The Inhibition Of Hepatitis B Virus Replication By Pokeweed Antiviral Protein

Part IPreparation of the pokeweed antiviral protein from seeds and assay of its toxicityObjective:To purified the pokeweed antiviral protein from seeds (PAP-S) from Phytolacca Americana and test its acute and subacute toxicity in mice.Methods:The PAP-S was purified from the seeds of Phytolacca Americana using ion exchange chromatography CM 50 and CM 52 and molecular sieve G 50. The molecular weight was tested by SDS/polyacrylamide-gel electrophoresis (SDS-PAGE) and the concentration of protein was checked by BCA Protein Assay Kit.80 male Kunming mice were divided into 8 groups randomly. In each group, PAP-S dissolved in 100μ1 of saline was injected intraperitoneally once at different doses ranging from 5.6 to 0.5 mg/kg of body weight, with a ratio between doses of 1.4. The health condition and mortality of mice was observed for 14 days after injection.40 mice were assigned to 4 groups randomly. PAP-S was injected intraperitoneally at different doses of 0,0.125,0.25,0.5 mg/kg of body weight per day for 14 days.24 hours after the last administration the mice were euthanized. Sera samples were collected to test the levels of alanine aminotransferase (ALT) and blood urea nitrogen (BUN). The tissue of liver and kidney were taken to be stained with haematoxylin and eosin for histological analysis.Results:The effluent representing PAP-S was freeze-dried to protein powder approximate to 200 mg and demonstrated a single 30-kDa band in SDS-PAGE. The LD50 values of PAP-S calculated from the number of deaths within 3 days and 2 weeks were 2.41 and 1.75 mg/kg respectively in the acute toxicity examination. In the assay of subacute toxicity, no pathological BUN levels were observed in mice in each group while the level of ALT in the group with the dose of 0.5mg/kg was slightly higher than the saline group (P>0.05). No obvious organ lesions were observed in liver and kidney by haematoxylin and eosin staining.Conclusion:The PAP-S was successfully prepared and its toxicity was not observed in low concentration.Part IIThe inhibitory effects of the PAP-S on hepatitis B virus replication in vivoObjective:To investigate the inhibitory effects of PAP-S on HBV replication in HBV transgenic mice.Methods:32 HBV transgenic mice, matched for the level of HBV DNA and HBsAg, were divided into negative control group, lamivudine group and 2 doses of PAP-S treatment group randomly. Discontinue the drugs for 15 days after 45 days of administration. Sera samples were collected at 15,30,45 days after first administration and 15 days after discontinuation for the concentration of HBsAg and the level of HBV DNA. Hematoxylin-eosin staining which processed for histological analysis and immunohistochemical staining was performed to test the number of HBsAg-positive hepatocytes. Real time RT-PCR was used to test the level of HBV mRNA in liver tissues.Results:The express of HBV DNA in groups which treated by 0.25 and 0.125 mg/kg of PAP-S was inhibited by 79.7%,68.9% at 45 day and the lamivudine group was inhibited by 75.7%. The serum HBsAg levels were decreased by 29.7%,36.0% and 23.3% in the lamivudine group and 2 doses of PAP-S groups respectively. The level of serum HBV DNA and HBsAg reduced continuously at 14 days after discontinuation in each group. No organ lesions were observed in liver and kidney by haematoxylin and eosin staining. The result of immunohistochemistry showed that in the 45 day, the amount of HBsAg positive liver cells in the negative control group was much more than the PAP-S group. The level of HBV mRNA in PAP-S groups was much less in negative control group (p<0.05).Conclusion:PAP-S could inhibit HBV replication in HBV transgenic mice effectively.PartⅢConstruction, expression and purfication of prokaryotic expression plasmids of total length, deleted mutant and HBc-fusion PAPObjective:To construct the total length, deleted mutant and HBc-fusion PAP vectors, then get the his-tagged recombinant proteins.Methods:The genes which encoding the full length and partial deleted mutant PAP were amplified by PCR with the template of the PGEM-PAP plasmid. The recombinant PAPs were confirmed by enzyme digestion and DNA sequencing.The recombinant PAP and the HBc plasmid were used as templates for PCR and primers were synthesized to amplify the entire coding sequence. PCR products were cloned into pMD18-T vector and identified by restriction digestion and sequencing. Both of the insert and the pET-28a vector were digested by the enzyme of BamH I and HindⅢ, and then the insert were inserted into the vector by T4 ligase.Total length and HBc-fusion PAP was purified by affinity chromatography using Ni-NTA column after induced by isopropyl-1-thio-(β-D-galactopyranoside (IPTG).Results:The total length, deleted mutant and HBc-fusion PAP vectors were confirmed by restriction digestion and sequencing. The relative molecular masses of purified proteins were determined by SDS-PAGE and proved to be the expected band.Conclusion:The prokaryotic expression plasmids of total length, deleted mutant and HBc-fusion PAP were successfully constructed, the total length and HBc-fusion PAP proteins were purified.Part IVThe anti-HBV effects of the total length and HBc-fusion PAP proteins in vitroObjective:To investigate the inhibitory effects of the total length and HBc-fusion PAP proteins on HBV replication in HBV transgenic mice.Methods:32 HBV transgenic mice were matched for the level of HBV DNA and HBsAg and divided into negative control group, lamivudine group, total length PAP12 protein group and total length PAP12-HBC group. Drugs were administrated for 45 days continuously. Sera samples were collected at 15,30,45 days after first administration and 15 days after discontinuation and the concentration of HBsAg and the level of HBV DNA were detected. Haematoxylin and eosin staining was used for observe the liver and kidney lesions, immunohistochemical staining were uesd to test the HBsAg in the liver and real time RT-PCR was used to test the level of HBV mRNA in liver tissuesResults:Compared to the negative control group, the express of HBV DNA in groups which treated with total length PAP 12 protein group and total length PAP12-HBc group was inhibited by 73%,77% at 45 day, and the serum HBsAg levels were decreased by 24.7%, 27.2% respectively. No organ lesions were observed in liver and kidney by haematoxylin and eosin staining. The result of immunohistochemistry showed that the amount of HBsAg positive liver cells in the negative control group was much more than the amount in the recombinant PAP group (p<0.05). The level of HBV RNA in recombined PAP groups was much less in negative control group.Conclusion:Total length and HBc-fusion prokaryotic protein of PAP could inhibit HBV replication in HBV transgenic mice without any toxicity in the effective concentration.