Oker-Blom C, Summers M D
Department of Entomology, Texas A & M University, College Station 77843-2475.
J Virol. 1989 Mar;63(3):1256-64. doi: 10.1128/JVI.63.3.1256-1264.1989.
To study protein processing in an insect Spodoptera frugiperda (fall armyworm; Sf9) cell line, a 26S cDNA encoding the sequence of Sindbis virus structural proteins (capsid protein, of 30 kilodaltons [kDa]; p62 [the precursor of E3 and E2], of 62 kDa; a 6-kDa peptide; and the E1 protein, of 56 kDa) was inserted into the genome of Autographa californica nuclear polyhedrosis virus (AcNPV) adjacent to the polyhedrin promoter. By immunoblot analysis with antisera directed against whole Sindbis virus and the individual structural proteins (capsid, E2, and E1), we have shown that polypeptides similar in size and antigenicity to those synthesized in Sindbis virus-infected BHK cells are expressed in Sf9 cells infected with the recombinant baculovirus Ac373-SV26. By pulse-chase labeling in the presence or absence of tunicamycin, by endo-beta-N-acetylglucosaminidase H (endo-H) treatment of the recombinant glycoproteins, and by N-terminal sequence analysis of the E1 envelope glycoprotein, we have further shown that the 26S transcription translation unit of Sindbis virus, although normally encoded by nonnuclear RNA, is expressed and proteolytically cleaved similarly, if not identically, in Sf9 cells as compared with BHK cells when a baculovirus expression vector is used.
为了研究昆虫草地贪夜蛾（秋粘虫；Sf9）细胞系中的蛋白质加工过程，将编码辛德毕斯病毒结构蛋白序列（30千道尔顿[kDa]的衣壳蛋白；62 kDa的p62[E3和E2的前体]；一个6 kDa的肽；以及56 kDa的E1蛋白）的26S cDNA插入到苜蓿银纹夜蛾核型多角体病毒（AcNPV）基因组中多角体蛋白启动子附近。通过用针对完整辛德毕斯病毒和各个结构蛋白（衣壳、E2和E1）的抗血清进行免疫印迹分析，我们已经表明，在感染重组杆状病毒Ac373 - SV26的Sf9细胞中表达了与在感染辛德毕斯病毒的BHK细胞中合成的那些大小和抗原性相似的多肽。通过在有或没有衣霉素的情况下进行脉冲追踪标记、对重组糖蛋白进行内切β - N - 乙酰葡糖胺糖苷酶H（内切H）处理以及对E1包膜糖蛋白进行N端序列分析，我们进一步表明，辛德毕斯病毒的26S转录翻译单元虽然通常由非核RNA编码，但当使用杆状病毒表达载体时，在Sf9细胞中与BHK细胞中表达和蛋白水解切割的方式相似（即使不完全相同）。
