Objective:To investigate the relationship between variation of HBV preS genes and clinical consequencesof HBV infection. Methods: We selected 3 groups (3 in each) of HBV infected individuals including those of acutein...Objective:To investigate the relationship between variation of HBV preS genes and clinical consequencesof HBV infection. Methods: We selected 3 groups (3 in each) of HBV infected individuals including those of acuteinfection with complete recovery, chronic HBV carriers and chronic severe hepatitis B. The preS genes were amplified from serum samples with half nested PCR. The PCR products were cloned into M13 vectors and 10 cloneswere randomly selected for each individual and sequenced with standard methods. A total of 90 clones were sequenced and analyzed by DNA homological comparison. Results: The characteristics of the preS genes were significantly different in HBV-infected individuals with different clinical consequences.①As for acute hepatitis B, thepreS function domains were stable and no changes in epitopes for T and B cells were found. ②For chronic HBVcarriers, the function domains within preS proteins showed potentially changes with epitope drifts of T and B cellsbecause of the high variation of HBV preS genes. ③For chronic severe hepatitis B, there were special changes inthe function domains of B cells. The abnormally high immune response would cause severe liver injury in these individuals. Conclusion: This study provides experimental evidence for researches about the possible association ofvariation of preS1/S2 functional regions with clinical consequences.展开更多
AIM: To prepare a cancer vaccine (H(22)-DC) expressing high levels of costimulatory molecules based on fusions of hepatocarcinoma cells (H(22)) with dendritic cells (DC) of mice and to analyze the biological character...AIM: To prepare a cancer vaccine (H(22)-DC) expressing high levels of costimulatory molecules based on fusions of hepatocarcinoma cells (H(22)) with dendritic cells (DC) of mice and to analyze the biological characteristics and induction of specific CTL activity of H(22)-DC. METHODS: DCs were isolated from murine spleen by metrizamide density gradient centrifugation, purified based on its characteristics of semi-adhesion to culture plates and FcR-,and were cultured in the medium containing GM-CSF and IL-4. A large number of DC were harvested. DCs were then fused with H(22) cells by PEG and the fusion cells were marked with CD11c MicroBeads. The H(22)-DC was sorted with Mimi MACS sorter. The techniques of cell culture, immunocytochemistry and light microscopy were also used to test the characteristics of growth and morphology of H(22)-DC in vitro. As the immunogen, H(22)-DC was inoculated subcutaneously into the right armpit of BALB/C mice, and their tumorigenicity in vivo was observed. MTT was used to test the CTL activity of murine spleen in vivo. RESULTS: DC cells isolated and generated were CD11c+ cells with irregular shape, and highly expressed CD80, CD86 and CD54 molecules. H22 cells were CD11c- cells with spherical shape and bigger volume, and did not express CD80, CD86 and CD54 molecules.H(22)-DC was CD11c+ cells with bigger volume, being spherical, flat or irregular in shape, and highly expressed CD80, CD86 and CD54 molecules, too. H(22)-DC was able to divide and proliferate in vitro, but its activity of proliferation was significantly decreased as compared with H(22) cells and its growth curve was flatter than H(22) cells. After subcutaneous inoculation over 60 days, H(22)-DC showed no tumorigenecity in mice, which was significantly different from control groups (P【0.01). The spleen CTL activity against H(22) cells in mice implanted with fresh H(22)-DC was significantly higher than control groups (P 【 0.01). CONCLUSION: H(22)-DC could significantly stimulate the specific CTL activity of murine spleen, which suggests that the fusion cells have already obtained the function of antigen presenting of parental DC and could present H(22)specific antigen which has not been identified yet, and H(22)-DC could induce antitumor immune response; although simply mixed H(22) cells with DC could stimulate the specific CTL activity which could inhibit the growth of tumor in some degree, it could not prevent the generation of tumor. It shows that the DC vaccine is likely to become a helpful approach in immunotherapy of hepatocarcinoma.展开更多
