新城疫病毒感染鸡肾脏组织蛋白质组学方法条件的优化

为研究新城疫病毒(NDV)与宿主组织细胞之间的相互作用关系,本研究以NDV疫苗(La Sota)接种的鸡肾脏组织为材料,对其差异蛋白的表达进行分析,通过条件的优化,建立了NDV感染鸡肾脏组织的双向凝胶电泳检测方法。采用ImageMaster2D Platinum version 6.0软件对电泳图谱分析后显示:24 cm IPG胶条(pH4～pH7)对应的凝胶中可检测到约1 400个蛋白点,蛋白点匹配率达90%,表明NDV感染鸡肾脏组织蛋白质组双向凝胶电泳模型稳定、分辨率高、重复性好,在对4个时间点对照和人工感染凝胶分析过程中发现差异蛋白点主要集中于接种后7 d,该样品的2-DE电泳图谱中共有29个差异表达明显的蛋白点,其中上调表达蛋白点有15个,下调表达蛋白点有14个,利用荧光定量PCR对其中4个差异蛋白在mRNA水平进行检测,结果与双向电泳结果一致,NDV接种鸡肾脏组织蛋白质组学方法的建立为NDV致病机理的研究提供有效的方法。

Optimization of proteomic method for chicken kidney tissues infected with Newcastle disease virus

To optimize the conditions of proteomic method in Newcastle disease virus(NDV) infected chickens,the kidney tissues from NDV vaccine La Sota strain infected chickens were collected for analysis of the differential protein expression.Under the optimized conditions of tissue sample treatment and pH range of isoelectro-focusing gel comparison,about 1,400 protein spots were visualized on 2-DE with 24 cm IPG strip(pH4 to pH7) and analyzed by the software ImageMaster2D Platinum version 6.0 for the match rate of protein spots between the NDV infected and the uninfected kidney tissue,which were up to 90% identity.The analysis of multiple 2-DE gels revealed that a total of 29 protein spots differentially expressed post 7 days with NDV infection,including 15 up-regulated and 14 down-regulated protein spots.Some of the differentially expressed proteins were further verified at mRNA level using real-time PCR technology.The optimized proteomic method was facilitated to further investigation of the interaction between NDV and its hosts.