Construction Of A Stable HBV Infected Cell Line By Gene Trapping Technic

Objective: To establish a cell model in vitro that stable expressing HBV by integrating HBA DNA into cell chromosome and prepare to further explore the regulation of related genes on HBV DNA replication. Methods: The HBV1.3 full length DNA was obtained by digested pGEM-HBV1.3 plasmid with HindIII and then was linked with PU-21 vector digested by HindIII.This was resulted in generation of a recombined plasmid named PU21-HBV plasmid. The recombined plasmid was introduced into HepG2 cells by electroporation method. The transfected cells were screened by G418. The cells that inserted and expressed HBV DNA were indentified by X-gal stainning, RT-PCR , Southern blot and ELISA. Results: The result of PU21-HBV plasmid sequence demonstrated that HBV1.3 DNA was linked correctly with PU-21 vector. A series of positive cell clons were obtained by G418 screening method followed by transfected PU21-HBV plasmid to HepG2 cells. The results of PCR, Southern blot,RT-PCR and ELISA exhibit that HBV DNA had successfully intergrated into the chromosomes of HepG2 cells and had functional HBV gene transcription. Conclusion: HBV1.3 DNA was inserted into HepG2...