DHBV Invasion Procedure And Expression PreS Protein In Ecoli. And The Application Of It In Evaluation Anti-HBV New Drug In Vivo And In Vitro

Hepatitis B virus(HBV)is a human hepadnavirus that causes acute and chronic hepatitis and hepatocellular carcinoma.The long-term objectives of our research are to design therapeutic strategies and to improve vaccines for human HBV infection.Our understanding of the immune response to HBV is incomplete,largely due to the narrow host restriction of this pathogen and the limitations of existing experimental models.Much of our current understanding of the viral life cycle of HBV infection is derived from studies of duck HBV(DHBV)infection in their natural hosts.Although the duck model has recognized limitations,it has proven to be an exceptionally useful system for the elucidation of hepadnaviral replication strategy and antiviral drug screening.One of the major advantages of the DHBV model is that its natural host,domestic duck,is inexpensive,easy to handle,and available from commercial breeders.It is necessary to develop a standard DHBV animal model to enhance our understanding of the replication strategy,natural history,and pathogenesis of HBV infection.So the invasion procedure of DHBV infection in model should be clarified.The contents was summaried as follows:1.Molecular characterization of DHBV isolates from Sichuan Mallard.To clone and analyze the genome of a duck hepatitis B virus(DHBV)isolated from a Sichuan Mallard,DHBV DNA-positive serum were collected from the investigation of the natural carrying rate of DHBV in adult ducks in Sichuan area.The complete genome of a DHBV strain was amplified by polymerase chain reaction(PCR)and sequencing.The natural carrying rate of DHBV in Sichuan adult ducks was 4%.The strain had DHBV genomes of 3,006 nucleotides with unique characteristic features.The present investigation shows Sichuan ducks are one of the best species for experimental DHBV infection.2.Expression preS protein in Ecoli.and preparation anti-preS antibody.To purify the recombinant preS peptide of duck hepatitis B surface antigen in E.coli and to investigate its physiochemical characters and immunogenicity.The PreS gene was amplified by PCR,and then inserted into prokaryotic expression vector pET32a(+).The recombinant plasmid was confirmed by sequence analysis and named pET32a(+) /DHBV-preS.The expression of recombinant proteins were induced with IPTG,and then the expression product were confirmed by Western blot analysis and further purified by affinity chromatography methods.Recombinant proteins of PreS displayed bands of Mr 37,000 on SDS-PAGE gel.The purity of the fusion protein was over 90%after purification by affinity chromatography method.The specific antibody titer could reach 1:32 in recombinant protein immunized rabbit.Purified fusion proteins laid a foundation for better understanding of the mechanism of DHBV PreS protein in viral endocytosis and were helpful for seeking the PreS-related protein.3.Quantification of DHBV by fluorescence quantitative PCR(FQ-PCR).To develop a fluorescence quantitative PCR based on TaqMan chemistry for quantification of DHBV.The PCR fragment of DHBV DNA was cloned into vector pMD18-T.The recombinant plasmid was purified and subsequently quantified as DHBV DNA strand.A pair of primers and fluorescent probe were designed from conserved sequence.The experimental conditions and reagents of amplification were sopisticatedly optimized in order to produce perfect amplification efficiency and reduce non-specific amplification. The standard curve indicated the linear relationship between CT(cycle threshold)and template concentration with a good correlation(r=0.993),sensitivity was 5 copies/L of DHBV genome.The FQ-PCR method for quantification of DHBV DNA is a simple,highly sensitive and specific method.4.Immunohistochemical assay for detection DHBsAg.To establish immune-ohistochemical assay for duck hepatitis surface antigen(DHBsAg)using anti-preS antibody.Liver,Pancreas,Kidney,Spleen,Cerebrum samples,taken from 12 days ducklings post-inoculation,were used to locate DHBsAg by IHC.The result illustrated that DHBsAg was usually restricted to randomly scatter cells showing strong cytoplasmic staining;Positive cells also were detected in Liver,Pancreas,Kidney,Spleen and Cerebrum.The methodology is helpful to illustrate the localization of virus specific antigen in situ at subcellsular level and for exploring important aspects in this family of hepadnavirus such as viral invasion procedure,viral replication,course of infection, pathogenesis and the response to antiviral therapy.These data provide further information about the pathogenesis and distribution of DHBV infection for animal model.5.The invasion procedure in DHBV infection model.To develop a standard DHBV animal model and use it as an in vivo experimental system to study DHBV invasion prcedure and antiviral strategies.One-day-old ducklings were subcutaneously inoculated DHBV serum and sacrificed during in different time from 2d to 70d respectively whose tissues were collected including liver,pancreas,kidney,spleen,cerebrum et al.These tissues were detected using developed immunohistochemistry.Quantitative analysis of DHBV DNA in serum and liver samples was performed with FQ-PCR.Pathological changes were observed and biochemical indexes were determined at different times.The results showed that positive signals can be detected from liver,pancreas,kidney,spleen and cerebrum during different time from 3dpi to 52dpi.DHBsAg was usually restricted to randomly scatter cells showing strong cytoplasmic staining.The most positive tissue samples was at 12d pi to 31dpi(post inoculation).DHBsAg positive signals appeared in liver at 4dpi,in kidney,pancreas and spleen at 6dpi.The max detection rate was the liver among the detected tissues while the min detection rate was the cerebrum.DHBsAg can not be detected from gullet,glandular stomach,lung,heart,genitals and muscle.Increase the serum levels of biochemical index,especially ALT,AST,SB,UN and CR.The liver and renal functions were changed in accordance with DHBV DNA level,pathological changes and DHBsAg changes.The Sichuan duck model with experimental DHBV infection of transfected supernatant is more suitable for the hepadnavirus biologic research.6.Antiviral treatment to the HepG2.2.15 and DHBV-infected ducklings. Nucleoside analogues provide a large reservoir of potentially active anti-hepatitis B virus (HBV)agents.To evaluated the novel nucleoside analogue(PNA)in HepG2 2.2.15 cell line and in the duck model of hepatitis B.The extracellular HBV DNA,hepatitis B e antigen(HBeAg)and hepatitis B surface antigen(HBsAg)concentrations in cell culture medium were determined by quantitative real-time PCR and ELISA,respectively.PNA appeared to downregulate the secretion of HBsAg and HBeAg as well as the release of HBV DNA from HepG2 2.2.15 in a dose- and time-dependent manner.Consistent with the HBV antigen reduction,PNA also reduced the extracellular HBV DNA level in a dose-dependent manner between 7.8 and 62.5μg/mL.PNA(25,50,or 100mg/kg, intraperitoneally,twice daily)can significantly lower the DHBV DNA levels in sera and livers.Histopathological changes in the treatment groups were significantly improved and the changes were associated with liver viral DNA levels.Immunohistological staining of the liver confirmed the duck hepatitis B surface antigen(DHBsAg)reduction by PNA The relapse in PNA-treatment groups is slighter than in 3TC-treatment group at 3 day withdrawl,In conclusion,the results demonstrate that PNA is a strong inhibitor of anti-HBV activity both in vitro and in vivo.