本文選題：結(jié)核分枝桿菌　切入點：murB基因　出處：《大連醫(yī)科大學(xué)》2010年碩士論文

【摘要】： 結(jié)核分枝桿菌(Mycobacterium tuberculosis),俗稱結(jié)核桿菌,是引起結(jié)核病的病原菌。20世紀(jì)80年代后期,由于耐藥菌株尤其是耐多藥結(jié)核菌(multi-drug resistant TB, MDR TB)的出現(xiàn)和流行,免疫缺陷病毒(HIV)/艾滋病(AIDS)的傳播與流行,以及世界人口移民流動增多等原因,使結(jié)核病疫情在世界范圍內(nèi)急劇惡化,各國結(jié)核病的發(fā)病率均呈回升趨勢。目前,結(jié)核病仍然是嚴(yán)重危害人類健康的傳染病之一。因而,研究新型抗結(jié)核藥物勢在必行。 細胞壁是結(jié)核分枝桿菌賴以生存所必需的結(jié)構(gòu)基礎(chǔ),所以結(jié)核分枝桿菌的細胞壁可以作為新藥研發(fā)的靶點。其細胞壁核心結(jié)構(gòu)由肽聚糖、聚阿拉伯糖半乳糖和分枝菌酸構(gòu)成,如果研發(fā)新型藥物可以破壞肽聚糖的結(jié)構(gòu),就會破壞結(jié)核分枝桿菌的細胞壁,導(dǎo)致細胞死亡。因為人體中不存在肽聚糖分子,所以肽聚糖可以作為研發(fā)新型抗結(jié)核藥物的重要靶標(biāo)。結(jié)核分枝桿菌基因組中murA-G七種基因編碼了MurA-G七種酶,參與了由底物UDP-N-乙酰葡萄糖胺合成肽聚糖的全過程,共八步反應(yīng)。MurA-G七種酶的任一種,都可以作為開發(fā)抗結(jié)核新藥的靶標(biāo)。 結(jié)核分枝桿菌MurB,即UDP-N-乙酰葡萄糖胺烯醇式丙酮酸還原酶是肽聚糖合成過程中第二步反應(yīng)所需的酶。為了深入研究MurB的活性,我們首先要克隆編碼這種酶的murB(Rv0482)基因,然后利用大腸桿菌高效表達MurB蛋白質(zhì),為進一步研究MurB酶的酶促反應(yīng)動力學(xué)及建立高效的抗結(jié)核藥物篩選提供物質(zhì)基礎(chǔ)。 本論文的目的是：(1)用PCR法從結(jié)核分枝桿菌基因組DNA中擴增Tb murB基因；(2)將Tb murB基因克隆到pMD18-T的克隆載體中,并對DNA序列進行測定；(3)將Tb murB基因亞克隆到pET29b表達載體中,并通過改變不同的誘導(dǎo)條件實現(xiàn)在表達菌株BL21(DE3)/pKJE7中表達目的蛋白MurB；(4)用親和層析法純化重組MurB蛋白質(zhì),并用Western Blotting方法鑒定MurB蛋白質(zhì)；(5)建立測定MurB酶活性的方法；(6)確定MurB酶的最佳反應(yīng)條件,并測定MurB酶的反應(yīng)動力學(xué)常數(shù)。 本論文所獲得的結(jié)果如下： 1.用PCR方法對目的基因Tb murB (Rv0482)擴增 從結(jié)核分枝桿菌H37Rv菌株基因組數(shù)據(jù)庫(http://genolist.pasteur. fr./TubercuList/)中獲得Tb murB基因的核苷酸序列(1110bp)。根據(jù)其核苷酸序列設(shè)計一對PCR引物,并在上游引物和下游引物的5’端分別引入NdeⅠ和XhoⅠ限制性內(nèi)切酶位點,以便將Tb murB基因克隆到pET29b表達載體的NdeⅠ和XhoⅠ位點。利用高保真LA Taq聚合酶,以H37Rv菌株基因組DNA為模板成功擴增了Tb murB基因。 2. pMD18-Tb murB的構(gòu)建及核苷酸序列測定 將純化的PCR產(chǎn)物與pMD18-T載體進行連接,然后將連接產(chǎn)物轉(zhuǎn)化入大腸桿菌NovaBlue菌株的感受態(tài)細胞中。用限制性內(nèi)切酶BamH I酶切的方法鑒定陽性重組質(zhì)粒。對pMD18-Tb murB中的TbmurB基因進行DNA序列測定,對DNA測序結(jié)果進行BLAST分析,將所測得的核苷酸序列與Tb murB基因進行序列比對,結(jié)果完全一致,表明在本實驗中利用PCR方法獲得的Tb murB為正確的基因,理論上能夠正確表達出Tb MurB蛋白。 