| ObjectiveVvhA,encoded by vvhA gene of V.vulnifcus,is an extracellular protein.VvhA is a haemolysin pore-forming toxin.Studies have showed that it plays an essential role in humsan infection.But few studies had shown the function of vvhA until now.Selecting V.vulfinicus Y066 as the research object,this study was to determine the immune response of vvhA using the molecular biology,immunology,flow cytometry and other methods.Methods1.V.vulfinicus strain Y066 was cultured at 37℃ on Columbia blood agar plate.After the genome DNA was extracted,the sequence of vvhA was amplified by using of the specific primers.The recombinant expression vector pET28a(+)-vvhA was constructed and sequenced by Life Technologies Corporation.2.After the amino acid sequence of VvhA was deduced through the nucleotide sequence,the bioinformatics and computational biology were analyzed by bioinformatics analysis methods.3.After the recombinant expression vector pET28a(+)-vvhA was transformed into E.coli Rosetta(DE3),the recombinant VvhA fusion protein was induced by IPTG and purified using Ni-nitrilotriacetic acid resin.The protein was confirmed by Western blot.The polyclonal antibody of VvhA was produced by immunizing mice and the antibody of serum was detected by ELISA.The animal model of V.vulfinicus Y066 infection was built for future study about the therapy effect of polyclonal antibody.4.A suicide vector named pBluescript-△vvhA::Kan was constructed for an isogenic mutant of V.vulfinicus Y066 vvhA by electroporation and then we got the vvhA-deleted mutant after the electroporation.5.The Thl/Th2 cell subsets was measured by animal infection model through FCM while the cytokines IFN-y were detected at the same time through ELISA,so that we can acquire the immune response by VvhA.Afterwards,we carried out pathological examination and separated the serum to analyze the toxin activity of VvhA protein.Results1.The vvhA was successfully cloned in pET28a(+)by the V.vulfinicus strain Y066.2.The vvhA gene in V.vulfinicus Y066 is 1416bp,consisted of 471 amino acids with 52.87kDa.VvhA is a hydrophilic protein,and the signal peptide was calculated at 1-20 amino acids by SignalP 4.1.The PSORTb shows that it is the extracellular secreted protein.3.The pET28a-vvhA was highly expressed in E.coli Rosetta(DE3)and the target protein was purified by Ni-nitrilotriacetic acid resin system.After the mice was immunized with purified protein,the result of ELISA indicated that the titer of the antisera was 1:51200.The animal model of V.vulfinicus Y066 infection shows that the survival rate was 50%.4.The suicide vector pBlueKM40-△vvhA::kan was constructed and we got the vvhA deleted mutant successfully.5.The Thl/Th2 cell were detected by FCM,and the cytokine IFN--y was detected at the same time.It indicates that the immune response level of Thl cell was improved apparently in wild strain group and the level of IFN-y were higher than vvhA-deleted mutant group,while there was no significance difference in Th2 cell subset.The pathological examination showed that more liver damage was found among wild strain and the serological test showed the same result(wild strain:ALT 160U/L,AST 717 U/L;vvhA deleted mutant:ALT 41 U/L,AST 147 U/L).Conclusions 1.The expression vector pET28a-vvhA was successfully constructed.2.VvhA is a hydrophilic,stable and extracellular secreted protein,with solid antigenicity.The result of CDD shows that VvhA contains leucocidin and RICIN superfamily,which involves in cytotoxicity and hemolytic activity.3.The VvhA protein was obtained after induced by IPTG.After the mice were immunized with purified protein the polyclonal antibody of VvhA was extracted.The animal experiment indicated that the polyclonal antibody was a kind of protective neutralization antibody.4.The vvhA deleted mutant was successfully constructed.5.The VvhA protein can mediate the Thl cell immune response at the early stage of infection through animal model,FCM and etc.This establishes the foundation for the study on pathogenic mechanism and vaccine preparation of VvhA protein. |
