OBJECTIVETo investigate the immune-functional epitopes and inflammation-inducing effects of the major outer envelope proteins of Leptospira interrogans.

METHODSNi-NTA affinity chromatography was established to extract the target recombinant proteins rOmpL1/1 and OmpL1/2, LipL32/1 and rLipL32/2, LipL41/1 and rLipL41/2 expressed by the different genotypes. By using Signal P-NN software in Signal P3.0 prediction server, EMBOSS software in propred MHC class-II binding peptide prediction-ProPred prediction server, the possible signal peptides, MHC-II binding peptides and lymphocyte B epitopes were analyzed. The IL-1, IL-8 and TNF-alpha secretion in human umbilical vein endothelial cell line EVC-304 induced by target recombinant proteins were measured by ELISA.

RESULTSUnder the inducement of IPTG, the constructed prokaryotic systems efficiently expressed rOmpL1/1 and rOmpL1/2, rLipL32/1 and rLipL32/2, and rLipL41/1 and rLipL41/2 with outputs of 30% and 15%, 40% and 35%, and 15% and 10% of the total bacterial proteins, respectively. Each of the purified target recombinant proteins showed a single protein band in SDS-PAGE. The signal peptides of OmpL1s, LipL32/1 and LipL32/2, and LipL41s were located at the N ends of 1-24, 1-21 and 1-24, and 1-24 amino acid residuals, respectively. OmpL1s, LipL32s and LipL41s displayed 2,2 and 1 same major epitopes of MHC-II binding peptides and lymphocyte B and OmpL1/2 had another one (59-78). The different dosages of rOmpL1s, rLipL32s and rLipL41s increased the secretion of IL-1alpha , IL-8 and TNF-alpha (P<0.05) in EVC-304 cells. The IL-1alpha levels reached the highest at the 24 h and then declined,while the IL-8 and TNF-alpha levels after 48 h treatment were higher that those after 24 h.

CONCLUSIONThe expression products in ompL1/1, lipL32 or lipL41 genotypes of L.interrogans contain similar immune functional epitopes. rOmpL1/1 and rOmpL1/2, rLipL32/1 and rLipL32/2, and rLipL41/1 and rLipL41/2 are able to directly induce inflammatory reaction in EVC-304 cells.