两种完整的Ara h 2亚型cDNA的分离与鉴定

Isolation and characterization of two complete Ara h 2 isoforms cDNA.

作者信息

Chatel Jean-Marc, Bernard Hervé, Orson Frank M

机构信息

Veterans Affairs Medical Center, Baylor College of Medicine, Houston, Tex., USA.

出版信息

Int Arch Allergy Immunol. 2003 May;131(1):14-8. doi: 10.1159/000070429.

DOI:10.1159/000070429

PMID:12759484

Abstract

BACKGROUND

Ara h 2 is a major peanut allergen recognized by IgE in more than 90% of patients. After electrophoretic separation the purified protein exists as a doublet, and sequences of one incomplete cDNA and one genomic clone for this allergen have been reported.

METHODS

Ara h 2 isoforms were purified and analyzed by mass spectroscopy, and PCR amplification products of Ara h 2 were cloned and sequenced.

RESULTS

Mass spectroscopy of purified Ara h 2 clearly identified a molecular doublet of 16,670 and 18,050 Daltons. Amplification of a peanut cDNA library using PCR primer pairs located at the amino- and carboxy-terminus revealed 2 bands separated by 50 base pairs, which we cloned and sequenced. Two types of complete cDNA clones were obtained, Ara h 2.01 and Ara h 2.02. Compared to Ara h 2.01 and the previously reported cDNA sequences, Ara h 2.02 is characterized by a 12 amino acid insertion starting at position 75 that contains a third repeat of the major IgE binding epitope DPYSPS.

CONCLUSION

We demonstrated the molecular and genetic characteristics of two Ara h 2 isoforms, revealing that one, Ara h 2.02, might be the more potent allergen.

摘要

背景

Ara h 2是一种主要的花生过敏原，90%以上的患者体内可检测到针对它的IgE。经电泳分离后，纯化的该蛋白呈现为双重态，且已报道了此过敏原的一个不完整cDNA和一个基因组克隆的序列。

方法

对Ara h 2亚型进行纯化并通过质谱分析，对Ara h 2的PCR扩增产物进行克隆和测序。

结果

纯化的Ara h 2的质谱分析明确鉴定出分子量分别为16,670和18,050道尔顿的双重态分子。使用位于氨基末端和羧基末端的PCR引物对扩增花生cDNA文库，发现两条相差50个碱基对的条带，我们对其进行了克隆和测序。获得了两种类型的完整cDNA克隆，即Ara h 2.01和Ara h 2.02。与Ara h 2.01及先前报道的cDNA序列相比，Ara h 2.02的特征在于从第75位开始有一个12个氨基酸的插入，其中包含主要IgE结合表位DPYSPS的第三个重复序列。