本文選題：分枝桿菌噬菌體 + 全基因組測序　； 參考：《西南大學》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菌,兩種常見的致病菌：結(jié)核分枝桿菌(Mycobacterium tuberculosi)以及麻風分枝桿菌(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世紀末基本消滅麻風的目標后,目前由麻風分枝桿菌引起的麻風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)核病則因為日益嚴重的耐藥性、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。控制結(jié)核病已成為科研及醫(yī)療工作者所面臨的巨大任務。 分枝桿菌噬菌體是一類可感染分枝桿菌屬細菌的噬菌體,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)。基于以上幾點,本論文主要從以下幾個部分進行研究： 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,進行了遺傳學上的解釋,分析認為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,如必須進行全基因組測序、花費較大等。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,我們設(shè)計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簇的引物被驗證是可行的。 作為細菌的病毒,噬菌體在進化過程中獲得了一些可控制宿主細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、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,宿主會有一些防御噬菌體的反應,比如,誘導信號傳導系統(tǒng)從全局應對噬菌體的感染,上調(diào)細胞壁合成基因試圖修復被噬菌體破壞的細胞壁。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DUF2637超家族結(jié)構(gòu)域。在恥垢分枝桿菌內(nèi)表達此蛋白質(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注入進重組菌體內(nèi)。進一步研究顯示其可抑制大腸桿菌的生長,是一個新發(fā)現(xiàn)的宿主關(guān)閉蛋白。通過蛋白截短實驗探索到D29gp34.1具有宿主毒性的核心作用區(qū)域為第26個氨基酸到第100個氨基酸區(qū)段的肽段。我們認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個分枝桿菌原噬菌體,其中11個是之前未報道的原噬菌體。通過對全部33個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 .
【學位授予單位】：西南大學
【學位級別】：博士
【學位授予年份】：2014
【分類號】：R378.911;Q93

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