AI Article Synopsis
- The pHA2 plasmid containing a BAC vector and GFP expression cassette was inserted into the UL23(TK) gene of Pseudorabies virus (PRV), resulting in a recombinant virus called rPRV-HA2.
- This recombinant virus was confirmed through plaque purification and was subsequently electroporated into E. coli to recover the PRV BAC (pPRV).
- Transfection of pPRV into VeroE6 cells led to successful viral replication, with no significant effects on viral characteristics despite modifications to the TK gene, demonstrating the BAC system's potential for gene manipulation and vaccine development.
Article Abstract
pHA2 plasmid sequence,with Bacterial Artificial Chromosome(BAC) vector and the GFP expression cassette, was introduced into the UL23(TK) gene of Pseudorabies virus(PRV)strain ZJ by homologous recombination,and the recombinant PRV (rPRV-HA2) was confirmed and isolated by plaque purification. The circular genome of rPRV-HA2 was electroporated into Escherichia coli strain DH10B and then the PRV BAC (pPRV) was recovered. The transfection of pPRV into VeroE6 cells resulted in productive infection. The rescued virus isolated following transfection was indistinguishable from rPRV-HA2 in cytopathic effects (CPE) and replication curve in vitro. The growth kinetics of the viruses indicated that partial deletion of TK gene and BAC vector insertion had no effect on the viral titre and plaque size in vitro. The PRV BAC system will enable quick and reliable manipulation of the viral genome for the functional investigation on the PRV genes and the development of PRV vector in vaccine.
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