Study on tropomyosin, a common and major allergen in Crustacea

甲壳类常见且主要过敏原原肌球蛋白的研究

基本信息

批准号：
13680157
负责人：
KIMOTO Masumi
金额：
$ 1.92万
依托单位：
Okayama Prefectural University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2001
资助国家：
日本
起止时间：
2001 至 2002
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-13680157/
关键词：
Shrimp Allergen Monoclonal antibody Sandwich ELISA Tropomyosin cDNA cloning Epitope Immunoblot IgE antibody モノクロナール抗体

项目摘要

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.

最近，在日本，许多患者因摄入虾而引起过敏。就我国的消费量而言，虾是一种重要的动物蛋白食品。为了阐明虾的致敏性，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过敏原，只发现了一种蛋白质，即原肌球蛋白。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，据报道是海鲜以及头足类、软体动物、甲壳类中常见的过敏蛋白。在本研究中，我们尝试从日本对虾中纯化原肌球蛋白（Pen j 1），并制备针对纯化的原肌球蛋白的单克隆和多克隆抗体。我们通过夹心酶联免疫吸附测定（ELISA）研究了烹饪过程中的低过敏性使用获得的单克隆和多克隆抗体建立系统。大多数原肌球蛋白是在沸水中提取的，这表明烹饪过程中的煮沸过程对虾的低过敏性有效。此外，我们研究了编码过敏原的cDNA的克隆，以揭示有关其完整一级结构和过敏原性的信息。以鲜虾制备的mRNA为模板，通过5'-和3'-RACE方法克隆该cDNA克隆，编码由284个氨基酸组成的蛋白质。此外，在大肠杆菌中表达了多种重组肽，以阐明表位，即与患者血清中 IgE 的结合区域。表位作图结果表明，原肌球蛋白多肽链上很宽的区域内出现了表位，并被分成几个片段。