Stable expression of siteª²directed mutated HBcAg in human immortalized Bª²lymphoblastoid cell line

    ZHANG Mingª²Xia, DIAO Zhiª²Hong, HOU Jinª²Lin, LUO Kangª²Xian

    Department of Infectious Diseases, Nanfang Hospital, Southern Medical University, Guangzhou 510515, China

    ¡¾Abstract¡¿ 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 fullª²length HBV genome carrying the HBcAg hotª²spots mutation V60, G87, L97. METHODS: V60, G87, L97 point mutation was introduced from HBV p3.8¢ò plasmid which contained 1.2 copy HBV genome using siteª²directed mutagenesis. The HBV genome was amplified by PCR from p3.8¢ò and p3.8¢òª² V60, G87, L97 plasmid, then the PCR product was inserted into EBOª²plpp eukaryotic expression vector. The recombinant vectors and the EBOª²plpp vector were transfected into lymphoblastoid cells with lipofectamine 2000 and selected with hygromycin. The steady expression of target genes was determined by RTª²PCR, 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 EBOª²HBV transfected cell lysate or culture supernatant. CONCLUSION: The siteª²directed mutated HBcAg plasmid can be expressed stably in human immortalized Bª²lymphoblastoid cell line. ......