Construction and characterization of BoHV-1 UL30-HA and UL42-FLAG expression plasmids. (A) Schematic representation of the BoHV-1 genome (140 kb), along with unique short (US), unique long (UL), terminal repeat (TR), and internal repeat (IR) sequences. Position of UL30 (POL) is located between 45237 nt and 48978 nt on the BoHV-1 genome. (B) UL30 (POL) gene with restriction sites HindIII and EcoRI and the HA tag at C terminus was cloned into the vector pcDNA4. (C) Position of UL42 located between 19596 nt and 20824 nt on the BoHV-1genome. (D) UL42 containing restriction sites HindIII and EcoRI and the FLAG tag at C terminus were cloned into pcDNA4. (E) Restrictive digestion with Hind III and EcoRI. (F) SDS-PAGE/Western blotting assays were performed to detect the indicated fusion proteins. MDBK cells were transfected with indicated plasmids. At 24 h post transfection, the cells were processed for SDS-PAGE/Western blotting assays and the fusion proteins pUL30-HA and pUL42-FLAG were detected with monoclonal antibodies against either HA or FLAG tags. A representative blot of three independent experiments was shown.