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

作　　者：谢宇舟[1,2] 李军[1,2] 冯世文[1,2] 彭昊[1,2] 潘艳[1,2] 禤雄标[1,2] 陈泽祥 许力干[1,2] 杨威[1,2] 

机构地区：[1]广西兽医研究所,广西南宁530001 [2]广西畜禽疫苗新技术重点实验室,广西南宁530001

出　　处：《中国兽医杂志》2017年第6期46-49,共4页Chinese Journal of Veterinary Medicine

基　　金：广西科技攻关项目(桂科攻0993009-1);广西基本科研业务费专项(桂科专项13-2);广西畜禽疫苗新技术重点实验室专项(13-051-27-A-4)

摘　　要：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%.

关 键 词：副猪嗜血杆菌 双向电泳 菌体蛋白 蛋白质组学 

分 类 号：S852.651[农业科学—基础兽医学]