【摘要】： 從世界范圍來(lái)看,結(jié)核病仍是一種發(fā)病率和死亡率極高的傳染性疾病,全球人口的三分之一感染了結(jié)核分枝桿菌,而且,其發(fā)病率也呈逐年上升的趨勢(shì)。如今,耐多藥結(jié)核病(MDR-TB)不斷增多,使原本可以治愈的結(jié)核病再次成為治療難點(diǎn)。因此,當(dāng)前的首要任務(wù)是從結(jié)核分枝桿菌自身尋找新的靶點(diǎn),以此來(lái)研發(fā)新的抗結(jié)核藥物。 靶點(diǎn)的選擇當(dāng)然離不開結(jié)核分枝桿菌堅(jiān)厚的細(xì)胞壁結(jié)構(gòu),它是結(jié)核分枝桿菌賴以生存的重要部件,細(xì)胞壁獨(dú)特的結(jié)構(gòu)成為近年來(lái)研究的焦點(diǎn),其核心層由三部分組成:靠近細(xì)胞膜的肽聚糖、中間的聚阿拉伯半乳糖及最外的分枝菌酸,聚阿拉伯半乳糖通過(guò)L-鼠李糖-D-N-乙酰葡糖胺雙糖銜接分子與肽聚糖相連。UDP-N-乙酰葡糖胺是N-乙酰葡糖胺的糖基供體,其合成須經(jīng)歷四步反應(yīng),GlmU以其雙功能參與其中乙酰化和尿苷化反應(yīng);同時(shí)基因敲除模型已證實(shí)glmU為細(xì)菌生長(zhǎng)的必需基因。另外,UDP-N-乙酰葡糖胺的生物合成在人體中有所不同,葡糖胺-1-磷酸乙酰轉(zhuǎn)移酶活性在人體中不存在。因此,GlmU,尤其是其乙酰轉(zhuǎn)移酶活性是一個(gè)潛在的藥物作用靶點(diǎn),研究其乙酰轉(zhuǎn)移酶特性有助于研發(fā)安全可靠且無(wú)毒性的抗結(jié)核藥物。 本論文目的是:(1)用pET16b表達(dá)載體在大腸桿菌BL21(DE3)中高效表達(dá)結(jié)核分枝桿菌GlmU蛋白; (2)采用親和層析技術(shù)純化GlmU蛋白,并用SDS-PAGE以及Western blotting鑒定所純化的GlmU蛋白;(3)對(duì)GlmU酶活性測(cè)定方法進(jìn)行比較,從中選擇可用于高通量篩選GlmU抑制劑的檢測(cè)方法;(4)對(duì)GlmU進(jìn)行酶促反應(yīng)動(dòng)力學(xué)研究,確定最佳反應(yīng)條件并測(cè)定其反應(yīng)動(dòng)力學(xué)常數(shù)Km值以及Vmax,便于日后篩選抑制劑。 本論文所獲的結(jié)果如下: 1.誘導(dǎo)GlmU蛋白在大腸桿菌BL21(DE3)中的高效表達(dá) 用1mM IPTG,30oC振蕩培養(yǎng)3.5小時(shí),誘導(dǎo)BL21(DE3)表達(dá)重組GlmU蛋白。用超聲法破碎細(xì)菌,對(duì)上清組分進(jìn)行SDS-PAGE和Western blotting分析。結(jié)果表明,在上清中存在高表達(dá)的GlmU蛋白,分子量為54.10 kD。 2.采用親和層析技術(shù)純化GlmU蛋白 pET16b表達(dá)載體使重組GlmU蛋白N端帶有組氨酸標(biāo)簽,因此,用組氨酸-Ni2+親和層析技術(shù)對(duì)GlmU蛋白進(jìn)行純化。用SDS-PAGE分析洗脫組分1-5,結(jié)果表明,洗脫組分2-5蛋白純度很高,無(wú)其他雜蛋白存在。用Western blotting分析洗脫組分3-5,結(jié)果表明,純化所得的蛋白為GlmU蛋白。用考馬斯亮藍(lán)法定量洗脫組分4的濃度為442μg/ml,該組分用于酶活性分析。 3.建立測(cè)定GlmU酶活性的測(cè)定方法,便于建立高通量篩選酶抑制劑的方法 GlmU乙酰轉(zhuǎn)移酶活性: (1) HPLC法:采用Nova-Pak C18 (3.9×150 mm,4μm)色譜柱,以磷酸鹽緩沖液(pH6.5)-甲醇(95:5)為流動(dòng)相,流速1.0 mLmin-1,在波長(zhǎng)259 nm處檢測(cè)產(chǎn)物HSCoA,其洗脫時(shí)間在9.6 min左右。 (2)化學(xué)顯色法:酶反應(yīng)體系中加入DTNB (Ellman’s Reagent),通過(guò)檢測(cè)巰基進(jìn)而測(cè)定產(chǎn)物HSCoA含量,用酶標(biāo)儀在405 nm處測(cè)吸光值的改變。 GlmU尿苷轉(zhuǎn)移酶活性: (1) HPLC法:采用Nova-Pak C18 (3.9×150mm,4μm)色譜柱,以20 mM三乙胺-醋酸緩沖液(pH 4.0)為流動(dòng)相,流速0.5mLmin-1,在波長(zhǎng)260nm處檢測(cè)反應(yīng)產(chǎn)物UDP-GlcNAc,其洗脫時(shí)間為7.8 min。 (2)化學(xué)顯色法:偶聯(lián)焦磷酸酶將反應(yīng)產(chǎn)物焦磷酸水解為磷酸,磷酸與鉬酸銨形成磷鉬酸復(fù)合物后使孔雀石綠顏色由黃綠變?yōu)樗{(lán)綠,用酶標(biāo)儀在630 nm處檢測(cè)吸光值變化,以測(cè)定所生成磷酸的含量。 4.研究GlmU蛋白酶促反應(yīng)動(dòng)力學(xué)特性 (1)初速度的確定: GlmU乙酰轉(zhuǎn)移酶活性:將反應(yīng)底物GlcN-1-P和AcCoA與不同濃度GlmU蛋白在37oC反應(yīng)不同時(shí)間,繪制酶濃度曲線和反應(yīng)時(shí)間進(jìn)程曲線。結(jié)果表明GlmU乙酰轉(zhuǎn)移酶反應(yīng)初速度酶濃度范圍為0.88μg/ml,時(shí)間范圍為5 min。 尿苷轉(zhuǎn)移酶活性:反應(yīng)底物GlcNAc-1-P、UTP和焦磷酸酶與不同濃度GlmU在37oC反應(yīng)不同時(shí)間,繪制酶濃度曲線和反應(yīng)進(jìn)程曲線。結(jié)果表明GlmU尿苷轉(zhuǎn)移酶反應(yīng)初速度酶濃度范圍為1.33μg/ml,時(shí)間范圍為5 min。 (2)確定GlmU兩種活性的最佳反應(yīng)條件: 分別改變反應(yīng)體系的溫度和pH值,測(cè)其產(chǎn)物生成量。GlmU乙酰轉(zhuǎn)移酶的最適溫度為30oC,最適pH值為8.0,且其活性不需要Mg2+的參與;GlmU尿苷轉(zhuǎn)移酶最適溫度為42oC,最適pH值為8.0,在沒(méi)有Mg2+存在時(shí),檢測(cè)不到尿苷轉(zhuǎn)移酶的活性,可見(jiàn)Mg2+是GlmU尿苷轉(zhuǎn)移酶的激活劑,其最適濃度為20 mM。 (3)在最佳反應(yīng)條件下測(cè)得GlmU酶動(dòng)力學(xué)常數(shù): 在初速度范圍內(nèi),采用最佳反應(yīng)條件,保證一種底物過(guò)量,改變另一種底物濃度采用雙倒數(shù)法測(cè)其Km值和Vmax。對(duì)乙酰轉(zhuǎn)移酶來(lái)說(shuō),AcCoA的Km值為0.224±0.07mM,最大速率Vmax為0.119±0.038 mMmin-1,GlcN-1-P的Km值為0.061±0.005mM,最大速率Vmax為0.081±0.003mMmin-1;對(duì)尿苷轉(zhuǎn)移酶來(lái)說(shuō),底物GlcNAc-1-P的Km值為0.044±0.005mM,最大速率Vmax為0.0054±0.0002mMmin-1,底物UTP的Km值為0.024±0.0015mM,最大速率為0.006±0.0001mMmin-1。 結(jié)論: GlmU在大腸桿菌BL21(DE3)中的可溶性表達(dá)為我們研究其酶促反應(yīng)動(dòng)力學(xué)特性提供了物質(zhì)保障;針對(duì)GlmU兩種酶活性所建立的化學(xué)顯色方法精確、簡(jiǎn)便,便于酶促反應(yīng)動(dòng)力學(xué)特性的研究及高通量篩選GlmU抑制劑;研究了GlmU酶促反應(yīng)動(dòng)力學(xué)特性,確定了最佳反應(yīng)條件及其反應(yīng)動(dòng)力學(xué)常數(shù)Km和Vmax。
[Abstract]:Tuberculosis is still an infectious disease with high morbidity and mortality worldwide. One third of the world's population is infected with Mycobacterium tuberculosis, and its incidence is on the rise year by year. Today, the increasing number of multidrug-resistant tuberculosis (MDR-TB) makes it difficult to treat tuberculosis that could be cured once again. Therefore, the primary task now is to find new targets from Mycobacterium tuberculosis itself to develop new anti-tuberculosis drugs.
