鲶鱼和秋刀鱼过敏蛋白提取及鉴定

Extraction and identification of allergenic protein from Silurus asotus and Cololabis saira

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。

Objective To extract and identify the allergenic proteins in Silurus asotus and Cololabis saira.Methods The 2 kinds of fish meat were degreased with isopropanol and extracted through phosphate buffer solution(PBS). Allergenic proteins of Silurus asotus and Cololabis saira were obtained by separating and purifying the dialyzed samples on Sephadex G-250. Bicinchoninic acid(BCA) kit was used to determine the protein content, sodium dodecyl sulfate-polyacrylamide gel electrophoresis(SDS-PAGE) and Western blot were used to identify the 2 kinds of fish allergenic proteins and verify their immunity. The species of allergenic proteins in the 2 kinds of fish species were identified by mass spectrometry and comparison with the National Center of Biotechnology Information(NCBI) database. Results The amount of isopropanol and treatment time had certain effect on defatting efficiency, and the optimum degreasing efficiency was obtained at 1:50(m:V) feed-liquid ratio for 24 h. Four kinds of allergenic proteins were identified in Silurus asotus, with molecular weights of 150, 51, 45 and 38 kDa, respectively. Two kinds of allergic proteins were identified in Cololabis saira,with molecular weights of 51 and 14 kDa, respectively. Conclusion This study has identified 4 kinds of allergenic proteins including myosin, β-enolase, actin and arginine kinase in Cololabis saira and 2 kinds of allergenic proteins including β-enolase and parvalbumin in Silurus asotus, which provides a theoretical basis for the study of fish allergens.