Cloning and expression of Corynebacterium diphtheriae toxin gene

Diphtheria is an acute bacterial disease caused by oxygenic strains of Corynebacteria. Fatality rate of this disease, between 5 and 10%. In children under 5 years and adults, the fatality rate may be as much as 20%. Outbreaks, although very rare, still occur worldwide, even in developed nations. Diphtheria toxin (DT) is the major virulence factor for these organisms. The aim of this study was cloning and expression diphtheria toxin gene to produce recombinant protein and application in next investigations. The bacterial DNA was extracted and amplification of diphtheria toxin gene was carried out with specific primers. This gene was cloned in pTZ57R/T vector and sub cloned into pETDuet-1 expression vector then recombinant plasmid was transformed into BL21 of Escherichia coli strain and induced by IPTG. Diphtheria toxin gene was amplified successfully and cloned in pTZ57R. Recombinant plasmid was digested by restriction enzymes and released fragment (diphtheria toxin gene) sub cloned in pETDuet-1 expression vector and expressed protein was analysed in serological assay. In this study, the diphtheria toxin gene was cloned in pETDuet-1 expression vector and confirmed by sequencing and restriction analysis then recombinant plasmid was transformed in BL21 expression cell.