Recombinant Expression, Monoclonal Antibody Preparation, and Tissue Localization Study of Dust Mite Allergen Der f 36.

Objective To prepare the dust mite allergen Der f 36 recombinant protein and its specific monoclonal antibodies, and to investigate the tissue localization characteristics of Der f 36 in dust mites. This study aims to provide experimental evidence for the precise diagnosis and immunotherapy of dust mite allergies.Methods A pcDNA3.1-Der f 36 eukaryotic expression plasmid was constructed, and recombinant protein rDer f 36 was expressed in HEK293 cells and purified. The immunoreactivity of rDer f 36 was evaluated using IgE-ELISA. Monoclonal antibodies against Der f 36 were prepared by hybridoma technology, and their specificity and cross-reactivity were analyzed using antibody chips. The distribution of Der f 36 within the dust mite was localized using immunofluorescence histochemistry.Results The rDer f 36 protein was successfully obtained. IgE-ELISA showed that the serum IgE positivity rate was 32.14% (9/28). Three highly specific monoclonal antibodies against Der f 36 were selected, exhibiting high titers and low cross-reactivity. Immunofluorescence localization demonstrated that Der f 36 was mainly distributed in the epithelial cells of the intestinal region of the dust mite, forming vesicular aggregates, suggesting its involvement in allergic reactions as a secreted protein.Conclusion The rDer f 36 prepared in this study showed significant IgE-binding activity, providing a valuable reagent for allergen component-based diagnosis. The monoclonal antibodies produced are highly specific and suitable for allergen detection and immunotherapy research. The specific distribution of Der f 36 in the mite's intestinal region supports its role as a secreted allergen in the sensitization mechanism.