Document Type : Original Articles
Authors
1
Department of Microbiology, NT.C., Islamic Azad University, Tehran, Iran.
2
Department of Microbiology, Razi Vaccine and Serum Research Institute, Agricultural Research, Education and Extension Organization (AREEO), Karaj, Iran.
3
Department of Immunology, Razi Vaccine and Serum Research Institute, Agricultural Research, Education and Extension Organization (AREEO), Karaj, Iran.
4
Department of Medical Vaccine, Razi Vaccine and Serum Research Institute, Agricultural Research, Education and Extension Organization (AREEO), Karaj, Iran.
10.32598/ARI.81.1.3450
Abstract
Introduction: Although considerable progress has been made in leptospiral vaccine development, their use is limited because of short-term and serovar-specific immunity. Thus far, many approaches have been used to identify heterologous and costeffective antigen(s) against leptospirosis. Recent advances have identified leptospiral immunoglobulin-like (Lig) proteins as promising candidates for vaccine development.
Materials & Methods: Hence, in this study, the recombinant LigA subunit consists of the ligA9, ligA10, ligA11, and ligA12 domains, were selected as conserved regions of the LigA protein. Immunoinformatics approaches, including I-TASSER, ProSA, DiscoTope v2.0, and Molprobity were utilized to check the conformational accuracy. Furthermore, 10 of the most efficient peptides for MHC-I and II grooves were predicted by the ElliPro, NetMHCpan 4.1 EL and NetMHCIIpan 4.1 EL servers.
Results: The Ramachandran plot showed acceptable conformations of the selected recombinant protein amino acid residues. The results showed that selected epitopes elicit both humoral- and cell-mediated immune responses. Hence, the selected epitopes were constructed in the pET41a+ plasmid and synthesized by General Biosystems. Recombinant plasmids were transferred to Escherichia coli Top10-DH5α and BL21 StarTM (DE3) competent cells for cloning and expression, respectively. Plasmid transformation and purification were confirmed using polymerase chain reaction (PCR) and enzymatic digestion. Recombinant LigA (r-LigA) was expressed in the presence of 0.5 M IPTG at 30˚C for 16 hours. The sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE) result revealed the production of 38-kDa protein, which accumulated mostly in inclusion bodies and was purified using the urea method and dialysis. Dot blotting of the r-LigA protein confirmed a high degree of accuracy of immunogenicity.
Conclusion: The present study revealed that r-LigA is a promising candidate for developing diagnostic and subunit leptospirosis vaccines.
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