本文選題：結(jié)核分枝桿菌　切入點(diǎn)：半胱氨酸合成　出處：《大連醫(yī)科大學(xué)》2010年碩士論文

【摘要】： 上世紀(jì)八十年代以來,由結(jié)核分支桿菌(Mycobacterium tuberculosi-s)感染而引發(fā)的結(jié)核病(Tuberculosis,TB)在世界范圍內(nèi)再次重現(xiàn)流行趨勢,嚴(yán)重危害公共健康。艾滋病與結(jié)核病之間的協(xié)同作用,以及多重耐藥性結(jié)核分枝桿菌的不斷增加,使得現(xiàn)有的抗結(jié)核治療方案不能達(dá)到有效治愈的目的�？菇Y(jié)核新藥的發(fā)現(xiàn)距今已超過四十年,現(xiàn)今迫切需要發(fā)現(xiàn)新的抗結(jié)核藥物靶點(diǎn),研制開發(fā)新型抗結(jié)核藥物。 絲氨酸乙酰基轉(zhuǎn)移酶CysE普遍存在于植物及微生物體中,但在哺乳類動物中沒有發(fā)現(xiàn)。該酶參與催化生物體中半胱氨酸合成的第一步反應(yīng)(如圖1所示),即以L-絲氨酸(L-Serine)及乙酰輔酶A(AcCoA)為底物催化生成氧-乙酰絲氨酸(O-acetyl-L-Ser)和輔酶A(CoASH),第二步反應(yīng)由氧-乙�；�-L-絲氨酸硫化氫解酶(O-acetyl-L-Ser sulfhydrylase)催化生成L-半胱氨酸(L-cysteine)。結(jié)核分支桿菌絲氨酸乙�；D(zhuǎn)移酶CysE屬于結(jié)核分枝桿菌正常生長所需酶類,其突變可影響菌株的正常生長,是一個潛在的新型抗結(jié)核藥物靶點(diǎn)。 本論文目的是：(1)用PCR方法以結(jié)核分枝桿菌基因組DNA為模板擴(kuò)增Tb cysE基因；(2)將Tb cysE基因克隆到pMD18-T克隆載體,并進(jìn)行DNA序列測定；(3)將Tb cysE基因亞克隆到表達(dá)載體pET29b中,并在大腸桿菌BL21(DE3)中誘導(dǎo)表達(dá)CysE蛋白；(4)采用親和層析方法純化CysE蛋白,并用Western blotting方法進(jìn)行鑒定；(5)建立測定CysE酶活性的方法；(6)確定CysE酶的最佳反應(yīng)條件并進(jìn)行相關(guān)反應(yīng)動力學(xué)常數(shù)Km、Vmax的測定。 本論文所獲的結(jié)果如下： 1.利用PCR方法擴(kuò)增目的基因Tb cysE 從結(jié)核分枝桿菌H37Rv菌株的基因組數(shù)據(jù)庫(網(wǎng)址為http:／／genolist.pasteur.fr／TubercuList)中獲得基因Tb cysE核苷酸序列(690bp)。據(jù)該序列設(shè)計一對PCR引物,并在上下游引物的5’端分別引入NdeⅠ和XhoⅠ限制性內(nèi)切酶位點(diǎn)(以便將Tb cysE基因克隆到pET29b表達(dá)載體的NdeI和XhoI位點(diǎn))。以菌株H37Rv基因組DNA為模板,用LA TaqDNA聚合酶成功擴(kuò)增出Tb cysE基因。 2.構(gòu)建重組克隆載體pMD18-Tb cysE并進(jìn)行核苷酸序列測定 將已純化PCR產(chǎn)物與pMD18-T載體連接,并轉(zhuǎn)入大腸桿菌NovaBlue菌株的感受態(tài)細(xì)胞中。用限制性內(nèi)切酶酶切鑒定陽性重組質(zhì)粒。對pMD18-Tb cysE中的Tb-cysE基因進(jìn)行核苷酸序列測定,并進(jìn)行Multalin.分析,結(jié)果表明所克隆的Tb-cysE基因與結(jié)核分枝桿菌H37Rv菌株基因組數(shù)據(jù)庫中Tb cysE基因的核苷酸序列一致,即利用PCR技術(shù)擴(kuò)增出的Tb cysE基因不存在堿基突變。 