Identification of a new allergen from Amaranthus retroflexus pollen, Ama r 2.

BACKGROUND Pollinosis from Amaranthus retroflexus pollen is a common cause of respiratory allergy in Iran with a high positive rate (68.8%) among Iranian allergic patients. The aim of the present study was to evaluate the allergenicity of the A. retroflexus pollen profilin. METHODS Using sera from twelve patients allergic to A. retroflexus pollen, IgE-binding proteins from the A. retroflexus pollen extract was identified by immunoblotting. The cDNA of A. retroflexus pollen profilin was amplified, then cloned into the pET-21b (+) vector, expressed in Escherichia coli, and finally purified by metal affinity chromatography. The IgE-binding capacity of the recombinant protein was then analyzed by the ELISA, immunoblotting, and inhibition assays, as well as by the skin prick test (SPT). RESULTS Immunoblotting results indicated a 14.6kDa protein with IgE-reactivity to 33% (4/12) among A. retroflexus pollen-allergic patients. Nucleotide sequencing of the cDNA revealed an open reading frame of 399 bp encoding for 133 amino acid residues which was belonged to the profilin family and designated as Ama r 2. A recombinant Ama r 2 (rAma r 2) was then produced in E. coli as a soluble protein which showed a strong IgE-reactivity via ELISA confirmed by the SPT. Inhibition experiments revealed high IgE cross-reactivities with the profilins from other plants. CONCLUSIONS The profilin from the A. retroflexus pollen, Ama r 2, was firstly identified as an allergen. Moreover, rAma r 2 was produced in E. coli as a soluble immunoreactive protein with an IgE-reactivity similar to that of its natural counterpart.