Green fluorescent protein reporter microplate assay for high-throughput screening of compounds against Mycobacterium tuberculosis.
Green fluorescent protein reporter microplate assay for high-throughput screening of compounds against Mycobacterium tuberculosis.
复制标题
绿色荧光蛋白报告基因微孔板测定，用于高通量筛选抗结核分枝杆菌化合物。
DOI：
10.1128/aac.42.2.344
复制
发表时间：
1998
影响因子：
4.9
通讯作者：
Franzblau,SG
中科院分区：
文献类型：
--
作者：
Collins,LA;Torrero,MN;Franzblau,SG
An optimal assay for high-throughput screening for new antituberculosis agents would combine the microplate format and low cost of firefly luciferase reporter assays and redox dyes with the ease of kinetic monitoring inherent in the BACTEC system. The green fluorescent protein (GFP) of the jellyfishAequorea victoriais a useful reporter molecule which requires neither substrates nor cofactors due to the intrinsically fluorescent nature of the protein. The gene encoding a red-shifted, higher-intensity GFP variant was introduced by electroporation intoMycobacterium tuberculosisH37Ra andM. tuberculosisH37Rv on expression vector pFPV2. A microplate-based fluorescence assay (GFP microplate assay [GFPMA]) was developed and evaluated by determining the MICs of existing antimycobacterial agents. The MICs of isoniazid, rifampin, ethambutol, streptomycin, amikacin, ofloxacin, ethionamide, thiacetazone, and capreomycin, but not cycloserine, determined by GFPMA were within 1 log2dilution of those determined with the BACTEC 460 system and were available in 7 days. Equivalent MICs of antituberculosis agents in the BACTEC 460 system for both the reporter and parent strains suggested that introduction of pFPV2 did not influence drug susceptibility, in general. GFPMA provides a unique tool with which the dynamic response ofM. tuberculosisto the existing and potential antituberculosis agents can easily, rapidly, and inexpensively be monitored.
