[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. HepG2 cell lines were employed to transfect with the construct. Hygromycin selection was done at a concentration of 150 micrograms/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 hen 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.


CONCLUSIONS
After the packaging signal was deleted and transfected into HepG2 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.
Hu
Da-rong
DR
Institute of Hepatology, Beijing Army General Hospital of PLA, Beijing 100700, China.
Sun
Dian-xing
DX
Xiong
Jin-hua
JH
Wu
Guang-hui
GH
Hu
Xue-ling
XL
Li
Juan
J
Fan
Gong-ren
GR
Han
Ju-qiang
JQ
D002460:Cell Line; D024882:Drug Resistance, Viral; D005822:Genetic Vectors; D016679:Genome, Viral; D006515:Hepatitis B virus / Q000187:drug effects / Q000235:genetics*; D006801:Humans; D006921:Hygromycin B / Q000494:pharmacology*; D009154:Mutation; D010957:Plasmids; D012190:Retroviridae / Q000235:genetics; D014162:Transfection; D019065:Virus Assembly
D006921:Hygromycin B