Expression, purification, and activity of novel allergen Tyr p 31 from Tyrophagus putrescentiae

Abstract

Allergen component products, such as recombinant proteins and epitope peptides of allergic components, are used as an adjunct to allergen-specific immunotherapy. We characterized a novel allergen, Tyr p 31, from Tyrophagus putrescentiae, a common allergenic mite. T. putrescentiae total RNA was amplified to Tyr p 31-encoding cDNA, which was inserted into pET28a(+). pET28a(+)-Tyr p 31 was then transformed into Rosetta 2 (DE3) pLysS cells and expressed under isopropyl β-D-thiogalactoside induction. Next, we visualized Tyr p 31 through sodium dodecyl sulfate polyacrylamide gel electrophoresis and Western blotting based on its theoretical molecular weight. Recombinant Tyr p 31 (rTyr p 31) was purified, and its secondary structure was noted to comprise α-helices, antiparallel coils, β-turns, parallel coils, and random coils. Our enzyme-linked immunosorbent assay and Western blotting results for T. putrescentiae–positive sera from children with allergic disorders demonstrated rTyr p 31-specific IgE-positivity rates of 72.41 % and 85.7 %, respectively. In BEAS-2B cells, rTyr p 31 increased IL-6 and IL-8 expression; furthermore, BEAS-2B cells treated with 30 μg/mL rTyr p 31 demonstrated 100 upregulated and 12 downregulated genes. In summary, we identified Tyr p 31, a novel T. putrescentiae allergen component, and noted rTyr p 31 to have a high IgE-binding rate and strong immunogenicity.