陸黎敏　黃建國　李慶章　高學(xué)軍

摘 要：該研究采用組織塊兒培養(yǎng)法，成功構(gòu)建了體外培養(yǎng)的奶牛乳腺上皮細(xì)胞模型，應(yīng)用CASY細(xì)胞分析儀檢測了不同濃度和作用時間下蛋氨酸和賴氨酸對奶牛乳腺上皮細(xì)胞活力的影響，利用高效液相色譜檢測了β-casein的分泌情況，確定了蛋氨酸和賴氨酸最佳劑量分別為0.6 mmol/L和1.2 mmol/L，最佳作用時間為24 h。建立奶牛乳腺上皮細(xì)胞核磷酸化蛋白質(zhì)組技術(shù)，應(yīng)用雙向電泳（2-DE）結(jié)合質(zhì)譜技術(shù)（MALDI-TOF-MS）鑒定蛋氨酸和賴氨酸對奶牛乳腺上皮細(xì)胞核磷酸化蛋白質(zhì)的差異影響，發(fā)現(xiàn)蛋氨酸處理組，mitogen-activated protein kinase 1（MAPK1）和eEF1B蛋白質(zhì)表達(dá)上調(diào)；并應(yīng)用熒光定量PCR和Western blotting技術(shù)驗證差異蛋白在轉(zhuǎn)錄水平和蛋白水平上的表達(dá)，與2-DE結(jié)果一致。實驗過程中構(gòu)建了真核表達(dá)載體pGCMV-IRES-EGFP-MAPK1、eEF1B，轉(zhuǎn)染至奶牛乳腺上皮細(xì)胞中，通過G418篩選獲得穩(wěn)定轉(zhuǎn)染的細(xì)胞株；然后通過基因沉寂和過表達(dá)等技術(shù)，減少或增加MAPK1、eEF1B的表達(dá)水平后，檢測泌乳信號轉(zhuǎn)導(dǎo)通路中重要調(diào)控蛋白的表達(dá)情況，發(fā)現(xiàn)蛋氨酸和賴氨酸營養(yǎng)素可以通過調(diào)節(jié)MAPK1、eEF1B介導(dǎo)mTOR信號轉(zhuǎn)導(dǎo)通路，影響Stat5的表達(dá)，進(jìn)而調(diào)節(jié)乳蛋白的表達(dá)。以上實驗結(jié)果為奶牛營養(yǎng)基因組學(xué)的研究提供了理論依據(jù)和技術(shù)方法。

關(guān)鍵詞：蛋氨酸 賴氨酸 細(xì)胞核磷酸化蛋白質(zhì)組 MAPK1 eEF1B 奶牛乳腺上皮細(xì)胞

Abstract：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.

Key Words：Methionine；Lysine；Nuclear phosphoproteomics；MAPK1；eEF1B；Dairy cow mammary epithelial cells

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