重组HBsAg免疫逃逸突变体的表达及其抗原性分析

为了深入研究乙型肝炎病毒S基因变异所致的包膜抗原抗原性的改变，从含ＨＢＶＤＮＡ双拷贝的质粒载体ｐＥｃｏｂ６，获得一个８３７ｂｐ的ＨＢＶ－Ｓ基因片段，将其插入至载体ｐＢｌｕｅｓｃｒｉｐｔ ＫＳ~＋的ＳｍａⅠ位点，通过体外寡核苷酸介导的人工定点突变分别获得第１４５位、１２６位和第１４５位＋１２６位氨基酸三种Ｓ基因变异型。然后将这些Ｓ基因变异片段克隆到真核表达载体ｐＭＥｐ４上，从而构建了含ＨＢＶ－Ｓ基因及其突变型的表达载体ＰＭＥｐ４ＨＢＶＳＭ。用其转染人肝癌细胞系ＨｅｐＧ２，获得稳定分泌ＨＢｓＡｇ及其变异体的抗性细胞系。经体外初步研究表明，ＨＢｓＡｇ １４５位氨基酸变异体可影响ＨＢｓＡｇ的“ａ”抗原决定簇的结构。

Expression of recombinant antibody-escape-mutants of HBsAg and its antigenic analysis

Amino acid 145 , 126 and 145 1 26 mutants of HBaAg were obtained by oligonucleotide-mediated site directed mutagenesis from a vector containing double copes of HBV DNA. The mutant genes of HBsAg were inserted into a eukaryotic expression vector pMEp4. The expression vector containintg the mutant genes were transfected into a human hepatocellular carcinoma cells (HepG2 ) ,and recombinant proteins were expressed . All three mutants had reactivity with polyclonal antibody against HBsAg in Western blot , but failed to react with monoclonal antibody against "a" epitopes of HBsAg except aa 126 mutant in ELISA. That indicates the mutation in aa 145 of HBsAg is critical for escaping protective antibody to HBV in nature.