Molecular cloning and sequence analysis of full-length cDNAs encoding new group of Cyn d 1 isoallergens

Background: Cyn d 1, the major allergen of Bermuda grass pollen, contains some acidic/basic isoforms. The N-terminal amino acid sequences of some acidic Cyn d I isoforms were found to be different from those of Cyn d I cDNA clones identified previously. Methods: A predicted 17-meric oligonucleotide probe was designed to fish the unidentified isoallergen cDNAs out of BGP cDNA library. The reactive clones were isolated and verified by sequencing. Two of them were expressed in the yeast Pichia pastoris to obtain recombinant Cyn d I proteins. Results: All four cDNA clones encode the full-length Cyn d I with mature proteins of 244 amino acid residues. A 97-99% identity was found among the deduced amino acids of these four clones while an 86% identity was elicited between the four clones and the ones previously identified. The predicted isoelectric focusing (pI) values of the newly identified Cyn d Is are acidic while pIs of the previously identified Cyn d Is are basic. The two recombinant acidic Cyn d 1 proteins possess the epitopes recognized by mouse and rabbit polyclonal anti-Cyn d 1 antibodies, and have human IgE-binding capacity as revealed by immunodot assay. Conclusions: The present study identified full-length cDNAs encoding new isoallergens of Cyn d 1, and separated Cyn d 1 gene into an acidic group and a basic group.