Allergy and Immunology Section, CSIR-Institute of Genomics and Integrative Biology, Delhi, India.
PLoS One. 2013;8(1):e54755. doi: 10.1371/journal.pone.0054755. Epub 2013 Jan 18.
The identification of B-cell epitopes is an important step to study the antigen- antibody interactions for diagnosis and therapy. The present study aimed to identify B- cell epitopes of osmotin using bioinformatic tools and further modify these regions to study the allergenic property. B-cell epitopes were predicted based on amino acid physicochemical properties. Three single point mutations M1, M2, and M3 and a multiple point mutant (M123) were selected to disrupt the IgE binding. These mutants were cloned, expressed and proteins purified to homogeneity. The IgE binding of the purified proteins was evaluated by ELISA and ELISA inhibition with patients' sera. Three regions of osmotin M1 (57-70 aa), M2 (72-85 aa) and M3 (147-165 aa) were identified as potential antibody recognition sites using in silico tools. The sequence similarity search of the predicted epitopes of osmotin using Structural Database of Allergenic proteins (SDAP) showed similarity with known allergens from tomato, kiwifruit, bell pepper, apple, mountain cedar and cypress. Mutants M1, M2 and M3 showed up to 72%, 60% and 76% reduction, respectively in IgE binding whereas M123 showed up to 90% reduction with patients' sera. The immunoblot of M123 mutant showed 40% reduction in spot density as compared to osmotin. All mutants showed decreased inhibition potency with M123 exhibiting lowest potency of 32% with osmotin positive pooled patients' sera. The three B- cell epitopes of osmotin predicted by in silico method correlated with the experimental approach. The mutant M123 showed a reduction of 90% in IgE binding. The present method may be employed for prediction of B- cell epitopes of allergenic proteins.
B 细胞表位的鉴定是研究抗原-抗体相互作用用于诊断和治疗的重要步骤。本研究旨在使用生物信息学工具鉴定 osmotin 的 B 细胞表位，并进一步修饰这些区域以研究变应原性。根据氨基酸的物理化学性质预测 B 细胞表位。选择三个单点突变 M1、M2 和 M3 以及一个多点突变（M123）来破坏 IgE 结合。这些突变体被克隆、表达和纯化为蛋白质。通过 ELISA 和 ELISA 抑制试验用患者血清评估纯化蛋白的 IgE 结合。使用计算机工具鉴定了 osmotin 的三个区域 M1（57-70 aa）、M2（72-85 aa）和 M3（147-165 aa）为潜在的抗体识别位点。使用结构数据库的变应原蛋白（SDAP）对 osmotin 预测表位的序列相似性搜索显示与番茄、猕猴桃、甜椒、苹果、山地雪松和柏木的已知过敏原具有相似性。突变体 M1、M2 和 M3 的 IgE 结合分别减少了 72%、60%和 76%，而 M123 与患者血清结合减少了 90%。与 osmotin 相比，M123 突变体的免疫印迹显示斑点密度降低了 40%。所有突变体的抑制能力均降低，其中 M123 与 osmotin 阳性患者血清的抑制能力最低，为 32%。通过计算机方法预测的 osmotin 的三个 B 细胞表位与实验方法相关。突变体 M123 的 IgE 结合减少了 90%。本方法可用于预测变应原性蛋白的 B 细胞表位。
