Screening And Identificaiton Of Linear B-cell Epitopes In Outer Membrane Protein U Of Vibrio Mimicus

Vibrio mimicus a causative agent of serious ascitic fluid disease of aquaticanimals. It can also cause human diarrhea and food poisoning through confactingaquatic environment and aquatic products. Because ascitic fluid disease has affectedon the healthy development of aquaculture nowdays. So, it is important significanceto establish a rapid diagnosis method and develope efficient vaccine againstV.mimicus. Screening suitable antigen and antigen epitopes is the most key problemto be solved. This paper, the linear B-cell epitopes in outer membrane protein U(OmpU) of V.mimicus were identified and their immunological activity were analysedby combining eptitope prediction and experiment which lay the foundation forestablishing epitope-based rapid diagnosis method and developing epitope vaccine.Firstly, in order to predict the possible linear B-cell epitopes in OmpU ofV.mimicus, the secondary structure, flexible regions, surface probability,hydrophilicity and antigenic index of OmpU were analyzed by DNAStar Proteansoftware based on OmpU sequence of V.mimicus strain HX4isolated in Anhuiprovince. Meanwhile, the model of3D structure of OmpU protein was constructedwith Molecular Operating Environment v2008, and10possible epitopes on the3Dstructure were visualized by Synchronize program.7epitopes which location at thesurface of3D structure were recognized as the most possible epitopes. Thesepredicted epitopes and one control non-specific peptide sequence were synthesized soas to identified.Secondly, the OmpUa containing major antigenic domain of OmpU protein wassubcloned into pAML-c4x vector and was expressed in prokaryotic cells, and thenexpression form of recombinant fusion protein were evaluated.According topAML-c4x reading frame, a pair of specific primers containing Sal I and Hind IIIrestriction enzyme sites were designed. The OmpUa was amplified from extractedDNA of V.mimicus strain HX4by PCR method and cloned into pMD18-T vector.pMD18-T-OmpUa was digested by SalI and HindIII, the purified OmpUa wasinserted into pAML-c4x and transformed into E.coli TB1. Gene engineering E.colistrain pAML-c4x-OmpUa/TB1was induced by IPTG at16℃, then the expressionproducts was identified and analyzed by SDS-PAGE and Western-blot. The result ofSDS-PAGE showed a apparent protein band with the relative molecular mass ofrecombinant protein MBP-OmpUa, the expression products mainly in the soluble form could react specifically with rabbit anti-MBP aserum by western-blot, whichindicates that recombinant fusion protein MBP-OmpUa was expressed successfully.Thirdly, rabbit anti-MBP-OmpUa antibody was prepared.The recombinantfusion protein MBP-OmpUa was purified with Amylose Resin affinitychromatography column and rabbit was immuned with purified MBP-OmpUa fusionprotein emulsified in Oil adjuvant A5fully two times. The results showed thatspecific antibody can be detected on the14th day after immunization, the titration ofrabbit anti-OmpUa-MBP antisera was1:4096000detected by indirect ELISA on24thday after immunization and the titer of agar diffusion assay was1:32, which couldfully meet the requirements of the following experiment.Finally, the predicted epitopes were determined by Peptide ELISA. ELISAplates were coated with each synthetic potential epitope peptide and control peptidewith a final concentration of40ug/100ul per well and detected by indrect ELISA withrabbit anti-MBP-OmpUa at1:400dilution and HRP-conjugated goat anti-rabbit IgGat1:7000dilution. The results showed the7peptide epitopes could all react withrabbit anti-MBP-OmpUa antibody but couldn’t react with rabbit anti-MBP antibody,and the control peptide couldn’t react with the each antibodies. The result suggestedthat the7peptide epitopes could recognized specifically by rabbit anti-MBP-OmpUaantibody, and retained immunoreactivity of natural OmpU, so they are the tureepitope in OmpU.Among these, epitope3showed strongest immunoreactivity thenfollow by epitope4(aa239-250), epitope6(aa183-195), epitope2(aa117-126),epitope1(aa25-33), epitope5(aa173-180) and epitope7(aa211-218).Above all, the7eptitopes located in OmpU amino acid regions25~33,56~66,90~98,100~106,117~125,173~180,184~192,211~218,239~250and284~295werescreened and identified by eptitope prediction with experimental validate method forthe first time.