摘要
为了进一步研究乙型肝炎病毒（ＨＢＶ）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（含双拷贝ＨＢＶＤＮＡ），得到约９００ｂｐ的ＨＢＶ－ｓ基因片断。将其插入到噬菌粒载体ｐＢｌｕｅｓｃｒｉｐｔＳＫ＋的Ｓｍａｌ位点上。然后通过体外寡核昔酸介导的人工定点突变获得一系列（共１２种）ｓ基因“免疫逃逸”突变型。再通过ＥＢ病毒真核表达载体ｎＭＥＰ４将ｓ基因突变型片断定向克隆到ｐＭＥＰ４上，从而构建了含ＨＢＶ－ｓ基因突变型的重组质粒ｐＭＥＰ４ＨＢＳＭ。用其转染人肝癌传代细胞系ＨｅｐＧ２，经潮霉素选择，３周后获得抗性细胞克隆。经用ＨＢＶ－ｓ单克隆抗体检测发现除含变异体１４５（即１４５位上甘氨酸为精氨酸替代）外，其余变异体ＨＢｓＡｇ均为阳性。经Ｗｅｓｔｅｒｎｂｌｏｔ杂交证实变异体１４５在分子量约为２４０００处也存有一特异性ＨＢｓＡｇ蛋白带。这一表达细胞系的成功建立，为ＨＢＶ免疫逃逸的诊断和治疗提供了基础。
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.
出处
《中华医学杂志》
CAS
CSCD
北大核心
1995年第7期396-398,共3页
National Medical Journal of China
