摘要
目的用亲电性反应元件(EPRE)调控的转虫荧光素酶基因细胞快速评价环境的污染程度。方法采用分子生物学的方法,将EPRE与TK基本启动子和编码虫荧光素酶(LUC)的基因相融合,构建成EPRE调控的LUC稳定表达载体,将其转染于HeLa细胞,经G418筛选出稳定的细胞株。该细胞经不同浓度的亚砷酸钠(NaAsO2)、氯化镉(CdCl2)、氯化汞(HgCl2)和顺丁烯乙酸二乙酯(DEM)作用后,用荧光素酶试剂盒物测LUC的表达量。结果DNA测序显示,EPRE调控的LUC稳定表达载体结构框架正确;LUC的表达量与受试物具有一定的剂量-效应关系,其中DEM的剂量-效应关系最明显。结论EPRE调控的转LUC基因细胞构建成功。
Objective To assess quickly the potential environmental pollution by using the luciferase transgenic cells. Methods Many electrophile compounds in the environment can generate oxidative stress, so that the transcription of certain protective genes is induced via specific DNA motifs called electrophile response elements （EPREs）. We have made a vector containing a single EPRE fused to the TK minimal promoter and the gene encoding firefly luciferase （EPRE-LUC） by adopting biomolecular techniques. From this vector the stable LUC expression vector regulated by EPRE had been successfully reconstructed. This reporter construct was transfected into HeLa cells, and the clones resistant to G418 were selected. The resistant cells were treated by the different concentrations of sodium arsenate （NaAsO2）, cadmium chloride （CdCl2）, mercury chloride （HgCl2） and diethyl mateate （DEM）. After that, the expression of luciferase was determined by luciferase assay kit. Results The correct construct frame of LUC reporter vector regulated by EPRE was identified by DNA sequencing; the dose-dependent relationships between the LUC expression and the test substance concentration were found. Among them, the relationship produced by DEM was the most significant. Conclusion The LUC transgenic cells regulated by EPRE have been successfully constructed.
出处
《环境与健康杂志》
CAS
CSCD
北大核心
2007年第7期483-486,共4页
Journal of Environment and Health
基金
国家自然科学基金资助项目(30471426)
扬州大学科技创新培育基金资助项目(2006CXJ017)
关键词
环境污染物
亲电性化学污染物
虫荧光素酶
转基因细胞
Environmental pollutants
Electrophile chemical pollutants
Firefly luciferase
Transgenic cell
