Objective     To construct eukaryotic expression plasmid pneo-CH9/3091 and establish a HepG2 cell line with stable expression of the plasmid.      Methods     Neomycin resistance gene (neo) obtained by PCR was inserted into plasmid pCH9/3091 containing 1.1-unit-length HBV genome and CMV promoter to construct recombinant plasmid pneo-CH9/3091. The recombinant plasmid

after being verified by enzyme digestion and sequencing

was transferred into human hepatocarcinoma cell line HepG2 by liposome-mediated transfection. The transfected HepG2 cells were screened with G418 to obtain positive clones. The expression levels of HBV surface antigen (HBsAg) and HBV e antigen (HBeAg) in the culture supernatant of the monoclonal cells were measured by ELISA

and HBV particles were detected by PCR. The expression of HBV core protein (HBcAg) in the cells was analyzed by Western blotting

and HBV DNA replication was checked by Southern blotting.      Results     Out of six HepG2 cell lines that had positive expression of HBsAg and HBeAg in the supernatant

three showed PCR-amplified HBV core gene. Western blot results further showed that HBV core protein was expressed only in cell line HepG2-H7. HBV DNA replicative intermediates were detected by Southern blotting in cell line HepG2-H7

indicating that HBV genome had been stably integrated into cell line HepG2-H7.      Conclusion     Cell line HepG2-H7 with stably integrated HBV genome is established. The cell line can produce HBV particles continually.