用逆转录病毒载体表达乙型肝炎病毒表面抗原  

EXPRESSION OF HEPATITIS B VIRUS SURFACE ANTIGEN IN MAMMALIAN CELLS USING A RETROVIRAL VECTOR

作　　者：杨毅力[1] 赵小侠[1] 龚春梅[2] 侯云德[1] 

机构地区：[1]中国预防医学科学院病毒学研究所病毒基因工程国家重点实验室 [2]中国医学科学院医学生物学研究所

出　　处：《病毒学报》1992年第3期223-227,共5页Chinese Journal of Virology

摘　　要：将含有乙型肝炎病毒(HBV)Pre-S及S基因开放读码框架的DNA片段克隆到逆转录病毒表达载体Pzip-neo SV(X)的5′-端长末端重复序列(LTR)下游BamH Ⅰ位点,构建成重组质粒Pzip-HBS。用磷酸钙沉淀技术将重组质粒转染到辅助包装细胞ψ-2中,经G418筛选,2周后获得的抗性细胞克隆能有效地表达HBsAg。5×10~6细胞培养48小时之后,收集上清,用放射免疫方法测得HBsAg产量达6283cpm,P/N值为32.6。Western blot证实,在分子量约23kD处有一条与乙型肝炎病毒表面抗体相结合的特异性蛋白带。Southern杂交显示,在细胞染色体上整合有HBsAg基因。电镜观察可见上清液中的HBsAg和重组的逆转录病毒颗粒。A expression HBsAg plasmid Pzip-HBS was constructed by insertion of a DNA fragment containing complete Pre-S and S gene open reading frams into BamH I site downstream 5 -LTR of a retroviral vector Pzip neo SV ( X ) . The plasmid Pzip-HBS was transf ected into ψ-2 packaging cells by calcium phosphate precipitation technique and cells resistant aga inst G418 can efficently expressing HBsAg after selecting with G418 for two weeks.The amount of HBsAg secreted by 5×10 8 cells was 6283 cpm/48h(P/N 32.6) assayed by radioimmunological method. Western blot showed that the synthesized HBsAg is 23kD protein. Southern blot showed that HBs Ag gene was integrated into transfected cells. HBsAg and retrovirai particles can be observed by electromicroscope in the culture medium.

关 键 词：逆转录病毒 载体 乙型肝炎病毒 抗原 

分 类 号：R394.8[医药卫生—医学遗传学]