Fig. 3: Generation and Confirmation of Recombinant MVA-MEU.

A Schematic representation of the recombinant shuttle vector construction. The MEU gene fragment was inserted into the pEPMVAdVI PH5 plasmid downstream of the PH5 promoter. B Generation of recombinant MVA-BAC in E. coli 1783 via a two-step recombination process. PCR amplification verified the successful insertion of the I-SecI-Kan-pH5/MEU-Flag fragment into the MVA genome of the BAC plasmid. Following arabinose-induced excision of the KanS cassette and a second recombination event at 42 °C, the correct clones were identified by PCR using DelVI-specific primers. C Rescue and propagation of recMVA-MEU. Recombinant viruses were rescued in DF-1 cells in the presence of RFV helper virus and recMVA-BAC plasmid. GFP expression, observed under fluorescence microscopy, confirmed expression of the BAC plasmid carrying the eGFP gene (Right panel). D Characterization of rescued viruses. PCR verified the presence of the transgene in the viral genome (Right panel). Western blot analysis detected distinct MEU bands in infected DF-1 cells (Right panel).