Construction Of Artificial Transcription Factor Of Specific Binding To X Gene Promoter Of Hepatitis B Virus

OBJECTIVE: To design a novel artificial transcription factor ofspecific binding to x gene promoter of hepatitis B virus,which coulddecrease the expression of HBV X gene to inhibite the replication andexpression of HBV DNA in HepG2.2.15cells.The study will provide theexperimental basis for gene therapy of hepatitis B virus.METHODS:（1）ZFP gene was synthesized and cloned into pEGFP-N1plasmids to construct the recombinant plasmids pEGFP-N1-ZFP.Recombinant plasmids were transformed into COS-7cells andthe expressed EGFP was observed under fluorescent microscope and theZFP expression was detected by RT-PCR and Western blot.(2)ATF wasconstructed successfully,containing a DNA-binding domain of ZFP and afunctional domain of KRAB.（3）Recombinant plasmids were transformedinto HepG2.2.15cells， detected the efficiency of suppression of thereplication and expression of HBV DNA by ELISA、immunocytochemistry、 RT-PCR.RESULTS:(1)Recombinant eukaryotic expression vectors of pEGFP-N1-ZFP were constructed and expressed successfully in cos7cells.(2)ATF was constructed successfully and which could expressnormally in eukaryotic cells; the EMSA results demonstrated that ATFcould bind specifically to HBV X gene promoter target sequence.(3)Therecombinant plasmids of pEGFP-N1-ATF can inhibite the replication andexpression of HBV DNA in HepG2.2.15cells.CONCLUSION:ATF can inhibite the replication and expression ofHBV DNA in HepG2.2.15cells.