结核分枝杆菌CysE的酶促反应特性分析

发布时间：2018-04-02 11:06

  本文选题：结核分枝杆菌　切入点：半胱氨酸合成　出处：《大连医科大学》2010年硕士论文

【摘要】： 上世纪八十年代以来,由结核分支杆菌(Mycobacterium tuberculosi-s)感染而引发的结核病(Tuberculosis,TB)在世界范围内再次重现流行趋势,严重危害公共健康。艾滋病与结核病之间的协同作用,以及多重耐药性结核分枝杆菌的不断增加,使得现有的抗结核治疗方案不能达到有效治愈的目的。抗结核新药的发现距今已超过四十年,现今迫切需要发现新的抗结核药物靶点,研制开发新型抗结核药物。 丝氨酸乙酰基转移酶CysE普遍存在于植物及微生物体中,但在哺乳类动物中没有发现。该酶参与催化生物体中半胱氨酸合成的第一步反应(如图1所示),即以L-丝氨酸(L-Serine)及乙酰辅酶A(AcCoA)为底物催化生成氧-乙酰丝氨酸(O-acetyl-L-Ser)和辅酶A(CoASH),第二步反应由氧-乙酰基-L-丝氨酸硫化氢解酶(O-acetyl-L-Ser sulfhydrylase)催化生成L-半胱氨酸(L-cysteine)。结核分支杆菌丝氨酸乙酰基转移酶CysE属于结核分枝杆菌正常生长所需酶类,其突变可影响菌株的正常生长,是一个潜在的新型抗结核药物靶点。 本论文目的是：(1)用PCR方法以结核分枝杆菌基因组DNA为模板扩增Tb cysE基因；(2)将Tb cysE基因克隆到pMD18-T克隆载体,并进行DNA序列测定；(3)将Tb cysE基因亚克隆到表达载体pET29b中,并在大肠杆菌BL21(DE3)中诱导表达CysE蛋白；(4)采用亲和层析方法纯化CysE蛋白,并用Western blotting方法进行鉴定；(5)建立测定CysE酶活性的方法；(6)确定CysE酶的最佳反应条件并进行相关反应动力学常数Km、Vmax的测定。 本论文所获的结果如下： 1.利用PCR方法扩增目的基因Tb cysE 从结核分枝杆菌H37Rv菌株的基因组数据库(网址为http:／／genolist.pasteur.fr／TubercuList)中获得基因Tb cysE核苷酸序列(690bp)。据该序列设计一对PCR引物,并在上下游引物的5’端分别引入NdeⅠ和XhoⅠ限制性内切酶位点(以便将Tb cysE基因克隆到pET29b表达载体的NdeI和XhoI位点)。以菌株H37Rv基因组DNA为模板,用LA TaqDNA聚合酶成功扩增出Tb cysE基因。 2.构建重组克隆载体pMD18-Tb cysE并进行核苷酸序列测定 将已纯化PCR产物与pMD18-T载体连接,并转入大肠杆菌NovaBlue菌株的感受态细胞中。用限制性内切酶酶切鉴定阳性重组质粒。对pMD18-Tb cysE中的Tb-cysE基因进行核苷酸序列测定,并进行Multalin.分析,结果表明所克隆的Tb-cysE基因与结核分枝杆菌H37Rv菌株基因组数据库中Tb cysE基因的核苷酸序列一致,即利用PCR技术扩增出的Tb cysE基因不存在碱基突变。 3.构建重组表达载体pET29b-Tb cysE 用NdeI和XhoI双酶切质粒pMD18-Tb cysE,回收纯化目的片段Tb-cysE基因,将其连接到表达载体pET29b的NdeI和XhoI位点,构建pET29b-Tb cysE表达质粒。用EcoRI和XhoI双酶切方法鉴定阳性重组质粒,将要表达的重组CysE蛋白的C端与质粒上的组氨酸标签形成融合蛋白。 4.用大肠杆菌BL21(DE3)菌株表达Tb CysE蛋白并纯化 将质粒pET29b-Tb cysE转化到BL21(DE3)感受态细胞中,用IPTG诱导表达Tb CysE蛋白。超声破碎BL21(DE3)诱导菌株细胞,对上清和沉淀组分进行SDS-PAGE和Western blotting分析,结果表明CysE蛋白在BL21(DE3)中可溶性表达。 采用组氨酸-镍柱亲和层析技术纯化CysE蛋白,对纯化蛋白进行蛋白质定量(考马斯亮蓝法),第1ml所纯化CysE蛋白的浓度为37ug/ml。SDS-PAGE和Western blotting分析结果表明CysE蛋白的纯度较高。 5.CysE酶活性测定方法的建立 向酶反应体系中加入DTNB(Ellman's Reagent),用酶标仪在405nm处测定光吸收值的变化,进而得出反应产物HSCoA生成量。 6.CysE酶的相关反应动力学研究 (1)初速度的确定： 将反应底物L-Ser、AcCoA与不同浓度CysE蛋白在37℃反应不同时间,绘制酶浓度曲线及反应时间进程曲线。结果表明CysE丝氨酸乙酰基转移酶反应初速度酶浓度范围为0.74ug/ml,时间范围为5min。 (2)CysE酶促反应的最佳反应条件的确定 分别改变反应的温度和反应体系的pH值,测定产物HSCoA生成量。CysE丝氨酸乙酰基转移酶催化的最适温度为37℃,最适pH值为7.5,该酶活性不需要Mg2+的参与。 (3)最佳反应条件下CysE酶促反应动力学常数的测定 在最佳反应条件及酶促反应初速度范围,保证一种底物过量,同时改变另一底物的浓度进行反应。采用双倒数法分别测定两底物的Km、Vmax值。底物AcCoA的Km值为0.0513350.00499mM,最大速率Vmax值为0.008189±O.00047mM-1min:L-Ser的Km值为0.026405±0.00063 mM,Vmax值为0.006455±0.00047mM-1min。 结论： 在本研究中,构建了pET29b-Tb cysE重组表达载体,并在大肠杆菌BL21(DE3)中成功表达出可溶性蛋白CysE。建立了CysE酶活性鉴定方法并已进行相关酶促反应动力学研究,确定了最佳反应条件及反应动力学常数Km及Vmax。
[Abstract]:Tuberculosis ( TB ) , which has been caused by Mycobacterium tuberculosis - s infection since the 1980 ' s , has once again reproduced the epidemic trend in the world , which seriously endangers public health . The synergistic effect between AIDS and tuberculosis , as well as the increasing of multiple drug - resistant Mycobacterium tuberculosis , makes the existing anti - TB treatment regimens not effective for the purpose of effective cure . The discovery of anti - tuberculosis drugs has more than 40 years now , and it is urgent to find new targets for anti - tuberculosis drugs and develop new anti - tuberculosis drugs .



