定向诱变方法研究结核分支杆菌KatG基因突变与异烟肼耐药机制的关系

目的：探讨结核分支杆菌KatG基因的常见点突变是否会导致与产生异烟肼耐药性相关的过氧化氢酶活性降低。方法：用定向诱变方法产生耐异肼的结核分支杆菌中最常见的KatG基因315突变体，造成该位点从丝氨酸（AGC）到苏氨酸（ACC）的突变，随后构建含KatG基因S315T突变体的质粒，转化进入大肠菌并实现高表达，对表达的蛋白进行过氧化氢酶活性的测定。结果：通过定向诱变方法成功获得KatG基因S315T突变体，并通过pET24b质粒转入大肠杆菌，KatG基因S315T突变体蛋白在大肠杆菌中得到高表达，对DatG S315T表达产物的过氧化氢酶活性进行检测，发现比野生株KatG基因表达产物酶活性下降了50％左右。结论：KatG基因315位从丝氨酸（AGC）到苏氨酸（ACC）的突变造成过与异烟肼耐药产生直接相关的氧化氢酶活性的下降。

The effect of point mutation of KatG gene on the catalase activity associated with INH resistance by site-directed mutagenesis

To determine whether specific missense mutations in the M. tuberculosis katG gene could result in a loss of enzymatic activity associated with resistance to isoniazid (INH). Methods The authors used site directed mutagenesis to induce the commonest mutation S315T in the KatG gene of INH resistant M. tuberculosis . Furthermore, we constructed a plasmid containing katG with the S315T mutation, expressed the recombinant protein in a catalase peroxidase deficient strain of Escherichia coli, and assessed its enzymatic activity via detecting the release of O 2 from whole cell orga nisms. Results The mutated katG gene S315T was successfully obtained by site directed mutagenesis and constructed in pET24b and transformed into E. Coli . The katG(S315T) protein was then expressed and the catalase activities of which were detected. It was found that the catalase activity of S315T katG mutant was reduced 50% compared with KatG(wt). Conclusions The results suggest that S315T leads to reducing the activity of catalase which is related with activating INH. The method to detect the gene function of KatG gene with point mutation will establish the basis of gene markers of INH resistance and further develop the technique for fast detecting clinical strains resistant to INH.