Tehran University of Medical Sciences Iranian Journal of Allergy, Asthma and Immunology 1735-1502 20 4 2021 08 07 484 493 Fahimeh Sadat Emami Mahmoudabadi Department of Biology, Faculty of Basic Science, Shahed University, Tehran, Iran Atina Vakili Department of Biology, Faculty of Basic Science, Shahed University, Tehran, Iran Shahram Nazarian Department of Biology, Faculty of Science, Imam Hussein University, Tehran, Iran Jafar Amani Applied Microbiology Research center, System Biology and Poisonings Institute, Baqiyatallah University of Medical Sciences, Tehran, Iran Seyed Latif Mousavi Gargari Department of Biology, Faculty of Basic Science, Shahed University, Tehran, Iran 2020 04 08 2021 02 07 Shigella and Salmonella cause serious problems in many subjects, including young children and the elderly, especially in developing countries. Chimeric proteins carrying immunogens increase immune response. In-silico tools are applied to design vaccine candidates. Invasion plasmid antigens D (ipaD) gene is one of the Shigella virulence factors. The N-terminal region of the IpaD plays a significant role in invading the host cell. Invasion protein H (invH) gene plays important role in bacterial adherence and entry into epithelial cells. A recombinant chimeric construct, containing IpaD and InvH was designed and used as a vaccine candidate against Shigella and Salmonella enteritidis. After bioinformatics assessments, the construct was designed, synthesized, and expressed in E.coli. Chimeric protein, IpaD, and InvH were purified with Ni-NTA chromatography. Purified proteins were confirmed with western blotting and then were injected into separate mice groups. The antibody titer was estimated with an enzyme-linked immunosorbent assay (ELISA). Mice were challenged with 10, 100, and 1000 LD50 of Salmonella, and the sereny test was performed for Shigella. The Codon adaptation index of the chimeric gene was increased to 0.84. Validation results showed that 97.9% of residues lie in the favored or additional allowed region of the Ramachandran plot. A significant antibody rise was observed in all test groups. The immunized mice with chimer and InvH could tolerate 100 LD50 of Salmonella. In the sereny test, the application of bacteria treated with immunized mice sera of both antigens showed no infection in Guinea pigs' eyes. The recombinant protein could protect animal models against Salmonella and Shigella and therefore can be considered as a suitable vaccine candidate against these two pathogens. https://ijaai.tums.ac.ir/index.php/ijaai/article/view/2779 https://ijaai.tums.ac.ir/index.php/ijaai/article/download/2779/1721