alexa Abstract | Fusion and cloning of the binding domains of botulinum neurotoxin type A and B in E. coli DH5α

European Journal of Experimental Biology
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Research Article Open Access

Abstract

Botulinum neurotoxins (BoNTs) are the most potent bacterial toxins that cause paralysis at femtomolar concentrations by blocking acetylcholine neurotransmitter release. Each BoNT is composed of a light chain (50 kDa) which acts as a metalloprotease, and a heavy chain (100 kDa). The carboxyl-terminal domain (HC) of heavy chain mediates binding to the specific cell surface receptors and has a considerable immunogenicity without any significant toxicity. In this study the binding domains of BoNT/A,B were joined together to produce a recombinant chimeric protein as a vaccine candidate. The relative genes were synthesized in pET28a(+) expression vector separately then appropriate primers were designed to amplify the binding domain gene of BoNT/B. 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 with NcoI and NdeI enzymes. At the same time the construct of pET28a (+) and binding domain A gene was digested with the same enzymes. Digested fragments were joined together by T4 DNA ligase. The recombinant construct then introduced into E.coli DH5α. Our enzymatic digestion, PCR amplification and sequencing of recombinant construct confirmed the correct fusion of BoNT/B-HC and BoNT/A-HC genes to produce a chimeric gene.

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Author(s): Bahram Hamidi Firouz Ebrahimi Abbas Hajizadeh and Hani Keshavarz Alikhani

Keywords

Botulinum neurotoxin, binding domain, chimeric gene, chimeric vaccine, Botulinum neurotoxin, binding domain, chimeric gene, chimeric vaccine

 
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