本文選題：分枝桿菌噬菌體 + 全基因組測序��； 參考：《西南大學(xué)》2014年博士論文

【摘要】：分枝桿菌屬是隸屬于放線菌目的一類細(xì)菌,兩種常見的致病菌：結(jié)核分枝桿菌(Mycobacterium tuberculosi)以及麻風(fēng)分枝桿菌(M. leprae)都是分枝桿菌屬內(nèi)的一員。自從1981年衛(wèi)生部提出20世紀(jì)末基本消滅麻風(fēng)的目標(biāo)后,目前由麻風(fēng)分枝桿菌引起的麻風(fēng)病在我國已得到有效的控制。而由結(jié)核分枝桿菌所引起的結(jié)核病則因?yàn)槿找鎳?yán)重的耐藥性、與HIV的共感染以及卡介苗的不穩(wěn)定性等呈現(xiàn)重新蔓延的趨勢�？刂平Y(jié)核病已成為科研及醫(yī)療工作者所面臨的巨大任務(wù)。 分枝桿菌噬菌體是一類可感染分枝桿菌屬細(xì)菌的噬菌體,對其研究起始于1950s。隨著研究的深入,分枝桿菌噬菌體己成為控制結(jié)核病的重要工具,比如：分枝桿菌噬菌體以及其衍生物已逐漸被開發(fā)為基礎(chǔ)研究中分枝桿菌的遺傳操作載體和臨床上(耐藥)結(jié)核菌的有效診斷工具；部分噬菌體組分也被研究,其中的一些被認(rèn)為具有開發(fā)為新一代抗結(jié)核藥物的潛力。但令人遺憾的是,現(xiàn)在基于分枝桿菌噬菌體開發(fā)出的結(jié)核菌分子操作載體以及結(jié)核病診斷工具的專利權(quán)都集中在歐美。因此為了對我國開發(fā)具有自主知識(shí)產(chǎn)權(quán)的結(jié)核病控制工具提供源頭上的創(chuàng)新,分離中國特有的分枝桿菌噬菌體并對其進(jìn)行全基因組測序勢在必行。另外,研究分枝桿菌噬菌體與宿主菌相互作用的機(jī)理、一些噬菌體宿主關(guān)閉蛋白的功能、分枝桿菌原噬菌體多樣性及其與結(jié)核菌致病性的關(guān)系都為利用噬菌體控制結(jié)核病提供了基礎(chǔ)�；谝陨蠋c(diǎn),本論文主要從以下幾個(gè)部分進(jìn)行研究： 首先我們分離得到了一株新的分枝桿菌噬菌體,并將其命名為SWU1。這株噬菌體感染恥垢分枝桿菌(M. smegmatis mc2155)后形成周圍有暈環(huán)的牛眼狀噬菌斑,電鏡觀察發(fā)現(xiàn)其為典型的蝌蚪狀。得到其全基因組序列后,分析發(fā)現(xiàn)其屬于分枝桿菌噬菌體A2群體。針對SWU1與L5(A2群體典型噬菌體)表型的不同,進(jìn)行了遺傳學(xué)上的解釋,分析認(rèn)為SWU1中的gp39可能具有多糖解聚酶的功能。 在分離分枝桿菌噬菌體SWU1的過程中,我們發(fā)現(xiàn)現(xiàn)有的分枝桿菌噬菌體分類鑒定方法具有較大的缺點(diǎn),如必須進(jìn)行全基因組測序、花費(fèi)較大等。為了解決這一難題,我們開發(fā)了一種新的基于保守基因的分枝桿菌噬菌體鑒定新方法。利用68個(gè)可獲取全基因組的分枝桿菌噬菌體,我們首先獲得分枝桿菌噬菌體的核心基因組。之后,我們分析了其中的核心基因,發(fā)現(xiàn)A類核心基因非常保守,最適合被用來鑒定分枝桿菌噬菌體。最后,針對A類核心基因的保守位點(diǎn),我們設(shè)計(jì)了針對不同分枝桿菌噬菌體簇的引物對。其中針對分枝桿菌噬菌體A2簇的引物被驗(yàn)證是可行的。 作為細(xì)菌的病毒,噬菌體在進(jìn)化過程中獲得了一些可控制宿主細(xì)菌正常生理代謝的蛋白質(zhì)。而這些蛋白質(zhì)本身或其作用位點(diǎn)、作用方式是開發(fā)新型抗生素的模型。為了更好的了解分枝桿菌噬菌體是如何侵染、控制其宿主菌的,我們首次利用RNA-seq技術(shù)研究了分枝桿菌噬菌體SWUl與其宿主恥垢分枝桿菌的互作。數(shù)據(jù)分析顯示,宿主會(huì)有一些防御噬菌體的反應(yīng),比如,誘導(dǎo)信號(hào)傳導(dǎo)系統(tǒng)從全局應(yīng)對噬菌體的感染,上調(diào)細(xì)胞壁合成基因試圖修復(fù)被噬菌體破壞的細(xì)胞壁。在此過程,噬菌體也劫持了菌體的蛋白質(zhì)、DNA及RNA的合成系統(tǒng),破壞了菌體的鐵攝取系統(tǒng)以幫助自身的繁殖。其中破壞分枝桿菌的鐵攝取系統(tǒng)也許是開發(fā)新型抗結(jié)核藥物的新理念。 依靠RNA-seq技術(shù)從全局上了解到宿主菌與噬菌體SWU1的相互作用后,我們更想知道的是單個(gè)蛋白質(zhì)在噬菌體侵染宿主菌過程中所發(fā)揮的功能。其中分枝桿菌噬菌體D29的gp34.1吸引了我們。這一蛋白存在一個(gè)DUF2637超家族結(jié)構(gòu)域。在恥垢分枝桿菌內(nèi)表達(dá)此蛋白質(zhì)后,發(fā)現(xiàn)其菌落形態(tài)改變,且重組菌獲得耐受噬菌體TM4的表型。噬菌體仍然可以正常的吸附并將DNA注入進(jìn)重組菌體內(nèi)。進(jìn)一步研究顯示其可抑制大腸桿菌的生長,是一個(gè)新發(fā)現(xiàn)的宿主關(guān)閉蛋白。通過蛋白截短實(shí)驗(yàn)探索到D29gp34.1具有宿主毒性的核心作用區(qū)域?yàn)榈?6個(gè)氨基酸到第100個(gè)氨基酸區(qū)段的肽段。我們認(rèn)為這一多肽片段具有開發(fā)為新型抗生素骨架的潛力。 有研究表明分枝桿菌基因組內(nèi)存在大量的原噬菌體,為了更好的了解噬菌體與分枝桿菌致病性的關(guān)系,我們對分枝桿菌基因組內(nèi)的原噬菌體進(jìn)行了系統(tǒng)的挖掘、比較并探討了其與分枝桿菌致病性的關(guān)系�？偣菜阉鞯搅�33個(gè)分枝桿菌原噬菌體,其中11個(gè)是之前未報(bào)道的原噬菌體。通過對全部33個(gè)原噬菌體的全基因組的比較：首次發(fā)現(xiàn)位于M.massiliense Strain M172基因組上的全長原噬菌體phil72_2可被歸為分枝桿菌噬菌體A簇；另外,發(fā)現(xiàn)了一組缺陷原噬菌體phiMmcs_1(phiMkms_1)、phiBN44_1以及phiMCAN_1具有同源性,可以被歸為同一類。
[Abstract]:Mycobacterium tuberculosis is a kind of bacterium belonging to Actinomyces , two common pathogenic bacteria : Mycobacterium tuberculosis and M . leprae are a member of the genus Mycobacterium .

