基于双向凝胶电泳/基质辅助激光解析离子化飞行时间质谱技术的腐蹄病奶牛血浆蛋白质组学轮廓分析

Plasma proteomics profile of cows with foot rot based on two-dimensional electrophoresis / matrix-assisted laser desorption / ionization time of flight mass spectrometry

目的利用双向凝胶电泳/基质辅助激光解析离子化飞行时间质谱(two-dimensional electrophoresis matrixassisted laser desorption/ionization time of flight mass spectrometry,2DE/MALDI-TOF-MS)技术,筛选、鉴定腐蹄病奶牛血浆差异表达蛋白,并进行血浆蛋白质组学轮廓分析。方法选取20头腐蹄病奶牛为实验组,20头健康奶牛为对照组,采血,分离血浆,2DE筛选差异表达蛋白点,MALDI-TOF-MS鉴定差异表达蛋白,并对其进行生物信息学分析(GO分析和KEGG pathway数据库)。根据2DE及MALDI-TOF-MS的实验结果,选取纤维蛋白原进行Western blot验证试验。结果患腐蹄病奶牛血浆中共筛选出63个差异蛋白点,其中表达上调蛋白点14个,表达下调蛋白点49个,选取33个点进行质谱分析,得到26个阳性结果,鉴定为11种蛋白,差异表达蛋白多与炎症反应过程相关。Western blot对差异表达蛋白的验证结果与2DE结果一致。结论奶牛腐蹄病使血浆蛋白的表达发生了变化,机体蛋白质组参与炎症反应、补体通路、凝血过程等多个机体生理和病理过程,为进一步探索奶牛腐蹄病发病机制提供理论依据。

Objective To screen and identify the differentially expressed proteins in plasma of cows with foot rot by twodimensional electrophoresis / matrix-assisted laser desorption / ionization time of flight mass spectrometry（2DE / MALDITOF-MS）and analyze the proteomics. Methods Blood samples were collected from twenty cows with foot rot, from which plasma was separated, screened for differentially expressed protein sites by 2DE, identified for differentially expression proteins by MALDI-TOF / TOF-MS, and analyzed for bioinformatics by GO analysis and DEGG pathway data bank. Based on the results of 2DE and MALDI-TOF-MS, fibrinogen was screened for verification test by Western blot. Results A total of 65 differentially expressed protein sites were screened in plasma, of which 14 were up-regulation protein sites while 49 were down-regulation protein sites. Thirty-three sites were selected for MS, of which 26 were positive and identified as 11 kinds of proteins. Most of differentially expressed proteins were involved in the inflammatory reactions. The verification result of differentially expressed proteins by Western blot was consistent with that by 2DE. Conclusion The foot rot in cows led to the change of proteins in plasma, and the proteome was involved in various physiological and pathological course such as inflammatory reactions, complement pathway and blood coagulation. It provided a theoretical basis for further study on the pathogenic mechanism of foot rot in cows.