含荧光素酶四膜虫B2086-LUC全细胞生物传感器的构建及其对重金属离子的响应

为了获得可用于快速检测环境中重金属污染的全细胞生物传感器,本研究将含有HA标签的萤火虫荧光素酶(LUC)基因重组到含有四膜虫金属硫蛋白MTT1启动子和微管蛋白终止子序列的载体pBX中,获得重组质粒pBX-LUC,用基因枪粒子轰击法将pBX-LUC转化入四膜虫细胞中,通过同源重组和巴龙霉素抗性筛选, LUC基因整合到四膜虫大核基因组MTT1 位点,获得含有LUC基因的细胞株B2086-LUC. B2086-LUC对重金属镉和汞的响应敏感,可检测的最低浓度为5~10ng/mL;对铜和锌的响应较弱,可检测的最低浓度为0.5~ 1mg/mL.因此,四膜虫B2086-LUC可作为环境中镉和汞污染快速检测的全细胞生物传感器.

Construction of whole cell biosensor Tetrahymena B2086-LUC containing luciferase and its response to heavy metal ion

To obtain new whole cell biosensor which can be used for rapidly detecting metal ion pollution in the environment, Tetrahymena thermophilia was used in this study. Luciferase (LUC) gene was recombinated into the vector pBX containing Tetrahymenna Metallothioneins gene MTT1 promoter and tubulin terminator. Plasmid pBX–LUC was constructed and transformed into Tetrahymena strain by particle bombardment. Then, the LUC gene replaced the Tetrahymena macronuclear MTT1 gene by homologous recombination under increasing paromomycin. The B2086-LUC strain was obtained. The B2086-LUC strain is sensitively responsed to heavy metals cadmium and mercury ion (5 ~ 10 ng/mL) and less sensitive for copper and zinc ion (0.5~1 mg/mL). The results showed genetically engineered whole cell biosensor Tetrahymena B2086-LUC strain can be used for rapidly detecting cadmium and mercury pollution in environment.