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.

研究目的：制备屋尘螨变应原Der f 36重组蛋白及其特异性单克隆抗体（monoclonal antibodies），并探究Der f 36在屋尘螨体内的组织定位特征。本研究旨在为屋尘螨过敏的精准诊断与免疫治疗提供实验依据。
方法：构建pcDNA3.1-Der f 36真核表达质粒（eukaryotic expression plasmid），于HEK293细胞中表达重组蛋白rDer f 36并进行纯化。采用IgE-ELISA法评估rDer f 36的免疫反应性（immunoreactivity）。通过杂交瘤技术（hybridoma technology）制备抗Der f 36的单克隆抗体，利用抗体芯片（antibody chip）分析其特异性与交叉反应性（cross-reactivity）。采用免疫荧光组织化学法（immunofluorescence histochemistry）对Der f 36在屋尘螨体内的分布进行定位。
结果：成功获得rDer f 36蛋白。IgE-ELISA检测显示，血清IgE阳性率为32.14%（9/28）。筛选得到3株抗Der f 36的高特异性单克隆抗体，其滴度高且交叉反应性低。免疫荧光定位结果显示，Der f 36主要分布于屋尘螨肠道区域的上皮细胞内，形成囊泡状聚集体，提示其作为分泌型蛋白参与过敏反应。
结论：本研究制备的rDer f 36重组蛋白具有显著的IgE结合活性，可为基于变应原组分的诊断提供极具价值的实验试剂。所获得的单克隆抗体特异性强，适用于变应原检测与免疫治疗相关研究。Der f 36在螨类肠道区域的特异性分布，进一步支持其作为分泌型变应原参与致敏过程的作用机制。