【作者】 刘姗姗；

【导师】 赵兴绪；

【作者基本信息】 甘肃农业大学 ， 发育生物学， 2015， 硕士

【摘要】 本研究采用二维凝胶电泳(2-DE)结合MADIL-TOF-TOF串联质谱法对荷斯坦奶牛妊娠早期血浆进行蛋白质组学分析鉴定,发现了6种差异蛋白在妊娠后表达量上调,分别是凝集素1前体、转甲状腺素蛋白前体、3,5-二碘水杨酸络合的牛血清白蛋白的晶体结构的A链、白蛋白、未命名蛋白产物及结合珠蛋白-25同源物1前体。凝集素1前体的表达量在发情及配种后呈现先升高后下降的趋势;转甲状腺素蛋白前体,白蛋白的表达量在发情及配种后呈先升高后下降再升高的趋势;3,5-二碘水杨酸络合的牛血清白蛋白的晶体结构的A链的表达量不断增加,未命名蛋白产物、结合珠蛋白-25同源物1前体的含量是先升高后保持稳定。妊娠期这6种蛋白的含量均高于妊娠前,这些蛋白在妊娠早期血浆中可能与免疫应答、甲状腺激素转运、防止淀粉样纤维形成和碘的运输有关。采用相同方法对荷斯坦奶牛分娩后不同时期的血浆进行蛋白质组学分析鉴定,发现5种差异蛋白。结合珠蛋白前体、结合珠蛋白在分娩后表达量先升高后下降至与分娩当天相近。免疫球蛋白γ2链C区(克隆32.2)在分娩后表达量先下降再升高之后保持一个平稳的趋势。分娩后3,5-二碘水杨酸络合的牛血清白蛋白的晶体结构的A链和B链表达量低于妊娠后45天的含量且在分娩后表达量相对稳定。这5种蛋白可能分别与产后应激、免疫应答和物质运输有关。采用二维凝胶电泳(2-DE)结合MADIL-TOF-TOF串联质谱法对荷斯坦奶牛妊娠早期乳汁进行蛋白质组学分析鉴定,发现5个差异蛋白点,分别是:3,5-二碘水杨酸络合的牛血清白蛋白的晶体结构的A链、免疫球蛋白γ1重链恒定区、免疫球蛋白G1重链恒定区、白蛋白、蛋白胨成分3。3,5-二碘水杨酸络合的牛血清白蛋白的晶体结构的A链在乳汁中含量的变化趋势为先降低再升高后降低。免疫球蛋白G1重链恒定区和免疫球蛋白γ1重链恒定区表达量显著降低,之后基本保持稳定的趋势。蛋白胨3的表达量虽有波动,但是妊娠早期高于发情当天。这些蛋白可能与物质运输、免疫应答和抗菌有关。采用相同方法对荷斯坦奶牛分娩后不同时期乳汁进行蛋白质组学分析鉴定,发现8种差异蛋白,分别是:α-S2酪蛋白前体、β-酪蛋白、κ-酪蛋白前体、免疫球蛋白重链前体、白蛋白、血清白蛋白前体、cGMP磷酸二酯酶的α亚基、角蛋白I型细胞骨架10。α-S2酪蛋白前体只在分娩当天乳样中检测到,β-酪蛋白在分娩后7天和分娩后35天检测到,且在分娩后35天的表达量比分娩后7天高,κ-酪蛋白前体在分娩当天表达量最高,之后表达量快速下降之后保持平稳。免疫球蛋白重链前体存在于分娩后7、15、28及35天的乳样中,虽有波动,但在分娩后35天的乳样中表达量最低。血清白蛋白前体在分娩后7天到分娩后35天表达量表现出先升高后降低的趋势,白蛋白在分娩后7天到分娩后35天的表达量呈先降低后升高再降低的趋势,分娩后35天的表达量最低。cGMP磷酸二酯酶的α亚基在分娩后7、15及28天的表达量呈先上升后下降的趋势。角蛋白I型细胞骨架10在分娩当天到分娩后28天表达量的变化趋势平稳。这些蛋白可能与初乳的被动免疫、物质运输和产奶有关。更多还原

