该文以青鱼为研究对象,研究肌浆蛋白在不同pH和离子强度条件下,IgG/IgE结合能力及结构的变化。通过间接酶联免疫吸附测定(indirect-enzyme linked immunosorbent assay,间接 ELISA)、十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(sodium dodecylsulphate polyacrylamide gel electrophoresis, SDS-PAGE)和免疫印迹试验测定肌浆蛋白IgG/IgE结合能力的变化,并结合二级结构变化、表面疏水性变化和内源荧光值变化所反映的蛋白结构改变来分析其变化机理。结果表明,pH从4.0升高到7.0会小幅度降低肌浆蛋白IgG/IgE结合能力,二级结构中α-螺旋增加而不规则卷曲减少,表面疏水性大幅减小且色氨酸疏水基向极性环境暴露。离子强度从0增加到1.0则不会对IgG/IgE结合能力产生显著影响。由此可知,肌浆蛋白IgG/IgE结合能力变化与其构象表位有关,初步判断主要由二级结构的改变引起,但仍需要进一步研究。
Freshwater fish is the main source of protein for Chinese. The increased fish consumption correlates with increased hypersensitive reactions, which could reduce consumer’s living quality. The aim of this article was to evaluate the IgG/IgE binding capacity and structure of sarcoplasmic protein of black carp (Mylopharyngodon piceus) exposed to different pH and ion strength. The IgG/IgE binding capacity was determined by indirect ELISA, SDS-PAGE and western blotting. And protein structure was determined by secondary structure, surface hydrophobicity and intrinsic fluorescence intensity. The results showed that the IgG/IgE binding capacity of sarcoplasmic protein decreased slightly with pH shifting from 4.0 to 7.0, and along with the increased α-helix and decreased random coil in the secondary structure. The surface hydrophobicity decreased significantly and the tryptophan hydrophobic base was exposed to the polar environment. However, the increase of ion strength from 0 to 1.0 had no significant impact on sensitization. The change of immunological properties of sarcoplasmic protein was related to its conformational epitopes, which was mainly caused by the change of secondary structure.
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