Figure 10

Generation of recombinant BV vector designed for display of scFv using the N18E2 signal peptide. Shown is the stepwise construction and isolation of BV-N18E2/scFv, a recombinant BV designed to expose at its surface scFv molecules of interest equiped with the signal octadecapeptide N18E2. Step (1): PCR amplification of the scFv coding sequence, using a Fw 5'-primer containing a Nhe I site and the N18E2 coding sequence, and Rev 3'-primer encoding a HA tag and a Hind III site. Step (2): insertion of N18E2-scFv-HA-encoding DNA fragment into the baculoviral intermediate plasmid pBlueBac4.5, digested with Nhe I and Hind III. Step (3): cotransfection of insect cells with pBlueBac4.5-N18E2-scFv-HA and linearized baculoviral DNA, and homologous recombination. Step (4): Isolation of plaques positive for recombinant baculoviral clone harboring N18E2-scFv-HA (e.g. blue plaque selection); amplification and isolation of recombinant BV expressing N18E2-scFv-HA, and displaying N18E2-scFv-HA on the baculoviral envelope.