發(fā)布時(shí)間：2018-06-17 11:47

  本文選題：釀酒酵母菌 + HMO2��； 參考：《重慶醫(yī)科大學(xué)》2014年碩士論文

【摘要】：背景高速泳動(dòng)族蛋白（HMGB）是一類(lèi)廣泛存在于真核生物的非組蛋白家族，有著高度保守的三維結(jié)構(gòu)，在轉(zhuǎn)錄調(diào)控、DNA修復(fù)、基因重組、細(xì)胞分化和胞外信號(hào)轉(zhuǎn)導(dǎo)等過(guò)程中扮演著重要的角色。作為HMGB蛋白質(zhì)超家族的成員之一，HMO2在進(jìn)化上高度保守且分布廣泛提示該蛋白質(zhì)具有重要的生物學(xué)功能，如ATP依賴(lài)性染色體重建、DNA損傷修復(fù)等。目前，釀酒酵母菌HMO2蛋白的三維結(jié)構(gòu)還沒(méi)有得到解析，其具體的功能機(jī)制也尚不清楚。 目的解析釀酒酵母菌重要蛋白質(zhì)HMO2的三維結(jié)構(gòu)，并基于其結(jié)構(gòu)研究功能，進(jìn)而了解其具體的功能機(jī)制，為進(jìn)一步的染色體重建和DNA損傷修復(fù)研究打下基礎(chǔ)。 方法將HMO2基因重組到pET22b載體上，構(gòu)建pET22b-HMO2重組表達(dá)質(zhì)粒，用大腸桿菌原核表達(dá)系統(tǒng)表達(dá)該蛋白，通過(guò)金屬離子親和層析柱（Ni-NTA）、陰離子交換層析（Resource Q）和凝膠排阻層析（分子篩Superdex200）進(jìn)行純化，，得到足夠純度的目的蛋白。用分子篩分析蛋白在溶液中的聚集狀態(tài)。通過(guò)氣相擴(kuò)散法，培養(yǎng)蛋白質(zhì)晶體，用X-射線(xiàn)衍射儀收集晶體衍射數(shù)據(jù)，再進(jìn)行數(shù)據(jù)分析處理以解析晶體三維結(jié)構(gòu)。 結(jié)果通過(guò)分子克隆技術(shù)成功構(gòu)建了重組質(zhì)粒pET22b-HMO2；通過(guò)蛋白表達(dá)純化相關(guān)技術(shù)成功獲得了純度達(dá)98%的目的蛋白質(zhì)HMO2；凝膠排阻層析（Superdex200）結(jié)果顯示HMO2蛋白質(zhì)在溶液中以二聚體形式存在。培養(yǎng)出晶型好、偏光能力強(qiáng)的母體蛋白質(zhì)單晶。在上海同步輻射光源收到晶體的衍射數(shù)據(jù)以供結(jié)構(gòu)解析使用。HMO2晶體屬于空間群P222或P212121，晶胞參數(shù)為a=39.35, b=75.69,c=108.03。根據(jù)HMO2的空間群和分子量（約24kDa），以及溶劑含量分析提示，每個(gè)晶體學(xué)不對(duì)稱(chēng)單位含有1個(gè)蛋白質(zhì)分子，表明一個(gè)VM值3.193Da-1。但是，多波長(zhǎng)反常散射方法仍沒(méi)能成功解析出蛋白質(zhì)三維結(jié)構(gòu)。
[Abstract]:Background High Speed Swimming Group protein (HMGB) is a nonhistone family of eukaryotes, with highly conserved three-dimensional structure, DNA repair and gene recombination in transcriptional regulation. Cell differentiation and extracellular signal transduction play an important role. As a member of the HMGB protein superfamily, HMO2 is highly conserved and widely distributed in evolution, which indicates that the HMGB protein has important biological functions, such as ATP-dependent chromosome reconstruction and DNA repair. At present, the three-dimensional structure of HMO2 protein of Saccharomyces cerevisiae has not been elucidated, and its specific functional mechanism is still unclear. Objective to analyze the three-dimensional structure of HMO2, an important protein of Saccharomyces cerevisiae, and to understand its specific functional mechanism based on its structure and function, so as to lay a foundation for further studies on chromosome reconstruction and DNA damage repair. Methods the recombinant expression plasmid pET22b-HMO2 was constructed by recombination of HMO2 gene into pET22b vector and expressed in E. coli prokaryotic expression system. The target protein was purified by metal ion affinity chromatography column (Ni-NTAA), anion exchange chromatography (Resource Q) and gel exclusion chromatography (molecular sieve Superdex200). Molecular sieve was used to analyze the aggregation state of proteins in solution. The protein crystals were cultured by gas phase diffusion method. The crystal diffraction data were collected by X-ray diffractometer, and the data were analyzed and processed to analyze the three-dimensional structure of the crystals. Results the recombinant plasmid pET22b-HMO2 was successfully constructed by molecular cloning, and the target protein HMO2 with 98% purity was obtained by protein expression and purification. The results of gel exclusion chromatography showed that HMO2 protein existed in the form of dimer in solution. The parent protein single crystals with good crystal form and strong polarizing ability were cultured. Diffraction data of crystals were received at Shanghai synchrotron radiation source for structural analysis. HMO2 crystals belong to the space group P222 or P212121. The cell parameters of the crystals are AZ39.35 and BX 75.69 cnc108.03. According to the spatial group and molecular weight of HMO2 (about 24 kDa) and the solvent content analysis, each crystallographic asymmetric unit contains one protein molecule, indicating a VM value of 3.193 Da-1. However, the multi-wavelength anomalous scattering method has not been able to successfully analyze the three-dimensional structure of protein.
【學(xué)位授予單位】：重慶醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2014
【分類(lèi)號(hào)】：R378

【共引文獻(xiàn)】

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1 王玉嬌;擬南芥AtHMGB15基因在花粉管萌發(fā)生長(zhǎng)過(guò)程中的功能研究[D];中國(guó)農(nóng)業(yè)大學(xué);2014年

相關(guān)碩士學(xué)位論文 前2條

1 陳艷麗;酶催化直接不對(duì)稱(chēng)aldol反應(yīng)和Biginelli反應(yīng)的研究[D];西南大學(xué);2013年

2 楊春蘭;胸苷酸合成酶基因多態(tài)性與兒童急性白血病易感性及HD-MTX毒副作用的相關(guān)性[D];重慶醫(yī)科大學(xué);2013年

本文編號(hào)：2030931