Immunogenicity Evaluation of a Recombinant C-terminus of Botulinum Neurotoxin Protein Type A Binding Domain Protein

Introduction: Botulinum neurotoxin is one of the most potent toxins. This neurotoxin is a biological weapon due to its high lethality and simplicity of preparation. The receptor-binding domain of botulinum neurotoxin is a promising vaccine candidate. In this study, the immunogenicity of the C-terminal domain of botulinum neurotoxin type A binding domain was investigated.Materials and Methods: The synthetic gene encoding ۲۸۵ C-terminal amino acids of BoNT/A binding domain fused to trxA gene, was constructed. The sequence was optimized codon usage for expression in E. coli and subcloned into pET-۱۷b expression vector. The recombinant protein was expressed using ۱ mM IPTG and purified by affinity chromatography on a column of Ni-NTA. The recombinant protein was confirmed by Western blotting and was used to immunize mice. The indirect ELISA and t-test were used to assess and compare antibody titers against recombinant protein.Results: The codon adaptation index of the contract was altered from ۰.۶۲ to ۰.۹۰ after optimization. The minimum energy of the predicted mRNA structure was (-۳۰۸.۳۹) kcal/mol. SDS-PAGE and Western blotting confirmed the ۴۴/۶ kDa recombinant protein. Following immunization, mice elicited significant IgG antibodies in serum compared to control mice (p<۰.۰۵).Conclusions: The results indicated a highly expressed and purified recombinant protein, which is able to evoke high antibody titers in mice. Future studies may develop the recombinant antigen as a potential immunogenic candidate against botulinum neurotoxin type A.