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

【摘要】：應(yīng)激的因素是各種各樣。在北方，尤其是冬天，寒冷應(yīng)激是動(dòng)物（特別是雛禽）最常見的應(yīng)激因素，嚴(yán)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(yīng)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(biāo)志蛋白，進(jìn)一步也可能作為藥物靶標(biāo)。因此通過動(dòng)物的體液如血液、組織等尋找高特異性、高敏感性的冷應(yīng)激標(biāo)志物具有重大意義。利用高通量的蛋白質(zhì)組學(xué)技術(shù)分析和檢測(cè)在冷應(yīng)激條件下雛雞血清蛋白質(zhì)表達(dá)水平的差異，得到在冷應(yīng)激影響下發(fā)生變化的關(guān)鍵蛋白，有助于揭示并驗(yàn)證蛋白質(zhì)與特定生理、病理過程中的相關(guān)性和探討冷應(yīng)激發(fā)生發(fā)展的分子機(jī)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(chǔ)上，從而減少高豐度蛋白對(duì)低豐度蛋白的遮蔽和提高分離技術(shù)的敏感性。選取冷應(yīng)激組雛雞和冷適應(yīng)組雛雞的血清為主要研究對(duì)象，以常溫組雛雞的血清作為對(duì)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(duì)冷應(yīng)激組和冷適應(yīng)組與常溫組的血清蛋白表達(dá)譜進(jìn)行圖像的差異分析。后進(jìn)行質(zhì)譜鑒定冷應(yīng)激相關(guān)的蛋白質(zhì)。 首先對(duì)30只雛雞的血清進(jìn)行了初步的分析，采用雙向凝膠電泳分離和考馬斯亮藍(lán)染色法來檢驗(yàn)樣品制備的質(zhì)量，并對(duì)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(duì)雛雞血清進(jìn)行初步差異分析后，進(jìn)一步對(duì)差異蛋白進(jìn)行了基質(zhì)輔助激光解吸電離飛行時(shí)間質(zhì)譜（MALDI-TOF-MS）鑒定和Westernblot驗(yàn)證。 結(jié)果表明：通過對(duì)常溫、冷應(yīng)激和冷適應(yīng)處理的雛雞血清進(jìn)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(gè)差異蛋白質(zhì)點(diǎn)。分析后發(fā)現(xiàn)蛋白表達(dá)水平上調(diào)的差異點(diǎn)和表達(dá)水平下調(diào)的差異點(diǎn)分別有11個(gè)和12個(gè)。將8個(gè)明顯和重復(fù)性好的差異蛋白點(diǎn)進(jìn)行MALDI-TOF-MS分析，，成功鑒定4個(gè)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(guān)蛋白質(zhì)，ALDOC蛋白除了在糖酵解和果糖代謝中起著關(guān)鍵和樞紐作用，還可能在冷應(yīng)激的發(fā)生及抵抗冷應(yīng)激為機(jī)體供能方面發(fā)揮重要作用。隨后，利用Western blot方法對(duì)差異蛋白ALDOC進(jìn)一步驗(yàn)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(xué)位授予單位】：黑龍江八一農(nóng)墾大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2015
【分類號(hào)】：S858.31

【參考文獻(xiàn)】

相關(guān)期刊論文 前10條

1 劉莉莉;初芹;徐青;王雅春;;動(dòng)物冷應(yīng)激的研究進(jìn)展[J];安徽農(nóng)業(yè)科學(xué);2012年16期

2 胡惠忠;付強(qiáng);盧晟盛;張明;陸陽清;蒙冰;盧克煥;;豬卵母細(xì)胞蛋白質(zhì)組雙向電泳體系的建立及初步分析[J];動(dòng)物學(xué)雜志;2008年04期

3 李雪華,李欣,徐守軍,丁彥青;血清蛋白質(zhì)組學(xué)研究中白蛋白和IgG去除方法的建立與評(píng)價(jià)[J];第一軍醫(yī)大學(xué)學(xué)報(bào);2004年11期

4 方文;肖靚靚;包懷恩;牟榮;;未成熟與成熟豬囊尾蚴蛋白雙向電泳的圖譜分析[J];貴州農(nóng)業(yè)科學(xué);2011年09期

5 楊永新;程廣龍;趙輝玲;江喜春;陳勝;;基于二維凝膠電泳分析健康泌乳奶牛血漿蛋白質(zhì)組的個(gè)體差異[J];中國(guó)畜牧獸醫(yī);2010年11期

6 袁學(xué)軍,牛靜華,吳海燕,徐洪鑫,李義春,孫秀軍;冷應(yīng)激對(duì)伊褐紅公雛雞外周血淋巴細(xì)胞數(shù)目變化的影響[J];黑龍江八一農(nóng)墾大學(xué)學(xué)報(bào);2002年01期

7 王玉海,王蔡麗,張文英,黃麗波,吳海燕;溫和冷應(yīng)激對(duì)貴妃雛雞脾臟、胸腺中肥大細(xì)胞數(shù)目的影響[J];黑龍江八一農(nóng)墾大學(xué)學(xué)報(bào);2005年04期

8 邳靜偉;崔繼良;;冷應(yīng)激對(duì)貴妃雛雞血液中某些生化指標(biāo)的影響[J];中國(guó)牧業(yè)通訊;2010年15期

9 陳龍;朱化彬;沙里金;韓偉東;王棟;王宗禮;郝海生;杜衛(wèi)華;程金華;;牛精子蛋白質(zhì)組學(xué)技術(shù)平臺(tái)的建立及凍融前后精子差異蛋白初步分析[J];中國(guó)生物工程雜志;2008年07期

10 廖國(guó)周;王桂瑛;程志斌;賈俊靜;葛長(zhǎng)榮;;云南騰沖雪雞肌肉蛋白質(zhì)組學(xué)研究[J];肉類研究;2013年07期

相關(guān)博士學(xué)位論文 前1條

1 姜昊;中國(guó)草原紅牛與長(zhǎng)白山黑牛眼肌差異蛋白質(zhì)組學(xué)研究[D];吉林大學(xué);2011年

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