结核分枝杆菌MurB的表达及其酶特性研究

发布时间：2018-03-25 12:11

  本文选题：结核分枝杆菌　切入点：murB基因　出处：《大连医科大学》2010年硕士论文

【摘要】： 结核分枝杆菌(Mycobacterium tuberculosis),俗称结核杆菌,是引起结核病的病原菌。20世纪80年代后期,由于耐药菌株尤其是耐多药结核菌(multi-drug resistant TB, MDR TB)的出现和流行,免疫缺陷病毒(HIV)/艾滋病(AIDS)的传播与流行,以及世界人口移民流动增多等原因,使结核病疫情在世界范围内急剧恶化,各国结核病的发病率均呈回升趋势。目前,结核病仍然是严重危害人类健康的传染病之一。因而,研究新型抗结核药物势在必行。 细胞壁是结核分枝杆菌赖以生存所必需的结构基础,所以结核分枝杆菌的细胞壁可以作为新药研发的靶点。其细胞壁核心结构由肽聚糖、聚阿拉伯糖半乳糖和分枝菌酸构成,如果研发新型药物可以破坏肽聚糖的结构,就会破坏结核分枝杆菌的细胞壁,导致细胞死亡。因为人体中不存在肽聚糖分子,所以肽聚糖可以作为研发新型抗结核药物的重要靶标。结核分枝杆菌基因组中murA-G七种基因编码了MurA-G七种酶,参与了由底物UDP-N-乙酰葡萄糖胺合成肽聚糖的全过程,共八步反应。MurA-G七种酶的任一种,都可以作为开发抗结核新药的靶标。 结核分枝杆菌MurB,即UDP-N-乙酰葡萄糖胺烯醇式丙酮酸还原酶是肽聚糖合成过程中第二步反应所需的酶。为了深入研究MurB的活性,我们首先要克隆编码这种酶的murB(Rv0482)基因,然后利用大肠杆菌高效表达MurB蛋白质,为进一步研究MurB酶的酶促反应动力学及建立高效的抗结核药物筛选提供物质基础。 本论文的目的是：(1)用PCR法从结核分枝杆菌基因组DNA中扩增Tb murB基因；(2)将Tb murB基因克隆到pMD18-T的克隆载体中,并对DNA序列进行测定；(3)将Tb murB基因亚克隆到pET29b表达载体中,并通过改变不同的诱导条件实现在表达菌株BL21(DE3)/pKJE7中表达目的蛋白MurB；(4)用亲和层析法纯化重组MurB蛋白质,并用Western Blotting方法鉴定MurB蛋白质；(5)建立测定MurB酶活性的方法；(6)确定MurB酶的最佳反应条件,并测定MurB酶的反应动力学常数。 本论文所获得的结果如下： 1.用PCR方法对目的基因Tb murB (Rv0482)扩增 从结核分枝杆菌H37Rv菌株基因组数据库(http://genolist.pasteur. fr./TubercuList/)中获得Tb murB基因的核苷酸序列(1110bp)。根据其核苷酸序列设计一对PCR引物,并在上游引物和下游引物的5’端分别引入NdeⅠ和XhoⅠ限制性内切酶位点,以便将Tb murB基因克隆到pET29b表达载体的NdeⅠ和XhoⅠ位点。利用高保真LA Taq聚合酶,以H37Rv菌株基因组DNA为模板成功扩增了Tb murB基因。 2. pMD18-Tb murB的构建及核苷酸序列测定 将纯化的PCR产物与pMD18-T载体进行连接,然后将连接产物转化入大肠杆菌NovaBlue菌株的感受态细胞中。用限制性内切酶BamH I酶切的方法鉴定阳性重组质粒。对pMD18-Tb murB中的TbmurB基因进行DNA序列测定,对DNA测序结果进行BLAST分析,将所测得的核苷酸序列与Tb murB基因进行序列比对,结果完全一致,表明在本实验中利用PCR方法获得的Tb murB为正确的基因,理论上能够正确表达出Tb MurB蛋白。 3.表达载体pET29b-Tb murB的构建 用Nde I和Xho I双酶切pMD18-Tb murB质粒,回收和纯化murB基因,将其连接到pET29b表达载体的NdeⅠ和XhoⅠ位点,构建pET29b-Tb murB表达质粒。用限制性内切酶Apa I酶切的方法鉴定阳性重组质粒。MurB蛋白的C端与质粒pET29b上的组氨酸标签形成融合蛋白。 4. Tb MurB蛋白在大肠杆菌BL21(DE3)/pKJE7中表达和纯化 将pET29b-Tb murB质粒转化到BL21(DE3)/pKJE7感受态细胞中,25℃振荡培养4小时,使其达到对数生长期。此时加入终浓度为0.5mg/mlL-阿拉伯糖(L-arabinose),25℃振荡培养3小时,使其充分表达分子伴侣dnaK、dnaJ及grpE。然后加入IPTG,终浓度达到0.4 mM,25℃振荡培养8小时,诱导携带pET29b-Tb murB质粒的BL21(DE3)/pKJE7菌株表达重组蛋白。用超声方法破碎诱导后的BL21(DE3)/pKJE7细菌,分别对上清和沉淀组分进行SDS-PAGE和Western blotting分析,结果表明MurB蛋白在BL21(DE3)/pKJE7菌株中可溶性表达。 采用组氨酸-Ni2+亲和层析技术纯化MurB蛋白,对纯化蛋白进行蛋白质定量(考马斯亮蓝法),第1 ml MurB蛋白的浓度为64.42μg/ml。SDS-PAGE和Western blotting分析结果表明MurB蛋白的纯度较高。 5.建立测定MurB酶活性的方法 将制备的反应底物UDP-N-乙酰葡萄糖胺和NADPH及FAD与纯化的MurB酶蛋白在37℃反应30分钟,迅速放于冰水混合物中终止反应,然后利用754紫外分光光度计检测340nm处的吸光度值,吸光度值明显减小,表明有产物生成,纯化的MurB蛋白质具有酶活性。 6.确定MurB酶的最佳反应条件 分别改变反应的温度和反应的pH值,利用754紫外分光光度计检测340nm处吸光度值的减小值,反应产物的生成量。结果表明MurB酶蛋白的最适反应温度是37℃,最适pH值是8.0。 7.测定MurB酶的反应动力学常数。 采用最佳酶促反应条件,即37℃,pH值8.0,酶浓度为1μg/ml,反应时间为5分钟。分别用不同的底物浓度,进行酶促反应,然后置于冰水中终止反应。利用754紫外分光光度计检测340nm处吸光度值的减小,反应产物的生成量。用双倒数作图法得出MurB的Km值和Vmax。对于底物UDP-N-乙酰葡萄糖胺烯醇丙酮酸,Km值为0.3752mmol/L, Vmax为0.6876 mmol/(L.min)。 结论： 在本研究中,我们构建了pET29b-Tb murB表达载体,在大肠杆菌BL21(DE3)/pKJE7中表达出大量的可溶性MurB蛋白。用纯化的蛋白研究酶的活性,确定了MurB的最佳反应条件并测定了MurB酶蛋白的反应动力学常数Km和Vmax,为建立完善的筛选抗结核药物的酶反应体系奠定了基础。
[Abstract]:Mycobacterium tuberculosis (Mycobacterium tuberculosis), commonly known as Mycobacterium tuberculosis, tuberculosis is caused by pathogenic bacteria in.20 in late 80s, due to drug resistant strains of multi drug resistant tuberculosis (especially multi-drug resistant TB, MDR TB) and popular, HIV / AIDS (HIV) (AIDS) of the spreading and prevalence of the world's population and immigration flow increase and other reasons, the tuberculosis epidemic situation deteriorated sharply in the world, the incidence of tuberculosis showed a recovery trend. At present, tuberculosis is still a serious infectious disease endangering human health. Therefore, research on new anti tuberculosis drugs is imperative.
