KatG基因产物消除结核杆菌对异烟肼耐药性的实验研究

Mycobacterium tuberculosis is still the most pathogens around the world. In recent years, the dramatic increase in the number of drug-resistant strains has complicated the management of tuberculosis, with resistance rates as high as 35% being reported both in China and worldwide. Isoniazid is one of the earliest used anti-TB drugs and remains to be an most important ingredient in all treatment regimens. However, the isoniazid-resistant MTB infections become more severe than ever. It has been found that at least three genes have been implicated in resistance to INH - katG, inhA and ahpc, and katG variation almost exist in 90% resistant strains. Further studies have found that INH is a prodrug, which need to be activated by peroxidase as well as catalyse. The peroxidase and catalase of Mycobacteria tuberculosis are encoded by the katG gene. The mutation of katG gene leads to the decrease of the activity of peroxidase and catalase. In this study, katG of M. Tuberculosis was cloned and overexpressed. The effect of katG product in activating isoniazid was analysed. Furthermore, both in vitro and in vivo expriment were performed to recover the sensitivity of katG-mutated tuberculosis to isoniazid．In this study, KatG gene was cloned by PCR technique and plasmid pET24b containing KatG was constructed and transferred into competent E coli. The catalase activity of KatG protein was detected to have the activity of catalase as well as peroxidase. Furthermore, 2 mutagenic oligonucleotides were used to introduce mutations at 463 and 315 codons in the wild-type M. tuberculosis katG gene using site-directed mutagenesis techniques. The presence of the desired mutation (eg, S315T) was confirmed by nucleotide sequence analysis. It was found that the enzymatic activity of S315T as well as R463L katG mutant were reduced. Using HPLC, it was found that matated KatG protein can not activate the isoniazid into isoniazid acid. On the contrary, the wild type katG protein can do.Furthermore, the initially cloned S315T pET24b was used to transform the S315T katG gene into pMD31, which was the vector to transform katG gene into highly INH-resistant catalase-negative M, smegmatis. The pMD31 katG clone with S315T mutant was electroporated into the M. smegmatis and the transformants were selected on middlebrook 7H9 agar containing kanamycin. It showed that M. smegmatis expressing M. tuberculosis condon 315 mutant katG gene did not restore the susceptibility of M. smegnatis to INH, whereas, M. smegmatis expressing M. tuberculosis wild-type katG gene could restore the susceptibility.

本课题拟将结核杆菌KatG基因克隆并转化入大肠杆菌，表达并纯化KatG蛋白，然后利用KatG蛋白进行体内与体外对INH耐药菌株耐药性消除的试验。本课题旨在探索消除细菌耐药性的峦揪叮杂诮徊讲鹘岷烁司訧NH耐药的分子机制以及为控制耐药菌株感染具有深远意义。....