S-periaxin蛋白分子聚合状态的研究
发布时间：2018-10-16 21:42
【摘要】：Periaxin蛋白是有髓施旺细胞特异表达的一种蛋白,该蛋白对维持髓鞘的稳定起着重要的作用,其突变将导致腓骨肌萎缩症4F亚型的发生。Periaxin基因由于mRNA不同的剪切方式可以编码两种长短不同的含PDZ结构域的蛋白,即L-periaxin和S-periaxin。L-periaxin具有PDZ domain、核输入信号NLS、Repeat domain、Acidic domain四个结构域,S-periaxin只含有PDZ结构域。虽然periaxin蛋白发现已经20多年,但S-periaxin蛋白的结构和功能仍不清楚。本文围绕S-periaxin蛋白展开以下工作。首先以大鼠来源的RSC96细胞的cDNA为模板,进行PCR扩增S-periaxin基因,将其克隆至表达载体pET-M-3C上。利用定点突变技术对S-periaxin的半胱氨酸残基进行突变,从而获得了不同的突变体pET-M-3C-S-periaxm (C88/G、C97/G、C139/G、C88、97/G、C88、139/G、 C97、139/G、C88、97、139/G)。将重组质粒转入Ecoli BL 21中,利用IPTG诱导表达,重组表达产物经Ni-NTA、Sephacryl S-200凝胶层析获得重组蛋白His-S-periaxin及其突变体蛋白。戊二醛交联分析体外His-S-periaxin蛋白的聚合状态,表明S-periaxin蛋白在体外易于形成一系列不同聚合度的同源聚合物。进一步免疫共沉淀也表明S-periaxin蛋白存在同源蛋白间互作。另外利用H202氧化、DTT还原、非还原SDS-聚丙烯酰胺凝胶电泳等方法分析,结果显示在氧化条件下,His-S-periaxin蛋白形成二聚体,此二聚体可以被DTT重新还原成单体。对S-periaxin中三个Cys进行分析发现,半胱氨酸残基(Cys)参与了二聚体的形成且Cys88和Cys139对氧化压力的敏感度比Cys97高。其次,通过PCR扩增mCherryl-159和mCherry 160-237两片段,将其分别连接到pQE-30、pET-28a载体上构建了基于mCherry的红色双分子荧光互补原核系统,并利用GFP蛋白间微弱的相互作用验证双分子荧光互补原核系统的可行性,进一步将S-periaxin及其突变体基因分别连接到双分子荧光互补原核载体上,结果显示在大肠杆菌细胞内S-periaxin蛋白也是通过半胱氨酸残基互作形成二聚体。此外我们将mCherry1-159和mCherry 160-237分别克隆到载体pEGFP-N1、pEGFP-C1上构建了基于mCherry的红色双分子荧光互补真核系统,将S-periaxin及其突变体基因连入该系统,结果显示S-periaxin蛋白在RSC96细胞内发生同源蛋白间互作。
[Abstract]:Periaxin protein is a protein specifically expressed by myelinated Schwann cells and plays an important role in maintaining the stability of myelin sheath. The mutation will lead to the development of 4F subtype in fibula muscular atrophy. Periaxin gene can encode two kinds of proteins with different length and length of PDZ domain because of the different shearing mode of mRNA. That is, L-periaxin and S-periaxin.L-periaxin have four domains of PDZ domain, kernel input signal NLS,Repeat domain,Acidic domain, and S-periaxin contains only PDZ domain. Although periaxin protein has been discovered for more than 20 years, the structure and function of S-periaxin protein are still unclear. In this paper, the following work is carried out around S-periaxin protein. Firstly, the cDNA of rat RSC96 cells was used as template, and the S-periaxin gene was amplified by PCR and cloned into the expression vector pET-M-3C. By using site-directed mutation technique, the cysteine residues of S-periaxin were mutated and different mutants pET-M-3C-S-periaxm were obtained. The recombinant plasmid was transferred into Ecoli BL 21 and expressed by IPTG. The recombinant protein His-S-periaxin and its mutant protein were obtained by Ni-NTA,Sephacryl S-200 gel chromatography. Glutaraldehyde crosslinking analysis showed that S-periaxin protein could easily form a series of homologous polymers with different degree of polymerization in vitro. Further immunoprecipitation also showed that S-periaxin protein had homologous protein interaction. In addition, H202 oxidation, DTT reduction, non-reduced SDS- polyacrylamide gel electrophoresis and other methods were used. The results showed that the His-S-periaxin protein formed dimer under the oxidation condition, and the dimer could be rereduced to monomer by DTT. By analyzing three Cys in S-periaxin, it was found that cysteine residue (Cys) was involved in the formation of dimer and that Cys88 and Cys139 were more sensitive to oxidation pressure than Cys97. Secondly, two fragments of mCherryl-159 and mCherry 160-237 were amplified by PCR and ligated to pQE-30,pET-28a vector to construct a red bimolecular fluorescent complementary prokaryotic system based on mCherry. The feasibility of bimolecular fluorescence complementary prokaryotic system was verified by weak interaction between GFP proteins. Furthermore, S-periaxin and its mutant genes were linked to bimolecular fluorescent complementary prokaryotic vectors, respectively. The results showed that S-periaxin protein also formed dimer through cysteine residue interaction in E. coli cells. In addition, mCherry1-159 and mCherry 160-237 were cloned into the vector pEGFP-N1,pEGFP-C1 to construct the red bimolecular fluorescence complementary eukaryotic system based on mCherry. The S-periaxin and its mutant genes were inserted into the system. The results showed that the S-periaxin protein interacted with each other in RSC96 cells.
