论文部分内容阅读
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 ,gp36与天然产物一致
Objective To express HIV-2 envelope glycoprotein gp10 5 and transmembrane glycoprotein gp36 in insect cell Sf9 by Bac / Bac expression system and lay the foundation for the development of AIDS vaccine and diagnostic reagent. Methods The full-length sequence of HIV-2 outer membrane protein gp10 5 and transmembrane protein gp36 were cloned into the downstream of the polyhedrin promoter in the baculovirus transposon p Fast Bac HTa and p Fast Bac HT HT respectively to construct the recombinant transposase p Fast Bac HTa-gpl05 and p FastBac HTb-gp36, the recombinant baculovirus was screened using the Bac to Bac expression system and HIV-2 gp105 and gp36 were expressed in insect cell Sf9. Results The results of SDS-PAGE analysis showed that the expression product of gp10 5 gene was a 6600 u glycoprotein while the gp36 gene expressed a 41000 u glycoprotein, which was consistent with the natural product. Western blot results show that both are antigen specific. Conclusion gp10 5 and gp36 were highly expressed in insect cells and both were glycosylated; gp10 5 was close to the natural product, and gp36 was consistent with the native product