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To obtain recombinant Epstein-Barr virus (EBV) particles with the neomycin resistance from BAC-EBV with hygrymicin (hyg) gene, homologous recombination was employed for altering the original hyg gene to neo resistance gene in the BAC-EBV genome. The kanamycin marker gene (kan) containing the recombinant protein FLP recognition sites (FRTs) was connected to the neo gene in the vector of pGEM-T using conventional cloning techniques. Both ends of the connective are the arms with 20bp of homology sequences responding to the ends next to the hyg gene. PCR and cloning techniques were further performed to extend homology arms to make them reach 89bp. The linear fragment kan-neo with 89bp of arms was then electroporated into the bacteria containing BAC-EBV genome. Under the mediation of the redαβγ system, the hyg gene in BAC-EBV was replaced by neo gene. A series of methods including antibiotic selection, PCR amplification, sequencing and restriction enzyme digestion were used for the identification of the mutated plasmids. The results showed that BAC-EBV/neo, was constructed as expected. Through the transfection of the BAC-EBV/neo plasmid, G418 selection, and GFP observation, we established 293-EBV/neo cell line. This research lays a foundation for the establishment of broader cell models in the study of EBV-associated pathogenic mechanisms.

Genetic Alteration of Selection Resistance on BAC-EBV Plasmids by Homologous Recombination and Establishment of the Stably Transfected Cell Line