The DNA fragments coding for preS2(120 146) and preS1(21 47) amplified by PCR were fused to both 5′ and 3′ ends of S gene at the position of amino acid 223. The fusion gene was placed downstream of the promoter P7.5...The DNA fragments coding for preS2(120 146) and preS1(21 47) amplified by PCR were fused to both 5′ and 3′ ends of S gene at the position of amino acid 223. The fusion gene was placed downstream of the promoter P7.5 of the universal vaccinia viral vector pGJP 5 and the recombinant vaccinia virus vS2SS1 was then selected by \%in vivo\% homogeneous recombination. Fusion protein S2SS1 could be expressed in the mammalian cells infected with vS2SS1. The investigation of expression, secretion, antigenicity and particle assembly of the S2SS1 protein demonstrated that S2SS1 protein could be assembled into particles which presented preS1, preS2 and S antigenicity and be efficiently secreted from the cells. It also showed that the level of its expression and secretion approached to that of the S protein expressed by the recombinant vaccinia virus.展开更多
A synthetic peptide, the N\|terminus of hepatitis B virus surface antigen Pre\|S1, was studied by two\| dimensional NMR techniques. A series of 1 H nuclear magnetic resonance experiments were used to complete the iden...A synthetic peptide, the N\|terminus of hepatitis B virus surface antigen Pre\|S1, was studied by two\| dimensional NMR techniques. A series of 1 H nuclear magnetic resonance experiments were used to complete the identification of spin systems and sequential assignments of this 28\|residue peptide. 157 distance constraints and 55 dihedral angle constraints were obtained. 20 structures with the lowest target function were selected by the distance geometry program DIANA. Energy minimization and the following 100 ps time\|averaged restrained molecular dynamics （TRMD） simulation in aqueous solution were performed for each conformer. After TRMD simulation, three locally convergent regions corresponding to residues 22\31, 36\40, 41\46 were found. The averaged pairwise root\|mean\|square deviation （RMSD） of backbone atoms for them were （1.71±0.49）?, （0.76±0.31）?, （1.05±0.52）?, respectively. Four reverse turns found in these regions, residues 22\25, 37\40, 41\44 and 43\46, correspond to several important antibody binding sites revealed in relevant immunological research.展开更多
Antigenic determinants expressed on the bacterial cell surface are of importance in the serological characterization and microbiological diagnosis. The bacterial strains carrying these identical or similar antigenic e...Antigenic determinants expressed on the bacterial cell surface are of importance in the serological characterization and microbiological diagnosis. The bacterial strains carrying these identical or similar antigenic epitopes might react with antibodies produced against other strains. In this study, strong immunogenicity and antigenic cross reactivity were demonstrated among V. cholerae O1, S. flexnerii 2a and H. influenzae b surface components. The enzyme linked immunosorbent assay （ELISA） results were supported by Western blot analysis, where at least 20 antigenic bands, were obtained in each of the reactions, when the surface components were reacted with the homologous antisera. The indirect ELISA results also demonstrated high degree of antigenic relatedness between the surface components of these species, where each surface component was reacted with the heterologous antisera. Western blot analysis also revealed cross reactions between the surface components suggesting common distribution of antigens/epitopes in these bacterial species. This study, thus, gave a clear idea of the level of antigenic sharing and variations among the pathogenic V. cholerae O1, S. flexneri 2a and H. influenzae b strains, which in future, may help in selecting a proper candidate for vaccines and immunodiagnostics development.展开更多
文摘Objective:To investigate the relationship between variation of HBV preS genes and clinical consequencesof HBV infection. Methods: We selected 3 groups (3 in each) of HBV infected individuals including those of acuteinfection with complete recovery, chronic HBV carriers and chronic severe hepatitis B. The preS genes were amplified from serum samples with half nested PCR. The PCR products were cloned into M13 vectors and 10 cloneswere randomly selected for each individual and sequenced with standard methods. A total of 90 clones were sequenced and analyzed by DNA homological comparison. Results: The characteristics of the preS genes were significantly different in HBV-infected individuals with different clinical consequences.①As for acute hepatitis B, thepreS function domains were stable and no changes in epitopes for T and B cells were found. ②For chronic HBVcarriers, the function domains within preS proteins showed potentially changes with epitope drifts of T and B cellsbecause of the high variation of HBV preS genes. ③For chronic severe hepatitis B, there were special changes inthe function domains of B cells. The abnormally high immune response would cause severe liver injury in these individuals. Conclusion: This study provides experimental evidence for researches about the possible association ofvariation of preS1/S2 functional regions with clinical consequences.
