評價與改良牛乳品質(zhì)的研究最終報告

王建武 隋順超 王慧敏

摘 要：該研究利用蛋白質(zhì)組學(xué)的方法對中國荷斯坦奶牛、美國荷斯坦奶牛、娟姍牛和中國水牛的牛奶蛋白質(zhì)進(jìn)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質(zhì)及乳脂肪球膜蛋白質(zhì)進(jìn)行了細(xì)致的研究，建立牛乳清蛋白差異表達(dá)譜、乳脂肪球膜差異蛋白質(zhì)表達(dá)譜，挖掘與乳品質(zhì)改良相關(guān)的功能性蛋白，揭示不同奶牛品種乳蛋白質(zhì)合成、分泌信號傳導(dǎo)通路的差異，為牛乳品質(zhì)的改良提供素材，同時通過牛乳蛋白質(zhì)組技術(shù)平臺和牛乳蛋白質(zhì)數(shù)據(jù)庫的建立，進(jìn)行轉(zhuǎn)基因牛奶蛋白質(zhì)表達(dá)差異的食品安全研究。在4種牛乳初乳和常乳的乳清及乳脂肪球膜蛋白質(zhì)中，利用高效液相色譜和質(zhì)譜聯(lián)用的方法，鑒定出1148種蛋白質(zhì)；將娟珊牛與水牛中表達(dá)的全部蛋白質(zhì)分別與荷斯坦牛進(jìn)行比較，在水牛中特異表達(dá)的蛋白質(zhì)有132種，在娟珊牛中特異表達(dá)的蛋白質(zhì)有131種。對這些特異表達(dá)的蛋白質(zhì)結(jié)合4種牛泌乳特性和牛奶的特性進(jìn)行分析，發(fā)現(xiàn)了大量與乳脂肪合成分泌相關(guān)的蛋白質(zhì)。采用同位素標(biāo)記相對和絕對定量（iTRAQ）定量蛋白質(zhì)組學(xué)質(zhì)譜技術(shù)對這些蛋白質(zhì)進(jìn)行表達(dá)量的檢測。對中國荷斯坦牛、美國荷斯坦牛、娟珊牛和水牛中初乳乳清和常乳乳清中168種具有定量結(jié)果的蛋白進(jìn)行聚類及主成分分析，并根據(jù)聚類及主成分分析比較美國荷斯坦牛、娟珊牛及水牛與中國荷斯坦牛蛋白表達(dá)差異之間的相關(guān)性。在乳脂肪球膜蛋白質(zhì)iTRAQ實驗中，在8種乳脂肪球膜蛋白質(zhì)樣品中共有710種蛋白質(zhì)具有定量的結(jié)果。我們以中國荷斯坦牛牛乳蛋白質(zhì)相對表達(dá)量為本底，其他3種牛奶蛋白質(zhì)相對含量與其比較。通過對差異表達(dá)蛋白的鑒定與功能分析我們找到了多個與脂代謝與炎癥反應(yīng)及其他疾病相關(guān)的蛋白，這些蛋白可以作為候選基因轉(zhuǎn)入牛乳中，提高牛奶品質(zhì)或增強(qiáng)奶?？共⌒?，從而能夠形成高品質(zhì)牛奶生產(chǎn)種群。經(jīng)建立了從樣品收集、前期處理、高效液相色譜與質(zhì)譜聯(lián)用（LC-MS/MS）分析到后期樣品質(zhì)譜數(shù)據(jù)的分析處理等一整套全面系統(tǒng)的對乳制品進(jìn)行蛋白質(zhì)組學(xué)分析的流程。通過已經(jīng)建立的乳蛋白質(zhì)組學(xué)技術(shù)平臺，我們還完成了對一種克隆牛奶、3種轉(zhuǎn)基因克隆牛奶的乳清蛋白質(zhì)表達(dá)譜的分析，為轉(zhuǎn)基因牛乳食品安全評價提供了科學(xué)數(shù)據(jù)，也為轉(zhuǎn)基因生物安全評價提供了技術(shù)分析手段。

關(guān)鍵詞：乳清蛋白差異表達(dá)譜 乳脂肪球膜差異蛋白質(zhì)表達(dá)譜 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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