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

Samiei Abianeh, Hossein; Nazarian, Shahram; Amani, Jafar; Rezaei, Amir; Rostami, Mehdi; Ramezani, Mohammad Reza

doi:10.30491/jabr.2023.377373.1586

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

Document Type : Original Article

Authors

Hossein Samiei Abianeh ¹^{, 2}

Shahram Nazarian ²

Jafar Amani ³

Amir Rezaei ²

Mehdi Rostami ⁴

Mohammad Reza Ramezani ²

¹ Department of Medical Biotechnology and Nanotechnology, Faculty of Medicine, Mashhad University of Medical Sciences, Mashhad, Iran

² Department of Biology, Faculty of Basic Sciences, Imam Hossein University, Tehran, Iran

³ Applied Microbiology Research Center, Systems Biology and Poisonings Institute, Baqiyatallah University of Medical Sciences, Tehran, Iran

⁴ Department of Clinical Biochemistry, Faculty of Medicine, Mashhad University of Medical Sciences, Mashhad, Iran

10.30491/jabr.2023.377373.1586

Abstract

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.

Keywords

Clostridium botulinum

Botulinum Neurotoxin Type A

Binding Domain

Recombinant Protein

Immunogenicity