Abstract
The construction of a hexahistidine-tagged version of the B fragment of diphtheria toxin (DTB) represents an important step in the study of the biological properties of DTB because it will permit the production of pure recombinant DTB (rDTB) in less time and with higher yields than currently available. In the present study, the genomic DNA of the Corynebacterium diphtheriae Park Williams 8 (PW8) vaccine strain was used as a template for PCR amplification of the dtb gene. After amplification, the dtb gene was cloned and expressed in competent Escherichia coli M15 cells using the expression vector pQE-30. The lysate obtained from transformed E. coli cells containing the rDTB PW8 was clarified by centrifugation and purified by affinity chromatography. The homogeneity of the purified rDTB PW8 was confirmed by immunoblotting using mouse polyclonal anti-diphtheria toxoid antibodies and the immune response induced in animals with rDTB PW8 was evaluated by ELISA and dermonecrotic neutralization assays. The main result of the present study was an alternative and accessible method for the expression and purification of immunogenically reactive rDTB PW8 using commercially available systems. Data also provided preliminary evidence that rabbits immunized with rDTB PW8 are able to mount a neutralizing response against the challenge with toxigenic C. diphtheriae.
Highlights
Diphtheria toxin (DT) is an A-B type protein toxin produced by Corynebacterium diphtheriae [1,2,3,4]
Amplification of the dtb gene of the C. diphtheriae Park Williams 8 (PW8) vaccine strain by PCR
The clarified rDTBPW8 lysate was purified in one step using nickel-coated agarose beads and the eluted material was submitted to analysis by SDS-PAGE
Summary
Diphtheria toxin (DT) is an A-B type protein toxin produced by Corynebacterium diphtheriae [1,2,3,4]. The B fragment (DTB) binds to the receptor on the host cell surface and mediates the translocation of the A fragment (DTA) through the cell membrane, which inactivates the protein synthesis elongation factor 2 in some mammalian cells [5,6,7,8,9]. The expression of rDTB in bacteria was initially considered difficult, due in part to the fact that DTB without DTA was found to be rapidly degraded during the process [13,14]. Attempts to produce immunogenically reactive rDTB in bacteria in a more accessible form using newer expression systems are of interest. The objective of the present study was to express the dtb gene of the Park Williams 8 (PW8) C. diphtheriae vaccine strain to produce the immunogenically reactive rDTBPW8 using commercially available expression and purification systems
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