Study On The Immune Functional Epitopes And Inflammation-induced Effects Of The Major Outer Envelope Proteins Of Leptospira Interrogans

Leptospirosis is a world-spread infectious anthropozoonosis caused by pathogenic Leptospira species. These highly invasive spirochetal pathogens are capable of infecting a broad range of mammalian host through either direct contact with an infected animal or indirect contact with soil or water contaminated with urine from a chronically infected animal. This disease is also one of the important infectious diseases that special strictly monitored in waterlogging and flood. The most serious clinical syndrome is Weil's syndrome. Although Leptospira virulence factors such as hemolysins, lipopolysaccharide, glycolipoprotein, peptidoglycan, heat shock proteins, flagellin, and others may contribute to the pathogenesis, their pathogenetic mechanisms have not been clearly understood.Leptospira is divided into Leptospira interrogans and Leptospira biflexa. The former is pathogenic for human and animals and the latter is saprophytic. There are many serogroups and serovars in Leptospira and the cross immune protection among different serovars of L.interrogans is weak or little. The presently used multivalent vaccine of leptospira in the world is made of death whole cells of the microorganism, usually composed of 3~5 dominant serovars of L.interrogans in local area. However,the side effect is obvious and the cross immune protection is limited.Recently, the whole genome sequence of L. interrogans strain lai was published by Chinese scientists(http://www.chgc.sh.cn/gn/). Besides ,in early Iiteratures,it is reported that some leptospiral proteins, such as OmpLl, LipL41 and LipL32, are found to be the main surface protein presented in all of the L. interrogans outer membranes. We have confirmed that there are ompLl, HpL32 and HpL41 in the genome of L. interrogans reference standard strains dominant in China, and their recombinate expression products showed satisfactory immunogenic and immunoreactivity.However the potential epitopes of OmpLl, LipL32 and LipL41,as well as their effects on inducing secretion of proinflammatory cytokine have not yet been reported.In our study, Ni-NTA affinity chromatography was established to extract the target recombinant proteins rOmpLl/1 and OmpLl/2, LipL32/l and rLipL32/2, and LipL41/l and rLipL41/2 expressed by the corresponding different genotypes. SDS-PAGE was applied to examine the expressions of the target recombinant proteins and purities of the extracts. By using Prediction Servants, the possible functional epitopes in molecules of the proteins were analyzed. By using a human umbilical vein endothelial cell line EVC-304 as the effective cell, the effects of the target recombinant proteins on inducing secretion of proinflammatory cytokine were measured by ELISA.MATERIALS AND METHODS1 .Bacterial strain and cell lineIhe prokaryotic expression system pET32a-ompLl/l -￡co//BL21DE3, pET32a- ompLl/2 -￡.co/iBL21DE3, pET32a- lipL32/l- ￡co//BL21DE3, pET32a- lipL32/2- ￡co//BL21DE3, pET32a- lipL41/l-￡.co/JBL21DE3 andpET32a- lipL41/2 -￡co//BL21DE3 were offered byour laboratory. Human umbilical vein endothelial cell line EVC-304 was purchased from Institute of Cell Biology ,Shanghai Institutes for Biological Sciences ,Chinese Academy of Sciences. The cells were cultured at 37°C in RPMI 1640, supplemented with 10% fetal calf serum, 100 U/ml penicillin, and 100 ug/ml streptomycin in a 5% CO2 humidified atmosphere.2. Expressions and identification of the target recombinant proteinsThe prokatyotic expression systems were cultivated in LB medium at 30°C with vigorous shaking. Once culture had reached mid-log phase,when the OD600 was 0.6~0.8, added IPTG to the culture at the dosage of 0.25 mmol.L"1.And induction at 30°C with vigorous shaking for 4 hours. The cultured bacteria were transferred to centrifuge tubes and spinned 15 minutes at 10,000 rpm at 4°C. The precipitate was suspended in sterile purified water for ultrasonically broking at 300V for 200 times. With the tag of 6*His ,the target recombinant proteins were collected by Ni-NTA affinity chromatography (BioAsia).10% SDS-PAGE was applied to examine the expressions of the target recombinant proteins and purities of the extracts.3. Analysis of signal peptides, MHC-II binding peptides and Lymphocyte B epitopesThe primary sequences of proteins were offered by our laboratory. By using Signal P-NN soft ware in Signal P3.0 Prediction