摘要
近10年来，结核病再度成为全球首要的死亡原因之一，每年约有3百万人死于此病。造成结核病复燃的重要原因是耐药结核杆菌的出现，延误诊断又是造成耐药菌株扩散的主要原因。因为传统的药敏筛选方法费时颇长，所以临床上迫切需要创建一种可对临床菌株进行快速筛查的有效手段。异烟肼（INH）作为抗结核联合化疗方案的基础，在结核病的防治中起着重要的作用，它的耐药情况是临床药敏筛查工作的重点。目前研究已明确，KatG基因突变是造成INH耐药的主要原因，而点突变、插入和缺失又是KatG基因突变的主要形式。确定与INH耐药直接相关的KatG突变基因型，可为研制临床耐药结核杆菌快速筛查试剂盒提供理论依据。而对KatG蛋白的结构研究将有助于指导点突变的筛查和新型抗痨药物的研制开发。
本研究利用基因重组技术成功克隆了结核杆菌KatG基因，在此基础上采用三步PCR法对KatG基因的6个位点进行了定向诱变。这6个位点均是在临床菌株中筛查到的，其中2个为国外报道（S315T、W321Q），4个为国内首次报道（R418Q、P441L、A456S和A476V）。后4个基因的意义尚未见深入的研究。此后，本课题又实现了这些重组蛋白的表达和纯化。通过本部分工作建立了快速稳定的诱变体系和重组KatG蛋白纯化体系，为进一步研究点突变对KatG蛋白结构和功能的影响，探讨INH耐药机制奠定基础。
在第二部分研究中，作者对前面所得野生型KatG和诱变型KatG重组蛋白的结构和功能进行了进一步的研究，测定了这些蛋白的过氧化氢-过氧化物酶活性，二聚体的形成情况和荧光光谱的变化。通过这些工作明确了6个突变点对KatG蛋白结构和功能的影响以及它们与INH耐药的相关性，从而为明确耐药结核杆菌基因谱打下基础。
在最后一部分研究中，作者以Haloarcula marismortui来源的过氧化氢-过氧化物酶（HmCP）为模板，利用SWISS-MODEL服务器，模拟了KatG蛋白的三维结构。根据这一模型，结合HmCP和细胞色素C氧化酶（CcP）的一些信息，对这6个点突变影响KatG蛋白结构和功能的机制作了一些解释和探讨。通过这些工作，为从结构上深入研究KatG基因提供了一种新的方法和有效手段，为下一步选择诱变点更为深入地研究KatG蛋白结构提供了指导。
第一部分、结核分支杆菌KatG基因的定点诱变及诱变蛋白的表达和纯化
本研究采用PCR方法克隆KatG基因并将其构建在表达载体pET24b中，然后利用这一克隆，以三步PCR法的方法，对KatG基因中的6个位点进行了定向诱变，在IPTG诱导下高效表达了野生型和诱变型KatG蛋白，通过镍亲和柱和Q-Sepharose Fast Flow凝胶纯化获得高纯度的表达产物。还原性SDS-PAGE电泳发现KatG蛋白单体的分子量约为82kDa，表达量随表达时间的延长明显增加，22℃表达21小时时，表达量占总菌体可溶性蛋白的65%以上，IPTG浓度对表达量的影响不大。6个诱变蛋白的表达均受温度影响，在37℃表达时，主要表现为包涵体，在22℃表达时才主要表现为可溶状态，存在于破菌上清中。本研究为下一步的酶学和结构研究奠定了基础。
第二部分、野生型和诱变型KatG基因表达产物功能和结构的比较分析
本部分研究对第一部分所得重组蛋白的结构和功能进行了比较研究。过氧化氢-过氧化物酶活性测定的结果表明，6个突变中有3个（W321G、R418Q和A456S）可导致KatG蛋白过氧化氢酶活性完全丧失，其余3个位点的突变亦导致KatG蛋白过氧化氢酶活性不同程度的下降。6个诱变蛋白的过氧化物酶活性均有不同程度的下降。所有突变均不影响二硫键的形成。KatG(R418Q)和KatG(A456S)的荧光强度较野生型KatG蛋白明显下降，而其它诱变蛋白没有明显改变。
第三部分、SWISS-MODEL在KatG蛋白结构研究中的应用
本研究通过SWISS-MODEL模拟出了KatG的三维模型，依据此模型对点突变的意义作了更为深入的探讨，结果发现这些点突变可能是通过多种机制来影响KatG蛋白的结构和功能的，其中包括破坏电子传递链、阻碍底物接近活性中心等。
总之，INH的作用机制和耐药机制都相当复杂，迄今为止的大量研究还不能对之作出完全的解释，尚有许多领域需要进一步探索。进行有目的的KatG定向诱变研究将有助于深化对KatG蛋白结构的认识，揭示INH耐药的机制，并为研制临床耐药结核杆菌快速筛查试剂盒和开发新的抗痨药物提供理论支持。
In the last decade,tuberculosis(TB) has reemerged as one of the leading causes of death (nearly 3 million death annually). One reason contributing to the increasing death rate is the emergence of new strains of M. tuberculosis resistant to one or all current antitubecular drugs. Delayed recognition of drug resistance, which result in delayed initiation of effective therapy ,is one of the major factors contributing to drug-resistant M.tuberculosis (DRMT) outbreak. The high infection and death rate caused by DRTB pose an urgent challenge to rapidly detect cases. Isoniazid (isonicotinic acid hydrazide,INH) is an important first-line anti-TB drug and form the backbone of all chemotherapy regimens. However, the mechanisms of action of INH, as well as the mechanisms conferring INH resistance, have not been completely understood yet. It is now clear that INH is a prodrug that requires the activation of bacterial catalase-peroxidase enzyme,KatG,which is coded by KatG gene. KatG is the only enzyme capable of activitating INH in TB. Consequently,The variations of KatG,including mutation,shift and depletion,is the most important reason conferring INH-resistance. Definiting the relationship between the genotype mutations and phenotype mutations in KatG is the foundation for rapidly screening the clinical anti-drug TB. Fourthemore, research about the structure of KatG will be helpful to understand the mechanism of INH-resistance and find new anti-TB drugs.
