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

Abstract

Background and Objectives: Botulism syndrome is caused by Clostridium botulinum neurotoxin. This neurotoxin has seven serotypes ranging from A to G. The best way to prevent botulism syndrome caused by Botulinum neurotoxin (BoNT), is using recombinant vaccine made from its binding domain (due to having sufficient epitopes to stimulate immune system). In this study, the binding domain of BoNT serotype A (BoNT/A), was investigated. Methods: Initially, BoNT/A gene with accession number CP000727.1, was obtained from GenBank and was codon optimized according to the codon usage of E. coli. Then, the sequence was synthesized in pET28a plasmid and then subcloned in pGEX-4T-1 expression plasmid. The subcloning was done using PCR with Pfu DNA polymerase and then double digestion with XmaI and XhoI restriction enzymes. E. coli BL21 strain was used as the expression host. The selected marker for pGEX-4T-1 was ampicillin. Results: PCR and restriction digestion with the mentioned enzymes confirmed the subcloning process. The assessment of gene expression was performed by SDS-PAGE and western blotting (using horse anti-BoNT/A (and then glutathione affinity chromatography was performed. Although, the subcloning was performed successfully, no protein expression was observed.  Conclusion: According to the findings of this study, it seems that other hosts, such as eukaryotic hosts should be used for recombinant expression of BoNT/A binding domain.

Keywords

• clostridium botulinum type a
• binding domain
• subcloning
• gene expression
• vaccines
• synthetic.