本文關(guān)鍵詞：結(jié)核分枝桿菌哺乳動物細(xì)胞入侵因子mce家族Rv0590A基因的性質(zhì)及功能研究　出處：《西南大學(xué)》2011年碩士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： mce Rv0590A 結(jié)核分枝桿菌 脂肪酸 Cc15

【摘要】：結(jié)核病是一種嚴(yán)重危害人類健康的傳染病,它是由結(jié)核分枝桿菌(Mycobacterium tuberculosis)引起的一種慢性疾病。結(jié)核病發(fā)病率的增加在一些資源短缺國家甚至工業(yè)發(fā)達(dá)國家都已是不爭的事實(shí)。人們對(Mycobacterium tuberculosis)的生物特性進(jìn)行廣泛研究,研發(fā)了鏈霉素、異煙肼、利福平等治療性藥物,使得人類對結(jié)核病的控制取得了可喜的成績。然而,化學(xué)藥物的不合理應(yīng)用,耐藥菌株的廣泛出現(xiàn),M. tuberculosis與人類免疫缺陷病毒HIV的共感染,人口流動的增加使得結(jié)核病疫情下降緩慢,在部分地區(qū)甚至有所回升。開發(fā)出對結(jié)核病有效的預(yù)防性、治療性疫苗和新型藥物是亟待解決的難題。尋找新的結(jié)核藥物作用靶點(diǎn)和開發(fā)新型抗結(jié)核藥物已成為當(dāng)前最受關(guān)注的熱點(diǎn)。 M. tuberculosis作為一種胞內(nèi)致病菌,主要存在于單核吞噬細(xì)胞內(nèi)。它的一個重要特征是能夠在宿主巨噬細(xì)胞內(nèi)存活和增殖,并抑制吞噬小體的酸化和成熟。M. tuberculosis的基因組包含4個哺乳動物細(xì)胞入侵因子(mce)操縱子(mce1-4),每個mce操縱子含有8-13個基因,它們相互毗連且在各操縱子中排列相似。每個操縱子中相對應(yīng)的基因具有相似的序列及有機(jī)組成,編碼與M. tuberculosis入侵以及在宿主巨噬細(xì)胞中存活相關(guān)的輸出蛋白。mce基因使得重組非致病菌E. coli能夠入侵哺乳動物并在其巨噬細(xì)胞中存活,與M. tuberculosis入侵宿主細(xì)胞并在其巨噬細(xì)胞內(nèi)的持久存活有著密切的關(guān)系。Rv0590A作為mce操縱子上一個未知功能的ORF,是mce2操縱子上一個獨(dú)有的基因,可能與Mce2R共同調(diào)控mce2的表達(dá)。Rv0590A與mce2B組成M. tuberculosis H37Rv的一個中斷編碼序列(ICDS),與其他18個ICDS共同構(gòu)成M. tuberculosis和Mycobacterium bovis (M. bovis)分化的依據(jù)之一。 本研究通過生物信息學(xué)對結(jié)核分枝桿菌H37Rv的Rv0590A基因的性質(zhì)及功能進(jìn)行研究,并對該基因編碼蛋白的結(jié)構(gòu)和功能進(jìn)行分析和預(yù)測,以期為該蛋白的研究提供生物信息學(xué)相關(guān)的參考依據(jù)。運(yùn)用TMHMM Serverv.2.0, Jpred, Vector NTI 9, ClustalⅩ, SignalP, swiss model等生物信息學(xué)資源對Rv0590A的基本性質(zhì)進(jìn)行了分析,包括對蛋白一級結(jié)構(gòu)(親水性/疏水性,跨膜結(jié)構(gòu)和信號肽等)二級結(jié)構(gòu)以及三級結(jié)構(gòu)等基本性質(zhì)的預(yù)測分析。結(jié)果表明：結(jié)核分枝桿菌H37RvRv0590A編碼蛋白無信號肽,不存在跨膜結(jié)構(gòu)區(qū)；其二級結(jié)構(gòu)是由2個α螺旋及2個β折疊片構(gòu)成。同時在一、二級結(jié)構(gòu)分析的基礎(chǔ)上,利用同源建模的方法完成了其三維結(jié)構(gòu)的建模。運(yùn)用http://string.embl.de/軟件預(yù)測Rv0590A蛋白相互作用網(wǎng)絡(luò),結(jié)果表明與其相互作用蛋白主要是同源及與其毗鄰的蛋白即mce2操縱子所編碼的蛋白及其調(diào)節(jié)子Rv0586。與報道的Rv0590A可能與Rv0586共同調(diào)節(jié)mce2的轉(zhuǎn)錄相互印證。利用比較基因組學(xué)分析該蛋白在分枝桿菌屬中的同源和分布情況,結(jié)果表明該基因在進(jìn)化上相對保守。 從GenBank數(shù)據(jù)庫中獲得結(jié)核分枝桿菌H37Rv的Rv0590A基因的核苷酸序列,設(shè)計(jì)兩對引物,分別以結(jié)核分枝桿菌H37Rv全基因組為模板,分別體外擴(kuò)增獲得Rv0590A基因。分別通過TA克隆,將PCR產(chǎn)物連接到pMD19-T Simple Vector,分別亞克隆至載體pET28a(+)和pcDNA3.1(+)中,經(jīng)菌落PCR以及序列測定證明成功構(gòu)建了重組質(zhì)粒pET28/Rv0590A和pcDNA3.1/Rv0590A。將重組質(zhì)粒pET28/Rv0590A轉(zhuǎn)化Escherichia coli BL21 (DE3),對該重組蛋白質(zhì)進(jìn)行誘導(dǎo)表達(dá)。擴(kuò)大培養(yǎng)后,采用Ni+凝膠親和層析方法純化獲得高純度的重組Rv0590A蛋白,western bloting驗(yàn)證Rv0590A融合表達(dá)。我們研究了過表達(dá)該蛋白對宿主菌的影響,包括過表達(dá)Rv0590A對宿主菌生長,脂肪酸組成以及對宿主菌抗氧化壓力的耐受性研究。將重組pcDNA3.1/Rv0590A轉(zhuǎn)染真核細(xì)胞U937,轉(zhuǎn)錄水平上檢測到Rv0590A的表達(dá)及Rv0590A對于U937的轉(zhuǎn)染所引起的細(xì)胞因子表達(dá)量的變化。本研究為揭示結(jié)核分枝桿菌Rv0590A的生理功能及其對mce2操縱子的特殊作用以及Mce家族的作用機(jī)制提供線索。
