Experimental Study On Inhibition Effect On HBV Replication By The Genome Of The Genome Of The HBV Defective Virus.

Many studies have been made in the field of gene therapy for HBV infection. Such gene therapy strategies have proved to be effective in vitro as DNA vaccine ribozyme antisense RNA interfering protein dominant negative and RNA interfering. Owing to the virus's variation, administrating repeatedly is needed. There is still a long distance from the clinical application because such problems are to be solved as safety specifity effectiveness and controllness about the vectors.We meant to generate a HBV defective virus to interfere with and inhibit the replication of HBV. The preliminary study was made to prepare for obtaining the HBV defective virus on generating the packaging cell line of the HBV defective virus highly expressing HBV S gene and HBV Pol gene. It was so difficult to highly express HBV Polymersase that we could not obtain the packaging cell line of the HBV defective virus highly expressing HBV S gene and HBV Pol gene. Two recombinant retrovirus vectors were constructed, that is the recombinant retroviral vector containing the genome of HBV and the recombinant retroviral vector containing the genome of the HBV defective virus. The two recombinant plasmids were transfected into the HepG2 cells , then the inhibition effect on HBV replication by the genome of the HBV defective virus was studyed by ELISA dot hybridization and semiquantitative PCR.In this study, we have carried out the following work of three parts: 1 preliminary study on generating the packaging cell line of the HBV defective virus highly expressing HBV S gene and HBV Pol gene; 2 Construction of the recombinant retroviral vector containing the genome of HBV and the recombinant retroviral vector containing the genome of the HBV defective virus; 3 Experimental study on the infection of the HBV replication by the genome of the HBV defective virus.1. Preliminary study on generating the packaging cell line of the HBV defective virus highly expressing HBV S gene and HBV Pol geneHBV Pol gene was amplified by PCR and cloned into the retroviral vector such as pMSCV neo and pLNCX2. The retroviral vectors containing HBV Pol gene, that were HBV Pol/pMSCVneo and HBV Pol/pLNCX2, were transfected into the HepG2 cells. The positive cells were selected in the presence of G418. HBV S gene was amplified by PCR and cloned into the retroviral vector pLHCX. The retroviral vector containing HBV S gene, that wasHBV S/pLHCX, was transfected into the HepG2 cells. The positive cells were selected in the presence of Hyg. The expression of HBV S gene could be detected by ELISA and Western blot. The expression of HBV Pol gene could be detected by RT-PCR, but could not detected by SDS-PAGE. The cloning and expressing the P ptotein was so difficult that we could not select the cell lines that highly expressed HBV Pol and S gene.Because of the difficulty of generating the packaging cells, it was difficult to obtain the HBV defective virus. As a result, we stopping obtaining the HBV defective virus but meant to explore gene therapy combating HBV infection by means of the idea of defective inhibition. In the experimental studies, the recombinant retroviral vector containing the genome of HBV and the recombinant retroviral vector containing the genome of HBV defective virus were constructed and the preliminary study was made on the inhibition effect on HBV replication by the genome of the HBV defective virus.2. Construction of the recombinant retroviral vector containing the genome of HBV and the recombinant retroviral vector containing the genome of the HBV defective virusThe primers was designed to amplify HBV C gene containing DR1 and DR2 which were necessary for the replication of HBV and X gene containing the terminal sequence by PCR. The two PCR fragments were subcloned into the pMD18-T vector respectively. After the digestion by EcoR I/Sal I or Sal I/Hpa I respectively, the two digestional fragments were ligated with the digestional fragment of pMSCVneo by EcoR I/Hpa I. In consequence, HBV CX/pMSCVneo, the recombinant retroviral vector containing the gen...