3.表達載體pET29b-Tb murB的構(gòu)建 用Nde I和Xho I雙酶切pMD18-Tb murB質(zhì)粒,回收和純化murB基因,將其連接到pET29b表達載體的NdeⅠ和XhoⅠ位點,構(gòu)建pET29b-Tb murB表達質(zhì)粒。用限制性內(nèi)切酶Apa I酶切的方法鑒定陽性重組質(zhì)粒。MurB蛋白的C端與質(zhì)粒pET29b上的組氨酸標(biāo)簽形成融合蛋白。 4. Tb MurB蛋白在大腸桿菌BL21(DE3)/pKJE7中表達和純化 將pET29b-Tb murB質(zhì)粒轉(zhuǎn)化到BL21(DE3)/pKJE7感受態(tài)細胞中,25℃振蕩培養(yǎng)4小時,使其達到對數(shù)生長期。此時加入終濃度為0.5mg/mlL-阿拉伯糖(L-arabinose),25℃振蕩培養(yǎng)3小時,使其充分表達分子伴侶dnaK、dnaJ及grpE。然后加入IPTG,終濃度達到0.4 mM,25℃振蕩培養(yǎng)8小時,誘導(dǎo)攜帶pET29b-Tb murB質(zhì)粒的BL21(DE3)/pKJE7菌株表達重組蛋白。用超聲方法破碎誘導(dǎo)后的BL21(DE3)/pKJE7細菌,分別對上清和沉淀組分進行SDS-PAGE和Western blotting分析,結(jié)果表明MurB蛋白在BL21(DE3)/pKJE7菌株中可溶性表達。 采用組氨酸-Ni2+親和層析技術(shù)純化MurB蛋白,對純化蛋白進行蛋白質(zhì)定量(考馬斯亮藍法),第1 ml MurB蛋白的濃度為64.42μg/ml。SDS-PAGE和Western blotting分析結(jié)果表明MurB蛋白的純度較高。 5.建立測定MurB酶活性的方法 將制備的反應(yīng)底物UDP-N-乙酰葡萄糖胺和NADPH及FAD與純化的MurB酶蛋白在37℃反應(yīng)30分鐘,迅速放于冰水混合物中終止反應(yīng),然后利用754紫外分光光度計檢測340nm處的吸光度值,吸光度值明顯減小,表明有產(chǎn)物生成,純化的MurB蛋白質(zhì)具有酶活性。 6.確定MurB酶的最佳反應(yīng)條件 分別改變反應(yīng)的溫度和反應(yīng)的pH值,利用754紫外分光光度計檢測340nm處吸光度值的減小值,反應(yīng)產(chǎn)物的生成量。結(jié)果表明MurB酶蛋白的最適反應(yīng)溫度是37℃,最適pH值是8.0。 7.測定MurB酶的反應(yīng)動力學(xué)常數(shù)。 采用最佳酶促反應(yīng)條件,即37℃,pH值8.0,酶濃度為1μg/ml,反應(yīng)時間為5分鐘。分別用不同的底物濃度,進行酶促反應(yīng),然后置于冰水中終止反應(yīng)。利用754紫外分光光度計檢測340nm處吸光度值的減小,反應(yīng)產(chǎn)物的生成量。用雙倒數(shù)作圖法得出MurB的Km值和Vmax。對于底物UDP-N-乙酰葡萄糖胺烯醇丙酮酸,Km值為0.3752mmol/L, Vmax為0.6876 mmol/(L.min)。 結(jié)論： 在本研究中,我們構(gòu)建了pET29b-Tb murB表達載體,在大腸桿菌BL21(DE3)/pKJE7中表達出大量的可溶性MurB蛋白。用純化的蛋白研究酶的活性,確定了MurB的最佳反應(yīng)條件并測定了MurB酶蛋白的反應(yīng)動力學(xué)常數(shù)Km和Vmax,為建立完善的篩選抗結(jié)核藥物的酶反應(yīng)體系奠定了基礎(chǔ)。
[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.

【學(xué)位授予單位】：大連醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2010
【分類號】：R378

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