The Inhibitory Effects Of Pokeweed Antiviral Protein On Hepatitis B Virus

PartⅠConstruction of the total length and deleted mutant PAP encoded by a eukaryotic expression plasmidObjective: To construct the total length and deleted mutant pokeweed antiviral protein (PAP) encoded by a eukaryotic expression plasmid so that they will make a firm base for investigating the active domain of PAP on anti-HBV.Methods: The coding genes of the total length and deleted mutant PAP were amplified from the plasmid PGEM-PAP by PCR and then were cloned into pMD18-T vector. The recombinant pMD18-T-PAPs were identified by PCR, enzyme digestion, and were confirmed by DNA sequencing at last. Then they were digested with BamHⅠand HindⅢand were correctly inserted into BamHⅠ/HindⅢrestriction sites of the CMV-driven expression vector pXF3H with a hemagglutinin fusion epitope tag. The recombinant pXF3H-PAPs were identified by PCR, enzyme digestion, and were confirmed by DNA sequencing at last.Results: The total length and deleted mutant PAP encoded by a eukaryotic expression plasmid pXF3H were successfully constructed. Restriction analysis indicated that they were correctly inserted into the pXF3H vector.Conclusion: The total length and deleted mutant PAP encoded by a eukaryotic expression plasmid were successfully constructed.PartⅡThe inhibitory effects of the total length PAP encoded by a eukaryotic expression plasmid on hepatitis B virus replication in vitroObjective: To explore the inhibitory effects of the total length PAP encoded by a eukaryotic expression plasmid on HBV at the DNA,RNA and protein level in vitro.Methods: The total length PAP encoded by a eukaryotic expression plasmid (pXF3H-PAP12) and the replication competent wild-type 1.3 fold over-length HBV plasmid(pHBV1.3) were cotransfected into HepG2 hepatoma cells using lipofectamine 2000 transfection reagent. The plasmid pHBV1.3 and pXF3H were cotransfected as blank controls. 3TC was used as positive controls. On day 3 after transfection, HBsAg and HBeAg were measured by using ELISA. Levels of HBV core-associated DNA and RNA were detected by using Southern and Northern blot, respectively.Results:After transfection with 0.5ug/ml,1.0ug/ml plasmid pXF3H-PAP12, the levels of HBV nucleocapside-associated DNA were reduced by 38.0% and 74.0% respectively(P﹤0.01), the levels of HBsAg in the media by 76.8% and 99.7% respectively(P﹤0.01), the levels of HBeAg by 72.7% and 99.3% respectively(P﹤0.01), compared with control. Transfection with 1.0ug/ml plasmid pXF3H-PAP reduced the levels of HBV nucleocapside- associated RNA by 69.0%(P﹤0.01).Conclusion: The total length PAP encoded by a eukaryotic expression plasmid (pXF3H-PAP12) could effectively inhibit HBV replication (at the DNA level) and gene expression (at the RNA and protein level) in vitro.PartⅢThe inhibitory effects of natural PAP on HBV replication in vivoObjective: To explore the antiviral effects of natural PAP in acute HBV infected mice.Methods:Immunocompetent BAL B/C mice were hydrodynamically injected with a replication competent plasmid (pAAV-HBV1.2) having 1.2-fold overlength of HBV DNA and a mouse model of acute HBV infection was sucessfully established.On day 1, the levels of HBsAg and HBeAg in blood serum were detected by using ELISA.The 35 mice injected with HBV plasmid were partnered according to the level of serum HBsAg and HBeAg and divided into two groups: the PAP treated group and the control group(12 mice per group).The PAP treated group were intraperitoneal injected 0.25mg/kg PAP once daily for 7 days (the control group were intraperitoneal injected physiologic saline). Before PAP injection and 1,3,5,7 day after PAP injection, the levels of serum HBsAg,HBeAg,anti-HBs and anti-HBe in blood serum were detected by using ELISA.The titers of HBV DNA were analyzed by fluorogenic quantitative PCR (FQ-PCR). 7 day after PAP injection, liver tissue was extracted and stained with HE to be observed with light microscope.HBsAg and HBcAg in the liver were assayed by immunohistochemical staining.Results: HBsAg in blood serum could be detected on day 1 after injection by ELISA in 30(85.7%) of 35 mice injected with pAAV-HBV1.2, and could not be detected in the other 5 mice. In the model,HBsAg and HBeAg were detected at the peak of expression on day 1,and dropped gradually,and were not detected on day 8. The titers of HBV DNA in blood serum were at the peak on day 2,and keep the level later, the titer was 1.9×104 copies/ml on day 8. The levels of HBsAg were reduced by 23%,47% and 68% respectively (P﹤0.05) on day 1,3,5 after PAP intraperitoneal,the levels of HBeAg by 36%,55% and 83% respectively (P﹤0.05) ,the titers of HBV DNA were reduced by 70.7%,86.9%,95.2% and 95.3%respectively (P﹤0.05).The expressions of HBsAg and HBcAg in the liver tissue were also reduced.Conclusion: 0.25mg/kg PAP could inhibit the expression of HBsAg and HBeAg, the replication of HBV DNA in blood serum and the expression of HBsAg and HBcAg in the liver in acute HBV infected mice.PartⅣThe anti-HBV effects of the total length and deleted mutant PAP encoded by a eukaryotic expression plasmid in vitroObjective: To compare the anti-HBV effects and the cytotoxicity of the total length and deleted mutant PAP encoded by a eukaryotic expression plasmid in vitro.Methods: The total length and two deleted mutant(C-deleted 25 amino acids,both N-deleted 69 amino acids and C-deleted 25 amino acids) PAP gene encoded by a eukaryotic expression plasmid (pXF3H-PAP12,pXF3H-PAP14,pXF3H-PAP34) was respectively transfected into HepG2 2.2.15 cells using lipofectamine 2000 transfection reagent.On day 3 after transfection, the medium and cells were collected.HBsAg and HBeAg were measured by using ELISA. The titers of HBV DNA were analyzed by fluorogenic quantitative PCR (FQ-PCR). HepG2 cells were used for determining cytotoxicity of the transfected plasmids by MTT assays.Results:After transfection with 2.0μg/ml plasmid pXF3H-PAP12,pXF3H-PAP14,pXF3H-PAP34, the levels of HBsAg in the media were reduced by 61.39%(P﹤0.05),56.3 %(P﹤0.05) and 7.8%(P﹥0.05) respectively, the levels of HBeAg by 84.2%(P﹤0.05),75.8%(P﹤0.05)and 11.0%(P﹥0.05) respectively, the titers of HBV DNA by 63.2%(P﹤0.05),61.7%(P﹤0.05) and 20.5%(P﹤0.05) respectively, compared with control. The inhibitory effects on cell growth were 28.74%(P﹤0.05),12.6%(P﹤0.05) and 4.78%(P﹥0.05) respectively by MTT assays.Conclusion: C-terminal 25 amino acid of PAP is related to cytotoxicity but not related to anti-HBV effect of PAP. N-terminal 69 amino acid of PAP is related to anti-HBV effect of PAP.