A precise excision of the complete Epstein-Barr virus genome in a plasmid based on a bacterial artificial chromosome

作者：Yu, Zhengyuan; Lu, Jianhong^*; Yu, Haibo; Yan, Qijia; Zuo, Lielian; Li, Guiyuan

来源：Journal of Virological Methods, 2011, 176(1-2): 103-107.

DOI：10.1016/j.jviromet.2011.06.015

• Homologous recombination
• Epstein-Barr virus genome
• Bacterial artificial chromosome
• Vector

The Maxi-EBV is a bacterial artificial chromosome (BAC)-based plasmid that contains the complete Epstein-Barr virus (EBV) genome of 172 kb. This plasmid also carries an additional cassette of 11.5 kb in size for the expression of a mini F factor, selection markers and GFP. In the intracellular study of EBV infection based on the Maxi-EBV system, a parallel control that only contains this 11.5 kb vector is desirable but unavailable. In order to construct the vector in this approach, a clean deletion of the complete EBV genome from the Maxi-EBV was performed. This was achieved by homologous recombination using the bacteriophage lambda Red system. Initially, an FRT-flanked kanamycin-resistance (Ran) fragment of 1.4 kb with 61 bp homologies on the ends was introduced into the Maxi-EBV plasmid, replacing the 172-kb EBV genome. The kan gene was then removed by Flp/FRT excision. The results of identification demonstrated that the mutation was generated precisely. The results highlight the feasibility for a genome as large as 172 kb to be replaced by a greatly shorter fragment and for a much smaller vector backbone to be retrieved. Cell lines derived from the transfection of the vector will subsequently be appropriate controls in the related study.

• 出版日期2011-9
• 单位中南大学