Cloning of Binding Domain of Clostridium botlllinum Toxin Type A in Escherichia coli

Neurotoxin of Clostridium botulinum is one of the most potent known toxins in nature, which can cause an often fatal food poisoning. However, recently the use ofbinding do main of the toxin has also been proposed as a candidate vaccine. In this study coding region for this do main was amplified from genomic DNA of Clostridium botulinum type A using a pair of primers containing BamHI and HindII!. The amplified l.3kb fragment corresponding to the gene ofinterest was cloned in pUCI8 after verification with different restriction enzymes. The construct was then transformed into competent Escherichia coli DH5a and the recombinant clone was selected on plates containing ampicillin. The plasmid purified from these clones was used for sequencing by dideoxy chain termination method. The sequence thus obtained was verified by comparison with published sequence, which showed the authencity of the PCR product. This fragment was subcloned in an IPTG inducible expression vector pQE30 using E. coli M 15 as a host and expression was analyzed by RT-PCR.