Document Type : Microbiology and Immunology
Authors
1
Department of Microbiology and Food Hygiene, Faculty of Veterinary Medicine, Lorestan University, Khorramabad, Iran
2
Department of Animal Sciences, Faculty of Agriculture, Lorestan University, Khorramabad, Iran
3
Department of Microbiology and Food Hygiene, Faculty of Veterinary Medicine, Lorestan University, Khorramabad, Iran.
4
Department of Basic Sciences, Faculty of Veterinary Medicine, Lorestan University, Khorramabad, Iran.
Abstract
Background: Salmonella Typhimurium, a Gram-negative bacterium, causes salmonellosis in humans and livestock. Beyond hygiene measures, vaccination with next-generation vaccines exhibiting minimal side effects represents a fundamental approach to combating this deadly disease.
Objective: This study aimed to design and construct a recombinant immunogenic chimera structure comprising the OmpL antigen from Salmonella Typhimurium and the HBHA adjuvant, and to evaluate its expression in a prokaryotic system.
Methods: For this purpose, the DNA extraction process was performed from Salmonella Typhimurium bacteria and also the B.C.G vaccine. Each of the OmpL and HBHA genes was amplified using flagded primers and connected to each other using the SOE-PCR reaction. Next, to connect the recombinant HBHA-OmpL chimera to the pET22b(+) expression vector, both were first subjected to double-digestion treatment and then ligated together using T4 ligase enzyme. Finally, the recombinant construct was transferred into E.coli (DH5α) and E.coli BL21 (DE3) using a heat shock process. After gene expression induction, protein purification was performed using a nickel NTA chromatography resin column. Finally, the quality of recombinant protein expression was evaluated using 12% SDS-PAGE gel.
Results: Amplification of HBHA and OmpL genes, construction of the chimeric structure, and transformation of the expression vector into E. coli DH5α and BL21(DE3) cells were validated by PCR, SOE-PCR, and colony-PCR, respectively. Successful expression and purification of the 49.5 kDa recombinant HBHA-OmpL protein was confirmes by 12% SDS-PAGE.
Conclusion: The recombinant HBHA-OmpL immunogenic construct was successfully expressed in a prokaryotic system, demonstrating feasibility for developing a candidate vaccine against Salmonella Typhimurium.
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