Molecular structure of BoHV-1ΔgEβgal virus. (a) Target region for the gE deletion in BoHV-1 genome. (b) gE ORF (US8) is flanked by gI (US7) and US9 genes. (c) Primers gE1 and gE2 were designed to amplify the left (L) fragment, and primers gE3 and gE4 were designed to amplify the right (R) fragment. The sequences of primers gE2, gE3 and gE4 were designed in order to create the depicted restriction sites to facilitate the cloning strategy. (d) pUCLRβgal recombination vector diagram restriction sites used for cloning and relative position of the elements decribed are shown (e) Confirmation gE-specific deletion by PCR. Lanes 2–4 primers specific to the gE sequence (gE7 and gE8) were used. Different templates were used for each lane shown: Lane 2 negative control (ultrapure sterile water); lane 3 BoHV-1ΔgEβgal DNA; lane 4 parental BoHV-1 LA DNA; lanes 1 molecular weight marker (100pb, Promega). (f) Molecular characterization of the recombinant BoHV-1ΔgEβgal strain. Southern blot analysis for gE gene. Viral DNA from BoHV-1ΔgEβgal (lane 1) and from parental BoHV-1 LA strain (lane 2) were digested with HindIII restriction enzyme, separated by 0.6% agarose gel electrophoresis, blotted to a membrane and probed with the gE probe. (g) Western blot for gE protein. Concentrated virus (BoHV-1ΔgEβgal, lane 1; and parental BoHV-1 LA, lane 2) was separated by electrophoresis in a 12% SDS-PAGE, blotted to a membrane and incubated with specific serum. Panel (i) gE-specific monoclonal antibody, panel (ii) gD-specific monoclonal antibody.