【作者】 宋宇；

【导师】 马郁芳；

【作者基本信息】 大连医科大学 ， 生物化学与分子生物学， 2010， 硕士

【摘要】 结核分枝杆菌(Mycobacterium tuberculosis)是引起结核病的病原体。从世界范围来看,结核病仍是一种发病率和死亡率极高的传染性疾病,全球人口的三分之一感染了结核分枝杆菌,而且,其发病率也呈逐年上升的趋势。其主要原因之一是由于耐多药(multi drug resistant, MDR)甚至是广泛耐药(extensive drug resistant, XDR)结核杆菌的出现,许多一线甚至一些二线抗结核药物都无法有效治疗结核病。因此,当前的首要任务是从结核分枝杆菌自身寻找新的靶点,以此来研发新的抗结核药物。分枝杆菌细胞壁的核心结构由分枝菌酸,聚阿拉伯半乳糖和肽聚糖构成。其中,肽聚糖是维持细菌细胞形态和渗透压平衡的机构基础,其组成及结构可能与G-细菌肽聚糖相似,即由N-乙酰葡糖胺和N-乙酰胞壁酸交替连接形成的多糖链与短肽链交叉连接形成网状大分子机构。UDP-乙酰胞壁酸是N-乙酰胞壁酸的糖基供体,两个酶催化由UDP-乙酰葡糖胺转变为UDP-乙酰胞壁酸的代谢途径。MurA (UDP-N-乙酰葡糖胺1-羧基乙烯基转移酶)是催化此条途径的第一个关键酶,由于此反应途径无代谢旁路,并且在哺乳动物中不存在此代谢途径,因此,催化此代谢途径的酶可能是抗结核药物的作用靶点。为了建立体外筛选抗结核药物的分子模型,我们首先要克隆编码结核分枝杆菌UDP-N-乙酰葡糖胺1-羧基乙烯基转移酶的murA基因,然后表达Tb MurA蛋白,为进一步研究UDP-N-乙酰葡糖胺1-羧基乙烯基转移酶的功能及建立筛选抗结核药物的酶反应体系提供物质保障。本论文的目的：(1)用pET29b表达载体在大肠杆菌BL21(DE3)中表达结核分枝杆菌MurA蛋白,优化表达条件以期获得可溶性蛋白；(2)分离、溶解、纯化及鉴定包含体形式的Tb MurA蛋白,并用纯化的Tb MurA蛋白免疫兔子以制备多克隆抗体,对抗体的效价和特异性进行检测；(3)用pVV 16表达载体在耻垢分枝杆菌mc2155中表达Tb MurA蛋白,并用亲和层析技术纯化Tb MurA蛋白,用SDS-PAGE以及Western blotting鉴定所纯化的Tb MurA蛋白；(4)用高效液相色谱(HPLC)法和化学显色法两种方法测定MurA蛋白的酶活性。本论文获得的结果：1.表达载体pET29b-Tb murA的构建用NdeⅠ和HindⅢ双酶切pMD18-Tb murA质粒,回收和纯化TbmurA基因,将其连接到pET29b表达载体的NdeⅠ和HindⅢ位点,构建pET29b-Tb murA表达质粒。用SmaⅠ酶切的方法鉴定阳性重组质粒。MurA蛋白的C端与pET29b表达载体上的6个连续的组氨酸标签形成融合蛋白,便于目的蛋白的检测和纯化。2. Tb MurA蛋白在大肠杆菌BL21(DE3)中的诱导表达及表达条件的优化将pET29b-Tb murA质粒转化到BL21(DE3)感受态细胞中,诱导携带pET29b-Tb murA质粒的BL21(DE3)表达重组蛋白。用超声方法破碎诱导后的BL21(DE3),对上清和沉淀组分进行SDS-PAGE和Western blotting分析,结果表明Tb MurA蛋白在BL21(DE3)中形成包含体。优化表达条件,通过降低IPTG的浓度、诱导温度及与分子伴侣共表达等,但没有改善目的蛋白的溶解性。3.包含体的分离、溶解、纯化及鉴定将包含体进行了分离,用溶解缓冲液溶解,获得了很好的溶解效果,获得了大量的可溶性蛋白。用组氨酸-Ni2+亲和层析技术纯化溶解后的Tb MurA蛋白。对纯化的蛋白进行蛋白质定量(考马斯亮蓝法),SDS-PAGE和Western blotting分析结果表明我们获得了纯Tb MurA蛋白。4.多克隆抗体的制备及效价、特异性检测将纯化的Tb MurA蛋白溶液制备成免疫用抗原,免疫家兔,制备抗血清,通过间接法酶联免疫吸附试验测得抗血清的抗体滴度为1：78125,采用抗血清及偶联碱性磷酸酶的山羊抗兔IgG二抗分别对在耻垢分枝杆菌中表达的TbMurA蛋白和野生耻垢分枝杆菌上清蛋白进行Western Blotting分析,结果表明制备的MurA多克隆抗体特异性较好。5.表达载体pVV16-Tb murA的构建用NdeⅠ和HindⅢ双酶切pMD18-Tb murA质粒,回收和纯化TbmurA基因,将其连接到pVV1 6表达载体的NdeⅠ和HindⅢ位点,构建pVV16-Tb murA表达质粒。用XhoI酶切的方法鉴定阳性重组质粒。TbMurA蛋白的C端与pVV1 6表达载体上的组氨酸标签形成融合蛋白,便于目的蛋白的检测和纯化。6. Tb MurA蛋白在耻垢分枝杆菌mc2155中的表达将pVV16-Tb murA质粒转化到mc21 55感受态细胞中,37℃震荡培养48小时表达目的蛋白,用超声方法破碎培养后的细菌,对上清和沉淀组分进行SDS-PAGE和Western blotting分析,结果表明Tb MurA蛋白在mc2155中可溶性表达。7.采用亲和层析技术纯化Tb MurA蛋白pVV16表达载体使重组MurA蛋白C端带有组氨酸标签,因此,用组氨酸-Ni2+亲和层析技术纯化Tb MurA蛋白。对纯化的蛋白进行蛋白质定量(考马斯亮蓝法),SDS-PAGE和Western blotting分析结果表明我们获得了纯Tb MurA蛋白。8.建立Tb MurA蛋白的酶活性测定方法(1)高效液相色谱法：采用Nova-Pak C18(3.9×150mm,4μm)色谱柱,以20 mM三乙胺-醋酸缓冲液(pH 4.0)为流动相,流速0.5mLmin-1,在波长260nm处检测反应底物UDP-GlcNAc的减少,其洗脱时间为6.3min。(2)化学显色法：反应产物之一为磷酸,磷酸与钼酸铵形成磷钼酸复合物后使孔雀石绿颜色由黄绿变为蓝绿,用酶标仪在630 nm处检测吸光值变化,以测定所生成磷酸的含量。结论：在本研究中,我们构建了pET29b-Tb murA表达载体,在大肠杆菌BL21(DE3)中表达出大量以包含体性形式存在的Tb MurA蛋白。我们用包含体中的Tb MurA蛋白制备了多克隆抗体,分别用ELISA和Western blotting进行检测和鉴定,多克隆抗体有较高的效价和特异性。我们构建了pVV16-Tb murA表达载体,在耻垢分枝杆菌mc2155中表达出以可溶性形式存在的Tb MurA蛋白,并鉴定了Tb MurA蛋白的酶活性。更多还原

