Construction And Performance Evaluation Of EGFP Vector For Demethylation Epigenetic Toxicity Assessment Of Environmental Pollutants

Objective: To construct eukaryotic expression vector for evaluation of the epigenetic demethylation of pollutants. Methods: PEGFP-C3 plasmid was extracted and purified after E. coli amplified intensively; and then was double digested with restriction enzyme digestion for 3.0kb long DNA strand and 1.7kb short DNA strand, which be collected and purified with agarose gel electrophoresis. The EGFP gene promoter within the short DNA strand were methylated completely with Msss.I, and then ligated to the long DNA strand for whole cycular pEGFP-C3 plasmid, which be gel purified for full-methylation status verification. The methylated pEGFP-C3 plasmid were transfected into HepG-2 cells and incubated with 5-AZA at series of concentrations for 30 hrs. Sodium bisulfite sequencing assay were carried out for quantification of methylation status of EGFP gene promoter, quantitative real-time quantitative PCR were used for quantification of EGFP gene expression, quantitative flow cytometry and fluorescence photographs of cultured cells were resorted for quantitative detection of green fluorescence intensity. The dose-response relationship between 5-AZA exposure and its ability to demethylation at different levels study of DNA methylation, EGFP gene mRNA expression, and GFP protein were explored. Results: Restriction enzymes co-digestion analysis and bisulfite sequencing results showed that the methylation status of pEGFP-C3 gene promoter is high. The tests of DNA methylation, EGFP gene mRNA expression, EGFP gene expression were conducted successfully after artificial treated pEGFP-C3 plasmid transfected into HepG2 hepatoma cells successfully. A good linear relationship were observed between 5-AZA's exposure and gene promoter methylation status, EGFP mRNA gene expression, GFP protein fluorescence intensity while 5-AZA's exposure gradient of 0.0016, 0.008, 0.004, 0.2 Um, respectively. The linear relationship for EGFP gene promoter methylation and 5-AZA is y =-0.1962x + 0.856; R2 = 0.800. The linear relationship for relative EGFP expression volume and 5-AZA dose is y = 1.359Ln(x) + 8.0869,R2 = 0.6569. The linear relationship for GFP mean fluorescence intensity of cells and 5-AZA is y = 10.402x + 6.0334; R2 = 0.829. Conclusion: The construction of a high-methylated green fluorescent protein reporter gene C3 vector is successful and a relative sensitive dose-response relationship existed between its green fluorescent protein gene expression in eukaryotic cells and positive pollutants demethylation ability. A Sensitive, stable pEGFP-C3 methylated vector is looking forward to playing important role and giving deep insight for study of epigenetic toxicity evaluation method for chemical demethylation ability test.