A quantitative and highly sensitive luciferase-based assay for bacterial toxins that inhibit protein synthesis.
A quantitative and highly sensitive luciferase-based assay for bacterial toxins that inhibit protein synthesis.
复制标题
一种基于荧光素酶的定量、高灵敏度检测方法，用于检测抑制蛋白质合成的细菌毒素。
DOI：
10.1099/jmm.0.46143-0
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发表时间：
2005
影响因子：
3
通讯作者：
Haslam,DavidB
中科院分区：
文献类型：
--
作者：
Zhao,Luyi;Haslam,DavidB
抑制蛋白质合成是细菌和植物毒素损伤人类细胞的常见机制。抑制蛋白质合成的毒素包括大肠杆菌志贺毒素、白喉毒素、假单胞菌毒素A和植物毒素蓖麻毒素。为了便于研究毒素的致病机制，并筛选毒素作用的抑制物，建立了一种高灵敏度的定量检测这些毒素对哺乳动物细胞作用的方法。将编码失稳荧光素酶的基因克隆到腺病毒表达载体中，获得了高滴度的病毒表达载体。在转导Vero细胞后，发现荧光素酶的表达与病毒感染的多样性呈线性关系。仅10个细胞就能检测到荧光素酶的表达。转导细胞用放线菌素或志贺毒素处理后，荧光素酶活性下降，半衰期为1~2小时。当毒素浓度低至1pgml−1时，荧光素酶的表达受到明显的抑制。用该方法测定了Vero、Hep2、Chang、A549、COS-1和HeLa细胞对三种不同毒素的敏感性。这些结果表明，基于荧光素酶的检测方法适用于多种细胞类型的研究，具有定量、高灵敏度和重复性好的特点。这些特征将促进毒素介导疾病的病理生理学研究，并允许高通量筛选细胞毒性抑制剂。
Inhibition of protein synthesis is a common mechanism by which bacterial and plant toxins injure human cells. Examples of toxins that inhibit protein synthesis include shiga toxins ofEscherichia coli, diphtheria toxin,Pseudomonasexotoxin A and the plant toxin ricin. In order to facilitate studies on toxin pathogenesis and to enable screening for inhibitors of toxin action, a quantitative and highly sensitive assay for the action of these toxins on mammalian cells was developed. The cDNA encoding destabilized luciferase was cloned into an adenoviral expression plasmid and a high-titre viral stock was prepared. Following transduction of Vero cells, luciferase expression was found to be linear with respect to viral multiplicity of infection. Luciferase expression by as few as 10 cells was readily detected. Treatment of transduced cells with either cycloheximide or shiga toxin resulted in a decrease in luciferase activity, with a half-life ranging from 1 to 2 h. Inhibition of luciferase expression was evident at toxin concentrations as low as 1 pg ml−1. The assay was adapted for use in 24-, 96- and 384-well plates, enabling rapid processing of large numbers of samples. Using this approach, susceptibility of Vero, Hep2, Chang, A549, COS-1 and HeLa cells to three different toxins was determined. These results demonstrate that the luciferase-based assay is applicable to the study of numerous cell types, is quantitative, highly sensitive and reproducible. These features will facilitate studies on pathophysiology of toxin-mediated diseases and allow high-throughput screening for inhibitors of cytotoxicity.