Screening and Identification of30kDa Heat-Modifiable Outer Membrane Protein in Vibrio Alginolyticus Strain SR1and Research on its Immune Protection to Paralichthys Olivaceus
|School||Ocean University of China|
|Keywords||Vibrio algnolyticus Heat‐modifiable outer membrane protein antigenity immuno‐protection OmpA|
Vibrio alginolyticus is the pathogen of some aquacultural animals such as fish、shrimps and shellfish,which could cause disease of aquacultural animals andhuman.One of the important method to prevent the incident of this disease is thedevelopment of vaccine and application of immunotherapy.Results from presentinvestigation suggest that heat‐modifiable protein is a kind of outer membrane ofbacteria in the nature of good immunogenicity and highly antigenic conservationwhich could be a potential vaccine candidate. A pathogenic Vibrio alginolyticus strainSR1which was isolated from the ascites of cultured turbot (Scophthalmus maximus)is employed in this research,the outer membrane protein of which is prepared andtreated by heat combined by the method of electrophoresis and immunoblotting.Aouter membrane protein with molecular weight of30kDa is found to beheat‐modifiable.Identification of the protein species was executed by means of2‐Delectrophoresis and FACS. At last, the products was used as a kind of immunogen tocheck out its immunoeffects on Japanese flounder（Paralichthys olivaceus）.⑴Major outer membrane proteins (MOMPs) of a pathogenic Vibrioalginolyticus strain SR1was prepared by Sarkosyl method combined withultracentrifugal purification. The MOMPs solubilized in sample buffer at differenttemperatures （28℃、37℃、50℃、60℃、70℃、100℃） were analyzed bysodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE). A majorouter membrane protein (MOMP) which have a molecular weight of30kDa was theonly high‐abundance protein when the MOMPs preparation was solubilized at28℃,but turn to a low‐abudance protein when solubilized at100℃with a arising of twonew protein bands with molecular weights of36kDa and37.5kDa. Purified30kDaMOMP present the same electrophoresis‐mobility when solubilized at28℃and 100℃. Mouse antiserum against the30kDa heat‐modifiable protein was prepared todo western blotting after SDS‐PAGE of MOMPs solubilized at different temperaturesmentioned above. MOMPs of strain SR1with molecular weights of30kDa,36kDaand37.5kDa were detected. It was proved the heat‐modifiability of30kDa MOMPwhich would present in molecular weights of30kDa,36kDa and37.5kDa whenheated.⑵SDS‐PAGE of MOMPs of11Vibrio strains solubilized at28℃and100℃respectively showed that30kDa MOMP of5strains of Vibrio alginolyticus and29.5kDa MOMP of V. parahaemolyticus were heat‐modifiable proteins. When all theMOMPs preparation of11strains were solubilized at28℃,western blot analysisshowed positive reactions to30kDa MOMP of5strains of V. alginolyticus and to29.5kDa MOMP of V. parahaemolyticus, but to30kDa、36kDa MOMP of5strainsof V. alginolyticus(and to a37.5kDa MOMP of SR1) and to29.5kDa、30.5kDaMOMP of V. parahaemolyticus when they were solubilized at100℃. No positivereaction was detected with any MOMPs of other5Vibrio species. ELISA ananlysisshowed positive results of Vibrio alginolyticus strans SR1、283、284、RB、RH2andV. parahaemolyticus strains458（P/N>2.1）,but negative results of other strains（P/N<2.1）.These results proved that the30kDa MOMP of V.alginolyticus strainSR1was highly conservative among different strains of V. alginolyticus.⑶Major outer membrane proteins (MOMPs) of Vibrio alginolyticus strain283、284、RB、RH2were prepared by “Sarkosyl method”. SDS‐PAGE of all the MOMPssolubilized at28℃、50℃、70℃、100℃respectively showed that a30kDa OMPwas the only high‐abundance protein when the MOMP preparation was solubilized at28℃and50℃, but turn to a low‐abudance protein when solubilized at70℃and100℃coupled with a arising of some new protein bands with high molecularweights, the heat‐mobility of which was resemble with that of SR1MOMPs. Westernblot analysis showed positive reactions respectively to30kDa MOMP,and to30kDa、36kDa OMP of every strains when they were solubilized at28℃and100℃.It was proved that all the30kDa OMP of the4strains were heat‐modifiableproteins,the same as30kDa OMP of SR1.In addition, digestion analysis with trypsin showed that all the36kDa OMP were digested with a arising of new protein bandwhich have a molecular weight of19.5kDa、18kDa、18kDa、18.5kDa、19.5kDarespectively. Immunoadsorption with the aim of locating the30kDa HMP in intactcell of5V. alginolyticus strains proved all the30kDa OMP exposed on the surface ofthe intact cell. Those results provided theoretical basis for the following researchabout the immuno‐protection of the30kDa HMP of SR1.⑷The flounder, immunized with purified30kDa OMP of SR1,were challengedby intrapertoneal injection with3live V. alginolyticus strains SR1、284and RH2at thefourth weekend of vaccination.The fish vaccinated with30kDa OMP had asignificantly higher survival rate than the unvaccinated fish.The specific serumantibody titre against30kDa OMP of SR1were measured using ELISA for6weeks,which had an increase and reached their peaks4weeks after immunization and thendecreased gradually.⑸IEF‐PAGE of30kDa HMP showed it comprising of3protein spots with thesame pI (pH≈4.66).One protein spots still present a molecular of30kDa but anothertwo had migrated to36kDa and37.5kDa respectively,which were identified asOmpA by MALDI‐TOF‐MS peptide mapping.The result of peptidoglycan associationassay proved that all the3protein were not associated with the PG.The digestionresults showed36kDa protein was digested by trypsin into a small protein with amolecular of19.5kDa,but30kDa and37.5kDa protein were trypsin‐resistant, stillpresent the same molecular weight as ever. Heat sensitivity research showed that withthe rising of treatment temperature and the extending of treatment time,30kDa and36kDa proteins still kept the same molecular weight and abundance,but the37.5kDaprotein was gradually decreasing its abundance with the arising of2new protein band22kDa and15kDa. In conclusion,the heat‐modifiable protein30kDa was OmpAand consisited of3conformer. with different characteristics.