Study on tropomyosin, a common and major allergen in Crustacea

最近，在日本，许多患者都患有因摄入虾而引起的过敏。从我国虾的消费量来看，虾是一种重要的动物蛋白食品。为了阐明虾的过敏性，我们用虾过敏的特应性皮炎患者的血清对虾中的过敏原进行了筛选，发现只有一种蛋白质，原肌球蛋白。这是一种蛋白质成分，形成脊椎动物和无脊椎动物的肌肉蛋白质，据报道，它是海鲜以及头足类、软体动物和甲壳类动物中常见的过敏蛋白。本研究试图从日本对虾中提纯原肌球蛋白(Pen j-1)，并制备抗原肌球蛋白的单抗和多克隆抗体。利用所获得的单抗和多克隆抗体建立的夹心酶联免疫吸附试验(EL ISA)系统，对烹调过程中的低变应原进行了研究。大部分原肌球蛋白是在沸水中提取的，这表明煮熟过程对虾的低过敏性是有效的。此外，我们对编码过敏原的cDNA的克隆进行了研究，以揭示其完整的一级结构和过敏性。采用5‘-RACE和3’-RACE方法，以新鲜对虾为模板，克隆了一个编码284个氨基酸的蛋白质。此外，在大肠杆菌中表达了一系列重组多肽，以确定患者血清中与IgE结合的表位。表位定位结果表明，这些表位在原肌球蛋白的多肽链上存在，并在很宽的区域内被分成几个片段。

Recently, in Japan, many patients are suffering from allergy eliciting by ingestion of shrimp. Shrimp is an important foodstuff of the animal protein in view of its consumption quantity in this country. In order to elucidate the allergenicity of shrimp, we screened the allergens in it using the sera of shrimp-sensitive patients with atopic dermatitis and found only one protein, tropomyosin. This is a protein component, which form the muscle proteins in vertebrates and invertebrates, and is reported as a common allergic protein in seafood as well as Cepharopoda, Mollusca, Crustacea. In the present study, we attempted to purify tropomyosin (Pen j 1) from Penaceus japonicus, and prepared the monoclonal and polyclonal antibodies raised against the purified tropomyosin We investigated hypoallergenicity during cooking process by means of a sandwich enzyme-linked immunosorbent assay (ELISA) system established using the monoclonal and polyclonal antibodies obtained. Most of tropomyosin was extracted in the boiling water, suggesting that boiling process in cooking is effective in hypoallergenicity of shrimp.Furthermore, we investigated the cloning of cDNA encoding the allergen to reveal information concerning its complete primary structure and allergenicity. The cDNA clone was cloned by means of 5'- and 3'-RACE method using mRNA prepared from fresh shrimp as a template and encoded a protein consist of 284 amino acids. Further, a various of recombinant peptides were expressed in E. coli to elucidate the epitopes, the binding region with IgE in patients' sera. The result of epitope mapping showed that the epitopes occurred and were divided into several segments over a very wide region on the polypeptide chain of tropomyosin.