Supplementary Material for: <b><i>In Silico</i></b> Identification and <b><i>in Vitro</i></b> Analysis of B and T-Cell Epitopes of the Black Turtle Bean (<b><i>Phaseolus Vulgaris L</i></b>.) Lectin

<b><i>Background/Aims:</i></b> The incidence of lectin allergic disease is increasing in recent decades, and definitive treatment is still lacking. Identification of B and T-cell epitopes of allergen will be useful in understanding the allergen antibody responses as well as aiding in the development of new diagnostics and therapy regimens for lectin poisoning. In the current study, we mainly addressed these questions. <b><i>Methods:</i></b> Three-dimensional structure of the lectin from black turtle bean (<i>Phaseolus vulgaris L.</i>) was modeled using the structural template of Phytohemagglutinin from <i>P. vulgaris</i> (PHA-E, PDB ID: 3wcs.1.A) with high identity. The B and T-cell epitopes were screened and identified by immunoinformatics and subsequently validated by ELISA, lymphocyte proliferation and cytokine profile analyses. <b><i>Results:</i></b> Seven potential B-cell epitopes (B1 to B7) were identified by sequence and structure based methods, while three T-cell epitopes (T1 to T3) were identified by the predictions of binding score and inhibitory concentration. The epitope peptides were synthesized. Significant IgE binding capability was found in B-cell epitopes (B2, B5, B6 and B7) and T2 (a cryptic B-cell epitope). T1 and T2 induced significant lymphoproliferation, and the release of IL-4 and IL-5 cytokine confirmed the validity of T-cell epitope prediction. Abundant hydrophobic amino acids were found in B-cell epitope and T-cell epitope regions by amino acid analysis. Positively charged amino acids, such as His residue, might be more favored for B-cell epitope. <b><i>Conclusion:</i></b> The present approach can be applied for the identification of epitopes in novel allergen proteins and thus for designing diagnostics and therapies in lectin allergy.**背景与目的**：近数十年来，凝集素（lectin）过敏性疾病的发病率逐年攀升，目前仍缺乏根治性治疗方案。明确过敏原的B细胞与T细胞表位，不仅有助于深入解析过敏原抗体应答机制，还可为凝集素中毒的新型诊断技术与治疗策略开发提供重要支撑。本研究主要围绕上述问题展开探索。 **方法**：本研究以菜豆（Phaseolus vulgaris L.）来源的植物血凝素（phytohemagglutinin, PHA-E，PDB编号：3wcs.1.A）为高同源性结构模板，对黑龟豆（菜豆，Phaseolus vulgaris L.）来源凝集素的三维结构进行同源建模。通过免疫信息学（immunoinformatics）方法筛选并鉴定B细胞与T细胞表位，随后采用酶联免疫吸附试验（enzyme-linked immunosorbent assay, ELISA）、淋巴细胞增殖实验及细胞因子谱分析对表位进行验证。 **结果**：本研究基于序列与结构联合分析方法，共鉴定出7个潜在B细胞表位（B1至B7）；通过结合得分与抑制浓度预测，筛选得到3个T细胞表位（T1至T3）。随后合成了上述表位肽段。实验结果显示，B细胞表位B2、B5、B6、B7以及T2（隐性B细胞表位）具备显著的IgE结合活性。T1与T2可诱导显著的淋巴细胞增殖，且IL-4、IL-5细胞因子的释放结果验证了T细胞表位预测的准确性。氨基酸组成分析表明，B细胞与T细胞表位区域富含疏水氨基酸；带正电荷的氨基酸（如组氨酸残基）可能更有利于B细胞表位的形成与功能发挥。 **结论**：本研究所采用的表位鉴定策略可推广应用于新型过敏原蛋白的表位挖掘，进而为凝集素过敏性疾病的诊断与治疗方案设计提供理论与技术支撑。