CN101861331B - 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 2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 2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(SIT)是变应性疾病的唯一病原疗法，能够顺利地改变这类疾病的特征性免疫参数。
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，尤其是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)的疫苗(3)。
如果IgG抗体对于致敏变应原是特异性的，则SIT的有利因素之一是诱导。这类(保护)抗体能够抑制抗原-IgE结合，特别是抑制IgE与Bet v 2抗原结合，改变该分子的三维构像(4，5)。包含具有未改变免疫原性的低变应原性重组蛋白的疫苗的开发将改善变应性疾病的治疗。
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(6，7)。Bet v2属于抑制蛋白家族，其是涉及调节真核细胞细胞骨架的遍在胞质蛋白质。它们与至少两种细胞(大)分子相互作用，该至少两种细胞(大)分子即磷脂酰肌醇-4，5-二磷酸，从而阻止C-γ磷脂酶(phospholypase)对该脂肪酸的水解(8)，和肌动蛋白，调控其的聚合作用(9)。抑制蛋白在成熟和萌发的花粉中的高表达暗示它们参与调节微丝前体，该微丝前体参与萌发过程(10)。抑制蛋白被认为是来自多种乔木和草本植物的花粉中的变应原，和多种水果和蔬菜中的变应原，因此抛开抑制蛋白仅在20％对花粉变应性的(过敏的)患者中发现的事实，它们被定义为“泛-变应原(pan-allergens)”(11，12)。
在大部分多种来源的植物抑制蛋白中的高序列同源性(高于60％)导致交叉致敏，不仅在来自相关植物(13)和不相关植物(14)的花粉之间，还在花粉和植物食品(aliment，15)之间，或在花粉和橡胶(latex)之间(16)。植物抑制蛋白和哺乳动物抑制蛋白之间的同源性较低，尽管证明了它们能够结合来自不同物种的肌动蛋白且显示出可交换性(17，18，19)。解释是所有的抑制蛋白具有相似的三维结构，如使用X-射线晶体学所示的(20，21，22)。
许多研究证实抑制蛋白的免疫等同物。事实上，已显示来自对确定的花粉致敏的患者的IgE能够结合不同来源的抑制蛋白，结合抑制蛋白的IgE可以相互抑制(16)。
不同花粉之间的高交叉反应性使得单独的抑制蛋白可用于变应反应诊断，重组Bet v 2通常用作选择抑制蛋白特异性IgE测定的变应原(23，24)。
存在多种测定抑制蛋白IgE表位的研究。
根据同时期相同小组进行的下述研究(26)，Vrtala(1996)在Phe44和Gln47诱变Bet v 2，将它们变成Tyr44、Glu47和Asn47，所述研究中鉴定了单克隆抗体4A6识别的线性表位。使用合成的十二肽(跨越Bet v2的整个氨基酸序列)对该抗体识别的表位作图。更有效结合该抗体的多肽含有氨基酸38-49和40-51之间的区域。IgG-肽结合中Gln47残基的重要性由下述证据证明：4A6不能够识别来自烟草(Nicotiana tabacum)和梯牧草(Phleum pratense)的抑制蛋白，其序列显示谷氨酸位于Bet v 2的Gln47。与Gln47→Glu47突变不同，从Phe44到Tyr44的改变或从GIn47到Asn47的改变不影响抗体的结合。如免疫印记和ELISA试验(25)所示，对重组Bet v 2应用相同的诱变(Gln47到Glu，或Asn和Phe44到Tyr44)不能够降低抑制蛋白和IgE之间的结合。
在1997年公开的随后的研究(22)中，通过克隆桦树抑制蛋白cDNA的随机片段鉴定了主要的IgE表位，该cDNA来自用对抑制蛋白变应性的患者血清分析的表达文库。证明三个区域更有反应性，即对应于位于氨基端的α螺旋(aa 1-30)的区域。羧基端的α螺旋(aa 106-132)的区域，以及包含残基30-50的片段的区域。
在随后的研究(23)中，对IgE表位的研究基于来自不同植物的抑制蛋白的理论结构模型与桦树或橡胶抑制蛋白晶体之间的比较。预测了11个可能的构象表位(包含相邻的氨基酸区域)，其中至少20％是暴露于表面的。氨基酸序列和构象模型的比较得到两类表位：物种特异性表位(由高变异性表征)和高度保守表位，后者更可能涉及不同植物抑制蛋白之间的交叉反应。氨基酸序列比对反映Fedorov研究(22)的结果，证明在抑制蛋白N-端具有两个可能的线性表位，在残基30-80之间有三个可能的线性表位，且其余两个在抑制蛋白C-端。所有这些区域在该研究中所评测的植物抑制蛋白中均是高度保守的。可能的构象表位的预测基于橡胶抑制蛋白Hev b8的3D模型的分析。该分析证明了选自表位中心的12个突出的残基。虽然所有可能的表位均是构想性的，它们包含线性序列以及保守或可变的残基。在该研究中没有报导证实所研究的表位的特异性IgE-结合能力的测试。
