Development of a recombinant Lactobacillus casei strain expressing Bovine rotavirus VP4 and VP7 proteins as an oral vaccine candidate against calf enteritis.

Rotavirosis is a leading cause of diarrhea in neonatal calves, resulting in significant economic losses in the livestock industry. Existing commercial vaccines exhibit limitations in inducing effective mucosal immunity and present challenges in field application. In this study, we aimed to develop a probiotic-based oral vaccine candidate by expressing the outer capsid proteins VP4 and VP7 of bovine rotavirus in Lactobacillus casei. Viral RNA was extracted from a previously characterized bovine rotavirus strain, and the VP4 (828 bp) and VP7 (1032 bp) gene segments were amplified by reverse transcription PCR (RT-PCR). The resulting amplicons were cloned into the pNZ2103 expression vector and introduced into L. casei via electroporation. Expression of the recombinant proteins was confirmed by SDS-PAGE and Western blot analyses using in-house polyclonal antibodies. Protein bands of the expected molecular weights (~27 kDa for VP4 and ~37 kDa for VP7) were successfully detected in the engineered L. casei strains. These findings demonstrate that L. casei is a viable host for the expression of rotavirus antigens and may serve as a promising live oral delivery system for mucosal immunization. This recombinant approach offers several advantages over traditional parenteral vaccines, including the potential for cold-chain independence, needle-free administration, and enhanced mucosal immune responses. Future research will focus on in vivo evaluation of immunogenicity and protective efficacy in relevant animal models.