基金Supported jby the Natural Science Foundation of Guangdong Province China,No.980180
文摘AIM: To prepare a cancer vaccine (H(22)-DC) expressing high levels of costimulatory molecules based on fusions of hepatocarcinoma cells (H(22)) with dendritic cells (DC) of mice and to analyze the biological characteristics and induction of specific CTL activity of H(22)-DC. METHODS: DCs were isolated from murine spleen by metrizamide density gradient centrifugation, purified based on its characteristics of semi-adhesion to culture plates and FcR-,and were cultured in the medium containing GM-CSF and IL-4. A large number of DC were harvested. DCs were then fused with H(22) cells by PEG and the fusion cells were marked with CD11c MicroBeads. The H(22)-DC was sorted with Mimi MACS sorter. The techniques of cell culture, immunocytochemistry and light microscopy were also used to test the characteristics of growth and morphology of H(22)-DC in vitro. As the immunogen, H(22)-DC was inoculated subcutaneously into the right armpit of BALB/C mice, and their tumorigenicity in vivo was observed. MTT was used to test the CTL activity of murine spleen in vivo. RESULTS: DC cells isolated and generated were CD11c+ cells with irregular shape, and highly expressed CD80, CD86 and CD54 molecules. H22 cells were CD11c- cells with spherical shape and bigger volume, and did not express CD80, CD86 and CD54 molecules.H(22)-DC was CD11c+ cells with bigger volume, being spherical, flat or irregular in shape, and highly expressed CD80, CD86 and CD54 molecules, too. H(22)-DC was able to divide and proliferate in vitro, but its activity of proliferation was significantly decreased as compared with H(22) cells and its growth curve was flatter than H(22) cells. After subcutaneous inoculation over 60 days, H(22)-DC showed no tumorigenecity in mice, which was significantly different from control groups (P【0.01). The spleen CTL activity against H(22) cells in mice implanted with fresh H(22)-DC was significantly higher than control groups (P 【 0.01). CONCLUSION: H(22)-DC could significantly stimulate the specific CTL activity of murine spleen, which suggests that the fusion cells have already obtained the function of antigen presenting of parental DC and could present H(22)specific antigen which has not been identified yet, and H(22)-DC could induce antitumor immune response; although simply mixed H(22) cells with DC could stimulate the specific CTL activity which could inhibit the growth of tumor in some degree, it could not prevent the generation of tumor. It shows that the DC vaccine is likely to become a helpful approach in immunotherapy of hepatocarcinoma.
文摘The DNA fragments coding for preS2(120 146) and preS1(21 47) amplified by PCR were fused to both 5′ and 3′ ends of S gene at the position of amino acid 223. The fusion gene was placed downstream of the promoter P7.5 of the universal vaccinia viral vector pGJP 5 and the recombinant vaccinia virus vS2SS1 was then selected by \%in vivo\% homogeneous recombination. Fusion protein S2SS1 could be expressed in the mammalian cells infected with vS2SS1. The investigation of expression, secretion, antigenicity and particle assembly of the S2SS1 protein demonstrated that S2SS1 protein could be assembled into particles which presented preS1, preS2 and S antigenicity and be efficiently secreted from the cells. It also showed that the level of its expression and secretion approached to that of the S protein expressed by the recombinant vaccinia virus.
文摘A synthetic peptide, the N\|terminus of hepatitis B virus surface antigen Pre\|S1, was studied by two\| dimensional NMR techniques. A series of 1 H nuclear magnetic resonance experiments were used to complete the identification of spin systems and sequential assignments of this 28\|residue peptide. 157 distance constraints and 55 dihedral angle constraints were obtained. 20 structures with the lowest target function were selected by the distance geometry program DIANA. Energy minimization and the following 100 ps time\|averaged restrained molecular dynamics （TRMD） simulation in aqueous solution were performed for each conformer. After TRMD simulation, three locally convergent regions corresponding to residues 22\31, 36\40, 41\46 were found. The averaged pairwise root\|mean\|square deviation （RMSD） of backbone atoms for them were （1.71±0.49）?, （0.76±0.31）?, （1.05±0.52）?, respectively. Four reverse turns found in these regions, residues 22\25, 37\40, 41\44 and 43\46, correspond to several important antibody binding sites revealed in relevant immunological research.
文摘Antigenic determinants expressed on the bacterial cell surface are of importance in the serological characterization and microbiological diagnosis. The bacterial strains carrying these identical or similar antigenic epitopes might react with antibodies produced against other strains. In this study, strong immunogenicity and antigenic cross reactivity were demonstrated among V. cholerae O1, S. flexnerii 2a and H. influenzae b surface components. The enzyme linked immunosorbent assay （ELISA） results were supported by Western blot analysis, where at least 20 antigenic bands, were obtained in each of the reactions, when the surface components were reacted with the homologous antisera. The indirect ELISA results also demonstrated high degree of antigenic relatedness between the surface components of these species, where each surface component was reacted with the heterologous antisera. Western blot analysis also revealed cross reactions between the surface components suggesting common distribution of antigens/epitopes in these bacterial species. This study, thus, gave a clear idea of the level of antigenic sharing and variations among the pathogenic V. cholerae O1, S. flexneri 2a and H. influenzae b strains, which in future, may help in selecting a proper candidate for vaccines and immunodiagnostics development.