更为近期的出版物涉及甜瓜(Cucumis melo)抑制蛋白IgE表位的鉴定(27)。通过测定跨越该蛋白质的整个氨基酸序列的肽的IgE反应性，鉴定了两个线性表位，其中E1被对甜瓜变应性的患者血清强烈识别，包含残基66-75和81-93，E2由氨基酸95-99和122-131组成。另外两个表位被较弱的IgE应答表征，即E3(残基2-10)和E4(35-45)。对应甜瓜抑制蛋白3D模型的表位E1和E2的肽的重叠表示具有良好定义的静电性质的两个区域：E1和E2，其分别和正电性和负电性蛋白质结构域相关。
来自文献的数据表示IgE抑制蛋白表位位于其中的分子部分，但是未指出涉及IgE结合的氨基酸。
发明内容
现已发现通过取代或缺失Bet v 2变应原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 2变应原的序列变体，且其特征在于：
1)与野生型Bet v 2变应原(SEQ ID NO：1)相比，对IgE的反应性降低；
2)氨基酸序列为：
a)与SEQ ID NO：1至少90％相同，优选至少93％相同，进一步优先至少97％相同；
b)与SEQ ID NO：1的序列比对表明SEQ ID NO：1在对应于(matching)Ser39、Lys 45、Lys88或Lys89的Ser或Lys残基处具有至少一个取代或缺失。
在优选的实施方案中，本发明的Bet v 2变应原的变体在所示位置具有1-3个中的多个取代或缺失，其中产生单、双或三取代和/或缺失变体。虽然可以在Bet v 2分子中同时存在不同氨基酸残基的取代和缺失，优选在指示位点通过一个或多个残基的单取代获得变体，特别是此类残基被中性、极性或酸性氨基酸置换的变体，所述中性、极性或酸性氨基酸选自Ala、Thr、Gly、Pro、Leu、Ile、Ser、Phe、Glu、Asp，更优选选自Ala、Thr、Ser、Gly、Glu、Asp。
本发明变体的实例示于SEQ ID NO：2(1个残基取代)、SEQ ID NO：3(1个残基取代)、SEQ ID NO：4(2个残基取代)、SEQ ID NO：5(3个残基取代)和SEQ ID NO：6(3个残基取代)。
相对于野生型对应物(counterpart)，本发明的Bet v 2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的反应性，其降低至少10％，优选至少50％，更优选至少90％，其中IgE反应性通过例如ELISA测定的方法检测。
来自变应性患者血清库的蛋白质SEQ ID NO：2-6的IgE反应性由ELISA测定检测(图1)。相对于野生型Bet v 2变应原(SEQ ID NO：1)，当(SEQID NO：2)、(SEQ ID NO：3)、(SEQ ID NO：4)和(SEQ ID NO：5和6)蛋白质与来自桦树花粉变应性患者的血清库孵育时，观察到IgE反应性平均降低92％(SEQ IDNO：2)、13％(SEQ ID NO：3)、97％(SEQ ID NO：4)和93％(SEQ ID NO：5和6)。
这些结果由REAST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。在存在1.45ng抑制物时，当血清用相同的蛋白质预处理过，野生型Bet v 2(SEQ ID NO：1)与来自变应性患者血清库的IgE的结合82.6％抑制，当血清用变体SEQ ID NO：2和SEQ ID NO：3预温育时，分别是40.4％和71％抑制，而当血清分别用相同量的双取代变体(SEQ IDNO：4)和三取代变体(SEQ ID NO：5和SEQ ID NO：6)预温育时，观察到的抑制仅为13.4％、4％和8.8％(图2)。
这些结果明确表明SEQ ID NO：1第39、45、88、89位的氨基酸参与IgE对Bet v 2变应原的识别。
另外，Balb/c小鼠免疫接种试验证明野生型Bet v 2变应原和低变应原性蛋白质SEQ ID NO：5(选自作为示例性突变变应原)均能诱导IgG-特异性免疫应答(图3)。抗SEQ ID NO：5抗体能够识别野生型-对应物SEQ IDNO：1(图4)，证明第45、88、89位Lys-残基的取代并不导致分子免疫原性的重大改变，且改变其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 2和SEQ ID NO：5。
另一方面，本发明提供Bet v 2片段相应的免疫活性肽，所述免疫活性肽包含至少一种上述取代和/或缺失。所述肽优选含有15-35个氨基酸残基、更优选15-20个氨基酸残基。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，通过诱变野生型Bet v 2cDNA序列(SEQ ID NO：7)可容易地制备本发明的取代和/或缺失变体。
编码SEQ ID NO：2-6所示的单、双和三取代变体的cDNA序列报告于SEQ ID NO：8-12。
另一方面，本发明提供编码本文公开的低变应原性Bet v 2变体的核酸分子、衍生自其的肽、包含该分子的表达载体，其中所述分子与真核细胞或原核细胞中控制其表达的遗传元件(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。
