本文關(guān)鍵詞：結(jié)核分枝桿菌ClpX和ClpP2蛋白酶的克隆表達(dá)及其性質(zhì)研究　出處：《西南大學(xué)》2010年碩士論文　論文類(lèi)型：學(xué)位論文

  更多相關(guān)文章： ClpX蛋白酶 ClpP2蛋白酶 結(jié)核分枝桿菌 氧化壓力耐受

【摘要】： 結(jié)核分枝桿菌所導(dǎo)致的結(jié)核病仍然是全球人類(lèi)健康的主要威脅,結(jié)核分枝桿菌每年引起兩三百萬(wàn)的人死亡。結(jié)核分枝桿菌是一種嚴(yán)格而成功的胞內(nèi)致病菌,在宿主的免疫系統(tǒng)細(xì)胞,主要是巨噬細(xì)胞內(nèi)存活并復(fù)制。蛋白酶一直被認(rèn)為是致病微生物的重要毒力因子。比較基因組學(xué)研究發(fā)現(xiàn)結(jié)核分枝桿菌主要有16大類(lèi)蛋白酶,其中許多成員都與結(jié)核分枝桿菌的致病性和在單核細(xì)胞、巨噬細(xì)胞中的持留性相關(guān)。蛋白質(zhì)的降解是所有生物生理調(diào)控和蛋白質(zhì)量控制所必需的。弄清蛋白質(zhì)降解系統(tǒng)對(duì)細(xì)胞內(nèi)蛋白質(zhì)質(zhì)量的控制,揭示致病菌在宿主免疫壓力下細(xì)胞內(nèi)蛋白質(zhì)穩(wěn)態(tài)的維持機(jī)制,有利于進(jìn)一步揭示結(jié)核分枝桿菌的致病機(jī)理,進(jìn)而為藥物開(kāi)發(fā)提供可能的靶標(biāo)。 結(jié)核分枝桿菌入侵人體和在巨噬細(xì)胞中持留性感染時(shí)往往會(huì)遭遇許多不利的環(huán)境壓力,諸如營(yíng)養(yǎng)缺乏、低PH值、氧化壓力及高溫壓力等。其中,氧化壓力和熱應(yīng)激條件下會(huì)產(chǎn)生大量蛋白質(zhì)的解折疊和聚集,如何降解這些錯(cuò)誤折疊的蛋白質(zhì),對(duì)維持結(jié)核分枝桿菌在壓力條件下的正常生長(zhǎng)起著至關(guān)重要的作用。細(xì)菌胞內(nèi)蛋白的水解由四種依賴(lài)ATP的蛋白酶執(zhí)行,分別是ClpXP,ClpYQ(HslUV),FtsH和Lon家族。在結(jié)核分枝桿菌中未發(fā)現(xiàn)HslUV和Lon蛋白家族,因此ClpXP蛋白酶在結(jié)核分枝桿菌中的功能顯得更加重要。 為了研究結(jié)核分枝桿菌可能的ClpX和ClpP2蛋白的基本功能,本研究提取結(jié)核分枝桿菌H37Rv的基因組,采用體外PCR擴(kuò)增的方法得到clpX(Rv2457c)和clpP2(Rv2460c)基因。將clpX和clpP2基因的PCR產(chǎn)物分別連入載體pET28a(+)和pET32a(+)中,經(jīng)菌落PCR及測(cè)序證明成功構(gòu)建了重組質(zhì)粒pET28-clpX和pET32-clpP2。將重組質(zhì)粒轉(zhuǎn)化大腸桿菌BL21(DE3),IPTG誘導(dǎo)表達(dá)重組蛋白,Ni親和層析純化重組蛋白,并進(jìn)行了ClpX蛋白酶的ATP酶活的體外初步檢測(cè)。為了進(jìn)一步了解ClpXP蛋白在生物體內(nèi)的活性,我們研究了過(guò)表達(dá)ClpXP蛋白酶對(duì)宿主菌的影響,包括：①過(guò)表達(dá)ClpX和ClpP2蛋白酶對(duì)宿主菌生長(zhǎng)的影響；②過(guò)表達(dá)ClpX和ClpP2蛋白酶體對(duì)宿主氧化應(yīng)激的影響。研究證明ClpX蛋白的過(guò)表達(dá)促進(jìn)了宿主菌的生長(zhǎng),共表達(dá)ClpX和ClpP2蛋白也能促進(jìn)宿主菌的生長(zhǎng),ClpP2蛋白的過(guò)表達(dá)能夠增加宿主細(xì)胞對(duì)過(guò)氧化氫的耐受。長(zhǎng)時(shí)間過(guò)表達(dá)ClpP2蛋白酶,純化到的ClpP2蛋白酶具有分子量大小有差異的兩條目的蛋白條帶,MALDI-TOF-TOF鑒定結(jié)果顯示兩條大小有差異的目的蛋白均為ClpP2重組蛋白。生物信息學(xué)分析顯示ClpP2蛋白酶存在酰胺化修飾位點(diǎn)、CAMP磷酸化位點(diǎn)等多個(gè)修飾位點(diǎn),可能是由于這些位點(diǎn)的修飾導(dǎo)致了蛋白質(zhì)分子量的變化。并用在線數(shù)據(jù)庫(kù)預(yù)測(cè)了ClpX和ClpP2蛋白可能的作用網(wǎng)絡(luò),進(jìn)一步分析了ClpXP蛋白酶在細(xì)胞內(nèi)的生理功能。 從實(shí)驗(yàn)結(jié)果來(lái)看,結(jié)核分枝桿菌可能的ClpX和ClpP2蛋白在細(xì)菌生長(zhǎng)和氧化壓力耐受中發(fā)揮著很重要的作用。對(duì)它們的深入研究可以揭示結(jié)核分枝桿菌對(duì)宿主氧化壓力耐受和體內(nèi)蛋白質(zhì)質(zhì)量控制的機(jī)制,為我們開(kāi)發(fā)新的抗結(jié)核靶標(biāo)提供思路。
[Abstract]:Caused by Mycobacterium tuberculosis is still a major threat to human health worldwide, Mycobacterium tuberculosis caused by two or three million people each year. The death of Mycobacterium tuberculosis is a kind of strict and successful intracellular pathogen in the cells of the host immune system, mainly macrophages survive and replicate. Protease has been considered the important virulence factors of pathogenic microorganisms. Comparative genomics of Mycobacterium tuberculosis found there are 16 main types of protease, many of the members are related to the pathogenicity of Mycobacterium tuberculosis and on monocytes and macrophages in the retention. The degradation of protein is required for all physiological regulation and protein quality control. The quality of protein in cells to protein degradation, reveal the pathogen in the host immune pressure cell protein homeostasis mechanisms conducive to One step reveals the pathogenesis of Mycobacterium tuberculosis and provides a possible target for drug development.
