两种新食物过敏原——来自甜椒（辣椒属）和番茄（番茄属）的肌动蛋白结合蛋白Cap a 2和Lyc e 1的克隆、分子及免疫学特性分析

Cloning and molecular and immunological characterisation of two new food allergens, Cap a 2 and Lyc e 1, profilins from bell pepper (Capsicum annuum) and Tomato (Lycopersicon esculentum).

作者信息

Willerroider M, Fuchs H, Ballmer-Weber B K, Focke M, Susani M, Thalhamer J, Ferreira F, Wüthrich B, Scheiner O, Breiteneder H, Hoffmann-Sommergruber K

机构信息

Department of Chemistry and Biochemistry, University of Salzburg, Salzburg, Austria.

出版信息

Int Arch Allergy Immunol. 2003 Aug;131(4):245-55. doi: 10.1159/000072136.

DOI:10.1159/000072136

PMID:12915767

Abstract

BACKGROUND

Profilins are recognised by IgE of about 20% of patients allergic to birch pollen and plant foods. They are ubiquitous intracellular proteins highly cross-reactive among plant species. Therefore, they were called panallergens and are made responsible for cross-sensitisation between plant pollen and food.

OBJECTIVES

The aim of the present study was to clone the cDNAs encoding profilins from bell pepper and tomato, to produce and purify the recombinant proteins and to compare their IgE-binding capacities to those of the natural proteins.

METHODS

cDNA clones coding for profilin were obtained by RT-PCR from total RNA of tomato and bell pepper fruits, sequenced and expressed as non-fusion proteins in ESCHERICHIA COLI. The recombinant profilins were subsequently purified and tested for IgE-binding and inhibition capacity with sera from 34 food-allergic patients. Possible oligomerisation of recombinant profilins was investigated by HPLC analysis and its influence on IgE binding assayed by ELISA.

RESULTS

The open reading frame from both profilins encompasses 393 bp with a predicted molecular mass of 14,184 kD and a pI of 4.44 for bell pepper profilin (Cap a 2) and 14,257 kD and a pI of 4.46 for the profilin from tomato (Lyc e 1). The two protein sequences display 91% identity, whereas tomato profilin from pollen shares only 75% identity with tomato fruit profilin. Eleven out of 34 food-allergic patients (32%) display IgE binding to both purified profilins. Preincubation of a serum pool with either purified rCap a 2 or rLyc e 1 nearly abolished IgE binding to natural Cap a 2 and Lyc e 1, respectively. In addition, purified recombinant Cap a 2 was able to inhibit IgE-binding to rLyc e 1 by approximately 50%, whereas rLyc e 1 completely blocked IgE-binding to rCap a 2 in cross-inhibition assays. HPLC analysis showed that in solution Cap a 2 and Lyc e 1 can be found predominantly as dimers, which can be partially reduced to monomers by addition of dithiothreitol (DTT). In ELISA DTT-treated Lyc e 1 displayed a clearly lower IgE-binding capacity than untreated profilin.

CONCLUSIONS

Purified rCap a 2 and rLyc e 1 proved to be valuable tools for studying cross-reactivity to profilins in patients allergic to pollen and food.

摘要

背景

在对桦树花粉和植物性食物过敏的患者中，约20%的患者血清IgE可识别肌动蛋白单体结合蛋白。它们是普遍存在的细胞内蛋白质，在植物物种间具有高度交叉反应性。因此，它们被称为泛过敏原，并被认为是导致植物花粉与食物间交叉致敏的原因。

目的

本研究旨在克隆辣椒和番茄中编码肌动蛋白单体结合蛋白的cDNA，制备并纯化重组蛋白，比较它们与天然蛋白的IgE结合能力。

方法

通过RT-PCR从番茄和辣椒果实的总RNA中获得编码肌动蛋白单体结合蛋白的cDNA克隆，进行测序并在大肠杆菌中表达为非融合蛋白。随后纯化重组肌动蛋白单体结合蛋白，并用34例食物过敏患者的血清检测其IgE结合和抑制能力。通过HPLC分析研究重组肌动蛋白单体结合蛋白可能的寡聚化，并通过ELISA检测其对IgE结合的影响。

结果

两种肌动蛋白单体结合蛋白的开放阅读框均为393 bp，预测分子量：辣椒肌动蛋白单体结合蛋白（Cap a 2）为14,184 kD，pI为4.44；番茄肌动蛋白单体结合蛋白（Lyc e 1）为14,257 kD，pI为4.46。两种蛋白序列的同源性为91%，而番茄花粉中的肌动蛋白单体结合蛋白与番茄果实中的肌动蛋白单体结合蛋白的同源性仅为75%。34例食物过敏患者中有11例（32%）的血清IgE与两种纯化的肌动蛋白单体结合蛋白均有结合。用纯化的rCap a 2或rLyc e 1预先孵育混合血清，几乎分别完全消除了血清IgE与天然Cap a 2和Lyc e 1的结合。此外，在交叉抑制试验中，纯化的重组Cap a 2能够抑制约50%的IgE与rLyc e l的结合，而rLyc e 1则完全阻断了IgE与rCap a 2的结合。HPLC分析表明，在溶液中Cap a 2和Lyc e 1主要以二聚体形式存在，加入二硫苏糖醇（DTT）后可部分还原为单体。在ELISA中，经DTT处理的Lyc e 1的IgE结合能力明显低于未处理的肌动蛋白单体结合蛋白。