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

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.