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[目的]建立应用荧光素酶报告基因筛检干扰维甲酸功能的环境化学物的方法。[方法]利用基因重组方法将3个重复序列RXR/RXR反应元件和2个重复序列RAR/RXR反应元件插入含荧光素酶报告基因的pGL4质粒上游,再将该质粒转染表达维甲酸受体的MLE-12细胞,加入全反式维甲酸或雌二醇后,检测荧光素酶活性。[结果]构建的RARE-TATA-pGL4报告基因质粒经限制性内切酶和DNA测序证实构建成功。经报告基因试验证实,与阴性对照组相比,维甲酸能使pGL4质粒的荧光素酶相对活性增加30.28倍,而雌二醇仅为1.4倍,说明雌二醇没有干扰作用。[结论]RARE-TATA-pGL4报告基因质粒可用于筛检环境中维甲酸样干扰物。
[Objective] To establish a method for screening environmental chemicals that interfere with retinoic acid function with luciferase reporter gene. [Method] Three repeat sequences of RXR / RXR and two repeat sequences of RAR / RXR were inserted into the upstream of pGL4 plasmid containing luciferase reporter gene by gene recombination method, then the plasmid was transfected to express retinoic acid receptor Of MLE-12 cells, after addition of all-trans retinoic acid or estradiol, luciferase activity was measured. [Result] The constructed RARE-TATA-pGL4 reporter gene plasmid was confirmed by restriction enzyme and DNA sequencing. The reporter gene test confirmed that compared with the negative control group, retinoic acid pGL4 plasmid luciferase activity increased 30.28 times, while estradiol was only 1.4 times, indicating that estradiol has no interference. [Conclusion] The RARE-TATA-pGL4 reporter gene plasmid can be used to screen the retinoid-like interferences in the environment.