Development of hygromycin-resistant packaging cell line for hepatitis B virus-derived vectors

Objective To cooperate with the study of HBV vector,hygromycin-resistant packaging cell line was developed that allows encapsidation of plasmids into HBV particles. Methods Free of packaging signal,HBV genome was inserted into plasmid pMEP4,which expresses the HBV structural proteins including core,pol and preS/S proteins. HepG 2 cell lines were employed to transfect with the construct. Hygromycin selection was done at a concentration of 150 μg/ml in the culture medium. The hygromycin-resistant clones with the best expressions of HBsAg and HBcAg were theoretically considered as packaging cell line and propagated under the same conditions. It was infected with recombinant retrovirus vector and then selected with G418 and hygromycin in the culture medium. The existence of recombinant HBV virion in the culture medium was examined by PCR. Results Hygromycin-resistant HBV packaging cell line was generated,which harbored an HBV mutant whose packaging signal had been deleted. Expressions of HBsAg and HBcAg were detectable. Infected with recombinant retrovirus pRV-CP,the hygromycin-resistant packaging cell line was found to secrete mutant HBV particles and no wild-type HBV was detectable in the culture medium. Conclusion After the packaging signal was deleted and transfected into HepG 2 cell lines,the partial HBV genome lost its ability to form wild-type HBV,but conserves cis-action providing structural proteins for the packaging of the replication-defective HBV.