本发明还包含用本发明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cDNA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 2变体可方便地用于制备药物组合物(例如片剂)，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 2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(28，29)。
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。
附图简述
图1：ELISA分析IgE与Bet v 2变应原和Bet v 2低变应原性变体的反应性；
图2：IgE与Betv 2变应原结合的抑制；
图3：突变IgE与各免疫原性蛋白质的应答；
图4：SEQ ID NO：5免疫的小鼠中的IgG应答。
实施例1-Bet v 2变应原编码cDNA的位点特异性诱变
Bet v 2变应原编码cDNA(SEQ ID NO：7)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(30)。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 v2 S39 | ggg ccc aga gcg ctt cct tcc cac ag |
| Bet v2 K45 | cct tcc cac agt tta cgc ctc agg aaa tc |
| Bet v2 K88-89 | gtc atc cgt gga ggg gag gga tct gga g |
实施例2-制备Bet v 2蛋白及其变体
根据标准方案(31，32)在大肠杆菌中克隆并表达野生型(SEQ ID NO.7)和诱变的(SEQ ID NO：8-12)Bet v 2cDNA。细胞离心收集，重悬于PBS1X(6.46mM NaH2PO4、1.47mM KH2PO4、136.89mM NaCl)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PBS 1X、6M尿素(变性缓冲液)中并在4℃下搅拌60分钟。溶解的重组蛋白从不溶的残渣中离心分离，用PBS 1X透析，滤过1μM的滤器并通过使用用聚-脯氨酸衍生化的琼脂糖柱(Sigma，Milan，意大利)亲和层析纯化。用PBS 1X、2M尿素洗涤后，用PBS 1X、8M尿素洗脱重组蛋白质，并在PBS 1X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Bet v 2变应原有RAST 3+和4+特异反应性的个体，然后集中这些血清并以这种形式使用。来自非变应性患者的血清库用作阴性对照。
实施例4-Bet v 2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(1μg)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％山羊血清、1mM EDTA、0.05％Tween 20、0.01％乙基汞硫代水杨酸钠，pH 7.4)封闭。70μl人血清RAST 3+和4+库(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-REAST抑制测定。Bet v 2变体抑制生物素化Bet v 2与血清库中包含的IgE的结合
1∶3稀释于稀释缓冲液(150mM磷酸盐缓冲液中的25％山羊血清、1mM EDTA、0.05％Tween 20、0.01％乙基汞硫代水杨酸钠，pH 7.4)中的对Bet v 2的人血清库RAST 4+和3+的整分试样(50μl)与野生型变应原及其突变体的连续稀释物(从67ng/ml开始)在25℃下预温育1.5小时。然后将混合物加入ELISA聚苯乙烯(polystirene)平板的小孔中并在25℃温育1.5小时，该小孔吸附人抗-IgE。用0.06M的磷酸缓冲液、0.05％Tween-20，pH6.5洗涤三次后，加入稀释缓冲液中的0.1ml生物素化Bet v 2抗原(85.3ng/ml)并在25℃温育1小时。洗涤三次后，25℃下加入过氧化物酶-链霉亲和素(0.1μg/ml)30分钟。用100μl 1N HCl进行比色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是无抑制物时450nm处测量的吸光度，而B是存在抑制物时的吸光度。
实施例6-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 IDNO：1，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，从颈静脉获取血样，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7-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 2和变体SEQ ID NO：5(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3和4显示通过分析每组的5只小鼠的血清获得的平均反应性。
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序列表
<110>洛法玛有限公司(LOFARMA S.P.A.)
