【作者】 吴永平；

【导师】 周继勇；

【作者基本信息】 浙江大学 ， 预防兽医学， 2009， 博士

【摘要】 鸡传染性法氏囊病(Infectious bursal disease, IBD)是由传染性法氏囊病毒(Infectious bursal disease virus, IBDV)引起的一种高度接触性传染病,主要侵害雏鸡和青年鸡的法氏囊器官,破坏法氏囊中的B淋巴细胞,导致免疫抑制,使病鸡更易感其它疫病。为进一步了解病毒与宿主的相互关系,我们利用差异蛋白质组学方法分析了IBDV感染前后,鸡法氏囊组织蛋白表达的变化。(一)家禽免疫器官二维凝胶电泳技术的建立和优化。二维凝胶电泳技术(2-DE)是研究生物体内蛋白表达和功能强有力的工具,通过对蛋白提取物的分析,能快速了解细胞内发生的变化。目前尚无2-DE技术用于分析家禽法氏囊组织的报道。样品制备是二维凝胶电泳中的关键环节。组织中含有多种杂质,这些杂质可引起2-DE图谱中的横纹或纵纹。所以,在组织样品制备过程中,如何增大蛋白质的溶解度和获得尽可能多的蛋白质种类是2-DE中具有挑战性的问题,这个问题也最终决定了2-DE技术的成败。本实验中,我们优化了鸡法氏囊组织的蛋白质组样品制备中的一些关键环节,包括样品裂解液、样品抽提方法、IPG胶条。发现由7 M urea、2 M thiourea、2%(w/v) CHAPS、50 mM DTT、0.2% Bio-Lyte 3/10、1mM PMSF、20 U/ml DNase I、0.25 mg/ml RNase A组成的裂解液结合超声+振荡的抽提方法能产生高质量的2-DE图谱。最后我们将优化的蛋白质组学样品制备方法应用于鸡的脾脏和胸腺,也获得了高质量的二维电泳图谱。稳定、有效的禽法氏囊蛋白质组二维电泳样品制备技术为研究禽免疫器官的差异蛋白质组学(differential proteomics)奠定了基础。(二)宿主细胞在不同病毒感染时,会做出各种防御反应。为了从分子水平上分析病毒与宿主之间的相互作用,本研究应用二维电泳(2-DE)结合基质辅助激光解吸电离飞行时间质谱技术(MALDI-TOF-MS)鉴定的差异蛋白质组学方法,研究分析了IBDV感染鸡后12h、24h、48h、72h和96h五个不同时间点法氏囊组织蛋白的动态表达变化。二维凝胶的差异表达分析显示,鸡感染IBDV后,其法氏囊组织共有96个蛋白点出现了显著的差异表达。采用MALDI-TOF/TOF质谱法对96个差异表达蛋白点进行质谱鉴定,结果成功鉴定了65个表达差异显著的蛋白点,对应54种蛋白,包括12种表达上调蛋白和42种表达下调的蛋白。数据库搜索和对鉴定蛋白的功能分析表明,这些差异蛋白主要涉及细胞骨架网络、应激反应、大分子合成、能量代谢、泛素-蛋白酶体通路、信号传导通路及一些功能未知的蛋白的变化,这些差异蛋白可能通过不同途径参与了IBDV感染机体的过程并发挥着不同的功能。采用荧光实时PCR方法在mRNA水平上分析了部分差异蛋白相应基因的变化情况。此外,用Western blot方法在蛋白水平上对部分差异蛋白进行了验证,结果与2-DE试验结果基本相符。这些差异蛋白的鉴定为进一步揭示IBDV致病的分子机制提供了丰富的实验数据和有用线索,对发现和开发潜在的生物标记分子提供了重要参考。更多还原

【Abstract】 Infectious bursal disease virus (IBDV), a member of the genus Avibirnavirus within the family Birnaviridae, is the causative agent of infectious bursal disease (IBD). IBDV causes severe immunodeficiency in young chickens by destroying immature antibody-producing B lymphocytes in the bursa of Fabricius (BF), which is followed by bursal atrophy. This renders the animal increasingly susceptible to disease, and decreases the effectiveness of vaccination against infectious diseases. To characterize the host response to IBDV infection, the differential proteomes of bursa of chicken, with and without IBDV infection, were analyzde at different time with two-dimensional gel electrophoresis (2-DE) followed by MALDI-TOF/TOF identification.Optimized two-dimensional gel electrophoresis protocol for isolation of soluble proteins from poultry immune organs.2-DE is a potent method to study protein expression and function in living organisms. It allows a fast overview of changes in cell processes by analysis of the entire protein extracts. However there is little information about the use of 2-DE in avian organs. In this study,2-DE sample preparation methodologies including removal of interfering compounds, protein extraction methods and extraction buffers, were optimized using bursa of Fabricius (BF) of chickens as a model system, when dealing with poultry immune organs. An extraction protocol involving in the extraction buffer IV containing 7M urea,2M thiourea,2%(w/v) CHAPS,50mM DTT,0.2% Bio-Lyte 3/10, 1mM PMSF,20U/ml DNase I,0.25 mg/ml RNase A and combination of sonication and vortex, gave the best 2-DE quality. Compared to non-frozen IPG strips, after IEF, the frozen IPG strips did not result in significant difference in the 2-DE patterns. In addition, this optimized protocol was also successfully applied in 2-DE analysis of spleen and thymus in chicken. Theses data imply that the optimized protocol is a potential useful tool for comparative proteomics analysis of avian immune tissues.Comparative proteomic analysis of infectious bursal disease virus-infect bursal of chicken. In different viral infection, host cells will make a variety of defense response. To understand the interaction between the host and virus at the molecular level, the comparative proteomes of IBDV-infected and mock-infected bursal of chicken at five different time points were analyzed using two-dimensional gel electrophoresis (2-DE) followed by MALDI-TOF/TOF protein identification. The analyses of multiple 2-DE gels indicated that a total of 65 protein spots (corresponding to 54 altered proteins) differentially expressed during IBDV infection, including 12 up-regulated proteins and 42 down-regulated proteins. Database search and identification of functional protein analysis showed that these altered proteins mainly related to cytoskeleton network, stress response, macromolecular biosynthesis, energy metabolism ubi-quitin-proteosome pathway, signal trasduction. These proteins may be involved, through various pathwaym, in the IBDV infection and play different functions. This work effectively provides a wealth of experimental data and useful clues to further reveal the molecular mechanism of IBDV pathogenicity, and provides an important reference for the discovery and development of potential molecular biomarkers.更多还原