Construction Of Pseudorabies Virus SH Strain Mutant With TK Gene Partial Deletion And Primary Study On Its Biological Property

Pseudorabies virus (PrV), or suid herpes virus I, is a member of Alphaherpesvirinae, herpesviridae, which can cause Aujeszky's disease in swine, bovine, sheep and wild animals. PrV infection could cause its natural and main storing host-swine (also its main infectious origination) a huge loses, the most huge economical lose in all swine infectious diseases except Classic Swine Fever and Food and Mouth Disease.The local PrV Shanghai isolate (PrV SH), isolated from the pigs with the clinical symptom in a swine farm at Shanghai suburbs is a virulent PrV strain. The analytical comparison of TK gene of PrV SH with that of the virulent Ea and Fa strains, both isolated from China, shows that there are great homologue and similarity between their nucleotide and deduced amino acid sequences. So we chose PrV SH to construct the relevant gene deleted vaccine, which may has great application prospective and economic value in China.PrV SH strain genome DNA was extracted and two pairs of primers were synthesized according PrV kaplan strain nucleotide sequence published on GenBank, one pairs of the primers used to amplify the left recombinant fragment (L) of 1075 bp containing the partial UL24 and partial TK, the other to amplify the right recombinant fragment(R) of 936 bp containing the partial TK and partial UL22. The left and right fragments were cloned into pBluescript IISK to obtain the transfer vector pBLR with a deletion of TK gene of 498 bp. The plasmid pBLR was cotransfected into RK13 cells with PrV SH strain , the recombinant rPrV-SH was obtained with screen tests using BrdU on the cell line of TK-143.In this research, we investigated the TK gene-deleted PrV strain rPrV-SH's growing properties in cultured ceils, its LD50 for mice, and the other its biological properties. Our results suggested that the deletion of TK gene on PrV SH might not affect its propagation in cultural cell, or the typical PRV plaque forming. Those experimental results suggested that the deletion of TK gene of PrV do not affect Its growth on RK cells. The LD50 values of PrV-SH and rPrV-SH strains for mice are 1.39×104PFU and >3×105PFU respectively. Those results demonstrated that the virulence of rPrV-SH was reduced, when compared with that of PrV-SH.