不同条件对鸭瘟病毒感染细胞蛋白质组二维电泳分析结果的影响  

Two-dimensional gel electrophoresis model for proteomic analysis of duck plague virus infected duck embryo fibroblasts

作　　者：贾仁勇[1] 程安春[1,2] 汪铭书[1,2] 郭宇飞[1,2] 徐超[1,2] 齐雪峰[1,2] 袁桂萍[1,2] 朱德康[1,2] 丁轲[1,2] 龚永强[1,2] 杨梅[1,2] 陈孝跃[1] 

机构地区：[1]四川农业大学动物科技学院禽病防治研究中心,四川雅安625014 [2]四川省动物疫病与人类健康重点实验室,四川雅安625014

出　　处：《中国兽医学报》2009年第3期272-276,共5页Chinese Journal of Veterinary Science

基　　金：国家自然科学基金资助项目(30771598);教育部"长江学者和创新团队发展计划"创新团队项目(PCSIRT0853);教育部"新世纪优秀人才支持计划"项目(NCET-06-0818);教育部高等学校科技创新工程重大项目培育资金资助项目(706050);四川省杰出青年基金项目(07ZQ026-132);四川省基础研究重大项目(07JY029-016/07JY029-017);四川省教育厅重大项目(07ZZ028/07ZA051);四川省重点建设学科基金资助项目(SZD0418)

摘　　要：本研究以鸭瘟病毒感染鸭胚成纤维细胞为材料,围绕影响二维电泳因素进行全面探讨,以建立和优化鸭瘟病毒感染细胞蛋白质组二维电泳模型。结果表明,样品经过冷丙酮处理,水化液DTT浓度为30 mmol/L都有利于等电聚焦;采用pH5～8 IPG窄胶条和混合两性载体电解质pH3～10/pH5～8为2/1比pH3～10 IPG宽胶条和单一两性载体电解质pH3～10分离蛋白时,各蛋白点间距较大,分辨率高,更有利于显示低丰度蛋白点;1.5 mg的蛋白上样量偏大,2～DE图像出现拖尾和水平条纹,部分相邻高丰度的蛋白重叠,且还掩盖了低丰度蛋白点。PDQuest7.40软件分析显示:17 cmpH5～8 IPG胶条电泳鸭瘟病毒感染细胞蛋白质组,银染可获得1 253个蛋白点,而考染却检测到388个蛋白点;重复试验仍获得清晰、稳定的2-DE图像,同一样本不同时期,考染可获得约348、331个蛋白点,蛋白点匹配率达88%,表明了鸭瘟病毒感染细胞蛋白质组二维电泳模型稳定、分辨率高、重复性好,为鸭瘟病毒蛋白组的进一步研究和新蛋白的发现提供了重要的研究方法。To establish and optimize two dimensional gel electrophoresis （2-DE） models for proteomic analysis of duck plague virus infecting duck embryo fibroblasts. Various protein extraction methods,Sample loading buffer strip range of IPG,sampling volume of protein, staining method and so on were compared and analyzed. Results： Protein extraction by acetone and reducing agent of 30 mmol/L DTT were easy to be focalized; Loading sampling buffer of mixturation with pH3-10 carrier ampholytes and pHS-8 carrier ampholytes at a 2 ： 1 ratio with the 17 cm IPG strip of pH 5-8 could improve the resolution. Protein sample of 0.8 mg was fit for Coomassie brilliant blue R-250 staining,while 1.5 mg was too high. It was found that 1 253 protein spots were obtained in 17 cm pH 5-8 gel strip by silver staining while around 388 were obtained by eoomassie brilliant blue staining. The spots of 348 and 331 protein were obtained in 17 cm pH 5-8 gel strip each different time in the sample with coomassie stains ,and the mean matc hing rate achieves to 88%. The findings showed that the better repetition, resolution and stability of 2-DE models will be a better method to supply further study of DPV proteomic,and served as an important method for discovering new proteins.

关 键 词：蛋白质组 二维电泳 鸭瘟病毒 成纤维细胞 

分 类 号：S858.32[农业科学—临床兽医学] S851.34[农业科学—兽医学]