【Abstract】 Proteome analysis in plasma from Holstein cows in early pregnancy were performed using two-dimensional gel electrophoresis(2-DE) method combined with MADIL-TOF-TOF tandem mass spectrometry. Six proteins were up-regulated after breeding, which including TPA: conglutinin 1 precursor, transthyretin precursor, Chain A, Crystal Structure Of Bovine Serum Albumin In Complex With 3,5-Diiodosalicylic Acid(CSBSACDA), ALB protein, unnamed protein product and protein HP-25 homolog 1 precursor. The expression of conglutinin 1 precursor firstly increased and then decreased. The contents of transthyretin precursor and ALB protein fluctuated after estrus and breeding. The expression of Chain A, CSBSACDAincreased constantly. The contents of unnamed protein product and protein HP-25 homolog 1 precursor firstly increased and then kept stable. The contents of 6 proteins during pregnancy were higher than non-pregnant period. These proteins in the early pregnant plasma may be related to the immune response, thyroid hormone transport, prevention of amyloid fibril formation and transportation of iodine. Using the same methods to analyze the proteome in plasma from Holstein cows in the different postpartum period, 5 differential proteins were found. The expression of haptoglobin precursorand haptoglobin after delivery firstly increased and then decreased as the labor day. The expression of Ig gamma-2 chain C region(clone 32.2)-bovine(fragment) decreased at first then increased and maintained a steady trend at last. The levels of Chain A, CSBSACDA and Chain B, CSBSACDA after delivery were lower than on the 45 th day in the early pregnancy, and remained stable. These five proteins may be associated with postpartum stress, immune response and material transportation respectively. Proteome analysis in milk from Holstein cows in early pregnancy were performed using two-dimensional gel electrophoresis(2-DE) method combined with MADIL-TOF-TOF tandem mass spectrometry. Five differential proteins were identified. They are Chain A, CSBSACDA, immunoglobulin gamma 1 heavy chain constant region, Ig G1 heavy chain constant region, ALB protein and component PP3. The change of Chain A, CSBSACDA in milk firstly decreased then increased and decreased again. The contents of immunoglobulin gamma 1 heavy chain constant region and Ig G1 heavy chain constant region significantly decreased, then remained stable trend. The expression of component PP3 fluctuated, but it was higher during pregnancy than non-pregnant period. These proteins may be related to transportation, immune response and antibacterial. Using the same methods to analyze the proteome in milk from Holstein cows in the different period after delivery, eight differential proteins were found, which were serum albumin precursor, albumin, Ig heavy chain precursor(B/MT.4A.17.H5.A5)–bovine, alpha-s2-like casein precursor, beta-casein, kappa-casein precursor, c GMP phosphodiesterase alpha subunit and Keratin, type I cytoskeletal10. Alpha-s2-like casein precursor was only detected in the milk sample on the labor day. Beta-casein was detected in milk samples on the 7th day and the 35 th day after delivery, and the level on the 35 th day was higher than the 7th day. Kappa-casein precursor was highly expressed on the labor day, and then decreased rapidly and kept stable. Ig heavy chain precursor(B/MT.4A.17.H5.A5)–bovine existed in the milk samples on the 7, 15, 28 and 35 th day. The levels fluctuated, however, the level on the 35 th day was the lowest. The expression of serum albumin precursor from 7th day to 35 th day after delivery firstly increased and then decreased. The change of expression of albumin was contrary to the serum albumin precursor, and the level on the 35 th day was the lowest. The contents of c GMP phosphodiesterase alpha subunit from 7th day to 28 th day after delivery firstly increased and then decreased. The change of type I cytoskeletal10 from the 0th day to 28 th day after delivery remained stable. These proteins may be related to the passive immunization of colostrums, material transportation and lactation.更多还原