PDF(Pubmed)
Abstract：
Baculovirus expression vector system (BEVS) is a powerful and versatile platform for recombinant protein production in insect cells. As the most frequently used baculovirus, Autographa californica multiple nucleopolyhedrovirus (AcMNPV) encodes 155 open reading frames (ORFs), including a considerable number of non-essential genes for the virus replication in cell culture. Studies have shown that protein production in BEVS can be improved by removing some viral dispensable genes, and these AcMNPV vectors also offer the possibility of accommodating larger exogenous gene fragments. In this study, we, respectively, deleted 14 DNA fragments from AcMNPV genome, each of them containing at least two contiguous genes that were known nonessential for viral replication in cell culture or functionally unknown. The effects of these fragment-deletions on virus replication and exogenous protein production were examined. The results showed that 11 of the 14 fragments, containing 43 genes, were dispensable for the virus replication in cultured cells. By detecting the expression of intracellularly expressed and secreted reporter proteins, we demonstrated that nine of the fragment-deletions benefited protein production in Sf9 cells and/or in High Five cells. After combining the deletion of some dispensable fragments, we obtained two AcMNPV vectors shortened by more than 10 kb but displayed an improved capacity for recombinant protein production. The deletion strategies used in this study has the potential to further improve the BEVS.
摘要：
杆状病毒表达载体系统(BEVS)是用于在昆虫细胞中产生重组蛋白的强大且通用的平台。作为最常用的杆状病毒，加州自拟多核多角体病毒(AcMNPV)编码155个开放阅读框(ORF),包括相当数量的非必需基因的病毒复制在细胞培养。研究表明，BEVS中的蛋白质生产可以通过去除一些病毒可有可无的基因来改善，这些AcMNPV载体还提供了容纳较大外源基因片段的可能性。在这项研究中,我们,分别,从AcMNPV基因组中删除了14个DNA片段，它们中的每一个都包含至少两个连续的基因，这些基因在细胞培养物中对病毒复制是非必需的或功能未知的。检查了这些片段缺失对病毒复制和外源蛋白产生的影响。结果表明，14个片段中的11个，包含43个基因,对于培养细胞中的病毒复制是可有可无的。通过检测细胞内表达和分泌的报告蛋白的表达，我们证明,在Sf9细胞和/或HighFive细胞中,有9个片段缺失有利于蛋白质的产生.在结合了一些可有可无的片段的缺失之后，我们获得了两个缩短了10kb以上的AcMNPV载体，但显示出提高的重组蛋白生产能力。本研究中使用的删除策略有可能进一步改善BEVS。
