Lipopolysaccharide (LPS) or glycolipid antigens of Leptospira interrogans have been candidates as serogroup or serotype specific antigen. In this study, therefore, we prepared the LPS and lipid antigens from L. interrogans serovars lai, icterohaemorrhagiae, copenhageni, canicola, pomona, grippotyphosa, and a Korean isolate 30R. The LPS antigens were analyzed by a polyacrylamide gel electrophoresis and lipid antigens by thin-layer chromatography, respectively. The seroreactivity of the antigens were also examined with homologous or heterologous antisera using an enzyme-linked immunosorbent assay. The LPS antigens from serovar lai and the strain 30R were closely related but different from serovar icterohaemorrhagiae. Particularly, the LPS antigens from serovars icterohaemorrhagiae and grippotyphosa were reactive only with the homologous antisera, thus indicating serovar specificity. However, the LPS antigens of the other serovars were reactive to the heterologous antisera. The lipid antigen of serovar icterohaemorrhagiae reacted only with the homologous antisera. In contrast, lipids of other serovars reacted broadly with heterologous antisera, particularly among serovars lai, copenhageni, canicola, pomona, and the strain 30R. The results thus indicated that the LPS and lipid antigens of L. interrogans may contain serovar-specific as well as cross-reactive epitopes.
Antigens, Bacterial/*analysis ; Chromatography, Thin Layer ; Comparative Study ; Electrophoresis, Polyacrylamide Gel ; Leptospira interrogans/*chemistry/immunology ; Lipids/*analysis/immunology ; Lipopolysaccharides/*analysis/immunology ; Support, Non-U.S. Gov't

OBJECTIVETo construct prokaryotic expression systems of ltB/ctB-lipL41/1 fusion genes, identify immunogenic and adjuvant activities of the products as well as to understand the frequencies of lipL41 gene that carrying and expressing in L. interrogans wild strains and specific antibody levels in sera from patients with leptospirosis.

METHODSPolymerase chain reaction (PCR) with linking primer was applied to construct the fusion genes ltB-lipL41/1 and ctB-lipL41/1. By routine molecular biological techniques, prokaryotic expression systems of the two fusion genes were constructed. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was used to examine expression of the target recombinant proteins rLTB-rLipL41/1 and rCTB-rLipL41/1. Both western blot and Ganglioside-enzyme linked immunosorbent assay (GM-ELISA) were used while immunogenic and adjuvant activities of rLTB-rLipL41/1 and rCTB-rLipL41/1 were measured. PCR and MAT were performed to detect lipL41 gene and expression of the gene in 97 wild strains of L. interrogans, respectively. Antibodies against product of lipL41 gene in serum samples from 228 leptospirosis patients were detected by ELISA.

RESULTSIn comparison with reported corresponding sequences, the similarities of ltB-lipL41/1 and ctB-lipL41/1 fusion genes to nucleotide and putative amino acid sequence were 99.6%-99.9% and 99.8%-100%, respectively. Expression outputs of both rLTB-rLipL41/1 and rCTB-rLipL41/1, mainly presenting with inclusion body, consisting approximate 10% of the total bacterial proteins. Both rLTB-rLipL41/1 and rCTB-rLipL41/1 could combine rabbit anti-rLipL41/1 serum as well as bovine GM1, respectively. 87.6% of the L. interrogans wild strains(85/97) having lipL41 gene while 84.5% (82/97) of the wild strains with rLipL41/1 or rLipL41/2 antiserum were positive for MAT with titers of 1:4-1:128. 84.6% (193/ 228), 78.5% (179/228) from the patients' serum samples were positive for rLipL41/1 and rLipL41/2 antibodies, respectively.

CONCLUSIONltB-lipL41/1 and ctB-lipL41/1 fusion genes and their prokaryotic expression systems were successfully constructed in this study. The two expressed fusion proteins showed qualified immunogenic and adjuvant activities. lipL41 gene was extensively distributed and frequently expressed in different serogroups of L. interrogans. rLTB-rLipL41/1 or rCTB-rLipL41/1 seemed to have had good potential to serve as an antigen in L. interrogans genus-specific vaccine.

Amino Acid Sequence ; Animals ; Antibody Specificity ; Antigens, Bacterial ; biosynthesis ; chemistry ; genetics ; immunology ; Cattle ; Enzyme-Linked Immunosorbent Assay ; Escherichia coli Proteins ; genetics ; Genetic Engineering ; methods ; Humans ; Leptospira interrogans ; genetics ; physiology ; Leptospirosis ; immunology ; Polymerase Chain Reaction ; Recombinant Fusion Proteins ; biosynthesis ; chemistry ; genetics ; immunology ; Sequence Analysis, DNA