Abstract

Background and Aim: Canine parvovirus (CPV) is a small non-enveloped ssDNA virus composed of viral capsid proteins 1, 2 and 3 (VP1, VP2 and VP3). This virus has a natural affinity to cancer cells via VP2 ligands/transferrin receptors (TfRs) attachment. It has shown that VP1 and VP3 are unnecessary for capsid formation and consequently the VP2 alone is sufficient for assembly of canine parvo-virus like particle (CP-VLP) for therapeutic aims. So, in this research our purpose was to construct a recombinant bacmid shuttle vector expressing VP2 of CPV using site-specific transposition mechanism in a Bac-to-Bac baculovirus expression system. Methods: The mini-Tn7 transposone located in pFastBac1 donor vector containing expression cassette of CPV-VP2 was used in this experimental study that had constructed in our previous study. Firstly, the presence of gene of interest in pFastBac1 donor vector was evaluated by PCR and enzymatic digestion analysis. Then the confirmed pVP2FastBac1 plasmid was transferred into E. coli DH10Bac competent cells and the site-specific transposition of VP2 into a bacmid shuttle vector was accomplished using helper plasmid. Finally, the accuracy of transposition process was evaluated by a PCR panel using specific primers and PUC/M13 universal primers. Results: The presence of the gene of interest in pFastBac1 donor vector was confirmed by PCR and enzymatic digestion analysis and VP2-containing recombinant bacmid was subsequently constructed successfully by site-specific transposition mechanism and verified by the mentioned PCR panel. Conclusions: In this study, we used the Bac-to-Bac system for site-specific transposition of VP2 gene from pVP2FastBac1 to a baculovirus derived bacmid shuttle vector. The constructed recombinant bacmid can express recombinant VP2 protein in insect cells.