Objective        To prepare a zinc finger protein eukaryotic expression vector of artificial transcription factor targeting HBV enhancer and determine its expression in eukaryotic cells and the effect on cell proliferation and other biological activities.       Methods        Using the zinc finger protein (ZFP) design tool Zinc finger tool 3.0 to obtain DNA-binding domain of the artificial transcription factors (ATF)

combing with an inhibitory effector structure domain KRAB

cloning total synthesis ATF gene into eukaryotic expression vector pcDNA3.1 (+)

confirmed correctness by using enzyme cleavage and sequencing. pcDNA3.1-ATF were transformed into HepG2 cells by X-tremeGENE HP. The mRNA and protein expression of ATF were detected by RT-PCR

immunofluorescence staining and Western blotting. The cell proliferation was tested by CCK-8 kit by using transfection of ATF expression vector at different concentrations. Dual luciferase report gene was used to detect whether ATF recognizing HBV sequence and exerted targeted inhibition.       Results        pcDNA3.1-ATF eukaryotic expression vector was successfully constructed and expressed in HepG2 cells. CCK-8 assay indicated that ATF had no obvious toxic effect on cell proliferation. Dual luciferase report gene analysis showed that ATF specifically combined with enhancer and then inhibited report gene expression.       Conclusion        pcDNA3.1-ATF eukaryotic expression vector can normally express and have no cytotoxic effect in HepG2 cells by genetic engineering

which can target HBV enhancer and have corresponding biological activities.