Expression of recombinant allergen, Der f 1, Der f 2 and Der f 4 using baculovirus-insect cell systems

INTRODUCTION: Specific immunotherapy is critical for alleviating symptoms associated with house dust mite allergy, such as asthma and rhinitis. However, this approach relies on crude extracts, which are often not of sufficient quality or purity and are not standardized. The use of recombinant allergens may enable safer, more effective treatment. MATERIAL AND METHODS: Using our previously constructed plasmids pET28a(+)-Der f 1, pET28a(+)-Der f 2 and pET28b(+)-Der f 4 as templates, the gene fragments coding for the allergens Der f 1, Der f 2 and Der f 4, respectively, of the dust mite Dermatophagoides farinae were amplified by PCR. Next the PCR-amplified DNAs were recovered, cloned into pFastBacHT A, and transformed into Escherichia coli DH10Bac. The resulting vectors were co-transfected into Spodoptera frugiperda Sf9 cells for expression. The recombinant allergens were purified by Ni(2+) affinity chromatography, and identified by SDS-PAGE and ELISA. RESULTS: The recombinant allergens were successfully expressed and purified from a baculovirus expression system introduced into Sf9 cells, which were verified as being of the correct predicted molecular weights by SDS-PAGE. Furthermore, the reactivity to recombinant allergens rDer f 1, rDer f 2, and rDer f 4 was 85.2%, 88.9%, and 44.4%, respectively, in 27 children with asthma and D. farinae allergy. CONCLUSIONS: Recombinant allergens from dust mites can be successfully generated using a baculovirus-insect expression system. Furthermore, these recombinant allergens can be used to detect mite sensitivity in sera, highlighting their utility in future work to understand and develop treatment for mite allergy.