鞘糖脂Gb3的配体结构对其胞内运输的影响

发布时间：2018-01-04 20:35

本文关键词：鞘糖脂Gb3的配体结构对其胞内运输的影响　出处：《东北师范大学》2007年硕士论文　论文类型：学位论文

【摘要】： 志贺(氏)菌毒素(Shiga toxin)是主要由痢疾志贺菌(Shigella dysenteriae)产生的一种重要的致病毒力因子,由具有酶活性的A亚基和结合作用的B亚基两部分构成。志贺(氏)菌毒素的受体为鞘糖脂Gb3(globotriaosylceramide),其广泛存在于哺乳动物细胞膜表面。志贺(氏)菌毒素经过复杂的胞吞途径进入细胞内部杀死细胞。志贺(氏)菌毒素与细胞表面的Gb3受体结合后,经过逆转运方式进入内质网,再由内质网最终到达细胞质中。在细胞质之中,志贺(氏)菌毒素的酶活部分使28S rRNA失活,从而抑制蛋白的合成。但是,目前的研究对于毒素在胞内转运的机制还有很多不清楚的地方。本论文旨在研究志贺(氏)菌毒素B亚基N端的作用,通过基因克隆的方法对B亚基N端进行修饰,并对修饰后B亚基的胞内运输进行研究。本论文首先以pGlyc-KDEL质粒为模板扩增出STxB的基因序列,并将其连接到带有GST的pGEX-4T-1骨架载体上,构建原核表达载体GST-STxB-pGEX-4T-1,并在大肠杆菌DH5α中通过IPTG诱导表达,然后利用谷胱苷肽Sepharose 4B凝胶亲和层析对重组的蛋白进行纯化。纯化的蛋白采用SDS-PAGE和Western blot进行检测,并且用高效液相Bio-sil SEC 250凝胶柱对重组的蛋白进行分析,以确定其多聚体结构。我们对重组纯化后的GST-STxB蛋白进行了胞内运输的研究。首先将N端修饰的蛋白GST-STxB和未做修饰的蛋白STxB(对照),分别与Hela细胞在4℃培养30min和37℃培养120min,然后利用间接免疫荧光法在荧光显微镜下观察蛋白的位置,结构表明N端修饰的GST-STxB蛋白的胞内运输受到严重影响,说明志贺(氏)菌毒素B亚基N端的结构,对其胞内运输起着重要的作用。流式细胞仪检测发现,GST-STxB蛋白与细胞表面受体Gb3结合率的下降是导致转运障碍的直接原因。
[Abstract]:Shiga toxin (s) (Shiga toxin) is mainly composed of Shigella dysenteriae (Shigella dysenteriae) is an important virulence factor production, is composed of two parts: a A subunit with enzyme activity and binding of the B subunit. Shiga toxin receptor (s) for glycosphingolipid Gb3 (globotriaosylceramide), which widely exists in the membrane of mammalian cells. Shiga toxin (s) after endocytosis complex inside the cell to kill cells. Shiga (Rockwell) combined with toxin and Gb3 receptors on the cell surface, through the reverse transport into the endoplasmic reticulum, the endoplasmic reticulum reach the cytoplasm in the cytoplasm, Shiga (Rockwell) part of the 28S inactivation of rRNA bacteria toxin enzyme, thereby inhibiting protein synthesis. However, the current study for many unclear mechanism of transport toxins in the cell.
The purpose of this study is to study the function of N terminal of shigella toxin B subunit, and to modify the N terminal of B subunit by gene cloning, and to study the intracellular transport of modified B subunit.
In this paper, pGlyc-KDEL was amplified from STxB gene sequence, and connect it to the pGEX-4T-1 with backbone vector GST, prokaryotic expression vector GST-STxB-pGEX-4T-1 was constructed and transformed into Escherichia coli DH5 alpha induced expression by IPTG, then using glutathione Sepharose 4B gel was purified by affinity chromatography on recombinant protein. The purified protein by SDS-PAGE and Western blot were detected by high performance liquid chromatography and Bio-sil SEC gel column of 250 recombinant proteins were analyzed to determine its multimeric structure.
We investigated the intracellular transport of recombinant purified GST-STxB protein. The N end of the modified protein GST-STxB and modified protein STxB (control), 30min and 120min were cultured in culture 37 C 4 C and Hela cells, and then observe the position of the protein under fluorescence microscopy using indirect immunofluorescence method the structure, showed that the N N-terminal modified GST-STxB protein intracellular transport have been seriously affected, that ('s) structure of Shiga toxin B subunit bacteria N end, plays an important role in the intracellular transport. Flow cytometry showed that GST-STxB protein and cell surface receptor Gb3 binding rate is decreased direct cause of transport barriers.

【学位授予单位】：东北师范大学
【学位级别】：硕士
【学位授予年份】：2007
【分类号】：R378

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