The Effects Of Salmonella Flagellin On Enhancing Immune Function Of Epitope Peptide In T Cell

【Objectives】 Bacterial flagellin a monomer subunit that polymerizes to form filament of bacterial flagella,is a ligand of TLR5 when it occurs extracellularly.Salmonella flagellin has been demonstrated to be a potent adjuvant for immune responses.When chemical conjugation the flagellin with peptides,it can enhanced immunogenicities of peptides.Firstly,we are trying to introduces a kind of chemical conjugation between peptide and protein.Secondly,use the conjuction formulation OVA323-rFliC to immunized BALB/c mice subcutaneously.At the same time compared adjuvant efficacy of rFliC with the mixed(OVA323+rFliC)group.Finally,we hope to built a foundation for the study of therapeutic vaccines against autoimmune diseases.【Methods】 The recombinant plasmid of p ET28a(+)-fli C-6His was trans-formed into E.coli BL21(DE3).After induction with isopropyl-d-1-thiogalactopyranoside,bacterial cells were harvested and disrupted by sonication.Sonicated cell supernatant was applied to a Ni-Sepharose column to purify histidine-tagged Salmonella flagellin in accordance with the manufacturer’s instruction.After desalting with a PD-10 column,the recombinant protein was treated with endotoxin removal resins The purified protein was examined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis(SDS-PAGE)and Western blot under non-reducing condition.The chemical linker of Sulfo-EMCS was used to add maleimide groups onto rFliC following the procedure described previously.The protein was treated by of 2 m M Sulfo-EMCS.The modified rFliC(rFliC-M)was examined by SDS-PAGE under non-reducing condition,and the content of maleimide group added on rFliC-M(moles of maleimide per mole of rFliC)was determined by indirect Ellman’s reaction.The study were carried out in BALB/c female mice aged 6–8 weeks,which were purchased from Shanghai SLAC Laboratory Animal(China)and randomly assigned into four experimental groups of three mice ina group.(1)PBS(2)OVA323+ rFliC: OVA323 and rFliC mixture in saline,(3)OVA323–rFliC: conjugate in saline,(4)OVA323+Freund: OVA323 formulated with complete or incomplete Freund’s adjuvant for the first and second immunization,respectively.For in vitro restimulation,splenocytes were harvested from mice after immunization and plated in duplicate in 96-well flat-bottom plates.Cultures were incubated for 72 h in the presence or the absence of OVA323 and analyzed for the presence of IL-4 and IFN-γ.The number of OVA323-specific IL-4 and IFN-γsecreting cells was counted using commercial ELISPOT assay kits.Then ues flow cytometry to analyze the percentage of CD4+IL-4+ or CD4+IFN-γ+ cells.To test for a significant level of enhancement of T cell reponses between PBS,OVA323+rFliC,OVA323-rFliC and OVA323+Freund,a Kruskal-Wallis One-Way ANOVA was performed;P values of < 0.025 were considered significant.If the Kruskal Wallis test was significant,then a post hoc analysis was performed with Bonferroni comparison;P values of < 0.05 were considered significant.【Results】 The recombinant FliC was highly expressed in a soluble form in E.coli and could be recognized by the commercial anti-FliC monoclonal antibodies.SDS-PAGE analysis showed that rFliC remained stable in the process of chemical modification,as only weak aggregates were observed after treated by different concentrations of Sulfo-EMCS.The number of maleimide group added on rFliC was measured by indirect Ellman’s reaction.Each mole of rFliC contained 5.55 moles of maleimide group The maleimide modified rFliC treated by 2 m M of Sulfo-EMCS was chosen to react with OVA323 peptide to generate OVA323–rFliC conjugate.The retarded mobility of rFliC presented on the SDS-PAGE gel after conjugation reaction indicated that the peptide was successfully coupled to the flagellin.The conjugation ratio of the conjugate was determined to be 5.84,which was close to the value of maleimide group added on rFliC.Through ELISA assay showed that the expression of IL-4,IFN-γ was significantly increase in OVA323-rFliC group compared with PBS group and OVA323+rFliC;The results of ELISPOT showed that the number of IL-4,IFN-γsecreting lymphocytes was significantly increase in OVA323-rFliC group compared with PBS group and OVA323+rFliC.The same result can be seen in the flow cytometry analyses.【Conclusions】 Through the current study,we can see that after chemical conguate of OVA323 and rFliC,the immune efficacy of T cells were enhanced,confirmed that rFliC can be used as adjuvant for T cells response.rFliC was a useful carrier adjuvant which can be used in the research in therapeutic vaccines in future.