<120>垂枝桦花粉主要变应原的低变应原性变体
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<223>来自垂枝桦(Betula verrucosa)的野生型Bet v 2
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Met Ser Trp Gln Thr Tyr Val Asp Glu His Leu Met Cys Asp Ile Asp
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<210>7
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<223>来自垂枝桦的野生型Bet v 2的编码序列
<400>7
atgtcgtggc aaacgtacgt ggatgaacat ttgatgtgcg atatcgacgg gcaagccagc 60
aactcgctgg catctgcgat cgtcggtcac gatggctctg tgtgggccca gagctcttcc 120
ttcccacagt ttaagcctca ggaaatcact ggtatcatga aggactttga ggagccgggt 180
catcttgctc cgacgggctt acaccttggg ggcataaaat acatggtcat ccagggagag 240
gctggtgctg tcatccgtgg aaagaaggga tctggaggta ttactataaa gaagactggt 300
caagctctcg tttttggcat ctatgaagag cctgtgacac caggacagtg caacatggtt 360
gttgagaggt tgggggatta ccttattgac cagggcctgt ag 402
<210>8
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<213>未知
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<223>Bet v 2突变体的编码序列
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atgtcgtggc aaacgtacgt ggatgaacat ttgatgtgcg atatcgacgg gcaagccagc 60
aactcgctgg catctgcgat cgtcggtcac gatggctctg tgtgggccca gagcgcttcc 120
ttcccacagt ttaagcctca ggaaatcact ggtatcatga aggactttga ggagccgggt 180
catcttgctc cgacgggctt acaccttggg ggcataaaat acatggtcat ccagggagag 240
gctggtgctg tcatccgtgg aaagaaggga tctggaggta ttactataaa gaagactggt 300
caagctctcg tttttggcat ctatgaagag cctgtgacac caggacagtg caacatggtt 360
gttgagaggt tgggggatta ccttattgac cagggcctgt ag 402
<210>9
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<212>DNA
<213>未知
<220>
<223>Bet v 2突变体的编码序列
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atgtcgtggc aaacgtacgt ggatgaacat ttgatgtgcg atatcgacgg gcaagccagc 60
aactcgctgg catctgcgat cgtcggtcac gatggctctg tgtgggccca gagctcttcc 120
ttcccacagt ttacgcctca ggaaatcact ggtatcatga aggactttga ggagccgggt 180
catcttgctc cgacgggctt acaccttggg ggcataaaat acatggtcat ccagggagag 240
gctggtgctg tcatccgtgg aaagaaggga tctggaggta ttactataaa gaagactggt 300
caagctctcg tttttggcat ctatgaagag cctgtgacac caggacagtg caacatggtt 360
gttgagaggt tgggggatta ccttattgac cagggcctgt ag 402
<210>10
<211>402
<212>DNA
<213>未知
<220>
<223>Bet v 2突变体的编码序列
<400>10
atgtcgtggc aaacgtacgt ggatgaacat ttgatgtgcg atatcgacgg gcaagccagc 60
aactcgctgg catctgcgat cgtcggtcac gatggctctg tgtgggccca gagctcttcc 120
ttcccacagt ttaagcctca ggaaatcact ggtatcatga aggactttga ggagccgggt 180
catcttgctc cgacgggctt acaccttggg ggcataaaat acatggtcat ccagggagag 240
gctggtgctg tcatccgtgg aggggaggga tctggaggta ttactataaa gaagactggt 300
caagctctcg tttttggcat ctatgaagag cctgtgacac caggacagtg caacatggtt 360
gttgagaggt tgggggatta ccttattgac cagggcctgt ag 402
<210>11
<211>402
<212>DNA
<213>未知
<220>
<223>Bet v 2突变体的编码序列
<400>11
