花生致敏蛋白Ara h1与咖啡酸互作对其抗原性的影响

Effects of interaction between peanut allergenic protein Ara h1 and caffeic acid on its antigenicity

为探寻适宜的花生脱敏方法,该文研究了花生致敏蛋白Ara h1与咖啡酸互作对其抗原性的影响,利用荧光光谱、紫外光谱和间接ELISA法对碱法、酶法、自由基法处理后的咖啡酸蛋白复合物抗原性变化进行了分析,并对碱法互作反应温度、反应时间、pH值、咖啡酸浓度进行优化。结果表明:在温度33.2℃、时间25 h、pH值8.67和咖啡酸浓度1.76 mg/mL时,咖啡酸与花生致敏蛋白Ara h1碱法互作后其抗原性降至69.31%,接枝量为119.16 nmol/mg。碱法处理后,咖啡酸与花生致敏蛋白Ara h1互作能降低致敏蛋白抗原性,研究结果可为花生脱敏处理提供参考。

Peanut is one of the eight major allergens identified by the Food and Agriculture Organization(FAO), because of the high prevalence, severe reactions, and the lack of reliable treatment. Thus, a large negative impact has posed on the peanut-allergic population and the peanut industry. Most current peanut desensitization treatments have presented nutritional damage and low safety, including physical heating, and genetic engineering. Alternatively, a new avenue of peanut desensitization can be the interactions between natural plants’ polyphenols and proteins. This study aims to explore the effect of interactions between peanut allergenic protein Ara h1 and caffeic acid on the antigenicity during peanut desensitization.Firstly, the peanut allergen Ara h1 was covalently treated with caffeic acid by three methods: alkaline, enzymatic, and free radical method. Then, the complex structural, antigenic changes, the reactive groups, and the binding equivalents of Ara h1-CA were analyzed by fluorescence spectroscopy, Ultraviolet(UV) spectroscopy, and indirect Enzyme-Linked Immunosorbent Assay(ELISA). The results showed that the alkali method performed the best in the three ones, indicating the highest binding equivalents, and the largest number of reactive groups participated in the reaction. In the UV spectrum, the blue shift of the graft was found to be the most outstanding from 280 to 273 nm in the alkali method, indicating the greatest change in UV absorption intensity. The fluorescence intensity of the alkali graft decreased significantly, and there was a slight red shift in the fluorescence spectra. The Immunoglobulin G(IgG) binding capacity of the alkali graft decreased to 76.8% by ELISA analysis, followed by the enzyme and the free radical method. There was no significant difference between the enzyme and free radical method. Therefore, the best reaction was optimized on the reaction temperature, reaction time, pH value, and caffeic acid concentration in the alkali interactions. The single-factor and quadratic regression orthogonal experiments showed that the optimal combination was achieved, where the reaction temperature was 33.2 ℃, the reaction time was 25 h, pH value was 8.67, and the caffeic acid concentration was 1.76 mg/mL. In this case, the antigenicity of Ara h1-CA decreased to 69.31%,and the grafting amount was 119.16 nmol/mg. Furthermore, it was found that the caffeic acid concentration and its interaction with the temperature were extremely remarkable to influence the Ig G-binding ability. The experiments showed that the interaction between caffeic acid and peanut allergenic protein Ara h1 reduced the antigenicity of peanut allergenic protein. The finding can provide a strong reference for the peanut desensitization and further influencing mechanism.