论文部分内容阅读
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。
In order to further study the antigenic change of envelope antigen induced by HBV S gene mutation, an 837bp HBV-S gene fragment was obtained from plasmid pEcob6 containing double copies of HBVDNA, and inserted into vector pBluescript KS ~ + SmaI sites were obtained. Three S gene variants of amino acids 145, 126 and 145 + 126 were obtained by in vitro oligonucleotide-directed mutagenesis. Then these S gene mutants were cloned into the eukaryotic expression vector pMEp4 to construct the expression vector PMEp4HBVSM containing HBV-S gene and its mutation. The human hepatoma cell line HepG2 was transfected with it to obtain a resistant cell line stably secreting HBsAg and its variants. Preliminary in vitro studies have shown that the 145 amino acid variant of HBsAg can affect the structure of the “a” epitope of HBsAg.