The Chemically Activated Fluorescent Gene Expression System For Dioxins Detection In Feed

The widespread nature of TCDD and related chemicals and their ability to bioaccumulate/biomagnify in the food chain and produce adverse effects in humans and animals has generated considerable concern worldwide, especially since dioxin food crisis in Belgium occurred in 1999.The methods for detecting dioxins and DLCs mainly involve: chromatography, immunoassay and bioassay. Quantitative extraction procedures coupled with high-resolution gas chromatography/mass spectrometry (HR GC/MS) is the international standard method for analying these chemicals. But it can not meet the demands for highthroughly detecting DLCs in feed and food with low cost.The aim of this study is take advantage of the AhR-dependent mechanism of action of dioxins to establish a bioassay, which can be use as rapid, low-cost and sensitive screening methods for the detection and relative quantification of dioxins in feed and animal product. We constructed a expression vector pGMD1.1 which includes dioxin response element(DRE), mouse mammary tumor virus(MMTV) promoter and EGFP(Enhanced Green Fluorescent Protein) reporter gene. Then, the rat hepatoma(H4DE) cell line was stable transfected with the construct pGMD1.1 using polybrene. Reconstructed clones exhibiting the highest ratio of EGFP expression after induced by TCDD were selected. Treatment of these cells with TCDD and relative chemicals results in induction of EGFP expression in a time-, dose-, AhR-, and chemical-specific manner, so the amount of dioxins in sample extracts can be identified by measuring the amount of EGFP.This study can be divided into two parts mainly.1. Construction of EGFP reporter plasmid under transcriptional control of DRE. The plasmid pMTV-luc and pDRE contain MMTV promoter and DRE respectively. Amplify MMTV promoter and DRE using PCR. The plasmid pEGFP-N1 was excluded CMV promoter by restriction enzyme digestion and then ligate with MMTV promoter and DRE step by step. The recombinant plasmid was transient transfected into H4IIE. TCDD and DMSO inducing the EGFP expression of transient transfection cell line respectively, the result shows that the Relative Fluorescent Units(RFU) of TCDD group is 10 times stronger than that of DMSO group. The data indicates the reconstructed plasmid(named pGMD1.1) was constructed successfully.2.The rat hepatoma(H4IIE) cells were transfected with the construct pGMD1.1. After growth in selective media for 4 weeks, resistant clones were isolated and screened for the induction of EGFP expression by TCDD(1 nM for 24h). Clones exhibiting the highest ratio of inducible to constitutive EGFP expression were further characterized. According to results, we selected reconstructed cell line H4EG.1b2 for further study. The stable clonal cells were plated in black, 96-well, clear-bottomed microtiter plate for high-through assay. The cells were maintained at 37℃and incubated with test chemicals at 33℃to maximize EGFP expression and inducible fluorescence. Dose-response relationship experiments for induction of EGFP by TCDD in the H4EG1b2 cells revealed a minimal detection limit(MDL) of～1pM, EC50 of～5pM, and maximal induction at 500pM. Compare this bioassay to luciferase-based cell bioassays, this bioassay not only has the same sensitivity and chemical specificity, but it is easier, more rapid and less expensive, and reporter gene activity can be measured in "real time", so this bioassay can meet the need for a high-throughput and low-cost screening analysis for dioxin and DLCs in feed and animal product.