鉴定粉尘螨新过敏原方法学创新及新发现Der f39促进肥大细胞迁移机制的研究

鉴定尘螨新过敏原并揭示其致敏机制是变态反应学基础性科学目标。申请人结合转录组和蛋白组学方法发现了Der f24（JACI，2015），但该方法存在斑点比对一致性差的缺陷。为此，继续研究并获得：①基于多层次测序方法构建了粉尘螨亚染色体级基因组和转录组精细图谱，直接从2D/IgE-免疫印迹斑点提取微量蛋白进行高分辨LC/MS-MS分析，两者结合新发现了过敏原Der f39并获得了WHO/IUIS命名；②发现了Der f39促进肥大细胞活化和迁移现象。在此基础上开展研究，拟解决科学问题：①过敏原鉴定创新方法的完善并发现命名尘螨新过敏原；②解析Der f39主要IgE-B细胞线性表位；③验证Der f39通过Ca2+/NF-κb信号通路和CaMKII/Cofilin/F-actin解聚信号通路分别促进肥大细胞活化和迁移的假说。本课题实施有望克服长期限制本领域发展的技术瓶颈，并探索过敏原致敏新机制。

The identification of new house dust mite (HDM) allergens and characterization of their sensitization mechanisms are key allergological objectives. The traditional two-dimensional electrophoresis identification method, which we used previously in our discovery of the allergen .Der f24, is not effective due to poor spot comparison consistency (JACI 2015, 135(2):539-48.). Here, we invent a promising new identification method and its application leading to the discovery of the novel HDM allergen Der f 39. Specifically, we combine two technologies, a demonstration of fine mapping of the sub-chromosomal genome and transcriptome of Dermatophagoides farinae based on multi-level sequencing and high-resolution LC/MS-MS (liquid chromatography/ tandem mass spectrometry) technology for very small-quantity protein samples extracted from an IgE-positive western blot spot on the PVDF membrane. We then show that the first allergen identified with this approach, Der f 39 named by WHO/IUIS, promotes mast cell activation and migration. Jumping off from these results, we have planned three aims: (1) to improve and perfect this innovative technology for the identification of novel HDM allergens; (2) to analyze the main IgE linear epitopes of Der f39; and (3) to test the hypothesis that Der f39 promotes mast cell activation and migration via the Ca2+/NF-κb and CaMKII/Cofilin/F-actin depolymerization signaling pathways, respectively. Completion of these aims will resolve the bottleneck of high-throughput allergenomic technology and lay the groundwork for identification of the full spectrum of dust mite allergens and the development of diagnostics and immunotherapeutic vaccines for HDM allergic diseases.