α-Lactalbumin (ALA) is the main allergen in bovine milk allergy. Due to differences in the principles of identification methods, variations in patient symptoms, and population characteristics, the identification of key amino acids in ALA epitopes is still incomplete. In this study, bioinformatics prediction was performed on key amino acids based on two potential IgE linear epitopes in ALA. Then, peptide AA66-77 was confirmed as the IgE linear epitope through alanine scanning mutagenesis and immunological analysis based on a peptide microarray, with glutamine (Q⁷³) and isoleucine (I⁷⁴) identified as key amino acids. Additionally, in vitro simulated digestion results indicated that the mutant peptide 73 (Q⁷³) and peptide 74 (I⁷⁴) were more easily digested, with the IgE binding capacity of digestion products significantly reduced at 60 or 90 min, while changes in the epitope were only observed at 120 min. Finally, the KU812 cell degranulation model showed that the mutant peptides induced significantly lower β-Hex release and IL-6 levels compared to the epitope, confirming the critical role of specific amino acids in allergenicity. These findings are significant for further research on the prevention and treatment of bovine milk allergy.

中文翻译：

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谷氨酰胺和异亮氨酸是 α-乳清蛋白主要 IgE 线性表位的关键氨基酸，通过免疫肽微阵列测定法鉴定中牛奶过敏

α-乳清蛋白 （ALA） 是牛奶过敏的主要过敏原。由于鉴定方法原理的差异、患者症状的差异和群体特征的差异，ALA 表位中关键氨基酸的鉴定仍不完整。在这项研究中，基于 ALA 中两个潜在的 IgE 线性表位对关键氨基酸进行了生物信息学预测。然后，通过基于肽微阵列的丙氨酸扫描诱变和免疫学分析，确认肽 AA66-77 为 IgE 线性表位，其中谷氨酰胺 （Q⁷³） 和异亮氨酸 （I⁷⁴） 被鉴定为关键氨基酸。此外，体外模拟消化结果表明，突变肽 73 （Q⁷³） 和肽 74 （I⁷⁴） 更容易消化，消化产物的 IgE 结合能力在 60 或 90 min 时显著降低，而表位的变化仅在 120 min 时观察到。最后，KU812 细胞脱颗粒模型显示，与表位相比，突变肽诱导的 β-Hex 释放和 IL-6 水平显著降低，证实了特定氨基酸在过敏性中的关键作用。这些发现对于进一步研究牛奶过敏的防治具有重要意义。