AIM: To investigate the change of immunological characteristics of HBsAg caused by the mutation at codon 145 of HBsAg using DNA-based immunization.METHODS: Plasmids expressing mutant and wild type envelope antigens we...AIM: To investigate the change of immunological characteristics of HBsAg caused by the mutation at codon 145 of HBsAg using DNA-based immunization.METHODS: Plasmids expressing mutant and wild type envelope antigens were transfected into human hepatocellular carcinoma cells via electrotransformation. The antigenicity of HBsAg was studied with EIA and immunocytochemical staining. Then plasmids were used to immunize 5 C57BL/6 mice. Sera of mice were detected for anti-HBs and anti-preS2with ELISA. RESULTS: The mutant HBsAg could be detected by native antibody in EIA and immunocytochemical study. But the A(450nm) value of the mutant HBsAg in the supernatant was apparently lower than that of the wild-type. Both mutant and native HBsAg expression plasmid could stimulate a strong humoral immune response to HBsAg and preS2 antigen in mice. Protective antibodies against HBsAg elicited by the native HBsAg occurred earlier than that elicited by the mutant HBsAg about one to two weeks. The occurrence of protective antibodies against preS2 antigen was one to two weeks earlier than that of anti-HBs.CONCLUSION: The amino acid substitution causes changes of the antigenicity and immunogenicity of HBsAg, but mutant HBsAg can still induce a protective humoral immune responsein mice.展开更多
AIM:To establish the transgenic mouse line harbouring complete hepatitis B virus (HBV) genome with mutant s gene (adr subtype).METHODS: Transgenic mice were generated by microinjecting HBV genome into fertilized eggs....AIM:To establish the transgenic mouse line harbouring complete hepatitis B virus (HBV) genome with mutant s gene (adr subtype).METHODS: Transgenic mice were generated by microinjecting HBV genome into fertilized eggs. Integration, expression, replication of HBV gene and histological changes in transgenic mice were estimated by genomic DNA PCR, serum DNA PCR, Southern blot, ELISA, HE staining, immunohistochemistry and transmission electron microscopy. Transgenic mice with HBsAg positive in serum were bred and analyzed.RESULTS: A total of 288 eggs survived from microinjections were transplanted into the oviducts of 13 pseudopregnant mice and 49 pups were produced. Twenty-six mice were identified to have the integrated HBV gene. Serum HBsAg and HBeAg were detected in 2 of 43 mice. HBsAg and HBcAg in cytoplasm or nuclei of hepatocytes were detected in 10 mice. Founders with HBsAg in serum were named lineages G145R-15 and G145R-18. Of the 16 F1 offsprings generated by G145R-15 founder, 12 were positive for HBV genome with PCR, 10 were positive for HBsAg and HBcAg with immunohistochemistry and 7 were positive for HBsAg and HBeAg with ELISA. Only 1 of 8 F1 offsprings generated by G145R-18 founder was survived and it was detected positive for HBV genome, HBsAg, HBcAg and HBeAg. Both of the two lineages had some pathological characteristics of mild chronic hepatitis B in the liver, such as swelling of hepatocytes and focal hepatocellular necrosis and parenchymal lymphomononuclear cell infiltrate.CONCLUSION: Transgenic mice harbouring HBV with mutant s gene can be generated. The HBV genes are integrated in the transgenic mice genome and can be expressed, replicated, packaged and excreted. HBV DNA can be stably transmitted in the transgenic mice.展开更多
基金Supported by National Natural Science Foundation of China,No.39970676
文摘AIM: To investigate the change of immunological characteristics of HBsAg caused by the mutation at codon 145 of HBsAg using DNA-based immunization.METHODS: Plasmids expressing mutant and wild type envelope antigens were transfected into human hepatocellular carcinoma cells via electrotransformation. The antigenicity of HBsAg was studied with EIA and immunocytochemical staining. Then plasmids were used to immunize 5 C57BL/6 mice. Sera of mice were detected for anti-HBs and anti-preS2with ELISA. RESULTS: The mutant HBsAg could be detected by native antibody in EIA and immunocytochemical study. But the A(450nm) value of the mutant HBsAg in the supernatant was apparently lower than that of the wild-type. Both mutant and native HBsAg expression plasmid could stimulate a strong humoral immune response to HBsAg and preS2 antigen in mice. Protective antibodies against HBsAg elicited by the native HBsAg occurred earlier than that elicited by the mutant HBsAg about one to two weeks. The occurrence of protective antibodies against preS2 antigen was one to two weeks earlier than that of anti-HBs.CONCLUSION: The amino acid substitution causes changes of the antigenicity and immunogenicity of HBsAg, but mutant HBsAg can still induce a protective humoral immune responsein mice.
基金Supported by National Natural Science Foundation of China,No.39970676
文摘AIM:To establish the transgenic mouse line harbouring complete hepatitis B virus (HBV) genome with mutant s gene (adr subtype).METHODS: Transgenic mice were generated by microinjecting HBV genome into fertilized eggs. Integration, expression, replication of HBV gene and histological changes in transgenic mice were estimated by genomic DNA PCR, serum DNA PCR, Southern blot, ELISA, HE staining, immunohistochemistry and transmission electron microscopy. Transgenic mice with HBsAg positive in serum were bred and analyzed.RESULTS: A total of 288 eggs survived from microinjections were transplanted into the oviducts of 13 pseudopregnant mice and 49 pups were produced. Twenty-six mice were identified to have the integrated HBV gene. Serum HBsAg and HBeAg were detected in 2 of 43 mice. HBsAg and HBcAg in cytoplasm or nuclei of hepatocytes were detected in 10 mice. Founders with HBsAg in serum were named lineages G145R-15 and G145R-18. Of the 16 F1 offsprings generated by G145R-15 founder, 12 were positive for HBV genome with PCR, 10 were positive for HBsAg and HBcAg with immunohistochemistry and 7 were positive for HBsAg and HBeAg with ELISA. Only 1 of 8 F1 offsprings generated by G145R-18 founder was survived and it was detected positive for HBV genome, HBsAg, HBcAg and HBeAg. Both of the two lineages had some pathological characteristics of mild chronic hepatitis B in the liver, such as swelling of hepatocytes and focal hepatocellular necrosis and parenchymal lymphomononuclear cell infiltrate.CONCLUSION: Transgenic mice harbouring HBV with mutant s gene can be generated. The HBV genes are integrated in the transgenic mice genome and can be expressed, replicated, packaged and excreted. HBV DNA can be stably transmitted in the transgenic mice.
