Construction of Marek's Disease Virus Serotype 814 Strain as an Infectious Bacterial Artificial Chromosome

The aim of this study was to construct the complete genome of Marek's Disease Virus Serotype 1 814 strain as an infectious bacterial artificial chromosome (BAC). Using self-designed selection marker Eco-gpt (1.3 kb) and BAC vector pBeloBAC11 (7.5 kb), we constructed the transfer plasmid pUAB-gpt-BAC11. The plasmid pUAB-gpt-BAC11 and MDV Total- DNA were cotransfected into secondary CEFs; we passaged the virus-containing cells eight rounds in selection medium and obtained purified recombinant viruses. Recombinant viral genomes were extracted and electroporated into E. coli, BAC clones were identified by restriction enzyme digestion and PCR analysis. Finally, we obtained thirty-eight BAC clones, DNA from various MDV-1 BACs was transfected into CEFs, and recombinant virus was reconstituted by transfection of MDV-BAC2 DNA. In this study, we successfully cloned the complete genome of MDV-1814 strain as an infectious bacterial artificial chromosome. With these cloned genomes in hand, A revolutionary MDV-DNA engineering platform utilizing RED/ET recombination system was constructed successfully, which acclerate the understanding of MDV gene functions and promote the using of MDV as a vector for expressing foreign genes. In addition, it may be possible to generate novel MDV-1 vaccines based on the BACs.