花生过敏原蛋白Ara h 2的定向改造及其致敏特性研究

In present proposal, oriented modification was applied on Ara h 2, the major allergen in peanut, to destroy its allergen epitopes. The structure-effect relationship of Ara h 2 could be discovered, based on clear changes on both structure and allergenicity. The result would indicate the direction for desensitization processing of allergens..In study on epitopes mapping and critical amino acid residues identification, peptides got defined variety on both structure and the potential allergenicity. But these results could not substitute the allergenicity change of whole allergen, considering the antigen presentation and advanced protein structure. Although the allergenicity of allergen had been broadly explored, the structural changes had not been well defined. So the structure-effect relationship of whole protein could be clarified..Here, a new pathway was developed in our proposal, oriented modified was applied on Ara h 2 to change the structure of allergen, so the structural change of the whole protein is clear. By amino acids substitution and Ig E binding, the critical amino acid residues of Ara h 2 and their key features could screened. Based on the physio-chemical properties of residues that affect the potential allergenicity, the mutants of Ara h 2 could be designed and expressed. The advanced structure of protein mutants were simulated by homology modelling before expression and characterize after expression. The allergenicity of Ara h 2 and its mutants would be detected by Ig E binding, cytokine production and simulation in animal experiments..In this study, the structure of allergen and protein mutants would be characterized in details, and the allergenicity of allergen and protein mutants would be evaluated comprehensively. When both structural change and change of allergenicity were clear, the structure-effect relationship of whole protein could be established. The result indicated the material basis of food allergen, which presented the target for desensitization processing of allergens.

研究拟通过定向改造揭示花生主要过敏原Ara h 2致敏性的结构基础，为蛋白脱敏加工指示方向。目前多肽结构与其潜在致敏性的关系已经阐明，但考虑到高级结构和致敏过程，多肽无法替代全蛋白的致敏反应。而已有全蛋白致敏反应研究中，未能阐明过敏原结构具体变化。本研究拟对Ara h 2全蛋白进行定向改造，掌握其结构的变化，揭示该蛋白的构效关系。研究首先利用血清识别系统筛选其关键氨基酸，并针对各关键氨基酸的特性进行系列氨基酸替代，寻找蛋白结构改造方向；确定改造方向，即氨基酸替代种类和组合后，结合同源建模设计Ara h 2蛋白的系列突变体，并表达纯化；表征Ara h 2及其突变体高级结构，分别从血清学、细胞学和实验动物水平系统评估和比较它们的致敏特性。由于定向改造的Ara h 2结构变化明确，致敏性评估全面，由此解析的过敏原构效关系，能够揭示蛋白致敏的结构基础，为食物脱敏的精准加工指示修饰靶位。

花生是八大过敏性食物之一，Ara h 2是它的主要过敏原蛋白，通过对该蛋白结构的定向改造有望降其致敏性，并探索过敏原结构与致敏性的关系。人们已经确定了Ara h 2上三个优势过敏原表位及其上关键氨基酸。在此基础上，本研究通过对过敏原上关键氨基酸进行突变，设计并合成突变肽。通过多肽阵列及 KU812 细胞模型评价肽段的Ig E结合能力变化。筛选获得了降低 Ara h 2 蛋白致敏性的关键突变点，将第34和38位的谷氨酰胺改为酪氨酸，用天冬氨酸替代第65和72位酪氨酸，即可破坏其优势过敏原线性表位。再结合二硫键的缺失，研究设计了五种突变体，与野生型 Ara h 2一起原核表达。首先对蛋白的结构分别进行计算机模拟和实验表征，它们的致敏性则通过血清学、细胞模型、动物模型进行评估。综合分析突变前后蛋白的结构和致敏性变化发现，构象性表位在Ara h 2表位中更占优势，芳香族残基暴露或遮蔽与致敏性相关性大。