Expression, Purification and evaluation of the Immunogenicity of RecombinantC-terminus of the Receptor-Binding Domain of Neurotoxin Botulinum Protein Type B (BoNT/B-HcC)

Abstract

Background and Aim: Botulism, a syndrome caused by food poisoning, results from use of food contaminated with the botulinum toxin, which is very dangerous and deadly. Botulism is caused by the effects of bacterial toxins on the terminals of the motor nerves. Botulinum neurotoxins are among the most potent toxins. The aim of this study was to investigate expression, purification and, evaluation of the immunogenicity of the recombinant BoNT/B-HcC protein as an immunogen candidate in mice. Materials and Methods: The C-terminus of the receptor-binding domain of botulinum neurotoxin type B(BoNT/B-HcC) was selected as the antigen for bioinformatics evaluations. The pET17b-BoNT/B-HcC plasmid was transferred to E. coli BL21(DE3) by heat shock. The recombinant protein was purified and analyzed by SDS-PAGE. After verification of the recombinant protein by western blot, immunization of mice was performed. Antibody titers of recombinant proteins were evaluated by indirect ELISA and the results were compared using t-test. Results: The codon adaptation index (CAI) of the optimized gene was 0.99. The percentage of codons having high-frequency distribution was improved to 78%. Restriction analysis confirmed the 1119 bp construct gene and the recombinant protein with a molecular weight of 43.8 kDa was expressed in the prokaryotic host. The total yield of purified protein was 23 mg of protein per liter of culture. Immunization of mice induced serum antibody response. Statistical analysis showed that the antibody titer was significantly different compared to that of the control sample. Conclusion: The designed recombinant antigen showed high antigenicity that could be considered as an immunogen against botulinum type B neurotoxin in future studies.

Keywords

• clostridium botulinum
• vaccine
• ‌ immunizations
• antibody
• catalytic domain