【学位授予单位】：山西大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R746.4
本文编号：2275706
[Abstract]:Periaxin protein is a protein specifically expressed by myelinated Schwann cells and plays an important role in maintaining the stability of myelin sheath. The mutation will lead to the development of 4F subtype in fibula muscular atrophy. Periaxin gene can encode two kinds of proteins with different length and length of PDZ domain because of the different shearing mode of mRNA. That is, L-periaxin and S-periaxin.L-periaxin have four domains of PDZ domain, kernel input signal NLS,Repeat domain,Acidic domain, and S-periaxin contains only PDZ domain. Although periaxin protein has been discovered for more than 20 years, the structure and function of S-periaxin protein are still unclear. In this paper, the following work is carried out around S-periaxin protein. Firstly, the cDNA of rat RSC96 cells was used as template, and the S-periaxin gene was amplified by PCR and cloned into the expression vector pET-M-3C. By using site-directed mutation technique, the cysteine residues of S-periaxin were mutated and different mutants pET-M-3C-S-periaxm were obtained. The recombinant plasmid was transferred into Ecoli BL 21 and expressed by IPTG. The recombinant protein His-S-periaxin and its mutant protein were obtained by Ni-NTA,Sephacryl S-200 gel chromatography. Glutaraldehyde crosslinking analysis showed that S-periaxin protein could easily form a series of homologous polymers with different degree of polymerization in vitro. Further immunoprecipitation also showed that S-periaxin protein had homologous protein interaction. In addition, H202 oxidation, DTT reduction, non-reduced SDS- polyacrylamide gel electrophoresis and other methods were used. The results showed that the His-S-periaxin protein formed dimer under the oxidation condition, and the dimer could be rereduced to monomer by DTT. By analyzing three Cys in S-periaxin, it was found that cysteine residue (Cys) was involved in the formation of dimer and that Cys88 and Cys139 were more sensitive to oxidation pressure than Cys97. Secondly, two fragments of mCherryl-159 and mCherry 160-237 were amplified by PCR and ligated to pQE-30,pET-28a vector to construct a red bimolecular fluorescent complementary prokaryotic system based on mCherry. The feasibility of bimolecular fluorescence complementary prokaryotic system was verified by weak interaction between GFP proteins. Furthermore, S-periaxin and its mutant genes were linked to bimolecular fluorescent complementary prokaryotic vectors, respectively. The results showed that S-periaxin protein also formed dimer through cysteine residue interaction in E. coli cells. In addition, mCherry1-159 and mCherry 160-237 were cloned into the vector pEGFP-N1,pEGFP-C1 to construct the red bimolecular fluorescence complementary eukaryotic system based on mCherry. The S-periaxin and its mutant genes were inserted into the system. The results showed that the S-periaxin protein interacted with each other in RSC96 cells.
【学位授予单位】：山西大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R746.4
【参考文献】
相关期刊论文 前1条
1 樊晋宇;崔宗强;张先恩;;双分子荧光互补技术[J];中国生物化学与分子生物学报;2008年08期
，本文编号：2275706
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