乙型肝炎病毒表面抗原免疫逃逸突变基因在人肝癌细胞的表达

为了进一步研究乙型肝炎病毒的Ｓ基因变异在免疫逃逸中的作用，我们通过酶切消化质粒载体ＰＥｃｏｂ６，得到约９００ｂｐ的ＨＢＶ－ｓ基因片断。将其插入到噬菌粒载体ｐＢｌｕｅｓｃｒｉｐｔＳＫ＋的ＳｍａＩ位点上。然后通过体外寡核苷酸介导的人工定点突变获得得一系列ｓ基因“免疫逃逸”突变型。

Expression of 12 antibody escape mutants of hepatitis Bvirus surface antigen gene in mammalian cell by usingan epstein-barr based vector

900bp DNA fragment of hepatitis B virus suriaceantigen gene by digesting BamHI and HpaI sites on theopen reading frame of HBV DNA on plasmid pEcob6containing double copes of HBV DNA was cloned intoSmaI site on the phagmide vector pBluescribt SK .From this recombinant vector, 12 hepatitis B virus sur-face antigen gene mutants were obtained by oligonu-cleotide-mediated site directed mutagenesis. The expression vector-pMEP4HBS mutants were constructed bysub-cloning all of these mutant fragments of hepatitis Bvirus surface antigen gene on pBluescritSK HBSM intoBsmHI and Kpnl sites on an EpsteinBarr virus based oneukaryotic expression vector-pMEp4. The vectorpMEp4HBSMs were transfected to human hepatocellular carcinoma cell lineHepGZ by calcium phosphate me-diated transfection, and resistant cell clones were obtained 3 weeks after selecting by hygromycine B. Theresults of detection of HBsAg excreted by resistant cellclones with monoclonal antibody to HBsAg showed thatall antiberly escape mutants of HBsAg except mutant145R, a substitution of arginine for glycine at aminoacid 145 position in HBsAg , were positive.