Stable expression of HBV C gene mutants in immortalized human B-cell lines.
- Author:
Ming-xia ZHANG
1
;
Fu-yuan ZHOU
;
Zhi-hong DIAO
;
Hai-tang HE
;
Jin-lin HOU
;
Kang-xian LUO
Author Information
- Publication Type:Journal Article
- MeSH: B-Lymphocytes; cytology; virology; Base Sequence; Blotting, Western; Cell Line, Transformed; Cell Transformation, Viral; DNA, Viral; genetics; Eukaryotic Cells; metabolism; Gene Expression Regulation, Viral; Genetic Vectors; Genome, Viral; genetics; Hepatitis B Core Antigens; genetics; metabolism; Hepatitis B virus; genetics; metabolism; Humans; Molecular Sequence Data; Point Mutation; Reverse Transcriptase Polymerase Chain Reaction; Transfection
- From: Journal of Southern Medical University 2006;26(6):725-729
- CountryChina
- Language:Chinese
-
Abstract:
OBJECTIVETo provide an cell model of immortalized lymphoblstoid B-cell lines for studying the biological characteristics of full-length hepatitis B virus (HBV) genome carrying the hot-spot mutations V60, G87, and L97.
METHODSV60, G87, and L97 mutation points were introduced into HBV p3.8 II plasmid containing 1.2 copy of HBV genome by means of site-directed mutagenesis. The HBV genome was amplified by PCR from p3.8 II and p3.8 II-V60, G87, L97 plasmid, and the PCR product was inserted into EBO-plpp eukaryotic expression vector. The recombinant vectors and the EBO-plpp vector were transfected into immortalized human lymphoblasts with lipofectamine 2000 and selected with hygromycin. Steady expression of the target genes was determined by RT-PCR, Western blotting and microparticle enzyme immunoassay.
RESULTSDNA sequence analysis indicated that the desired mutation was introduced into wild-type HBV DNA. HBsAg, HBeAg and HBcAg could be detected in EBO-HBV-transfected cell lysate or culture supernatant.
CONCLUSIONTransfectants that stably express HBV mutant antigen may provide a cell model to study the biological characteristics of HBV carrying hot-spot mutation in vitro.
