食品与发酵工业 >

2022 , Vol. 48 >Issue 13: 163 - 172

DOI: https://doi.org/10.13995/j.cnki.11-1802/ts.031171

研究报告

牦牛酥油乳脂肪球膜蛋白分离纯化和蛋白组鉴定及功能分析

  • 谈婷 ,
  • 罗毅皓 ,
  • 孙万成
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  • (青海大学 农牧学院,青海 西宁,810016)
第一作者：硕士研究生(罗毅皓副教授为通信作者,E-mail:291649347@qq.com)

收稿日期: 2022-02-17

  修回日期: 2022-03-14

  网络出版日期: 2022-08-03

基金资助

国家自然科学基金项目(22167020)

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Isolation, purification, proteome identification and functional analysis of yak butter milk fat globular membrane protein

  • TAN Ting ,
  • LUO Yihao ,
  • SUN Wancheng
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  • (College of Agriculture and Animal Husbandry, Qinghai University, Xining 810016, China)

Received date: 2022-02-17

  Revised date: 2022-03-14

  Online published: 2022-08-03

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摘要

为探究牦牛酥油脂肪球膜(milk fat globule membrane,MFGM)蛋白纯化前后的组成及功能差异,通过表面活性剂聚乙二醇辛基苯基醚溶液和钠离子磷酸盐缓冲溶液提取牦牛酥油中MFGM蛋白,利用AKTA pure 25M蛋白质纯化系统结合阴离子交换层析柱DEAE-Sepharose FF(DEAE琼脂糖凝胶),对MFGM蛋白脂肪酸结合蛋白3(fatty acid binding protein 3,FABP 3)纯化工艺进行优化,得出最优条件:洗脱体积50 mL,缓冲液pH 7.4,洗脱速度1.6 mL/min,洗脱液浓度0.50 mol/L,该条件下,FABP 3得率达83.19 %。以牦牛酥油MFGM蛋白粗提物和纯化物为研究对象,通过十二烷基硫酸钠-聚丙烯酰胺凝胶电泳和液相色谱质谱联用技术,结合无标记相对定量分析,得出主要MFGM蛋白为FABP 3,共定量到31种MFGM蛋白,其中纯化MFGM蛋白和粗提MFGM蛋白差异蛋白20种,并通过基因本体功能注释、KEGG代谢通路分析对鉴定到的蛋白进行功能及作用的生物学途径探讨。

关键词： 牦牛酥油; 脂肪球膜蛋白; 分离纯化; 蛋白组成; 功能分析

本文引用格式

谈婷 , 罗毅皓 , 孙万成 . 牦牛酥油乳脂肪球膜蛋白分离纯化和蛋白组鉴定及功能分析[J]. 食品与发酵工业, 2022 , 48(13) : 163 -172 . DOI: 10.13995/j.cnki.11-1802/ts.031171

Abstract

In order to explore the protein composition and functional differences of milk fat globule membrane (MFGM) proteins, the MFGM proteins in yak butter were extracted with the surfactant polyethylene glycol octyl phenyl ether solution and sodium ion phosphate buffer solution. The purification process of MFGM proteins fatty acid binding protein 3 (FABP 3) was optimized by DEAE-Sepharose FF (DEAE agarose gel) using AKTA pure 25M protein purification system. The optimum conditions were as follows: elution volume was 50 mL and buffer pH was 7.4 with the eluent concentration of 0.50 mol/L; elution rate was 1.6 mL/min. Under above conditions, the yield of FABP 3 was 83.19%. The main MFGM protein was FABP 3, and a total of 31 MFGM proteins were identified with the method of SDS-PAGE and liquid chromatography-mass spectrometry combined with unlabeled relative quantitative analysis. There were 20 MFGM differential proteins could be purified and crude extracted. The biological pathways of function and action of the identified proteins were discussed by gene body functional annotation and KEGG metabolic pathway analysis.

Key words： yak butter; milk fat globular membrane protein; separation and purification; protein composition; functional analysis

