Jia Ping-ping, Zhao Li-li, Li Xiao-yu, Zhang Quan, Liu Zhen-long, Wang Xin, Yu Li-yan, Zhao Li-xun, Cen Shan
Laboratory of Immunobiology, Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences, Beijing 100050, China.
Yao Xue Xue Bao. 2011 Apr;46(4):377-82.
With the emergence of drug resistant tuberculosis, it is very urgent to find novel anti-tuberculosis drugs, especially novel anti-drug-resistant tuberculosis drugs. Because of the slow growth and the need to work in a biosafty environment of Mycobacterium tuberculosis, the development of evaluation of drug effect is severely impeded. In order to solve these issues, non-pathogenic fast-growing Mycobacterium smegmatis is introduced as test organism. The inhA is one of a target of isoniazid (INH) overexpression or mutation of this gene in Mycobacterium tuberculosis conferring resistant to INH. A recombinant plasmid bearing inhA was constructed and electroporated into Mycobacterium smegmatis, using shuttle expression vector pMV261. Transformants were induced to express a protein of inhA, identified by SDS-PAGE. Results show that Mycobacterium smegmatis containing inhA plasmids exhibited 100-fold or greater increased resistance to INH, but it conferred no increased resistance to others first-line anti-tuberculosis drugs. Resazurin microtiter assay plate testing of Mycobacterium smegmatis susceptibility to drugs is a rapid, simple, and inexpensive method and could decrease color background of drugs by detecting fluorescence. It will be benefit for high-throughout screening of drugs of anti-isoniazid-resistant Mycobacteria.
随着耐药结核病的出现，寻找新型抗结核药物，尤其是新型抗耐药结核病药物变得非常迫切。由于结核分枝杆菌生长缓慢且需要在生物安全环境中操作，药物效果评估的发展受到严重阻碍。为了解决这些问题，引入了非致病性快速生长的耻垢分枝杆菌作为测试菌株。inhA是异烟肼（INH）的靶点之一，该基因在结核分枝杆菌中的过表达或突变会导致对INH耐药。使用穿梭表达载体pMV261构建了携带inhA的重组质粒，并将其电穿孔导入耻垢分枝杆菌。通过SDS-PAGE鉴定诱导转化子表达inhA蛋白。结果表明，含有inhA质粒的耻垢分枝杆菌对INH的耐药性增加了100倍或更多，但对其他一线抗结核药物的耐药性没有增加。刃天青微量滴定板法检测耻垢分枝杆菌对药物的敏感性是一种快速、简单且廉价的方法，通过检测荧光可以降低药物的颜色背景。这将有利于高通量筛选抗异烟肼耐药分枝杆菌的药物。
