Construction Of LoxP Site-Containing Recombinant Pseudorabies Virus

Swine pseudorabies virus (PRV) is a double-stranded DNA virus, which has been used to construct gene deleted vaccines against swine pseudorabies and as a vector for construction of live vector vaccines against other animal diseases. However, the present strategy for construction of recombinant PRV is based mainly on homologous recombination or bacterial artificially chromosome cloning, which is technically difficulty and time-consuming. The Cre/loxP system is a site-specific recombination system derived from bacteriphage P1, which has been used as a powerful gene manipulation tool in a variety of organisms.To simplify construction of recombinant PRV, two pairs of primers were designed according to the unique long region sequence of PRV Bartha K61strain for amplification of left and right homologous arms. Using the PRV DNA genome as the template, the two homologous arms of expected sizes were amplified by PCR and subcloned into pMD-18vector, resulting in homologous recombination vector pMD-TK. Using eukaryotic expression vector pEGFP-N1as the template, the enhanced green fluorescent protein (EGFP) reporter gene, flanked by two loxP sites, was amplified by PCR and inserted into the pMD-TK, resulting in recombinant transfer vector pMD-TK-EGFP/loxP. After transfection of PK15cells with the pMD-TK-EGFP/loxP vector and the genomic DNA of PRV Bartha K61strain, the recombinant PRV-GFP-loxP (rPRV-GFP-loxP) was generated. After five cycles of plaque purification, the purified virus was obtained, which could infect and express GFP reporter gene in PK-15cells. In the presence of Cre enzyme, the GFP expression cassette can be removed, producing a recombinant PRV with single loxP site in the TK locus to facilitate construction of rPRVs.