Target selection is of course inseparable from the strong cell wall structure of Mycobacterium tuberculosis, which is an important part of the survival of Mycobacterium tuberculosis. The unique structure of cell wall has become the focus of recent research. The core layer consists of three parts: peptidoglycan near the cell membrane, polyarabinogalactose in the middle and mycobacterial acid in the outermost. Arabinogalactose is linked to peptidoglycan via L-rhamnose-D-N-acetylglucosamine disaccharide conjugation molecule. UDP-N-acetylglucosamine is a glycosyl donor of N-acetylglucosamine, and its synthesis undergoes four steps. GlmU participates in acetylation and ureaylation with its bifunctional function. At the same time, gene knockout model has confirmed that glmU is necessary for bacterial growth. In addition, the biosynthesis of UDP-N-acetylglucosamine is different in humans, and the activity of glucosamine-1-phosphate acetyltransferase does not exist in humans. Therefore, GlmU, especially its acetyltransferase activity, is a potential drug target. Studying the characteristics of UDP-N-acetylglucosamine acetyltransferase is helpful to develop safe, reliable and non-toxic anti-knotting drugs. Nuclear drugs.
The purpose of this paper is: (1) to express the GlmU protein of Mycobacterium tuberculosis in E. coli BL21 (DE3) by pET16b expression vector; (2) to purify the GlmU protein by affinity chromatography, and to identify the purified GlmU protein by SDS-PAGE and Western blotting; (3) to compare the methods for determining the activity of GlmU enzyme, and select the high-throughput screening method. GlmU inhibitor detection method was selected; (4) GlmU enzymatic reaction kinetics was studied to determine the optimal reaction conditions and determine the reaction kinetics constant Km value and Vmax, so as to facilitate the screening of inhibitors in the future.
The results obtained in this paper are as follows:
1. induce the high expression of GlmU protein in Escherichia coli BL21 (DE3).
BL21 (DE3) was induced to express recombinant GlmU protein by 1 mM IPTG and 30 oC shaking culture for 3.5 hours. The supernatant was analyzed by SDS-PAGE and Western blotting. The results showed that there was a high expression of GlmU protein in the supernatant with a molecular weight of 54.10 kD.
