Characterization of a novel allergen Der f 25, homologous to triose-phosphate isomerase, from Dermatophagoides farinae

Dermatophagoides farinae, as one species of house dust mites, was known to be rich in IgE-reactive components that contribute to allergy in China and worldwide. This study aims to characterize a novel allergen, the D. Farinae-derived Der f 25. In this study, total RNA was extracted from Dermatophagoides farinae and the full-length of Der f 25 gene was amplified by RT-PCR with the primers designed according to the sequence of Der f 25. The target gene was cloned into pUC57 vector following sequencing identification, sub-cloned into pET32a(+), and transformed into E. coli BL21 (DE3) for expression. The recombinant protein was purified by Ni-NTA affinity chromatography and analyzed by SDS-PAGE. Its allergenicity was identified by skin prick tests, Western blotting and ELISA. The sequencing indicated the cloned Der f 25 contains 744 bp encoding 247 amino acids which displaying strong homology with triose-phosphate isomerase Dermatophagoides farinae (Gen Bank AGC56216.1). Patients from 16 of 42 house dust mite allergy subjects (38%) showed positive reaction to Der f 25. And 4 positive sera obtained showed strong IgE binding ability to the recombinant protein Der f 25. IgE binding to the house dust mites extracts can be inhibited by recombinant Der f 25 in a dose-dependent manner. In conclusion: A new allergen (Der f 25) was identified from Dermatophagoides farinae which also has the similar immunologic activity to natural Der f 25 protein.