甘露糖—小清蛋白美拉德反应产物的免疫活性

Study on the immunoreactivity of mannose-parvalbumin conjugate

为探讨以甘露糖为还原糖进行美拉德反应对小清蛋白免疫活性的影响,通过将鲢重组小清蛋白(r PV)和甘露糖混合物在干热条件下(100°C)反应100 min制得糖化的r PV(Mr PV),采用Tricine-SDS-PAGE和斑点杂交(Dot-blotting)分析糖化前后r PV的聚合特性、免疫活性及消化稳定性的变化;采用圆二色谱仪和扫描电子显微镜分析糖化对r PV的二级结构及微观结构的影响。结果显示,r PV糖化后形成大分子量的片状聚集体;甘露糖糖化修饰的r PV免疫活性及消化稳定性均显著下降;美拉德反应导致r PV二级结构中的α-螺旋和无规则卷曲的含量有一定程度的减少,而β-折叠的含量显著增加。研究表明,以甘露糖为还原糖进行美拉德反应可有效降低r PV的免疫活性及消化稳定性,这可能与糖化后r PV聚集体的形成及二级结构的改变有关。

In order to determine the influence of Maillard reaction on the structural and immunological properties of parvalbumin, the major allergen of silver carp（Hypophthalmichthys molitrix）, recombinant silver carp PV（r PV）was used and incubated with mannose at 100 ℃ for 100 min, and glycated PV（M-r PV） was then obtained after dialysis. Tricine-SDS-PAGE and Dot-blotting was performed to elucidate the change of mobility and immunoreactivity of r PV after glycation. Simulated gastrointestinal digestion experiment was carried out to compare the digestibility of r PV and M-r PV. Circular dichroism spectroscopy（CD） and scanning electron microscopy（SEM） were used to determine the change of the secondary structure and the microstructure of r PV after Maillard reaction. The results showed that M-r PV had significantly high amounts of protein-bound mannose of approximately 150 μg per microgram M-r PV as measured by the anthrone sulfuric acid method. Dimers and polymers formed in the glycated sample, which might be attributed to the covalent bonds formed during Maillard reaction as analyzed by reducing Tricine-SDS-PAGE. Formation of compact aggregates after Maillard reaction was further confirmed by SEM, which might block the access of immunoglobulins to the epitopes, and therefore decrease the immunoreactivity of M-r PV, as demonstrated by dot-blotting using mouse anti-silver carp parvalbumin monoclonal antibody. M-r PV exhibited less resistance against gastrointestinal digestion in vitro as revealed by Tricine-SDS-PAGE and dot-blotting. Maillard reaction also showed a minor effect on the secondary structures of r PV with a decrease of α-helix and an increase of β-strand content. The aggregation formation and change in the secondary structure of M-r PV might explain the decrease of r PV immunoreactivity and digestibility after Maillard reaction. Our work might be useful for understanding the effects of Maillard reaction on the decrease of immunoreactivity of food allergens in general.