Molecular cloning, expression and characterization of Pru a 1, the major cherry allergen.
Molecular cloning, expression and characterization of Pru a 1, the major cherry allergen.
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DOI：
10.1016/s0161-5890(97)00072-2
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发表时间：
1997-06
影响因子：
3.6
通讯作者：
S. Scheurer;K. Metzner;D. Haustein;S. Vieths
中科院分区：
文献类型：
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作者：
S. Scheurer;K. Metzner;D. Haustein;S. Vieths
高比例的桦树花粉过敏患者在摄入几种水果和蔬菜后会出现食物过敏反应。以前的工作证明了来自甜樱桃(Prunus Avium)的18000个过敏原Mr 18000和来自桦树花粉的主要过敏原Bet v 1的共同表位。N端氨基酸序列分析表明，该变应原与Bet v1的同源性为67%。推导的氨基酸序列全长为MR17700，与Bet v1有59.1%的同源性。与其他树种花粉和植物食物中的相关过敏原以及来自食用植物如欧芹、土豆和大豆的应激诱导蛋白也发现了40%到60%的高度同源性。将樱桃蛋白的编码DNA克隆到载体pET-16b中，并在大肠杆菌BL21(DE3)中以His-Tag融合蛋白的形式表达。SDS-PAGE分析表明，非融合蛋白与天然变应原的表观分子质量完全相同。用免疫印迹和酶过敏吸附试验检测对樱桃过敏的患者血清时，融合蛋白显示出高的IgE结合效力。重组融合蛋白具有较高的生物学活性，并能诱导樱桃过敏患者嗜碱性粒细胞释放组胺。其中17 19例患者的血清中含有抗该变应原的IgE，因此将其归类为主要变应原，命名为Pru a1。重组Pru a1模拟了樱桃提取物的大部分致敏效力，因此可作为研究花粉相关食物变应原的分子和免疫学特性的有用工具。
A high percentage of birch pollen allergic patients experiences food hypersensitivity reactions after ingestion of several fruits and vegetables. Previous work demonstrated common epitopes on an allergen of Mr18 000 from sweet cherry (Prunus avium) and Bet v 1, the major allergen from birch pollen. N-terminal amino acid sequencing showed a sequence identity of 67% with Bet v 1. Here we report the cloning and cDNA sequencing of this cherry allergen. The entire deduced amino acid sequence described a protein of Mr17 700 with 59.1 % identity to Bet v 1. High degrees of identity in the range of 40 to 60% were also found with related allergens from other kinds of tree pollen and plant foods as well as with stress-induced proteins from food plants such as parsley, potato and soya. The coding DNA of the cherry protein was cloned into vector pET-16b and expressed in E. coli strain BL21(DE3) as a His-tag fusion protein. As shown by SDS-PAGE, the apparent molecular masses of the nonfusion protein and the natural allergen were identical. The fusion protein showed high IgE binding potency when sera from patients allergic to cherry were tested by immunoblotting and enzyme allergosorbent tests. Moreover, it cross-reacted strongly with IgE specific for the natural counterpart and for Bet v 1. The high biological activity of the recombinant fusion protein was further confirmed by the induction of a strong histamine release in basophils from cherry-allergic patients. Since sera from 17 19 of such patients contained IgE against this allergen it was classified as a major allergen and named Pru a 1. Recombinant Pru a 1 mimics most of the allergenic potency of cherry extract and hence could be a useful tool for studying the molecular and immunological properties of pollen related food allergens.