Construction and expression of eukaryotic plasmids containing lamivudine-resistant or wild-type strains of Hepatitis B Virus genotype C.
OBJECTIVE
To construct eukaryotic expression plasmids of full-length Hepatitis B Virus (HBV) genotype C genome, which contain lamivudine-resistant mutants (YIDD, YVDD) or wild-type strain (YMDD), and to observe the expression of HBV DNA and antigens [hepatitis B surface antigen (HBsAg) and hepatitis B e antigen (HBeAg)] of the recombinant plasmids in HepG2 cells.


METHODS
Three HBV full-length genomes were amplified from the plasmids pMD18T-HBV/YIDD, pMD18T-HBV/YVDD and pMD18T-HBV/YMDD, using PCR. Three recombinant plasmids were generated by inserting each of the PCR products into the eukaryotic expression vector pcDNA3.1 (+), between the EcoRI and HindIII sites. After being characterized by restriction endonuclease digestion, and DNA sequence analysis, the recombinant plasmids were transfected into HepG2 cells. At 48 and 72 h post-transfection, the levels of intracellular viral DNA replication were detected by real-time PCR, and the expression of HBsAg and HBeAg in the cell culture supernatant was determined by ELISA.


RESULTS
Restriction endonuclease digestion and DNA sequence analysis confirmed that the three recombinant plasmids were correctly constructed. After transfecting the plasmids into HepG2 cells, high levels of intracellular viral DNA replication were observed, and HBsAg and HBeAg were secreted into the cell culture supernatant.


CONCLUSIONS
Eukaryotic expression plasmids pcDNA3.1 (+)-HBV/YIDD, pcDNA3.1 (+)-HBV/YVDD or pcDNA3.1 (+)-HBV/YMDD, which contained HBV genotype C full-length genome, were successfully constructed. After transfection into HepG2 cells, the recombinant plasmids efficiently expressed HBV DNA, HBsAg and HBeAg. Our results provide an experimental basis for the further study of HBV lamivudine-resistant mutants.
Xu
Wei-Zhen
WZ
Department of Micro-biology, Harbin Medical University, Harbin 150081, Heilongjiang Province, China.
Fang
Yong
Y
Li
Di
D
Wang
Yan
Y
Shang
Qing-Long
QL
Li
Gui-Qiu
GQ
Teng
Xu
X
Gu
Hong-Xi
HX
D000998:Antiviral Agents / Q000494:pharmacology*; D045744:Cell Line, Tumor; D004279:DNA, Viral / Q000096:biosynthesis; D024882:Drug Resistance, Viral / Q000235:genetics*; D015967:Gene Expression Regulation, Viral; D005822:Genetic Vectors; D016679:Genome, Viral; D005838:Genotype; D006514:Hepatitis B Surface Antigens / Q000096:biosynthesis / Q000235:genetics; D006513:Hepatitis B e Antigens / Q000096:biosynthesis / Q000235:genetics; D006515:Hepatitis B virus / Q000187:drug effects / Q000235:genetics* / Q000254:growth & development / Q000276:immunology; D006801:Humans; D019259:Lamivudine / Q000494:pharmacology*; D009154:Mutation; D010957:Plasmids; D011994:Recombinant Proteins / Q000096:biosynthesis; D017422:Sequence Analysis, DNA; D013997:Time Factors; D014162:Transfection; D014779:Virus Replication
D000998:Antiviral Agents; D004279:DNA, Viral; D006514:Hepatitis B Surface Antigens; D006513:Hepatitis B e Antigens; D011994:Recombinant Proteins; D019259:Lamivudine