桦树花粉主要过敏原Bet v 1过敏原性形成的分子机制研究

DCs are the most potent APCs and function as innate gatekeepers of adaptive immune responses, which have the unique ability to induce proliferation of naive T cells and direct them towards TH2 or TH1 immune responses, and elicit TReg cells responses, resulting in allergic reaction or immune tolerance. According to our previous work, the process of antigen/allergen recognition and presentation by DCs plays a critical role in the allergic reaction and confers immune tolerance. Nowadays, even many studies have been done on the signal pathways of DCs, the molecular mechanism of the formation of allergenicity is still unclear..This project aims to reveal the underlying molecular mechanism of the allergenicity of Bet v 1, which is the main allergen of birch pollen. Using the online software NetMHCⅡ 2.2, the strong binding sites of Bet v 1 with MHC-Ⅱ are predicted, according to which a hypoallergenic Bet v 1 mutant is constructed (designed as mBet v 1). Then, PBMCs from patients will be stimulated by Bet v 1 and mBet v 1, and DCs and TReg cells will be isolated and applied to RNA sequencing and proteome analysis. According to the results of RNA sequencing and proteome analysis, we will figure out the critical genes, signal pathways and effector molecules in the formation of allergenicity of Bet v 1 and give a whole view of the molecular mechanism of allergenicity of Bet v 1. This work will contribute to the study of allergic reaction and provide new prospects to the prevention and therapy of allergy related diseases.

临床研究表明，花粉过敏症患者接受脱敏治疗形成免疫耐受后，其特异性IgE并无显著降低，同时本实验室发现对已形成免疫耐受的过敏病例作体外激发试验，结果仍为阳性。据此推测：过敏原性（免疫耐受）并非在肥大细胞/嗜碱性粒细胞水平形成，而是由DCs等细胞的抗原提呈过程决定。目前国际上对DCs细胞在过敏原性形成中的作用及过敏原性形成的分子机制仍不清楚。本研究以桦树花粉主要过敏原Bet v 1作为研究对象，通过在线软件NetMHCⅡ 2.2对其主要MHC-Ⅱ类分子抗原表位进行预测，并构建低过敏原性突变体mBet v 1；通过转录组及蛋白组分析，对分别受Bet v 1及mBet v 1刺激后的DCs及TReg细胞中关键信号通路进行剖析。目标：理清DCs及TReg在过敏原形成中的作用及各信号通路及因子的相互关系，给出一个系统、完整及清晰的过敏原性形成的分子机制蓝图，为相关疾病的预防与治疗提供新思路及理论基础。

众所周知，过敏症患者接受脱敏治疗形成免疫耐受后，其特异性IgE并无显著降低。因此，我们认为过敏原性（免疫耐受）并非在肥大细胞/嗜碱性粒细胞水平形成，而是由DCs等细胞的抗原提呈过程决定。为了探讨其分子机制，本课题以桦树花粉过敏原Bet v 1为研究对象，构建了MHC Ⅱ低结合力的突变蛋白。发现，MHCII锚定位点氨基酸的突变导致了其α-螺旋结构含量的增加。结构的改变促使其IgE结合能力下降，DC活化能力减弱，免疫小鼠血清中IgG含量增加，机体更倾向于Th1型的免疫反应。此外，我们还用原核系统构建和表达纯化了HLA-DRB1*07:01蛋白，为进一步测定蛋白质与MHC Ⅱ的结合力提供了材料。本课题获得的低致敏原性的蛋白质和HLA-DRB1*07:01蛋白将为推进临床脱敏治疗及过敏相关疾病的预防和治疗，新药及新疗法的开发，提供新的思路及理论依据