title: Cloning and Expression of the Olea europaea allergen Ole e 5, the pollen Cu/Zn superoxide dismutase.
abstract: 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.
authors.affiliation: Department of Infectious, Parasitic and Immune-Mediated Diseases, Istituto Superiore di Sanità, Rome, Italy. c.butteroni@iss.it
authors.forename: Cinzia
authors.initials: C
authors.lastname: Butteroni
authors.forename: Claudia
authors.initials: C
authors.lastname: Afferni
authors.forename: Bianca
authors.initials: B
authors.lastname: Barletta
authors.forename: Patrizia
authors.initials: P
authors.lastname: Iacovacci
authors.forename: Silvia
authors.initials: S
authors.lastname: Corinti
authors.forename: Barbara
authors.initials: B
authors.lastname: Brunetto
authors.forename: Raffaella
authors.initials: R
authors.lastname: Tinghino
authors.forename: Renato
authors.initials: R
authors.lastname: Ariano
authors.forename: Raphael C
authors.initials: RC
authors.lastname: Panzani
authors.forename: Carlo
authors.initials: C
authors.lastname: Pini
authors.forename: Gabriella
authors.initials: G
authors.lastname: Di Felice
mesh_terms: D000485:Allergens / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics* / Q000276:immunology; D000595:Amino Acid Sequence; D052179:Antigens, Plant; D001483:Base Sequence; D003001:Cloning, Molecular; D004797:Enzyme-Linked Immunosorbent Assay; D004926:Escherichia coli / Q000235:genetics; D006801:Humans; D015151:Immunoblotting; D007073:Immunoglobulin E / Q000097:blood; D008969:Molecular Sequence Data; D031658:Olea / Q000201:enzymology / Q000235:genetics* / Q000276:immunology; D010940:Plant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics* / Q000276:immunology; D012313:RNA / Q000737:chemistry / Q000235:genetics; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000276:immunology; D020133:Reverse Transcriptase Polymerase Chain Reaction; D016415:Sequence Alignment; D013482:Superoxide Dismutase / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics* / Q000276:immunology
chemical_list: D000485:Allergens; D052179:Antigens, Plant; C110947:Ole e 5 protein, Olea europaea; D010940:Plant Proteins; D011994:Recombinant Proteins; D007073:Immunoglobulin E; D012313:RNA; D013482:Superoxide Dismutase