Blomia tropicalis is a common house dust mite in tropical regions. Although the predominant sensitizations to Blomia allergens were clearly demonstrated in some South-East Asian countries as Singapore or Malaysia, the prevalence of B.tropicalis hypersensitivities as well as the identification of the major allergens remains to be elucidated in Thailand. The goal of the study is the cloning and expression of recombinant Blo t 7 (rBlo t 7) allergen in Pichia pastoris.
The cDNA encoding mature Blo t 7 was amplified from B. tropicalis total cDNA template using specific primers derived from the allergen mRNA sequence (GenBank accession number AAQ24545.1). The cDNA was subsequently cloned into the pPicZα A P.pastoris expression vector, downstream to the a mating factor leader sequence for protein secretion. P. pastoris KM71H cells were transformed with linearized recombinant pPicZα A-Blo t7 plasmid by electroporation. rBlo t 7 expression was subsequently assayed in zeocin-resistance colonies following methanol induction. The recombinant allergen was purified to homogeneity by anion-exchange and gel filtration chromatographies.
The amplified cDNA of mature Blo t 7 encoded 195 amino acids. Protein sequence alignment with the mature Blo t 7 reference primary structure (UniprotKB/TrEMBL, accession number A1KXI4) evidenced four point mutations but also a twenty amino acids insertion. Multiple alignments showed that this Blo t 7 sequence segment is a conserved domain shared with other group 7 mite allergens (Tyr p 7, Aca s 7, Lep d 7, Gly d 7, Der p 7 and Der f 7). Mature rBlo t 7 was successfully expressed and secreted from P.pastoris following induction with 0.5% methanol at 25°C. The expressed protein migrated onto SDS-PAGE as a 25kD band. The difference with the predicted molecular weight (21.18 kD) could be attributed to glycan structures from the predicted N-glycosylation site (NTT, aa 187-189).
The correction of the mature Blo t 7 cDNA sequence together with the successful recombinant allergen production in P. pastoris offers great opportunities to initiate the component-resolved diagnosis of B. tropicalis allergy in Thailand.