【作者】 常耀光； 梅轩玮； 孙孟辉； 韩瑾； 薛长湖；

【Author】 Chang Yaoguang;Mei Xuanwei;Sun Menghui;Han Jin;Xue Changhu;College of Food Science and Engineering,Ocean University of China;

【机构】 中国海洋大学食品科学与工程学院；

【摘要】 特异性结合蛋白是多糖研究的关键工具,可服务于多糖的可视化分析、特异性检测、高通量分析等场景。通过动物免疫制备抗体是特异性结合蛋白获取的典型策略,然而多糖的免疫原性相较蛋白等生物大分子弱、抗体制备难度大,导致多糖特异性结合蛋白匮乏。本研究以微生物基因组中的糖结合域(Carbohydrate-binding module)序列为切入点,综合运用多种生物信息学策略高效挖掘潜在的多糖结合蛋白基因,以异源过量表达技术进行高效获取,以微阵列、生物膜干涉、亲和电泳等技术考察结合蛋白的特性。分别获得褐藻胶、卡拉胶、岩藻聚糖、透明质酸、香菇多糖等多糖的特异性结合蛋白。将特异性结合蛋白与荧光蛋白进行融合表达,并以荧光融合蛋白作为探针,成功实现了香菇中香菇多糖、海带中褐藻胶、红藻中卡拉胶、海参中岩藻聚糖的荧光显微观察。将结合蛋白与侧流免疫层析法及酶联免疫吸附法等连用,实现了透明质酸等多糖的快速鉴定及特异定量。上述研究丰富了多糖特异性结合蛋白的"工具箱",有助于多糖的进一步研究与开发。更多还原

【Abstract】 Specific binding proteins are key tools for polysaccharide investigations,and could serve in the visualization,specific detection,and high-throughput analysis of polysaccharides.The preparation of antibodies through animal immunization is a typical strategy for the acquisition of specific binding proteins.However,compared to proteins and other biomolecules,polysaccharides are often poor immunogens,andtheir antibodies are therefore difficult to prepare,which leads to the scarcity of polysaccharide-specific binding proteins.In this report,taking the carbohydrate-binding module in microbial genomes asthe entry point,based on various bioinformatics strategies,thepotential polysaccharide-binding protein genes were efficiently explored.The expressed proteins were efficiently obtained by heterologous overexpression techniques,and further characterized by microarray,bio-layer interferometry,and affinity gel electrophoresis assays.Theproteins exhibited the specific binding capacity to the targetpolysaccharides,e.g.,alginate,carrageenan,and sulfated fucan,hyaluronic acid,lentinan.The specific binding proteins were fused with a fluorescent protein,and the in situ visualizations of lentinan,alginate,carrageenan,and sulfated fucan in foods were realized employing the fluorescent fusion proteins as probes.Furthermore,the on-site identification and specific quantification of polysaccharides(e.g.,hyaluronic acid) were implemented by integrating the binding proteins into the lateral flow immunoassay and enzyme-linked immunosorbent assay respectively.This study enriches the " toolbox" of polysaccharide-specific binding proteins and would contribute to further investigations and development of polysaccharides.更多还原