Rahimirad Nazanin, Kavoosi Saeedeh, Shirzad Hadi, Sadeghizadeh Majid
Department of Genetics, Faculty of Biological Sciences, Tarbiat Modares University, Tehran, Iran.
Department of Genetics, Faculty of Medical Sciences, Tarbiat Modares University, Tehran, Iran.
Iran J Pharm Res. 2019 Spring;18(2):686-695. doi: 10.22037/ijpr.2019.1100687.
Cell-based biosensors (CBBs) are becoming important tools for biosecurity a lications and rapid diagnostics in food microbiology for their unique capability of detecting hazardous materials. Pollutants, such as heavy metals and chemicals, are now considered as a global threat and are associated with detrimental health outcomes. Fast and accurate detection of pollutants is essential to reduce these threats. In this study, the enhancer sequence of human cytomegalovirus (hCMV) IE genes cloned upstream of gene in PGL4.26 plasmid, in order to increase basal luciferase activity. This recombinant vector was transfected into Huh7 cell line and after 21 days of treatment with Hygromycin B selectable marker, stable cell line was generated. After several passages, cells containing this vector showed high luciferase activity in normal conditions without any induction following to overexpression of gene. Huh7-CMV-luc cell line was able to detect the slightest changes in ATP level, due to the effect of different toxins on the cell which disrupt cellular respiration and ATP production processes. In order to investigate the sensitivity of the cell line, the cells were incubated with 0.1-10 μM of chemical toxins affecting ATP production. These toxins affect luciferase activity in a dose dependent manner, with maximal sensitivity approximately about 0.2 μM to toxin concentrations. Additionally, this biosensor provided a rapid detection as early as 4 h in response to the toxicants. Whole cell biosensors like huh7-CMV-luc cell line can be considered as a powerful tool for the sensitive and efficient monitoring of general toxins, drugs, and environmental pollutants.
基于细胞的生物传感器（CBBs）因其独特的有害物质检测能力，正成为生物安全应用和食品微生物学快速诊断的重要工具。重金属和化学物质等污染物如今被视为全球威胁，并与有害健康的后果相关联。快速准确地检测污染物对于减少这些威胁至关重要。在本研究中，人巨细胞病毒（hCMV）IE基因的增强子序列被克隆到PGL4.26质粒中基因的上游，以增加基础荧光素酶活性。该重组载体被转染到Huh7细胞系中，在用潮霉素B选择标记处理21天后，产生了稳定细胞系。经过几次传代后，含有该载体的细胞在正常条件下显示出高荧光素酶活性，在基因过表达后无需任何诱导。由于不同毒素对细胞的影响会破坏细胞呼吸和ATP产生过程，Huh7-CMV-luc细胞系能够检测到ATP水平的最微小变化。为了研究该细胞系的敏感性，将细胞与影响ATP产生的0.1 - 10 μM化学毒素一起孵育。这些毒素以剂量依赖的方式影响荧光素酶活性，对毒素浓度的最大敏感性约为0.2 μM。此外，这种生物传感器对毒物的响应最早在4小时就能实现快速检测。像huh7-CMV-luc细胞系这样的全细胞生物传感器可被视为灵敏高效监测一般毒素、药物和环境污染物的强大工具。
