A dairy cow mammary epithelial cell line was established through culture method of tissue block and detection of cell biological characters. Effects of methionine and lysine on viabilities and β-casein of DCMECs were evaluated by CASY and HPLC, and determined the optimal dose of methionine and lysine was 0.6mmol/L and 1.2mmol/L, the best time was 24h. A nuclear phosphoproteomics of DCMECs was successfully established, five proteins for which expression was significantly increased in methionine-treated DCMECs were selected, The 5 up-regulated expressed phosphoproteins included Staphylococcal nuclease domain-containing protein 1(SND1), Septin-6, Glycyl-tRNA synthetase (GARS), Twinfilin-1 and eukaryotic elongation factor1-beta (eEF1B); Six proteins for which expression was significantly increased in lysine-treated DCMECs were selected, The 6 up-regulated expressed phosphoproteins included SKIV2L2、sec-related protein D、T-complex protein 1 subunit delta、protein disulfide-isomerase A3、coronin actin binding protein 1C and mitogen-activated protein kinase 1(MAPK1); the expression levels of these were verified by quantitative real-time PCR (qRT-PCR) and Western blotting analysis, which were consisted with the results of 2-DE. In this research, eukaryotic expression vector pGCMV-IRES-EGFP-MAPK1 and pGCMV-IRES-EGFP-eEF1B were constructed by stably transfected into DCMECs after geneticin (G418) selection. The gene functions of MAPK1 and eEF1B were identified by the RNA interference and gene overexpression, methionine and lysine as energy substrates can promote expression of Stat5a gene and increase lactation of DCMECs by MAPK1 and eEF1B. MAPK1 and eEF1B also regulates the expression of key mediators of the PRLR/JAK2/STAT5 and mTOR signaling pathway. We used Co-immunoprecipitation and GST-pull down assay here to identify the interacting proteins of MAPK1, including ARPC4、β-enolase、Annexin A2、Small G protein signaling modulator 1and Polymerase I and transcript release factor, the interacting proteins of eEF1B are 14-3-3theta、Heat shock protein、eEF1A1、Small nuclear ribonucleoprotein polypeptide A and 40S ribosomal protein S4. The results of this research have enriched the study content of nutritional genomics of dairy cow, and have important basic theory and research methods.%该研究采用组织块儿培养法，成功构建了体外培养的奶牛乳腺上皮细胞模型，应用CASY细胞分析仪检测了不同浓度和作用时间下蛋氨酸和赖氨酸对奶牛乳腺上皮细胞活力的影响，利用高效液相色谱检测了β-casein的分泌情况，确定了蛋氨酸和赖氨酸最佳剂量分别为0.6 mmol/L和1.2 mmol/L，最佳作用时间为24 h。建立奶牛乳腺上皮细胞核磷酸化蛋白质组技术，应用双向电泳（2-DE）结合质谱技术（MALDI-TOF-MS）鉴定蛋氨酸和赖氨酸对奶牛乳腺上皮细胞核磷酸化蛋白质的差异影响，发现蛋氨酸处理组，mitogen-activated protein kinase 1（MAPK1）和eEF1B蛋白质表达上调；并应用荧光定量PCR和Western blotting技术验证差异蛋白在转录水平和蛋白水平上的表达，与2-DE结果一致。实验过程中构建了真核表达载体pGCMV-IRES-EGFP-MAPK1、eEF1B，转染至奶牛乳腺上皮细胞中，通过G418筛选获得稳定转染的细胞株；然后通过基因沉寂和过表达等技术，减少或增加MAPK1、eEF1B的表达水平后，检测泌乳信号转导通路中重要调控蛋白的表达情况，发现蛋氨酸和赖氨酸营养素可以通过调节MAPK1、eEF1B介导mTOR信号转导通路，影响Stat5的表达，进而调节乳蛋白的表达。以上实验结果为奶牛营养基因组学的研究提供了理论依据和技术方法。
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