Document Type : Original Article
Department of Medical Biotechnology and Nanotechnology, Faculty of Medicine, Mashhad University of Medical Sciences, Mashhad, Iran
Department of Biology, Faculty of Basic Sciences, Imam Hossein University, Tehran, Iran
Applied Microbiology Research Center, Systems Biology and Poisonings Institute, Baqiyatallah University of Medical Sciences, Tehran, Iran
Department of Clinical Biochemistry, Faculty of Medicine, Mashhad University of Medical Sciences, Mashhad, Iran
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 285 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-17b expression vector. The recombinant protein was expressed using 1 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 0.62 to 0.90 after optimization. The minimum energy of the predicted mRNA structure was (-308.39) kcal/mol. SDS-PAGE and Western blotting confirmed the 44/6 kDa recombinant protein. Following immunization, mice elicited significant IgG antibodies in serum compared to control mice (p<0.05).
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.