Author(s): Ramos JD, Teo AS, Ou KL, Tsai LC, Lee BW
BACKGROUND: The complementary DNA (cDNA) encoding for Blo t 11, a 102 kD allergen from Blomia tropicalis (Bt) was isolated, expressed and characterized previously. This study aimed to isolate the native Blo t 11 allergen and compare its allergenicity with the recombinant forms. METHODS: Native Blo t 11 (nBlo t 11) was isolated from crude Bt extract by immuno-affinity chromatography, analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot, and verified by MALDI-TOF MS. Recombinant full-length Blo t 11 (rFL-Blo t 11) and its immunodominant peptide (fD) were expressed as glutathione S-transferase (GST)-fusion proteins in Escherichia coli. Immunoglobulin E (IgE) reactivity of the Blo t 11 allergens were determined by enzyme-linked immunosorbent assay (ELISA) and skin prick test. The inhibition capacity of the nBlo t 11 against fD and vice versa was determined by absorption studies. RESULTS: Affinity purified nBlo t 11 was susceptible to degradation with the major degraded product resolved at approximately 66 kD. The nBlo t 11 was confirmed by immunoblot analysis and MALDI-TOF MS that generated 13 peptides with complete identity to the deduced amino acid sequence of Blo t 11. Comparative in vitro and in vivo allergenicity tests and the cross inhibition studies between the native and recombinant Blo t 11 showed that recombinant fD, but not the rFL-Blo t 11, has comparable IgE reactivity with the native counterpart. CONCLUSIONS: This comparative study confirmed that the recombinant peptide fD contains the main immunodominant region of Blo t 11. This recombinant peptide, instead of the full-length protein, is a good candidate for diagnostic and therapeutics development for mite allergy.