Cloning of the gene encoding neurotoxin heavy chain of Clostridium botulinum in E. coli
Subject Areas : Medical Microbiology
Abbas Doosti
1
(Biotechnology Research Center, Islamic Azad University, Shahrekord Branch, Shahrekord, Iran.)
Keywords: Cloning, Botulism, Neurotoxin, Heavy Chain,
Abstract :
Background and Objectives: Botulism is a food poisoning problem in which the transmission of neuromuscular stimuli is disrupted due to the activity of botulinum neurotoxins (BoNT) produced by Clostridium botulinum. The botulinum neurotoxins are the most known toxic substances that cause flaccid paralysis due to preventing the release of the neurotransmitter acetylcholine at neuromuscular junctions. The aim of this study was cloning the heavy chain (HC) domain of Clostridium botulinum neurotoxin gene in E. coli as a gene vaccine candidate. Material and Methods: In this study, the heavy chain of neurotoxin of Clostridium botulinum was fully amplified based on PCR and the specific primers. DNA fragment of heavy chain of neurotoxin gene was cloned by T/A cloning technique in PCR 8/GW/TOPO vector and the clone was transformed into E. coli. Results: Cloning of 2530 bp of neurotoxin was confirmed by PCR. The results of next step showed that the heavy chain of Clostridium botulinum neurotoxin was successfully cloned in E. coli . Final confirmation of construct was done by BamHI and HindIII restriction enzymes. Conclusion: According to the results, the amplified gene can be used as a target for gene vaccines against botulism in the future.
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