环境镉离子生物检测工程菌株的构建及应用
Construction and application of bio-engineered bacteria for detection of environmental cadmium
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摘要:目的 以绿色荧光蛋白(GFP)基因为报告基因, 和重金属镉特异性启动子PcadA构建成基因工程重组体, 用于重金属镉的特异性检测。方法 采用聚合酶链(PCR)方法从质粒pPcadA上扩增得到重金属镉离子特异性诱导启动子PcadA, 以绿色荧光蛋白基因为报告基因, 以大肠埃希菌-枯草杆菌穿梭载体pNW33N构建了绿色荧光蛋白镉离子诱导表达载体。以枯草芽孢杆菌1A751为宿主菌, 采用感受态转化法将表达载体导入1A751, 成功构建镉离子检测菌株。结果 通过酶切鉴定、PCR和DNA序列分析, 重组载体构建成功, 并在有镉离子存在的环境中得到表达。结论 该工程菌株能够以绿色荧光蛋白基因为报告基因检测培养体系中最低浓度达0.1μmol/L的镉离子; 利用该菌株有望建立起一种方便快捷的重金属镉的检测方法, 有较高的应用价值。Abstract:Objective A new method was developed to detect environmental cadmium, in which green fluorescent protein gene(GFP)and cadmium-specific promoter PcadA were ligated to form a new construct.Methods Cadmium-specific promoter PcadA was obtained by PCR from p PcadA and was ligated into E.coli-B.subtilis shuttle vector pNW33N, GFP as reporting gene, and, 1A751 strain as the host.Results After PCR and DNA sequence analysis, the targeted DNA was successfully inserted into the vector and was expressed as anticipated.Conclusion By using the bio-engineered construct, a novel cadmium detection method is expected to be established.
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