Cloning, Expression And Immunogenicity Analysis Of Major Outer Membrane Proteins Of Vibrio Parahaemolyticus

Vibrio parahaemolyticus has been listed one of the pathogenic vibrios endangeringnet-cage cultured large yellow croaker(Pseudosciaena crocea) in coastal areas of China.Immunoglobulins of large yellow croaker was purified after salting out with gradientconcentration of saturate ammonium sulfate and filtering with Sephadex G200, thenrecovered from SDS-PAGE gel. And polycolonal antibody was prepared with Newzealand rabbits for ELISA and Western blotting detection. Outer membrane proteins(OMP) were extracted from four strains of V. parahaemolyticus by method of detergenttreatment with Sarkosyl and separated by SDS-PAGE. Three different antiserum, rabbitantiserum against formalin-killed-cells (FKC) of V. parahaemolyticus zj2003 (apathogenic strain isolated from diseased large yellow croaker, from Xiangshan bay,Zhejiang province), rabbit antiserum against OMP of the bacteria and large yellowcroaker convalescent antiserum were prepared for western blotting of the OMPpreparation. SDS-PAGE profiles appeared 5-6 main bands with molecular weights(MW) about 21kDa to 54kDa, four common bands with MW of about 21kDa, 28 kDa,and 35 kDa were present among the strains. Western blotting results showed that theOMP preparations were differently recognized by various antiserum. Both of the twomost abundant bands with MW of about 28kDa and 35kDa were recognized bY rabbitantiserum against OMP, which indicated that the main outer membrane proteins ofV.parahaemolyticus are immunogenic. The other main band with MW of about 21kDawas the dominantly one that reacted with rabbit antiserum against FKC; and mainly fourbands with MW of about 21kDa, 28kDa, 35kDa and 44kDa were recognized by the fishconvalescent sera, it is suggested such OMPs should have been exposed to the host'simmune system during vaccination and in vivo infection and may be of some significancefor vaccine development. The protein band with MW of about 21 kDa reacted with all theantiserum, which well displayed the strong immunogenicity of this protein.The immunogenic OMP of Vibrio parahaemolyticus zj2003 with MW of about21kDa was preliminary confirmed as ompW by method of N terminal amino acidresidues sequencing. Primers were designed to amplify the mature peptide codingsequence of the protein, and the sequence was sent to GenBank under the accessionnumber DQ425109. The sequence was ligated into prokaryotic expression vector pET30a,then the recombinant plasmid pET30a-ompW was transferred to Escherichia coli BL21,the strain was induced to express the recombinant protein by addition of IPTG.SDS-PAGE showed the recombinant protein appeared the expected MW of 27kDa. The recombinant protein was expressed in fusion bodies and purified with Ni-IDA affinitychromatography. Rabbit antiserum prepared against the purified protein well recognizedthe protein, which indicated that the protein was appropriately expressed. And therecombinant protein also reacted with rabbit antiserum against FKC of V.parahaemolyticus and large yellow croaker convalescent sera. The results suggested thatomp W was strong immunogenic and may act as target gene for species specific rapididentification of V.parahaemolyticus and may be considered a powerful vaccine Candidateagainst the bacteria.Four OMPs and two iron-regulated outer membrane proteins (IROMPs) ofV.parahaemolyticus zj2003, ompV, ompK, ompU, TolC, psuA and pvuA were cloned andthe genes were registered in GenBank (the accession number DQ016303, DQ016304,DQ006287, DQ141606, DQ141607 and DQ141608, respectively). Then the maturepeptide coding sequences of the proteins were ligated into prokaryotic expression vectorpET30a, the recombinant plasmids pET30a-OMP were transferred to E.coli BL21 and thestrains were induced to express the recombinant proteins by addition of IPTG. Therecombinant proteins were expressed in fusion bodies and purified with Ni-IDA affinitychromatography. Except ompU, the other five recombinant proteins showed expectedMW in SDS-PAGE, 32kDa, 33 kDa, 48 kDa, 80kDa and 78 kDa, respectively.Recombinant ompU moved more slowly in SDS-PAGE, appeared a MW of 47kDa, somehigher than the calculated value (39.6kDa). Western blotting results showed that threerecombinant proteins, ompK, ompU and TolC reacted with rabbit antiserum against OMPof V. parahaemolyticus, while the other three proteins not. It is suggested that the formerthree proteins were expressed under routinely culture condition and they remained thereactivity of native proteins. Combined with results of SDS-PAGE, it is indicated thatompV, psuA and pvuA were not expressed under the present culture condition or notenough expressed to elicit antibody response. Further study must carry out to solve theproblem for expression of these proteins. And rabbit antiserum against ompK of V.harveyicross-reacted with recombinant ompK of V.parahaemolyticus, which indicated thehomology of the protein between the two vibrio species. That's may be of somesignificance for vaccine development.The purified seven OMPs of V. parahaemolyticus were classified into four groupsconsidering of their biological function: (1) ompW&ompV; (2) ompK; (3) ompU&TolC;(4) psuA & pvuA. The protein or proteins combination was administrated to large yellowcroaker by method of intraperitoneall injection (i. p.) with the dose of 100μg per fish. Specific antibody level was detected by indirect ELISA during 4-8 weeks postvaccination and challenge experiment was undertaken for determination of relativepercent survival (RPS) of the immunized fish. Western blotting was carried out with largeyellow croaker convalescent sera. The results showed that antibody titers to theimmunized recombinant proteins increased 4-8 weeks post vaccination. The recordedRPS of vaccinated groups reached above 80%. And the fish convalescent sera reactedwith all recombinant proteins except TolC. It is suggested that six OMPs ofV.parahaemolyticus ompV, ompW, ompK, ompU, psuA and pvuA, are immunogenicduring in vivo infection, and may be of great significance for vaccine development.Especially, fish group immunized with recombinant ompK achieved a high protection of90%. For the wide expression of this conserved protein among vibrio species, ompKmust be paid enough attention for vaccine development against vibriosis.Partial sequence of pvuA of Vibrio parahaemolyticus zj2003, was ligated intoeukaryotic expression vector pCI together with EGFP, the gene encoding enhanced greenfluorescent protein, resulted the recombinant plasmid pCI-pvuAl-EGFP with a targetORF of 1551bp, encoding a fusion protein with the expected MW of 57.16kDa. EGFPexpression by Vero cells transferred with the recombinant vector was observed withfluorescent microscope. The DNA vaccine prepared with the purified plasmids wasadministrated to large yellow croaker by intramuscular injection (i.m., 20μg per fish).Challenge experiment with live bacteria resulted with a RPS of 50% 4 weeks postvaccination. And antibody against the recombinant pvuA was detected by Westernblotting with antiserum of the immunized fish. It is indicated that the target Protein wasexpressed in vivo and elicited antibody reponse.