Cloning, fusion and expression ctB and N-terminal ipaD gene cassette in E. coli

Shigella dysenteriae one of the main causes of diarrhea in humans، but there is no vaccine against it. IpaD protein is one of the most important virulence factors in pathogenic shigella. The cloning N-terminal ipaD genes with ctB genes that have a role in adjuvant and carrier as recombinant vaccine can caused enhance the mucosal immune response. designing primers for genes ctB and ipaD were carried out based on the construction of gene cassettes، respectively. PCR reactions were performed to amplify the fragments and amplified fragments were cloned into pGEM-Teasy vector. Both the vector cut by restriction enzymes HindIII and XhoI and ipaD gene to gene ctB finally were Fusion. The ctB-ipaD gene cassette and expression vector pET28a (+) cuted by SalI and HindIII restriction enzymes. The cloning ctB-ipaD cassette was performed in the expression vector and expression of gene cassettes. In this study، the N-terminal ipaD as vaccine candidate antigen was genetically linked to the C terminal of ctB which has a carrier and adjuvant role. Fusing ctB-ipaD in the expression vector pET28a (+) is confirmed by sequencing، PCR and digested with restriction enzymes. The recombinant proteins produced is confirmed by SDS-PAGE and Western blot. According to previous and similar studies، product cassette ctB-ipaD and expression its was expected. Is hoped to protein expression of this gene cassette and the production of antibodies could be achieved the candidate vaccine against Shigella.