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g

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ｇｐ１０５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ｇｐ１０５和跨膜蛋白ｇｐ３６全基因序列克隆到杆状病毒转座载体ｐＦａｓｔ Ｂａｃ ＴＨａ和ｐＦａｓｔ Ｂａｃ ＴＨｂ中我角体启动子下游，构建成重组转座载体ｐＦａｓｔ Ｂａｃ ＨＴａ－ｐｇ１０５和ｐＦａｓｔ Ｂａｃ 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ｇｐ１０５和ｇｐ３６。结果 ＳＤＳ－ＰＡＧＥ分析结果表明，ｐｇ１０５基因表达产物为－６６０００ｕ糖蛋白，ｐｇ３６基因则表达－４１０００ｕ糖蛋白，与天然产物一致。Ｗｅｓｔｅｒｎ ｂｌｏｔ结果显示：

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

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