用细菌/杆状病毒系统在昆虫细胞中表达HIV-2外膜糖蛋白gp105和跨膜糖蛋白gp36

Expression of external glycoprotein gp105 and transmembrane glycoprotein gp36 of human immunodeficiency virus type 2 in insect cell by Bac to Bac system

目的用细菌 /杆状病毒 (Bac to Bac)表达系统在昆虫细胞 Sf9中表达 HIV- 2外膜糖蛋白 gp10 5和跨膜糖蛋白 gp36 ,为研制艾滋病疫苗和诊断试剂奠定基础。方法分别将 HIV- 2外膜蛋白 gp10 5和跨膜蛋白 gp36全基因序列克隆到杆状病毒转座载体 p Fast Bac HTa和 p Fast Bac HTb中多角体启动子下游 ,构建成重组转座载体 p Fast Bac HTa- gp10 5和 p FastBac HTb- gp36 ,利用细菌 /杆状病毒 (Bac to Bac)表达系统筛选重组杆状病毒 ,并在昆虫细胞 Sf9中表达 HIV- 2的 gp10 5和gp36。结果 SDS- PAGE分析结果表明 ,gp10 5基因表达产物为一 6 6 0 0 0 u糖蛋白 ,gp36基因则表达一 410 0 0 u糖蛋白 ,与天然产物一致。 Western blot结果显示 :两者均具有抗原特异性。结论 gp10 5和 gp36在昆虫细胞中得到了高效表达 ,并均被糖基化 ;gp10 5接近天然产物 。

ObjectiveTo develop effective vaccine and diagnostic agents of AIDS, the external glycoprotein gp105 and transmembrane glycoprotein gp36 of human immunodeficiency virus type 2 were expressed in insect cells Spodoptera frugiperda (Sf9) using baculovirus expression system : Bac to Bac system MethodsThe recombinant transfer vector pFast Bac HTa gp105 and pFast Bac HTb gp36 were constructed by cloning HIV 2 gp105 gene and gp36 gene into the end of polyhedrin promoter of baculovirus transfer vector pFast Bac HTa and pFast Bac HTb respectively HIV 2 external glycoprotein gp105 and transmembrane glycoprotein gp36 were expressed in insect cells Spodoptera frugiperda (Sf9) by transposing these two foreign genes to shuttle vector bacmid respectively using Bac to Bac system ResultsThe resulting products determined by SDS PAGE showed that recombinant baculovirusescontaining gp105 gene expressed a 66 kDa glycoprotein, whereas those contai ning gp36 gene generated a 41 000u glycoprotein, the apparent molecular weight of which is equivalent to that of natural gp36 Western blot indicated that both gp105 and gp36 showed antigenic specificity Conclusionsgp105 and gp36 has been expressed in insect cells Spodoptera frugiperda (Sf9) using baculovirus expression system