[Expression of 12 antibody escape mutants of hepatitis B virus surface antigen gene in mammalian cell by using an Epstein-Barr based vector].

ABSTRACT

A 900bp DNA fragment of hepatitis B virus surface antigen gene by digesting BamHI and HpaI sites on the open reading frame of HBV DNA on plasmid pEcob6 containing double copes of HBV DNA was cloned into SmaI site on the phagmide vector pBluescribt SK +. From this recombinant vector, 12 hepatitis B virus surface antigen gene mutants were obtained by oligonucleotide-mediated site directed mutagenesis. The expression vector-pMEP4HBS mutants are constructed by sub-cloning all of these mutant fragments of hepatitis B virus surface antigen gene on pBluescritSK+HBSM into BsmHI and KpnI sites on an Epstein-Barr virus based on eukaryotic expression vector-pMEp4. The vector pMEp4HBSMs were transfected to human hepatocellular carcinoma cell line-HepG2 by calcium phosphate mediated transfection, and resistant cell clones were obtained 3 weeks after selecting by hygromycine B. The results of detection of HBsAg excreted by resistant cell clones with monoclonal antibody to HBsAg showed that all antibody escape mutants of HBsAg except mutant 145R, a substitution of arginine for glycine at amino acid 145 position in HBsAg, were positive.