Abstract
Tetanus is an infective disease caused by the Gram-negative bacilli, the Clostridium tetani which releases its toxin causing muscular spasm which may terminate with death. Current tetanus vaccine, in the time it is effective, it is laborious and costly to produce and needs alum as an adjuvant. The aim of this work was to produce non-toxic part of tetanus toxin genetically joined to the potent adjuvant cholera toxin subunit B ( CTB) to be used as a vaccine. The CTB was genetically linked to heavy chain of tetanus toxin ( Hc) gene to replace alum. Computational analysis of the proposed CTB-Hc gene cassette ( insert) to test restriction sites, primary, secondary and tertiary structure of proposed protein, open reading frames, protein folding and interaction and testing of antigenicity were done. Design of final synthetic insert containing restriction sites in both ends was done and submitted to GenBank. The insert was cloned into pUC57 cloning plasmid and then subcloned into pQE-30 expression plasmid. M15 bacteria were transformed with CTB-Hc-pQE30 recombinant vector and recombinant CTB- Hc protein was expressed and purified. Identity of purified protein was tested using SDS-PAGE. The gene construct submitted to GenBank was given the accession No: KX022510. Analysis of expected fusion protein showed that the proposed protein is immunogenic and in right 3D structure with no interference of both proteins on antigenic determinant of each other. The insert in both pUC57 and pQE-30 was found to be at right orientation and open reading frames. Analysis of recombinant CTB- Hc fusion protein showed that the protein at right expected molecular weight.
In conclusion, construction of DNA segment encoding for the non-toxic Hc gene genetically linked to the highly immunogenic non-toxic CTB gene in one cassette was possible. This lead to the production of recombinant tetanus-cholera double protein in one expression system with a save and economic feedback. Further studies to test doses and prolongation of the vaccine are needed.