Characterization of a hybrid protein designed with segments of allergens from Blomia tropicalis and Dermatophagoides pteronyssinus

An Engineered Hybrid Protein from Dermatophagoides pteronyssinus Allergens Shows Hypoallergenicity

“…Similar to other hybrid molecule of 22.8 kDa that was developed by our group (MAVAC-BD-2) [43], DPx4 induced IgG blocking antibodies in immunized mice, showed comparatively lower human serum IgE reactivity and basophil activation than the purified allergens and mite extract. However, there are major differences between these molecules in allergen representation and antigenic composition; DPx4 contains antigenic segments of four allergens from only D. pteronyssinus , with a larger contribution from Der p 1, while MAVAC-BD-2 contains antigenic segments of four allergens from this mite species and three allergens from B. tropicalis , with the largest contribution of Der p 2 and Blo t 5.…”

“…The plasmid containing of DPx4 insert was transformed into competent E. coli BL21 (DE3) cells (Invitrogen Corporation, Carlsbad, CA, USA) by electroporation. The expression and purification was performed, as described elsewhere [43]. In brief, the culture was induced with 1 mM IPTG for 4 h at 37 °C.…”

“…26 sera were randomly chosen from the allergic patients to compare the IgE reactivity to DPx4 with that of Der p 1, Der p 2, and D. pteronyssinus extract for non-denaturing dot blot experiments, as described previously [43]. Three sera from non-allergic individuals and buffer alone were used as the controls.…”

“…The bound IgE and IgG antibodies were detected by incubation with 100 μL of alkaline phosphatase-conjugate anti human IgE (Sigma A3525) or anti-IgG (Sigma A3187). The substrate p-nitrophenyl phosphate (Sigma) diluted in 10% diethanolamine was added for the development of reactivity, and OD was measured in an ELISA reader at 405 nm [43]. The quantification of IgE was performed by using the standard curve of human IgE (Merk Millipore, Billerica, MA, USA).…”

“…DPx4-specific IgG1 and IgG2a in serum were determined by ELISA while using MaxiSorp plate wells (Nunc, Thermo Fisher) that were coated with 1 µg/mL of DPx4. In addition, we evaluated whether IgG antibodies induced by immunization reacted with mite extract by dot blot assay, using nitrocellulose membrane that was dotted with 5 µg of allergen extract, as described in [43]. An ELISA inhibition test was performed using MaxiSorp plate wells that were coated with 5 µg of D. pteronyssinus extract and serum pool from the immunized mice with DPx4 to test whether the IgG antibodies could inhibit the human IgE binding to D. pteronyssinus extract, as described previously [43].…”