Cre/LoxP Mediated Report Gene Deletion from Pseudorabies Virus
A GFP expression cassette flanked by LoxP at two sites with the same orientation was amplified by PCR and inserted into the EcoRI site of pSKLR.Based on homologous recombination,the GFP expression recombinant virus S03109 was constructed using PrV RongA as a parental strain.After serial passage of S03109 on HEK-293T cell which was transfected with Cre encoding plasmid pOG231,the recombinant virus S0419 was obtained after plaque purification.Fluorescence microscope,Western blot analysis and PCR were employed to detect the expression of GFP. The results showed that S03109 infected-cells could express GFP but S0419 could not.PCR amplification and sequencing of S0419 TK gene indicated that the GFP expression cassette between two LoxP sites were deleted and only one LoxP site was left in TK locus.The sequencing result was submitted to GenBank(AY822465). The LD_(50) of S03109 and S0419 for BALB/c mice both exceeded 3×10~5 PFU.Using S03109 and S0419 to immunize BALB/c mice,the mean survival rates were 67.5% and 70% respectively after wild virus challenge.As a conclusion,the report gene GFP expression cassette was removed successfully from PrV genome by using Cre/LoxP recombinant system.