| Vibrio mimicus is one of the main pathogenic bacteria that harm aquatic animals.It not only leads to serious ascitic fluid disease of fish and Eriocheir sinensis,but also causes human diarrhea and food toxicosis through aquatic animals and aquatic products.Ascitic fluid disease is one of common epidemic diseases in fish and Eriocheir sinensis cultivation in Anhui province.Controlling occurrence and spread of ascitic fluid disease has become the key to sustainable development of aquaculture.Bacterial adhesion to host cells surface by outer membrane protein is an important elementary step of initialing infection, otherwise bacteria cannot stay there,produce toxin or penetrate deeper tissue and cause infection.Exploiting outer membrane protein vaccine with antiadhesion is one of the most effective measures for the immune prevention and treatment bacterial disease.Therefore, in this study outer membrane protein OmpU of V.mimicus is taken as the target protein.In order to lay the foundation for developing OmpU genetic engineering vaccine,ompU from V.mimicus isolated in Anhui was cloned and prokaryotic expressed,and antigenicity and immuneprotection of GST-OmpU fusion protein was detected.Firstly,ompU from V.mimicus isolated in Anhui was amplified,cloned,sequenced and analyzed.Two pairs of specific primers which aimed at the complete gene and the partial gene of OmpU were designed based on the complete sequence of ompU from V.cholerae. ompU was amplified by PeR from genomic DNA of V.mimicus and cloned into pMD18-T vector,and then sequenced and biological information analyzed.The results showed that ompU partial gene fragment of strain HX4,04-5,04-13 and 04-14 were all 849bp in length Through the blast analysis software,the homology of the corresponding ompU shared higher conservative property among different Anhui strains of V.mimicus than the referent strains of V.cholerae.They were 94.2%-100%and 83.4%-87.6%respectively.OmpU gene of strain HX4 contained an open reading frame(ORF)about 1038 bp,encoding 346 amino acids with a predicted molecular weight of 36.95 kDa,and the N-terminal 22 amino acids composed the signal peptide.The N terminal amino acid sequence was similar to HMW adhesin sequence of Hemophilus influenzae pneumonia.There were two obvious transmembrane territories between No.5aa～21aa and No.201aa～218aa in amino acids sequence;the second structure of OmpU contains moreα-helix regions,β-sheet regions, but littleβ-turn regions and ciols regions.No.20aa-34aa,36aa-67aa,73aa-81aa, 88aa-112aa,123aa-134aa,143aa-160aa,164aa-174aa,177aa-200aa,217aa-227aa, 244aa-259aa,275aa-288aa,295aa-303aa and 314aa-340aa may be potential epitopes of OmpU predicted by DNAStar software.These results indicated OmpU of V.mimicus was an adhesion porin with better antigenicity and conservation.Secondly,recombinant plasmid pGEX-4T-OmpU was constructed and prokaryotic expressed.A new pair of primers including restriction enzyme cleavage position spot BamHI and EcoRI was designed.The ompU was amplified by PeR and then inserted into the expressive plasmid pGEX-4T-1.The constructed recombinant plasmid pGEX-4T-OmpU was transformed into E.coIi BL21(DE3)and expressing products were identified and analyzed.The results showed that the fusion proteins GST-OmpU were highly expressed in E.coli BL21 after induced with 1.0mmol/L IPTG at 37℃with 4h. Most of GST-OmpU was expressed in the form of inclusion bodies.Finally,antigenicity and immuneprotection of recombinant fusion proteins GST-OmpU were detected by Western blot analysis and Immuneprotection experiment. After the Sodium dodecyl sulfate-polyacrylamide gel electrophoresis(SDS-PAGE), different density natural Omps,the purified recombinant OmpU as well as the induced expressive bacteria were shifted to the PVDF film and detected by Western blot analysis. The results showed mouse anti-GST-OmpU serum and mouse anti-Omps serum was possible to distinguish mutually between recombinant OmpU and natural Omps.These results suggested that expression recombinant OmpU retained certain antigenicity of natural Omps.Simultaneously,the experiment mice were immunized with fusion protein GST-OmpU,after four immunizations,the immunized mice were infected with 50LD50 V.mimicus and the immunity protection ratio was 60%.It indicated OmpU was one of protection antigen of V.mimicus.Above all,ompU of V.mimius was successfully cloned,sequenced and analyzed. There was a high homology at the molecular level between Anhui isolation strains and other V.cholerae referent strains.The OmpU of V.mimicus was a kind of adhesion porins with antigenicity and conservative.At the same time,Recombinant plasmids pGEX-4T-OmpU was constructed successfully.The fusion protein GST-OmpU,retained the natural OmpU antigenicity and immunity protection,might be regarded as candidate antigen of OmpU vaccine. |