参考文献

[1] 孙美青, 马莺, 程金菊. 牦牛酥油理化性质研究[J]. 中国乳品工业, 2016, 44(3):9-11, 19.
SUN M Q, MA Y, CHENG J J. Physical and chemical properties of yak butter[J]. China Dairy Industry, 2016, 44(3):9-11, 19.
[2] HEID H W, KEENAN T W. Intracellular origin and secretion of milk fat globules[J]. European Journal of Cell Biology, 2005, 84(2-3): 245-258.
[3] D’ALESSANDRO A, SCALONI A, ZOLLA L. Human milk proteins: An interactomics and updated functional overview[J]. Journal of Proteome Research, 2010, 9(7):3 339-3 373.
[4] FONG B Y, NORRIS C S, MACGIBBON A K H. Protein and lipid composition of bovine milk-fat-globule membrane[J]. International Dairy Journal, 2007, 17(4): 275-288.
[5] 曹春杰. 乳脂肪球膜蛋白的富集分离及定量分析研究[D]. 天津: 天津科技大学, 2020.
CAO C J. Enrichment and quantitative analysis of milk fat globule membrane[D]. Tianjin: Tianjin University of Science & Technology, 2020.
[6] 苏薇, 杨洁, 沈晓丽, 等. 驴乳中蛋白质组分的分离纯化与鉴定[J]. 食品科学, 2010, 31(23):44-48.
SU W, YANG J, SHEN X L, et al. Purification and identification of donkey’s milk protein fractions[J]. Food Science, 2010, 31(23):44-48.
[7] 马莺, 姬晓曦, 李琳. 一种牦牛乳乳脂肪球膜蛋白的分离方法: CN102863526A[P]. 2013-01-09.
MA Y, JI X X, LI L. A method for separation of milk fat globule protein from yak milk: CN102863526A [P]. 2013-01-09.
[8] 刘法辉, 陈红兵, 高金燕. 牛奶中β-酪蛋白和κ-酪蛋白的分离纯化[C]// 中国食品科学技术学会第六届年会暨第五届东西方食品业高层论坛. 北京: 中国食品科学技术学会, 2009: 108.
LIU F H, CHEN H B, GAO J Y. Isolation and purification of β -casein and κ -casein from milk [C]// Proceedings of the 6th Annual Conference of China Institute of Food Science and Technology and the 5th International Forum on Food Science and Technology. Beijing: Chinese Institute of Food Science and Technology, 2009: 108.
[9] 张冬, 易华西, 陈玉洁, 等. 离子交换树脂吸附脂肪球膜蛋白及其影响因素的研究[J]. 食品科技, 2019, 44(8):268-273.
ZHANG D, YI H X, CHEN Y J, et al. Research on adsorption of milk fat globule membrane protein by ion exchange resin[J]. Food Science and Technology, 2019, 44(8):268-273.
[10] 张尔贤, 林文华, 俞丽君, 等. 牛奶黄嘌呤氧化酶的纯化及性质研究[J]. 汕头大学学报(自然科学版), 1998, 13(1):25-31.
ZHANG E X, LIN W H, YU L J, et al. Milk xanthine oxidase: Purification and properties[J]. Journal of Shantou University (Natural Science Edition), 1998, 13(1):25-31.
[11] NGUYEN H T H, ONG L, HOQUE A, et al. A proteomic characterization shows differences in the milk fat globule membrane of buffalo and bovine milk[J]. Food Bioscience, 2017, 19:7-16.
[12] 李墨翰, 张秀敏, 宋婉莹, 等. 驴初乳与常乳乳脂球膜蛋白质组的对比分析(英文)[J]. 食品科学, 2022, 43(8):104-112.
LI M H, ZHANG X M, SONG W Y, et al. Comparative proteomic analysis of milk fat globule membrane proteins between donkey colostrum and mature milk[J]. Food Science, 2022, 43(8):104-112.
[13] 叶清, 杨梅, 梁肖娜, 等. 人初乳与牛初乳中乳脂肪球膜蛋白质组成的对比研究[J]. 乳业科学与技术, 2016, 39(5):13-18.
YE Q, YANG M, LIANG X N, et al. Comparative study on milk fat globule membrane protein composition of human and bovine milk[J]. Journal of Dairy Science and Technology, 2016, 39(5):13-18.
[14] 刘小帅. 内蒙古民族乳制品的概述及发展前景展望[J]. 消费导刊, 2008(18):199.
LIU X S. Overview and development prospect of Inner Mongolia national dairy products [J]. Consumer Guide, 2008(18): 199.
[15] 洪蕾, 杨丽杰, 张洋, 等. 精酥油的制作与脂肪酸组成分析[J]. 食品工业, 2013, 34(3):190-192.
HONG L, YANG L J, ZHANG Y, et al. The processing technology and fatty acid composition analysis of pure butter[J]. The Food Industry, 2013, 34(3):190-192.
[16] 柏韵, 李然, 张振, 等. 中国对虾虾壳制备几丁质工艺研究[J]. 食品工业科技, 2017, 38(9):174-180.
BAI Y, LI R, ZHANG Z, et al. Optimum technology of chitin preparation from Penaeus chinensis[J]. Science and Technology of Food Industry, 2017, 38(9):174-180.