3.構(gòu)建重組表達(dá)載體pET29b-Tb cysE 用NdeI和XhoI雙酶切質(zhì)粒pMD18-Tb cysE,回收純化目的片段Tb-cysE基因,將其連接到表達(dá)載體pET29b的NdeI和XhoI位點(diǎn),構(gòu)建pET29b-Tb cysE表達(dá)質(zhì)粒。用EcoRI和XhoI雙酶切方法鑒定陽性重組質(zhì)粒,將要表達(dá)的重組CysE蛋白的C端與質(zhì)粒上的組氨酸標(biāo)簽形成融合蛋白。 4.用大腸桿菌BL21(DE3)菌株表達(dá)Tb CysE蛋白并純化 將質(zhì)粒pET29b-Tb cysE轉(zhuǎn)化到BL21(DE3)感受態(tài)細(xì)胞中,用IPTG誘導(dǎo)表達(dá)Tb CysE蛋白。超聲破碎BL21(DE3)誘導(dǎo)菌株細(xì)胞,對上清和沉淀組分進(jìn)行SDS-PAGE和Western blotting分析,結(jié)果表明CysE蛋白在BL21(DE3)中可溶性表達(dá)。 采用組氨酸-鎳柱親和層析技術(shù)純化CysE蛋白,對純化蛋白進(jìn)行蛋白質(zhì)定量(考馬斯亮藍(lán)法),第1ml所純化CysE蛋白的濃度為37ug/ml。SDS-PAGE和Western blotting分析結(jié)果表明CysE蛋白的純度較高。 5.CysE酶活性測定方法的建立 向酶反應(yīng)體系中加入DTNB(Ellman's Reagent),用酶標(biāo)儀在405nm處測定光吸收值的變化,進(jìn)而得出反應(yīng)產(chǎn)物HSCoA生成量。 6.CysE酶的相關(guān)反應(yīng)動力學(xué)研究 (1)初速度的確定： 將反應(yīng)底物L(fēng)-Ser、AcCoA與不同濃度CysE蛋白在37℃反應(yīng)不同時間,繪制酶濃度曲線及反應(yīng)時間進(jìn)程曲線。結(jié)果表明CysE絲氨酸乙酰基轉(zhuǎn)移酶反應(yīng)初速度酶濃度范圍為0.74ug/ml,時間范圍為5min。 (2)CysE酶促反應(yīng)的最佳反應(yīng)條件的確定 分別改變反應(yīng)的溫度和反應(yīng)體系的pH值,測定產(chǎn)物HSCoA生成量。CysE絲氨酸乙酰基轉(zhuǎn)移酶催化的最適溫度為37℃,最適pH值為7.5,該酶活性不需要Mg2+的參與。 (3)最佳反應(yīng)條件下CysE酶促反應(yīng)動力學(xué)常數(shù)的測定 在最佳反應(yīng)條件及酶促反應(yīng)初速度范圍,保證一種底物過量,同時改變另一底物的濃度進(jìn)行反應(yīng)。采用雙倒數(shù)法分別測定兩底物的Km、Vmax值。底物AcCoA的Km值為0.0513350.00499mM,最大速率Vmax值為0.008189±O.00047mM-1min:L-Ser的Km值為0.026405±0.00063 mM,Vmax值為0.006455±0.00047mM-1min。 結(jié)論： 在本研究中,構(gòu)建了pET29b-Tb cysE重組表達(dá)載體,并在大腸桿菌BL21(DE3)中成功表達(dá)出可溶性蛋白CysE。建立了CysE酶活性鑒定方法并已進(jìn)行相關(guān)酶促反應(yīng)動力學(xué)研究,確定了最佳反應(yīng)條件及反應(yīng)動力學(xué)常數(shù)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，

本文編號：1700101