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

开发用于生产 BAC 衍生重组 Epstein-Barr 病毒的包装细胞

基本信息

批准号：
18590445
负责人：
KANDA Teru
金额：
$ 2.59万
依托单位：
Hokkaido University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2006
资助国家：
日本
起止时间：
2006 至 2007
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-18590445/
关键词：
Epstein-Barr virus bacterial artificial chromosome packaging system 293 cells B-lymphocyte transformation

项目摘要

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.

细菌人工染色体（BAC）系统可用于EB病毒（EBV）基因组工程。我们的目的是建立一个包装系统，以生产重组EBV来源于BAC克隆。我们测试了携带EBV辅助病毒的两种细胞系作为包装细胞的有用性;（1）P3 HR-1细胞（来源于人伯基特淋巴瘤细胞）和（2）B 95 -8细胞及其衍生物（来源于绒猴淋巴母细胞样细胞系）。但是，结果表明，我们无法避免辅助病毒DNA和BAC克隆DNA之间的重组。因此，为了获得纯的重组EBV，我们改变了策略，建立了一个不含辅助病毒的系统。为此，我们克隆了EB病毒B 95 -8株的全长基因组，并将BAC克隆DNA导入人293细胞。这种策略使我们能够有效地建立重组病毒生产细胞。所得重组EBV在转化B淋巴细胞的效率以及表达转基因的能力方面上级先前报道的293衍生的重组EBV。这些结果表明，B 95 -8株EBV的BAC克隆和293细胞的组合使用是生产高滴度纯重组EBV的有效方法。