Salem Tamer Z, Seaborn Craig P, Turney Colin M, Xue Jianli, Shang Hui, Cheng Xiao-Wen
Department of Microbiology, 32 Pearson Hall, Miami University, Oxford, Ohio, United States of America.
Biomedical Sciences, University of Science and Technology at Zewail City, Giza, Egypt.
PLoS One. 2015 Dec 14;10(12):e0145019. doi: 10.1371/journal.pone.0145019. eCollection 2015.
The simian virus 40 polyadenylation signal (SV40 polyA) has been routinely inserted downstream of the polyhedrin promoter in many baculovirus expression vector systems (BEVS). In the baculovirus prototype Autographa californica multiple nucleopolyhedrovirus (AcMNPV), the polyhedrin promoter (very late promoter) transcribes its gene by a viral RNA polymerase therefore there is no supporting evidence that SV40 polyA is required for the proper gene expression under the polyhedrin promoter. Moreover, the effect of the SV40 polyA sequence on the polyhedrin promoter activity has not been tested either at its natural polyhedrin locus or in other loci in the viral genome. In order to test the significance of adding the SV40 polyA sequence on gene expression, the expression of the enhanced green fluorescent protein (egfp) was evaluated with and without the presence of SV40 polyA under the control of the polyhedrin promoter at different genomic loci (polyherin, ecdysteroid UDP-glucosyltransferase (egt), and gp37). In this study, spectrofluorometry and western blot showed reduction of EGFP protein for all recombinant viruses with SV40 polyA, whereas qPCR showed an increase in the egfp mRNA levels. Therefore, we conclude that SV40 polyA increases mRNA levels but decreases protein production in the BEVS when the polyhedrin promoter is used at different loci. This work suggests that SV40 polyA in BEVSs should be replaced by an AcMNPV late gene polyA for optimal protein production or left untouched for optimal RNA production (RNA interference applications).
在许多杆状病毒表达载体系统（BEVS）中，猿猴病毒40多聚腺苷酸化信号（SV40 polyA）通常被插入到多角体蛋白启动子的下游。在杆状病毒原型苜蓿银纹夜蛾多粒包埋核型多角体病毒（AcMNPV）中，多角体蛋白启动子（极晚期启动子）通过病毒RNA聚合酶转录其基因，因此没有证据支持在多角体蛋白启动子控制下正确的基因表达需要SV40 polyA。此外，SV40 polyA序列对多角体蛋白启动子活性的影响在其天然多角体蛋白基因座或病毒基因组的其他基因座中均未得到测试。为了测试添加SV40 polyA序列对基因表达的重要性，在不同基因组基因座（多角体蛋白、蜕皮甾体UDP-葡萄糖基转移酶（egt）和gp３７）的多角体蛋白启动子控制下，评估了有无SV40 polyA时增强型绿色荧光蛋白（egfp）的表达。在本研究中，荧光分光光度法和蛋白质免疫印迹法显示，所有含有SV40 polyA的重组病毒的EGFP蛋白均减少，而定量PCR显示egfp mRNA水平增加。因此，我们得出结论，当在不同基因座使用多角体蛋白启动子时，SV40 polyA会增加BEVS中的mRNA水平，但会降低蛋白质产量。这项工作表明，BEVS中的SV40 polyA应该被AcMNPV晚期基因多聚腺苷酸化信号取代以实现最佳蛋白质生产，或者保持不变以实现最佳RNA生产（RNA干扰应用）。