Servant offered by Technical university of Denmark dtu, Propred MHC class-II binding peptide prediction-ProPred Prediction Servant offered by Institute of Microbial Toechnology, Chandigarh, India and ANTIGENIC program in EMBOSS soft ware, the possible signal peptides, MHC-IIbinding peptides and Lymphocyte B epitopes in molecules of the proteins were analyzed respectively.4. Detection ofproinflammatory cytokineDetection EVC-304 cells was added to 96-well microtiter plates with 5><103cells every well, then were cultured at 37°C for 24h in RPMI 1640, supplemented with 10% fetal calf serum, 100 U/ml penicillin, and 100 ug/ml streptomycin in a 5% CO2 humidified atmosphere.The rOmpLl/1, rOmpLl/2, rLipL32/l, rLipL32/2, rLipL41/l and rLipL41/2 were prepared with RPMI 1640, supplemented with 2% fetal calf serum, at different concentrations. Culture medium was changed to the recombinant proteins solution ,at the final concentrations of 1 and 10 ug every well, with triplicate samples of every protein at different concentrations. Cells were proceeded to culture at 37°C for 24h and 48h respectively in a 5% CO2 humidified atmosphere. The amount of IL-la,IL-8 and TNF-a were determined in the supernatants by measuring the absorbance at 490 ran with a commercially available ELISA, following the manufacturer's instructions (TPI).The product of recombinant TNF-a(SibEnzyme) at the concentrations of 0.1 ug every well and RPMI 1640, supplemented with 2% fetal calf serum, acted as positive and negative control respectively. Standard curves were drew according to the standard of IL-la,IL-8 and TNFot With the absorbance at 490 nm of the standard preparation of IL-la,IL-8 and TNFa as Y-axis, and their concentrations as X-axis, standard curves were drew, and calculation formula of cytokines were obtained by Excel soft ware.5. Analysis of dataThe results of proinflammatory cytokine secreted by EVC-304 cells ,under the inducement of rOmpLl/1, rOmpLl/2, rLipL32/l, rLipL32/2, rLipL41/l and rLipL41/2 respectively were analyzed by t-test, using SPSS 9.0 soft ware.RESULTS1. Analysis of signal peptides and their cleavage sitesThe signal peptides of OmpLls, LipL32/l 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, and the cleavage sites lies between the N ends of 21-22 or 24-25 amino acid residuals correspondingly.2. Analysis of MHC-II binding peptides and Lymphocyte B epitopesOmpLls, LipL32s and LipL41s displayed 3, 3 and 1 major epitopes of MHC-II binding peptides, which can be bound by more than half of 51 alleles in query, among these , there are 3, 2 and 1 epitopes overlapping with lymphocyte B epitopes at the same time.3. Expressions and identification of the target recombinant proteinsUnder the inducement of IPTG, the constructed prokaryotic systems could efficiently express rOmpLl/1 and rOmpLl/2, rLipL32/l and rLipL32/2, and rLipL41/l and rLipL41/2 with their 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 fragment after SDS-PAGE.4. Inflammation-induced effects of the major outer envelope proteins of Leptospira interrogans.The concentration of IL-la, IL-8 and TNF-awere calculated espectively as follows: yIL-la=46.51X-6.32, yIL-8=42.46X-3.50 and yTNFa=362.64X-24.79.The different dosages of rOmpLls, rLipL32s and rLipL41s could induce EVC-304 cells to obviously increase the synthesis and secretion of IL-la, IL-8 and TNF-a (PO.05),furthermore the different genotypic products of the same gene showed similar effects (P>0.05). The IL-la levels reached the highest at the 24 h and then declined, while the IL-8 and TNF-a levels were gradually elevated in pace with increase of the treatment time. Their effects on inducing secretion of proinflammatory cytokine could be ordered from OmpLls,LipL41s to LipL32s, according to their intension.Conclusion1. In this study, the prokaryotic expression system of ompLl/1, ompLl/2, UpL32/l, UpL32/2, HpL41/l and HpL41/2 showed high expression efficiency.2. The target recombinant proteins can be collected by Ni-NTA affinity chromatography in high purity in short time.3. The expression products between the tested different ompLl/1, HpL32 or lipLAl genotypes of L. interrogans contain similar immune functional epitopes.4. Inflammation-induced models of the major outer envelope proteins of Leptospira interrogans on human umbilical vein endothelial cell has been established.5. rOmpLl/1 and rOmpLl/2, rLipL32/l and rLipL32/2, and rLipL41/l and rLipL41/2 were able to directly induce inflammatory reaction in EVC-304 cells.