Luciferase in vivo expression technology: Use of recombinant mycobacterial reporter strains to evaluate antimycobacterial activity in mice
Luciferase in vivo expression technology: Use of recombinant mycobacterial reporter strains to evaluate antimycobacterial activity in mice
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DOI：
10.1128/aac.40.2.400
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发表时间：
1996-02-01
影响因子：
4.9
通讯作者：
Stover, CK
中科院分区：
文献类型：
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作者：
Hickey, MJ;Arain, TM;Stover, CK
由于结核分枝杆菌生长缓慢，而且必须在严格的生物安全条件下工作，这严重阻碍了针对结核分枝杆菌的新药和疫苗的开发。这些困难对进行结核分枝杆菌动物研究构成了相当大的障碍。我们研究了一种称为荧光素酶体内表达的新方法，使用一种增强的荧光素酶表达分枝杆菌菌株，是否可以用于评估小鼠的抗真菌活性。在牛分枝杆菌卡介苗（bacillus calmett - guerin， BCG）菌株中构建了高水平表达萤火虫荧光素酶（lux基因）的载体，并选择了一株重组卡介苗报告菌株（rBCG-lux），该菌株高水平表达了lux基因产物，并在小鼠中具有复制能力。通过直接测定器官匀浆中荧光素酶的发光来监测rBCG-lux报告菌株在小鼠体内的生长，并评价了该方法在评估抗细菌化合物体内功效方面的实用性。标准抗细菌药物的活性在感染rBCG-lux报告菌株的小鼠中直接表现出来，脾脏发光明显减少，具有统计学意义。此外，在bcg免疫小鼠中抗细菌免疫也很明显，与未接种小鼠相比，明显观察到rBCG-lux生长受到抑制。荧光素酶在体内表达用于体内抗真菌活性评估，与标准CFU测定相比，在时间、劳动、费用和统计学意义上都有优势，但允许在7天或更短的时间内评估小鼠的抗真菌药物和免疫力。因此，该技术的使用可以大大加快对生长缓慢的致病性分枝杆菌动物模型中抗生素和免疫原的评估过程。
The development of new drugs and vaccines directed against Mycobarterium tuberculosis is severely impeded by the slow growth of this organism and the need to work under stringent biosafety conditions, These difficulties pose considerable obstacles when animal studies with M. tuberculosis are performed. We investigated whether a novel approach termed luciferase in vivo expression, using an enhanced luciferase-expressing mycobacterial strain, could be used to evaluate antimycobacterial activity in mice. Vectors that expressed firefly luciferase (lux gene) at high levels in the bacillus Calmette-Guerin (BCG) strain of Mycobacterium bovis were constructed for use in vivo, One recombinant BCG reporter strain (rBCG-lux) was selected for high-level expression of the lux gene product and for its ability to replicate in mice. Methodology to monitor in vivo growth of the rBCG-lux reporter strain in mice by direct assay of luciferase luminescence in organ homogenates was developed, The utility of this approach for assessing the in vivo efficacies of antimycobacterial compounds was evaluated. The activities of standard antimycobacterial drugs were directly apparent in mice infected with the rBCG-lux reporter strain by statistically significant reductions in spleen luminescence, In addition, antimycobacterial immunity was also evident in BCG-immunized mice, in which suppression of rBCG-lux growth in comparison with that in naive mice was clearly observed. The use of luciferase in vivo expression for the in vivo evaluation of antimycobacterial activity compared favorably with standard CFU determinations in terms of time, labor, expense, and statistical significance but permitted the evaluation of antimycobacterial drugs and immunity in mice in 7 days or less. Thus, the use of this technology can greatly accelerate the process of evaluation of antibiotics and immunogens in animal models for the slowly growing pathogenic mycobacteria.