Construction and purification of a recombinant porcine pseudorabies virus co-ex-pressing the AD protein of the TGEV and the CS region of the PEDV S protein
To construct a recombinant virus strain rPRV-AD-CS co-expressing the CS region of TGEV AD protein and PEDV S protein,and to lay the groundwork for the development of a safe and effective triple vaccine for the prevention of TGEV,PEDV and PRV infections.A pair of spe-cific primers were designed based on the sequence of CS neutralization epitope region(499-789 aa)of porcine epidemic diarrhea virus(PEDV)S protein,and the PEDV CS region was PCR amplified by introducing BamHI and HindⅢ at its 5'end,respectively.The amplified CS fragment was in-serted into the BamHI and HindⅢ sites of the pBApo-EF1α DNA eukaryotic expression vector,and then the CS region-containing expression cassette was amplified and inserted into the PRV transfer plasmid pG-AD-EGFP containing the AD gene of transmissible gastroenteritis virus(TGEV)to construct the transfer plasmid pG-AD-CS-EGFP.The PRV triple gene deletion strain rPRV NY-gE-/gI-/TK-genome was cotransfected with the transfer plasmid pG-AD-CS-EGFP using transfection reagent ZLip2000 to obtain the recombinant virus rPRV-AD-CS-EGFP carrying TGEV AD,PEDV CS region and green fluorescent protein marker genes.The ex-pression cassette of recombinant virus rPRV-AD-CS,the genetic stability evaluation was carried out after sequencing,and the recombinant virus rPRV-AD-CS,the parental strain Bartha-K61 and the PRV strong strain were inoculated in mice for safety evaluation.The recombinant virus was co-transfected with CRISPR/Cas9-EGFP knockout plasmid in ST cells,and the recombinant virus rPRV-AD-CS without green fluorescent marker was obtained after three rounds of virus null spot purification.rPRV-AD-CS was confirmed to express CS exogenous protein in ST cells by Western blot and IFA.It had good genetic stability and safety.In conclusion,recombinant virus rPRV-AD-CS was successful constructed,which was genetically stable and safe.