异烟肼耐药耻垢分枝杆菌的建立及药物敏感性的测定  

Establishment and drug susceptibility test of isoniazid resistant Mycobacterium smegmatis

作　　者：贾平平 赵莉莉 李晓宇 张全 刘振龙 王鑫 余利岩 赵立勋 岑山 

机构地区：[1]中国医学科学院医药生物技术研究所免疫生物学室,北京100050 [2]中国医学科学院医药生物技术研究所菌种保藏中心,北京100050 [3]中国医学科学院医药生物技术研究所发酵工程室,北京100050

出　　处：《药学学报》2011年第4期377-382,共6页Acta Pharmaceutica Sinica

基　　金：国家"重大新药创制"科技重大专项(2009ZX09303-005)

摘　　要：耐药结核病的涌现,使发展新型抗耐药结核药物变得尤为迫切。本研究选择生长快且无致病性的耻垢分枝杆菌为研究对象,探索快速评价药物抗异烟肼耐药结核分枝杆菌的能力。inhA是异烟肼的作用靶点,由于inhA的突变或者过表达可以引起结核分枝杆菌对异烟肼耐药性的产生。通过将inhA克隆入pMV261中,构建过表达inhA的耻垢分枝杆菌。结果显示,过量表达inhA的耻垢分枝杆菌对异烟肼的敏感性下降了100倍以上。建立了利用刃天青为指示剂的抗异烟肼耐药株的快速药效评价方法,可快速对药物的活性进行定性或定量评价,为进行新型抗结核药物的高通量筛选和药效评价奠定基础。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.

关 键 词：耻垢分枝杆菌 inhA过表达 异烟肼耐药 评价方法 

分 类 号：R965.1[医药卫生—药理学]