Cooksey R C, Crawford J T, Jacobs W R, Shinnick T M
Division of Bacterial and Mycotic Diseases, Centers for Disease Control and Prevention, Atlanta, Georgia 30333.
Antimicrob Agents Chemother. 1993 Jun;37(6):1348-52. doi: 10.1128/AAC.37.6.1348.
We developed a rapid method to screen the efficacy of antimicrobial agents against Mycobacterium tuberculosis. A restriction fragment carrying a promoterless firefly luciferase gene was cloned into a 4,488-bp shuttle vector, pMV261, and luciferase was expressed under the control of a mycobacterial heat shock promoter. The resulting plasmid, pLUC10, was introduced by electroporation into the avirulent strain M. tuberculosis H37Ra. Luciferase assays of sonic lysates of Triton X-100-treated cells of M. tuberculosis H37Ra(pLUC10) yielded bioluminescence in excess of 1,000 relative light units/approximately 10(9) tubercle bacilli, compared with 0.0025 for the same number of parental cells. A 48-h microdilution antimicrobial agent-screening assay using this strain was developed.
我们开发了一种快速方法来筛选抗微生物剂对结核分枝杆菌的疗效。将携带无启动子萤火虫荧光素酶基因的限制性片段克隆到一个4488碱基对的穿梭载体pMV261中,荧光素酶在分枝杆菌热休克启动子的控制下表达。将所得质粒pLUC10通过电穿孔导入无毒力的结核分枝杆菌H37Ra菌株。对经Triton X - 100处理的结核分枝杆菌H37Ra(pLUC10)细胞的超声裂解物进行荧光素酶测定,产生的生物发光超过1000相对光单位/约10⁹结核杆菌,而相同数量的亲本细胞为0.0025。利用该菌株开发了一种48小时的微量稀释抗微生物剂筛选试验。