斑点鲈鱼(Lateolabraxmaculatus)是中国第二大海水养殖鱼类,是与食物有关的过敏反应的主要诱因。然而,对黄斑乳杆菌过敏原的研究有限。本研究旨在表征黄斑乳杆菌的泛过敏原小白蛋白。纯化两种约11kDa的蛋白质,并通过质谱确认为小白蛋白。通过免疫印迹测定评价IgG和IgE结合活性。结合蛋白质组学研究β-小白蛋白的分子特征,基因组学,和免疫信息学方法。结果表明,β-小白蛋白由109个氨基酸组成,分子量为11.5kDa,是表现出强IgE结合能力的主要变应原。计算机模拟分析和斑点印迹分析证实了七个线性B细胞表位主要分布在α螺旋和钙结合环上。此外,分析了26种常用鱼类之间的交叉反应性。内部产生的反L黄斑鱼小白蛋白多克隆抗体识别26种鱼类的100%,证明了交叉反应性和更好的结合能力比抗鳕鱼小白蛋白抗体。一起,这项研究提供了一种通过多组学方法表征过敏原的有效方案,并支持黄斑乳杆菌的小白蛋白作为鱼类过敏原测定和过敏诊断的候选物。
Spotted seabass (Lateolabrax maculatus) is the second largest maricultural fish species in China and is the main trigger of food-related allergic reactions. Nevertheless, studies on the allergens of L. maculatus are limited. This study aimed to characterize pan-allergen
parvalbumin from L. maculatus. Two proteins of about 11 kDa were purified and confirmed as parvalbumins by mass spectrometry. The IgG- and IgE-binding activities were evaluated through an immunoblotting assay. The molecular characteristics of β-
parvalbumin were investigated by combining proteomics, genomics, and immunoinformatics approaches. The results indicated that β-
parvalbumin consists of 109 amino acids with a molecular weight of 11.5 kDa and is the major allergen displaying strong IgE-binding capacity. In silico analysis and a dot blotting assay confirmed seven linear B cell epitopes distributed mainly on α-helixes and the calcium-binding loops. In addition, the cross-reactivity among 26 commonly consumed fish species was analyzed. The in-house generated anti-L. maculatus
parvalbumin polyclonal antibody recognized 100% of the 26 fish species, demonstrating cross-reactivity and better binding capacity than the anticod
parvalbumin antibody. Together, this study provides an efficient protocol to characterize allergens with multiomics methods and supports parvalbumin from L. maculatus as a candidate for fish allergen determination and allergy diagnosis.