结核分枝杆菌Pup-蛋白酶体系统蛋白的表达纯化研究
发布时间：2018-08-15 14:48
【摘要】：结核分枝杆菌(Mycobacterium tuberculosis Mtb)中类泛素蛋白Pup (Prokaryotic ubiquitin-like protein)-蛋白酶体系统是胞内蛋白降解重要机制,在去酰胺酶Dop(Deamidase of Pup)和连接酶PafA(Proteasome accessory factor A)、ATP酶Mpa(Mycobacterial proteasome ATPase)等辅助因子的作用下,Pup可以共价标记多种功能蛋白,并介导被标记蛋白通过蛋白酶体降解,靶蛋白涉及物质中间代谢、信号通路、毒性和抗毒性因子、细胞壁和细胞膜组份等多个方面,并且与结核分枝杆菌的致病性相关,被认为是新的结核病治疗药物靶点,因此本领域研究具有深远的意义。 本文以Mtb为研究对象,优化并克隆出pup基因,同时克隆出dop、pafA和底物蛋白fabD基因,将这些基因加上用于纯化的标签构入表达载体,得到pGEX-2T-his6-pup, pET21cc-dop-his6, pET21cc-fabD-his6, pET21cc-pafA-his6, pBAD-pafA-his6重组表达质粒。通过重组蛋白技术,在大肠杆菌BL21中诱导表达出GST-His6-Pup、Dop-His6、FabD-His6和PafA-His6蛋白,并分别通过常规和变复性等方法纯化出这些蛋白,用蛋白免疫印迹等技术鉴定了这些蛋白,为建立Pup-蛋白酶体体外蛋白降解系统提供了蛋白。合成Pup的N端十肽免疫兔,得到能识别Pup蛋白且有较高效价的Pup多克隆抗体。对Mtb的非致病同源菌耻垢分枝杆菌(Mycobacterium smegmatis, Msm)的dop基因敲除进行了初步探索,为进一步研究Pup-蛋白酶体系统参与的细胞调控功能做准备。
[Abstract]:Ubiquitin protein Pup (Prokaryotic ubiquitin-like protein)-proteasome system is an important mechanism of intracellular protein degradation in Mycobacterium tuberculosis (Mycobacterium tuberculosis Mtb). In the presence of auxiliary factors such as deaminase Dop (Deamidase of Pup) and ligase PafA (Proteasome accessory factor A) ATPase Mpa (Mycobacterial proteasome ATPase), Pup can covalently label many functional proteins and mediate the degradation of labeled proteins through proteasome. The target proteins are involved in the intermediate metabolism of substances and the signal pathway. Toxic and antitoxic factors, cell wall and cell membrane components, and related to the pathogenicity of Mycobacterium tuberculosis, are considered as new drug targets for tuberculosis treatment, so the research in this field has far-reaching significance. In this paper, the pup gene was optimized and cloned from Mtb, and the fabD gene was cloned into the expression vector pGEX-2T-his6-puppet, pET21cc-dop-his6, pET21cc-fabD-his6, pET21cc-pafA-his6, and pBAD-pafA-his6 recombinant expression plasmid was obtained by adding the tag of these genes to the recombinant expression vector pGEX-2T-his6-puppet, pET21cc-dop-his6, pET21cc-pafA-his6, and the recombinant expression plasmid pET21cc-pafA-his6. GST-His6-PupDop-His6-Dop-His6FabD-His6 and PafA-His6 proteins were induced and expressed in E. coli BL21 by recombinant protein technique. These proteins were purified by routine and renaturation methods, and identified by Western blot. It provides protein for protein degradation system of Pup-proteasome in vitro. The N-terminal decapeptide of Pup was synthesized to immunize rabbits to obtain Pup polyclonal antibody which could recognize Pup protein and had higher titer. The knockout of the dop gene of (Mycobacterium smegmatis, Msm), a non-pathogenic homologous bacterium of Mtb, was preliminarily explored in order to further study the cellular regulatory function involved in the Pup-proteasome system.
【学位授予单位】：北京林业大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R378.911
[Abstract]:Ubiquitin protein Pup (Prokaryotic ubiquitin-like protein)-proteasome system is an important mechanism of intracellular protein degradation in Mycobacterium tuberculosis (Mycobacterium tuberculosis Mtb). In the presence of auxiliary factors such as deaminase Dop (Deamidase of Pup) and ligase PafA (Proteasome accessory factor A) ATPase Mpa (Mycobacterial proteasome ATPase), Pup can covalently label many functional proteins and mediate the degradation of labeled proteins through proteasome. The target proteins are involved in the intermediate metabolism of substances and the signal pathway. Toxic and antitoxic factors, cell wall and cell membrane components, and related to the pathogenicity of Mycobacterium tuberculosis, are considered as new drug targets for tuberculosis treatment, so the research in this field has far-reaching significance. In this paper, the pup gene was optimized and cloned from Mtb, and the fabD gene was cloned into the expression vector pGEX-2T-his6-puppet, pET21cc-dop-his6, pET21cc-fabD-his6, pET21cc-pafA-his6, and pBAD-pafA-his6 recombinant expression plasmid was obtained by adding the tag of these genes to the recombinant expression vector pGEX-2T-his6-puppet, pET21cc-dop-his6, pET21cc-pafA-his6, and the recombinant expression plasmid pET21cc-pafA-his6. GST-His6-PupDop-His6-Dop-His6FabD-His6 and PafA-His6 proteins were induced and expressed in E. coli BL21 by recombinant protein technique. These proteins were purified by routine and renaturation methods, and identified by Western blot. It provides protein for protein degradation system of Pup-proteasome in vitro. The N-terminal decapeptide of Pup was synthesized to immunize rabbits to obtain Pup polyclonal antibody which could recognize Pup protein and had higher titer. The knockout of the dop gene of (Mycobacterium smegmatis, Msm), a non-pathogenic homologous bacterium of Mtb, was preliminarily explored in order to further study the cellular regulatory function involved in the Pup-proteasome system.
【学位授予单位】：北京林业大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R378.911
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