Prediction And Identification Of Antigenic Epitopes In Genus-specific Out Membrane Proteins LipL41 Of Leptospira Interrogans

Background and Objective:Leptospirosis,caused by infection of Leptospira interrogans,is a widespread zoonosis in the world,especially in paddy areas,so China is one of its major epidemic areas.These highly invasive spirochetal pathogens are capable of infecting a broad range of mammalian host,survive and propagate in kidney,contaminate soil and water.People often infected through either direct contact with an infected animal or indirect contact with soil or water contaminated with urine from a chronically infected animal.So this is the diseases especially monitored and controlled during periods of floods and water logging.Inoculating vaccine is the most effective measure to prevent and control Leptospirosis.But the serogroups of L.interrogans are numerous,and what's more important,there is a low or even no cross-protection among them.The epidemic dominant serogroups of the L.interrogans were found to be distinct in different areas, and often changed with the mammalian hosts.Therefore,the epidemically dominant serogroups of L.interrogans have been selected for preparing polyvalent vaccine production both domestically and aboard.However,this kind of vaccine offers little protection against other serogroups of microbe that were not selected in the vaccine. In addition,there were serious side effects for this kind of vaccine.A multivalence outer membrane vaccine,developed by seolars of our country,have finely immunity and little side effects,but still had no cross-protection among different serogroups of L.interrogans。Some of the outer membrane proteins(OMPs)which located on the out membrane of L.interrogans,like LipL41,are to be think as leptospiral genus-specific protective antigens,and they are the hope for developing a universal vaccine to prevent and control Leptospirosis.LipL41 is a surface-exposed leptospiral lipoprotein,consist by 355-amino-acid polypeptide.Haake et al(2000)have cloned lipL41,and thought that lipL41 genes exist in the DNAs of all pathogenic Leptospira but not in saprophytic one.Based on the results of our recent studies,all the 15 strains that represent the 15 serogroups of L. interrogans mostly epidemic in China were positive for lipL41 genes,but can be divided into lipL41/1 and lipL41/2 genotypes.Well the lipL41/1 genotypes exist in the major epidemic L.interrogans serogroups such as Icterohaemorrhagiae,Canicola, Autumnalis,Pomona,Grippotyphosa,Hebdomadis and Sejroe,and Leptospirosis infected by the L.interrogans which belong to lipL41/2 genotype are not often to been seen in China.Other research indicated that most L.interrogans strains which come from clinical infection patient have lipL41 gene,express LipL41 protein,and the corresponding antibody could also been detected.In the recently years,a novel vaccine strategy constructing multiple antigenic peptides(MAP)on one carrier molecule was developed and might be a promising way to conquer the deficiency of nocross-protection between different serogroups. Screening efficient epitopes is the first step for developing MAP vaccine.Studying the epitopes could not only reveal the molecular mechanism and feature of immune response induced by protein antigen,but also could be applied for investigation of novel molecular vaccine.Here,we intend to screen the T and B cell epitopes on the LipL41/1 and LipL41/2 molecules ofL.interrogans,in the hope of lay a foundation for developing the MAP vaccine.Sequence compared show that there are some amino acids mutation between two genotypes,and some of the mutiation have obvious influence on the prediction reasults,we could develop our understand about the characteristic of immune reaction and the possible influence of the mutation to epitopes though research them,well may be also helpful in evaluating the vaccine's effection in the future. Methods:Based on bioinformatic technique,the combined epitopes of T and B lymphcytes in LipL41/1 and LipL41/2 molecules were screened.Nucleotide fragments of each epitopes were amplified using PCR and then constructed their phage display systems.PEG/NaCl precipitation method and SDS-PAGE were used to extract the recombinant phage and identify the target recombinant PⅢ(rPⅢ).Using antisera against rLipL41/1,rLipL41/2,L.interrogans serogroup Icterohaemorrhagiae strain Lai and Lptospirosis patients' sera as the first antibodies,respectively,Western Blot assays were performed to determine the immunoreactivity and reactive ability of the epitopes with different antisera.Results:Six combined epitopes of LipL41/1 and two combined epitopes of LipL41/2 were selected out by the predicting procedure.All the amplified epitope fragments were accurately inserted into the region at N end of phage PⅢprotein and successfully expressed.All of the antisera could recognize each of the epitopes,but the hybridization signals intensity are different.On the one side the signals intensity of different epitopes in one antisera are various,on the other hands,the relative signals intensity of the same epitopes in different antisera are changeable too.Beside, the amino acid sequence mutation in or near by the epitopes could infect their antigenicity,either increased the affinity with their own antibody or decreased it. Above all,compromise the result of the 8 epitopes in difference antisera and their practical importance,the epitopes LipL41/1-30 and LipL41/233 are more stronger, and others either too small or too changeable.Conclusion:In this study,for identification of epitopes,phage display systems of the major LipL41 T and B cell combined epitope peptides are successfully constructed. The applied bioinformatic techniques can be used to efficiently predict antigenic epitopes.All eight combined epitopes in this study are efficiently antigenic,and the epitopes LipL41/1-30 and LipL41/1-233 have a potential for developing Leptospiral MAP vaccuine.