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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烟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 ,转化进入大肠杆菌并实现高表达 ,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 pET2 4b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KatGS315T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 5 0 %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
Objective To investigate whether common point mutations in the Mycobacterium tuberculosis KatG gene lead to a decrease in catalase activity associated with isoniazid resistance. Methods The most common KatG gene 315 mutant in M. tuberculosis resistant to isoniazid was generated by site directed mutagenesis, resulting in the mutation of this site from serine (AGC) to threonine (ACC). Subsequently, a plasmid containing the S315T mutant of KatG gene was constructed and transformed into Escherichia coli for high expression. The catalase activity of the expressed protein was measured. Results The S315T mutant of KatG gene was successfully obtained by directional mutagenesis and transferred into Escherichia coli via pET2 4b plasmid. The S3G15T mutant protein was highly expressed in E. coli. The catalase activity of the KatGS315T expression product was detected, and it was found that the enzyme activity of the KatG gene expression product decreased by about 50% compared with that of the wild-type strain. Conclusion The mutation of serine (AGC) to threonine (ACC) at position 315 of KatG gene resulted in the decrease of catalase activity directly related to the production of isoniazid resistance