CN1454213A - 尘螨属2组的变应原性蛋白质的变体 - Google Patents

尘螨属2组的变应原性蛋白质的变体 Download PDF

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CN1454213A
CN1454213A CN01815422A CN01815422A CN1454213A CN 1454213 A CN1454213 A CN 1454213A CN 01815422 A CN01815422 A CN 01815422A CN 01815422 A CN01815422 A CN 01815422A CN 1454213 A CN1454213 A CN 1454213A
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M·斯图拉罗
A·维奥蒂
P·法拉贾尼
G·米斯特雷洛
D·龙卡罗洛
S·扎诺塔
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Abstract

欧洲屋尘螨种的变应原变体，其具有降低的变应原性。

Description

尘螨属2组的变应原性蛋白质的变体
本发明涉及螨的欧洲屋尘螨(Dermatophagoides pteronyssinus)种变应原的新变体。
更具体地，本发明涉及变应原Der p 2的低变应原性变体的氨基酸序列，其通过将编码所述变应原的核苷酸序列进行位点专一诱变而获得。该低变应原性变体可用于由尘螨引起的变应性病理现象的特异性免疫治疗。
发明背景
变态反应为接触变应原后由产生的IgE类抗体而引起的速发型超敏反应。IgE与位于效应细胞(嗜碱性粒细胞和肥大细胞)表面的特异性受体结合，并且当再次暴露于该变应原时，它们诱导所述细胞脱颗粒，释放介质(如组胺和白三烯)，引起下列公知的变态反应症状：鼻炎、结膜炎、特应性皮炎和哮喘。
尘螨属的欧洲屋尘螨和美洲屋尘螨(Dermatophagoides farinae)为存在于室尘中的两种相似的螨。来自这些节肢动物的变应原在临床上具有显著的重要性。
至今已鉴定出了九种不同类型的螨变应原，其中主要的两类为Der p 1(Der f 1)和Der p 2(Der f 2)，它们中的每一种均与约80％变应性受试者的IgE发生免疫反应。
变应原Der p 2(其核苷酸序列在GenBank中的存取代码为AF276239)为由129个氨基酸残基组成的分子量为约14kD的蛋白质，其含有为免疫原性所必需的三个二硫键[1，2]。它与除Derf2(GenBank存取代码D10449)外的任一其它已知蛋白质无序列同源性。
变态反应的唯一病原学治疗表现为特异性脱敏免疫治疗(SIT)。该治疗在于将引起变态反应的物质施用渐增的剂量，由此在患者体内对该物质诱导逐渐的脱敏反应(3)。
虽然免疫治疗构成了对变态反应的一种已建立的治疗方案，但它并非完全没有危险(4)。
由于在此类治疗期间可发生更为严重的副作用，研究人员正着重研究应用非注射途径(经口/舌下途径)来施用疫苗以及产生蛋白质的用作疫苗的低变应原性变体，其中所述低变应原性变体是通过化学处理或位点专一诱变来改变变应原而获得的[5]。
详细公开
编码欧洲屋尘螨的主要变应原Der p 2的新cDNA已通过RT-PCR分离，其核苷酸序列在SEQ ID N.1报导。它在8个位置处不同于GenBank中的序列。分离的克隆所编码的在SEQ ID N.3中报导的蛋白质有两个残基Ala16和Ser63不同于变应原Der p 2。
现在已发现成熟蛋白质Der p 2的变应性效应可通过在存在赖氨酸残基的下列位置至少之一处进行氨基酸序列的改变而减弱：33、48、82、96、100、126，其中所述成熟蛋白质Der p 2通过从SEQ ID N.3去除残基1-16而产生。
此处“改变”是指在特定位置处替换一个或多个残基，优选地用中性和极性氨基酸替换，或者删除存在于天然形式中的一个或多个Lys残基，或者同时替换和删除两个或多个残基。
替换所致突变在上述6个位置处的每一位置导入了丙氨酸残基。最优选的变体是在SEQ ID N.4中所示的6处替换同时存在。
本发明也包含蛋白质Der p 2的变体，其与Der p 2的同源性大于85％，该变体在其氨基酸序列的相应位置处具有如上所述的针对Der p 2的相同替换/删除模式，该变体尤其是蛋白质Der f 2。
本发明还包含来自Der p 2的氨基酸序列或来自其同源序列并含有至少一个上述替换/删除的免疫活性肽。
在进一步的方面，本发明涉及编码如上所述的Der p 2突变变体的核酸分子、编码其同源变体的核酸分子、或编码来自于它们的肽的核酸分子。
本发明的序列变体易于从成熟Der p 2的cDNA或其同源变体的cDNA开始制备，在序列变体中编码信号肽的区域缺失且已导入目标突变。
编码具6处替换的优选变体(SEQ ID N.4)的cDNA序列(诱变处理的碱基用黑体表示)在SEQ ID N.2中报导。
对欧洲屋尘螨产生变应性的受试者血清中，本发明所产生的重组蛋白质具有减轻的IgE反应性。特别是使用对螨产生变应性且具有RAST 4+反应性的受试者的合并血清进行蛋白质印迹试验表明：与在大肠杆菌(Escherichia coli)中产生的正常蛋白质相比，SEQ ID N.4中所报导变体的IgE反应性平均降低了约90％[图1]。该结果用可确定天然构象中蛋白质表位包括构象性表位的ELISA试验证实[图2]。
本发明还涉及这样的表达载体，其包含编码任一以上定义的低变应原性变体的核酸分子。
