雏鸡法氏囊蛋白质组学双向电泳技术的建立及其初步分析

Establishment of Two-dimensional Gel Electrophoresis for Proteomic of Bursa of Fabricius

为了建立并优化鸡法氏囊蛋白质组学的双向电泳技术体系,以不同日龄雏鸡的法氏囊组织为研究对象,用固相pH梯度胶条进行等电聚焦、SDS-PAGE垂直电泳,采用不同的样品制备方法,对上样量、水化、等电聚焦、胶条平衡和凝胶染色方法等进行一系列优化,并应用PDQuest8.0.1软件对图谱进行初步分析。结果显示法氏囊组织在pH 5~8范围1、7 cm的2-DE胶上可以得到很好的分离,胶体考染后经PDQuest软件分析,在正常法氏囊组织可检测到800个以上蛋白点,不同2-DE图谱间蛋白点平均匹配率为83.5%,不同日龄雏鸡法氏囊存在有明显表达差异的蛋白质点37个,其中表达上调蛋白点17个,表达下调蛋白点11个,新增蛋白点5个,消失蛋白点4个。试验建立的鸡法氏囊组织蛋白质组双向电泳技术为法氏囊发育进化及其免疫功能的研究提供了新技术和方法。

To establish a method of two-dimensional electrophoresis （2-DE） in good repetition and high resolution for proteomic analysis of bursa of Fabricius, the bursa of Fabrieius from specific pathogen free chickens at different days old were taken to analyses by 2-DE. The first-dimension- al electrophoresis was immobilized pH gradients （IPG） strips isoeleetric focusing and second-di- mensional electrophoresis was vertical electrophoresis of SDS-PAGE. The conditions for sample preparation, rehydration, isoelectric focusing, equilibration, staining and other steps were modi- fied. The results were analyzed with the software PDQuest8. 0.1. More than 800 spots on 17 cm IPG strip （pH 5-8） for normal bursa of Fabricius tissue were detected with this method, and the match rate of protein spots was 83. 5%. Comparative analysis of bursa of Fabricius at different ages in multiple 2-DE gels revealed 37 differentially expressed protein spots, including 17 up-reg- ulated protein spots,ll down-regulated protein spots,5 new emerging protein spots and 4 disap- pear protein spots. The method of 2-DE for proteomic of bursa of Fabricius has been established. It would provide a basis for further elucidation of development and immune function of bursa of Fabrieius.