Preparation Of Artificial Transcription To HBV And Its Targeted Inhibitory Effect On HBV Replication

Objective：To constructe an artificial zinc-finger transcription factorseukaryotic expression vector specifically recogniting and binding HBVenhancer, detecting its expression in eukaryotic cells、the effect on cellproliferation and analysis of biological activities, it could bind to HBVenhancer and inhibit HBV DNA replication and expression, the study willprovide theoretical basis for the prevention and therapy of hepatitis B virus.Methods:（1） ATF eukaryotic expression vector pcDNA3.1-ATFwere designed and constructed.（2） pcDNA3.1-ATF were transformedinto HepG2cells, ATF mRNA and protein expression were detected byRT-PCR、 immunofluorescence and Western-blot; The cell proliferationwas tested by CCK-8by using different concentrations of ATF expressionvector transfection.; ATF could recognize HBV sequence and targeteinhibition by detecting dual luciferase report gene.（3） ThepcDNA3.1-ATF were transformed into HepG2.2.15cells, ATF expressionwere tested by immunofluorescence and evaluated its influence on cellgrowth, respectively, the inhibition effect on HBV DNA replication andexpression were detected by using fluorescence quantitative PCR and Western-blot.Results:(1) pcDNA3.1-ATF eukaryotic expression vector weresuccessfully constructed.（2） pcDNA3.1-ATF vector could expresse inHepG2cells; the effect of ATF on cell proliferation was tested by CCK-8without obvious toxic effect; dual luciferase report gene analysis indicatedthat ATF could specifically combine with enhancer and inhibited reportgene expression.（3）pcDNA3.1-ATF expression vector were transformedinto HepG2.2.15cells, it successfully demonstrated that ATF couldinhibit HBV DNA replication and expression in HepG2.2.15cells, aftertransfection72h, The inhibition efficiency was the highest. The resultsindicated that ATF could specifically bind to HBV EnhI and show itscorresponding biological activities.Conclusion: It is possible that pcDNA3.1-ATF eukaryotic expressionvector can normally express and have no cytotoxic effect by the method ofgenetic engineering,which can bind to HBV enhancer and inhibit HBVDNAreplication and expression.