【Abstract】 Mycobacterium tuberculosis is the pathogen that causes tuberculosis. As a global issue, tuberculosis (TB) is still an infectious disease with extremely high morbidity and mortality, catched by nearly one thirds of worldwide populations, moreover the incidence is rising yearly. Major problem is the emergence of multi-drug and extra-drug resistant strains makes invalid of many anti-TB drugs. Therefore, identifying new targets from M. tuberculosis itself and developing new anti-tuberculosis drugs may become a chief task currently.The core of the mycobacterial cell wall consists of mycolic acid, arabinogalactan, and peptidoglycan for connection. One of the best known and most validated targets for antibacterial therapy is the machinery for peptidoglycan biosynthesis. The biosynthetic pathway of Mycobacterium tuberculosis peptidoglycan is a complex two-stage process. The first stage, which occurs in the cytoplasm, is the formation of the monomeric building block N-acetylglucosamine-N-acetylmuramyl pentapeptide. The first committed step in the pathway is the transfer of an enolpyruvate residue from phosphoenolpyruvate (PEP) to position 3 of UDP-N-acetylglu-cosamine. This reaction is catalysed by the enzyme UDP-N-acetylglucosamine enolpyruvyl transferase (MurA). MurA is ubiquitous to both Gram-positive and Gram-negative bacteria, and has no homologue in mammalian cells. Therefore, MurA is a potential target to develop anti-TB drugs.Our long-term objective is to establish a molecule model of screening anti-TB drug. Therefore, we must first clone the gene murA which encodes the enzyme MurA, and then express soluble MurA protein. Soluble MurA will be utilized for further purification and study the kinetics of MurA as well as development of enzyme assay to screen anti-Tb drugs.The objectives of this study:(1) to express M. tuberculosis MurA protein by pET29b express vector in E.coli BL21(DE3) and optimize the expression conditions in order to obtain the soluble MurA; (2) to separate and solubolize inclusion body-forming MurA protein and purify MurA protein by affinity chromatography. To identify the purified MurA protein by SDS-PAGE and Western blotting and prepare anti-MurA polyclonal antibody by injecting rabbit with purified MurA protein and detect the serum specificity and its titer; (3) to express M. tuberculosis MurA protein by pVV16 express vector in M.smegmatis mc2155; (4) to establish methods to detect the activity of MurA enzyme.The results are followings:1. Expression vector pET29b-Tb murA was constructed.pMD18-Tb murA was digested by NdeⅠand HindⅢ, then Tb murA gene fragment was purified and ligated into the NdeⅠand HindⅢsites of vector pET29b to generate pET29b-Tb murA. The C-terminus of MurA protein was fused with histidine tag in pET29b vector.2. Tb MurA protein was expressed in E.coli BL21(DE3) and the expression conditions was optimized.pET29b-Tb murA were transformed into BL21(DE3) competent cells. BL21(DE3) cells carring pET29b-Tb murA were induced. Induced BL21(DE3) cells were sonicated and both supernatant and pellet fractions were analyzed by SDS-PAGE and Western blotting. MurA protein was expressed in E.coli BL21(DE3),but they were insoluble in inclusion body. Optimize the expression conditions, for example, to reduse the concen-tration of IPTG, to induce under lower temperature