The cell wall structure of Mycobacterium tuberculosis foundation necessary for survival, so the cell wall of Mycobacterium tuberculosis can be developed as a new drug target. The core of the cell wall structure by peptidoglycan, poly Arabia sugar galactose and mycolic acids, if new drugs can destroy the structure of cell wall peptidoglycan, will destruction of Mycobacterium tuberculosis, causing cell death. Because peptidoglycan molecules do not exist in the human body, so the peptidoglycan is a potential drug target to develop new anti tuberculosis drugs. The genome of Mycobacterium tuberculosis murA-G seven gene encoding MurA-G seven enzymes involved in the whole process, the substrate UDP-N- GlcNAc synthesis of peptidoglycan. A total of eight step reaction.MurA-G seven enzymes, can be used as the development of new anti tuberculosis drug targets.
Mycobacterium tuberculosis MurB, namely UDP-N- acetylglucosamine phosphoenolpyruvate reductase is required for the second step reaction in the synthesis of peptidoglycan enzymes. In order to study the activity of MurB, we first cloned murB encoding this enzyme (Rv0482) gene, then the expression of MurB protein in E.coli, for the further study of MurB enzyme the enzymatic reaction kinetics and the establishment of efficient anti tuberculosis drugs provide material basis for screening.
The purpose of this paper is: (1) the amplification of Tb murB gene from genomic DNA of Mycobacterium tuberculosis by PCR method; (2) the cloning vector of Tb murB gene was cloned into pMD18-T, and the DNA sequence was determined; (3) the Tb murB gene was subcloned into pET29b expression vector, and through change of different induction conditions in the expression strain BL21 (DE3) /pKJE7 protein expressed MurB; (4) to purify MurB protein by affinity chromatography, and using Western Blotting method to identify MurB protein; (5) to establish a method for determination of MurB activity; (6) to determine the optimum reaction conditions of MurB enzyme reaction. The kinetic constants and determination of MurB enzyme.
The results obtained in this paper are as follows:
1. amplification of Tb murB (Rv0482) of the target gene by PCR
From the database of H37Rv strain genome of Mycobacterium tuberculosis (http://genolist.pasteur. fr./TubercuList/) Tb nucleotide sequence of murB gene obtained in (1110bp). According to the design of a pair of PCR primers and its nucleotide sequence, upstream primer and downstream primer 5 'end respectively into Nde I and Xho I restriction endonuclease sites, so that the Tb murB gene was cloned into the pET29b expression vector of Nde I and Xho I sites. By using high fidelity LA Taq polymerase, H37Rv strain genomic DNA as template successfully amplified Tb murB gene.
Construction and nucleotide sequencing of 2. pMD18-Tb murB
The PCR product with pMD18-T vector were connected, and then connect the competent cells were transformed into Escherichia coli strain NovaBlue. The positive recombinant with restriction enzyme BamH I digestion method. The identification of plasmid TbmurB gene pMD18-Tb in murB by DNA sequencing, BLAST analysis of DNA sequencing results, the the nucleotide sequence of murB gene and Tb measured the sequences were the same as that in the experiment using PCR Tb murB method to obtain the correct gene theory to the correct expression of Tb MurB protein.
Construction of the 3. expression vector pET29b-Tb murB
