Expression of the Blomia tropicalis paramyosin Blo t 11 and its immunodominant peptide in insect cells.

ABSTRACT

Blo t 11, a dust-mite (Blomia tropicalis) paramyosin, is an allergen with significant IgE reactivity that has potential as a diagnostic/therapeutic reagent for house-dust-mite allergy. The present study describes the successful expression of Blo t 11 and its immunodominant peptide fD in insect cells using a baculovirus expression system. The Blo t 11 and fD genes were cloned into the pMelBacA vector and the resulting vectors were co-transfected into Sf9 insect cells with Bac-N-Blue DNA. Plaque assay was used to select for recombinant virus that was then used to infect High Five insect cells for protein expression. Secreted proteins were harvested by immuno-affinity purification using monoclonal antibodies to Blo t 11. Purified proteins were analysed by immunoblotting, N-terminal sequencing and ELISA. Immunoblot analyses revealed the full-length Blo t 11 cDNA expressed as a minor protein band of approx. 200 kDa and two major protein bands of approx. 60 and 70 kDa. Clones expressing fD cDNA fragment produced a protein of approx. 30 kDa that was confirmed to be fD by N-terminal sequencing. Approx. 4-7.5 mg/l of fD and 1 mg/l of Blo t 11 were obtained by affinity purification. ELISA results showed that human IgE reactivity to these recombinant allergens was lower as compared with that of the native Blo t 11, suggesting that these baculovirus-expressed allergens exhibiting reduced allergenicity could be useful for the development of immunotherapeutic reagents for mite allergy.