Molecular cloning and immunological characterization of the house dust mite allergen Der f 7

Abstract

The allergen Der p 7 from Dermatophagoides pteronyssinus has been defined by molecular cloning and shown to be an important specificity in 50% of mite-allergic patients. This study compares the cDNA sequence and serological reactivity of Der f 7 from D. farinae with Der p 7. cDNA encoding Der f 7 was amplified by polymerase chain reaction, sequenced and expressed as a fusion with glutathione-S-transferase for IgE and monoclonal antibody binding studies. Der f 7 cDNA encoded a 213 polypeptide containing a predicted 17 amino acid leader sequence, no cysteines and a single N-glycosylation site similar to Der p 7. The predicted 196 residue mature polypeptide had 86% identity to Der p 7 and a calculated molecular weight of 22,348Da. No homologues were found in searches of the data banks. The Der f 7 fusion protein showed a single band of 46 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and reacted with IgE antibodies in 19/41 (46%) of sera from asthmatic children. The degree of binding was usually 30% of that to Der p 7 consistent with the exposure of the patients to D. pteronyssinus. Monoclonal antibodies (WH9 and WH22) against Der p 7 reacted with Der f 7 but inhibition studies showed a 10-fold difference in reactivity. Der f 7 has a predicted 213 residue polypeptide with 86% homology and serological crossreactivity to Der p 7.