克氏原螯虾是我国重要的淡水甲壳类水产品，其引发的食物过敏病例屡见不鲜。申请人前期研究发现，精氨酸激酶（arginine kinase，AK）是甲壳类水产品主要过敏原，也是引起免疫交叉反应的潜在过敏原，且其致敏性强弱与空间结构的变化存在密切关联，因此AK的抗原表位鉴定和结构特征分析是揭示其致敏机理的关键。但淡水甲壳类水产品AK的抗原表位研究少见报道，免疫交叉反应性及构效关系的研究也不深入。本项目以克氏原螯虾为研究对象，针对AK制备特异性纳米抗体，解析AK的晶体结构，完成AK的抗原表位分析；明确AK与特纳米抗体的相互作用位点，揭示抗原-抗体相互作用随AK结构改变的规律；从制备的纳米抗体中筛选高亲和力的表位特异性纳米抗体，创建AK的纳米抗体精准检测方法。本项目旨在阐明AK致敏性的分子机理和结构基础，完善甲壳类水产品AK的过敏原性研究，为甲壳类食品过敏原的检测和改性研究提供理论依据。

Procambarus clarkii have served as an important freshwater aquaculture resource in China. In our previously research, arginine kinase (AK) was identified as a major allergen of shellfish, property analysis of AK revealed AK may cause cross-reactivity among invertebrates. Meanwhile, the molecular structure of AK has an important influence on its allergenicity. The epitope mapping and structure analysis of AK is crucial for the allergenicity determination. the epiotpe of AK in freshwater shellfish has not been described yet, and the cross-reactivity and structure-activity relationship is also unclear. In this project, AK specific nanobody will be prepared, and the crystal structure of P. clarkii AK will be determined for the epitope mapping; the analysis of allergen-antibody interaction will be carried out to verify the mechanism of the structure-activity relationship of AK. Furthermore, high affinity epitope specific nanobody will be screened, for the establishment of a detection method based on the epitope specific nanobody. From the above, the molecular mechanism and structural basic of the cross-reactivity of AK will be clearified, meanwhile, the allergenicity and structure-activity relationship of AK will be fully illustrated. We believe the results of this program are helpful to complete the fundamental research of shellfish allergens, as well as to establish the basis for the allergen detection and hypoallergenic food development.