本文選題：結(jié)核分枝桿菌 + 脂阿拉伯甘露聚糖��； 參考：《復(fù)旦大學(xué)》2006年博士論文

【摘要】： 結(jié)核分枝桿菌是引起人類疾病的重要的病原菌之一。目前全球人口中大約1／3的人口已感染結(jié)核分枝桿菌，每年約有200萬人死于結(jié)核。而且耐藥菌的數(shù)量的增加，因而急需發(fā)展新型的抗結(jié)核藥物。分枝桿菌的細(xì)胞壁成份獨(dú)特，也是目前一些一線抗結(jié)核藥物的作用靶點(diǎn)。因而分枝桿菌細(xì)胞壁合成途徑是理論設(shè)計(jì)抗結(jié)核藥物的有吸引力靶點(diǎn)。 磷脂酰肌醇及其代謝衍生物磷脂酰肌醇甘露糖，脂甘露聚糖，脂阿拉伯甘露聚糖是分枝桿菌細(xì)胞壁上主要和重要的磷酸脂／脂聚糖。磷脂酰肌醇和磷脂酰肌醇甘露糖在膜的穩(wěn)定性，進(jìn)而在細(xì)胞存活上起著重要作用。脂阿拉伯甘露聚糖，尤其是帶有甘露糖帽的脂阿拉伯甘露聚糖，是慢性生長(zhǎng)的分枝桿菌細(xì)胞壁上的重要糖脂，如結(jié)核分枝桿菌。帶有甘露糖帽的脂阿拉伯甘露聚糖還有多種免疫調(diào)節(jié)功能，例如抑制巨噬細(xì)胞激活，抑制巨噬細(xì)胞和樹狀突細(xì)胞產(chǎn)生IL-12，調(diào)節(jié)結(jié)核分枝桿菌調(diào)節(jié)的巨噬細(xì)胞凋亡。所有這些活性都有利于巨噬細(xì)胞內(nèi)細(xì)菌的存活。 結(jié)核分枝桿菌的pimA基因編碼一個(gè)甘露糖基轉(zhuǎn)移酶，介導(dǎo)來自GDP-甘露糖的甘露糖加到脂載體磷脂酰肌醇的2位上，而磷脂酰肌醇又是合成磷脂酰肌醇甘露糖，脂甘露聚糖和脂阿拉伯甘露聚糖的前體物。已有研究推測(cè)pimA基因是分枝桿菌生長(zhǎng)的必要成份。本研究中，將來自結(jié)核分枝桿菌的pimA基因克隆到pET28a載體中，得到的重組質(zhì)粒在大腸肝菌BL21(DE3)中表達(dá)重組蛋白，再用Ni-NTA柱親和層析純化重組蛋白。其純度和分子量分別通過HPLC和MALDI-TOF測(cè)定。圓二色儀結(jié)果表明PimA蛋白是折疊的。酶活測(cè)定結(jié)果表明Mg~(2+)是PimA酶活反應(yīng)所需的。其K_m是18±2uM，V_(max)是0.1±0.05nmol／min／ug。 肌醇單磷酸酶是肌醇合成途徑中的一個(gè)關(guān)鍵酶，肌醇是合成磷脂酰肌醇的前體物，而磷脂酰肌醇是結(jié)核分枝桿菌的重要成份。本研究中，將來自結(jié)核分枝桿菌的Rv2131c基因克隆到pET28a載體中，重組質(zhì)粒在大腸肝菌BL21(DE3)中表達(dá)重組蛋白，再用Ni-NTA柱親和層析純化重組蛋白。Rv2131c產(chǎn)物具有肌醇單磷酸酶活性，但是其底物特異性比其它細(xì)菌和真核生物的肌醇單磷酸酶廣。 Rv2131c產(chǎn)物還可以特異性的水解1，，6-二磷酸果糖。這個(gè)二聚體蛋白是一個(gè)雙功能酶，除了可以水解1-磷酸肌醇外，還可以水解1，6-二磷酸果糖。以1-磷酸肌醇為底物其K_m值為0.22±0.03mM，以1，6-二磷酸果糖為底物，其K_m值為0.45±0.05mM。為了進(jìn)一步了解其結(jié)構(gòu)的功能的關(guān)系，又構(gòu)建了不同的突變體，包括D40N，L71A，D94N突變體，并且表達(dá)，純化了相應(yīng)的突變體蛋白。D40N和D94N突變蛋白幾乎同時(shí)失去了兩種活性，而L71A突變蛋白的兩種活性都沒發(fā)生大的變化，但是其對(duì)Li~+離子抗性是野生型的12倍。因而根據(jù)Rv2131c其底物特異性及其存在相應(yīng)的保守序列，將Rv2131c編碼的酶歸為第四類1，6-二磷酸果糖磷酸酶(FBPase Ⅳ)。
[Abstract]:Mycobacterium tuberculosis is one of the important pathogens causing human disease. At present, about 1 / 3 of the population of the global population has been infected with Mycobacterium tuberculosis, and about 2 million people die from tuberculosis every year. And the number of drug-resistant bacteria is increasing, so it is urgent to develop a new anti tuberculosis drug. Therefore, the cell wall synthesis pathway of mycobacteria is an attractive target for the theoretical design of anti tuberculosis drugs.
Phosphatidylinositol and its metabolites, phosphatidylinositol mannose, fat mannan, and fat Arabia mannan, are the major and important phosphate / fat glycans on the cell walls of mycobacteria. The stability of phosphatidylinositol and phosphatidyl inositol mannose plays an important role in cell survival. Fat Arabia mannan, In particular, the fat Arabia mannan with mannose cap is an important sugar fat on the cell wall of the chronic growing mycobacterium, such as Mycobacterium tuberculosis. The fat Arabia mannan with mannose cap has a variety of immune regulating functions, such as inhibiting macrophage activation, inhibiting macrophages and dendritic cells to produce IL-12, regulating knot Mycobacterium tuberculosis regulates macrophage apoptosis. All these activities are beneficial to bacterial survival in macrophages.
