Abstract
BACKGROUND: Pathogenic strains of spirochetes of Leptospira spp. cause a globally distributed zoonotic disease called leptospirosis. The disease has several clinical manifestations, ranging from asymptomatic and subclinical infection to fatal and severe forms. HYPOTHESIS/OBJECTIVES: The aim of this study was to produce a recombinant Leptospiral immunoglobulin-like surface protein-A (r-LigA) antigen of Leptospira interrogans in a prokaryotic expression system and to assess its efficacy in a mouse model. MATERIALS AND METHODS: The optimal epitopes of the LigA protein were identified via bioinformatics studies. The pET32a(+)-LigA plasmid construct was cloned into E. coli Top10-DH5α, expressed in E. coli pLysS strains, and subjected to different IPTG concentrations at different times and temperatures. The expressed r-LigA was purified using nickel-affinity (Ni-NTA) chromatography from the insoluble fraction and reassessed by SDS-PAGE, western blotting, dot blotting, and Bradford assay. Female Balb/C mice were immunised subcutaneously with r-LigA alone or emulsified in Freund's adjuvant and subsequently boosted at 2 and 4 weeks. Specific antibody levels were evaluated by indirect ELISA. RESULTS: Bioinformatics analysis identified the key antigenic region of LigA spanning amino acids 852 to 1210. Colony PCR and digestion confirmed the successful transformation. Induction using 0.5 mM IPTG at 30°C for 5 h was found to be optimal. Overexpression of r-LigA under optimised conditions accumulated proteins as inclusion bodies. Purification of r-LigA under native conditions using optimised Ni-NTA yielded 1050 µg/mL protein and high immunogenicity by effectively stimulating the immune system in female Balb/C mice. CONCLUSIONS: These findings support r-LigA as a strong candidate for future leptospirosis diagnostic tools and subunit vaccine development.