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目的 克隆与结核分支杆菌耐异烟肼密切相关的KatG基因 ,实现其在大肠杆菌中的表达并对其酶活性进行初步检测。方法 构建含KatG基因的表达质粒 pET2 4b KatG ,转化大肠杆菌BL2 1 (DE3)菌株 ,获得稳定的高表达菌株后对表达产物进行过氧化氢酶活性的初步检测。结果 限制性内切酶分析结果显示所构建的 pET2 4b KatG表达质粒已成功克隆了KatG基因。含pET2 4b KatG表达质粒的大肠杆菌在导丙基硫代 β D 半乳糖苷(IPTG)诱导下表达 ,对表达产物进行SDS PAGE ,发现KatG表达产物相对分子质量约为 80× 1 0 3 ,表达产物约占总蛋白量的 1 7.7%。表达产物检测具有过氧化氢酶活性。结论 克隆并在工程菌中表达了异烟肼耐药密切相关的KatG基因 ,表达产物具备过氧化氢酶活性。可藉此方法研究结核分支杆菌的耐药机制。
Objective To clone the KatG gene, which is closely related to isoniazid - resistant Mycobacterium tuberculosis, and to express it in Escherichia coli. Methods The KatG gene expression plasmid pET2 4b KatG was constructed and transformed into E. coli BL21 (DE3) strain. The stable high expression strain was obtained and the catalase activity was detected. Results The restriction endonuclease analysis showed that the KatG gene was successfully cloned from pET2 4b KatG expression plasmid. E.coli harboring pET2 4b KatG expression plasmid was induced by IPTG, and SDS PAGE showed that the relative molecular mass of KatG expression product was about 80 × 10 3 The product accounts for about 7.7% of the total protein. Expression product detection has catalase activity. Conclusion The KatG gene, which is closely related to isoniazid resistance, was expressed in the engineered bacteria and expressed as catalase. This method can be used to study the drug resistance mechanism of Mycobacterium tuberculosis.