Enhanced baculovirus vectors with higher titers and increased genome stability

描述（由申请人提供）：杆状病毒表达载体系统（BEVS）已成功用于生产数千种用于疫苗、治疗和结构功能研究的蛋白质。BEVS的一个限制是杆状病毒倾向于积累转座子插入到fp25k基因中，导致“少多面体（FP）”表型。这种突变改变了病毒生产的平衡，从封闭源性病毒（在组织培养中不具有传染性）到出芽病毒（用于杆状病毒表达的病毒形式）。这些高水平的出芽病毒对BEVS用户是有利的，但FP突变体也缺乏多面蛋白启动子的转录，多面蛋白启动子驱动靶基因的表达。杆状病毒还会迅速积累有缺陷的干扰颗粒（DIP），这与靶基因表达的急剧下降有关，因为靶基因和/或表达所需的病毒基因被删除。促进DIP形成的一个因素是转位到fp25k。该项目的目标是开发杆状病毒表达载体，允许操纵fp25k基因。通过在病毒扩增过程中减少或消除FP25K的表达，我们期望获得5到10倍高的芽化病毒滴度，因为细胞的大部分复制潜力将用于产生芽化病毒，而不是封闭衍生的病毒。当需要靶蛋白表达时，可以恢复FP25K的表达。这将大大降低大规模生产杆状病毒表达蛋白的成本，因为高滴度的BV库存将更容易生产，有害突变的发生将减少。paratech将采取两种互补的方法来实现这一目标。一种方法是产生具有可诱导的fp25k基因的病毒，该基因可以在扩增过程中关闭，在靶蛋白表达过程中激活。另一种方法利用在fp25k中缺失的病毒与表达fp25k的细胞系结合。无论在设计阶段还是对最终用户而言，每种方法都有其优点和缺点。本提案中描述的实验将确定哪个提供更高水平的BV产生，多面体连接表达和稳定的基因组维护。最终，这里开发的系统将与paratech的vankyrin表达技术相结合，该技术将BEVS中的多面体驱动表达从2倍增加到20倍。

DESCRIPTION (provided by applicant): The baculovirus expression vector system (BEVS) has been successfully utilized to produce thousands of proteins for use as vaccines, therapeutics, and for structure-function studies. One limitation of BEVS is the propensity of baculoviruses to accumulate transposon insertions into the fp25k gene leading to the "few polyhedra (FP)" phenotype. This mutation shifts the balance of virus production from occlusion-derived viruses, which are not infectious in tissue culture, to budded viruses, which are the form of virus that is used in baculovirus expression. These higher levels of budded virus would be advantageous for BEVS users, but FP mutants are also deficient in transcription from the polyhedrin promoter, which drives expression of target genes. Baculoviruses also rapidly accumulate defective interfering particles (DIP), which are linked to a sharp decrease in target gene expression due to deletion of the target gene and/or viral genes needed for its expression. One factor that promotes DIP formation is transposition into fp25k. The goal of this project is to develop baculovirus expression vectors that allow for manipulation of the fp25k gene. By reducing or eliminating expression of FP25K during virus amplification, we expect to obtain 5- to10- fold higher levels of budded virus titers because most of the replicative potential of the cel would go to producing budded virus instead of occlusion-derived virus. Then FP25K expression would be restored when target protein expression is desired. This would significantly lower costs for large-scale production of baculovirus-expressed proteins because high titer BV stocks would be easier to produce and the occurrence of deleterious mutations would be reduced. ParaTechs will pursue two complementary approaches to achieve this goal. One involves the production of a virus with an inducible fp25k gene that can be turned off during amplification and activated during target protein expression. The other approach utilizes a virus with a deletion in fp25k coupled with a cell line that expresses FP25K. Each of these approaches has advantages and disadvantages, both in the design phase and for the end user. Experiments described in this proposal will determine which provides higher levels of BV production, polyhedrin-linked expression, and stable genome maintenance. Ultimately, the system developed here would be combined with ParaTechs vankyrin expression technology, which increases polyhedrin-driven expression in BEVS from 2- to 20-fold.