In silico characterization and heterologous expression of the recombinant YkuE protein from Corynebacterium pseudotuberculosis for immunobiological purpose

Caseous lymphadenitis (CLA), caused by Corynebacterium pseudotuberculosis , remains a major challenge for small ruminant production due to the limited efficacy and questionable safety of currently available vaccines, as well as the inefficacy of antibiotic treatment once abscesses are established. These limitations reinforce the need for novel antigens that may improve both prophylactic and diagnostic strategies. In this study, the YkuE protein, previously identified as a promising antigen through reverse vaccinology, was comprehensively characterized using in silico immunoinformatics approaches and experimental validation. The computational analyses predicted YkuE as a stable, non-allergenic, and moderately antigenic protein containing multiple B- and T-cell epitopes, supporting its immunogenic potential. Structural modeling and refinement confirmed the reliability of the three-dimensional structure, while molecular docking analysis revealed a strong interaction between YkuE and Toll-like receptor 2 (TLR2), suggesting its involvement in innate immune recognition. The ykuE gene was cloned into the pET28a expression vector and heterologously expressed Escherichia coli BL21 Star (DE3). The recombinant YkuE protein was predominantly recovered in the insoluble fraction and exhibited an apparent molecular mass of approximately 37 kDa. Purified rYkuE was specifically recognized by sera from sheep and goats naturally infected with C. pseudotuberculosis , while no reactivity was observed with negative sera. Together, these findings highlight recognition by YkuE as a naturally targeted antigen in infected small ruminants and support its potential application in the development of recombinant vaccines and immunodiagnostic tools for CLA control.