锯缘青蟹主要过敏原的纯化与鉴定

以锯缘青蟹为研究对象,从免疫鉴定、分离纯化、抗体制备和免疫学分析等方面对其主要过敏原进行研究。首先利用过敏者血清的免疫印迹法,确定锯缘青蟹的主要过敏原为分子量约38kD的蛋白。然后通过制备丙酮粉、等电点沉淀、硫酸铵沉淀及加热处理对分子量为38kD的主要过敏蛋白进行了高度纯化。该蛋白的pI约为4.5,与虾的原肌球蛋白Pena1性质相近,证实了锯缘青蟹的主要过敏原为原肌球蛋白。通过免疫新西兰大白兔,制备了原肌球蛋白的抗血清,采用Protein A Sepharose亲和层析柱对动物抗体进行了纯化。该抗血清效价高,经4×105倍稀释后仍能与抗原进行反应。该抗体与甲壳类动物及软体动物的原肌球蛋白具有较强的免疫交叉反应,可用于食品过敏原检测。  

PURIFICATION AND IDENTIFICATION OF THE MAJOR ALLERGEN OF MUD CRAB

IgE-mediated hypersensitive reactions to ingestion of crustacean are among the most serious forms of food allergies. Sensitized individuals can develop urticaria, angioedema, asthma, and even life-threatening anaphylaxis. Mud crab (Scylla serrata) is a kind of crab constitutes a promising fishery industry in China. Because of the increasing consumption of such kind of crab, the occurrence of hypersensitive reactions is extending year by year. Although tropomyosin is assumed to be a major allergen in crustaceans, few experimental data are available on allergens in crabs in China, such as Mud crab. Thus, it is important to confirm whether tropomyosin is the major allergen of Mud crab or not. In order to characterize and confirm the biochemical quality of Mud crab allergen, we report herein the isolation, identification, and determination of the major allergen of Mud crab. Crude cooked extract of Mud crab muscle was used as antigen and sera samples from 11 crustacean-allergic patients used as antibody investigated by Western-blot. Allergen with molecular mass about 38 kD was detected by all of 11 sera with crustacean allergy by Western-blot, suggesting this protein was the major allergen of Mud crab. The 38 kD protein was purified to homogeneity by isoelectric point precipitation, ammonium sulfate fractionation, and heating, and it was further characterized as tropomyosin. Polyclonal antibody against Mud crab-tropomyosin was prepared and further purified by Protein A Sepharose affinity column. The antibody cross reacted positively with tropomyosins from other crustaceans, suggesting its potential application in the detecting of the major allergen tropomyosin in crustacean foods. The identification and characterization of the major allergen in Mud crab will facilitate not only the elucidation of cross-reactions to crustaceans but also the advance of the diagnosis and treatment of seafood allergy. The 38 kD tropomyosin allergen in Mud crab as identified in the present study will benefit further allergic studies not only in this species of crab but also in other species of aquatic products.