基于表位掩蔽和构象折叠理论的糖基化修饰降低OVA致敏性的分子机制

前期研究发现，糖基化修饰可以大大降低卵清蛋白（OVA）的过敏性，且与表位掩蔽和构象变化密切相关，但是其内在分子机制尚不清楚。针对这一科学问题，本项目拟基于卵清蛋白的表位掩蔽和构象折叠理论，分别从糖基化修饰位点、修饰程度和构象变化的角度出发，以OVA和还原单糖为研究对象，采用温和条件进行糖基化修饰，并监测其修饰程度；通过ELISA方法分析糖基化OVA的过敏性（IgG/IgE 结合能力）；利用MALDI MS、紫外、荧光、圆二、HDX-MS等方法分析糖基化OVA的聚合度和高级构象变化；采用Orbitrap LC-MS、18O稳定同位素标记等手段对糖基化修饰位点和修饰程度进行定性和定量分析；结合OVA的3D结构和过敏表位，揭示糖基化修饰位点-修饰程度-高级构象-过敏性之间的关系，从线性表位掩蔽和构象折叠方面阐述糖基化修饰降低OVA过敏性的内在机制，为低敏或脱敏蛋白的定向修饰和开发奠定理论基础。

The preliminary study found that glycosylation can greatly reduce the sensitization of ovalbumin (OVA), which is closely related with the epitope masking and conformation changing, but its molecular mechanism is not clear. According to this scientific problem, this project aims to monitor the glycosylation between OVA and glucose under mild conditions from three aspects (modification sites, the dgree of modification and conformational change), which is based on the theory of epitope masking and conformation folding. Then we analyse the sensitivity of glycated OVA, the ability of binding with IgG/IgE, by Elisa; explore the change rules of advanced conformation and calculate the degree of polymerization by means of MALDI MS, ultraviolet spectrometry, fluorescence spectrometry, circular dichroism spectroscopy, HDX-MS; determine the glycosylation site and the degree of modification of qualitatively and quantitatively by using Orbitrap LC-MS, 18O isotope labeling and other methods; reveal the relationship between the modification sites, advanced conformation and Allergic reaction according to the 3D structure of OVA; finally, we will elaborate the intrinsic mechanism of glycosylation modification to reduce OVA allergen from the aspects of linear epitope masking and conformation folding, to lay a theoretical foundation for the directional modification of the desensitized protein.