Yang Yang, Cao Min-Jie, Alcocer Marcos, Liu Qing-Mei, Fei Dan-Xia, Mao Hai-Yan, Liu Guang-Ming
College of Food and Biological Engineering, Fujian Collaborative Innovation Center for Exploitation and Utilization of Marine Biological Resources, Jimei University, 43 Yindou Road, Xiamen 361021, Fujian, China.
School of Biosciences, Sutton Bonington Campus, Loughborough LE12 5RD, UK.
Mol Immunol. 2015 Jun;65(2):310-20. doi: 10.1016/j.molimm.2015.02.010. Epub 2015 Feb 28.
Arginine kinase (AK) is a panallergen present in crustaceans, which can induce an immunoglobulin (Ig) E-mediated immune response in humans. The aim of this work was to map and characterize the antigenic epitopes of Scylla paramamosain AK. Specific-protein-A-enriched IgG raised in rabbits against purified S. paramamosain AK was used to screen a phage display random peptide library. Five AK mimotope clones were identified among 20 random clones after biopanning. Four conformational epitopes D3A4K43M1A5T49T44I7, L31K33V35T32E11E18F14S34D37, V177G172M173D176Q178T174L181K175L187, and R202L170Y203E190P205W204L187T206Y145 were identified with the program LocaPep, and mapped to S. paramamosain AK. The key amino acids of these conformational epitopes were D3, K33, T174, and W204, respectively. On the basis of biopanning, six IgE-specific peptides were mapped with synthetic overlapping peptides using the sera from crab-allergic patients, and four seropositive peptides (amino acids 113-127, 127-141, 141-155, and 204-218) were confirmed as linear epitopes in a degranulation assay in RBL-2H3 cells. Stability experiments showed that the structural integrity of AK is essential for its allergenicity, and the intramolecular disulfide bond at Cys201-Cys271 is essential for its structural stability.
精氨酸激酶（AK）是甲壳类动物中存在的一种泛过敏原，可在人类中诱导免疫球蛋白（Ig）E介导的免疫反应。这项工作的目的是绘制和表征拟穴青蟹AK的抗原表位。用针对纯化的拟穴青蟹AK在兔中产生的特异性蛋白A富集IgG来筛选噬菌体展示随机肽库。经过生物淘选后，在20个随机克隆中鉴定出5个AK模拟表位克隆。使用LocaPep程序鉴定出四个构象表位D3A4K43M1A5T49T44I7、L31K33V35T32E11E18F14S34D37、V177G172M173D176Q178T174L181K175L187和R202L170Y203E190P205W204L187T206Y145，并将其定位到拟穴青蟹AK上。这些构象表位的关键氨基酸分别为D3、K33、T174和W204。基于生物淘选，使用蟹过敏患者的血清通过合成重叠肽绘制了六个IgE特异性肽，并且在RBL-2H3细胞的脱颗粒试验中确认了四个血清阳性肽（氨基酸113 - 127、127 - 141、141 - 155和204 - 218）为线性表位。稳定性实验表明，AK的结构完整性对其致敏性至关重要，并且Cys201 - Cys271处的分子内二硫键对其结构稳定性至关重要。
