热带无爪螨三维基因组学和过敏原组学研究

官方已公布粉尘螨有32组分、屋尘螨有23组分过敏原。热带无爪螨是全球分布的重要致敏螨种之一，才命名14组分过敏原，挖掘其余组分具有重要性和可行性。申请者人工培养热带无爪螨并已完成转录组测序、获得多组分过敏原重组蛋白。在此基础上，本课题拟对该螨进行以下研究：①分离虫卵进行PacBio SMART和Illumina Hiseq测序，并用Hi-C辅助组装获得三维基因组；②取全培养混合物（卵、幼螨、若螨和成螨等），提取蛋白质进行双向电泳，用阳性血清进行免疫印迹，质谱鉴定单组分；③整合蛋白质组学、全长转录组、全基因组数据获得过敏原单组分基因序列，通过分子克隆技术获得重组蛋白，制备蛋白芯片，检测单组分血清IgE结合率；④分别取卵、幼螨、若螨、成螨和雌、雄螨进行二代测序，比较过敏原单组分表达量的不同。本研究有望在全球首次解析螨虫三维基因组学和热带无爪螨过敏原组学信息，为开发组分诊断试剂奠定基础。

There are many mite species in house dust. A total of 32 and 23 groups of allergen were named officially for Dermatophagoides farinae (D. farinae) and Dermatophagoides pteronyssinus (D. pteronyssinus), respectively. Blomia tropicalis, a very important mite species in the globe, merely presents 14 groups of allergens deposited in the official site for the systematic allergen nomenclature, all which is approved by the World Health Organization and International Union of Immunological Societies (WHO/IUIS) Allergen Nomenclature Sub-committee. No doubt, it is of great value, also viability, to resolve other groups from Blomia tropicalis. The authors have succeeded in culture of Blomia tropicalis, finished its RNA-Seq analysis, obtained a few recombinant allergens. Here, we apply funding to do the following research work: ① The Illumina HiSeq and Pacbio were used for sequencing the eggs in purity, and the three-dimensional genome was mapped with the aid of Hi-C technology to catch the interactions for all DNA in Chromatin. ② The whole culture, including eggs, larva, nymphs, adults and media, were extracted for proteins mixture that was then submitted for two-dimensional electrophoresis and immunoblotting to identify novel allergens. ③ By the integration of the genome and proteomics, the nucleotide sequences for each individual allergen were obtained, and their corresponding recombinant proteins were produced by molecular cloning technology. Then, the protein chip, made of by those recombinant allergens, was intended to detect IgE binding rates for individual allergens. ④ The RNA-Seq was conducted for egg, larva, nymphs, adults, male and female mites, the expression levels were compared for individual allergens. The present study will resolve three-dimensional genome of a mite, so as to pioneer the genome of Blomia tropicalis all over the world. And what is more, it will be a firm foundation to further explore a kit for component-resolved diagnosis of mite allergic disorders.