蜡样芽孢杆菌总蛋白双向电泳方法的建立和优化

Establishment and optimization of the two-dimensional gel electrophoresis for total protein in Bacillus cereus

目的探索建立有效的蜡样芽孢杆菌(Bacillus cereus)蛋白质组双向电泳体系,为进一步揭示其促进氧化葡萄糖酸杆菌(Gluconobacter oxydans)产酸的作用机制奠定基础。方法以培养的B.cereus为材料,比较蛋白质制备超声破壁时间、新型细胞裂解液、pH梯度和不同上样量对B.cereus蛋白双向电泳结果的影响。结果与结论采用15 min超声破壁提取B.cereus总蛋白,选用新型蛋白质裂解,用长24 cm、pH 4～7的IPG胶条,采用80μg上样量进行等电聚焦,于60 V 15 min、120 V 6 h条件下进行SDS-PAGE垂直电泳,可以获得背景清晰、重复性好的双向电泳图谱,建立一套用于B.cereus蛋白质组分析的双向电泳方法。

Objective To establish a two-dimensional gel electrophoresis （2-DE） of proteome in Bacillus cereus and to explore how B. cereus stimulates the growth of Gluconobacter oxydans to produce 2-keto-L-gulonic acid （2-KLG） after ente- ring the stationary phase. Methods Different parameters, including protein preparation by different ultrasonic broken time, new type lysis, pH gradient and different sample of B. cereus protein, were used to evaluate the effect of 2-DE of pro- teins in B. cereus. Results and Conclusion The clear background and reproducibility of 2-DE are established under condi- tions of 15 minutes of ultrasonic broken extraction, selection of protein cleavage I , pH 4-7,24 cm 1PG slrips, 80 μg load- ing volume, and isoelectric focus at 60 V 15 min, and 120 V 6 h.