【摘要】：結(jié)核分枝桿菌(Mycobacterium tuberculosis Mtb)中類泛素蛋白Pup (Prokaryotic ubiquitin-like protein)-蛋白酶體系統(tǒng)是胞內(nèi)蛋白降解重要機(jī)制,在去酰胺酶Dop(Deamidase of Pup)和連接酶PafA(Proteasome accessory factor A)、ATP酶Mpa(Mycobacterial proteasome ATPase)等輔助因子的作用下,Pup可以共價(jià)標(biāo)記多種功能蛋白,并介導(dǎo)被標(biāo)記蛋白通過蛋白酶體降解,靶蛋白涉及物質(zhì)中間代謝、信號通路、毒性和抗毒性因子、細(xì)胞壁和細(xì)胞膜組份等多個(gè)方面,并且與結(jié)核分枝桿菌的致病性相關(guān),被認(rèn)為是新的結(jié)核病治療藥物靶點(diǎn),因此本領(lǐng)域研究具有深遠(yuǎn)的意義。 本文以Mtb為研究對象,優(yōu)化并克隆出pup基因,同時(shí)克隆出dop、pafA和底物蛋白fabD基因,將這些基因加上用于純化的標(biāo)簽構(gòu)入表達(dá)載體,得到pGEX-2T-his6-pup, pET21cc-dop-his6, pET21cc-fabD-his6, pET21cc-pafA-his6, pBAD-pafA-his6重組表達(dá)質(zhì)粒。通過重組蛋白技術(shù),在大腸桿菌BL21中誘導(dǎo)表達(dá)出GST-His6-Pup、Dop-His6、FabD-His6和PafA-His6蛋白,并分別通過常規(guī)和變復(fù)性等方法純化出這些蛋白,用蛋白免疫印跡等技術(shù)鑒定了這些蛋白,為建立Pup-蛋白酶體體外蛋白降解系統(tǒng)提供了蛋白。合成Pup的N端十肽免疫兔,得到能識別Pup蛋白且有較高效價(jià)的Pup多克隆抗體。對Mtb的非致病同源菌恥垢分枝桿菌(Mycobacterium smegmatis, Msm)的dop基因敲除進(jìn)行了初步探索,為進(jìn)一步研究Pup-蛋白酶體系統(tǒng)參與的細(xì)胞調(diào)控功能做準(zhǔn)備。
[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.
【學(xué)位授予單位】：北京林業(yè)大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2011
【分類號】：R378.911

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本文編號：2184532