Isolation, Cloning and Fusion of N-Terminal Region of ipaD Shigella dysenteriea and Ricin Toxin B Subunit

Shigella dysenteriae is an important pathogen that induces acute enteric infection in hu‑ mans. IpaD antigen plays an important role in the creation and induction of infection by Shigella. The challenges of using ipad in designing mucosal vaccines against Shigella are the limited potency and lack of transfer to mucosal tissues. By linking IpaD antigen to Ricin toxin B subunit as carrier and adjuvant we can overcome the challenges of producing mu‑ cosal vaccines. This study aimed to construct a gene cassette containing the N‑terminal region of ipaD gene connected to the RTB gene as a new approach for generating mucosal vaccines against Shigella. Genomic DNA of Ricins communis was extracted by the CTAB method. Using specific prim‑ ers a fragment containing 819 nucleotides of the RTB gene with linker was amplified. PCR was performed to amplify the ipaD gene. Each amplified fragment was cloned into the pGEM‑T vector. In order to construct a gene cassette ipaD was ejected from the vector pGEM‑ipaD with enzymesXhoI / HindIII. Subsequently the recombinant plasmid pGEM‑ RTB was cut with XhoI / HindIII enzymes. During the process of ligation ipaD was inserted into the pGEM‑RTB recombinant plasmid to construct RTB‑ipaD gene cassette and then the gene cassette was sequenced by the sequencer. After confirming the cloning of N‑ terminal region of ipaD and RTB genes accurate construction of the gene cassette was confirmed by PCR Nested‑PCR restriction enzyme and sequencing. Base on the latest information this is the first report of designing and construction of RTB‑ ipaD gene cassette which can be used for generating a suitable candidate mucosal vaccine agaist shigellosis. This study also creates a strategic direction for the production of fusion proteins and immunological studies.