Molecular cloning of the mountain cedar (Juniperus ashei) pollen major allergen, Jun a 1

Background: Cedar pollens cause allergic disease in diverse geographic area s. We have recently purified and characterized the major mountain cedar (Ju niperus ashei) pollen allergen, Jun a 1. Objective: A full-length complementary DNA for Jun a 1 was cloned and seque nced, and the recombinant protein was expressed, Methods: Messenger RNA from mountain cedar pollen was purified and Jun a 1 sequences were established with use of reverse transcriptase-PCR and primer s based on the N-terminal amino acid sequence of Jun a 1 and the homologous protein Cry j 1. Portions of the nucleotide sequence were confirmed by com parison with N-terminal amino acid sequencing of the intact tryptic fragmen ts of the purified native protein. Recombinant Jun a 1 was cloned into pET 30, expressed in BL21, and purified by HPLC, and its allergenicity mas anal yzed by Western blotting with patient sera. Results: Jun a 1 possesses a high level of amino acid sequence homology wit h Cha o 1 and Cry j 1, the major allergens of Japanese cypress and Japanese cedar. The amino acid sequence of a region with putative pectate lyase act ivity mas identical to that of Cry j 1 and Cha o 1. Jun a 1 contained 2 pot ential N-glycosylation sites that mere distinct from those found in Cry j 1 , The IgE from patient sera bound recombinant Jun a 1 in Western blot analy sis. Conclusion: The high degree of homology of Jun a 1 with Cha o 1 and Cry j 1 may explain the cross-reactivity of conifer pollens. Differences in N-glyc osylation suggest little overlap of glycopeptide epitopes.