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World J Gastroenterol. Jun 21, 2008; 14(23): 3733-3738
Published online Jun 21, 2008. doi: 10.3748/wjg.14.3733
Construction and expression of eukaryotic plasmids containing lamivudine-resistant or wild-type strains of Hepatitis B Virus genotype C
Wei-Zhen Xu, Yong Fang, Di Li, Yan Wang, Qing-Long Shang, Gui-Qiu Li, Xu Teng, Hong-Xi Gu
Wei-Zhen Xu, Yong Fang, Di Li, Yan Wang, Qing-Long Shang, Gui-Qiu Li, Xu Teng, Hong-Xi Gu, Department of Microbiology, Harbin Medical University, Harbin 150081, Heilongjiang Province, China
Author contributions: Xu WZ and Li D contributed equally to this study; Xu WZ and Li D designed the research, preformed the study and wrote the paper; Fang Y, Wang Y and Shang QL performed the study; Li GQ and Teng X analyzed the data; Gu HX was an instructor.
Correspondence to: Hong-Xi Gu, Department of Micro-biology, Harbin Medical University, No. 157, Baojian Road, Nangang District, Harbin 150081, Heilongjiang Province, China. hxgu2432@163.com
Telephone: +86-451-86685122
Fax: +86-451-86685122
Received: March 3, 2008
Revised: April 23, 2008
Accepted: April 30, 2008
Published online: June 21, 2008
Abstract

AIM: 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.

CONCLUSION: 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.

Keywords: Hepatitis B virus; Lamivudine-resistant mutant; Wild-type strain