分子营养 Molecular Nutrition

奶牛不同乳腺健康状态下乳清蛋白的差异性表达研究

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  • 1. 奶牛营养学北京市重点实验室, 北京农学院动物科学技术学院, 北京 102206;
    2. 中国农业科学院北京畜牧兽医研究所, 北京 100193
汪悦(1993-),女,青海西宁人,硕士研究生,研究方向为动物营养与免疫。E-mail:wangyue9313@163.com

收稿日期: 2018-07-12

  网络出版日期: 2019-02-18

基金资助

十三五国家重大科技专项(2016YFDO700201);北京市农业局北京市现代农业产业技术体系奶牛创新团队

Differentially Expression of Whey Protein in Different Mammary Glands of Dairy Cows

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  • 1. Key Laboratory of Dairy Nutrition in Beijing, College of Animal Science and Technology, Beijing University of Agriculture, Beijing 102206, China;
    2. Beijing Institute of Animal Science and Veterinary Medicine, Chinese Academy of Agricultural Sciences, Beijing 100193, China

Received date: 2018-07-12

  Online published: 2019-02-18

摘要

本试验旨在通过研究奶牛不同乳腺健康状态下乳清蛋白差异性表达,探究奶牛乳腺健康状况对乳清蛋白的影响以及病原微生物蛋白诱发炎症的分子机制,为诊断奶牛乳腺健康程度以及早期炎症的发生提供潜在生物标记物。选择102头荷斯坦奶牛[胎次2~3胎、泌乳天数(152±27)d、产奶量(27±3)kg/d]进行牛奶样本采集,根据乳体细胞数(SCC)及细菌学鉴定结果将牛奶样本分为6组,分别为细菌培养阳性组(传染型、环境型和机会型)和细菌培养阴性组[培养阴性-低SCC组(SCC<100 000个/mL)、培养阴性-中等SCC组(SCC为100 000~400 000个/mL)和培养阴性-高SCC组(SCC>400 000个/mL)]。采用十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)与非标记定量蛋白质组学等技术对6组乳清样本中的蛋白质功能及表达水平进行鉴定。结果表明:1)奶牛健康乳腺与炎症乳腺之间的差异主要是由蛋白质的表达水平造成的。2)试验总共鉴定出272种蛋白质,其中有58种蛋白质显示出显著调节变化。与细菌培养阴性乳清样本相比,细菌培养阳性乳清样本中有20种蛋白质表达显著上调(P<0.05),其中导管素-4(CATHL4)、导管素-3(CATHL3)、导管素-2(CATHL2)、α-球蛋白抑制因子H4(ITIH4)、丝氨酸蛋白酶抑制剂A3-1(SERPINA3-1)、前列腺素H-2 D异构酶(PTGDS)、血清淀粉样蛋白A3(SAA3)和免疫球蛋白κv3-20(IGKV3-20)在培养阳性乳清样本中的表达量超过培养阴性乳清样本的约8倍,且上调蛋白质的功能多数与特异性免疫反应有关。显著或极显著下调的蛋白质有38种(P<0.05或P<0.01),其中血小板糖蛋白4(CD36)、补体蛋白CD59(CD59)、κ酪蛋白(CSN3)、嗜乳脂蛋白亚家族1成员A1(BTN1A1)、ATP结合盒转运蛋白G2(ABCG2)、围脂滴蛋白2(PLIN2)、包膜糖蛋白2(GP2)、聚泛素-C(UBC)和异柠檬酸脱氢酶1(IDH1)呈现极显著下调(P<0.01),BTN1A1与脂质合成和分泌有关,PLIN2和GP2与转运有关,ABCG2具有酶活性,CD59与免疫系统有关。3)在细菌培养阳性乳清样本中鉴定到21种菌体蛋白,这些菌体蛋白的存在既验证了细菌培养鉴定结果,又从菌体蛋白特性和功能角度阐明了病原微生物诱发炎症的分子机制。综上所述,通过对奶牛不同乳腺炎症状态下乳蛋白差异表达研究,根据不同炎症类型奶样中乳蛋白质及菌体蛋白的表达变化及功能揭示了不同乳腺健康状态下乳腺内的生物学现象,此外为诊断乳腺健康特别是早期乳腺炎症的发生提供潜在生物标记物。

