Dermatophagoides farinae allergen Der f 9: Cloning, expression, purification, characterization and IgE-binding in children with atopic asthma.

BACKGROUND: The house dust mite species Dermatophagoides farinae releases allergens that cause allergies and asthma worldwide. This study sought to clone and express the full-length cDNA encoding the group 9 allergen of D. farinae (Der f 9).

METHODS: The published sequence of Der f 9 was used to design primers for RT-PCR and RACE to obtain the full-length cDNA encoding Der f 9. After removal of signal peptide sequence, Der f 9 was then sub-cloned into plasmid pET-28b (+), and the plasmid was transformed into Escherichia coli BL21 (DE3) cells for expression. The recombinant protein was purified by Nickel affinity chromatography, identified by SDS-PAGE, Western blotting, dot blotting, and MALDI-TOF, and tested by ELISA for IgE reactivity with sera from children with asthma. Bioinformatics analyses were used to identify features of Der f 9.

RESULTS: By RT-PCR, 3'-RACE, and 5'-RACE, the full-length sequence of Der f 9 was generated, which was confirmed by nucleotide sequencing. The mature Der f 9 was expressed successfully in E. coli, which was identified by SDS-PAGE. The recombinant allergen was purified by chromatography and confirmed by SDS-PAGE, Western blotting, dot blotting, and MALDI-TOF. Sera from 56.7% (17/30) of mite-allergic patients reacted with the purified recombinant Der f 9.

CONCLUSIONS: The successful production of recombinant Der f 9 protein revealed the importance of Der f 9 in mite allergy, and provides a foundation for further study of this allergen in diagnosis and treatment of symptoms. Pediatr Pulmonol. 2017;52:282-292. © 2016 Wiley Periodicals, Inc.