |
| CN103874509B (zh) * | 2011-08-03 | 2016-08-17 | 洛法玛有限公司 | 来自苹果的主要变应原Mal d 1的低变应原性变体 |
Also Published As
| Publication number | Publication date |
|---|---|
| WO2007073907A1 (en) | 2007-07-05 |
| NO20082881L (no) | 2008-09-25 |
| EP2172215B1 (en) | 2013-10-16 |
| JP2009521910A (ja) | 2009-06-11 |
| CA2634955A1 (en) | 2007-07-05 |
| EP1973567B1 (en) | 2010-02-17 |
| DK1973567T3 (da) | 2010-05-31 |
| DE602006012376D1 (de) | 2010-04-01 |
| EP2172215A2 (en) | 2010-04-07 |
| EA200801463A1 (ru) | 2008-12-30 |
| MX2008008578A (es) | 2008-09-26 |
| US8945574B2 (en) | 2015-02-03 |
| UA93894C2 (ru) | 2011-03-25 |
| ITMI20052517A1 (it) | 2007-06-30 |
| CA2634955C (en) | 2017-02-28 |
| ES2341367T3 (es) | 2010-06-18 |
| CN101351224B (zh) | 2013-04-03 |
| EP2172215A3 (en) | 2010-05-26 |
| ATE457738T1 (de) | 2010-03-15 |
| US20090304752A1 (en) | 2009-12-10 |
| ES2441217T3 (es) | 2014-02-03 |
| BRPI0620749A2 (pt) | 2011-11-22 |
| DK2172215T3 (da) | 2014-01-20 |
| PT1973567E (pt) | 2010-05-12 |
| EP1973567A1 (en) | 2008-10-01 |
| AU2006331005A1 (en) | 2007-07-05 |
| AU2006331005B2 (en) | 2012-02-02 |
| EA015446B1 (ru) | 2011-08-30 |
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