【摘要】：本研究主要圍繞二硫鍵在人朊病毒蛋白分子huPrP23-231中的作用展開，首先通過(guò)體外對(duì)純化的原核重組人正常朊蛋白硫醇基團(tuán)的氧化還原過(guò)程，測(cè)定二硫鍵的改變對(duì)其生化特性的影響；進(jìn)而探究了N端八肽重復(fù)區(qū)序列對(duì)二硫鍵形成的硫醇基團(tuán)的氧化還原過(guò)程介導(dǎo)的聚集和纖維化的敏感性，最后進(jìn)行了部分二硫鍵相關(guān)突變體質(zhì)粒的構(gòu)建，和真核細(xì)胞轉(zhuǎn)染。蛋白沉淀實(shí)驗(yàn)顯示重組正常人朊病毒蛋白經(jīng)過(guò)硫醇基團(tuán)的氧化還原過(guò)程明顯增加了其聚集性，而八肽重復(fù)區(qū)缺失和插入型突變體蛋白則沒有明顯變化；硫磺素T(Thioflavin T，ThT)實(shí)驗(yàn)測(cè)定發(fā)現(xiàn)，經(jīng)過(guò)硫醇基團(tuán)的氧化還原過(guò)程重組PrP蛋白的纖維形成增多，同樣的八肽重復(fù)區(qū)突變體蛋白無(wú)明顯變化；圓二色譜（Circular Dichroism,CD）測(cè)定顯示，處理后的重組PrP蛋白二級(jí)結(jié)構(gòu)發(fā)生改變，其β-折疊結(jié)構(gòu)比例顯著增多，八肽重復(fù)區(qū)突變體蛋白的β-折疊則減少；蛋白酶K消化實(shí)驗(yàn)也進(jìn)一步顯示硫醇基團(tuán)的氧化還原后PrP的蛋白酶K抵抗能力有所增加，而八肽重復(fù)區(qū)突變體蛋白的蛋白酶K抗性增加更明顯。這些結(jié)果提示二硫鍵的形成可明顯地改變PrP的二級(jí)結(jié)構(gòu)，促進(jìn)朊蛋白聚集和成纖維過(guò)程，而且正常的5個(gè)八肽重復(fù)區(qū)序列對(duì)蛋白穩(wěn)定的構(gòu)象轉(zhuǎn)變是必需的。為進(jìn)一步研究二硫鍵的作用，構(gòu)建了二硫鍵相關(guān)的突變體質(zhì)粒pQE30-C214G、pQE30-C179G/C214G、pQE30-M166C/E221C，并進(jìn)行了測(cè)序和酶切鑒定。正在進(jìn)行的和后續(xù)實(shí)驗(yàn)準(zhǔn)備通過(guò)原核表達(dá)、純化并進(jìn)行硫醇基團(tuán)的氧化還原過(guò)程，進(jìn)行上述參數(shù)測(cè)定；并且在真核細(xì)胞中轉(zhuǎn)染構(gòu)建的二硫鍵相關(guān)突變質(zhì)粒pcDNA3.1-C179G/C214G、pcDNA3.1-M166C/E221C，測(cè)定可能引起的細(xì)胞毒性和細(xì)胞保護(hù)作用。
[Abstract]:This study focused on the role of disulfide bond in human prion protein molecule huPrP23-231. Firstly, the effect of the change of disulfide bond on the biochemical properties of purified human normal prion protein mercaptan was determined by redox process in vitro. Furthermore, the sensitivity of N-terminal octapeptide repeat sequence to the redox process of mercaptan group formed by disulfide bond was investigated. Finally, the partial disulfide bond associated mutants were constructed and transfected into eukaryotic cells. Protein precipitation test showed that recombinant human prion protein increased its aggregation through mercaptan redox process, while octapeptide repeats deletion and insertion mutant protein did not change significantly. It was found that the fiber formation of the recombinant PrP protein increased during the redox of mercaptan group, but the same octapeptide repeat mutant protein did not change, and the Circular dichroism (CD) assay showed that the recombined PrP protein had no significant change after the redox of mercaptan group. After treatment, the secondary structure of recombinant PrP protein changed, the proportion of 尾 -fold structure increased significantly, and the 尾 -folding of octapeptide repeat mutant protein decreased. The protease K digestion experiment also further showed that the protease K resistance of PrP was increased after redox of mercaptan group, but the protease K resistance of octapeptide repeat mutant protein was more obvious. These results suggest that the formation of disulfide bonds can obviously change the secondary structure of PrP, promote the aggregation and fibrogenesis of prion proteins, and that the normal sequence of five octapeptide repeats is necessary for the stable conformational transition of proteins. In order to further study the role of disulfide bond, a mutant particle pQE30-C214G, pQE30-C179G / C214G / pQE30-M166C / E221C, was constructed and sequenced and digested. Ongoing and subsequent experiments were prepared to purify and redox mercaptan groups by prokaryotic expression and to determine the above parameters; The recombinant plasmid pcDNA3.1-C179G / C214GN pcDNA3.1-M166C / E221C was transfected into eukaryotic cells to determine the cytotoxicity and cytoprotective effect.
【學(xué)位授予單位】：河北大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2012
【分類號(hào)】：R346

【共引文獻(xiàn)】

相關(guān)期刊論文 前1條

1 孫晗;石琦;王紹彬;郭飛;解武玲;陳操;劉存歧;董小平;;硫醇基團(tuán)的氧化還原過(guò)程對(duì)人正常朊蛋白聚集和纖維化特征的影響[J];病毒學(xué)報(bào);2012年04期

相關(guān)博士學(xué)位論文 前1條

1 王朝云;利用RNAi干擾PrP及其突變體的研究[D];中國(guó)疾病預(yù)防控制中心;2011年

相關(guān)碩士學(xué)位論文 前1條

1 任科;膜蛋白Flotillin-1與細(xì)胞膜朊蛋白PrP~C內(nèi)吞相關(guān)性研究[D];中國(guó)疾病預(yù)防控制中心;2013年

，

本文編號(hào)：2194270