Recombinant multiepitope protein for diagnosis of leptospirosis

Abstract

Leptospirosis is an emerging infectious disease and is considered to be the most widespread zoonotic disease in the world. It can be misdiagnosed because manifestations of this febrile disease vary from mild flu-like symptoms to severe illness involving vital organs such as the liver and lungs. Therefore, accurate diagnosis for differentiation of leptospirosis from other pyrogenic infections prevailing in the same locality is imperative for proper treatment. Here, we report a customized recombinant leptospirosis multiepitope protein (r-LMP) that can specifically detect the immunoglobulin class of anti-leptospirosis antibodies in patient sera. Immunodominant epitopes from leptospire outer membrane proteins OmpL1, LipL21, and LipL32 were predicted and confirmed using phage display and immunity reaction. On the basis of the sequences of the identified epitopes, five major immunodominant epitopes were selected to construct a synthetic gene, recombinant lmp. The recombinant lmp gene was doubled and expressed in Escherichia coli. The recombinant protein was purified and used as an antigen to develop an enzyme-linked immunosorbent assay for detection of special immunoglobulin M (IgM) or IgG in sera from patients with leptospirosis or other febrile illnesses and healthy subjects. The results showed that the r-LMP protein recognized IgG and IgM in all the sera that were microscope agglutination test positive, and there were no cross-reactions with other patient sera. This approach of creating customized antigens coupled to overexpression and simple purification offers a promising alternative option for leptospirosis diagnosis, with the potential to circumvent the drawbacks of whole-leptospirosis-antigen-based assays.

FIG. 1.

Design of recombinant multiepitope protein. (A) Complete amino acid sequence of one copy of the recombinant multiepitope protein. The epitope amino acids are shown in normal font, and the tetraglyclyl linkers are in bold font and underlined. (B) Computer-generated representation of the codon protein unit. The protein amino acid sequence was imported into the three-dimensional position-specific scoring matrix Web server (3D-PSSM) and visualized using ViewerLite software.

FIG. 2.

Expression of recombinant protein in E. coli BL21(DE3) plysS. Results are shown for localization of r-LMP protein in induced cell lysates. An aliquot of the induced cell lysate prepared by sonication in 1× PBS buffer was separated into supernatant and pellet fractions and analyzed by SDS-PAGE. Lane 1, protein ladder; lane 2, pET28a; lanes 3 and 4, supernatant fractions; lanes 5 and 6, pellet fractions. The position of the r-LMP fusion protein is indicated by arrows.

FIG. 3.

Purification and characterization of r-LMP protein. (A) SDS-PAGE analysis of purified r-LMP protein. Lane 1, protein ladder; lane 2, pET28a; lane 3, pET28a-2LMP induced by 1.0 mM IPTG; lanes 4 to 7, pET28a-2LMP induced by 1.0 mM IPTG, eluted with the NTA-0, NTA-40, NTA-100, and NTA-1000 buffers, respectively. (B) Western blot analysis of r-LMP protein by use of a Leptospira interrogans monoclonal antibody. Lanes 1 to 3, hybridized strip of pET28a, pET28a-2lmp, and purified multiepitope protein with an anti-leptospire antibody. The positions of the hybridized strip of r-LMP fusion protein and the antibody are indicated by arrows.