Development of packaging cells for production of BAC-derived recombinant Epstein-Barr virus

细菌人工染色体(BAC)系统可用于在E.coll中进行EB病毒基因组工程。我们的目标是建立一种包装系统，以生产来自BAC克隆的重组EBV。我们测试了两种携带EBV辅助病毒的细胞系作为包装细胞的用途：(1)P3HR-1细胞(来自人Burkitt淋巴瘤细胞)和(2)B95-8细胞及其衍生物(来自绒猴淋巴母细胞系)。然而，结果表明，辅助病毒DNA和BAC克隆DNA之间的重组是不可避免的。因此，为了获得纯净的重组EBV，我们改变了策略，建立了一个没有辅助病毒的系统。为此，我们克隆了B95-8株EBV的全长基因组，并将BAC克隆DNA导入人293细胞。这一策略使我们能够高效地建立重组病毒生产细胞。得到的重组EBV在转化B淋巴细胞的效率和表达转基因的能力方面优于先前报道的293来源的重组EBV。这些结果表明，B95-8株EBV的BAC克隆与293细胞的联合使用是制备高滴度纯重组EBV的有效方法。

Bacterial artificial chromosome (BAC) system is useful for engineering the genome of Epstein-Barr virus (EBV) genome in E. coll. We aimed to establish a packaging system to produce recombinant EBVs derived from the BAC clone. We tested two cell lines harboring helper viruses of EBV for their usefulness as packaging cells; (1)P3HR-1 cells (derived from human Burkitt's lymphoma cells) and (2)B95-8 cells and their derivatives (derived from marmoset lymphoblastoid cell lines). However, the results revealed that we could not avoid the recombination between the helper virus DNA and the BAC clone DNA. Therefore, in order to get pure recombinant EBVs, we switched our strategy to establish a system that is free from helper virus. For this purpose, we cloned the full length genome of B95-8 strain EBV, and introduced the BAC clone DNA into human 293 cells. This strategy enabled us to efficiently establish recombinant virus-producing cells. The resultant recombinant EBVs are superior to previously-reported 293-derived recombinant EBVs in their efficiency to transform B-lymphocyte as well as in their ability to express transgenes. These results demonstrate that the combinational usage of the BAC clone of B95-8 strain EBV and 293 cells is an efficient way to produce high-titer pure recombinant EBVs.