Martínez-Solís María, Gómez-Sebastián Silvia, Escribano José M, Jakubowska Agata K, Herrero Salvador
Department of Genetics, Universitat de València, Burjassot, Spain; Estructura de Recerca Interdisciplinar en Biotecnologia i Biomedicina (ERI BIOTECMED), Universitat de València, Burjassot, Valencia, Spain.
Alternative Gene Expression S.L. (ALGENEX) , Madrid , Spain.
PeerJ. 2016 Jun 28;4:e2183. doi: 10.7717/peerj.2183. eCollection 2016.
The baculovirus expression vector system (BEVS) has been widely used to produce a large number of recombinant proteins, and is becoming one of the most powerful, robust, and cost-effective systems for the production of eukaryotic proteins. Nevertheless, as in any other protein expression system, it is important to improve the production capabilities of this vector. The orf46 viral gene was identified among the most highly abundant sequences in the transcriptome of Spodoptera exigua larvae infected with its native baculovirus, the S. exigua multiple nucleopolyhedrovirus (SeMNPV). Different sequences upstream of the orf46 gene were cloned, and their promoter activities were tested by the expression of the GFP reporter gene using the Autographa californica nucleopolyhedrovirus (AcMNPV) vector system in different insect cell lines (Sf21, Se301, and Hi5) and in larvae from S. exigua and Trichoplusia ni. The strongest promoter activity was defined by a 120 nt sequence upstream of the ATG start codon for the orf46 gene. On average, GFP expression under this new promoter was more than two fold higher than the expression obtained with the standard polyhedrin (polh) promoter. Additionally, the orf46 promoter was also tested in combination with the polh promoter, revealing an additive effect over the polh promoter activity. In conclusion, this new characterized promoter represents an excellent alternative to the most commonly used baculovirus promoters for the efficient expression of recombinant proteins using the BEVS.
杆状病毒表达载体系统（BEVS）已被广泛用于生产大量重组蛋白，并且正成为生产真核蛋白最强大、最稳健且最具成本效益的系统之一。然而，与任何其他蛋白质表达系统一样，提高该载体的生产能力很重要。在感染其天然杆状病毒——甜菜夜蛾多核多角体病毒（SeMNPV）的甜菜夜蛾幼虫转录组中，orf46病毒基因被鉴定为丰度最高的序列之一。克隆了orf46基因上游的不同序列，并使用苜蓿银纹夜蛾多核多角体病毒（AcMNPV）载体系统，通过绿色荧光蛋白（GFP）报告基因在不同昆虫细胞系（Sf21、Se301和Hi5）以及甜菜夜蛾和粉纹夜蛾幼虫中的表达来测试它们的启动子活性。最强的启动子活性由orf46基因ATG起始密码子上游120 nt的序列确定。平均而言，在这个新启动子控制下的GFP表达比使用标准多角体蛋白（polh）启动子获得的表达高出两倍多。此外，还测试了orf46启动子与polh启动子的组合，结果显示对polh启动子活性有累加效应。总之，这个新鉴定的启动子是使用BEVS高效表达重组蛋白时，最常用杆状病毒启动子的一个极佳替代品。
