Bacillus mucilaginosus荚膜多糖脱除与全蛋白双向电泳体系的建立

Dissolution of Expolysaccharide and Two-Dimensional Electrophoresis Construction of Bacillus mucilaginosus

为了建立胶质芽孢杆菌全蛋白的双向电泳技术体系,分别比较了荚膜多糖去除方法,不同菌体蛋白提取方法,不同类型IPG胶条以及不同等电聚焦程序对双向电泳图谱的影响。结果表明,预先使用低浓度的盐酸多次处理能够完全去除荚膜多糖,然后采用TCA-丙酮与酚抽提结合法提取胶质芽孢杆菌全蛋白的效果最佳。采用17 cm pH 4~7的线性IPG胶条,在程序Ⅱ下获得了背景清晰,分辨率较高,且无明显横纵条纹的胶质芽孢杆菌全蛋白双向电泳图谱。采用以上条件,所得图谱约分离得到700个蛋白质点,该双向电泳图谱的构建为胶质芽孢杆菌双向电泳研究奠定了基础。

In order to establish two-dimensional electrophoresis（2-DE） technology system of total proteins of Bacillus mucilaginosus,2-DE maps obtained from different microbial expolysaccharide dissolution methods,protein extraction methods,pH ranges of IPG strips and isoelectric focusing（IEF） programs were compared. The results showed that hydrochloric acid solution could dissolute expolysaccharide effectively,and phenol extraction protocol combined with TCA/acetone was more beneficial to total protein extraction from Bacillus mucilaginosus. The 2-DE maps without obvious vertical and horizontal stripes,with low background,high resolution and good reproducibility were obtained using the following optimized parameters：17 cm IPG strip with pH 4~7,program Ⅱ（100 V30 min,1000 V 3 h,8000 V 5 h,8000 V 60000 Vh,500 V 20 h）. Analyzed by the PDQuest software,about 700 protein spots were isolated from the 2-DE protein map. The construction of 2-DE technology system will make a major contribution to the further study of B. mucilaginosus.