马立克氏病病毒超强毒感染鸡羽髓蛋白质组分析

Proteomics analysis of feather pulp from chickens infected with very virulent strain of Marek's disease virus

【目的】羽毛是细胞游离马立克氏病病毒(Marek's disease virus,MDV)释放的部位,为了解感染MDV后鸡羽中宿主基因表达的变化及对病毒感染的应答,进行了MDV感染鸡的羽髓蛋白质组学分析。【方法】1日龄无特定病原体(specific pathogen free,SPF)鸡人工感染MDV超强毒RB1B株(1000PFU),感染后21d采集鸡羽毛,提取羽髓蛋白,以17cm,pH5-8的IPG胶条进行二维电泳,以未感染病毒的SPF鸡羽髓蛋白为对照,使用PDQuest软件对二维电泳图谱进行差异蛋白分析,并选取部分差异斑点进行质谱鉴定。【结果】PDQuest软件分析发现攻毒组和对照组表达差异大于两倍的蛋白点有41个,其中攻毒组表达上调的蛋白点25个,下调的蛋白点7个,新出现的蛋白点有9个。质谱分析共成功鉴定了21个斑点,对应于20个蛋白。如载脂蛋白AI(apolipoprotein AI)、14-3-3 sigma(两个斑点均为该蛋白)、癌蛋白18(stathmin)等。【结论】功能预测表明这些蛋白涉及到宿主的抗病毒应答、物质代谢、细胞骨架成分、细胞增殖相关等方面。

[Objective] Feather follicle epithelium（FFE） and feather of chicken are sites that produce and release enveloped infectious Marek＇s disease virus（MDV）.In order to investigate host responses,the feather pulp from chickens infected with MDV was analyzed by proteomics.[Methods] Forty-eight one-day old specific pathogen free（SPF） chickens were randomly divided into two groups.One group of birds（n=24） were inoculated intraperitoneally with 1000 plaque-forming unit（PFU） of the RB1B strain of MDV,the rest（n=24） kept as uninfected control.Feather pulp were extracted from feather tips collected from chickens of infected and control group at 21 days post infected（dpi）,and dissolved in two dimensional electrophoresis（2-DE） sample buffer.The soluble proteins were separated by 2-DE,6 images（2 groups×3 images） of 2-DE were used to analyze the differentially expressed proteins with PDQuest 8.0.1.Some of spots changed significantly were further analyzed by mass spectrometry.[Results] 41 spots,which expression level changed above two fold,were detected.Among of them,25 of these spots were up-regulated,7 spots down-regulated,9 spots newly induced expression in group infected with MDV.21 spots,corresponding to 20 proteins,were successfully identified by mass spectrometry.These differential expressed proteins are apolipoprotein A I,14-3-3 sigma（two spots are the same protein）,stathmin,and so on.[Conclusion] Bioinformatics analysis indicates these differential proteins are mainly associated with host response,metabolism,cytoskeleton,and cell proliferation.