所述载体可为基因工程中常用的质粒、粘粒、病毒、噬菌体或任一其它载体，并且除了本发明的核酸分子外，还可包括用于调控表达的真核或原核元件，如起始和终止转录的调节序列、增强子、启动子、信号序列等。
而且，本发明包含转化入本发明的载体或用本发明的载体转染的原核或真核宿主细胞。大体而言，将用原核细胞如大肠杆菌或枯草芽孢杆菌(Bacillus subtilis)，或者用真核细胞如酿酒酵母(Saccharomycescerevisiae)来克隆载体并表达cDNA。
本发明的蛋白质变体可如原样产生或以融合蛋白产生。
由于降低的IgE反应性，所述变体可用来制备疫苗，用于免疫治疗由尘螨引起的变态反应的治疗目的。
本发明进一步的方面因此涉及这样的药物组合物，其包含有效量的本发明的低变应原性变体，任选地与尘螨属或相似属的螨的其它天然或改变了的变应原组合，并包含可药用赋形剂。
在一个优选的实施方案中，所述药物组合物为用于预防性或治疗性处理变应性疾病如支气管哮喘、变应性鼻炎、变应性皮炎、变应性结膜炎的疫苗。疫苗接种的原则和操作过程为本领域技术人员熟知并在如(7)和(8)进行了描述。
下列实施例更详细地阐述了本发明。
实施例
如果没有另外指出，下列实施例中所用的方法为Sambrook，Fritsch ET Maniatis“分子克隆：实验室手册”(“Molecular cloning.A laboratorymanual”)第二版，1-2-3卷，CSH Lab Press 1989中所述的方法。
实施例1-RT-PCR分离Der p 2的cDNA
用欧洲屋尘螨的mRNA通过RT-PCR产生相应的cDNA，该反应的引物为多聚dT寡核苷酸，用反转录酶催化该反应。此后，通过PCR(聚合酶链反应)选择性扩增对应于变应原Der p 2的cDNA，使用了两条特异性引物，每条引物具有15个核苷酸，对应于编码该蛋白质的基因区的末端。将Der p 2的cDNA克隆入载体(pBluescript-Stratagene)，用于在大肠杆菌细胞中扩增，并用自动测序仪根据Sanger法测序。
实施例2—编码变应原Der p 2的cDNA的位点专一诱变
通过PCR扩增(聚合酶链反应)克隆在原核载体(pBluescript)中的同一cDNA来进行编码变应原Der p 2的cDNA的位点专一诱变。用作PCR反应引物的寡核苷酸具有所需的碱基替换。对每一诱变而言，使用了与两条DNA链的相应区结合的所述寡核苷酸的互补对。扩增后，原始的未改变模板通过用限制性酶Dpn I酶促消化进行选择性降解。然后用诱变处理过的分子转化大肠杆菌细胞。从单个的细菌菌落所得的克隆根据Sanger法测序以证实碱基的正确改变且不存在cDNA的非特异性突变。
实施例3—蛋白质Der p 2和其变体的产生
当来自Der p 2的正常cDNA以及对应于SEQ ID N.2的诱变处理过的cDNA克隆入表达载体(pCALn-Stratagene)后，按照标准方法于大肠杆菌细胞中表达，其为当培养物处于指数生长(O.D.600nm＝0.6)时，向其中加入IPTG(异丙基-β-D-硫代半乳糖吡喃糖苷)诱导cDNA表达。诱导蛋白质合成2小时后通过超声裂解细菌细胞和通过离心去除细胞微粒来分离重组蛋白质。通过亲和层析从上清中纯化蛋白质，所使用的柱中基质与钙调蛋白结合，其中的钙调蛋白与融合至变应原的CBP部分(钙调蛋白结合蛋白)相互作用。
实施例4—Der p 2变体变应原性的蛋白质印迹试验
根据Towbin所述的技术(6)，将等量的正常重组变应原和SEQ ID N.4所示的诱变处理过的变体通过聚丙酰胺凝胶电泳并随后通过电印迹转移至硝酸纤维素膜上进行分析。
将膜在含5％奶粉的TBS(饱和缓冲液)中孵育1小时，然后与一种对螨产生变应性且具有反应性RAST 4+的患者血清过夜孵育。用含0.05％吐温-20的TBS洗三次后，将膜与过氧化物酶连接的抗人IgE抗血清孵育1小时、进一步漂洗、并用检测系统检测与膜结合的IgE抗体，其中所述检测系统基于使用含H2O2的DAB(二氨基联苯胺)溶液作为过氧化物酶的底物。
实施例5—ELISA检测Der p 2变体对IgE的反应性
将在碳酸盐/碳酸氢盐50mM缓冲液，pH9.6中的等量(0.1μg)正常变应原和其诱变处理过的变体于4℃孵育16小时而吸附于聚苯乙烯板的孔中，用于ELISA试验。然后用洗涤液(含0.05％吐温-20的60mM磷酸盐缓冲液pH6.5)洗抗原，并用稀释液(在磷酸盐缓冲液150mM pH7.4中的25％马血清、EDTA 1mM、0.05％吐温20、0.01％硫柳汞)饱和游离位点。具有RAST4+反应性的人合并血清以1∶2的比率在稀释缓冲液中系列稀释制备。将等量(100μl)的多个血清稀释液加至每一样本并在25℃孵育2小时。漂洗三次后，加入在稀释缓冲液中1∶1500稀释的过氧化物酶连接的抗人IgE抗血清，在25℃孵育1.5小时。漂洗三次后，加入100μl Ultra Blu试剂(Intergen，Milford，Mass.)并在25℃孵育15分钟进行显色反应。该反应通过加入100μl 1N HCl终止，并于450nm处用分光光度计评估。
参考文献
1)Chua K.Y.，Doyle C.R.，Simpson R.J.，Turner K.J.，Stewart G.A.，Thomas W.R.，(1990)″通过IgE空斑免疫测定分离编码主要螨变应原Derp II的cDNA″.Int.Arch.Allergy Appl.Immunol.91(2)：118-123
