评价与改良牛乳品质的研究最终报告

王建武 隋顺超 王慧敏

摘 要：该研究利用蛋白质组学的方法对中国荷斯坦奶牛、美国荷斯坦奶牛、娟姗牛和中国水牛的牛奶蛋白质进行了定性及定量的研究，其中包括初乳和常乳的牛奶乳清蛋白质及乳脂肪球膜蛋白质进行了细致的研究，建立牛乳清蛋白差异表达谱、乳脂肪球膜差异蛋白质表达谱，挖掘与乳品质改良相关的功能性蛋白，揭示不同奶牛品种乳蛋白质合成、分泌信号传导通路的差异，为牛乳品质的改良提供素材，同时通过牛乳蛋白质组技术平台和牛乳蛋白质数据库的建立，进行转基因牛奶蛋白质表达差异的食品安全研究。在4种牛乳初乳和常乳的乳清及乳脂肪球膜蛋白质中，利用高效液相色谱和质谱联用的方法，鉴定出1148种蛋白质；将娟珊牛与水牛中表达的全部蛋白质分别与荷斯坦牛进行比较，在水牛中特异表达的蛋白质有132种，在娟珊牛中特异表达的蛋白质有131种。对这些特异表达的蛋白质结合4种牛泌乳特性和牛奶的特性进行分析，发现了大量与乳脂肪合成分泌相关的蛋白质。采用同位素标记相对和绝对定量（iTRAQ）定量蛋白质组学质谱技术对这些蛋白质进行表达量的检测。对中国荷斯坦牛、美国荷斯坦牛、娟珊牛和水牛中初乳乳清和常乳乳清中168种具有定量结果的蛋白进行聚类及主成分分析，并根据聚类及主成分分析比较美国荷斯坦牛、娟珊牛及水牛与中国荷斯坦牛蛋白表达差异之间的相关性。在乳脂肪球膜蛋白质iTRAQ实验中，在8种乳脂肪球膜蛋白质样品中共有710种蛋白质具有定量的结果。我们以中国荷斯坦牛牛乳蛋白质相对表达量为本底，其他3种牛奶蛋白质相对含量与其比较。通过对差异表达蛋白的鉴定与功能分析我们找到了多个与脂代谢与炎症反应及其他疾病相关的蛋白，这些蛋白可以作为候选基因转入牛乳中，提高牛奶品质或增强奶牛抗病性，从而能够形成高品质牛奶生产种群。经建立了从样品收集、前期处理、高效液相色谱与质谱联用（LC-MS/MS）分析到后期样品质谱数据的分析处理等一整套全面系统的对乳制品进行蛋白质组学分析的流程。通过已经建立的乳蛋白质组学技术平台，我们还完成了对一种克隆牛奶、3种转基因克隆牛奶的乳清蛋白质表达谱的分析，为转基因牛乳食品安全评价提供了科学数据，也为转基因生物安全评价提供了技术分析手段。

关键词：乳清蛋白差异表达谱 乳脂肪球膜差异蛋白质表达谱 功能性蛋白

Abstract：Proteomic approaches has been used for conducting series qualitative and quantitative studies in the milk protein of Chinese Holstein cows，U.S.Holstein cows， Jersey cattle and Chinese Buffalo's milk proteins， including the structural research on whey protein and fat globule membrane protein in both colostrum and regular milk， the establishment of differential expression profiles on whey protein and fat globule membrane protein.We also detected functional proteins that have the potential character to improve the milk quality， revealed the different protein synthesis and secretion signaling pathways on different dairy cows. Evaluation studies about the safety of milk from GM dairy cattle have also been carried out by milk proteome technology and the construction of milk protein database.1，148 kinds of proteins has been identified in the whey and fat globule membrane proteins using high-performance liquid chromatography combined with mass spectrometry methods. Comparing all the proteins that expressed in Jerseys Cattle or Chinese buffalo with the Holstein cattle， there are 132 kinds of proteins that are expressed in Chinese buffalo， while 131 different proteins are expressed in Jerseys Cattle. We found out a large number of proteins related to the synthesis and secretion of milk fatWe also detected the expression level of these proteins by isotope labeling the relative and absolute quantification （iTRAQ） quantitative proteomics mass spectrometry. We did clustering and principal component analysis on 168 different kinds of proteins that are quantitatively determined in colostrum and regular whey milk， and further found the correlation of expression differences between U.S.Holsteins，Jerseys or Chinese Buffalo with Chinese Holstein cattle. A total number of 710 kinds of protein in 8 samples of milk fat globule membrane proteins are given the quantitative results using iTRAQ experiment. A set of comprehensive proteomic analysis for dairy products have been established， including sample collecting，pre-processing，high performance liquid chromatography （HPLC） combined mass spectrometry （LC-MS/MS） analysis and post-sample data processing procedure.We also completed the whey protein expression profile analysis for the milk of one cloned cattle， three kinds of transgenic cloned milk.These analyses will provide the scientific data to the safety evaluation of GM milk products， provides the technical tools to the safety evaluation for GM animals.

Key Words：Whey Protein Differential Expression Profiles；Fat Globule Membrane Protein Differential expression Profiles；Functional Proteins

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