| Since the discovery of HBs Ag by Blumberg in 1963, HBV infection is a major health problem which affects millions of people worldwide. Many patients develop persistent infection that may lead to chronic hepatitis, cirrhosis and hepatocellular carcinoma.The study of HBV biology and development of new therapies have been slow to evolve due to the lack of a practical and convenient small animal model.Major process in the understanding of hepadnavirus infection,replication and viral assembly was generated through the analysis of viral life cycle in their natural hosts,but these animal models lack various aspects of human disease.Attempts have been made to establish animal models for the study of HBV infection in rats and in transgenic or immunodeficient mice.In all the existing rodent systems the generation of each animal is conducted through a technically difficult, multi-step process.Hence,the development of a small animal model for hepatitis virus life cycle and for the evaluation of novel drugs for treatment of human infection.Zhang developed a simple hydrodynamic based procedure for efficient transient transfection of liver cells in vivo by means of plasmid DNA injections into the tail vein of mice.Viruses have evolved to coexist with the host by employing a range of strategies for evading the host immune system, avoiding immune surveillance and eliminating from the host, thus create a favorable environment for virus persistence and successfully infect the immunocompetent host to cause disease. Human Cytomegalovirus(HCMV)and Epstein-Barr virus(EBV) are members of the herosviridae family,they are the only human herpesviruses that encode a viral homolog of IL-10. Virus-encoded IL-10 homologs are likely to enable modulation of the local immune response so as to enhance the capacity to replicate, disseminate and persisit in an immunocompetent individual.We want to induce mouse chronic HBV infection through giving HBV immunosuppression function by constructing of a replication-competent HBV vector.HBV vectors with theoreticly immunosuppression function(expressions of Epstein–Barr virus interleukin 10(ebvIL-10), Cytomegalovirus interleukin 10(cmvIL-10)) will be constructed and confirmed for the performance in mouse cells. Finally, the viral particle from the recombinant HBV vectors with immunosuppressive function is planed to be used for the infection of mouse in vivo. We adapted the hydrodynamic-based procedure for the establishment of a simple small animal model for HBV infection.Characteristics of mouse model of HBV infection will be observed.Part 1 Construction of HBV vectors separately expressing cmvIL-10 and ebvIL-10Objective: To construct HBV vectors separately expressing cmvIL-10 and ebvIL-10, study on molecular biological characteristics of the two plasmids and analysis whether they could exert immunosuppression function as theoreticly do.Methods:1 Two HBV vectors with theoreticly immunosuppression function(expressions of cmvIL-10, ebvIL-10) will be constructed by gene engineering method.we will construct a replication-competent HBV vector on the basis of wild-type HBV expression plasmid,the overlapping region of HBV Core and Polymerase will be separated and then two exogenous gene(cmvIL-10, ebvIL-10)will be inserted between them.2 The replication efficacy of these two HBV vectors were analyzed with procedures annotated as followed. With the pCH-3093 plasmid as control, pCH-cmvIL-10, and pCH-ebvIL-10 were transfected into Hep G2 cells respectively. Replication efficacy of newly-constructed HBV vectors were determined by Southern blotting to detect HBV DNA in cell lysates and supernatant of c?lture medium respectively and by Native western blotting to detect three HBV core protein. Expression of the HBV m RNA level were detected by Northern blotting.3 With pCH-3093 plasmid as control, pCH-cmvIL-10 and pCH-ebvIL-10 were transfected into 293 cells respectively,at 24 h after transfection,the supernatant was collected and treated with LPS-induced murine monocytes,MHC-II m RNA and TNF-? m RNA was analyzed by real-time fluorescent quantitative PCR.Results:1 HBV vectors containing exogenous genes of pCH-cmvIL-10 ?pCH-ebvIL-10 were validated by restriction endonuleases digestion and sequencing followed.2 Replication efficacy of newly-constructed HBV vectors were marginally decreased compared with wild type HBV vector.3 Because the insertion of exogenous genes,both of the two newly-constructed HBV vectors' pg RNA electrophoresis were slow compared with the wild type HBV,while sg RNAs were similar?4 HBV core protein in Hep G2 cells transfected with pCH-cmvIL-10?pCH-ebvIL-10 were similar with that of Hep G2 cells transfected with wild type HBV vector, which indicated that our HBV vectors should not interfere with the formation of HBV core proteins.5 Both MHC-II m RNA and TNF-? m RNA analyzed by real-time fluorescent quantitative PCR decreased in different extent.Conclusions:1 Reconstructed HBV-expressing vectors by separating the overlapping region of P gene and C gene and subsequent insertion of exogenous genes:cmvIL-10?ebvIL-10,could produce plenty of HBV DNA when transfected into Hep G2 cells, which consolidated our newly-reconstructed vectors as replicative ones.2 Both of the two HBV vectors possess the capacity to exert potent immunosuppressive functions in murine.Part 2 Hydrodynamic-based procedure for the establishment of mouse model of HBV infectionObjective: to establish mouse model of HBV infection with newly-reconstructed vectors: pCH-cmvIL-10?pCH-ebvIL-10.Methods:1 Balb/c male mice(6-8weeks old,22-25g) were purchased from Hebei Medical University.All animals were cared for in compliance with institutional guidelines.Animals were fed rat chow ad libitum and drank tap water,and were kept with a 12 h light-dark cycle at constant temperature and humidity. They were divided randomly into three groups:1.pCH-3093 group;2.pCH-cmvIL-10 group;3.pCH-ebvIL-10 group.2 Mice were injected with plasmid DNA(15ug per mouse)diluted in saline to a volume equivalent to 8% of the total bodyweight of each animal via the tail vein within 5-7 seconds.3 On the indicated days,mice were sacrificed,blood was withdrawn and sera were used for assessments.HBs Ag and HBe Ag were detected using AXSYM systems.Immunohitochemistry analysis of liver sections by specific antibodies for detection of HBc Ag expression.Results : After hydrodynamic injection with reconstructed HBV-expressing vector pCH-ebvIL-10 to Balb/c mice,murine serum of HBs Ag and HBe Ag together with HBc Ag highly expression in liver sections were observed, and infection time was longer than the wild type HBV group. The expression of HBV was low in pCH-cmvIL-10 group.Conclusions: A mouse model of HBV acute infection with reconstructed HBV vector pCH-ebvIL-10 was successfully constructed with prolongated infection time. |
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