线粒体膜间隙内蛋白质间相互作用网络的初步研究

发布时间：2018-04-10 05:33

本文选题：线粒体膜间隙　切入点：酵母双杂交　出处：《安徽医科大学》2008年硕士论文

【摘要】： 目的:以人源线粒体膜间隙内蛋白为主要研究对象,发掘它们之间的相互作用,构建其相互作用网络。方法:采用PCR技术扩增以线粒体膜间隙内蛋白为主的蛋白编码基因,分别将其构建酵母双杂交质粒pDBLeu和pPC86的重组载体;应用酵母双杂交技术,分别采用共转酵母法和阵列组合法进行相互作用蛋白的筛选,挑取部分阳性结果用免疫共沉淀或(和)GST-Pull down验证,并对个别酵母双杂交阴性但有生物学意义的相互作用进行免疫共沉淀鉴定;采用生物信息学分析方法对所获得的相互作用数据可信度进行分析,并运用Cytoscape软件构建出相互作用网络图,在此基础上通过文献调研对相互作用的生物学意义进行深入挖掘。结果:成功构建了38个诱饵和41个猎物融合基因的酵母双杂交载体。除去1个有自激活活性的诱饵后,通过酵母双杂交技术筛选和免疫共沉淀鉴定共得到涉及36个蛋白间的50对相互作用,其中2对为已知相互作用,从新相互作用中挑取6对以免疫共沉淀或(和)GST-Pull down进行验证,结果4对阳性,另2对没有发现相互作用。对Y2H结果采用生物信息学分析,我们发现有13对相互作用(26%)的诱饵和猎物基因名共同出现在PubMed文献中;没有相同的基因敲除表型的相互作用对;12对相互作用蛋白(24.5%)共享第4级或以上GO功能注释;最后整合多种参数对每对相互作用进行综合评分,27对相互作用(55%)具有高可信度。应用Cytoscape软件可视化分析相互作用网络的结果显示:我们获得的相互作用对原有网络进行了有效补充和拓展,在此基础上通过文献调研我们挖掘出相互作用的多种重要生物学意义。结论:线粒体膜间隙内蛋白间存在复杂的相互作用网络,这些新相互作用的发现为探讨线粒体膜间隙内一些生物学过程及其发生机制提供了重要线索。
[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.
【学位授予单位】：安徽医科大学
【学位级别】：硕士
【学位授予年份】：2008
【分类号】：R341

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