Oker-Blom C, Pettersson R F, Summers M D
Department of Entomology, Texas, A&M University, College Station.
Virology. 1989 Sep;172(1):82-91. doi: 10.1016/0042-6822(89)90109-8.
To study the capability of Spodoptera frugiperda (fall armyworm; Sf9) cells to synthesize and process mature rubella virus (RV) proteins, a cDNA encoding the structural envelope glycoproteins, E1 (58 kDa) and E2 (42-47 kDa) were inserted into the genome of Autographa californica nuclear polyhedrosis virus (AcNPV) and expressed during infection under the transcriptional regulation of the polyhedrin gene promoter. By immunoblot analysis with antibodies directed against purified RV, the individual proteins E1 and E2, and human convalescent serum, a polyprotein precursor migrating with an apparent molecular weight of 90-95 kDa was identified in Sf9 cells infected with the recombinant baculovirus, Ac701-RVE. In addition, two proteins migrating somewhat faster than authentic viral E1 and E2 were resolved. Pulse-chase labeling experiments in the absence and presence of tunicamycin, as well as treatment of the recombinant proteins with endo-beta-N-acetyl-D-glucosaminidase H indicated that the recombinant proteins are glycosylated and that the E1 and E2 apoproteins, respectively, were similar in size as compared to their in vitro synthesized counterparts. The recombinant protein products were further detected by some monoclonal antibodies directed against RV. The results presented here indicate that a polyprotein containing the envelope glycoproteins of RV is expressed and proteolytically cleaved in lepidopteran insect cells to form two proteins which resemble authentic E1 and E2. The baculovirus may therefore be suitable for the abundant expression of RV antigen.
为研究草地贪夜蛾（秋粘虫；Sf9）细胞合成和加工成熟风疹病毒（RV）蛋白的能力，将编码结构包膜糖蛋白E1（58 kDa）和E2（42 - 47 kDa）的cDNA插入苜蓿银纹夜蛾核多角体病毒（AcNPV）基因组，并在多角体蛋白基因启动子的转录调控下于感染期间表达。通过用针对纯化RV、单个蛋白E1和E2以及人恢复期血清的抗体进行免疫印迹分析，在感染重组杆状病毒Ac701 - RVE的Sf9细胞中鉴定出一种表观分子量为90 - 95 kDa的多蛋白前体。此外，还分辨出两种迁移速度比天然病毒E1和E2稍快的蛋白。在有无衣霉素存在的情况下进行脉冲追踪标记实验，以及用内切β - N - 乙酰 - D - 葡糖胺糖苷酶H处理重组蛋白，结果表明重组蛋白被糖基化，并且E1和E2脱辅基蛋白的大小分别与其体外合成的对应物相似。一些针对RV的单克隆抗体进一步检测到了重组蛋白产物。此处呈现的结果表明，含有RV包膜糖蛋白的多蛋白在鳞翅目昆虫细胞中表达并被蛋白水解切割，形成两种类似于天然E1和E2的蛋白。因此，杆状病毒可能适合大量表达RV抗原。
