氟喹诺酮类抗生素受体蛋白的制备、分析方法的建立及区域环境水体的生态风险评估

Fluoroquinolones (FQs) are widely used as prophylactic agents and medicine for treatment of diseases in human and domestic animals, which may be discharged into environmental waters with urine, excrement and aquaculture wastewater. Therefore, it is of great importance to develop a suitable analytical method for monitoring the occurrences and assessing the exposure and risk of FQs in environmental waters. In this study, we try to express gyrase subunits A (gyrA) from E.Coil in periplasmic space of E. coli, and investigate the molecular recognition mechanism of gyrA protein binding to FQs by using enzyme assay; after preparation and purification of Europium chelate labeled gyrA, a simple, sensitive time-resolved fluoroimmunoassay (TRFIA) was established for measuring total FQs concentration in aquatic system. Based on the results of which, risk assessment to FQs in regional environmental waters was achieved using total FQs concentration as main indicator. And our study will provide important background data for risk assessment and investigation of the antibiotics in the aquatic environments.

氟喹诺酮(Fluoroquinolones，FQs)是一类被广泛使用的人畜共用抗生素，可随尿液、粪便、养殖废水等排放到环境水体中，造成水体污染，进而威胁水生态系统的安全。FQs种类众多，建立能够广泛识别多种FQs的高效可靠的分析方法，对评估其生态风险具有重要意义。本课题拟以大肠杆菌促旋酶的gyrA亚基作为研究对象，通过筛选FQs的敏感菌株，体外克隆、表达天然gyrA基因；借助实验和分子模拟技术手段阐明其与FQs的分子识别机制，指导定向突变、进化，筛选出广泛识别FQs的重组gyrA受体蛋白；在上述工作的基础上，以稀土离子铕-螯合剂标记gyrA受体蛋白，建立基于受体蛋白gyrA的、可测定水体中FQs总浓度的、简单、灵敏、高通量的生物分析方法；同时，以总浓度为重要指标，对区域水体中FQs的生态风险进行评估。本研究成果，将为研究水环境中典型抗生素的污染状况与生态风险评估提供技术支撑。

通过体外克隆并表达gyrA受体蛋白（约100KD），合成两种FQs包被原，其中OVA-NOR与gyrA配对成功。经竞争性ELISA方法鉴定，gyrA蛋白能够广谱性的识别12种喹诺酮类抗生素，其IC50在112.3~217.7 ng/mL。建立了基于受体蛋白gyrA的竞争性标准曲线，其检测CIP的LOD为24.6 ng/mL。基于此，构建了间接竞争ELISA方法，用于分析环境水体中FQs的总浓度。所测得总浓度与预测无影响浓度值(the predicted no effect concentration, PNEC)比较，可对区域环境水体中FQs的污染状况进行评估。本研究对江苏镇江、扬州等地环境水体的污染状况进行了系统地调查研究，结果表明，两个地区FQs对水环境无显著影响。