2. purification of GlmU protein by affinity chromatography
The recombinant GlmU protein was purified by histidine-Ni2+ affinity chromatography. The elution fraction 1-5 was analyzed by SDS-PAGE. The results showed that the purity of the elution fraction 2-5 was high and no other impurity proteins were found. The elution fraction 3-5 was analyzed by Western blotting. GlmU protein was obtained. The concentration of elution component 4 was 442 ug/ml by Coomassie brilliant blue method. The component was used for enzyme activity analysis.
3. establish a method for the determination of GlmU enzyme activity and facilitate the establishment of a high throughput screening method for enzyme inhibitors.
GlmU acetyltransferase activity:
(1) HPLC method: Nova-Pak C18 (3.9 x 150 mm, 4 micron) column was used. Phosphate buffer (pH 6.5) -methanol (95:5) was used as mobile phase. The flow rate was 1.0 mLmin-1. The elution time of HSCoA was about 9.6 min at 259 nm.
(2) Chemical colorimetry: DTNB (Ellman's Reagent) was added into the enzyme reaction system, and the content of HSCoA was determined by detecting the thiol group. The absorbance was measured at 405 nm by enzyme labeling instrument.
GlmU uridine transferase activity:
(1) HPLC method: The reaction product UDP-GlcNAc was detected by Nova-Pak C18 (3.9 *150 mm, 4 micron) column with 20 mM triethylamine-acetic acid buffer (pH 4.0) as mobile phase at a flow rate of 0.5 mL min-1 at a wavelength of 260 nm. The elution time was 7.8 min.
(2) Chemical colorimetry: pyrophosphatase was used to hydrolyze pyrophosphate into phosphoric acid, phosphoric acid and ammonium molybdate formed phosphomolybdic acid complex, then the color of malachite green changed from yellow green to blue green. The absorbance value of malachite green was measured at 630 nm to determine the content of phosphoric acid.
4. the kinetics of GlmU protease reaction was studied.
(1) determination of initial velocity:
GlmU acetyltransferase activity: GlcN-1-P and AccCoA were reacted with different concentrations of GlmU protein in 37oC for different time, and the enzyme concentration curve and reaction time curve were drawn.
Urotransferase activity: substrate GlcNAc-1-P, UTP and pyrophosphatase reacted with different concentrations of GlmU at 37oC for different time, and plotted the enzyme concentration curve and reaction process curve. The results showed that the initial reaction rate of GlmU urease enzyme concentration range was 1.33 ug/ml, the time range was 5 minutes.
(2) determine the best reaction conditions for GlmU two activities:
The optimum temperature of GlmU acetyltransferase is 30oC, the optimum pH is 8.0, and the activity of GlmU acetyltransferase does not need the participation of Mg2 +; the optimum temperature of GlmU urease transferase is 42oC, and the optimum pH is 8.0. In the absence of Mg2 +, the activity of urease transferase can not be detected. It can be seen that Mg2 + is GlmU uridine. The optimum concentration of activator of transferase is 20 mM.
(3) GlmU enzyme kinetic constants were measured under the best reaction conditions.
The Km value and Vmax of AcCoA were measured by double reciprocal method. For acetyltransferase, the Km value of AcCoA was 0.224+0.07mM, the maximum rate of Vmax was 0.119+0.038 mMmin-1, the Km value of GlcN-1-P was 0.061+0.005mM, and the maximum rate of Vmax was 0.081+0.003mM. For urease, the Km value of substrate GlcNAc-1-P was 0.044+0.005mM, the maximum rate Vmax was 0.0054+0.0002mmin-1, the substrate UTP Km value was 0.024+0.0015mM, and the maximum rate was 0.006+0.0001mmmmin-1.
Conclusion:
The soluble expression of GlmU in E. coli BL21 (DE3) provides a material guarantee for us to study the kinetic characteristics of enzymatic reaction; the chemical coloration method for GlmU enzymatic reaction is accurate, simple, convenient for the study of kinetic characteristics of enzymatic reaction and high throughput screening of GlmU inhibitors; the kinetic characteristics of enzymatic reaction of GlmU are studied. The optimum reaction conditions and the kinetic constants Km and Vmax. were determined.
【學(xué)位授予單位】：大連醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2009
【分類號(hào)】：R378;Q78

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