低频超声-羰氨缩合降低小清蛋白致敏性的分子机制研究

前期研究表明，低频超声-羰氨缩合能显著降低鲢鱼小清蛋白（PV）的致敏性，但其分子机制尚不清楚，亟待深入研究。本项目以PV和还原糖为研究对象，通过调控不同影响因素（超声参数和羰氨反应条件）制备低频超声-羰氨缩合修饰的PV，利用ELISA、免疫印迹等方法研究其致敏性的变化规律；借助Orbitrap-MS、氨基酸分析等技术精确测定修饰后PV的氨基酸组成和羰氨结合位点，通过对比分析不同影响因素下PV的羰氨结合位点和致敏性，建立影响因素-羰氨结合位点-致敏性的相关性；采用HDX-MS、MALDI-MS等技术研究修饰后PV多级结构、聚集状态和空间构象的变化，解析低频超声-羰氨缩合对PV抗原表位（线性表位和构象性表位）的影响规律。结合PV的3D结构和抗原表位，揭示影响因素-致敏性-抗原表位之间的相关性，最终从抗原表位方面阐明低频超声-羰氨缩合降低PV致敏性的分子机制，为低敏性鱼类制品的开发提供理论。

Our previous research showed that low-frequency ultrasound combined with Maillard reaction (MR) reduced the allergenicity of silver carp parvalbumin (PV). However, the mechanism is not clear, and needs to be further studied. PV and reducing sugar will be chosen as materials in this project. Firstly, preparation of modified PV by low-frequency ultrasound combined with MR through regulation of different influencing factors, such as ultrasonic parameters and conditions of MR. Methods such as Enzyme-linked immunosorbent assay and Western blotting will be used to research the influences of low-frequency ultrasound combined with MR on allergenicity of PV. Orbitrap-MS and Amino acid analyzer will be employed to investigate the influence of low-frequency ultrasound combined with MR modification on the binding sites and amino acid composition of PV. Comparative analysis of the binding sites and allergenicity of PV under different influencing factors, then establishing the correlation among influencing factors, allergenicity and binding sites of modified PV. Multi-structure, aggregation and conformational structure of modified PV were studied by using HDX-MS and MALDI-MS, then low-frequency ultrasound combined with MR modification affected antigen epitopes of PV was analyzed, included linear epitopes and conformational epitopes. Based on 3D structure and antigen epitopes, the correlation between the influencing factors, allergenicity and antigen epitopes was revealed. Finally, the mechanism of the reduction in the allergenicity of PV induced by low-frequency ultrasound combined with MR, will be clarified from the antigen epitopes. This program will provide the theoretical basis for the preparation of fish products of low allergenicity.