原肌球蛋白(tropomyosin, TM)是凡纳滨对虾中最主要的过敏原,为获得高纯度TM,该文以凡纳滨对虾为原料,采用丙酮去脂、PBS粗提、热处理除杂、硫酸铵沉淀和柱层析法进行提取纯化虾肉中TM。经SDS-PAGE确定其分子质量,通过Western blot和液质联用进行鉴定,同时利用生物信息学方法分析TM的理化性质并预测其空间结构。结果表明采用此法纯化的TM纯度为98%,分子质量为36 kDa,且Western blot实验显示仅36 kDa处出现阳性反应。经生物信息学分析TM蛋白为亲水性热稳定蛋白,二级结构以α-螺旋为主,无复杂的空间结构。
Tropomyosin (TM) is the most important allergen in Litopenaeus vannamei. To obtain high-purity TM, L. vannamei was used as raw material. The extraction and purification procedures were performed using acetone, PBS, heat, ammonium sulfate precipitation and column chromatography. The molecular weight was determined by SDS-PAGE. The protein was identified by Western blot and LC-MS/MS. Meanwhile, bioinformatics methods were used to analyze the physical and chemical properties and predict the spatial structure of TM. The purity of TM using this extraction and purification method was 98%, and the molecular weight was 36 kDa. Only one positive band at 36 kDa was obtained in the Western blot assay. According to bioinformatics analysis, TM is a hydrophilic heat-stable protein, and α-helix is dominated in the secondary structure without complicated spatial structure.
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