Designing Two Individual AcMNPV Polyhedrin-Plus Bac-to-Bac Expression System in order to Express GFP and CPV-VP2 in Insect Cells.

The importance of viral protein-2 (VP2) of canine parvovirus (CPV) in binding to human cancer cells, production of veterinary vaccines and diagnostic kits has motivated several researches on producing this protein. Our purpose was to construct recombinant bacmid shuttle vectors expressing VP2 of CPV using Bac-to-Bac baculoviral expression system. Mini-Tn7 transposones engineered in pFastBac1 donor vectors were used to construct expression cassettes of GFP and CPV-VP2. The plasmids were transferred into DHBac competent cells. Site-specific transposition of the genes into bacmid was accomplished using helper plasmid. Occurrence of Transposition was confirmed via PCR using specific primers and PUC/M universal primers. The recombinant bacmid DNAs were transfected into Sf9 cells using cationic lipids to generate new recombinant baculoviruses expressing GFP and CPV-VP2. GFP and VP2 expressions were evaluated by fluorescence microscopy and western analysis, respectively. Cloning, subcloning and recombination processes of both GFP and VP2 were accomplished and verified. Accuracy of transfection process was confirmed by GFP fluorescence microscopy.VP2 expression was verified by SDS-PAGE and western analysis. Two Bac-to-Bac expression systems were designed to produce recombinant VP2 and GFP in insect cells.