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 20 th -120 th ), intermediate (amino acid sequence 120 th -150 th ), and C-terminus (CrLipL32, amino acid sequence 160 th -260 th ) 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.

Production and characterization of immunoglobulin G anti-rLipL32 antibody as a biomarker for the diagnosis of leptospirosis

“…Several Leptospira proteins, such as LipL32, LigA, OmpL1, and LipL21, have been used as antigens for antibody detection [ 10 ]. Among the OMPs, LipL32 is widely used as a biomarker for antigen detection and is a potential candidate for vaccine and therapy production [ 27 ]. Therefore, this protein can be used in the development of promising diagnostic tools [ 33 ].…”

“…The OMPs of Leptospira are divided into three classes: Lips (lipoproteins), including LipL42, LipL32, and LipL24; transmembrane proteins, including OmpL1; and peripheral proteins, including LipL42 [ 23 ]. LipL32 is an outer membrane lipoprotein of pathogenic Leptospira , measuring 32-kDa, expressed at high levels during infection, highly conserved, and the most abundant lipoprotein in Leptospira interrogans [ 21 , 24 – 27 ]. This protein can be used as a diagnostic antigen in patients with acute and convalescent leptospirosis [ 28 ].…”