condition and to coexpress with chaperons, and so on. The Tb MurA still formed inclusion bodies.3. Inclusion body-forming Tb MurA protein was separated, solubolized, purified and detected.The inclusion body-forming Tb MurA protein was separated, solubolized with different buffers and obtained lots of soluble Tb MurA protein. Tb MurA protein was purified by histidine-Ni2+ affinity chromatography. The elution fraction was quantified by coomassie brilliant blue method. The purified MurA protein was confirmed by SDS-PAGE and Western blotting.4. Anti-Tb MurA polyclonal antibody was prepared and identified.Purified Tb MurA was prepared with particular means for injecting rabbit. Antiserum was separated and assayed for antibody titer as 1:78125 by enzyme-linked immunosorbent assay. Tb MurA protein expressed in mc2155 and protein of M. smegmatis mc2155 was detected by antiserum followed by antirabbit-IgG-conjugated with alkaline phosphatase. The result showed anti-MurA polyclonal antibody had high specificity.5. Expression vector pVV16-Tb murA was constructed.pMD18-Tb murA was digested by NdeⅠand HindⅢ, then murA gene fragment was purified and ligated into the NdeⅠand HindⅢsites of vector pVV16 to generate pVV16-Tb murA. The C-terminus of Tb MurA protein was fused with histidine tag in pVV16 vector.6. Tb MurA protein in M. smegmatis mc2155 was expressed and identified.pVV16-Tb murA was transformed into M. smegmatis mc2155 competent cells to express the Tb MurA protein. The mc2155 cells were broken by sonication, and the proteins from both supernatant and pellet fractions were analyzed by SDS-PAGE and Western blotting. The results showed that Tb MurA protein in mc2155 was soluble expressed.7. Tb MurA protein was purified by affinity chromatography.The C-terminus of Tb MurA was fused with histidine tag in pVV16, therefore Tb MurA was purified by histidine-Ni2+ affinity chromatography. The elution fraction was quantified by coomassie brilliant blue method. The purified Tb MurA protein was confirmed by SDS-PAGE and Western blotting.8. The enzyme assay of Tb MurA was established.(1) HPLC:UDP-G1cNAc was separated with Nova-Pak C18 at a flow rate of 0.5mLmin-1 under 20 mM triethylamine-acetic acid buffer (pH 4.0) and was monitored at 260 nm. The peak of UDP-G1cNAc was appeared at 6.3 min approximately. Its reduction indicated that MurA has activity. (2) Chemical colorimetric assays:The inorganic phosphate produced in the reation was detected. The reaction of inorganic phosphate and ammonium molybdate results in the formation of molybdenum blue, which turns malachite green from yellow to blue. The OD value at 630 nm was detected.Conclusions:In this study, we constructed pET29b-Tb murA expression vector and overexpressed recombinant Tb MurA protein in inclusion body and anti-MurA polyclonal antibody was prepared.We constructed pVV16-Tb murA expression vector and expressed considerable soluble recombinant Tb MurA protein in M. smegmatis mc2155.We established Tb MurA enzyme assay and identified Tb MurA enzyme activity.更多还原