[Abstract]:Tuberculosis is a serious infectious disease, which is caused by Mycobacterium tuberculosis (Mycobacterium tuberculosis) is a chronic disease caused by tuberculosis. The increasing incidence of resource shortage in some countries even industrial developed countries is an indisputable fact. People of (Mycobacterium tuberculosis) is widely studied biological characteristics the research of streptomycin, isoniazid, rifampin therapeutic drugs, which in human tuberculosis control has made gratifying achievements. However, the unreasonable application of chemical drugs, drug resistant strains appeared widely, CO infection with the human immunodeficiency virus M. tuberculosis HIV, the increase of population mobility the TB epidemic decreased slowly, even rebounded in in some areas. The development of effective prevention of tuberculosis, therapeutic vaccines and new drugs is urgent to solve the problem. Looking for new The target of tuberculosis drugs and the development of new anti tuberculosis drugs have become the most popular hot spots.
M. tuberculosis is an intracellular pathogen mainly exists in mononuclear phagocytes. Which is an important feature in macrophage survival and proliferation, and inhibit the invasion of mammalian cell contains 4 factors of phagosome acidification and mature.M. tuberculosis genome (MCE) operon (mce1-4), each the MCE operon containing 8-13 gene, and they join each other in each operon corresponding to each array. Similar genes in operons have similar sequences and organic composition, and tuberculosis and M. encoding invasion protein.Mce gene related to the output of survival in host macrophages and the recombinant non pathogenic E. coli can invade mammals and survive in the macrophages, M. and tuberculosis in the invasion of host cells and macrophages in the long-term survival is closely linked to the.Rv0590A as MCE control On an unknown function of the ORF, is a unique gene of the mce2 operon, encoding a sequence may interrupt the expression of.Rv0590A and mce2B and Mce2R to regulate the composition of the mce2 M. tuberculosis H37Rv (ICDS), and the other 18 ICDS tuberculosis and Mycobacterium bovis are composed of the M. (M. bovis) is one of the basis of differentiation.
This study through the research on the nature and function of bioinformatics of H37Rv of Mycobacterium tuberculosis Rv0590A gene, and the structure and function of the gene encoding protein were analyzed and predicted, in order to study the protein bioinformatics provides relevant reference. Using TMHMM Serverv.2.0, Jpred Vector, NTI 9, Clustal x SignalP, Swiss, model and other bioinformatics resources on the basic properties of Rv0590A are analyzed, including the protein primary