AIM: To investigate whether the formation of aggregated HBx has a potential linking with its cellular responses.METHODS: Recombinant HBx was expressed in Escherichia coli and purified by Ni-NTA metal-affinity chromato...AIM: To investigate whether the formation of aggregated HBx has a potential linking with its cellular responses.METHODS: Recombinant HBx was expressed in Escherichia coli and purified by Ni-NTA metal-affinity chromatography.Anti-HBx monoclonal antibody was developed for immunocytochemical detection. Bicistronic expression vector harboring full-length DNA of HBx was employed for transfection of human HepG2 ceils. Immunocytochemical staining was used to examine the intracellular HBx aggregates in cells. The effects of HBx aggregation on cell cycle and apoptosis were assessed by flow cytometry.RESULTS: Immunocytochemical staining revealed most of the HBx was formed intracellular aggregate in cytoplasm and frequently accumulated in large granules. Flow cytometry analysis showed that HepG2 cells transfected with vector harboring HBx significantly increased apoptosis and largely accumulated in the G0-G1 phase by maintenance in serum medium for 36 hours. Control cells without HBx aggregates in the presence of serum entered S phase and proliferated more rapidly at the same time.EGFP fluorescence in HBx expression cells was significantly decreased.CONCLUSION: Our observations show that cells with HBx aggregate undergo growth arrest and apoptosis, whereas control cells without HBx remain in growth and progression into S phase. Our data may provide helpful information to understand the biological effects of HBx aggregates on cells.展开更多
AIM:A strategy for viral vaccine design is the use of conserved peptides to overcome the problem of sequence diversity. At present it is still unclear whether conserved peptide is safe as a candidate vaccine. We repor...AIM:A strategy for viral vaccine design is the use of conserved peptides to overcome the problem of sequence diversity. At present it is still unclear whether conserved peptide is safe as a candidate vaccine. We reported it here for the first time not only to highlight the biohazard issue and safety importance for viral peptide vaccine, but also to explore the effect of a fully conserved peptide on HBV replication within the carboxyl terminus of HBx.METHODS:We synthesized the fully conserved peptide (CP) with nine residues, FVLGGCRHK. HBV-producJng 2.2.15 cells were treated with or without 3.5μM CP for 36 hours.Quantitative detection of viral DNA was performed by realtime PCR. HBV antigens were determined by enzyme-linked immunoadsorbent assay (ELISA). Quantitative analyses of p53 and Bax proteins were based on immunofluorescence.Flow cytometry was performed to detect cell cycle and apoptosis.RESULTS: Both extracellular and intracellular copies of HBV DNA per ml were significantly increased after incubation with 3.5μM of CP.HBsAg and HBeAg in the cultured medium of CP-treatment cells were as abundant as untreated control ceils. CP influenced negatively the extracellular viral gene products, and 3.5μM CP could significantly inhibit intracellular HBsAg expression. In response to CP, intracellular HBeAg displayed an opposite pattern to that of HBsAg, and 3.5μM CP could efficiently increase the level of intracellular HBeAg.Flow cytometric analyses exhibited no significant changes on cell cycle, apoptosis, p53 and Bax proteins in 2.2,15 cells with or without CP.CONCLUSION: Together with the results generated from the synthetic peptide, we address that the conserved region,a domain of HBx, may be responsible for modulating HBV replication. As conserved peptides from infectious microbes are used as immunogens to elicit immune responses, their latent biological hazard for human beings should be evaluated.展开更多
文摘AIM: To investigate whether the formation of aggregated HBx has a potential linking with its cellular responses.METHODS: Recombinant HBx was expressed in Escherichia coli and purified by Ni-NTA metal-affinity chromatography.Anti-HBx monoclonal antibody was developed for immunocytochemical detection. Bicistronic expression vector harboring full-length DNA of HBx was employed for transfection of human HepG2 ceils. Immunocytochemical staining was used to examine the intracellular HBx aggregates in cells. The effects of HBx aggregation on cell cycle and apoptosis were assessed by flow cytometry.RESULTS: Immunocytochemical staining revealed most of the HBx was formed intracellular aggregate in cytoplasm and frequently accumulated in large granules. Flow cytometry analysis showed that HepG2 cells transfected with vector harboring HBx significantly increased apoptosis and largely accumulated in the G0-G1 phase by maintenance in serum medium for 36 hours. Control cells without HBx aggregates in the presence of serum entered S phase and proliferated more rapidly at the same time.EGFP fluorescence in HBx expression cells was significantly decreased.CONCLUSION: Our observations show that cells with HBx aggregate undergo growth arrest and apoptosis, whereas control cells without HBx remain in growth and progression into S phase. Our data may provide helpful information to understand the biological effects of HBx aggregates on cells.
文摘AIM:A strategy for viral vaccine design is the use of conserved peptides to overcome the problem of sequence diversity. At present it is still unclear whether conserved peptide is safe as a candidate vaccine. We reported it here for the first time not only to highlight the biohazard issue and safety importance for viral peptide vaccine, but also to explore the effect of a fully conserved peptide on HBV replication within the carboxyl terminus of HBx.METHODS:We synthesized the fully conserved peptide (CP) with nine residues, FVLGGCRHK. HBV-producJng 2.2.15 cells were treated with or without 3.5μM CP for 36 hours.Quantitative detection of viral DNA was performed by realtime PCR. HBV antigens were determined by enzyme-linked immunoadsorbent assay (ELISA). Quantitative analyses of p53 and Bax proteins were based on immunofluorescence.Flow cytometry was performed to detect cell cycle and apoptosis.RESULTS: Both extracellular and intracellular copies of HBV DNA per ml were significantly increased after incubation with 3.5μM of CP.HBsAg and HBeAg in the cultured medium of CP-treatment cells were as abundant as untreated control ceils. CP influenced negatively the extracellular viral gene products, and 3.5μM CP could significantly inhibit intracellular HBsAg expression. In response to CP, intracellular HBeAg displayed an opposite pattern to that of HBsAg, and 3.5μM CP could efficiently increase the level of intracellular HBeAg.Flow cytometric analyses exhibited no significant changes on cell cycle, apoptosis, p53 and Bax proteins in 2.2,15 cells with or without CP.CONCLUSION: Together with the results generated from the synthetic peptide, we address that the conserved region,a domain of HBx, may be responsible for modulating HBV replication. As conserved peptides from infectious microbes are used as immunogens to elicit immune responses, their latent biological hazard for human beings should be evaluated.
