Screening, Identification And Immunological Characteirstics Of Mimotopes From OmpU Antigen Of Vibrio Mimicus

The ascites fluid disease in aquatic animals is a serious important disease causedby Vibrio mimicus. Antibiotics are commonly used as controlling the disease.However, antibiotic resistance of bacteria is caused by long-time using antibiotics,which lead to bad antimicrobial treatment. Therefore, it is an urgent problem fordevelopment a new vaccine to control ascitic fluid disease on the basis of looking forprotective epitopes. The outer membrane protein U (OmpU) of Vibrio mimicus is aadhesin protein with good antigenicity and highly conservation, which can be used asvaccine candidate, but its effective protective epitopes is not clear so far. In this study,seven immunodominant mimotopes of OmpU protein were screened and identificatedby means of the phage peptide library technique using purified rabbitanti-recombinant OmpUa polyclonal antibodies, then their immunogenicitiesand protective immunity were analyzed, respectively. Our studies provide afoundation for developing adhesin epitopes vaccine against ascites fluid disease inaquatic animals.In order to otain the molecular target for screening Vibrio mimicus OmpU proteinB-cell mimotopes, rabbit anti-recombinant OmpUa polyclonal antibody was purifiedby saturated ammonium sulfate precipitation and affinity chromatography. The resultsshowed that the purified antibody, which possessed95%purity,1.5mg/mLconcentration and1:4096000titer detected by indirect ELISA, could used asscreening target.To screening and identification the OmpU protein B-cell mimotopes, The phageclones binding to purified polyclonal antibody against recombinant OmpUa wereselected from the12-mer random peptide library. By3rounds of sereening，the phageinput and output percentage was an upward trend, and the phages were got enrichment.Nine positive phage clones were identified by sandwich ELISA and competitiveELISA from68phage clones selected randomly, and then were sequenced andanalyzed. The results showed that nine clones presented seven peptides sequences(HDSSKFPYVPSY, FNLHAWPTLDT, HAATVDKIENMR, GLNWSLSPADLI,DTAYEGNIARLM, FSSDSSKVHYPN and SVSEGMKPSPRP). Compared with thesequence of OmpU protein, there were no more than three continuous amino acidresidues similarity with the sequence of OmpU protein. The results indicated thatthey were mimotopes. Positive phage clones C17, C60and C67presented commonmotif DSSK-P, and they were high homology with the OmpU protein amino acid residues (293-316aa), indicating that D, S, S, K, P constitute the key amino acids ofOmpU protein B cell peptides.90healthy KM mice, which were divided into ten groups on average, wereimmunized three times with seven positive phage clones(1012PFU phage per mice),original phage M13and inactivated Suspension of Vibrio mimicus respectively. on the7th day after the third immunization, different antibody levels in sera were detectedby ELISA, at the same time The Experimental mice were challenged with1011CFU/mL suspension of Hx4strain. The results showed all mice produced highspecific antibodies with1:51200to1:409600titer. Survival rate of immunized micewith C21clone was70%,80%with C20and C25clone,100%with C17, C24, C60,C66clone and inactivated Suspension, while all mice died in the control group ofbacteriophage M13. These results indicated that seven phage clones which carryingOmpU protein B-cell mimotopes possesed good immunogenicity and protectiveeffect, and four mimotopes presented by C17, C24, C60and C66positive phageclones could be regarded as a vaccine candidate epitopes.In summary, for the first time,7protective mimotopes of the OmpU protein ofVibrio mimicus were screened and identificated using phage random peptide librarytechnology in this study. The results of our research possesed independent intellectualproperty rights, and provide a foundation for developing safe and effective adhesinepitopes vaccine.