The mycobacterial phage is a kind of phage that can infect the bacterium of Mycobacterium , which starts from the research . With the development of the research , the mycobacterium phage has become an important tool for controlling tuberculosis , such as Mycobacterium tuberculosis phage and its derivative has been developed into the genetic operation carrier of mycobacterium in the foundation study and the effective diagnostic tool for clinical ( resistant ) mycobacterium tuberculosis ;
Some of the bacteriophage components are also studied , some of which are thought to have the potential to develop a new generation of antitubercular drugs . However , it is regrettable that the Mycobacterium tuberculosis molecular manipulation vector and the patent right of tuberculosis diagnostic tools are now concentrated in Europe and America . In addition , it is necessary to study the mechanism of the development of tuberculosis control tools with independent intellectual property rights .

A new phage of Mycobacterium smegmatis mc2155 was isolated and named SWU1 . The phage infected with Mycobacterium smegmatis mc2155 was infected with M . smegmatis mc2155 . It was found that it was a typical tadpole . After the whole genome sequence was obtained , it was found that it belonged to Mycobacterium tuberculosis phage A2 population . In this paper , genetic explanation was made for the phenotype of SWU1 and L5 ( typical phage ) . It was suggested that gp39 in SWU1 might have the function of polysaccharide depolymerase .

In order to solve this problem , we have developed a new method for the identification of Mycobacterium tuberculosis phage . In order to solve this problem , we have developed a new method for the identification of mycobacterial phage based on conserved genes .

In order to better understand how to infect and control the host bacteria of the host bacteria , we first use RNA - seq technology to study the interaction between the mycobacterial phage SWUl and its host smegmatis . In this process , the phage also holds the protein , DNA and RNA synthesis system of the bacteria , destroying the iron intake system of the bacteria to help its own reproduction .

From the global perspective on the interaction of host bacteria with phage SWU1 , we would like to know the function of a single protein in the process of phage infection host bacteria . The protein has a DUF2637 superfamily domain . The protein has a DUF2637 superfamily domain .

A large number of prophage were found in the genome of Mycobacterium tuberculosis . In order to better understand the relationship between the pathogenicity of phage and Mycobacterium tuberculosis , we compared and discussed the relationship between phage and mycobacterial pathogenicity . In total , 33 Mycobacterium prophage were searched , 11 of them were previously unreported . By comparison of the whole genome of all 33 prophage : the first finding that the full - length prophage phil72 _ 2 located on the genome of M . massiliense Strain M172 can be classified as Mycobacterium tuberculosis phage A cluster ;
In addition , it has been found that phiMkms _ 1 ( phiMkms _ 1 ) , philist44 _ 1 and phiMCAN _ 1 have homology and can be classified into the same class .
【學(xué)位授予單位】：西南大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2014
【分類號(hào)】：R378.911;Q93

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相關(guān)期刊論文 前5條

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