Fusion and cloning of BoNT/ A & B binding domains

Botulinum neurotoxins (BoNTs) are potent bacterial toxins that cause paralysis at femtomolar concentrations by blocking acetylcholine release in motor neurons. Each BoNT is composed of a 50 kDa light chain of that acts as a metalloprotease، and of a 100 kDa heavy chain. The carboxy terminal domain of heavy chain (HC) that mediates the binding to specific cell surface receptors. The HC domain has considerable immunogenicity without any significant toxicity. In this study the BoNT/A& B-HC were joined together for producing a recombinant chimeric protein as a vaccine candidate. For this، the relative genes were synthesized in pET28a (+) expression vector separately. Then appropriate primers were designed to amplify the BoNT/B-HC gene. The restriction sites for NcoI and NdeI endonuclease were placed at 5'' end of forward and reverse primers respectively. The PCR products then were digested whit NcoI and NdeI enzymes. At the same time the construct of pET28a (+) –BoNT/A-HC gene was digested by the same enzymes. Then these digested fragments were joined together by T4 DNA ligase. The recombinant construct then introduced into E. coli DH5α strain. Our enzymatic digestion، PCR amplification and sequencing of recombinant construct confirmed correct fusion of BoNT/B-HC and BoNT/A-HC genes