Isolation and Characterization of Two Complete Ara h 2 Isoforms cDNA

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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.