Abstract: Objective To provide an cell model of immortalized lymphoblstoid B-cell lines for studying the biological characteristics of full-length hepatitis B virus (HBV) genome carrying the hot-spot mutations V60, G87, and L97. Methods V60, G87, and L97 mutation points were introduced into HBV p3.8Ⅱ plasmid containing 1.2 copy of HBV genome by means of site-directed mutagenesis. The HBV genome was amplified by PCR from p3.8Ⅱ and p3.8Ⅱ- V60, G87, L97 plasmid, and the PCR product was inserted into EBO-plpp eukaryotic expression vector. The recombinant vectors and the EBO-plpp vector were transfected into immortalized human lymphoblasts with lipofectamine 2000 and selected with hygromycin. Steady expression of the target genes was determined by RT-PCR, Western blotting and microparticle enzyme immunoassay. Results DNA sequence analysis indicated that the desired mutation was introduced into wild-type HBV DNA. HBsAg, HBeAg and HBcAg could be detected in EBO-HBV- transfected cell lysate or culture supernatant. Conclusion Transfectants that stably express HBV mutant antigen may provide a cell model to study the biological characteristics of HBV carrying hot-spot mutation in vitro.