本文選題：冷應激 + 雛雞��； 參考：《黑龍江八一農(nóng)墾大學》2015年碩士論文

【摘要】：應激的因素是各種各樣。在北方，尤其是冬天，寒冷應激是動物（特別是雛禽）最常見的應激因素，嚴重影響畜牧業(yè)和畜禽的生產(chǎn)性能及抗病能力。血清中富含豐富的蛋白質(zhì)可以反應正常的生理狀態(tài)和疾病狀況下的生理狀態(tài)，可能是重要的疾病診斷的標志蛋白，進一步也可能作為藥物靶標。因此通過動物的體液如血液、組織等尋找高特異性、高敏感性的冷應激標志物具有重大意義。利用高通量的蛋白質(zhì)組學技術(shù)分析和檢測在冷應激條件下雛雞血清蛋白質(zhì)表達水平的差異，得到在冷應激影響下發(fā)生變化的關鍵蛋白，有助于揭示并驗證蛋白質(zhì)與特定生理、病理過程中的相關性和探討冷應激發(fā)生發(fā)展的分子機理。 本研究在建立血清高豐度蛋白去除的蛋白質(zhì)組的基礎上，從而減少高豐度蛋白對低豐度蛋白的遮蔽和提高分離技術(shù)的敏感性。選取冷應激組雛雞和冷適應組雛雞的血清為主要研究對象，以常溫組雛雞的血清作為對照，結(jié)合雙向凝膠電泳技術(shù)（two-dimensional gelelectrophoresis,2-DE）；利用圖像分析軟件PDQuest8.0對冷應激組和冷適應組與常溫組的血清蛋白表達譜進行圖像的差異分析。后進行質(zhì)譜鑒定冷應激相關的蛋白質(zhì)。 首先對30只雛雞的血清進行了初步的分析，采用雙向凝膠電泳分離和考馬斯亮藍染色法來檢驗樣品制備的質(zhì)量，并對雙向電泳條件的優(yōu)化和比較去除高豐度蛋白的效果。成功建立了雛雞血清蛋白的雙向電泳方法。在對雛雞血清進行初步差異分析后，進一步對差異蛋白進行了基質(zhì)輔助激光解吸電離飛行時間質(zhì)譜（MALDI-TOF-MS）鑒定和Westernblot驗證。 結(jié)果表明：通過對常溫、冷應激和冷適應處理的雛雞血清進行2-DE分析，獲得了比較完整的差異蛋白質(zhì)數(shù)據(jù)，共找到23個差異蛋白質(zhì)點。分析后發(fā)現(xiàn)蛋白表達水平上調(diào)的差異點和表達水平下調(diào)的差異點分別有11個和12個。將8個明顯和重復性好的差異蛋白點進行MALDI-TOF-MS分析，，成功鑒定4個差異蛋白。獲得的差異蛋白為葡萄糖、能量代謝通路供能相關蛋白質(zhì)，ALDOC蛋白除了在糖酵解和果糖代謝中起著關鍵和樞紐作用，還可能在冷應激的發(fā)生及抵抗冷應激為機體供能方面發(fā)揮重要作用。隨后，利用Western blot方法對差異蛋白ALDOC進一步驗證，結(jié)果與2-DE的結(jié)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.
【學位授予單位】：黑龍江八一農(nóng)墾大學
【學位級別】：碩士
【學位授予年份】：2015
【分類號】：S858.31

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