| 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 andthe 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 KatGThe 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...
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