基于SOS反应及氧化应激反应相关启动子的辐射生物传感器研究

Radiation Biosensor Based on Promoter of SOS Reaction and Oxidative Stress Reaction

近年来的动物实验结果表明电磁辐射的危害主要是具有神经系统毒性、诱发肿瘤和生殖系统损伤等,广域、隐蔽和累积效应是辐射的特点,除对机体进行直接损伤外,还可导致间接损伤,即通过产生活性氧(ROS)和自由基攻击生物大分子。为了迅速和简便地检测辐射毒性的大小建立了新型的辐射生物传感器,构建了携带SOS反应和氧化应激反应相关的SulA、RecA、Cda和SoxR四种启动子融合经过密码子简并性优化的增强型绿色荧光蛋白(enhanced green fluorescent protein,EGFP)报告因子的工程菌传感器,并对这些生物传感器进行了γ射线辐照处理,筛选出了针对γ射线响应较好的,优选RecA工程菌传感器。利用PCR和Overlap PCR克隆获得了启动子-报告因子融合基因,并插入表达载体PUC19中,转化入宿主大肠杆菌DH5α,通过提取质粒进行双酶切和测序验证后,将构建成功的工程菌传感器首先进行化学毒性试剂刺激,一旦化学试剂刺激结果阳性便进行物理辐射刺激。结果显示,构建成功的4种工程菌传感器均对物理辐射产生应答,且随物理辐射剂量的增加(0~30Gy),绿色荧光强度逐渐增强。运用合成生物学手段,成功建立基于生物损伤修复效应和氧化应激反应的辐射生物传感器,具有制备简便、结果可视性等优点,能满足快速、广范围、在线监测的需求,在细胞毒性物、辐射环境乃至空间射线的损伤能力测定方面具有良好的应用前景。

The radiation environment exists everywhere in the living space,the results of animal experiments in recent years show that the harm of electromagnetic radiation are mainly concentrated on nervous system toxicity,inducing tumors(especially brain tumors,leukemia)and reproductive system damage.Radiation exposure has the characteristics of wide-area,concealed and cumulative effect.It acts on living organisms,causing a large amount of reactive oxygen species(ROS)in cells.By-products of normal aerobic physiological metabolism in the cells can also generate free radicals,thereby causing damage to the body.In other words,radiation can not only directly act on biological molecules and cause damage to the body,but also indirectly act on the body by acting on biological water and so on to produce free radicals.In order to detect the magnitude of radiation toxicity quickly and easily,there been established radiation biosensors.Engineered bacteria sensors carrying SoxR,RecA,Cda and Sul A four promoters and enhanced green fluorescent protein(EGFP)fusion gene related to SOS reaction and oxidative stress reaction were constructed,that is,the promoterreporter system.First,the four biosensors were treated with chemical damage agents,they all expressed a large amount of green fluorescent protein after stimulation,and thenγ-ray irradiation was performed.According to the treatment,the sensor with the highest sensitivity was the RecA promoter engineering bacteria sensor under radiation.The promoter-reporter fusion gene obtained by PCR and overlap PCR,and inserted into the vector PUC19,then transformed into E.coli DH5α.After double-enzyme digestion and sequencing verification,the successful engineered bacteria sensors were disposed of chemical oxidant and physical radiation.The results showed that the four engineering bacteria sensors successfully responded to the oxidant hydrogen peroxide and physical radiation,and the green fluorescence intensity gradually increased with the increase of physical radiation dose(0-30 Gy).Among them,the green fluorescence of Rec A engineered bacteria sensor was the most obvious after stimulation compared with the other sensors.The use of synthetic biology methods to establish physical radiation sensors based on biological effects successfully,with simple preparation,visibility of results,meeting fast,wide range,online monitoring needs,solving the problem of excessive background value in chemical sensors.It has a good application prospect in the measurement of radiation,radiation on the ground and even in the space.