健康奶牛与临床型乳腺炎奶牛乳腺线粒体蛋白组差异表达分析

Proteomic analysis of mitochondria of mammary tissue from healthy cows and clinical mastitic cows

运用亚细胞蛋白质组学的研究策略,分离纯化亚细胞结构再进行蛋白质组学研究,可提高低丰度蛋白在双向凝胶电泳中的检出率。通过对比分析乳腺炎奶牛乳腺与正常奶牛乳腺线粒体蛋白质组的表达变化,为奶牛乳腺炎的生物学治疗及抗病育种工作筛选出目基因和蛋白。超速离心法分离线粒体,双向凝胶电泳分离蛋白,PDQuest7.4软件分析差异蛋白斑点,高效液相色谱串联离子阱质谱鉴定差异蛋白。从奶牛乳腺线粒体蛋白2-DE图谱中筛选出17个差异表达的蛋白质斑点,质谱鉴定出17个差异表达蛋白(6个蛋白在奶牛乳腺炎发生过程中下调,8个上调,1个只在正常情况下表达,2个只在乳腺炎乳腺组织中表达)。筛选出的差异蛋白质涉及到细胞的能量代谢、蛋白质合成、mRNA的加工成熟及调亡调控等许多方面,表明奶牛乳腺炎发生时乳腺线粒体组织结构和代谢状态都发生了明显的变化。

The sub-cellular proteomic strategy can complement the separation power of two-dimensional electrophoresis,A method for sub-cellular fractionation is introduced before electrophoresis is conducted , and the more protein spots are displayed in 2-DE profiling,A comparative analysis proteomic profiling of mitochondria was performed between healthy mammary gland and which infected clinical mastitis, To find more valuable information involved in mastitis development in dairy cows, mitochondria could be isolated and purified with ultracentrifuge , Two-dimensional electrophoresis were applied to the separation of mitochondrial protein,imagines analysis for the selection differentially - expressed protein spots by PDQUest7.4 software,and ion trap mass spectrometry equipped with surveyor HPLC system for the identification of proteins. 18 protein spots from eleetrophoresis gels were selected as dif- ferentially-expressed protein for MS analysis which resulted in identification for 17 proteins. Among the 17 differen- tially-expressed proteins,8 shows a up-regulated expression and 6 shows a down-regulated expression, 1 protein only expressed in healthy mammary gland,2 proteins only expressed in mammary gland , which infected with clinical mastitis. These differentially-expressed proteins found in this work have a wide coverage of functions which are related to en- ergy metabolism, protein biosynthesis, pre -mRNA splicing and processing, apoptosis regulation. These results imply that mammary gland has experienced a fundamental change in structure and metabolism pattern in mastitie of cow.