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利用乙型肝炎病毒ＤＮＡ开放框架上的ＢａｍＨＩ和ＨｐａＩ位点，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ＳｍａＩ位点上。然后通过体外寡核苷酸介导的人工定点突变获得一系列（共１２种）Ｓ基因“免疫逃避”突变型。再通过ＥＢ病毒真核表达载体ｐＭＥＰ４上的ＢａｍＨＩ和Ｋｐｎｌ位点将噬菌粒ｐＢｌｕｅｓｃｒｉｐｓｋ＋上的Ｓ基因突变型片断定向克隆到ＰＭＥＰ４上，从而构建了含乙肝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，三周后获得抗性细胞克隆。经用抗ＨＢｓ单克隆抗体（含针对ＨＢｓＡｇ“ａ”抗原决定簇）检测除含变异体１４５（即１４５位上甘氨酸为精氨酸替代）外其余抗变异体ＨＢｓＡｇ均为阳性。经Ｗｅｓｔｅｒｎｂｌｏｔ证实变异体１４５，在分子量约为２３ＫＤ处有一特异ＨＢｓＡｇ蛋白带。
Using the BamHI and HpaI sites on the HBV DNA open framework, the plasmid vector PEcob6 (containing double copies of HBVDNA) was digested and digested to obtain an about 900 bp HBV-S gene fragment. This was inserted into the SmaI site of the PBluescriptsk + phagemid vector. Then a series of (12 total) S gene “immune escape” mutants were obtained by in vitro oligonucleotide-mediated manual site-directed mutagenesis. The S gene mutant fragment of the phagemid pBluescripsk + was cloned into PMEP4 through the BamHI and Kpnl sites on the EBV eukaryotic expression vector pMEP4 to construct a recombinant plasmid pMEP4HBSM containing the S gene of hepatitis B gene. The transfected human hepatoma cell line HepG2 was selected by hygromycin, and the resistant cell clone was obtained after three weeks. All anti-variant HBsAg positive were detected by anti-HBs monoclonal antibody (including against HBsAg “a” epitope) except for variant 145 (ie glycine at position 145 was arginine replacement). Variant 145 was confirmed by Western blot, with a specific HBsAg protein band at a molecular weight of about 23KD.