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为了进一步研究乙型肝炎病毒（ＨＢＶ）的ｓ基因变异在免疫逃逸中的作用，我们通过酶切消化质粒载体ｐＥｃｏｂ６（含双拷贝ＨＢＶＤＮＡ），得到约９００ｂｐ的ＨＢＶ－ｓ基因片断。将其插入到噬菌粒载体ｐＢｌｕｅｓｃｒｉｐｔＳＫ＋的Ｓｍａｌ位点上。然后通过体外寡核昔酸介导的人工定点突变获得一系列（共１２种）ｓ基因“免疫逃逸”突变型。再通过ＥＢ病毒真核表达载体ｎＭＥＰ４将ｓ基因突变型片断定向克隆到ｐＭＥＰ４上，从而构建了含ＨＢＶ－ｓ基因突变型的重组质粒ｐＭＥＰ４ＨＢＳＭ。用其转染人肝癌传代细胞系ＨｅｐＧ２，经潮霉素选择，３周后获得抗性细胞克隆。经用ＨＢＶ－ｓ单克隆抗体检测发现除含变异体１４５（即１４５位上甘氨酸为精氨酸替代）外，其余变异体ＨＢｓＡｇ均为阳性。经Ｗｅｓｔｅｒｎｂｌｏｔ杂交证实变异体１４５在分子量约为２４０００处也存有一特异性ＨＢｓＡｇ蛋白带。这一表达细胞系的成功建立，为ＨＢＶ免疫逃逸的诊断和治疗提供了基础。
To further investigate the role of s gene mutation in hepatitis B virus (HBV) in immune escape, we obtained an approximately 900 bp HBV-s gene fragment by digesting the plasmid vector pEcob6 (containing double copies of HBVDNA). This was inserted into the Smal site of the phagemid vector pBluescriptSK +. Then a series of (12 kinds) s gene “immune escape” mutants were obtained by in vitro oligonucleotide-mediated manual site-directed mutagenesis. The mutant gene of s gene was cloned into pMEP4 by eukaryotic expression vector nMEP4 of EB virus to construct recombinant plasmid pMEP4HBSM with HBV-s gene mutation. The transfected human hepatoma cell line HepG2 was selected by hygromycin, and the resistant cell clone was obtained after 3 weeks. The detection of HBV-s monoclonal antibody detected in addition to variant 145 (ie, glycine at position 145 for arginine substitution), the remaining variant HBsAg were positive. Western blot analysis confirmed that variant 145 also contained a specific HBsAg protein band at a molecular weight of about 24,000. The successful establishment of this expression cell line provides the basis for the diagnosis and treatment of HBV immune escape.