阴外动脉注射LPS奶牛的血浆蛋白质组变化研究

摘要

为了检测阴外动脉注射低剂量脂多糖(LPS)后泌乳期奶牛血浆蛋白的变化,以揭示LPS刺激机体应答的分子机制.本研究选择6头泌乳中后期的健康奶牛,均经阴外动脉注射LPS(0.01μg·kg-1体质量)后分别在不同时间点(注射后0、3、6、9、12、24、36、48、60、72、84和96 h)采集奶样和血样,采用双向电泳与质谱相结合的方法,双向电泳技术分离血浆蛋白,考马斯亮蓝G-250染色后用PDQuest 8.0.1软件自动检测差异蛋白斑点,并经MALDI-TOF-TOF进行质谱鉴定.结果,牛奶体细胞评分在注射LPS后上升,6h达到最大值,此后逐渐下降,24 h恢复到注射前水平.0、6、12和24 h血浆蛋白凝胶图谱分析后发现,8个蛋白点在6、12和24 h表达量升高,质谱鉴定为4种蛋白质(VD-结合蛋白前体、丝氨酸蛋白酶抑制剂A3-6、α1-抗胰蛋白酶和丝氨酸蛋白酶抑制剂A3-1前体).这些蛋白在LPS注射后6、12和24 h表达量显著高于0 h(P＜0.05),但它们在6～24 h之间的表达量无显著差异.VD-结合蛋白前体、丝氨酸蛋白酶抑制剂A3-6、α1-抗胰蛋白酶和丝氨酸蛋白酶抑制剂A3-1前体在机体免疫调节中具有重要作用,这些蛋白的鉴定为揭示奶牛机体对LPS刺激应答的分子机制提供了基础资料.%The aims of this study were to investigate the effects of injecting low dose bacterial lipopolysaccharide (LPS) into external pudic artery on plasma proteins in lactating dairy cow, and explore the mechanism of host response to LPS challenge. All 6 cows were injected LPS(0.01 μg · kg-1 body weight). Two-dimensional electrophoresis gel (2-DE) coupled with MALDI-TOF-TOF spec-trometry was used. Plasma proteins at 0, 6, 12 and 24 h after LPS injecting were separated by two-dimensional electrophoresis. After visualization proteins with Coomassie Brilliant Blue G-250, the different protein spots were detected by PDQuest 8.0.1 software and differential proteins were identified by MALDI-TOF-TOF spectrometry. The result showed that milk SCS started to increase after injecting LPS and peaked at 6 h, and then gradually decreased to the level before injecting LPS at 24 h. Eight protein spots were identified to be four proteins including vi- tamin D-binding protein precursor, serpin A3-6, alpha-1 antitrypsin and serpin A3-1 precursor. These protein expression levels were up-regulated at 6, 12 and 24 h in cows after injecting LPS, but there was not significantly different among 6, 12 and 24 h. Vitamin D-binding protein precursor, serpin A3-6, alpha-1 antitrypsin and serpin A3-1 precursor play important roles in immune response, the identification of this proteins may be helpful to elucidate the molecular mechanism of host response to LPS challenge.