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