[Stable cell line for secretion of replication-defective hepatitis B virus vector expressing blasticidin resistant gene].

OBJECTIVE

To construct a stable cell line with permanent secretion of recombinant hepatitis B virus (HBV) vector, which express blasticidin resistant gene.

METHODS

Replication-defective HBV vector, pCH-BsdR, which express blasticidin resistance gene was constructed by deleting the HBV genes and inserting the blasticidin resistance gene into the S region. The G418-resistant, the packaging signal deleted HBV helper plasmid, pcDNA3.1-CH3142, and the HBV vector pCH-BsdR were cotransfected into HepG2 cells. Cell clones were selected by the adding of both blasticidin and G418, then serial detection were done.

RESULTS

After 36 cell clones were picked and expanded. Three cell clones were defined as the best. Quantity of their HBV DNA were 4.1 x 10(6), 3.6 x 10(6) and 1.2 x 10(6) copies/ml, respectively. Enveloped recombinant, but not wild type HBV were confirmed in the culture medium.

CONCLUSIONS

The stable cell lines can realize large preparation of recombinant HBV virions. This will contribute to the use of HBV vector for gene therapy and HBV susceptible cell lines screening.