本文選題：乙�；�　切入點(diǎn)：去乙酰化酶　出處：《四川農(nóng)業(yè)大學(xué)》2005年碩士論文

【摘要】：包括人類基因組在內(nèi)的近一百個物種的基因組測序已經(jīng)基本完成。如何利用這些基因組的數(shù)據(jù),對蛋白進(jìn)行大規(guī)模的研究,已經(jīng)成為現(xiàn)代生物學(xué)所面臨的一個巨大挑戰(zhàn)。目前蛋白質(zhì)組學(xué)的主要研究內(nèi)容和方法主要有對于蛋白表達(dá)譜的研究、對于蛋白的細(xì)胞內(nèi)定位研究、對于蛋白間相互作用和信號途徑建立的研究以及對于蛋白的生物學(xué)功能的研究。 乙酰化酶和去乙�；冈谏顒舆^程中起著重要作用。目前對于乙�；负腿ヒ阴；傅难芯窟�基本上是傳統(tǒng)的單個蛋白的研究方法。利用基因組的數(shù)據(jù),系統(tǒng)化地研究乙酰化酶和去乙�；�,一方面可以擴(kuò)展對乙�；负腿ヒ阴；腹δ艿牧私�,另一方面如果能成功地系統(tǒng)化地進(jìn)行乙酰化酶和去乙�；傅难芯�,完全可以擴(kuò)大到激酶和酯酶等其它較大的蛋白家族或超家族的研究。 GNAT家族是蛋白乙酰化酶中極為重要的一個家族。研究表明,該家族的乙酰化酶參與了細(xì)胞生長調(diào)控、基因轉(zhuǎn)錄激活和DNA損傷修復(fù)。 本研究在對人類乙�；负腿ヒ阴；傅难芯窟^程中,得到了以下結(jié)果: 1、通過搜索比對等生物信息學(xué)方法系統(tǒng)地歸納和整理了人類乙酰化酶和去乙�；�,并構(gòu)建了它們的系統(tǒng)發(fā)生樹,為進(jìn)一步研究功能打下了基礎(chǔ)。 2、建立起一個快速、高效地研究人類乙�；负腿ヒ阴；傅钠脚_。 3、在對乙�；窯NAT家族進(jìn)行研究分析的過程中,找到一個人GNAT家族的新成員MAK3。MAK3含有一個保守的乙�；附Y(jié)構(gòu)域,并且不同物種中的MAK3蛋白序列高度保守。我們通過半定量和定量RT-PCR檢測了在不同組織中MAK3的mRNA水平。并且把MAK3分別克隆到了真核和原核表達(dá)質(zhì)粒中,通過免疫印跡技術(shù)證實(shí)了MAK3蛋白的表達(dá)。我們還進(jìn)一步用親和層析的方法成功純化了細(xì)菌中表達(dá)的MAK3蛋白,并對純化后的MAK3蛋白的酶活性進(jìn)行了測定。這些研究結(jié)果為進(jìn)一步深入研究MAK3的生物功能打下了基礎(chǔ)。 4、在對乙�；窯NAT家族進(jìn)行研究分析的過程中,找到另一個人GNAT家族的新成員FLJ13848。FLJ13848含有一個保守的乙�；附Y(jié)構(gòu)域,并且不同物種中的FLJ13848蛋白序列高度保守。我們把FLJ13848分別克隆到了真核和原核表達(dá)質(zhì)粒中,通過免疫印跡技術(shù)證實(shí)了FLJ13848蛋白的表達(dá)。并且用免疫染色的方法觀察到了FLJ13848在真核細(xì)胞內(nèi)的定位。我們還進(jìn)一步用親和層析的方法成功純化了細(xì)菌中表達(dá)的FLJ13848蛋白,并對純化后的FLJ13848蛋白的酶活性進(jìn)行了測定。另外,FLJ13848對轉(zhuǎn)錄因子AP-1的激活作用同時被檢測到。這些研究結(jié)果為進(jìn)一步深入研究FLJ13848的生物功能打下了基礎(chǔ)。
[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.
【學(xué)位授予單位】：四川農(nóng)業(yè)大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2005
【分類號】：R341

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