Search

Background: Recombinant DNA technology does provide pure, well-defined and reproducible products to be used for clinical purposes, by cloning and expressing the cDNA of allergens present in a specific extract. Ole e 5 is a pollen allergen of Olea europaea with an IgE-binding frequency of about 35%, which has been identified as a superoxide dismutase (SOD). The aim of this study was to clone the cDNA of Ole e 5, to express Ole e 5 in Escherichia coli and to characterize its immunoreactivity. Methods: cDNA of Ole e 5 was amplified by nested 3′-RACE PCR and cloned in pGEX vector 6P expression vector. After sequencing of some clones and homology analysis, the rOle e 5 was produced in an E. coli strain as a fusion protein with GST and purified. Then, the protein immunoreactivity was evaluated by patients’ IgE binding (ELISA, ELISA inhibition, and immunoblotting) and by rabbit anti-rOle e 5 binding (immunoblotting and immunoblotting inhibition). Results: The sequence analysis of Ole e 5 cDNA confirmed that Ole e 5 is a Cu/Zn SOD, with an identity from 90 to 80% with SOD from other species. rOle e 5 was recognized by IgE from 39% of olive pollen-allergic patients tested; moreover, this binding was inhibited by the olive pollen extract. An anti-rOle e 5 antiserum raised in rabbit strongly reacted with a natural component of about 16-kDa molecular weight present in the olive pollen extract; moreover, this binding was inhibited by the recombinant protein. Conclusions: Ole e 5 is the first Cu/Zn SOD identified as an allergen in a pollen source. Due to the widespread presence of this enzyme, rOle e 5 allergen, cloned and expressed in a complete form in E. coli, could represent a good tool to investigate the allergen cross-reactivity between O. europaea pollen and other allergenic sources, such as plant foods and other pollens. Copyright © 2005 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

Abstract: Sensitization to Cupressaceae pollen has become one of the most important causes of pollinosis in Western countries during winter and early spring. However, the characterization of the extracts, the allergens involved and the cross-reactivity with other pollen sources still remain poorly studied; in the case of Cupressus arizonica only two allergens have been described so far. A new allergen from C. arizonica pollen, Cup a 4, was cloned and expressed in Escherichia coli as an N-terminally His-tag recombinant protein that was characterized biochemically, immunologically and by circular dichroism spectroscopy. The new allergen has high sequence identity with Prickly Juniper allergen Jun o 4 and contains four EF-hand domains. The recombinant protein has structural similarities with other calcium binding allergens such as Ole e 3, Ole e 8 and Phl p 7. Cup a 4 is expressed in mature pollen grains and shares antigenic properties with the recombinant form. Sera from 9.6% C. arizonica allergic patients contain specific IgE antibodies against recombinant Cup a 4. [ABSTRACT FROM AUTHOR]