Sequence analysis and expression of a cDNA clone encoding a 98-kDa allergen in Dermatophagoides farinae

Background The important dust mite allergens identified to date are of mole cular weights ranging from 14 to 60 kDa. Our previous protein study indicat ed that the 98-kDa native paramyosin in Dermatophagoides farinae mite showe d IgE reactivity with 82% of the mite-sensitive asthmatic patients suggesti ng that it is a novel major mite allergen. This study described the isolati on and characterization of the cDNA clone encoding the 98-kDa mite allergen . Methods A Dermatophagoides farinae cDNA library was constructed in lambda Z APII vector and the library was immunoscreened with a monoclonal antibody 6 42. The cDNA insert was sub-cloned into M13 sequencing vector for single-st randed sequencing. The whole cDNA insert was expressed in pGEX-2T Escherich ia coli expression system as a fusion protein with GST. The allergenicity o f the recombinant peptides was tested by skin tests and IgE immunoassay. Th e IgE and IgG immunoassays were performed with sera from 20 mite-allergic p atients. Results The cDNA clone Df642 was 2134 bp long, coding for a polypeptide of 711 amino acid residues. Protein sequence analysis and alignment confirmed that the deduced polypeptide is a mite paramyosin which is truncated slight ly at the N- and C-terminuses. In vivo skin tests and in vitro IgE-binding study showed that 62% (13/21) and 50% (10/20) of the mite-sensitive asthmat ic patients reacted positively with the recombinant Dermatophagoides farina e paramyosin, respectively. Conclusion The study indicated that 98-kDa mite paramyosin is an important allergen.