Construction And Expression Of Recombinant PRV PK Gene Deleted Transfer Vector

Aujeszky's disease is an acute infectious disease and characteristic as fever and nervous system disease. It can infect livertock and many other animals. Pseudorabies virus (PRV) is the causative agent of Aujeszky's disease, which results in significant losses in pig husbandry. Protein kinase (PK) gene is one of the main virulent genes of PRV, and it is essential to the propagation of PRV in the nerve tissues, but dispensable for virus replication and infection in other tissues. So the PK gene deleted strain can be used for creation of PRV recombinants virus expressing foreign genes.In this study, the genomic DNA of pseudorabies virus TK/gI gene-deleted strain was extracted and digested by AscI, the 8.7kb fragment, which containing the whole PK gene was reclaimed. The plasmid P8-AA was constructed by cloning the 8.7kb fragment into the AscI site of pPolyII. The fragment from plasmid pEGFP-G harboring the EGFP gene and glycoprotein gene (G) of Rabies virus expression cassette was inserted into the SphI and NdeI sites of P8-AA, formed a transfer vector P8-EGFP-G and the antiplasmid of P8-EGFP-G (P8-EGFP-G-r). The plasmid of P8-EGFP and the antiplasmid of P8-EGFP (P8-EGFP-r) were construct by insert the EGFP gene expression cassette into the SphI and NdeI sites of P8-AA.Then, we used the plasmid pEGFP-c1, pEGFP-G, P8-EGFP, P8-EGFP-r, P8-EGFP-G, and P8-EGFP-G-r to transient transfect the PK-15 cells respectively , The fluorescence can be seen in plasmid pEGFP-cl, pEGFP-G, P8-EGFP-r, and P8-EGFP-G-r. The results showed that the transfer vector P8-EGFP-r and P8-EGFP-G-r can be used to homologous recombination to construct recombinants virus. Meanwhile the results showed that there is a negative regulation sequence in the plasmid P8-EGFP-G. and P8-EGFP. The purified genomic DNA of TK~-/gI~- deleted strain and transfer vector P8-EGFP-r were used to co-transfect PK cells using lipofection. After 24 hours of co-transfecting we can see the fluorescence of recombinant virus. After three cycles of bacteriophage plague purification we select the...