Establishment Of Immunocompetent Hepatitis B Virus Chronically Infected Mouse Model And Its Application In Antiviral Research

Objects:1. In order to get a HBV replication competent clone of B genotype which is epidemic in Chinese, a plasmid that contain HBV 1.3 copies genome was constructed. It can provide a useful tool to study HBV biology, establish HBV replication cell model and animal model.2. Based on the genotype B HBV repliation competent clone, the HBV 1.3 copies genome was subcloned to a adenovirus associated virus vector, pAAV-MCS, expecting to establish a immunocompetent HBV chronically infected murine model by hydrodynamic injection the pAAV-HBV1.3 plasmid into tail vein. Meanwhile, pAAV-HBV1.2 plasmid which Huang LR had established genotype A HBV chronic infection in mice was employed as control.3. The shRNA expression retroviral vector and nonviral vector which targeting HBV X gene were hydrodynamic injected into murine tail vein directly, to verify if retroviral vector based RNAi could supress HBV replication in longer period. So it gived some light on the study method about specific gene's function in a simple way.Methods: 1. The two corresponding sequences of HBV genome were amplified separately, than the target sequences were ligated in vitro. After the plasmid was verified by enzyme digestion, it was transfected into Huh7 cell line. The HBsAg and HBeAg concentration in culture supernatant were quantified. The transcript and replication intermediate of HBV were detected by Northern Blot and Southern Blot separately. On the other hand, the plasmid was hydrodynamic injected into BALB/cJ mice. Then the sera HBV DNA was quantified by real-time PCR, HBcAg in liver tissue was detected by immunohistochemistry staining.2. The HBV 1.3 copies genome DNA sequence was ampified by PCR, Then the PCR product was digested by Not I restriction enzyme and subcloned into pAAV-MCS vector. It was verified to be replicable by transfecting Huh7 cell in vitro. Then the pAAV- HBV1.3 plasmid was hydrodynamic injected into tail vein of C57 BL/6 mice. Mice sera and liver tissues were collected at indicated time point in the following 48 weeks or more. The samples were detected by ELISA, southern blot, real-time quantified PCR, reverse transcriptional PCR and et al. At the same time, the pAAV-HBV1.2 plasmid, which Huang et al used to establish HBV chronic infection mice model, was employed in parallel.3. The HBV X gene specific RNA interference target sequences were desigened. Then the corresponding shRNA expression retroviral vectors were constructed. These vectors were cotransfected into Huh7 cell line with pAAV-HBV1.3 plasmid we constructed and the best RNAi target expression vector was selected. The HBx 285nt specific shRNA expression nonviral vector was also constructed. Both retroviral and noviral HBx285 shRNA expression vectors were injected into C57 BL/6 mice with same quantity pAAV-HBV1.3 plasmid to study if retroviral vector in this transduction way can be functional in a longer period than nonviral vector. Results:1. After be transfected into Huh7 cell line, the HBsAg and HBeAg can be secreted in culture supernatant, and the transcript and replication intermediate of HBV also can be detected. The HBV DNA was detected with high titer in mice sera and HBcAg was expressed in hepatocytes of mice.2. Both pAAV-HBV1.3 and pAAV-HBV1.2 plasmid can be used to establish HBV chronic infection in mice. The pAAV-HBV1.2 treated mice can sustain sera HBsAg postive at least 48 weeks. The HBsAg can be postive until 36 weeks postinjection in mice hydrodynamic injected with pAAV-HBV1.3 we constructed, but the viral load was higher than the pAAV-HBV1.2 mice in 0.6-1.6log value, sustaining in 10~5-10~7copies/mL level. The HBsAg postive percentage were 14.3% and 44.4% in pAAV-HBV1.3 and pAAV-HBV1.2 injected mice at 24 weeks postinjection. The serum HBsAg positive sustaining time of pAAV-HBV1.2 injected mice was longer than pAAV-HBV1.3 counterparts.3. The retroviral vector can enlong its shRNA antiviral effect time than nonviral vector. Sera HBsAg positive percentage, HBsAg quantity, serum HBV DNA concentration and HBcAg positive hepatocytes number can lowered significantly at least 4 weeks after retroviral vector injected mice versus about almost 1 week in nonviral vector mice.Conclusions:1. The replication competent HBV clone of genotype B we constructed can replicate in vitro and in vivo.2. The genotype B and A HBV chronically infected murine models are successfully established.3. The retroviral shRNA expression vector can prolong the time of HBV replication supression by RNAi in hydrodynamic tail vein injection.