Cloning, Fusion, and Expression of Domain a-1 Protective Antigen (PA20) of Bacillus anthracis and N-Terminal ipaD Gene of Shigella in E. coli

Abstract

Background and Objectives: Genus Shigella is one of the main causes of diarrhea in human and Bacillus anthracis is the cause of anthrax (a zoonotic disease). IpaD protein is one of the most important virulence factors of Shigella . The cloning of N-terminal ipaD gene along with pa20 gene that have a role in immunization, can be considered as a recombinant vaccine candidate. In this study, we investigated the expression of the recombinant protein domain a-1 protective antigen (PA20) of Bacillus anthracis and N-terminal ipaD gene of Shigella in E. coli . Methods: In this experimental study, primers were designed for pa20 gene and PCR was performed to amplify this fragment. Then, the amplified fragment was cloned into pGEM-T Easy Vector Systems. The pa20 gene was cut using restriction enzymes EcoRI and XhoI and finally, pa20 gene was fused to ipaD gene. pET28a (+) Vector containing the gene cassette ipaD-pa20 was prepared and transformed into E. coli strain BL21 (DE3), and the expression of gene cassette was studied. Results: In this study, the ipaD-pa20 fusion genes in the expression vector pET28a(+), were confirmed by PCR and enzyme digestion. Also, the produced recombinant protein were confirmed by SDS-PAGE and Western blotting. Conclusion: Considering the detection of PA antigen by PA20 antibody and its apoptosis induction and immunization property of the produced IpaD antigen, it can be proposed as a recombinant vaccine candidate against types of Shigella and Bacillus anthracis.