Construction of recombinant pseudorabies virus strain expressing PEDV S and PoRV VP7 protein

Abstract:

[Backgroud] Porcine epidemic diarrhea, porcine rotavirus disease and pseudorabies are three important infectious diseases that seriously endanger the global pig industry. Mixed infections often lead to more serious losses on the farm. [Objective] A homologous recombination technique was used to construct a triple genetic engineering attenuated strain of pig pseudorabies co-expressing s gene and vp7 gene. [Methods] Through the sequence alignment and protein structure analysis of PEDV s and PoRV vp7 genes, 475?804 aa of s gene and 17?339 aa of vp7 gene were selected as candidate regions for the construction of strains. Three cloning vectors of pMD-S, pMD-VP7 and pMD-VP7.S and one transfer vector of pEGFP-VP7.S were constructed, respectively. Plaque purification was employed to obtain recombinant strain. [Results] A genetically engineered pseudorabies strain co-expressing S protein and VP7 protein was constructed. The strain was serially passaged for 20 generations, and both vp7 and s genes were detected without gE gene. Western blotting experiments confirmed foreign gene expression by recombinant virus. The TCID50 of the parent strain and the recombinant strain was determined by the Reed-Muench method to be 10?7.59/0.1 mL and 10?7.25/0.1 mL, respectively. [Conclusion] Recombinant pseudorabies strain PRV (CM) was obtained, which stably carried the two foreign genes, but with the deletion of virulence genes. The recombinant strain owned a similar proliferation characteristic with the parent strain. The study laid the foundation for further study on development of PRV, PEDV and PoRV triple genetic engineering vaccine.