2)Smith A.M.，Chapman M.D.，(1996)″IgE与通过定点突变产生的变应原变体的结合降低：二硫键对主要的室尘螨变应原Der p 2的抗原结构的作用″，分子免疫学(Mol.Immunol.)33(4-5)：399-405
3)Theodoropoulos D.S.，Lockey R.F.，(2000)″变应原免疫治疗：世界卫生组织的指导方针、更新和建议″。Allergy Asthma Proc.21(3)：159-166
4)Ohashi Y.，Nakai Y.，Tanaka A.，Kakinoki Y.，Washio Y.，Ohno Y.，Yamada K.，Nasako Y.，(1998)。″用标准化美洲屋尘螨提取物进行免疫治疗期间出现不利全身反应的危险因子″。Acta Otolaryngol。Suppl.538：113-117
5)Ferreira F.，Ebner C.，Kramer B.，Casari G.，Briza P.，Kungl A.J.，Grimm R.，Jahn-Schmid B.，Breiteneder H.，Kraft D.，Breitenbach M.，Rheinberger H.J.，Scheiner O.，(1998)。″通过定点诱变改变变应原的IgE反应性：低变应原性变体用于免疫治疗的潜在用途″。FASEB J.12：231-242
6)Towbin J.，Staehelin T.，Gordon J.，(1979)。″蛋白质从聚丙烯酰胺凝胶至硝酸纤维素膜上的电泳转移：步骤和一些应用″。美国国家科学院院报(Proc.Natl.Acad.Sci.USA)，76：4350-4354
7)Paul，(1989)，“基础免疫学”(″Fundamental Immunology″)，Raven press，纽约。
8)Cryz，S.J.(1991)，“免疫治疗和疫苗”(″Immunotherapy andVaccines″)，VCH Verlagsgesellschaft。
                        序列表<110>国家研究委员会(CONSIGLIO NAZIONALE DELLE RICERCHE)<120>尘螨属2组的变应原性蛋白质的变体<130>Cons Naz Ric<140><141><160>4<170>PatentIn Ver.2.1<210>1<211>444<212>DNA<213>欧洲屋尘螨(Dermatophagoides pteronyssinus)<400>1aaaatgatgt acaaaatttt gtgtctttca ttgttggtcg cagccgttgc cgctgatcaa 60gtcgatgtca aagattgtgc caatcatgaa atcaaaaaag ttttggtacc aggatgccat 120ggttcagaac catgtatcat tcatcgtggt aaaccattcc aattggaagc cgttttcgaa 180gccaaccaaa actcaaaaac cgctaaaatt gaaatcaaag cttcaatcga tggtttagaa 240gttgatgttc ccggtatcga tccaaatgca tgccattata tgaaatgtcc attggttaaa 300ggacaacaat atgatattaa atatacatgg aatgttccga aaattgcacc aaaatctgaa 360aatgttgtcg tcactgttaa agttatgggt gatgatggtg ttttggcctg tgctattgct 420actcatgcta aaatccgcga ttaa                                        444<210>2<211>390<212>DNA<213>欧洲屋尘螨<400>2gatcaagtcg atgtcaaaga ttgtgccaat catgaaatca aaaaagtttt ggtaccagga 60tgccatggtt cagaaccatg tatcattcat cgtggtgcac cattccaatt ggaagccgtt 120ttcgaagcca accaaaactc agcaaccgct aaaattgaaa tcaaagcttc aatcgatggt 180ttagaagttg atgttcccgg tatcgatcca aatgcatgcc attatatgaa atgtccattg 240gttgcaggac aacaatatga tattaaatat acatggaatg ttccggcaat tgcaccagca 300tctgaaaatg ttgtcgtcac tgttaaagtt atgggtgatg atggtgtttt ggcctgtgct 360attgctactc atgctgcaat ccgcgattaa                                  390<210>3<211>145<212>PRT<213>欧洲屋尘螨<400>3Met Tyr Lys Ile Leu Cys Leu Ser Leu Leu Val Ala Ala Val Ala Ala1               5                  10                  15Asp Gln Val Asp Val Lys Asp Cys Ala Asn His Glu Ile Lys Lys Val
