Jundishapur J Microbiol. 2019 May; 12(5):e66502. Published online 2019 May 4. doi: 10.5812/jjm.66502. Research Article Antigenicity Identification of a Novel Recombinant Multi-Epitope Antigen Based on FlaA and UreB Antigens of Helicobacter pylori Zeinab Hamzehloo 1 , Ghasem Mosayebi 2 , Behzad Khansarinejad 2 , Mina Zolfaghari 2 and Hamid Abtahi 2, * 1 Department of Biotechnology, School of Medicine, Arak University of Medical Sciences, Arak, Iran 2 Molecular and Medical Research Center, Arak University of Medical Sciences, Arak, Iran * Corresponding author: Molecular and Medical Research Center, Arak University of Medical Sciences, Arak, Iran. Email: abtahi@arakmu.ac.ir Received 2018 January 20; Revised 2019 March 10; Accepted 2019 March 14. Abstract Background: Helicobacter pylori is the main cause of stomach ulcers and gastric cancer. Hence, the diagnosis, treatment, and pre- vention of H. pylori infection can considerably reduce the fatality. Objectives: This study aimed to construct a dual-antigen protein by combining the antigenic regions of UreB and FlaA of H. pylori and determine its antigenicity as a promising vaccine and serodiagnosis candidate. Methods: The antigenic regions of FlaA and UreB were detected by immunological bioinformatics, amplified and joined together by polymerase chain reaction (PCR) with special primers containing linker sequences. Then, it was cloned into pET-32a and after ex- pression and purification of the recombinant multi-epitope protein (rFlaA-UreB), its antigenicity was evaluated by immunoblotting using the sera of infected patients. Results: DNA sequencing and enzyme digestion analysis showed the rFlaA-UreB gene was successfully inserted into pET32a. The recombinant protein was produced and purified via affinity chromatography and its molecular weight was similar to what had been predicted. Moreover, data indicated that rFlaA-UreB was recognized by all patients’ sera and its sensitivity and specificity were high. Conclusions: Although the developed recombinant multi-epitope protein was very smaller and lighter than the natural forms of these two critical antigens, they all had close antigenic properties. Therefore, this recombinant protein can be an important antigen in the diagnosis and vaccination against H. pylori. Keywords: Flagellum, Recombinant Protein, Urease, Helicobacter pylori 1. Background Most people in the world have a history of infection with Helicobacter pylori, a Gram-negative, spiral-shaped, and microaerophilic bacillus that resides the human gas- tric and duodenal mucosa. Persistent, chronic infection with H. pylori induces stomach ulcers and gastric cancer (1- 4). Helicobacter pylori is recognized as a class I human car- cinogenic agent and one of the etiological agents in hu- man gastric adenocarcinoma (5). Thus, it seems that pre- vention, early detection, and treatment of this infection can decrease morbidity and mortality rates in infected pa- tients. Unfortunately, current treatment against H. pylori infection is associated with some drawbacks such as re- infection, increased antibiotic resistance, side effects, pa- tient compliance, and high cost (6, 7). Therefore, vaccina- tion against this bacterium could be a potential way to con- trol the H. pylori infection. Epitope-based vaccines are a new strategy for increas- ing immune response to pathogens. Some advantages of an epitope vaccine include (i) increased safety, (ii) in- creased immunogenicity of predefined epitopes, and (iii) ability to focus immune responses on conserved epitopes (8). However, research has proven that immunity created against H. pylori using a single antigen does not lead to ef- fective protection, rather effective immunity against this infection is achieved via a combination of various antigens (9-11). Some commercial diagnostic kits used to detect H. pylori infection utilize a combination of antigens because this strategy provides high sensitivity and specificity com- pared to a single antigen (12-15). Based on previous studies, UreB and FlaA are excellent candidates for the production of vaccines and diagnostic antigens. UreB (the B subunit of urease and one of the four subunits of this enzyme) elicits an immune response; Copyright © 2019, Author(s). This is an open-access article distributed under the terms of the Creative Commons Attribution-NonCommercial 4.0 International License (http://creativecommons.org/licenses/by-nc/4.0/) which permits copy and redistribute the material just in noncommercial usages, provided the original work is properly cited.