Mycobacterium tuberculosis infection to invade the body and stay in macrophages will often encounter many adverse environmental stresses, such as lack of nutrition, low pH, oxidative stress and high temperature pressure. Which will produce a large number of protein unfolding and aggregation of oxidative stress and heat stress conditions, the degradation of these misfolded proteins. To maintain normal growth of Mycobacterium tuberculosis under pressure plays a vital role. The hydrolysis of intracellular bacterial protein by four ATP dependent proteases, including ClpXP, ClpYQ, FtsH and Lon (HslUV) family. HslUV and Lon proteins were found in Mycobacterium tuberculosis, so the function of ClpXP protease in Mycobacterium tuberculosis is becoming more and more important.
In order to study the basic function of Mycobacterium tuberculosis may ClpX and ClpP2 protein, the extraction of Mycobacterium tuberculosis H37Rv genome, using the method of in vitro amplification of PCR clpX (Rv2457c) and clpP2 (Rv2460c) gene. The product of PCR clpX and clpP2 gene were inserted into vector pET28a (+) and pET32a (+) in the colony PCR and sequencing proved that recombinant plasmid pET28-clpX was successfully constructed and the recombinant plasmid pET32-clpP2. was transformed into E. coli BL21 (DE3), recombinant protein expression was induced by IPTG, purified recombinant protein Ni affinity chromatography, and has carried on the preliminary detection of ATP enzyme ClpX protease activity in vitro. In order to further understand the activity of the ClpXP protein in organism inside, we investigated the effects of overexpression of ClpXP protein on host bacteria, including: the impact of ClpX overexpression and ClpP2 protease on the growth of the host bacteria; the expression of ClpX and ClpP2 proteasome in night The main effect of oxidative stress. The study shows that overexpression of ClpX promotes the growth of the host bacteria, co expression of ClpX and ClpP2 protein can also promote the growth of the host bacteria, overexpression of ClpP2 can increase the tolerance of host cells to hydrogen peroxide. Long time overexpression of ClpP2 protease, the purified ClpP2 protease with molecular weight there are differences between the two protein bands, the identification results of MALDI-TOF-TOF showed that two size different purpose protein was the recombinant ClpP2 protein. Bioinformatics analysis showed that ClpP2 protease exists amidation sites, multiple modification sites CAMP phosphorylation sites, may be due to modification of these sites leads to changes in protein molecular weight. And forecast the network ClpX and the role of ClpP2 protein in online databases, further analysis of the physiological function of ClpXP protease in cells.
From the experimental results, Mycobacterium tuberculosis ClpX and ClpP2 protein may play an important role in bacteria growth and oxidative stress tolerance. On their further study can reveal the Mycobacterium tuberculosis on host oxidative stress tolerance and protein quality control system, we provide ideas for the development of new anti tuberculosis targets.

【學(xué)位授予單位】：西南大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2010
【分類(lèi)號(hào)】：R378

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