凡纳滨对虾过敏原精氨酸激酶的质谱鉴定及其免疫交叉反应研究

目的:鉴定凡纳滨对虾分子质量40 k D过敏原,并分析其在有壳水产食物中的免疫交叉反应。方法:运用基质辅助激光解析串联飞行时间质谱仪(matrix assisted laser desorption ionization/time of flight mass spectrometry,MALDI-TOF/TOF-MS)鉴定凡纳滨对虾40 k D过敏原组分;使用软件BLAST、Clustal X2、MEGA 5.0分析该蛋白氨基酸序列的种间同源性;制备抗凡纳滨对虾过敏原精氨酸激酶的多克隆抗体,并与17种常见有壳水生动物粗提液进行Western blotting,以分析该过敏原的免疫交叉反应。结果:凡纳滨对虾40 k D过敏原为精氨酸激酶(arginine kinase,AK);其氨基酸序列同源性分析显示AK在虾类(96%~100%)、蟹类(91%~93%)、贝类(49%~52%)、蟑螂(83%)中具有很高的同源性;Western blotting结果显示抗AK多抗与17种不同虾类、蟹类、贝类的AK均能发生反应。结论:精氨酸激酶在甲壳类动物、软体动物、甚至昆虫中具有高度保守性,且能引起强免疫交叉反应,是有壳水生动物中的一种泛过敏原。

Proteomics and Immunological Analysis of Arginine Kinase,an Important Shrimp Allergen from Litopenaeus vannamei

This study aimed to identify the 40-kD allergen of Litopenaeus vannamei and to analyze its immune crossreactivity
among shellfish species. Several proteomics and immunological experiments were performed. By using matrixassisted
laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF/TOF-MS), the 40 kD allergen from
Litopenaeus vannamei was identified as arginine kinase (AK). Its amino acid sequence was compared with that of known
proteins by BLAST, Clustal X2 and MEGA 5. The results revealed that AK had high homology among shrimp (96%－100%),
crab (91%－93%), cockroach (83%) and mollusca (49%－52%)．The purified AK was injected subcutaneously into mice
to produce specific polyclonal antibodies for analyzing its immune cross-reactivity by Western blotting. It was shown that
the proteins with relative molecular mass (Mr) of about 40 kD corresponding to AKs from 17 species of shellfish could be
recognized by the AK-specific polyclonal sera. The results of both proteomics and immunological analysis have shown that
AK is a pan-allergen among crustacean and mollusca.