van Bokhoven H, Wellink J, Usmany M, Vlak J M, Goldbach R, van Kammen A
Department of Molecular Biology, Agricultural University, Wageningen, The Netherlands.
J Gen Virol. 1990 Nov;71 ( Pt 11):2509-17. doi: 10.1099/0022-1317-71-11-2509.
The baculovirus expression system has been used to produce non-structural proteins encoded by bottom-component RNA (B-RNA) of cowpea mosaic virus (CPMV). For this, cDNAs containing the 60K, 87K, 110K and 170K protein coding sequences were each provided with an ATG start codon and the cDNA containing the 60K coding sequence with a TAA stop codon immediately downstream of the coding sequence. Recombinant baculoviruses were retrieved which harboured the modified B-cDNA sequences under the control of the polyhedrin promoter of Autographa californica nuclear polyhedrosis virus (AcNPV). Upon infection of Spodoptera frugiperda cells with these recombinant baculoviruses, proteins were produced which were indistinguishable from the viral proteins found in CPMV-infected plants as judged by their migration in polyacrylamide gels and their reactivity with CPMV-specific antisera. Specific processing of CPMV polyproteins in cells infected with the 110K- and 170K-encoding baculovirus recombinants proved that the CPMV-encoded 24K protease activity contained in these polyproteins is active in these cells. Approximately 10% of the 110K protein was processed into 87K and 24K proteins and the 170K protein almost completely into the 110K, 87K, 84K, 60K and 24K polypeptides. In S. frugiperda cells infected by recombinant AcNPVs harbouring the 87K or 110K coding sequences, the CPMV-specific proteins amounted to 10 to 20% of the total cellular protein content, whereas in cells infected by recombinants encoding the 60K and 170K polypeptides the amounts of CPMV-specific proteins synthesized were much lower. Northern blot analysis indicated that the low-level synthesis of the 60K and 170K polypeptides was not due to inferior transcription of the cloned genes but was probably the result of inefficient translation of the RNAs derived from these constructs. It is concluded that plant virus genes can be efficiently expressed in an animal cell expression system to yield proteins that are structurally and, in at least one case (24K protein), functionally identical to the authentic plant virus proteins.
杆状病毒表达系统已被用于生产由豇豆花叶病毒（CPMV）底部组分RNA（B-RNA）编码的非结构蛋白。为此，分别为包含60K、87K、110K和170K蛋白编码序列的cDNA提供了一个ATG起始密码子，并且为包含60K编码序列的cDNA在编码序列下游紧邻处提供了一个TAA终止密码子。回收了重组杆状病毒，其在苜蓿银纹夜蛾核型多角体病毒（AcNPV）多角体蛋白启动子的控制下含有修饰的B-cDNA序列。用这些重组杆状病毒感染草地贪夜蛾细胞后，产生的蛋白质通过在聚丙烯酰胺凝胶中的迁移及其与CPMV特异性抗血清的反应性判断，与在CPMV感染植物中发现的病毒蛋白无法区分。在用编码110K和170K的杆状病毒重组体感染的细胞中CPMV多蛋白的特异性加工证明，这些多蛋白中包含的CPMV编码的24K蛋白酶活性在这些细胞中是有活性的。大约10%的110K蛋白被加工成87K和24K蛋白，而170K蛋白几乎完全被加工成110K、87K、84K、60K和24K多肽。在被携带87K或110K编码序列的重组AcNPV感染的草地贪夜蛾细胞中，CPMV特异性蛋白占细胞总蛋白含量的10%至20%，而在被编码60K和170K多肽的重组体感染细胞中，合成的CPMV特异性蛋白量要低得多。Northern印迹分析表明，60K和170K多肽的低水平合成不是由于克隆基因转录不佳，而是可能是这些构建体来源的RNA翻译效率低下的结果。得出的结论是，植物病毒基因可以在动物细胞表达系统中高效表达，以产生在结构上以及至少在一种情况下（24K蛋白）在功能上与真实植物病毒蛋白相同的蛋白质。