atgtcgtggc aaacgtacgt ggatgaacat ttgatgtgcg atatcgacgg gcaagccagc 60
aactcgctgg catctgcgat cgtcggtcac gatggctctg tgtgggccca gagctcttcc 120
ttcccacagt ttacgcctca ggaaatcact ggtatcatga aggactttga ggagccgggt 180
catcttgctc cgacgggctt acaccttggg ggcataaaat acatggtcat ccagggagag 240
gctggtgctg tcatccgtgg aggggaggga tctggaggta ttactataaa gaagactggt 300
caagctctcg tttttggcat ctatgaagag cctgtgacac caggacagtg caacatggtt 360
gttgagaggt tgggggatta ccttattgac cagggcctgt ag 402
<210>12
<211>402
<212>DNA
<213>未知
<220>
<223>Bet v 2突变体的编码序列
<400>12
atgtcgtggc aaacgtacgt ggatgaacat ttgatgtgcg atatcgacgg gcaagccagc 60
aactcgctgg catctgcgat cgtcggtcac gatggctctg tgtgggccca gagcgcttcc 120
ttcccacagt ttaagcctca ggaaatcact ggtatcatga aggactttga ggagccgggt 180
catcttgctc cgacgggctt acaccttggg ggcataaaat acatggtcat ccagggagag 240
gctggtgctg tcatccgtgg aggggaggga tctggaggta ttactataaa gaagactggt 300
caagctctcg tttttggcat ctatgaagag cctgtgacac caggacagtg caacatggtt 360
gttgagaggt tgggggatta ccttattgac cagggcctgt ag 402
Claims (11)
1.低变应原性蛋白质，其是Bet v2主要变应原的序列变体，其中所述蛋白质：
a)与由SEQ ID NO：1所示的野生型Bet v2变应原相比，对IgE的反应性降低；和
b)通过至少一个在SEQ ID NO：1的位置Ser39、Lys45、Lys88或Lys89的Ser或Lys残基的取代从SEQ ID NO：1获得，其中所述Ser或Lys残基被取代为选自Ala、Thr、Ser、Gly、Glu和Asp的氨基酸残基。
2.根据权利要求1的低变应原性蛋白质，其选自SEQ ID NO：2-6。
3.核酸分子，其编码权利要求1或2的蛋白质。
4.根据权利要求3的核酸分子，其选自SEQ ID NO：8-12。
5.载体，其含有权利要求3或4的核酸分子。
6.宿主细胞，其含有权利要求5的载体。
7.药物组合物，其含有有效量的权利要求1或2的低变应原性蛋白质，以及可药用载体。
8.药物组合物，其含有有效量的权利要求1或2的低变应原性蛋白质，以及赋形剂。
9.根据权利要求7或8的组合物，其为疫苗形式。
10.权利要求1或2的低变应原性蛋白质在制备用于由来自垂枝桦物种的植物花粉导致的变应性疾病的预防性或治疗性治疗的药物组合物中的用途。
11.根据权利要求10的用途，其中所述变应性疾病选自支气管哮喘和变应性结膜炎。
Applications Claiming Priority (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| IT001819A ITMI20071819A1 (it) | 2007-09-19 | 2007-09-19 | Varianti ipoallergeniche dell'allergene maggiore bet v 2 di polline di betula verrucosa |
| ITMI2007A001819 | 2007-09-19 | ||
| PCT/EP2008/007726 WO2009036949A1 (en) | 2007-09-19 | 2008-09-16 | Hypoallergenic variants of the major allergen from betula verrucosa pollen |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN101861331A CN101861331A (zh) | 2010-10-13 |
| CN101861331B true CN101861331B (zh) | 2014-07-16 |
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| Application Number | Title | Priority Date | Filing Date |
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| CN200880116680.6A Active CN101861331B (zh) | 2007-09-19 | 2008-09-16 | 垂枝桦花粉主要变应原的低变应原性变体 |
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| Country | Link |
|---|---|
| US (1) | US8591907B2 (zh) |
| EP (1) | EP2201031B1 (zh) |
| CN (1) | CN101861331B (zh) |
| AU (1) | AU2008300864B2 (zh) |
| CA (1) | CA2700047C (zh) |
| DK (1) | DK2201031T3 (zh) |
| EA (1) | EA019696B1 (zh) |
| ES (1) | ES2460573T3 (zh) |
| IT (1) | ITMI20071819A1 (zh) |
