Cloning, sequencing and expression in Escherichia coli of Pha a 1 and four isoforms of Pha a 5, the major allergens of canary grass pollen

Background: The pollen of canary grass, which was introduced as a pasture grass from Europe, is a major allergen source in the external environment of southern Australia. This study was performed to characterize the major recombinant allergens of canary grass pollen. It is anticipated that recombinant allergens may be useful in diagnosis and immunotherapy of grass pollen induced allergies. Objective: To clone major canary grass pollen allergens and assess their nucleotide and amino acid sequence homologies with other grass pollen allergens. This sequence information may then be useful in T and B cell epitope mapping studies. Methods: A canary grass pollen λgt11 cDNA expression library was constructed and screened with sera of grass- pollen-sensitive patients. IgE-reactive clones were isolated, sub-cloned into Escherichia coli, sequenced and, along with the deduced amino acid sequences, compared with other sequences in nucleotide and amino acid databases. Results: One of the clones encoded the group 1 allergen of canary grass pollen, Pha a 1, with a deduced amino acid sequence identity of 88.8% with Lol p 1, from rye-grass pollen, 88.1% with Hol l 1, from velvet grass pollen and 86.6% with Phl p 1, from timothy grass pollen. The other clones (e.g. clones, 5, 14, 28, 29) encoded polymorphic forms of Pha a 5. These polymorphic forms showed between 60.6-95.5% nucleotide and 40.1-81.7% deduced amino acid sequence identities with each other. Moreover, they shared significant sequence identity with other group 5 allergens from rye-grass, timothy and Kentucky bluegrass pollens. Conclusions: Group 1 and four isoforms of group 5 allergens of canary grass pollen have been cloned and upon sequencing demonstrated strong nucleotide and amino acid sequence identities with other group 1 and 5 grass pollen allergens.