Study on tropomyosin, a common and major allergen in Crustacea

最近，在日本，许多患者因食用虾而引起过敏。鉴于虾在我国的消费量，它是一种重要的动物蛋白食品。为了阐明对虾的致敏性，我们利用对虾敏感的特应性皮炎患者的血清筛选了其中的致敏原，只发现了原肌球蛋白一种蛋白。这是一种蛋白质成分，在脊椎动物和无脊椎动物中形成肌肉蛋白，据报道是海产品以及头足类、软体动物、甲壳类动物中常见的过敏蛋白。本研究从日本绒螯虾（Penaceus japonicus）中分离纯化原肌球蛋白（penj1），制备单克隆和多克隆抗体，并利用获得的单克隆和多克隆抗体建立夹心酶联免疫吸附试验（ELISA）体系，研究了蒸煮过程中的低致敏性。大部分原肌球蛋白是在沸水中提取出来的，说明蒸煮过程对降低虾的致敏性是有效的。此外，我们研究了编码该过敏原的cDNA的克隆，以揭示其完整的初级结构和过敏原的信息。以鲜虾制备的mRNA为模板，采用5′-和3′- race方法克隆了该cDNA克隆体，并编码了一个包含284个氨基酸的蛋白。此外，在大肠杆菌中表达了多种重组肽，以阐明患者血清中与IgE结合的表位。表位定位的结果表明，在原肌球蛋白多肽链的一个非常宽的区域内出现了表位，并被划分为几个片段。

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.