Expression and Purification of LigA Antigen, a Surface Lipoprotein in Pathogenic Leptospira Serovars

Chalesh, Aida; Khaki, Pejvak; Moradi Bidhendi, Sohaila; Taghizadeh Tarnabi, Morteza; Tebianian, Majid

doi:10.22092/ari.2025.367948.3450

Expression and Purification of LigA Antigen, a Surface Lipoprotein in Pathogenic Leptospira Serovars

Articles in Press

Document Type : Original Articles

Authors

¹ Department of Microbiology, North Tehran Branch, 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 Razi Vaccine and Serum Research Institute

⁴ Medical Vaccine Department, Razi Vaccine and Serum Research Institute, Agricultural Research, Education and Extension Organization (AREEO), Karaj, Iran

10.22092/ari.2025.367948.3450

Abstract

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 cost-effective antigen(s) against the leptospirosis. Recent advances have identified leptospiral immunoglobulin-like (Lig) proteins as promising candidates for vaccine development. Hence, in this study, recombinant LigA subunit consists of the ligA9, ligA10, ligA11, and ligA12 domains selected as conserved regions of LigA protein. Immunoinformatics approaches. I-TASSER, ProSA, DiscoTope v2.0, and Molprobity were exploited to check the conformational accuracy. Furthermore 10 of the most efficient peptides for MHC-I and II grooves were predicted by ElliPro servers, NetMHCpan 4.1 EL, and NetMHCIIpan 4.1 EL. Ramachandran plot showed acceptable conformation of the selected recombinant protein amino acid residues. The result showed that selected epitopes eliciting both humoral-and cell-mediated immune responses. Hence, construct of the selected epitopes designed in the pET41a+ plasmid and synthesized by General Biosystems. Recombinant plasmids were transferred to E. coli Top10-DH5α and BL21 StarTM (DE3) competent cells for cloning and expression, respectively. Plasmid transformation and purification were confirmed using polymerase chain reaction and enzymatic digestion. Recombinant LigA (r-LigA) expressed in the presence of 0.5 M IPTG at 30˚C for 16 hours. The result of the SDS-PAGE revealed the production of 38-kDa protein that accumulated mostly in inclusion bodies and was purified using the urea method and dialysis. SDS-PAGE shows. r-LigA protein dot blotting confirmed a high degree of accuracy of immunogenicity. The present study revealed that r-LigA is a promising candidate for developing diagnostic and subunit leptospirosis vaccines.

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