基于碳水化合物结合结构域的香菇多糖特异性荧光探针的构建与应用

Construction and Application of a Lentinan-Specific Fluorescent Probe Based on a Carbohydrate-binding Module

为构建得到香菇多糖特异性荧光探针以实现香菇多糖的原位可视化观察,本研究利用生物信息学技术从β-1,3-D-葡聚糖酶TlGluA中挖掘得到一个潜在具有香菇多糖结合能力的碳水化合物结合结构域,并对其进行了重组表达及分离纯化,重组蛋白命名为LBM,进一步利用微量滴定板法研究了LBM的结合特异性。结果表明LBM能够结合香菇多糖,但未表现出对大麦β-葡聚糖、凝结多糖或魔芋葡甘露聚糖的结合能力,这证实该蛋白对香菇多糖具有良好的结合特异性。此外,通过将LBM与绿色荧光蛋白EmGFP融合表达构建得到首个香菇多糖特异性荧光探针,命名为EmGFP-LBM,基于该探针实现了香菇多糖在香菇中的原位可视化观察。研究发现,香菇多糖不仅存在于菌丝细胞壁中,还以散在状态分布于菌丝细胞间隙与细胞内部。本研究成功构建得到首个香菇多糖特异性荧光探针,为香菇多糖的原位可视化观察提供了关键工具。

This study aimed to obtain a lentinan-specific fluorescent probe for the in situ visualization of lentinan.A carbohydrate-binding module with potential lentinan-binding capacity was discovered in aβ-1,3-D-glucanase by using the bioinformatics techniques,and further recombinantly expressed.The binding specificity of the expressed protein,which was named as LBM was determined by the microtiter plate assays.The results indicated that LBM displayed a desired specificity for lentinan.The protein exhibited positive binding signals to lentinan,while could not bind to several examined polysaccharides including barleyβ-glucan,curdlan,or konjac glucomannan.Furthermore,the first lentinan-specific fluorescent probe was successfully constructed by fusing LBM with a green fluorescent protein EmGFP,based on which the in situ visualization of lentinan in Lentinula edodes was realized.It was shown that lentinan was not only presented in the mycelial cell wall,but also amorphously distributed in the interstitial space and inside the cell.The construction of the EmGFP-LBM provided a promising tool for the in situ visualization of lentinan.