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蛋白质，用作疫苗、治疗剂和结构-功能研究。BEVS的一个限制是杆状病毒倾向于将转座子插入fp 25 k基因中，导致“少多角体（FP）”表型。这种突变将病毒生产的平衡从在组织培养中不具有感染性的闭塞衍生病毒转移到出芽病毒，出芽病毒是用于杆状病毒表达的病毒形式。这些较高水平的出芽病毒对于BEVS使用者是有利的，但是FP突变体也缺乏来自多角体蛋白启动子的转录，该启动子驱动靶基因的表达。杆状病毒还快速积累缺陷型干扰颗粒（DIP），其与由于靶基因和/或其表达所需的病毒基因的缺失而导致的靶基因表达的急剧降低有关。促进DIP形成的一个因素是转座到fp 25 k中。该项目的目标是开发杆状病毒表达载体，允许操作的fp 25 k基因。通过在病毒扩增过程中减少或消除FP 25 K的表达，我们预期获得5- 10倍更高水平的出芽病毒滴度，因为细胞的大部分复制潜力将产生出芽病毒而不是闭塞衍生的病毒。然后当需要靶蛋白表达时，FP 25 K表达将恢复。这将显著降低大规模生产杆状病毒表达蛋白的成本，因为高滴度BV原液将更容易生产，并且有害突变的发生将减少。ParaTechs将采取两种互补的方法来实现这一目标。其中一个涉及生产具有诱导型fp 25 k基因的病毒，该基因可以在扩增过程中关闭，并在靶蛋白表达过程中激活。另一种方法利用在fp 25 k中缺失的病毒与表达FP 25 K的细胞系偶联。这些方法中的每一种都有优点和缺点，无论是在设计阶段还是对于最终用户。本提案中描述的实验将确定哪种方法提供更高水平的BV生产、多角体蛋白连接的表达和稳定的基因组维持。最终，这里开发的系统将与ParaTechs vankyrin表达技术相结合，该技术将BEVS中多面体驱动的表达从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.