副猪嗜血杆菌菌体蛋白的提取及蛋白质双向电泳方法建立

Establishment of two-dimensional electrophoresis assay for proteome analysis of Haemophilus parasuis

为了建立副猪嗜血杆菌菌体蛋白的双向电泳方法,获得背景清晰、蛋白分辨率高的双向电泳图,对细菌裂解方法、蛋白上样量、IPG胶条选择等关键步骤进行优化。结果显示,采用超声(9.9 s/停顿9.9 s,200 W)冰浴破碎细菌,5 min后,裂解液溶解沉淀,4℃过夜裂解提取菌体蛋白,以0.6 mg菌体蛋白上在p H值3~10非线性IPG胶条上进行电泳,最后考马斯亮蓝G250染色,获得的蛋白质点清晰,效果最好,随机挑取14个蛋白点进行质谱鉴定,证实均为副猪嗜血杆菌菌体蛋白。

To establish a two - dimensional electrophoresis （2 - DE） assay for proteome analysis of Haemophilus parasuis and to get an electrophoretogram with a clear background, high protein point resolution and good repeatability, 2 - DE program was optimized, and various sample preparation methods, different loading quantities and IPG strip selection were studied. Samples were ultra - sounded with ice bath in 5 minute （9.9 s/every 9.9 s） followed by deposited at 4℃ at 24 hours. 0.6 mg total protein was loaded onto pH3 - 10 nolinear IPG strip for 2 - DE followed by staining with Coomassie Brilliant Blue G - 250 nitrate. 14 proteins were randomly chosen to identify by mass spectrum, and detection rate was 100%.