本文引用格式

汪悦, 熊本海, 蒋林树 . 奶牛不同乳腺健康状态下乳清蛋白的差异性表达研究[J]. 动物营养学报, 2019 , 31(2) : 775 -791 . DOI: 10.3969/j.issn.1006-267x.2019.02.034

Abstract

The objective of this study was to explore the effects of dairy breast health conditions on milk proteins and the molecular mechanisms of inflammation induced by pathogenic microorganisms by investigating the differential expression of whey proteins in dairy cows under different breast health status, which provided a potential biomarker to diagnose the early inflammation of udder. A total of 102 healthy Holstein dairy cows[2 to 3 parity, lactation days (152±27) d, milk yield (27±3) kg/d] were selected for milk sample collection. Milk samples were divided into 6 groups according to somatic cell count (SCC) and bacteriological identification results, they were bacterial culture-positive groups (contagious, environmental and opportunistic) and bacterial culture-negative groups[culture-negative-low SCC group (SCC<100 000 cells/mL), culture-negative-medium SCC group (SCC 100 000 to 400 000 cells/mL) and culture-negative-high SCC group (SCC>400 000 cells/mL)]. The sodium dodecyl sulfate polyacrylamide gel electropheresis (SDS-PAGE) and non-labeled quantitative proteomics were used to identify protein function and expression levels in 6 whey samples. The results showed as follows:1) the difference between healthy breast and inflammatory mammary glands in dairy cows was mainly caused by the protein expression level. 2) A total of 272 proteins were identified, of which 58 proteins showed significant regulatory changes. Compared with bacterial culture-negative whey samples, 20 proteins were significantly up-regulated expressed in bacterial culture-positive whey samples, and the expressions of cathelicidin-4 (CATHL4), cathelicidin-3(CATHL3), cathelicidin-2(CATHL2), inter-alpha-trypsin inhibitor heavy chain H4, (ITIH4), serpin A3-1 (SERPINA3-1), prostaglandin-H2 D-isomerase (PTGDS), serum amyloid A protein (SAA3) and immunoglobulin kappa variable 3-20 (IGKV3-20) in bacterial culture-positive whey samples were more than about 8 times than those in bacterial culture-positive whey samples, and most of the functions of the up-regulated proteins were related to specific immune responses. The 38 proteins significantly down-regulated expressed(P<0.05 or P<0.01), the platelet glycoprotein 4 (CD36), CD59 molecule (CD59), kappa-casein (CSN3), butyrophilin subfamily 1 member A1 (BTN1A1), ATP-binding cassette sub-family G member 2 (ABCG2), perilipin-2 (PLIN2), glycoprotein 2 (GP2), polyubiquitin-C (UBC) and isocitrate dehydrogenase 1 (IDH1) were significantly down-regulated(P<0.01), BTN1A1 was related to the lipid synthesis and secretion, PLIN2 and GP2 were related to the transport, ABCG2 had enzyme activity, CD59 was related to the immune system. 3) A total of 21 bacterial proteins were identified in the bacterial culture-positive whey samples, the existence of these bacterial proteins not only verified the results of bacterial culture identification, but also elucidated the molecules that induced inflammation by the pathogenic microorganisms from the perspective of the bacterial protein properties and functions. In summary, the differentially expression of milk proteins under different breast health conditions can reveals the biological phenomena in the mammary gland under different breast health conditions from the perspective of milk protein expression changes and function, in addition, it expects to provide some potential biomarkers for diagnosing the breast health, especially the early breast inflammation.

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