【作者】 杨龙蛟；

【导师】 夏成；

【作者基本信息】 黑龙江八一农垦大学 ， 临床兽医学， 2010， 硕士

【摘要】 背景:乳热(milk fever, MF)是以奶牛产犊前后低钙血症(hypocalcaemia)、瘫痪甚至昏迷为主要特征的一种钙代谢紊乱性疾病。它是奶牛围产期的主要疾病之一,我国每年因其而导致的直接经济损失为5869.8万元人民币,极大的影响了奶牛业的健康发展。鉴于奶牛乳热病因复杂,发生机制尚有不明之处。因此,本实验开展乳热奶牛血浆比较蛋白组学研究,为今后深入探讨奶牛乳热发生机制提供新的依据。目的:应用荧光差异双向凝胶电泳(two-dimensional fluorescence-based difference gel electrophoresis,2D-DIGE)和质谱分析技术分离和鉴定乳热奶牛与健康奶牛之间的血浆差异表达蛋白。并验证所鉴定出的差异蛋白质,为寻找与奶牛乳热发生发展相关的蛋白提供新的线索。方法:采集乳热奶牛与健康奶牛血浆各8份,分别作为实验组与对照组,同组等量混合,用AurumTM Serum Protein Mini Kit试剂盒去除血浆中白蛋白和IgG等高丰度蛋白,再用Pd-10除盐试剂盒进一步过滤,再对处理后的混合血浆样品分别进行荧光差异凝胶双向电泳,用DeCyDer差异分析软件对差异蛋白点进行确认,然后用4800 MALDI-TOF/TOF Analyzer进行质谱分析获得肽质量指纹图谱,而后用NCBI数据库检索蛋白质,再应用Western-blotting验证差异蛋白。结果:获得了重复性好的乳热和健康奶牛的血浆蛋白2D-DIGE图谱,用DeCyDer分析软件比对实验组与对照组电泳图,筛选出8个表达量显著改变的蛋白点,经MALDI-TOF/TOF质谱分析获得8个蛋白点的质谱信息,用NCBI数据库检索鉴定8个蛋白点为5种蛋白,其中3个点为Serum albumin表达下调,2个点为serpin peptidase inhibitor表达上调,其余3个点分别为fibrinogen beta chain表达下调、endopin 2B表达上调和Ig gamma-2 chain C region表达下调。并应用Western blotting初步证实患乳热奶牛血浆serpin peptidase inhibitor表达呈上调趋势,但是在围产期其他时间点当血钙降低或正常时血浆serpin peptidase inhibitor表达呈现或上调或下调的变化。结论:本实验首次应用荧光标记差异凝胶电泳技术、质谱技术和生物信息学技术分离和鉴定了与奶牛乳热相关的5种血浆差异表达蛋白及其表达状况,并初步证实血浆serpin peptidase inhibitor与奶牛乳热的关系。然而,今后尚需进一步证实5种差异表达蛋白与乳热和血钙之间的关系,为阐明奶牛乳热发生机制和判定病情与防治效果提供了新的方向。更多还原

【Abstract】 Background:Milk fever is a metabolic diseas which is mainly characteristic of hypocalcaemia, paresis, and even coma. It is one of the major metabolic disorders in dairy cows during transition period, whichi can cause a direct loss of 58.698 million RMB per year in China, have a tremendous affluence on health development of dairy industry. In view of complicated etiology and unclear mechanism of milk fever in dairy cows, therefore plasma comparative proteomics of milk fever was administrated in this experiment to offer a new theoretical basis on mechanism of milk fever in dairy cows in future.Objective:To find differentially expressed plasma proteins of dairy cows with milk fever, and offer a new path on studying mechanism of milk fever in dairy cows.Methods : A comparative proteomics approach, fluorescence two-dimensional differential in-gel electrophoresis (2D DIGE) was used to compare the change in plasma protein between 8 cows with milk fever and 8 cows with health control. Protein spots expression levels showed statisitically significant (P < 0.05) and AR > 1.5 on every gel were selected by DeCyder differential in-gel analysis (DIA) soft ware and one-way analysis of variance (ANOVA). Peptide mass fingerprinting was used by matrix-assisted laser desorption/ionization– time of flight (MALDI-TOF) mass spectrometry (MS) to identify all difference protein spots. One identified protein was validated by western blot analysis.Results: A well repetitiveness plasma 2D-DIGE spectrogram profile was obtained from dairy cows with or without milk fever. There were 8 protein spots consistent with statistical requests in Cy3 and Cy5 labeled plasma samples between milk fever cows and control cows by DIA soft ware. Five proteined were identified as downregulation of serum albumin for 3 protein spots, fibrinogen beta chain and Ig gamma-2 chain C region for 1 protein spot, upregulation of serpin peptidase inhibitor for 2 protein spots, endopin 2B for 1 protein spot in milk fever cows using MALDI-TOF/MS. Meanwhile, upregulation of plasma serpin peptidase inhibitor was validated by western blot in milk fever cows and health cows, but upregulation or downregulation of plasma serpin peptidase inhibitor all occurred as plasma Ca was low and normal in periparturient cows.Conclusions: Five plasma differential expression proteins were isolated and identified using methods of 2D-DIGE and MALDI-TOF/MS, relationship between serpin peptidase inhibitor and Ca in plasma was confirmed initially by western blot on dairy cows with or without milk fever. However, It need still further research on relationship between five plasma differential proteins and plasma Ca during transiton period, offer a new strategy to interpret mechanism of milk fever amd to assess state of an illness and effect of prevention and cure in future.更多还原