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中国农学通报

中国农学通报 ›› 2013, Vol. 29 ›› Issue (26): 19-24.doi: 10.11924/j.issn.1000-6850.2013-1350

所属专题： 生物技术； 畜牧兽医

• 畜牧 动物医学 蚕 蜂 • 上一篇    下一篇

感染新型鸭呼肠孤病毒的番鸭肝脏蛋白质组双向电泳方法的建立

黄梅清 朱果真 陈仕龙 郑敏 程晓霞 陈少莺   

  • 收稿日期:2013-05-13 修回日期:2013-07-11 出版日期:2013-09-15 发布日期:2013-09-15
  • 基金资助:
    村民关联度与农地利用的关系研究—以甘肃河西走廊为例;国家863项目;福建省农业科学院创新团队

Establishment of Two-dimensional Electrophoresis for Proteme of Liver from Muscovy Duck Experimentally Infected with Norvel Duck Reovirus

  • Received:2013-05-13 Revised:2013-07-11 Online:2013-09-15 Published:2013-09-15

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摘要： 为建立感染新型鸭呼肠孤病毒(NDRV)的番鸭肝脏蛋白质组双向电泳(2-DE)方法。本研究通过对人工感染NDRV的雏番鸭肝脏病变的观察，对肝脏蛋白质提取裂解液的配方、样品上样量、一向等电聚焦(IEF)参数等条件的对比和优化，建立了有效的番鸭肝脏2-DE方法。结果表明：采用攻毒后病变最典型的第5天的番鸭肝脏作为研究样品；采用研磨-超声-裂解液（7 mol/L尿素、2 mol/L硫脲、4% CHAPS、65 mmol/L DTT、40 mmol/L Tris-base、0.5% IPG buffer）法提取肝脏蛋白质，结合2D clean-up kit以及去拖尾DeStreak试剂纯化蛋白，按1100 μg上样，设置IEF参数为：50 V 12 h、200 V 1.5 h、500 V 1 h、1000 V 1 h、8000 V 3 h、8000 V 60000 V h、500 V维持，采用胶体考马斯亮蓝染色，能获得分辨率高、重复性好的2-DE图谱。应用建立的2-DE方法和PDQest 8.0分析软件，发现26个与正常番鸭肝脏蛋白表达水平超过3倍的差异蛋白点。该方法的建立为进一步寻找NDRV感染宿主组织相关蛋白以及水禽呼肠孤病毒差异蛋白质组学研究提供了技术支持。

关键词: 抑菌活性, 抑菌活性

Abstract: The aim was to establish an efficient Two-Dimensional Electrophoresis (2-DE) ptotocol for proteomic method of livers from muscovy ducks which were experimentally infected with Norvel Duck Reovirus (NDRV). The conditions which included the reagent of Lysis buffer, amount of loaded protein and parameters of Isoelectric Focusing (IEF) were compared and optimized. The pathological changes in livers were simultaneously observed. The results showed that the livers with characteristic pathology were chosen to be the samples, which were from muscovy ducks at the fifth day after inoculated with NDRV. The high-resolution and good-repeatability 2-DE images were obtained with 1100 μg of liver protein samples which were extracted by Lysis-buffer, purified by 2D clean-up kit, separated by 2-DE under suitable electrophoresis parameters, and stained with colloidal Coomassie brilliant blue G-250 solution. Analyzed with the software PDQest 8.0, 26 differentially expressed proteins spots were detected between the NDRV-NP03 infected group and the mock-infected group, of which the expression levels were more than 3 times in expression level. The established 2-DE method would pave the way for the further study on the host cell protein and differential proteomic analysis of waterfowl reovirus.