应用2-DE技术对冷应激前后雏鸡血清蛋白质的差异分析

发布时间：2018-07-02 06:55

本文选题：冷应激 + 雏鸡　；参考：《黑龙江八一农垦大学》2015年硕士论文

【摘要】：应激的因素是各种各样。在北方，尤其是冬天，寒冷应激是动物（特别是雏禽）最常见的应激因素，严重影响畜牧业和畜禽的生产性能及抗病能力。血清中富含丰富的蛋白质可以反应正常的生理状态和疾病状况下的生理状态，可能是重要的疾病诊断的标志蛋白，进一步也可能作为药物靶标。因此通过动物的体液如血液、组织等寻找高特异性、高敏感性的冷应激标志物具有重大意义。利用高通量的蛋白质组学技术分析和检测在冷应激条件下雏鸡血清蛋白质表达水平的差异，得到在冷应激影响下发生变化的关键蛋白，有助于揭示并验证蛋白质与特定生理、病理过程中的相关性和探讨冷应激发生发展的分子机理。本研究在建立血清高丰度蛋白去除的蛋白质组的基础上，从而减少高丰度蛋白对低丰度蛋白的遮蔽和提高分离技术的敏感性。选取冷应激组雏鸡和冷适应组雏鸡的血清为主要研究对象，以常温组雏鸡的血清作为对照，结合双向凝胶电泳技术（two-dimensional gelelectrophoresis,2-DE）；利用图像分析软件PDQuest8.0对冷应激组和冷适应组与常温组的血清蛋白表达谱进行图像的差异分析。后进行质谱鉴定冷应激相关的蛋白质。首先对30只雏鸡的血清进行了初步的分析，采用双向凝胶电泳分离和考马斯亮蓝染色法来检验样品制备的质量，并对双向电泳条件的优化和比较去除高丰度蛋白的效果。成功建立了雏鸡血清蛋白的双向电泳方法。在对雏鸡血清进行初步差异分析后，进一步对差异蛋白进行了基质辅助激光解吸电离飞行时间质谱（MALDI-TOF-MS）鉴定和Westernblot验证。结果表明：通过对常温、冷应激和冷适应处理的雏鸡血清进行2-DE分析，获得了比较完整的差异蛋白质数据，共找到23个差异蛋白质点。分析后发现蛋白表达水平上调的差异点和表达水平下调的差异点分别有11个和12个。将8个明显和重复性好的差异蛋白点进行MALDI-TOF-MS分析，，成功鉴定4个差异蛋白。获得的差异蛋白为葡萄糖、能量代谢通路供能相关蛋白质，ALDOC蛋白除了在糖酵解和果糖代谢中起着关键和枢纽作用，还可能在冷应激的发生及抵抗冷应激为机体供能方面发挥重要作用。随后，利用Western blot方法对差异蛋白ALDOC进一步验证，结果与2-DE的结果相一致。
[Abstract]:The factors of stress are various. In the north, especially in winter, cold stress is the most common stress factor in animals (especially young birds), which seriously affects the production performance and disease resistance of livestock and poultry. The rich protein in the serum can reflect the normal physiological state and the physiological state under the condition of disease, which may be an important marker of disease diagnosis, and may also be used as a drug target. Therefore, it is of great significance to search for high specific and sensitive cold stress markers through animal body fluids such as blood and tissue. Using high-throughput proteomics technique to analyze and detect the difference of serum protein expression in chicks under cold stress conditions, the key proteins changed under cold stress were obtained, which was helpful to reveal and verify the protein and specific physiology. The correlation of pathological process and the molecular mechanism of cold stress. In this study, the proteome of serum high abundance protein was established to reduce the masking of high abundance protein to low abundance protein and to improve the sensitivity of separation technique. The serum of chicks in cold stress group and cold acclimated group was selected as the main research object, and the serum of chicks in normal temperature group as control, combined with two-dimensional gel electrophoresis2-DE (2-DE). Image analysis software PDQuest 8.0 was used to analyze the difference of serum protein expression profiles between cold stress group and cold acclimated group and normal temperature group. The proteins related to cold stress were identified by mass spectrometry. At first, the serum of 30 chicks was analyzed preliminarily, and the quality of the samples was tested by two-dimensional gel electrophoresis and Coomassie brilliant blue staining, and the optimization of the conditions of two-dimensional electrophoresis and the effect of removing high abundance protein were compared. A two-dimensional electrophoresis method for chicken serum protein was successfully established. After preliminary differential analysis of chicken serum, the differential proteins were further identified by matrix assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF-MS) and verified by Western blot. The results showed that complete differential protein data were obtained by 2-DE analysis of chicken serum under normal temperature, cold stress and cold adaptation, and 23 differential protein spots were found. The results showed that there were 11 points and 12 points of difference in up-regulation and down-regulation of protein expression level, respectively. Eight distinct and reproducible differential protein spots were analyzed by MALDI-TOF-MS and 4 differentially expressed proteins were successfully identified. The differentially obtained proteins are glucose, and the energy supply related protein, ALDOC, plays a key and pivotal role in glycolysis and fructose metabolism. It may also play an important role in the occurrence of cold stress and the resistance to cold stress. Then, the differential protein ALDOC was further verified by Western blot method, and the results were consistent with that of 2-DE.
【学位授予单位】：黑龙江八一农垦大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S858.31

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