The serine acetyl transferase CysE catalyzes the production of L - cysteine ( L - cysteine ) catalyzed by O - acetyl - L - Ser sulfhydrase .



The aim of this paper is to : ( 1 ) amplify the Tb cysE gene by PCR method using Mycobacterium tuberculosis genomic DNA as template ;
( 2 ) cloning the Tb cysE gene to a pMD18 - T cloning vector and carrying out DNA sequence determination ;
( 3 ) subcloning the Tb cysE gene into an expression vector pET29b , and inducing expression of the CysE protein in E . coli BL21 ( DE3 ) ;
( 4 ) The CysE protein was purified by affinity chromatography and identified by Western blotting .
( 5 ) establishing a method for measuring CysE enzyme activity ;
( 6 ) determining the optimal reaction conditions of the CysE enzyme and carrying out the determination of the relevant reaction kinetic constants Km and Vmax .



The results obtained in this paper are as follows :



1 . Amplification of target gene Tb cysE by PCR



The gene Tb cysE nucleotide sequence ( 690 bp ) was obtained from the genomic database of Mycobacterium tuberculosis H37Rv strain ( http://genolist . pasteur . fr / cucuList ) . A pair of PCR primers was designed according to the sequence , and Nde I and Xho I restriction enzyme sites were respectively introduced at the 5 ' end of the upstream and downstream primers ( in order to clone the Tb cysE gene into the NdeI and Xho sites of the pET29b expression vector ) . Using the genomic DNA of strain H37Rv as a template , the Tb cysE gene was amplified successfully with LA Taq DNA polymerase .



2 . constructing the recombinant cloning vector pMD18 - Tb cysE and carrying out nucleotide sequence determination



The purified PCR product was ligated with pMD18 - T vector and transferred into competent cells of Escherichia coli NovaBlue strain . The positive recombinant plasmid was identified by restriction endonuclease digestion . The results showed that the cloned Tb - cysE gene was consistent with the nucleotide sequence of Tb cysE gene in the genomic database of Mycobacterium tuberculosis H37Rv strain .



3 . Construction of Recombinant Expression Vector pET29b - Tb cysE



The recombinant plasmid pMD18 - Tb cysE was digested with NdeI and pMD18 - Tb cysE , and the purified target fragment Tb - cysE was purified and ligated into the NdeI and Xl sites of the expression vector pET29b to construct pET29b - Tb cysE expression plasmid .



4 . Expression of Tb CysE protein by E . coli BL21 ( DE3 ) strain and purification



The plasmid pET29b - Tb cysE was transformed into BL21 ( DE3 ) competent cells to induce the expression of Tb CysE protein by IPTG .



CysE protein was purified by histidine - nickel column affinity chromatography . The purified protein was subjected to protein quantification ( Coma brilliant blue method ) . The concentration of CysE protein purified in 1ml was 37ug / ml . SDS - PAGE and Western blotting analysis showed that the purity of CysE protein was higher .



5 . Establishment of the method for measuring the activity of CysE enzyme



DTNB ( Ellman ' s Reagent ) was added to the enzyme reaction system , and the change of the light absorption value was measured at 405 nm with a microplate reader , and then the reaction product HSCoA generation amount was obtained .



6 . Study on the Related Reaction Kinetics of CysE



( 1 ) Determination of initial speed :



The reaction substrate L - Ser , AcCoA and CysE protein with different concentrations were reacted at 37 鈩，

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