Determination of the immunogenic region in the LipL32 protein of Leptospira

Abstract

Leptospirosis is a zoonotic disease caused by the pathogenic species of Leptospira. The initial symptoms include fever, myalgia, nausea, skin rash, chills, and headache, which can be misdiagnosed. LipL32 is the highly conserved and abundant outer membrane protein (OMP) of Leptospira, which is used as an antigen in serodiagnostic assays. We used three in silico methods to predict the immunodominant regions in the full-length LipL32 protein. We identified three regions, namely the N-terminus (NrLipL32, amino acid sequence 20th-120th), intermediate (amino acid sequence 120th-150th), and C-terminus (CrLipL32, amino acid sequence 160th-260th) regions. The full-length protein and two larger fragments were cloned into the pET22b plasmid and expressed in Escherichia coli BL21 (DE3). The purified proteins were used as antigens in an ELISA to detect Leptospira-specific antibodies. The CrLipL32 ELISA showed the highest sensitivity for IgM (73.3%) and IgG (65%), followed by the full-length rLipL32 ELISA (IgM 68% and IgG 60%). The full-length rLipL32 ELISA showed high specificity (IgM 85% and IgG 75%), followed by the NrLipL32 ELISA (IgM 75% and IgG 60%). The intermediate fragment showed very low sensitivity (IgM 17% and IgG 2%). The sensitivity of the rLipL32 ELISA could be enhanced by adding other OMPs of Leptospira.