[17] 孙琳. 去纯化标签的重组七鳃鳗rLj-RGD3(H~-)的中试纯化工艺研究[D]. 大连:辽宁师范大学, 2014.
SUN L. Pilot-scale purification of rLJ-RGD3 (H~-) from Lampreys lampreys rLJ-RGD3 (H~-) without label purification [D]. Dalian: Liaoning Normal University, 2014.
[18] BRIJESHA N, APARNA H S. Comprehensive characterization of bioactive peptides from buffalo (Bubalus bubalis) colostrum and milk fat globule membrane proteins[J]. Food Research International, 2017, 97:95-103.
[19] 王贤理, 蒙凯, 蔡其浩. 一种载脂蛋白e检测试剂: CN101799475A[P].
WANG X L, MENG K, CAI Q H. A novel apolipoprotein E detection reagent : CN101799475A[P].
[20] 李玮轩. 不同泌乳期驴乳与牛乳乳清和乳脂肪球膜蛋白质组学差异研究[D]. 沈阳:沈阳农业大学, 2020.
LI W X. Study on proteomics of whey and milk fat globule between donkey and cow in different lactation stages [D]. Shenyang: Shenyang Agricultural University, 2020.
[21] SHENG Q H, LI J C, ALAM M S, et al. Gross composition and nutrient profiles of Chinese yak (Maiwa) milk[J]. International Journal of Food Science & Technology, 2008, 43(3):568-572.
[22] 姬晓曦. 乳源乳脂肪球膜蛋白组学分析及其对细胞凋亡的作用机制[D]. 哈尔滨:哈尔滨工业大学, 2017.
JI X X. Proteomics analysis of milk adipose globule membrane from milk and its mechanism on cell apoptosis [D]. Harbin: Harbin Institute of Technology, 2017.
[23] LI H, XU W L, MA Y, et al. Separation and purification of the bovine milk fat globule membrane protein and its effect on improvement of C2C12mouse skeletal muscle cell proliferation[J]. New Journal of Chemistry, 2017, 41(14):6 530-6 539.
[24] 常花香. 不同泌乳期马乳乳蛋白和乳脂球膜蛋白差异蛋白组学的研究[D]. 乌鲁木齐: 新疆农业大学, 2021.
CHANG H X. Differential proteomics of protein and milk fat globular membrane in different lactation stages of horses[D]. Urumqi: Xinjiang Agricultural University, 2021.
[25] 蒋允昌, 冯虎, 高啸, 等. 膝关节骨性关节炎核心基因和关键通路的鉴别[J]. 徐州医科大学学报, 2020, 40(4):274-277.
JIANG Y C, FENG H, GAO X, et al. Identification of key genes and key pathways in knee osteoarthritis[J]. Journal of Xuzhou Medical University, 2020, 40(4):274-277.
[26] 陈彦同, 周明旺, 李盛华, 等. NONFH与脂质代谢紊乱[J]. 生命的化学, 2020, 40(4):542-547.
CHEN Y T, ZHOU M W, LI S H, et al. Advances in mechanism of lipid metabolism disorder in NONFH[J]. Chemistry of Life, 2020, 40(4):542-547.
[27] LI A C, BINDER C J, GUTIERREZ A, et al. Differential inhibition of macrophage foam-cell formation and atherosclerosis in mice by PPARalpha, beta/delta, and gamma[J]. The Journal of Clinical Investigation, 2004, 114(11):1564-1576.
[28] FINCK B N, KELLY D P. Peroxisome proliferator-activated receptor alpha (PPARalpha) signaling in the gene regulatory control of energy metabolism in the normal and diseased heart.[J]. Journal of Molecular & Cellular Cardiology, 2002, 34(10): 1 249-1 257.
[29] 杨智, 刘昭前. PPARγ在脂肪细胞分化和糖脂代谢中的作用[J]. 国际病理科学与临床杂志, 2008, 28(1):14-18.
YANG Z, LIU Z Q. Role of PPARγ in adipocyte differentiation and glucose and lipid metabolism[J]. International Journal of Pathology and Clinical Medicine, 2008, 28(1):14-18.
[30] 高文荣, 张麟, 余婷婷, 等. 解偶联蛋白-1(UCP1)的研究进展及其应用前景[J]. 生物过程, 2012, 2(3): 128-137.
GAO W R, ZHANG L, YU T T, et al. The research progress and application prospect of uncoupling protein 1 (UCP1)[J]. Bioprocess, 2012, 2(3):128-137.
[31] 王秋惠, 徐星科, 汪夏, 等. 黄连碱调节UCP1基因表达改善肥胖的分子机制研究[J]. 中国药学杂志, 2021, 56(21): 1 712-1 721.
WANG Q H, XU X K, WANG X, et al. Molecular mechanism of berberine regulating UCP 1 gene expression to improve obesity[J]. Chinese Journal of Pharmacy, 2021, 56(21): 1 712-1 721.
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