The pimA gene of Mycobacterium tuberculosis encodes a mannose based transferase, which mediates the mannose from GDP- mannose to the fat carrier phosphatidylinositol 2, and phosphatidylinositol is the precursor of the synthesis of phosphatidyl inositol mannose, fat mannan and fat Arabia mannan. In this study, the pimA gene from Mycobacterium tuberculosis was cloned into the pET28a vector and the recombinant plasmid was expressed in the Escherichia coli BL21 (DE3), and the recombinant protein was purified by Ni-NTA column affinity chromatography. The purity and molecular weight of the recombinant plasmid were determined by HPLC and MALDI-TOF respectively. The circular two color instrument showed PimA eggs. The results of enzyme activity test showed that Mg~ (2+) is required for PimA enzyme reaction. Its K_m is 18 + 2uM, V_ (max) is 0.1 + 0.05nmol / min / ug..
Inositol mono phosphatase is a key enzyme in the inositol synthesis pathway. Inositol is the precursor of the synthesis of phosphatidylinositol, and phosphatidylinositol is an important component of Mycobacterium tuberculosis. In this study, the Rv2131c gene from Mycobacterium tuberculosis was cloned into the pET28a vector, and the recombinant plasmid was expressed in the BL21 (DE3) of the Escherichia coli (DE3). Ni-NTA column affinity chromatography was used to purify the recombinant protein.Rv2131c products with inositase activity, but its substrate specificity was wider than that of inositol monophosphatase from other bacteria and eukaryotes.
The Rv2131c product can also hydrolyze 1,6- two phosphate fructose specifically. This two polymer protein is a bifunctional enzyme that can hydrolyze 1,6- two phosphoric acid in addition to the hydrolysis of the inositol of 1- phosphate. The K_m value of 1- phosphoric inositol as the substrate is 0.22 + 0.03mM, and 1,6- two phosphate is the substrate, and its K_m value is 0.45 + 0.05mM. for further development. To understand the functional relationship of its structure, different mutants were constructed, including D40N, L71A, and D94N mutants, and the corresponding mutant protein.D40N and D94N mutant protein were purified almost simultaneously with two kinds of activity, while the two activity of the L71A mutant protein had not changed greatly, but the resistance to Li~+ ion was wild type. In accordance with the substrate specificity of Rv2131c and its corresponding conservative sequence, the Rv2131c encoded enzyme was classified as the fourth class of 1,6- two phosphate fructose phosphatase (FBPase IV).
【學(xué)位授予單位】：復(fù)旦大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2006
【分類號(hào)】：R346

【參考文獻(xiàn)】

相關(guān)期刊論文 前2條

1 初乃惠,朱莉貞,葉志忠,苑松林,王靜云,許家璉,馬麗萍;重組人白細(xì)胞介素-2輔助治療復(fù)治肺結(jié)核近期療效觀察[J];中華結(jié)核和呼吸雜志;2003年09期

2 陸宇,段連山;抗結(jié)核藥物的研究進(jìn)展和發(fā)展趨勢(shì)[J];中國(guó)抗生素雜志;2005年04期

本文編號(hào)：1909434