基于iTRAQ技术的脂多糖刺激下奶牛乳腺上皮细胞的蛋白组学分析  

Analysis of Expression Protein Profiles of Bovine Mammary Epithelial Cell Based on Isobaric Tag for Relative and Absolute Quantification Proteomics and Bioinformatics Technique

作　　者：李莲[1] 唐娟[1] 吴洁[1] 王根林[1] 

机构地区：[1]南京农业大学动物科技学院,南京210095

出　　处：《畜牧兽医学报》2016年第9期1853-1860,共8页ACTA VETERINARIA ET ZOOTECHNICA SINICA

基　　金：国家自然科学基金(31372290);中央高校基本科研业务费专项资金(KJQN201607)

摘　　要：基于iTRAQ多重标记串联质谱技术系统研究脂多糖刺激下乳腺上皮细胞蛋白的差异表达,并对与乳蛋白、乳脂肪相关差异表达基因进行验证。培养纯化后的乳腺上皮细胞,经脂多糖(LPS)处理后提取细胞蛋白,采用iTRAQ标记和联合二维液相色谱-串联质谱(2D-LC-MS/MS)检测分析得到各组间细胞差异表达蛋白,并采用蛋白印迹和酶联免疫法(ELISA)对差异蛋白进行验证,然后对与乳蛋白、乳脂肪相关基因的相互作用蛋白进行PPI(Protein protein interaction)预测。乳腺上皮细胞共鉴定出2 487个蛋白,其中差异蛋白共341个,与对照组相比,163个蛋白表达上调,178个蛋白表达下调。iTRAQ测定结果显示,LPS处理组与对照组相比,αs1-酪蛋白(CSN1S1)、κ-酪蛋白(CSN3)和脂肪酸合酶(FASN)基因蛋白表达水平有统计学意义;经蛋白印迹试验结果表明,LPS诱导组αs1-酪蛋白表达水平同对照组比较,明显降低(P<0.05);应用ELISA方法测定细胞培养液中αs1-酪蛋白、κ-酪蛋白和脂肪酸合酶的浓度结果显示,LPS诱导组细胞培养液中上述蛋白的浓度明显低于对照组(P<0.05)。综上表明,LPS诱导的炎症反应可影响乳腺上皮细胞的乳蛋白和乳脂肪的合成。The aim of the present study was to screen differential expression proteins associated with inflammatory model of bovine mammary epithelial cells （BMECs） induced by lipopolysaccha- ride （LPS） using isobaric tags for relative and absolute quantification （iTRAQ） combined with 2- dimensional liquid chromatography-tandem mass spectrometry （2D-LC-MS/MS）. Normal and LPS-treated bovine mammary epithelial cells were analyzed using iTRAQ combined with 2D-LC- MS/MS. Bioinformatics analyses of differentially expressed proteins were performed by means of Gene Ontology and PPI （Protein protein interaction）analysis. According to iTRAQ results,a total of 2 487 proteins were identified. Compared with normal BMECs, 163 proteins were significantly up-regulated while 178 proteins were significantly down-regulated in response to ImPS treatment. The bioinformaties analyses highlighted the effects of LPS treatment on milk proteins and fat syn- thesis such as αsl-casein, κ-casein and fatty acid synthase. Down-regulation of αsl-casein （CSN1S1） in BMECs was confirmed by Western blotting αsl-casein,κ-casein （CSN3） and fatty acid synthase （FASN） in culture medium were confirmed by ELISA. The results showed that LPS treatment significantly decreased the expression of asl-casein in BMECs （P〈0.05） ,and also decreased the concentration of αsl-caseins,κ-casein and fatty acid synthase in culture medium （P〈 0.05）. In conclusion,LPS treatment could affect the synthesis of protein and fat in BMECs.

关 键 词：牛 乳腺上皮细胞 iTRAQ技术 蛋白质组学 脂多糖 

分 类 号：S823.2[农业科学—畜牧学]