二硫键对人朊蛋白生化特性和功能的影响
发布时间：2018-08-20 16:28
【摘要】：本研究主要围绕二硫键在人朊病毒蛋白分子huPrP23-231中的作用展开，首先通过体外对纯化的原核重组人正常朊蛋白硫醇基团的氧化还原过程，测定二硫键的改变对其生化特性的影响；进而探究了N端八肽重复区序列对二硫键形成的硫醇基团的氧化还原过程介导的聚集和纤维化的敏感性，最后进行了部分二硫键相关突变体质粒的构建，和真核细胞转染。蛋白沉淀实验显示重组正常人朊病毒蛋白经过硫醇基团的氧化还原过程明显增加了其聚集性，而八肽重复区缺失和插入型突变体蛋白则没有明显变化；硫磺素T(Thioflavin T，ThT)实验测定发现，经过硫醇基团的氧化还原过程重组PrP蛋白的纤维形成增多，同样的八肽重复区突变体蛋白无明显变化；圆二色谱（Circular Dichroism,CD）测定显示，处理后的重组PrP蛋白二级结构发生改变，其β-折叠结构比例显著增多，八肽重复区突变体蛋白的β-折叠则减少；蛋白酶K消化实验也进一步显示硫醇基团的氧化还原后PrP的蛋白酶K抵抗能力有所增加，而八肽重复区突变体蛋白的蛋白酶K抗性增加更明显。这些结果提示二硫键的形成可明显地改变PrP的二级结构，促进朊蛋白聚集和成纤维过程，而且正常的5个八肽重复区序列对蛋白稳定的构象转变是必需的。为进一步研究二硫键的作用，构建了二硫键相关的突变体质粒pQE30-C214G、pQE30-C179G/C214G、pQE30-M166C/E221C，并进行了测序和酶切鉴定。正在进行的和后续实验准备通过原核表达、纯化并进行硫醇基团的氧化还原过程，进行上述参数测定；并且在真核细胞中转染构建的二硫键相关突变质粒pcDNA3.1-C179G/C214G、pcDNA3.1-M166C/E221C，测定可能引起的细胞毒性和细胞保护作用。
[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.
【学位授予单位】：河北大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R346
本文编号：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.
【学位授予单位】：河北大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R346
【共引文献】
相关期刊论文 前1条
1 孙晗;石琦;王绍彬;郭飞;解武玲;陈操;刘存歧;董小平;;硫醇基团的氧化还原过程对人正常朊蛋白聚集和纤维化特征的影响[J];病毒学报;2012年04期
相关博士学位论文 前1条
1 王朝云;利用RNAi干扰PrP及其突变体的研究[D];中国疾病预防控制中心;2011年
相关硕士学位论文 前1条
1 任科;膜蛋白Flotillin-1与细胞膜朊蛋白PrP~C内吞相关性研究[D];中国疾病预防控制中心;2013年
，本文编号：2194270
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