Expression and Immunogenicity Analysis of Recombinant Leptospira Interrogans Surface Protein LigA in Mouse Model

TL;DR

This study produced a recombinant protein from Leptospira interrogans and showed it can stimulate an immune response in mice, suggesting it could be used for vaccines or diagnostics.

Contribution

The study demonstrates the successful production and immunogenicity of r-LigA in a mouse model for potential leptospirosis vaccine development.

Findings

The optimal epitope of LigA was identified between amino acids 852 and 1210.

r-LigA was purified at 1050 µg/mL and showed high immunogenicity in mice.

The recombinant protein is a promising candidate for leptospirosis diagnostics and vaccines.

Abstract

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.

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.

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.

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.

These findings support r‐LigA as a strong candidate for future leptospirosis diagnostic tools and subunit vaccine development.

Implemented strategies in designing, production, purification and immunogenicity of the recombinant Leptospiral immunoglobulin‐like surface protein‐A (r‐Lig A) as a vaccine candidate.

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