| MX (1) | MX2010002997A (zh) |
| WO (1) | WO2009036949A1 (zh) |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| FI20115374A0 (fi) * | 2011-04-18 | 2011-04-18 | Teknologian Tutkimuskeskus Vtt Oy | Uudet hypoallergeenit |
| RU2732013C1 (ru) * | 2019-12-11 | 2020-09-10 | Федеральное бюджетное учреждение науки "Государственный научный центр прикладной микробиологии и биотехнологии" Федеральной службы по надзору в сфере защиты прав потребителей и благополучия человека (ФБУН ГНЦ ПМБ) | Способ сенсибилизации планшета для иммуноферментного анализа нерастворимыми белковыми антигенами |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2007073907A1 (en) * | 2005-12-29 | 2007-07-05 | Lofarma S.P.A. | Hypoallergenic variants of the major allergen from betula verrucosa pollen |
-
2007
- 2007-09-19 IT IT001819A patent/ITMI20071819A1/it unknown
-
2008
- 2008-09-16 EP EP08802256.1A patent/EP2201031B1/en active Active
- 2008-09-16 ES ES08802256.1T patent/ES2460573T3/es active Active
- 2008-09-16 MX MX2010002997A patent/MX2010002997A/es not_active Application Discontinuation
- 2008-09-16 AU AU2008300864A patent/AU2008300864B2/en active Active
- 2008-09-16 DK DK08802256.1T patent/DK2201031T3/da active
- 2008-09-16 CA CA2700047A patent/CA2700047C/en active Active
- 2008-09-16 US US12/679,207 patent/US8591907B2/en active Active
- 2008-09-16 WO PCT/EP2008/007726 patent/WO2009036949A1/en active Application Filing
- 2008-09-16 CN CN200880116680.6A patent/CN101861331B/zh active Active
- 2008-09-16 EA EA201000360A patent/EA019696B1/ru not_active IP Right Cessation
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2007073907A1 (en) * | 2005-12-29 | 2007-07-05 | Lofarma S.P.A. | Hypoallergenic variants of the major allergen from betula verrucosa pollen |
Non-Patent Citations (1)
| Title |
|---|
| Susanne Vrtala et al.High-Level Expression in Escherichia cloi and Purification of Recombinant Plant Profilins:comparison of IgE-Binding Capacity and Allergenic Activity.《BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS》.1996,第226卷(第1039期),第42-50页. * |
Also Published As
| Publication number | Publication date |
|---|---|
| AU2008300864B2 (en) | 2014-02-27 |
| DK2201031T3 (da) | 2014-05-05 |
| EP2201031B1 (en) | 2014-03-19 |
| WO2009036949A1 (en) | 2009-03-26 |
| EA019696B1 (ru) | 2014-05-30 |
| US20100310590A1 (en) | 2010-12-09 |
| CN101861331A (zh) | 2010-10-13 |
| MX2010002997A (es) | 2010-06-01 |
| CA2700047A1 (en) | 2009-03-26 |
| AU2008300864A1 (en) | 2009-03-26 |
| EP2201031A1 (en) | 2010-06-30 |
| EA201000360A1 (ru) | 2010-10-29 |
| ITMI20071819A1 (it) | 2009-03-20 |
| CA2700047C (en) | 2017-01-17 |
| ES2460573T3 (es) | 2014-05-13 |
| US8591907B2 (en) | 2013-11-26 |
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