Construction And In Vitro Reproduction Feature Of A Recombinant Pseudorabies Virus With BAC Plasmid Insertion In The TK Gene

Pseudorabies virus (PRV), or Aujeszky's virus, is a member of alpha herpesvirinae I, Herpeaviridae, which can cause pseudorabies disease in swine, bovine, sheep and wild animals. Pseudorabies is an acute infection disease and characterized by high fever, extreme itching (except swine) and encephalomyelitis. Especially, porcine Pseudorabies has become one of the most economically important diseases in worldwide swine industry. PRV genome is a double-stranded DNA of approximately 150 kb and encodes 70～100 proteins. Like other alpha herpesvirus, PRV has some typical chatacteristics of alpha herpesvirus such as latent infection and neurotropism.For a long time, the main method studying on the functional genes of herpesvirus was to construct genetic-deleting mutants. However, it was tedious to produce recombinant mutants using homologous recombinant in infected cells. In 1997, Messerle et al., German researchers, firstly constructed infectious clone of MCMV based on bacterial artificial chromosomes (BACs). This technique actualized that the genomes of infectious virus could be stored, propagated or modificated by BAC-plasmid pattern in E. coli. and it was convenient and rapid to manipulate the technique. This technique lead to advancement in studying on gene functions of herpesvirus, and provided new ways to develop high-performance herpesviral-vetor system.In this study, a transfer vector pUCTK-EGFP-BAC11 was constructed by inserting the EGFP expression cassette with loxP site and BAC plasmid pBeloBAC11 between two homologous arms containing partial TK gene of PRV Bartha K61 strain and a recombinant virus (rv-PRVBAC) was achieved by cotransfecting the transfer vector and PRV genome into Vero cell. After 11 passages of phage assay based on EGFP expression, the recombinant virus was purified and confirmed to contain BAC plasmid. The reproduction feature of rv-PRVBAC in vitro was investigated by phage assay and single-step growth curve mensuration, the results showed that the reproduction rate of rv-PRVBAC was indistinguishable to that of parental virus Bartha K61, indicating the large fragment of BAC plasmid insertion in the TK gene didn't influence the reproduction rate of rv-PRVBAC in vitro cell culture. After 18 passages on Vero cell, the EGFP was expressed stably and genes on the BAC plasmid or genes on the virus genome adjacent to the insertion locus could be amplified by PCR, indicating the BAC plasmid and EGFP expression cassette can be passed down with PRV stably.