In our study,KatG gene was successfully cloned and expressed. Three-steps PCR technique was adopted to introduce six specific mutations in the KatG gene. Four of these were reported in China firstly and their significances have never been evaluated. Then, the wild type and mutated KatG genes were expressed in E.coli BL-21 (DE3). The products were purified and the catalase-peroxidase activities were determined. After that, The disulphide-bridge formation of these recombinant proteins and
the spectroscopic changes were observed. Finally, in order to study the structure of KatG, SWISS-MODEL were used to simulate the three-dimensions model for this protein. This is the first time in the world that the method was utilized in this field. Compared with HmCP (a catalase-peroxidase from Haloarcula marismortui) and CcP(cytochrome c peroxidase from S. cerevisiae), the model provided some new information about the effect of mutations on the structure of KatG.
Part one,Site-directed mutagenesis of the KatG gene of Mycobacterium tuberculosis and the overexpression and purification of the recombinant proteins.
KatG gene was cloned by PCR technique and plasmid pET24b containing KatG was constructed. Then the three-steps PCR technique was utilized to introduce six specific mutations into the KatG gene. This technique is stable, effective and efficient. The presence of the desired mutations were confirmed by nucleotide sequence analysis. Thereafter, plasmids containing wild-type KatG gene and mutated ones were transformed into E.coli and overexpressed. The recombinant KatG is over 65% of the total soluble thallus protein. Finally， the products were purified by Ni+-NTA resin and Q-Sepharose Fast Flow gel. The purified proteins show a single band in SDS-PAGE.
Part two,The comparison of function and structure between wild-type KatG and mutated KatG
The catalase-peroxidase activities of these recombinant proteins were determined., The steady-state tryptophan fluorescence spectre were measured. Futhermore, the reduced and non-reduced SDS-PAGE electrophoresis was performed to observe the formation of the disulphide-bridge. It was found that three recombinant
proteins,KatG(W321G)、KatG(R418Q) and KatG(A456S), showed little catalase-peroxidase activity.The enzyme activity of the other three mutations also decreased compared with KatG(wt). All the six substitutions did not affect the disulphide-bridge formation. The fluorescence intensity of KatG(R418Q) and KatG(A456S) decreased over 10% compared with KatG(wt) while other four had no apparent changes.
Par
引文
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