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 human. IpaD antigen، play an important role in the creation and induction of infection by Shigella. The challenges of use ipad in design mucosal vaccines against Shigella is limited potency and non transferred to mucosal tissues. by linking IpaD antigen to Ricin toxin B Subunit as carrier and adjuvant we can eliminate challenges of production of mucosal vaccines. This study aimed to construct a gene cassette containing the N-terminal region of ipaD gene which was connected to the RTB gene as a new approach generating of mucosal vaccines against Shigella. genomic DNA of Ricins communis was extracted by CTAB method. Using specific primers، the fragment of 819 nucleotide of RTB gene with linker were amplified. PCR reactions for amplification of the ipaD gene were carried out. Each of the amplified fragments was cloned into the pGEM-T vector. In order to construct a gene cassette، ipaD was ejected from the vector pGEM-ipaD with enzymes XhoI / 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 last information، this study is a first report of design، construction of RTB-ipaD gene cassette، that can use for generating of a suitable candidate mucosal vaccine for shigellosis. This study also creates a strategic direction for the production of fusion proteins and immunological studies.