Stable expression of site-directed mutated HBcAg in human immortalized Blymphoblastoid cell line

AIM: To get the stimulator and target cells of CTL response in chronic hepatitis B patients so as to provide an experimental cellular model for studying the biological characteristics of full-length HBV genome carrying the HBcAg hot-spots mutation V60, G87, L97. METHODS: V60, G87, L97 point mutation was introduced from HBV p3.8Ⅱ plasmid which contained 1.2 copy HBV genome using site-directed mutagenesis. The HBV genome was amplified by PCR from p3.8Ⅱ and p3.8Ⅱ- V60, G87, L97 plasmid, then the PCR product was inserted into EBO-plpp eukaryotic expression vector. The recombinant vectors and the EBO-plpp vector were transfected into lymphoblastoid cells with lipofectamine 2000 and selected with hygromycin. The steady expression of target genes was determined by RT-PCR, Western Blot and microparticle enzyme immunoassay. RESULTS: The DNA sequence analysis indicated that the desired mutation was introduced from wild HBV DNA. The HBsAg, HBeAg and HBcAg can be detected in EBO-HBV transfected cell lysate or culture supernatant. CONCLUSION: The site-directed mutated HBcAg plasmid can be expressed stably in human immortalized B-lymphoblastoid cell line.