A Study Of Artificial Transcription Factor That Specifically Recognizes Core Promoter Inhibits Hepatitis B Virus In Vitro

Objective To design an artificial transcription factor (ATF) which canrecognize and bind to hepatitis B virus (HBV) core promoter (Cp)specifically and observe its inhibitory effects on HBV expression.Methods (1) The optimal target sequence was determined after Thesequences of HBV Cp were analyzed and compared, the amino acidsequence of zinc finger protein (ZFP) which specifically recognizes it wasdesigned then;(2) The amino acid sequence of ZFP was reverse-translatedinto the nucleic acid sequence, then a nuclear localization signal and aKRAB repression domain were added in it after codon optimization, theacquired entire nucleic acid sequence of ATF was used to construct therecombinant plasmid pcDNA3.1(+)-ATF;(3) pcDNA3.1(+)-ATF wastransfected into eukaryotic cell COS-7, whether the ATF expressed normallywas detected by RT-PCR and Western blot at mRNA and protein levelsrespectively;(4) pcDNA3.1(+)-ATF was transfected into HepG2.2.15cell,HBV DNA, mRNA and HBeAg&HBsAg were detected by qPCR, RT-PCRand ELISA respectively. Results (1) The ATF mRNA and protein of experimental groupexpressed normally as compared with control groups in COS-7;(2) HBVDNA, mRNA and HBeAg&HBsAg of experimental group decreased indifferent degrees as compared with control groups in HepG2.2.15.Conclusion The ATF based on designed ZFP was constructedsuccessfully. It was confirmed by experiments that the constructed ATFexpressed normally in eukaryotic cells and could inhibit HBV expression,and the research results may provide new technical means and theoreticalbasis for the prevention and treatment of hepatitis B infection.