PMD18-Tb murB cut with plasmid Nde I and Xho I double enzyme, recovery and purification of the murB gene, and cloned into pET29b expression vector of Nde I and Xho I sites, construction of pET29b-Tb murB expression plasmid. Histidine tag identification method of.MurB protein positive recombinant plasmid by restriction endonuclease Apa I digested C terminal with plasmid pET29b on the formation of fusion protein.
Expression and purification of 4. Tb MurB protein in Escherichia coli BL21 (DE3) /pKJE7
The conversion of pET29b-Tb to murB plasmid BL21 (DE3) /pKJE7 competentcells, 25 DEG C for 4 hours, to reach the logarithmic growth phase. The final concentration was 0.5mg/mlL- was added to the Arabia sugar (L-arabinose), 25 DEG C for 3 hours, the full expression of molecular chaperone dnaK, dnaJ and grpE. adding IPTG the final concentration reached 0.4 mM, 25 DEG C for 8 hours, by carrying the pET29b-Tb murB plasmid BL21 (DE3) expression of recombinant protein of /pKJE7 strain. After induction of BL21 broken by ultrasonic method (DE3) /pKJE7 bacteria, respectively on the supernatant and pellet fractions were analyzed by SDS-PAGE and Western blotting, the results showed that MurB protein in BL21 (DE3) expression of soluble /pKJE7 strain.
The histidine -Ni2+ purified MurB protein affinity chromatography, the purified protein for protein quantification (Kaumas Bradford method), the concentration of first ml MurB protein is 64.42 g/ml.SDS-PAGE and Western blotting analysis showed that MurB protein with high purity.
5. to establish a method for the determination of MurB enzyme activity
The prepared substrate UDP-N- acetyl glucosamine and NADPH and FAD and purification of MurB protein in 37 DEG C for 30 minutes, quickly put in ice water mixture in the termination reaction, then use 340nm absorbance measured 754 UV absorbance value decreased significantly, that is the product formation, MurB protein purification with enzyme activity.
6. determine the optimum reaction conditions for MurB enzyme
The temperature of reaction and the pH value of reaction were respectively changed. The reduction value of absorbance value at 340nm and the product of reaction product were detected by 754 UV spectrophotometer. The results showed that the optimum reaction temperature of MurB enzyme protein was 37 C, and the best pH value was 8.0..
7. the kinetic constants of the MurB enzyme were determined.
The optimal enzymatic reaction conditions, which is 37 DEG C, the pH value is 8, the enzyme concentration is 1 g/ml, the reaction time is 5 minutes. Respectively with different concentration, enzymatic reaction, and then placed in ice water. Reduce the termination reaction measured 340nm absorbance value 754 by UV spectrophotometry, generation the amount of reaction product. Using double reciprocal Tufade MurB values of Km and Vmax. for the substrate UDP-N- GlcNAc enolpyruvate, Km = 0.3752mmol/L, Vmax 0.6876 mmol/ (L.min).
Conclusion:
In this study, we constructed pET29b-Tb murB expression vector in Escherichia coli BL21 (DE3) expression of a large number of soluble MurB protein in /pKJE7. With the study of enzyme activity of purified protein, the optimum reaction conditions of MurB and MurB enzyme kinetic parameters Km and Vmax were determined, which laid the foundation for the the enzyme reaction system to establish and improve the screening of anti tuberculosis drugs.

【学位授予单位】：大连医科大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R378

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