简单异尖线虫一种新型热稳定变应原的纯化及cDNA克隆

Purification and cDNA cloning of a new heat-stable allergen from Anisakis simplex.

作者信息

Kobayashi Yukihiro, Shimakura Kuniyoshi, Ishizaki Shoichiro, Nagashima Yuji, Shiomi Kazuo

机构信息

Department of Food Science and Technology, Tokyo University of Marine Science and Technology, Konan-4, Minato-ku, Tokyo 108-8477, Japan.

出版信息

Mol Biochem Parasitol. 2007 Oct;155(2):138-45. doi: 10.1016/j.molbiopara.2007.06.012. Epub 2007 Jun 30.

DOI:10.1016/j.molbiopara.2007.06.012

PMID:17689675

Abstract

The nematode Anisakis simplex is a representative parasite for marine animals and occasionally causes not only anisakiasis but also allergic reactions in sensitized subjects. Besides the known allergens, a number of unidentified allergens have been suggested to still exist in A. simplex. In this study, a new heat-stable allergen of 15kDa (named Ani s 8) was purified from the third stage larvae of A. simplex by gel filtration on Sephacryl S-300, anion-exchange HPLC on Mono Q and reverse-phase HPLC on TSKgel Phenyl-5PW RP. Analysis by fluorescence ELISA showed that 7 of 28 Anisakis-allergic patients had elevated serum levels of IgE to Ani s 8. On the basis of the determined partial amino acid sequence, the complete sequence of Ani s 8 (composed of 150 amino acid residues) was elucidated by cDNA cloning, in which as many as 32 homologs of the cDNA encoding 10 isoforms of Ani s 8 were detected. Ani s 8 shares amino acid sequence homology (up to 36%) with several members of the SXP/RAL-2 protein family, including Ani s 5 (15kDa) previously identified as an A. simplex allergen. Inhibition ELISA data demonstrated the IgE cross-reactivity between Ani s 8 and Ani s 5.

摘要

线虫简单异尖线虫是海洋动物的代表性寄生虫，不仅偶尔会引发异尖线虫病，还会在敏感个体中引起过敏反应。除了已知的过敏原外，有迹象表明简单异尖线虫中仍存在许多未鉴定的过敏原。在本研究中，通过在Sephacryl S-300上进行凝胶过滤、在Mono Q上进行阴离子交换HPLC以及在TSKgel Phenyl-5PW RP上进行反相HPLC，从简单异尖线虫的第三期幼虫中纯化出一种新的15kDa热稳定过敏原（命名为Ani s 8）。荧光ELISA分析表明，28名对异尖线虫过敏的患者中有7人的血清IgE水平升高至Ani s 8。基于所确定的部分氨基酸序列，通过cDNA克隆阐明了Ani s 8的完整序列（由150个氨基酸残基组成），其中检测到多达32个编码Ani s 8的10种同工型的cDNA同源物。Ani s 8与SXP/RAL-2蛋白家族的几个成员具有氨基酸序列同源性（高达36%），包括先前被鉴定为简单异尖线虫过敏原的Ani s 5（15kDa）。抑制ELISA数据证明了Ani s 8与Ani s 5之间的IgE交叉反应性。