Key Laboratory of Chemical Biology and Molecular Engineering of the Ministry of Education, Institute of Biotechnology, Shanxi University, Taiyuan, China.
Acta Biochim Biophys Sin (Shanghai). 2011 Jun;43(6):441-7. doi: 10.1093/abbs/gmr036. Epub 2011 May 12.
Allergic protein TBb, a major allergen in tartary buckwheat, was divided into four epitope-containing fragments and was named F1, F2, F3, and F4, respectively. Results of immunological assays revealed that F2 had the strongest IgE-binding activity to patient's sera, which indicated that it might contain the linear IgE-binding epitope of TBb. According to the results of sequence analysis and molecular modeling of tartary buckwheat allergen, three mutants of F2 gene (R139A, R141A, and D144A) were reconstructed using site-directed mutagenesis, and each mutant was expressed in Escherichia coli BL21 (DE3). Following purification by Ni(2+) affinity chromatography, enzyme-linked immunosorbent assay and dot blot were performed for wild-type F2 and its mutants using sera from buckwheat-allergic patients and a negative control (non-allergic patient). Results showed that mutants R139A and D144A had weaker IgE-binding activity to patient's sera than wild-type F2, implying that Arg(139) and Asp(144) might be involved in the allergic activity of TBb. However, R141A had the weakest IgE-binding activity, suggesting that Arg(141) may be the critical amino acid of TBb. This is the first report on the epitope mapping and identification of TBb. Our findings will contribute to the production of TBb hypoallergens and to allergen-specific immunotherapy for tartary buckwheat allergy.
过敏蛋白 TBb 是苦荞中的主要过敏原，被分为四个含表位的片段，分别命名为 F1、F2、F3 和 F4。免疫检测结果显示，F2 对患者血清的 IgE 结合活性最强，这表明它可能含有 TBb 的线性 IgE 结合表位。根据苦荞过敏原序列分析和分子建模的结果，采用定点突变技术构建了 F2 基因的三个突变体（R139A、R141A 和 D144A），并在大肠杆菌 BL21（DE3）中进行了表达。经 Ni(2+)亲和层析纯化后，采用苦荞过敏患者血清和阴性对照（非过敏患者）对野生型 F2 及其突变体进行酶联免疫吸附试验和点印迹试验。结果表明，突变体 R139A 和 D144A 对患者血清的 IgE 结合活性比野生型 F2 弱，表明 Arg（139）和 Asp（144）可能参与了 TBb 的过敏活性。然而，R141A 的 IgE 结合活性最弱，表明 Arg（141）可能是 TBb 的关键氨基酸。这是首次对 TBb 进行表位作图和鉴定的报告。我们的研究结果将有助于生产 TBb 的低过敏原性，并为苦荞过敏的过敏原特异性免疫治疗做出贡献。