         20                  25                  30Leu Val Pro Gly Cys His Gly Ser Glu Pro Cys Ile Ile His Arg Gly
     35                  40                  45Lys Pro Phe Gln Leu Glu Ala Val Phe Glu Ala Asn Gln Asn Ser Lys
 50                  55                  60Thr Ala Lys Ile Glu Ile Lys Ala Ser Ile Asp Gly Leu Glu Val Asp65                  70                  75                  80Val Pro Gly Ile Asp Pro Asn Ala Cys His Tyr Met Lys Cys Pro Leu
             85                  90                  95Val Lys Gly Gln Gln Tyr Asp Ile Lys Tyr Thr Trp Asn Val Pro Lys
        100                 105                 110Ile Ala Pro Lys Ser Glu Asn Val Val Val Thr Val Lys Val Met Gly
    115                 120                 125Asp Asp Gly Val Leu Ala Cys Ala Ile Ala Thr His Ala Lys Ile Arg
130                 135                 140Asp145<210>4<211>129<212>PRT<213>欧洲屋尘螨<400>4Asp Gln Val Asp Val Lys Asp Cys Ala Asn His Glu Ile Lys Lys Val  1               5                  10                  15Leu Val Pro Gly Cys His Gly Ser Glu Pro Cys Ile Ile His Arg Gly
         20                  25                  30Ala Pro Phe Gln Leu Glu Ala Val Phe Glu Ala Asn Gln Asn Ser Ala
     35                  40                  45Thr Ala Lys Ile Glu Ile Lys Ala Ser Ile Asp Gly Leu Glu Val Asp
 50                  55                  60Val Pro Gly Ile Asp Pro Asn Ala Cys His Tyr Met Lys Cys Pro Leu65                  70                  75                  80Val Ala Gly Gln Gln Tyr Asp Ile Lys Tyr Thr Trp Asn Val Pro Ala
             85                  90                  95Ile Ala Pro Ala Ser Glu Asn Val Val Val Thr Val Lys Val Met Gly
        100                 105                 110Asp Asp Gly Val Leu Ala Cys Ala Ile Ala Thr His Ala Ala Ile Arg
    115                 120                 125Asp

Claims (12)

1.Der p 2的天然形式变体，或者为与Der p 2比较序列同一性高于85％的同源形式，其中至少一个存在于Der p 2位置33、44、82、96、100、126处的Lys残基被替换和/或删除。
2.如权利要求1中所述的变体，其中所述天然形式选自Der p 2和Derf 2。
3.如权利要求1-2中所述的变体，其中所述残基用中性或极性氨基酸替换。
4.如权利要求1-3中所述的变体，其中所述残基用丙氨酸替换。
5.如权利要求1-4中所述的变体，其具有序列SEQ ID N.4。
6.包含权利要求1-5的变体的免疫活性部分的肽，其中存在至少一个如权利要求1中所述的替换/删除。
7.编码如权利要求1-5中所述的蛋白质变体的核酸分子，或编码如权利要求6中所述的肽的核酸分子。
8.如权利要求7中所述的核酸分子，其序列为SEQ ID N.2。
9.包含权利要求7-8的核酸分子的载体。
10.用权利要求9的载体转导的宿主细胞。
11.药物组合物，其包含有效量的如权利要求1-5中所述的蛋白质变体或如权利要求6中所述的肽，并包含可药用赋形剂。
12.如权利要求11中所述的组合物，其为疫苗形式。
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