Abstract:  In order to construct a gE and TK double genes deletion mutant without a reporter gene, the recombinant virus rPRV-gE^-/GFP with loxP sites was first constructed using PRV JSZ as a parental strain and an enhanced green fluorescent protein (EGFP) gene as a reporter gene. The recombinant virus rPRV-gE^- was obtained by treating the genomic DNA of rPRV-gE^-/GFP with Cre recombinase and transfecting PK15 cells. Then, the recombinant virus rPRV-gE^-/TK^- was constructed using rPRV-gE^- as a parental strain. The results of fluorescence detection and PCR amplification showed that a gE gene deletion mutant and a gE/TK double genes deletion mutant were constructed successfully. The characteristics of the viruses revealed that rPRV-gE^- had a similar growth curve in PK15 cells when compared with its wild type strain, but rPRV-gE^-/TK^- had a relatively slower growth speed. The mouse LD₅₀ of rPRV-gE^-/TK^- was more than 1×10⁵ TCID₅₀, which was significantly higher than that of rPRV-gE^- and wild type strain. The mice immunized with the rPRV-gE^-/TK^- provided an 80% protection against wild type virus challenge. These results indicated that a multiple gene deletion vaccine against PRV can be constructed by repeat use of Cre/loxP system, and it will provide a platform for the development of the recombinant attenuated PRV vaccine and its vectored vaccine.