structure (hydrophilic / hydrophobic transmembrane structure and signal peptide) analysis of two level structure and prediction of three level structure basic properties. The results showed that H37RvRv0590A encoding protein of Mycobacterium tuberculosis no signal peptide and transmembrane domain does not exist; the secondary structure is composed of 2 alpha helix and 2 beta sheet. At the same time, based on analysis of two level structure, using homology modeling methods. The modeling of the three dimensional structure. Using http://string.embl.de/ software to predict the network Rv0590A protein interactions, results show that the interacting proteins are transcription and protein homologous adjacent to the mce2 operon encoding protein and its regulation on Rv0586. and reported Rv0590A and Rv0586 regulate mce2 corroborated by comparative genomics analysis. The protein in Mycobacterium tuberculosis and the distribution, the results show that the gene is relatively conservative in evolution.
Obtain the nucleotide sequence of Rv0590A gene of Mycobacterium tuberculosis H37Rv from the GenBank database, two pairs of primers were designed respectively by Mycobacterium tuberculosis H37Rv genome as template, respectively. Rv0590A gene was amplified by PCR were cloned by TA, the product of PCR Simple connected to the pMD19-T Vector, were subcloned into vector pET28a (+) and pcDNA3.1 (+), colony PCR and sequence assay demonstrated that the recombinant plasmid pET28/Rv0590A and the recombinant plasmid pET28/Rv0590A was transformed into pcDNA3.1/Rv0590A. Escherichia coli BL21 (DE3), the recombinant protein expression was induced. After culture expansion, to obtain high purity of recombinant protein Rv0590A affinity chromatography purification method using Ni+ gel, Western bloting verification Rv0590A fusion expression. We study the effect of overexpression of the protein of the host bacteria, including overexpression of Rv0590A on the growth of the host bacteria, fatty acid Study on composition and tolerance to oxidative stress. The host strain of recombinant pcDNA3.1/Rv0590A eukaryotic cell U937 transcription level detected on cytokine expression of Rv0590A and Rv0590A caused by U937 for transfection of expression. This study provide clues to reveal the physiological function of the Mycobacterium tuberculosis strain Rv0590A and its special function of mce2 the Mce family and operon mechanism.

【學(xué)位授予單位】：西南大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2011
【分類號】：R378

【共引文獻(xiàn)】

相關(guān)碩士學(xué)位論文 前2條

1 王建有;系統(tǒng)性紅斑狼瘡患者血清趨化因子MCAF和RANTES的測定及其臨床意義[D];浙江大學(xué);2001年

2 嚴(yán)艷玲;溫陽活血通絡(luò)法聯(lián)合醋酸潑尼松治療系統(tǒng)性硬化癥的臨床及實(shí)驗(yàn)研究[D];湖北民族學(xué)院;2014年

，

本文編號：1398736