Subcloning and Assessment of the Expression of Synthetic Botulinum Neurotoxin Type B Binding Domain (BD/B)

Botulism syndrome is caused by Clostridium botulinum neurotoxin. This neurotoxin has 7 serotypes, from A to G. The best way to prevent botulism syndrome caused by BoNT/B is immunization with recombinant binding domains of BoNT/B (due to containing sufficient epitopes to stimulate the immune system). In this study, the expression of the BoNT/B binding domain as a candidate vaccine, was investigated.
At first, the sequence of the BoNT/B binding domain gene was obtained from NCBI website with accession number of EF028399.1. After codon optimization, the gene was synthesized in pUC18 vector. Then, the gene was subcloned in pET32a() expression vector that carries an ampicillin selection marker. PCR, enzymatic digestion, and sequencing were used to confirm subcloning accuracy, and E. coli BL21(DE3) was used for the expression analysis. The accuracy of protein expression was evaluated by electrophoresis and western blotting.
PCR reaction, enzymatic digestion, and sequencing confirmed that the gene of interest has been subcloned appropriately in the vector. The expression analysis by SDS-PAGE and subsequently western blotting, showed that the protein of interest is not expressed in the mentioned expression strain.
Although the gene was successfully subcloned in pET32a() vector, no significant expression of the gene was observed.