基于纳米抗体的Macadamia过敏原致敏机制研究和痕量检测

Food safety issue correlated to the intake of tree nuts or tree nut comprised food mainly lie on the allergic disease caused by allergens. As a global warning, the allergic diseases related to tree nuts show the raised tendency, which is strengthening the threat to the public health and the development of the food industry connected to tree nuts. In case of the allergy to tree nuts, the prior strategy is to avoid the intake, which is based on the highly sensitive detection system of the allergen. Whereas, the clinical therapeutic strategy against unfortunate intake of tree nut allergen needs base on the detailed sensitization mechanism related to the space structure and epitopes of allergen. The industry related to tree nuts expanded dramatically over past decade, and the tree nut resulted allergic disease has drawn significant attention from basic research and clinical evaluation. Based on the consumer orientation and previous research basis, the current project is focusing on Macadamia. Camelid derived Nanobody is the smallest antibody fragment to date, and possesses unique properties compared with conventional antibody. As the perfect candidate of chaperon molecule and neutralized antibody, it is proposed to determine the structure and epitopes of Macadamia allergen by utilizing Nanobody in current project. The allergen from Macadamia general extracts will be purified by Nanobody to form complex for the crystallization. The structure will be determined by X-ray diffraction and structure replacement. The overlapped peptides will be designed, and mutant amino acids will be introduced to the allergen for the epitope mapping, especially the space epitopes related to the sensitization variation after heat processing. Nanobodies against natural and heat induced allergen will be developed for the establishment of highly sensitive detection system for the detection of traceable Macadamia allergen quantitatively and qualitatively. The revealed sensitization mechanism and established detection system will provide research basis for the diagnosis and therapy of allergic disease. The project proposal will provide research scheme for other food allergens based on Nanobodies.

坚果相关的食品安全问题主要是坚果过敏原引起的过敏性疾病，是坚果类食品发展的瓶颈。坚果过敏的首要策略是过敏原避免，需基于高灵敏检测体系构建；过敏临床治疗需基于过敏原致敏机制研究，包括过敏原结构和致敏表位分析。本项目基于国内消费导向和前期研究，以Macadamia为研究对象，基于纳米抗体特殊的理化性质，从Macadamia过敏原特异性纳米抗体制备出发，运用纳米抗体-过敏原纯化体系、复合物结晶技术和X-ray衍射来解析Macadamia过敏原结构，基于结构信息和纳米抗体结合位点，指导多肽序列设计和关键氨基酸突变，通过纳米抗体结合和中和抗体竞争性结合验证Macadamia过敏原线性表位和热加工致敏性改变相关的空间表位；采用天然和热加工过敏原共筛选技术，基于共识别纳米抗体，构建Macadamia过敏原高灵敏检测体系，为我国食品过敏原致敏机制研究提供技术体系，为我国食品过敏原的高灵敏检测提供检测体系。

坚果作为食物八大过敏原之一，其相关的食品安全问题主要是坚果过敏原引起的过敏性疾病。本项目基于国内消费导向和前期研究，以Macadamia为研究对象，基于纳米抗体特殊的理化性质，从Macadamia过敏原特异性纳米抗体制备出发，运用纳米抗体-过敏原纯化体系、基于生物信息学手段的结构拟合和表位分析，通过质谱鉴定纳米抗体靶向过敏原，并基于天然和热加工过敏原共识别验证，筛选共识别纳米抗体，构建Macadamia过敏原高灵敏检测体系，为我国食品过敏原致敏机制研究提供技术体系。本研究基于无偏策略，进行动物免疫和特异性纳米抗体的筛选，验证了无偏策略在过敏原特异性纳米抗体制备方面的应用。首先，分析了总提取蛋白在还原和非还原条件下分布及条带差异，结果显示与已有结果一致的蛋白分布，在还原和非还原条件下呈较大的蛋白分布差异，揭示高含量的二硫键及聚合物存在。之后以总蛋白为抗原免疫年轻羊驼，成功构建了一个纳米抗体免疫文库，多样性高于107，成功插入率在75%左右，之后进行了多轮富集筛选，成功筛选到了多个阳性克隆，进一步的验证确定32个能够与总蛋白抗原结合的纳米抗体，经酶联免疫吸附和免疫印迹验证了6个抗体能够识别变性抗原，揭示其靶向抗原线性表位。利用免疫共沉淀和液质对纳米抗体的靶向抗原进行确证，用Sequest针对Uniprot DB进行序列比对确定过敏原为Vicilin-like antimicrobial peptides 2-3（MiAMP2），其结构和表位信息尚未报道，进一步的表位分析能够为致敏性及检测体系构建提供研究基础。基于所制备的纳米抗体构建了ELISA免疫检测方法，检测范围为0.442-2800 μg/mL，检测限为27.1 ng/mL。所构建方法的回收率在87%-91.9%之间，变异系数低于15%。基于生物信息学手段预测了所制备纳米抗体与过敏原蛋白的结合表位，并分析了过敏原蛋白的致敏表位，揭示其结构和表位信息。本研究应用无偏策略，制备了针对过敏原的特异性纳米抗体，为后续Macadamia过敏原免疫分析方法的构建提供研究基础。