本文選題：結核分枝桿菌 + dTDP-鼠李糖��； 參考：《大連醫(yī)科大學》2005年博士論文

【摘要】：結核分枝桿菌(M．tuberculosis)是引起結核病的病原體。由于耐多藥菌株的出現(xiàn)和HIV的協(xié)同作用，使得一度被控制的結核病的發(fā)病率在全球范圍內顯著回升，因此，尋找新一代抗結核藥物已迫在眉睫。細胞壁是分枝桿菌賴以生存的結構基礎，其核心結構由分枝菌酸、聚阿拉伯糖半乳糖及肽聚糖三種組分組成。分枝菌酸和聚阿拉伯糖半乳糖通過銜接雙糖(L-鼠李糖-D-N-乙酰葡糖胺)共價連接到肽聚糖大分子上，銜接雙糖尤其是鼠李糖可作為研發(fā)新一代抗結核藥物的理想靶標。銜接雙糖中鼠李糖的糖基供體是dTDP-鼠李糖，四種酶(Rm1A-D)參與了由底物葡萄糖-1-磷酸合成dTDP-鼠李糖的過程，，編碼這四種酶的rm1A-D基因存在于結核分枝桿菌基因組中不同的位置，其中rm1B和rm1C同屬于一個操縱子。盡管我們已發(fā)現(xiàn)在結核分枝桿菌中不存在dTDP-鼠李糖生物合成的補救代謝途徑，但仍有必要從理論上證明編碼結核分枝桿菌dTDP-鼠李糖合成酶系的rm1A-D基因為結核分枝桿菌的生長相關基因。 本論文的目的是用基因剔除方法證明結核分枝桿菌中編碼Rm1B(dTDP-D-葡萄糖-4，6-脫氫酶)和Rm1C(dTDP-4-酮基-6-脫氧-D-葡萄糖3，5表異構酶)的rm1B和C基因是分枝桿菌生長過程中所必需的基因，這將為dTDP-鼠李糖作為研發(fā)抗結核新藥的理想靶標提供更有力的證據。 利用與結核分枝桿菌有相同細胞壁結構，但生長快速且無致病性的恥垢分枝桿菌(Mycobacterium smegmatis，Sm)mc~2155菌株作為實驗模型。用攜帶Sm rm1B：：Kan~R突變基因的條件復制性質粒轉化mc~2155菌株，在42℃條件下篩選出mc~2155突變菌株M1(其基因組中同時存在Sm rm1B和Sm rm1B：：Kan~R)。用攜帶結核分枝桿菌M．tuberculosis，Tb)rm1B和rm1C基因的營救質粒轉化mc~2155 M1突變菌株，Tb rm1B和Tb rm1C基因的表達產物可補償mc~2155 M1突變菌株中突變的Sm rm1B以及突變的Sm rm1C基因的功能，在30℃條件下篩選出mc~2155 M2突變菌株(其基因組中只存在Sm rm1B：：Kan~R，即Sm rm1B基因剔除；同時Sm rm1B：：Kan~R下游的Sm rm1C基因發(fā)生框移突變)。分別在30℃和42℃條件下測定
[Abstract]:Mycobacterium tuberculosis M. tuberculosis is the pathogen of tuberculosis. Due to the emergence of multi-drug resistant strains and the synergistic effect of HIV, the incidence of once controlled tuberculosis has increased significantly in the world. Therefore, it is urgent to find a new generation of anti-tuberculosis drugs. The cell wall is the structural basis for the survival of Mycobacterium. Its core structure consists of three components: mycoic acid, polyarabinose galactose and peptidoglycan. Mycoic acid and polyarabinose galactose are covalently linked to peptidoglycan macromolecules by linking disaccharide L- rhamnose-D-N- acetyl glucosamine, which can be used as an ideal target for the development of a new generation of antituberculotic drugs. The glycosyl donor linking rhamnose in disaccharide is dTDP- rhamnose. Four enzymes Rm1A-D) are involved in the synthesis of dTDP- rhamnose from glucose-1-phosphate substrate. The rm1A-D gene encoding these four enzymes exists in different positions in the genome of Mycobacterium tuberculosis. Rm1B and rm1C belong to the same operator. Although we have found that there is no rescue metabolic pathway for dTDP- rhamnose biosynthesis in Mycobacterium tuberculosis, However, it is necessary to prove theoretically that the rm1A-D gene encoding the dTDP- rhamnose synthase system of Mycobacterium tuberculosis is related to the growth of Mycobacterium tuberculosis. The purpose of this paper is to prove that rm1B and C genes of Mycobacterium tuberculosis encode Rm1BP- dTDP-glucose-4nd6- dehydrogenase and Rm1CndTDP-4-keto-6-deoxy-D- glucose-3 isoisomerase) are essential genes in the growth of Mycobacterium tuberculosis. This will provide stronger evidence for dTDP- rhamnose as an ideal target for new anti-TB drugs. Mycobacterium smegmatissimus smmccf2155, which had the same cell wall structure as Mycobacterium tuberculosis, but grew rapidly and without pathogenicity, was used as the experimental model. Mc~2155 strain was transformed by conditional replicative plasmid carrying Sm rm1B::Kan~R mutant gene. Mc~2155 mutant strain M1 was screened at 42 鈩

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