[Stable expression of HBV C gene mutants in immortalized human B-cell lines].
OBJECTIVE
To 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.


METHODS
V60, 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.


RESULTS
DNA 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.


CONCLUSIONS
Transfectants that stably express HBV mutant antigen may provide a cell model to study the biological characteristics of HBV carrying hot-spot mutation in vitro.
Zhang
Ming-xia
MX
Department of Infectious Diseases, Nanfang Hospital, Southern Medical University, Guangzhou 510515, China. zhangmx@fimmu.com
Zhou
Fu-yuan
FY
Diao
Zhi-hong
ZH
He
Hai-tang
HT
Hou
Jin-lin
JL
Luo
Kang-xian
KX
D001402:B-Lymphocytes / Q000166:cytology / Q000821:virology*; D001483:Base Sequence; D015153:Blotting, Western; D002461:Cell Line, Transformed; D002472:Cell Transformation, Viral; D004279:DNA, Viral / Q000235:genetics; D005057:Eukaryotic Cells / Q000378:metabolism; D015967:Gene Expression Regulation, Viral; D005822:Genetic Vectors; D016679:Genome, Viral / Q000235:genetics; D006512:Hepatitis B Core Antigens / Q000235:genetics* / Q000378:metabolism; D006515:Hepatitis B virus / Q000235:genetics* / Q000378:metabolism; D006801:Humans; D008969:Molecular Sequence Data; D017354:Point Mutation; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014162:Transfection
D004279:DNA, Viral; D006512:Hepatitis B Core Antigens