Molecular characterization, expression in Escherichia coli, and epitope analysis of a two EF-hand calcium-binding birch pollen allergen, Bet v 4.
Molecular characterization, expression in Escherichia coli, and epitope analysis of a two EF-hand calcium-binding birch pollen allergen, Bet v 4.
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两个 EF 手钙结合桦木花粉过敏原 Bet v 4 的分子特征、在大肠杆菌中的表达和表位分析。
DOI：
10.1006/bbrc.1997.6860
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发表时间：
1997
影响因子：
3.1
通讯作者：
Rudolph Valenta
中科院分区：
文献类型：
--
作者：
A. Twardosz;B. Hayek;S. Seiberler;L. Vangelista;Lena Elfman;Hans Grönlund;Dietrich Kraft;Rudolph Valenta
桦树花粉是早春最有效的 I 型过敏反应诱发剂。使用桦树花粉过敏患者的血清 IgE，从噬菌体 lambda gt11 构建的桦树花粉表达 cDNA 文库中分离出两个 cDNA 克隆（克隆 6 和克隆 13）。克隆6编码9.3 kD的两个EF手钙结合蛋白，命名为Bet v 4，与来自杂草和草花粉的EF手钙结合过敏原具有显着的端到端序列同源性。重组 Bet v 4 以 β-半乳糖苷酶融合蛋白的形式表达，可与约 20% 的花粉过敏个体的血清 IgE 发生反应。通过 EGTA 处理消除过敏原结合钙导致 IgE 与 Bet v 4 的结合大幅减少，表明蛋白质结合钙对于维持 IgE 表位是必需的。克隆 13（缺乏 16 个 N 端氨基酸的 Bet v 4 片段）的 IgE 结合能力大大降低，表明 N 端对蛋白质 IgE 结合能力有显着贡献。通过 IgE 抑制实验证明，重组 Bet v 4 与天然 Bet v 4 和同源梯牧草花粉过敏原共享 IgE 表位。因此，重组 Bet v 4 可被视为相关的交叉反应植物过敏原，可用于诊断和治疗患有多价植物过敏的患者。
Birch pollen belongs to the most potent elicitors of Type I allergic reactions in early spring. Using serum IgE from a birch pollen allergic patient, two cDNA clones (clone 6 and clone 13) were isolated from a birch pollen expression cDNA library constructed in phage lambda gt11. Clone 6 encoded a 9.3 kD two EF-hand calcium-binding protein, designated Bet v 4, with significant end to end sequence homology to EF-hand calcium-binding allergens from weed and grass pollen. Recombinant Bet v 4, expressed as beta-galactosidase fusion protein, reacted with serum IgE from approximately 20% of pollen allergic individuals. Depletion of allergenbound calcium by EGTA treatment lead to a substantial reduction of IgE-binding to Bet v 4, indicating that protein-bound calcium is necessary for the maintenance of IgE-epitopes. The greatly reduced IgE-binding capacity of clone 13, a Bet v 4 fragment that lacked the 16 N-terminal amino acids, indicated that the N-terminus contributes significantly to the proteins IgE-binding capacity. By IgE-inhibition experiments it was demonstrated that recombinant Bet v 4 shared IgE-epitopes with natural Bet v 4 and a homologous timothy grass pollen allergen. Recombinant Bet v 4 may therefore be considered as a relevant crossreactive plant allergen, which may be used for diagnosis and treatment of patients suffering from multivalent plant allergies.