| GB virus C (GBV-C), also named Hepatitis G virus (HGV), is a new transfusion transmissible, single-stranded, positive-sense RNA virus identified in the sera of hepatitis patients. HGV has a similar genomic organization to those of viruses in the Flaviviridae family, especially hepatitis C virus (HCV), and contains an open reading frame (ORF) encoding a about 2900-amino-acid (aa) precursor polyprotein sharing 25.5% overall identity at the amino acid level with HCV. The putative HGV precursor polyprotein region is expected to be organized similarly to the C-E1-E2-NS2-NS3-NS4A-NS4B-NS5A-NS5B structure of HCV although the putative core protein has not been confirmed to exist. Interestingly, there are significant differences between HGV and HCV. In this study, we obtained HGV transgenic mouse strain in which HGV gene can be transmitted stablely and expressed in a higher level, which provide a useful system for studying HGV in molecular cellular and whole level. Using this technical platform, we also performed serial research on HGV replication and expression in vivo and in vitro, HGV pathogenicity, the differences of replication and expression between HGV and HCV as well as vaccine evaluation.1. Studies on Hereditary breeding and characteristics of hepatitis G virus transgenic miceTransgenic mouse is a useful system for studying viruses. Utilizing the full-length HGV cDNA clone (HGVqz), which was spliced by our lab, the HGV transgenic founder mice carrying the exogenous gene containing the CMV promoter and the whole genome of HGV were established by microinjection. In this study, the established founder mice (#2,#4,#8,#9) were used to produce Offsprings. The positive young mice were screened by PCR and southern blot. The concentrations of HGV antigen in serum and the HGV protein expression in the tissues of transgenic mice were detected by ELISA and immunohistological chemistry, respectively. At present, the exogenous genes could be transmitted stablely from #2founder mouse to F1,F1 to F2, F2 to F3, F3 to F4, F4 to F5 and from #8 founder mouse to F1, F1 to F2, F2 to F3,however, the transgene were lost in the offsprings of 4 and 9 founder mice.The positive and negative strands of HGV RNA could be detected in many tissues of HGV transgenic mice, such as liver, kidney, lung and spleen. The immunohistological chemistry of transgenic mouse tissues indicated that HGV gene could be expressed in liver, kidney, lung and spleen. The HGV antigen level was higher in liver than in other tissues. ELISA analysis showed that HGV antigen was present in mouse serum, however, the HGV antibodies were not be detected in serum of transgenic mice. The pathological analysis suggested that there were no obvious evidence of pathological changes in transgenic mouse tissues. There were nodifferences between normal and transgenic mice in serum ALT level.2. Cloning and functional analysis of promoter pagC from attenuated Salmonella typhimuriumThis study describes the cloning and function of promoter pagC(PpagC) from Salmonella typhimurium. The expression plasmid containing the in vivo-inducible promoter pagC and HGV-NS3 gene was introduced into the attenuated Salmonella typhimurium SL7207 to investigate the function of PpagC. The expressed HGV-NS3 protein was detectable by SDS-PAGE and Western blotting in the recombinant bacteria in the presence of low concentration of Mg2+ (<50mmol/L). When the concentration of Mg2+ reached to 50mmol/L, the amount of expressed HGV-NS3 was decreased significantly. The recombinant bacteria were multiplied in LB medium containing 50mmol/L of Mg2+ and used as a DNA vaccine to orally inoculate C57 mice for three times. The results of serum antibodies, T lymphocyte proliferative response and cytotoxic T lymphocyte response of immunized mice showed that the oral vaccine could iuduce strong humoral and cellular immune responses in mice, which indicates that the PpagC is a strong in vivo-inducible promoter and can be used in attenuated Salmonella typhimurium to construct an effective oral vaccine...
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