[Construction of recombinant eukaryotic expression plasmid containing 1.3-fold-overlength genome of HBV and its expression in HepG2 cells].
OBJECTIVE
To transfer 1.3-fold-overlength genome of HBV expression plasmid into HepG2 cells, and observe the dynamic changes of viral replication as well as expression in the transfected cells.


METHODS
4.1 kb fragment of HBV genome, derived from pGEM-HBV, was cloned into Hind III site of the eukaryotic expression vector pCDNA3.1 to construct the recombinant plasmid pHBV. Then HepG2 hepatoma, cells were transfected with pHBV, using Lipofectamine2000 transfection reagent. After 24, 48, 72 hours, the levels of HBsAg and HBeAg in the supernatant of HepG2 cells were determined with Abbott MEIA Kit. Intracellular viral DNA and RNA were analyzed by Southern and northern blot hybridization. In addition, viral-specific proteins (HBsAg and HBcAg) were assayed by immunofluorescence staining.


RESULTS
The expression vector pCDNA3.1 was constructed successfully. The levels of HBsAg were 5.36+-0.25, 13.42+-1.24, 7.52+-0.43, and the values of HBeAg were 9.16+-0.32, 22.75+-1.49, 15.96+-1.03 after 24, 48, 72 hours, respectively. All expected HBV replicative intermediates and specific transcripts were verified by Southern and northern blot analysis. The HBsAg-positive cells peaked after 24 hours, and then dropped slowly. HBsAg positive staining scattered in the cytoplasm, whereas HBcAg lied maily in the cytoplasm apart from nuclears.


CONCLUSIONS
This recombinant plasmid, which initiates viral replication efficiently in infected cells, is expected as a novel tool for investigating HBV replication in vitro.
Tang
Ni
N
Institute for Viral Hepatitis, Chongqing University of Medical Sciences, Chongqing 400010, China.
Huang
Ai-long
AL
Zhang
Bing-qiang
BQ
Yan
Ge
G
Xiang
Ming-que
MQ
Pu
Dan
D
Guo
Hui
H
D006528:Carcinoma, Hepatocellular / Q000473:pathology / Q000821:virology*; D004279:DNA, Viral / Q000235:genetics*; D005057:Eukaryotic Cells / Q000821:virology; D015967:Gene Expression Regulation, Viral; D016679:Genome, Viral; D006515:Hepatitis B virus / Q000235:genetics* / Q000502:physiology; D006801:Humans; D008113:Liver Neoplasms / Q000473:pathology / Q000821:virology*; D010957:Plasmids / Q000235:genetics; D014162:Transfection; D014407:Tumor Cells, Cultured; D014779:Virus Replication
D004279:DNA, Viral