Background: Cashew (Anacardium occidentale L.) is a commercially important plant. Cashew nuts are a popular food source that belong to the tree nut family. Tree nuts are one of the eight major food allergens identified by the Food and Drug Administration in the USA. Allergies to cashew nuts cause severe and systemic immune reactions. Tree nut allergies are frequently fatal and are becoming more common. Aim: We aimed to identify the key allergenic epitopes of cashew nut proteins by correlating the phage display epitope prediction results with bioinformatics analysis. Design: We predicted and experimentally confirmed cashew nut allergen antigenic peptides, which we named Ana o 2 (cupin superfamily) and Ana o 3 (prolamin superfamily). The Ana o 2 and Ana o 3 epitopes were predicted using DNAstar and PyMoL (incorporated in the Swiss-model package). The predicted weak and strong epitopes were synthesized as peptides. The related phage library was built. The peptides were also tested using phage display technology. The expressed antigens were tested and confirmed using microtiter plates coated with pooled human sera from patients with cashew nut allergies or healthy controls. Results: The Ana o 2 epitopes were represented by four linear peptides, with the epitopes corresponding to amino acids 108–111, 113–119, 181–186, and 218–224. Furthermore, the identified Ana o 3 epitopes corresponding to amino acids 10–24, 13–27, 39–49, 66–70, 101–106, 107–114, and 115–122 were also screened out and chosen as the key allergenic epitopes. Discussion: The Ana o 3 epitopes accounted for more than 40% of the total amino acid sequence of the protein; thus, Ana o 3 is potentially more allergenic than Ana o 2. Conclusions: The bioinformatic epitope prediction produced subpar results in this study. Furthermore, the phage display method was extremely effective in identifying the allergenic epitopes of cashew nut proteins. The key allergenic epitopes were chosen, providing important information for the study of cashew nut allergens.

目的 提取和鉴定鲶鱼和秋刀鱼中的过敏蛋白.方法 两种鱼肉用异丙醇进行脱脂,通过磷酸缓冲液(phosphate buffer solution,PBS)进行提取,葡聚糖凝胶Sephadex G-250对透析后的样品进行分离纯化得到鲶鱼及秋刀鱼过敏蛋白;二喹啉甲酸(bicinchoninic acid,BCA)试剂盒测定蛋白含量,通过十二烷基硫酸钠聚丙烯酰胺凝胶电泳(sodium dodecyl sulfate-polyacrylamide gel electrophoresis,SDS-PAGE)、蛋白质免疫印迹对两种鱼过敏蛋白进行身份识别并验证其免疫性,通过质谱及美国国家生物技术信息中心(National Center of Biotechnology Information,NCBI)数据库对比确定两种鱼中过敏蛋白的种类.结果 异丙醇用量及处理时间对脱脂效果有一定影响,在1:50(m:V)料液比条件下脱脂24 h效率最佳;经鉴定鲶鱼中共有4种过敏蛋白,分子量大小分别为150、51、45、38 kDa;秋刀鱼中共有两种过敏蛋白,分子量大小分别为51、14 kDa.结论 该研究鉴定出秋刀鱼中过敏蛋白为 β-烯醇化酶和小清蛋白,鲶鱼中过敏蛋白为肌球蛋白、β-烯醇化酶、肌动蛋白和精氨酸激酶,为鱼类过敏原研究提供理论基础.

In order to definite the molecular structure and allergic characteristics of cashew nuts, the main allergic proteins were isolated and purified by organic solvent defatting, salting out, dialysis and Sephadex G-150 gel. The allergenic proteins were definitely by UV, BCA kit, SDS-PAGE and Western-Blotting. The amino acid sequence and the DNA sequence of cashew allergic protein were obtained by using the NCBI database and protein mass spectrum sequencing. The structure of cashew allergic protein was predicted by DNAstar bio-information software and circular dichroism (CD). The results showed that the Ana o 2 and Ana o 3 were the main cashew allergenic proteins with molecular weight of 33 and 17 kD. The results of CD and Protean program in DNAstar predicted that there were α-helical, β-folded regions and corner regions which was a hydrophilic protein with strong sensitization because of the β-corners and random curls were found in 54% in Ana o 2. The secondary structure had only a small amount of β-folded region and corner region, accounted for 12%, but had mainly α-helix and random coil structure. It was also hydrophilic allergic protein, and the formation of allergenic epitopes was relatively weak. This study would provide reference for further clarifying the structure of the main allergens of natural cashew nuts and exploring the sensitization mechanism of cashew nuts.