Construction, expression, and characterization of a multi-epitope protein based on envelope glycoproteins B, C, and D of Varicellovirus bovinealpha1/5 in Escherichia coli.

Varicellovirus bovinealpha1 (BoAHV-1) and 5 (BoAHV-5) are important pathogens associated with respiratory, reproductive, and neurological disorders in cattle, remaining globally relevant since their first reports in the 1950s and 1960s. Envelope glycoproteins B, C, and D play critical roles in viral attachment and fusion, making them key targets for immune responses and promising candidates for vaccine development. This study aimed to design a multi-epitope protein using immunoinformatic approaches, incorporating highly conserved, antigenic B- and T- cell epitopes with strong predicted MHC-binding affinity from glycoproteins B, C, and D of BoAHV-1/5, followed by in silico characterization and heterologous expression in Escherichia coli. The construct was designed using bioinformatics tools, cloned into the pET-24a vector, and expressed in a prokaryotic system. The resulting protein consists of 321 amino acids, with a predicted molecular weight of 33.24 kDa, high antigenicity (1.1543) and non-allergenicity. Molecular docking analyses indicated strong interactions with bovine Toll-like receptors TLR2/6 and TLR4. The protein was successfully expressed in E. coli and detected by anti-His6x antibody in Western blot, showing the expected molecular weight (~33 kDa). It was also recognized by bovine serum containing neutralizing antibodies against BoAHV-1/5. Overall, these findings support the feasibility of the multi-epitope construct and provide a foundation for future investigations of its immunogenicity and protective efficacy studies in animal models.