结核分枝杆菌katG基因突变对异烟肼耐药性的影响

Trp328Phe mutation in the katG gene of Mycobacteria tuberculosis induces isoniazid resistance

目的探讨结核分枝杆菌katG基因突变TGG328TTT(Trp328Phe)对异烟肼耐药性的影响。方法以结核分枝杆菌标准株H37Rv和临床突变株DNA为模板,采用PCR扩增野生型和双突变型(含Trp328Phe与Met420Thr)katG基因,采用重叠延伸PCR技术构建单突变型(Trp328Phe)katG基因。将各katG基因重组至质粒pET22b(+),再导入表达宿主菌BL21(DE3)pLySs中,IPTG诱导目的蛋白表达,亲和层析法对重组KatG蛋白进行分离纯化。通过各重组KatG蛋白与过氧化氢和邻联大茴香胺反应,检测受试菌过氧化氢酶和过氧化物酶的活性。结果成功获得纯化的KatG蛋白。相比野生型KatG(19.05U/mg和7.05×10-3 U/mg),单突变型KatG(10.50U/mg和2.23×10-3 U/mg)过氧化氢酶和过氧化物酶活性分别下降44.9%和68.3%,双突变型KatG(11.88U/mg和3.31×10-3 U/mg)则分别下降37.6%和53.0%。结论 katG基因(Trp328Phe)突变引起KatG酶活性部分缺失,与异烟肼耐药有关,可作为耐药菌株基因检测的标志用于基因芯片的开发。双突变时KatG蛋白活性较高,提示katG-328TTT具有代偿性突变的作用。

Objective To investigate the effect of Trp328Phe mutation in the katG gene of Mycobacteria tuberculosis on isoniazid resistance. Methods The wild type katG gene from H37Rv and the katG double mutant carrying the Trp328Phe and Met420Thr mutation from isoniazid-resistant clinical isolates were amplified using PCR. Based on the katG double mutant, a katG single mutant that carried only the Trp328Phe mutation was generated by overlap extension PCR. After these target genes were cloned into pET22b（＋） plasmids, the recombinant plasnaids were transferred into BL21（DE3） pLySs （Escherichia coli ） as an expression host. The expression of the target proteins was then induced by the addition of IPTG. Different KatG proteins were purified using affinity chromatography. Catalase activity was assayed spectrophotometrieally by monitoring the degradation of H2 O2 at A240 and peroxidase activity was determined by measuring the oxidation of o-dianisidine at A460. Results KatG enzymes were successfully purified. Compared to the wild type KatG protein （19.05 U/rag and 7.05× 10 ^-3 U/mg）, the KatG single mutant had 44.9% less eatalase activity （10.50 U/mg） and 68.3% less peroxidase activity （2.23× 10^-3 U/mg） while the KatG double mutant had 37.6% less catalase activity （11.88 U/mg） and 53.0% less peroxidase activity （3.31×10 ^-3 U/mg）. Conclusion The Trp328Phe mutation in the katG gene results in a partial reduction in catalase activity and peroxidase activity, suggesting that it is associated with isoniazid resistance. Therefore, that mutation could serve as a genetic marker to detect isoniazid-resistant strains and it could be used to develop gene chips. Greater activity of the KatG double mutant indicates that katG-328TTT may serve as a compensatory mutation.