Construction Of X-gene Defected Hepatitis B Virus Vector Plasmid For Liver Cells Gene Therapy

Background:Chronic viral disease such as hepatitis B can be viewed as acquired genetic disease,therefore the application for gene therapy is worthy of research. Now it is undertaken. Vector as one of the key factor for Gene therapy of hepatitis B is required liver specific and lowest toxic. So,We tried to reconstruct the HBV whole gene group,and convert it to a vector. Methods:we destroyed HBV X gene by insert an linker which contains a Monocloning Site,then insert IFNa gene and clone the whole gene group to PCDNA3 plasmid which can express in Mammalian Cell. Also,we construct another HBV express plasmid which contain double-HBV gene group and normal X gene for transfection contrast. Hep3B cells were transfected by reconstructed plasmid. After 3-6 days,HBV virus particles from culture medium were isolated and detected by real time-PCR. Results:Plasmids PCDNA3-ES-HBV2,PCDNA3-KN- F1F2,and PCDNA3-KN-F1F2-IFN were constructed successfully;the former two were transfected to Hep3B cells. After 3-6 days,HBVDNA level detected of transfected cells was much higher(108copy/ml) than the negative transfected cells(less than103copy/ml). Conclusion:The reconstructed Plasmids can express HBV particles in Hep3B cell line and the HBVDNA replication in such cell line was at a high level. The reconstructed plasmids can be used for further study.