DNA损伤效应主动监测的抗氧化基因缺失微生物传感器的构建及性能评价  

Development and performance evaluation of an antioxidant gene-knockout microbial sensor for active monitoring of DNA damage effects

作　　者：俞悦 李安一 王文甲 姜浩 邓玉林[1] 李晓琼[1] 吕雪飞[1] 戴荣继[2] Yu Yue;Li Anyi;Wang Wenjia;Jiang Hao;Deng Yulin;Li Xiaoqiong;Lyu Xuefei;Dai Rongji(School of Medical Technology,Beijing Institute of Technology,Beijing 100081,China;School of Life Science,Beijing Institute of Technology,Beijing 100081,China)

机构地区：[1]北京理工大学医学技术学院,北京100081 [2]北京理工大学生命学院,北京100081

出　　处：《航天医学与医学工程》2024年第2期73-77,共5页Space Medicine & Medical Engineering

基　　金：载人航天工程航天医学实验领域项目(HYZHXM04001)。

摘　　要：目的活性氧基团(ROS)水平升高会引起生物体的DNA氧化损伤,监测DNA的氧化损伤程度,能够实现ROS损伤效应的有效评价。基于微生物传感器监测DNA损伤效应可以定量评价氧化损伤程度,但微生物本身具有的ROS清除机制,会影响监测灵敏度。本研究旨在敲除细菌ROS清除机制的关键基因,构建抗氧化基因缺失微生物传感器,实现对DNA损伤效应的灵敏监测,评价ROS对生物体的损伤效应。方法本研究基于λ-Red同源重组的方法敲除细菌抗氧化损伤相关基因ahpCF、katE与katG,构建抗氧化基因缺失微生物传感器,并评价传感器对萘啶酮酸钠和紫外照射的响应。结果成功构建ΔahpCF、ΔahpCF/ΔkatE与ΔahpCF/ΔkatE/ΔkatG三种抗氧化基因缺失的微生物传感器,工程菌ΔahpCF/ΔkatE/ΔkatG对DNA损伤试剂萘啶酮酸钠的响应灵敏度最高,检测限为0.40μmol/L,另外,1.80 min的紫外照射(254 nm)可诱导工程菌产生显著的荧光表达效应。结论本研究构建了抗氧化基因缺失微生物传感器,实现了对DNA损伤试剂和紫外照射等DNA损伤效应的主动灵敏监测,可为未来空间辐射效应的评价提供一种主动、有效、灵敏的潜在监测方法。Objective The oxidative damage of DNA can be caused by excessive levels of Reactive oxygen species(ROS).Monitoring of DNA oxidative damage enables effective evaluation of ROS damage effects.Although the detection of DNA damage effects based on microbial sensor allows quantitative analysis of oxidative damage,the ROS clearance mechanism existed in bacterial will affect the sensitive of detection.The work of this article is to knockout the key genes of ROS clearance mechanism and construct an antioxidant gene-knock-out microbial sensor.The microbial sensor can realize sensitive monitoring of DNA damage effects and then evaluates the damage effects of cells by ROS.Methods The antioxidant damage genes of bacterial ahpCF,katE and katG were knocked out byλ-Red homologous recombination and antioxidant gene-knockout microbial sensor was constructed.The nalidixic acid sodium salt and UV irradiation were used to characterize the performance for monitoring of DNA damage effects.Results The antioxidant gene-knockout microbial sensorsΔahpC,ΔahpCF/ΔkatEG andΔahpCF/ΔkatE/ΔkatG were constructed successfully.The results showed that the microbial sensorΔahpCF/ΔkatE/ΔkatGl had the highest sensitive of damage effects and the limit of detection for nalidixic acid sodium salt was 0.40μmol/L.In addition,1.80 min of UV irradiation(254 nm)was sufficient to induce a significant fluorescent expression effect in the engineered bacteria.Conclusion In this article,antioxidant gene-knockout microbial sensors had been constructed to realize active and sensitive monitoring of DNA damage effects such as DNA damage reagents and UV irradiation.The sensors could provide an active,effective,and sensitive potential monitoring method for future evaluation of radiation effects in space.

关 键 词：DNA损伤效应 基因敲除 微生物传感器 空间辐射 萘啶酮酸钠 紫外照射 

分 类 号：R85[医药卫生—航空、航天与航海医学] TP212[医药卫生—临床医学]