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.