B Gu1, H Ren, D Zhang. 1. Institute for Viral Hepatitis, Chongqing Medical University, Chongqing 400010.
Abstract
OBJECTIVE: To study the immunological characteristics of two inserting variants of HBsAg as further to elucidate the role of inserting mutants in the development of HBsAg-negative chronic hepatitis B. METHODS: The inserting mutants of HBsAg were constructed by PCR-mediated mutagenesis in vitro. Epstein-Barr virus vector pCEP4 and retrovirus vector PXT1 carrying the mutant genes of HBsAg were transfected into mammalian cells. The affinity of polyclonal and monoclonal antibodies against HBsAg for mutant antigens were studied by ELISA and western blot. RESULTS: Two insertions were introduced into s gene, and a six nucleotide insertion introduced two aminoacids (Arg and Ala) between codons 122 and 123 of the s polypeptide, whereas a nine nucleotide insertion introduced three aminoacids(Arg, Gly and Ala) between codons 123 and 124. These s genes were subcloned into two eukaryotic expression vectors (pCEP4 and PXT1), and the recombinant eukaryotic expression plasmids had been constructed, which could express s polypeptide in mammalian cells. Cell lines expressing wild-type or mutant-type HBsAg stably were obtained after transfection of these vectors into mammalian cells. Two two inserting variants failed to react with polyclonal and monoclonal antibodies against HBsAg by ELISA and Western blot. CONCLUSION: The insertions may result in a conformational change of the s protein, and affect its antigenicities, suggesting that the inserting mutations may be critical for immunoescape of HBV and in part responsible for chronicity of HBsAg-negative patients.
OBJECTIVE: To study the immunological characteristics of two inserting variants of HBsAg as further to elucidate the role of inserting mutants in the development of HBsAg-negative chronic hepatitis B. METHODS: The inserting mutants of HBsAg were constructed by PCR-mediated mutagenesis in vitro. Epstein-Barr virus vector pCEP4 and retrovirus vector PXT1 carrying the mutant genes of HBsAg were transfected into mammalian cells. The affinity of polyclonal and monoclonal antibodies against HBsAg for mutant antigens were studied by ELISA and western blot. RESULTS: Two insertions were introduced into s gene, and a six nucleotide insertion introduced two aminoacids (Arg and Ala) between codons 122 and 123 of the s polypeptide, whereas a nine nucleotide insertion introduced three aminoacids(Arg, Gly and Ala) between codons 123 and 124. These s genes were subcloned into two eukaryotic expression vectors (pCEP4 and PXT1), and the recombinant eukaryotic expression plasmids had been constructed, which could express s polypeptide in mammalian cells. Cell lines expressing wild-type or mutant-type HBsAg stably were obtained after transfection of these vectors into mammalian cells. Two two inserting variants failed to react with polyclonal and monoclonal antibodies against HBsAg by ELISA and Western blot. CONCLUSION: The insertions may result in a conformational change of the s protein, and affect its antigenicities, suggesting that the inserting mutations may be critical for immunoescape of HBV and in part responsible for chronicity of HBsAg-negative patients.