结核分枝杆菌脂阿拉伯甘露聚糖合成途径相关基因的克隆和功能研究

发布时间：2018-05-19 08:37

本文选题：结核分枝杆菌 + 脂阿拉伯甘露聚糖　；参考：《复旦大学》2006年博士论文

【摘要】： 结核分枝杆菌是引起人类疾病的重要的病原菌之一。目前全球人口中大约1／3的人口已感染结核分枝杆菌，每年约有200万人死于结核。而且耐药菌的数量的增加，因而急需发展新型的抗结核药物。分枝杆菌的细胞壁成份独特，也是目前一些一线抗结核药物的作用靶点。因而分枝杆菌细胞壁合成途径是理论设计抗结核药物的有吸引力靶点。磷脂酰肌醇及其代谢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杆菌细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稳定性，进而在细胞存活上起着重要作用。脂阿拉伯甘露聚糖，尤其是带有甘露糖帽的脂阿拉伯甘露聚糖，是慢性生长的分枝杆菌细胞壁上的重要糖脂，如结核分枝杆菌。带有甘露糖帽的脂阿拉伯甘露聚糖还有多种免疫调节功能，例如抑制巨噬细胞激活，抑制巨噬细胞和树状突细胞产生IL-12，调节结核分枝杆菌调节的巨噬细胞凋亡。所有这些活性都有利于巨噬细胞内细菌的存活。结核分枝杆菌的pimA基因编码一个甘露糖基转移酶，介导来自GDP-甘露糖的甘露糖加到脂载体磷脂酰肌醇的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体物。已有研究推测pimA基因是分枝杆菌生长的必要成份。本研究中，将来自结核分枝杆菌的pimA基因克隆到pET28a载体中，得到的重组质粒在大肠肝菌BL21(DE3)中表达重组蛋白，再用Ni-NTA柱亲和层析纯化重组蛋白。其纯度和分子量分别通过HPLC和MALDI-TOF测定。圆二色仪结果表明PimA蛋白是折叠的。酶活测定结果表明Mg~(2+)是PimA酶活反应所需的。其K_m是18±2uM，V_(max)是0.1±0.05nmol／min／ug。肌醇单磷酸酶是肌醇合成途径中的一个关键酶，肌醇是合成磷脂酰肌醇的前体物，而磷脂酰肌醇是结核分枝杆菌的重要成份。本研究中，将来自结核分枝杆菌的Rv2131c基因克隆到pET28a载体中，重组质粒在大肠肝菌BL21(DE3)中表达重组蛋白，再用Ni-NTA柱亲和层析纯化重组蛋白。Rv2131c产物具有肌醇单磷酸酶活性，但是其底物特异性比其它细菌和真核生物的肌醇单磷酸酶广。 Rv2131c产物还可以特异性的水解1，，6-二磷酸果糖。这个二聚体蛋白是一个双功能酶，除了可以水解1-磷酸肌醇外，还可以水解1，6-二磷酸果糖。以1-磷酸肌醇为底物其K_m值为0.22±0.03mM，以1，6-二磷酸果糖为底物，其K_m值为0.45±0.05mM。为了进一步了解其结构的功能的关系，又构建了不同的突变体，包括D40N，L71A，D94N突变体，并且表达，纯化了相应的突变体蛋白。D40N和D94N突变蛋白几乎同时失去了两种活性，而L71A突变蛋白的两种活性都没发生大的变化，但是其对Li~+离子抗性是野生型的12倍。因而根据Rv2131c其底物特异性及其存在相应的保守序列，将Rv2131c编码的酶归为第四类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).
【学位授予单位】：复旦大学
【学位级别】：博士
【学位授予年份】：2006
【分类号】：R346

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