Abstract
Abstract Tetanus is an infective disease caused by the Gram-negative bacilli, the Clostridium tetani which release 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. Cholera toxin subunit B (CTB), the potent adjuvant, was genetically linked to the heavy chain of tetanus toxin (Hc) gene to replace alum. Fused CTB-Hc genes were expressed to get recombinant choleratetanus protein as a vaccine. To get the CTB-HC recombinant protein, synthetic CTB-Hc fused genes were designed for synthesis. Computational analysis of the proposed CTB-Hc gene cassette to test restriction sites, primary, secondary and tertiary structure of proposed protein, open reading frames, protein folding and interaction, removal of toxic part of tetanus toxin and cholera toxin and testing of antigenicity were done. Design of final synthetic CTB-Hc gene cassette containing restriction sites in both ends was done and submitted to GenBank. CTB-Hc gene cassette was cloned into pUC57 cloning plasmid. CTB-Hc gene cassette was 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 theh 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. CTBHc gene cassette in both pUC57 and pQE-30 was found to be at right orientation and open reading frame. Analysis of recombinant CTB-Hc fusion protein showed that the protein at right expected molecular weight. In conclusion, construction of DNA segment encoding for non-toxic heavy chain of tetanus toxin gene genetically linked to highly immunogenic non-toxic cholera toxin subunit B gene in one cassette was possible. This lead to the production of recombinant Tetanus-Cholera Double protein Vaccine in one expression system with save and economic feedback.
