结核分枝杆菌哺乳动物细胞入侵因子mce家族Rv0590A基因的性质及功能研究

发布时间：2018-01-08 21:23

  本文关键词：结核分枝杆菌哺乳动物细胞入侵因子mce家族Rv0590A基因的性质及功能研究　出处：《西南大学》2011年硕士论文　论文类型：学位论文

  更多相关文章： mce Rv0590A 结核分枝杆菌 脂肪酸 Cc15

【摘要】：结核病是一种严重危害人类健康的传染病,它是由结核分枝杆菌(Mycobacterium tuberculosis)引起的一种慢性疾病。结核病发病率的增加在一些资源短缺国家甚至工业发达国家都已是不争的事实。人们对(Mycobacterium tuberculosis)的生物特性进行广泛研究,研发了链霉素、异烟肼、利福平等治疗性药物,使得人类对结核病的控制取得了可喜的成绩。然而,化学药物的不合理应用,耐药菌株的广泛出现,M. tuberculosis与人类免疫缺陷病毒HIV的共感染,人口流动的增加使得结核病疫情下降缓慢,在部分地区甚至有所回升。开发出对结核病有效的预防性、治疗性疫苗和新型药物是亟待解决的难题。寻找新的结核药物作用靶点和开发新型抗结核药物已成为当前最受关注的热点。 M. tuberculosis作为一种胞内致病菌,主要存在于单核吞噬细胞内。它的一个重要特征是能够在宿主巨噬细胞内存活和增殖,并抑制吞噬小体的酸化和成熟。M. tuberculosis的基因组包含4个哺乳动物细胞入侵因子(mce)操纵子(mce1-4),每个mce操纵子含有8-13个基因,它们相互毗连且在各操纵子中排列相似。每个操纵子中相对应的基因具有相似的序列及有机组成,编码与M. tuberculosis入侵以及在宿主巨噬细胞中存活相关的输出蛋白。mce基因使得重组非致病菌E. coli能够入侵哺乳动物并在其巨噬细胞中存活,与M. tuberculosis入侵宿主细胞并在其巨噬细胞内的持久存活有着密切的关系。Rv0590A作为mce操纵子上一个未知功能的ORF,是mce2操纵子上一个独有的基因,可能与Mce2R共同调控mce2的表达。Rv0590A与mce2B组成M. tuberculosis H37Rv的一个中断编码序列(ICDS),与其他18个ICDS共同构成M. tuberculosis和Mycobacterium bovis (M. bovis)分化的依据之一。 本研究通过生物信息学对结核分枝杆菌H37Rv的Rv0590A基因的性质及功能进行研究,并对该基因编码蛋白的结构和功能进行分析和预测,以期为该蛋白的研究提供生物信息学相关的参考依据。运用TMHMM Serverv.2.0, Jpred, Vector NTI 9, ClustalⅩ, SignalP, swiss model等生物信息学资源对Rv0590A的基本性质进行了分析,包括对蛋白一级结构(亲水性/疏水性,跨膜结构和信号肽等)二级结构以及三级结构等基本性质的预测分析。结果表明：结核分枝杆菌H37RvRv0590A编码蛋白无信号肽,不存在跨膜结构区；其二级结构是由2个α螺旋及2个β折叠片构成。同时在一、二级结构分析的基础上,利用同源建模的方法完成了其三维结构的建模。运用http://string.embl.de/软件预测Rv0590A蛋白相互作用网络,结果表明与其相互作用蛋白主要是同源及与其毗邻的蛋白即mce2操纵子所编码的蛋白及其调节子Rv0586。与报道的Rv0590A可能与Rv0586共同调节mce2的转录相互印证。利用比较基因组学分析该蛋白在分枝杆菌属中的同源和分布情况,结果表明该基因在进化上相对保守。 从GenBank数据库中获得结核分枝杆菌H37Rv的Rv0590A基因的核苷酸序列,设计两对引物,分别以结核分枝杆菌H37Rv全基因组为模板,分别体外扩增获得Rv0590A基因。分别通过TA克隆,将PCR产物连接到pMD19-T Simple Vector,分别亚克隆至载体pET28a(+)和pcDNA3.1(+)中,经菌落PCR以及序列测定证明成功构建了重组质粒pET28/Rv0590A和pcDNA3.1/Rv0590A。将重组质粒pET28/Rv0590A转化Escherichia coli BL21 (DE3),对该重组蛋白质进行诱导表达。扩大培养后,采用Ni+凝胶亲和层析方法纯化获得高纯度的重组Rv0590A蛋白,western bloting验证Rv0590A融合表达。我们研究了过表达该蛋白对宿主菌的影响,包括过表达Rv0590A对宿主菌生长,脂肪酸组成以及对宿主菌抗氧化压力的耐受性研究。将重组pcDNA3.1/Rv0590A转染真核细胞U937,转录水平上检测到Rv0590A的表达及Rv0590A对于U937的转染所引起的细胞因子表达量的变化。本研究为揭示结核分枝杆菌Rv0590A的生理功能及其对mce2操纵子的特殊作用以及Mce家族的作用机制提供线索。
[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.

【学位授予单位】：西南大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R378

【共引文献】

相关硕士学位论文 前2条

1 王建有;系统性红斑狼疮患者血清趋化因子MCAF和RANTES的测定及其临床意义[D];浙江大学;2001年

2 严艳玲;温阳活血通络法联合醋酸泼尼松治疗系统性硬化症的临床及实验研究[D];湖北民族学院;2014年

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