人乙酰化酶和去乙酰化酶研究体系的建立及对两个新乙酰化酶的克

发布时间：2018-03-26 20:05

本文选题：乙酰化酶　切入点：去乙酰化酶　出处：《四川农业大学》2005年硕士论文

【摘要】：包括人类基因组在内的近一百个物种的基因组测序已经基本完成。如何利用这些基因组的数据,对蛋白进行大规模的研究,已经成为现代生物学所面临的一个巨大挑战。目前蛋白质组学的主要研究内容和方法主要有对于蛋白表达谱的研究、对于蛋白的细胞内定位研究、对于蛋白间相互作用和信号途径建立的研究以及对于蛋白的生物学功能的研究。乙酰化酶和去乙酰化酶在生命活动过程中起着重要作用。目前对于乙酰化酶和去乙酰化酶的研究还基本上是传统的单个蛋白的研究方法。利用基因组的数据,系统化地研究乙酰化酶和去乙酰化酶,一方面可以扩展对乙酰化酶和去乙酰化酶功能的了解,另一方面如果能成功地系统化地进行乙酰化酶和去乙酰化酶的研究,完全可以扩大到激酶和酯酶等其它较大的蛋白家族或超家族的研究。 GNAT家族是蛋白乙酰化酶中极为重要的一个家族。研究表明,该家族的乙酰化酶参与了细胞生长调控、基因转录激活和DNA损伤修复。本研究在对人类乙酰化酶和去乙酰化酶的研究过程中,得到了以下结果: 1、通过搜索比对等生物信息学方法系统地归纳和整理了人类乙酰化酶和去乙酰化酶,并构建了它们的系统发生树,为进一步研究功能打下了基础。 2、建立起一个快速、高效地研究人类乙酰化酶和去乙酰化酶的平台。 3、在对乙酰化酶GNAT家族进行研究分析的过程中,找到一个人GNAT家族的新成员MAK3。MAK3含有一个保守的乙酰化酶结构域,并且不同物种中的MAK3蛋白序列高度保守。我们通过半定量和定量RT-PCR检测了在不同组织中MAK3的mRNA水平。并且把MAK3分别克隆到了真核和原核表达质粒中,通过免疫印迹技术证实了MAK3蛋白的表达。我们还进一步用亲和层析的方法成功纯化了细菌中表达的MAK3蛋白,并对纯化后的MAK3蛋白的酶活性进行了测定。这些研究结果为进一步深入研究MAK3的生物功能打下了基础。 4、在对乙酰化酶GNAT家族进行研究分析的过程中,找到另一个人GNAT家族的新成员FLJ13848。FLJ13848含有一个保守的乙酰化酶结构域,并且不同物种中的FLJ13848蛋白序列高度保守。我们把FLJ13848分别克隆到了真核和原核表达质粒中,通过免疫印迹技术证实了FLJ13848蛋白的表达。并且用免疫染色的方法观察到了FLJ13848在真核细胞内的定位。我们还进一步用亲和层析的方法成功纯化了细菌中表达的FLJ13848蛋白,并对纯化后的FLJ13848蛋白的酶活性进行了测定。另外,FLJ13848对转录因子AP-1的激活作用同时被检测到。这些研究结果为进一步深入研究FLJ13848的生物功能打下了基础。
[Abstract]:The genome sequencing of nearly 100 species, including the human genome, has been basically completed. How to use the data from these genomes to conduct large-scale research on proteins, It has become a great challenge in modern biology. At present, the main research contents and methods of proteomics include the study of protein expression profile and the intracellular localization of protein. Studies on protein interactions and signaling pathways, as well as on the biological functions of proteins. Acetylases and deacetylase play an important role in the process of life activity. At present, the study of acetylases and deacetylases is basically a traditional method of studying individual proteins. The systematic study of acetylase and deacetylase can, on the one hand, expand the understanding of the functions of acetylase and deacetylase, on the other hand, if the study of acetylase and deacetylase is successful and systematic, It can be extended to other larger protein families, such as kinase and esterase, or superfamily. The GNAT family is a very important family of protein acetylases. It has been shown that the GNAT family is involved in cell growth regulation, gene transcription activation and DNA damage repair. During the study of human acetylase and deacetylase, the following results were obtained:. 1. Human acetylase and deacetylase were systematically summarized and sorted by searching and comparing bioinformatics methods, and their phylogenetic tree was constructed, which laid a foundation for further research. A rapid and efficient platform for the study of human acetylase and deacetylase was established. 3. In the course of studying and analyzing the GNAT family of acetylases, we found that a new member of the GNAT family, MAK3.MAK3, contains a conserved domain of acetylase. The MAK3 protein sequences in different species were highly conserved. We detected the mRNA level of MAK3 in different tissues by semi-quantitative and quantitative RT-PCR, and cloned MAK3 into eukaryotic and prokaryotic expression plasmids, respectively. The expression of MAK3 protein was confirmed by Western blotting. We further purified the expressed MAK3 protein by affinity chromatography. The enzyme activity of purified MAK3 protein was determined. These results laid a foundation for further study on the biological function of MAK3. 4. In the course of studying and analyzing the GNAT family of acetylases, we found that another new member of the GNAT family, FLJ13848.FLJ13848, contains a conserved domain of acetylase. The sequence of FLJ13848 protein in different species was highly conserved. We cloned FLJ13848 into eukaryotic and prokaryotic expression plasmids, respectively. The expression of FLJ13848 protein was confirmed by Western blotting, and the localization of FLJ13848 in eukaryotic cells was observed by immunostaining. We further purified the expressed FLJ13848 protein by affinity chromatography. The enzyme activity of purified FLJ13848 protein was also determined. In addition, the activation of transcription factor AP-1 by FLJ13848 was also detected. These results laid a foundation for further study on the biological function of FLJ13848.
【学位授予单位】：四川农业大学
【学位级别】：硕士
【学位授予年份】：2005
【分类号】：R341

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