A construct competent to support the replication of hepatitis B virus of genotype B.
- Author:
Lei LI
1
;
Zhen-Hua ZHANG
;
Hong SHEN
Author Information
- Publication Type:Journal Article
- MeSH: Animals; Cell Line, Tumor; Cloning, Molecular; Disease Models, Animal; Genetic Vectors; Genotype; Hepatitis B; metabolism; virology; Hepatitis B Antigens; genetics; metabolism; Hepatitis B virus; genetics; metabolism; Liver; metabolism; virology; Mice; Mice, Inbred BALB C; Plasmids; Polymerase Chain Reaction; Transfection; Virus Replication
- From: Chinese Journal of Hepatology 2009;17(1):16-20
- CountryChina
- Language:Chinese
-
Abstract:
OBJECTIVETo construct a vector that is competent to support the replication of hepatitis B virus (HBV) of genotype B.
METHODSThe HBV genome of genotype B was amplified by PCR and ligated into pBlueskript II KS(+) vector, the resulting plasmid was verified by enzyme digestion and DNA sequencing. After transfection of this plasmid into Huh7 cells, the HBsAg and HBeAg antigens in culture medium were quantified by ELISA, the transcripts and replication intermediates of HBV were detected by northern blot and southern blot respectively. On the other hand, the plasmid was hydrodynamically injected into BALB/cJ mice via tail vein. Then the HBV DNA in serum was quantified by real-time PCR, and HBcAg expression in liver tissue was detected by immunohistochemistry.
RESULTSAfter transfection of the plasmid into Huh7 cells, the HBsAg and HBeAg antigens were detected in the culture medium, the transcripts and replication intermediates of HBV were detected in the cells. High titer of HBV DNA was detected in the serum of hydrodynamic-injected mice. Immunostaining indicated that HBcAg was expressed in hepatocytes of injected mice.
CONCLUSIONThis construct is competent to support the replication of hepatitis B virus of genotype B.
