Cloning, subcloning and recombination of VP2 gene of canine parvovirus intobaculoviral shuttle vector enabling its expression in insect cells

[摘要] The VP2 protein of canine parvovirus (CPV) is the main part of capsid and attachment ligands for entry into specific and cancerous cells through transferrin receptors (TfRs). Expression of VP2 alone results in assembly of a typically-sized virus like particle (VLP) in insect cells for therapeutic purposes. So, in this research our purpose was to construct a recombinant bacmid shuttle vector expressing VP2 of CPV using site-specific transposition mechanism in Bac-to-Bac baculovirus expression system. The full-length of CPV-VP2 gene (1755 bp) was isolated by PCR amplification using specific primers and cloned firstly into RBC T/A cloning vector and then subcloned into the corresponding restriction sites of pFastBac1 donor plasmid vector. Then the accuracy of cloning process in these vectors 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 bacmid shuttle vector was accomplished using the helper plasmid. Finally, the accuracy of transposition process was evaluated by a PCR panel using specific primers and PUC/M13 universal primers. Cloning, subcloning and recombination of VP2 gene of canine parvovirus into baculoviral shuttle vector were performed and confirmed successfully. In other words, VP2-containing recombinant bacmid was constructeed successfully. In this study, we used the Bac-to-Bac system for site-specific transposition of VP2 gene from pVP2FastBac1 to baculovirus derived bacmid shuttle vector. The constructed recombinant bacmid can express recombinant VP2 protein in insect cells.

[学科分类] 药理学