Isolation and molecular characterization of the first genomic clone of a major peanut allergen, Ara h 2

Abstract

Background: Peanuts have been identified as potent food allergens responsible for life-threatening IgE reactions among hypersensitive individuals. With the current increase of peanut allergies, there is an urgent need to molecularly characterize the genes encoding the target proteins and to understand the nature of their regulation.

Objectives: The objectives of this study were to isolate, sequence, and characterize at least one full-length genomic clone encoding the major peanut allergen Ara h 2.

Methods: A peanut genomic library, constructed in a Lambda Fix II vector, was screened with an 80-bp oligonucleotide probe constructed on the basis of the 5' end of a published Ara h 2 cDNA partial sequence. One putative positive lambda clone was isolated, digested with Bam HI to release its 16-kb insert, and confirmed by means of dot blot and Southern hybridization. The positive clone was subcloned in pBluescript SK+ vector, sequenced, and characterized.

Results: Sequence analysis revealed a full-length genomic clone with an open reading frame starting with an initiation codon (ATG) at position 1 and ending with a termination codon (TGA) at position 622. One putative polyadenylation signal (AATAAA) is identified at positions 951 in the 3' untranslated region, and 6 additional stop codons are located at positions 628, 769, 901, 946, 967, and 982 downstream from the start codon. In the 5' promoter region, a putative TATA box (TATTATTA) is located at position -72 upstream from the start codon. The deduced amino acid sequence has 207 residues and includes a putative signal peptide of 21 residues.

Conclusions: The results reveal for the first time information on the structure of a major peanut allergen, Ara h 2. Comparison of the cDNA and genomic sequences revealed the absence of an intron but the presence of 2 isoforms of Ara h 2 or different members of the same gene family.