Background and aims: Tetanus toxin, the product of Clostridium tetani, is the causative agent of fatal disease tetanus. Nowadays, toxoid form of this toxin used as a vaccine against this disease. Tetanus toxin is composed of three chains: L, HN and Hc. Hc chain is immunogenic and binding domain of toxin. Owing to this fact, Hc is considered as candidate for vaccine. The aim of this study was the design and development Hc gene and its synthesis, expression and expression optimization and purification of recombinant protein as vaccine candidate. Methods: In this laboratory study, after bioinformatics study and gene optimization, the Hc chain gene was ordered to synthase and clone in pET28a vector. The integrity of Hc gene was confirmed by restriction digestion methods. Then, this construct was transformed in E.coli. Subsequently, Gene expression, recombinant protein purification was performed and this protein was confirmed by western blotting technique. Results: The Hc gene was confirmed using bioinformatics and molecular analysis. A 1356 bp band was observed in agarose gel and a 50 kDa band was detected in SDS-PAGE and nitrocellulose membrane (western blot). Recombinant protein was produced in both soluble and insoluble forms (inclusion body). These two forms of proteins were purified in high level by native and denatured methods. Conclusion: Regarding to more advantages of recombinant Hc protein to tetanus toxoid, it seems Hc protein as a suitable vaccine candidate can be replaced with tetanus toxoid vaccine. [ABSTRACT FROM AUTHOR]