S-periaxin蛋白分子聚合狀態(tài)的研究
發(fā)布時間：2018-10-16 21:42
【摘要】：Periaxin蛋白是有髓施旺細(xì)胞特異表達(dá)的一種蛋白,該蛋白對維持髓鞘的穩(wěn)定起著重要的作用,其突變將導(dǎo)致腓骨肌萎縮癥4F亞型的發(fā)生。Periaxin基因由于mRNA不同的剪切方式可以編碼兩種長短不同的含PDZ結(jié)構(gòu)域的蛋白,即L-periaxin和S-periaxin。L-periaxin具有PDZ domain、核輸入信號NLS、Repeat domain、Acidic domain四個結(jié)構(gòu)域,S-periaxin只含有PDZ結(jié)構(gòu)域。雖然periaxin蛋白發(fā)現(xiàn)已經(jīng)20多年,但S-periaxin蛋白的結(jié)構(gòu)和功能仍不清楚。本文圍繞S-periaxin蛋白展開以下工作。首先以大鼠來源的RSC96細(xì)胞的cDNA為模板,進(jìn)行PCR擴(kuò)增S-periaxin基因,將其克隆至表達(dá)載體pET-M-3C上。利用定點突變技術(shù)對S-periaxin的半胱氨酸殘基進(jìn)行突變,從而獲得了不同的突變體pET-M-3C-S-periaxm (C88/G、C97/G、C139/G、C88、97/G、C88、139/G、 C97、139/G、C88、97、139/G)。將重組質(zhì)粒轉(zhuǎn)入Ecoli BL 21中,利用IPTG誘導(dǎo)表達(dá),重組表達(dá)產(chǎn)物經(jīng)Ni-NTA、Sephacryl S-200凝膠層析獲得重組蛋白His-S-periaxin及其突變體蛋白。戊二醛交聯(lián)分析體外His-S-periaxin蛋白的聚合狀態(tài),表明S-periaxin蛋白在體外易于形成一系列不同聚合度的同源聚合物。進(jìn)一步免疫共沉淀也表明S-periaxin蛋白存在同源蛋白間互作。另外利用H202氧化、DTT還原、非還原SDS-聚丙烯酰胺凝膠電泳等方法分析,結(jié)果顯示在氧化條件下,His-S-periaxin蛋白形成二聚體,此二聚體可以被DTT重新還原成單體。對S-periaxin中三個Cys進(jìn)行分析發(fā)現(xiàn),半胱氨酸殘基(Cys)參與了二聚體的形成且Cys88和Cys139對氧化壓力的敏感度比Cys97高。其次,通過PCR擴(kuò)增mCherryl-159和mCherry 160-237兩片段,將其分別連接到pQE-30、pET-28a載體上構(gòu)建了基于mCherry的紅色雙分子熒光互補(bǔ)原核系統(tǒng),并利用GFP蛋白間微弱的相互作用驗證雙分子熒光互補(bǔ)原核系統(tǒng)的可行性,進(jìn)一步將S-periaxin及其突變體基因分別連接到雙分子熒光互補(bǔ)原核載體上,結(jié)果顯示在大腸桿菌細(xì)胞內(nèi)S-periaxin蛋白也是通過半胱氨酸殘基互作形成二聚體。此外我們將mCherry1-159和mCherry 160-237分別克隆到載體pEGFP-N1、pEGFP-C1上構(gòu)建了基于mCherry的紅色雙分子熒光互補(bǔ)真核系統(tǒng),將S-periaxin及其突變體基因連入該系統(tǒng),結(jié)果顯示S-periaxin蛋白在RSC96細(xì)胞內(nèi)發(fā)生同源蛋白間互作。
[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.
【學(xué)位授予單位】：山西大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2014
【分類號】：R746.4
本文編號：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.
【學(xué)位授予單位】：山西大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2014
【分類號】：R746.4
【參考文獻(xiàn)】
相關(guān)期刊論文 前1條
1 樊晉宇;崔宗強(qiáng);張先恩;;雙分子熒光互補(bǔ)技術(shù)[J];中國生物化學(xué)與分子生物學(xué)報;2008年08期
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