Abstract
Blomia tropicalis has been recognized as a cause of allergic diseases in the tropical and subtropical regions. Here we report the immuno-characterization of its group 2 allergen, Blo t 2. Allergen Blo t 2 was amplified from the cDNA of B. tropicalis using degenerate primers, expressed in Escherichia coli as a recombinant protein and purified to homogeneity. The mature protein of Blo t 2 was 126 amino acids long with 52% sequence identity to Der p 2 and apparent molecular mass of 15 kDa. Circular dichroism spectroscopy showed that Blo t 2 is mainly a beta-sheeted protein. We confirmed the presence of three disulfide bonds in recombinant (r) Blo t 2 protein using electrospray mass spectrometry. Thirty-four percent of dust-mite allergic individuals from the Singapore showed specific IgE binding to rBlo t 2 as tested using immuno dot-blots. IgE-cross reactivity assays showed that Blo t 2 had between 20–50% of unique IgE-epitopes compared to Der p 2. IgE binding of native and recombinant forms of Blo t 2 were highly concordant (r2 = 0.77, p < 0.0001) to rBlo t 2. Dose-dependent in vitro histamine was observed when rBlo t 2 was incubated with whole blood of Blo t 2 sensitized individuals, demonstrating that it is a functional allergen. Nine naturally occurring isoforms of Blo t 2 were identified in this study, each having between 1–3 amino acid variations compared to the reference clone. Blo t 2 is a clinically relevant allergen of B. tropicalis as it has unique IgE epitopes compared to major group 2 allergens from Dermatophagoides spp.
Highlights
Blomia tropicalis has been recognized as a cause of allergic diseases in the tropical and subtropical regions
We amplified the group 2 allergen from B. tropicalis using a combination of two techniques, reverse transcription polymerase chain reaction (RT-PCR) and random amplification of cDNA ends (RACE) (Random Amplification of cDNA Ends)
A homology search was performed using the sequence obtained against the non-redundant database in NCBI using the BLAST-X algorithm[24], which showed a match to group 2 dust mite allergens, with the closest homology being to Ale o 2, with 60% amino acid sequence identity
Summary
Blomia tropicalis has been recognized as a cause of allergic diseases in the tropical and subtropical regions. Blo t 2 is a clinically relevant allergen of B. tropicalis as it has unique IgE epitopes compared to major group 2 allergens from Dermatophagoides spp. Dust-mite sensitized individuals living in these countries show varying degrees of skin prick reactivity to crude protein extracts of B. tropicalis, ranging from over 90% among individuals in Singapore, circa 70% among individuals in China and Taiwan to as low as 38% in Florida[9]. We report the identification and characterization of Blo t 2, in terms of its IgE-reactivity and cross-reactivity within the Singaporean population, histamine release capacity, presence in the indoor environment, and the frequency of naturally occurring isoforms of Blo t 2
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