Establishment of a Reporter Gene-based Assay for Identification of Estrogenic Compounds in HeLa Cells and Its Application in Detection of Aquatis Products

KeyWord:HeLa cells  estrogenic activity  reporter genes  luciferase  aquatic products

Abstract:

      Based on a recombinant cell strain for high sensitivity detection of estrogenic activity,estrogen synthesis technology is a revolutionary technology of the detection of the biological efficacy.It has the advantages of high efficiency and high sensitivity characteristic.The HeLa cells(the cells without endogenous hormone receptor) were used to recombine the human estrogen receptor ¦Á(hER¦Á) or estrogen receptor ¦Â(hER¦Â),and then build a stable expression of cell to detect the luciferase report gene.pERE-Luc plamid was generated by inserting estrogen response element(ERE) fragment into MCS of pGL3-promoter vector.HeLa cells were cotransfected with pERE-Luc using so fast transfection reagent.The cells were then treated with 17¦Â-estradiol(E2),diethylstilbestrol and progesterone£¬and expression of the reporter gene in the cell lysates was assayed using Dual-Luciferase reporter assay system.The pERE-Luc plasmid was constructed.Luciferase activities of pERE-Luc-ransfected HeLa cells showed that the correlations between RFU of Luc activity and E2,diethylstilbestrol and progesterone standard sample concentration was nonlinear£¬r2 was 0.879 8,0.955 0 and 0.926 2,respectively.E2 at 1.0¡Á10-13 mol£¯L induced the expression of reporter gene and that at 1.0¡Á10-9 mol£¯L resulted in the peak luciferase activity.Diethylstilbestrol luciferase activity was larger than 1.0¡Á10-10 mol£¯L and progesterone was larger than 1.0¡Á10-11 mol£¯L.The estrogenic activity of progesterone was more potent than that of diethylstilbestrol.The averages of the reporter gene emitted were 0.947 4,0.312 3 and 0.579 6,the relative standard deviation were 13.7%,3.7% and 3.4% after treated with E2,diethylstilbestrol and progesterone at 1.0¡Á10-10 mol£¯L.Results showed that good quality reproducibilities of luciferase reporter gene expression were obtained after treated with E2,diethylstilbestrol and progesterone.The spiked recoveries of E2 were over 75% in four fishes and the relative standard deviations(RSDs) were between 7.3%and 11.6% .The proposed reporter gene-based assay is effective and reliable.