本文選題：線粒體膜間隙　切入點(diǎn)：酵母雙雜交　出處：《安徽醫(yī)科大學(xué)》2008年碩士論文

【摘要】： 目的:以人源線粒體膜間隙內(nèi)蛋白為主要研究對象,發(fā)掘它們之間的相互作用,構(gòu)建其相互作用網(wǎng)絡(luò)。 方法:采用PCR技術(shù)擴(kuò)增以線粒體膜間隙內(nèi)蛋白為主的蛋白編碼基因,分別將其構(gòu)建酵母雙雜交質(zhì)粒pDBLeu和pPC86的重組載體;應(yīng)用酵母雙雜交技術(shù),分別采用共轉(zhuǎn)酵母法和陣列組合法進(jìn)行相互作用蛋白的篩選,挑取部分陽性結(jié)果用免疫共沉淀或(和)GST-Pull down驗(yàn)證,并對個(gè)別酵母雙雜交陰性但有生物學(xué)意義的相互作用進(jìn)行免疫共沉淀鑒定;采用生物信息學(xué)分析方法對所獲得的相互作用數(shù)據(jù)可信度進(jìn)行分析,并運(yùn)用Cytoscape軟件構(gòu)建出相互作用網(wǎng)絡(luò)圖,在此基礎(chǔ)上通過文獻(xiàn)調(diào)研對相互作用的生物學(xué)意義進(jìn)行深入挖掘。 結(jié)果:成功構(gòu)建了38個(gè)誘餌和41個(gè)獵物融合基因的酵母雙雜交載體。除去1個(gè)有自激活活性的誘餌后,通過酵母雙雜交技術(shù)篩選和免疫共沉淀鑒定共得到涉及36個(gè)蛋白間的50對相互作用,其中2對為已知相互作用,從新相互作用中挑取6對以免疫共沉淀或(和)GST-Pull down進(jìn)行驗(yàn)證,結(jié)果4對陽性,另2對沒有發(fā)現(xiàn)相互作用。對Y2H結(jié)果采用生物信息學(xué)分析,我們發(fā)現(xiàn)有13對相互作用(26%)的誘餌和獵物基因名共同出現(xiàn)在PubMed文獻(xiàn)中;沒有相同的基因敲除表型的相互作用對;12對相互作用蛋白(24.5%)共享第4級(jí)或以上GO功能注釋;最后整合多種參數(shù)對每對相互作用進(jìn)行綜合評(píng)分,27對相互作用(55%)具有高可信度。應(yīng)用Cytoscape軟件可視化分析相互作用網(wǎng)絡(luò)的結(jié)果顯示:我們獲得的相互作用對原有網(wǎng)絡(luò)進(jìn)行了有效補(bǔ)充和拓展,在此基礎(chǔ)上通過文獻(xiàn)調(diào)研我們挖掘出相互作用的多種重要生物學(xué)意義。 結(jié)論:線粒體膜間隙內(nèi)蛋白間存在復(fù)雜的相互作用網(wǎng)絡(luò),這些新相互作用的發(fā)現(xiàn)為探討線粒體膜間隙內(nèi)一些生物學(xué)過程及其發(fā)生機(jī)制提供了重要線索。
[Abstract]:Aim: to explore the interaction between human mitochondrial intermembrane proteins and construct their interaction networks.Methods: protein encoding genes with mitochondrial intermembrane proteins were amplified by PCR, and the recombinant vectors of yeast two-hybrid plasmids pDBLeu and pPC86 were constructed, respectively, and yeast two-hybrid technique was used.The interaction proteins were screened by co-transfer yeast method and array combination method, and some of the positive results were detected by immunoprecipitation or (and) GST-Pull down.Some yeast two-hybrid negative but biological interactions were identified by immunoprecipitation, and the reliability of the interaction data was analyzed by bioinformatics analysis.The interaction network diagram is constructed by using Cytoscape software, and the biological significance of interaction is excavated through literature research.Results: yeast two-hybrid vectors of 38 decoys and 41 prey fusion genes were successfully constructed.After removing one decoy with self-activating activity, 50 pairs of interactions involving 36 proteins were identified by yeast two-hybrid screening and immunoprecipitation, 2 of which were known interactions.Six pairs were selected from the new interaction to be verified by immunoprecipitation or (and) GST-Pull down. The results showed that 4 pairs were positive and the other 2 pairs had no interaction.Based on bioinformatics analysis of Y2H results, we found that 13 pairs of decoys and prey gene names appeared in PubMed literature.No identical knockout phenotypic interaction was found to be highly reliable for the 12 pairs of interacting proteins (24.5) sharing the fourth or higher go functional annotations, and finally integrating multiple parameters for a comprehensive score of each pair of interactions.The results of visual analysis of interaction networks by using Cytoscape software show that the interactions we obtained complement and extend the existing networks effectively.On this basis, we excavate the important biological significance of interaction through literature research.Conclusion: there are complex interaction networks among proteins in the mitochondrial membrane space. The discovery of these new interactions provides important clues for the study of some biological processes and their pathogenesis in the mitochondrial membrane space.
【學(xué)位授予單位】：安徽醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2008
【分類號(hào)】：R341

【共引文獻(xiàn)】

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本文編號(hào)：1729889