二硫鍵對人朊蛋白生化特性和功能的影響
發(fā)布時間：2018-08-20 16:28
【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，首先通過體外對純化的原核重組人正常朊蛋白硫醇基團的氧化還原過程，測定二硫鍵的改變對其生化特性的影響；進而探究了N端八肽重復區(qū)序列對二硫鍵形成的硫醇基團的氧化還原過程介導的聚集和纖維化的敏感性，最后進行了部分二硫鍵相關突變體質粒的構建，和真核細胞轉染。蛋白沉淀實驗顯示重組正常人朊病毒蛋白經過硫醇基團的氧化還原過程明顯增加了其聚集性，而八肽重復區(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)實驗測定發(fā)現(xiàn)，經過硫醇基團的氧化還原過程重組PrP蛋白的纖維形成增多，同樣的八肽重復區(qū)突變體蛋白無明顯變化；圓二色譜（Circular Dichroism,CD）測定顯示，處理后的重組PrP蛋白二級結構發(fā)生改變，其β-折疊結構比例顯著增多，八肽重復區(qū)突變體蛋白的β-折疊則減少；蛋白酶K消化實驗也進一步顯示硫醇基團的氧化還原后PrP的蛋白酶K抵抗能力有所增加，而八肽重復區(qū)突變體蛋白的蛋白酶K抗性增加更明顯。這些結果提示二硫鍵的形成可明顯地改變PrP的二級結構，促進朊蛋白聚集和成纖維過程，而且正常的5個八肽重復區(qū)序列對蛋白穩(wěn)定的構象轉變是必需的。為進一步研究二硫鍵的作用，構建了二硫鍵相關的突變體質粒pQE30-C214G、pQE30-C179G/C214G、pQE30-M166C/E221C，并進行了測序和酶切鑒定。正在進行的和后續(xù)實驗準備通過原核表達、純化并進行硫醇基團的氧化還原過程，進行上述參數測定；并且在真核細胞中轉染構建的二硫鍵相關突變質粒pcDNA3.1-C179G/C214G、pcDNA3.1-M166C/E221C，測定可能引起的細胞毒性和細胞保護作用。
[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.
【學位授予單位】：河北大學
【學位級別】：碩士
【學位授予年份】：2012
【分類號】：R346
本文編號：2194270
[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.
【學位授予單位】：河北大學
【學位級別】：碩士
【學位授予年份】：2012
【分類號】：R346
【共引文獻】
相關期刊論文 前1條
1 孫晗;石琦;王紹彬;郭飛;解武玲;陳操;劉存歧;董小平;;硫醇基團的氧化還原過程對人正常朊蛋白聚集和纖維化特征的影響[J];病毒學報;2012年04期
相關博士學位論文 前1條
1 王朝云;利用RNAi干擾PrP及其突變體的研究[D];中國疾病預防控制中心;2011年
相關碩士學位論文 前1條
1 任科;膜蛋白Flotillin-1與細胞膜朊蛋白PrP~C內吞相關性研究[D];中國疾病預防控制中心;2013年
，本文編號：2194270
本文鏈接：http://sikaile.net/xiyixuelunwen/2194270.html
最近更新
教材專著
