Study On The Adjuvanticity Of Different Domains Of E.coli Flagellin Protein FliC

Bacterial flagellin is a new type of immune adjuvant that has been intensly investigated in recent years.Flagellin exerts its immune adjuvant acitivity by inducing proinflammatory responses through the TLR5 signal pathway.However,whether the complete structure is required for its immune adjuvant activity fully exerted remains controversial.In addition,how the structural features of FliC contribute to this is also unknown.In this study,the main structural subunit FaeG of F4ac fimbriae derived from ETEC(Enterotoxigenic E.coli,ETEC)was used as the model antigen.Then,we genetically fused FaeG gene to different domains of E.coli flagellin FliCH7 gene:full-length FliCH7(aa1-585aa),N-terminal conserved region FliCN(aa1-174aa),N-terminal and middle hypervariable region FliCNV(aa1-496aa)to construct the chimeric genes.The fusion proteins were expressed and purified,and their immune adjuvant activities were evaluated by testing TLR5 receptor activity in vitro and measured the anti-FaeG specific antibody level after immunizing the mice.The purpose of this study is to further elaborate the mechanism of the flagellin immune adjuvant activity,and provide a reference for the more efficient use of flagellin in applied practice.1.Constuction?expression and identification of chimeric genesIn this study,we genetically fused FaeG gene(the main subunit of F4ac fimbriae derived from enterotoxigenic E.coli C83902(O8:K88:H19))to different domains of hemorrhagic E.coli EDL933(O157:H7)flagellin FliCH7 gene by overlap extension PCR(SOE-PCR)to construct the three chimeric genes:FliC(aa1-aa585)-FaeG,FliCN(aa1-aa174)-FaeG,and FliCNV(aa1-aa496)-FaeG.They were cloned into pET28a(+)expression vector and further transformed into E.coli BL21(DE3).The fusion proteins were expressed and induced by IPTG.Further,the fusion proteins were verified by combined methods of SDS-PAGE and Western blot PCR results showed that three chimeric genes with size of 2619 bp,1386 bp,and 2352 bp,respectively.SDS-PAGE results indicated that the proteins with molecular mass of 90 KDa,48 KDa,and 80 KDa,respectively,the expected size of the three fusion proteins.Western blot results showed that FliC-FaeG and FliCNv-FaeG fusion proteins can be recognized by both anti-FaeG monoclonal antibody and anti-FliCH7 serum,while FliCN-FaeG fusion protein can be just recognized by anti-FaeG monoclonal antibody.The TLR5 receptor binding activity of the fusion protein result showed that FliC-FaeG fusion protein can induce higher levels of IL-8 and TNF-?than FliCN-FaeG and FliCNv-FaeG fusion proteins in human intestinal epithelial cell line Caco-2 cells.The successful construction and identification of the fusion proteins will gain insight into the mechanism of E.coli flagellin immunoadjuvant activity and the relationship between different domains and their immunoadjuvant activities.2.Study on the immune adjuvant activity of different domains of E.coli flagellinIn order to study the possible differences in adjuvanticity of different domains of E.coli flagellin in vivo and the mechanism,we immunized 8-week-old BALB/c male mice with the purified FliC-FaeG,FliCN-FaeG and FliCNv-FaeG fusion protein by subcutaneously route,and specific anti-FaeG-IgG antibody in each mice was detected by ELISA method.The results showed that anti-FaeG antibodies were detected in mice from each immunized group from the seventh day after the first immunization,and the antibody level increased steadily as after booster immunization.The level of anti-FaeG antibody was significantly higher in FaeG+Freund's adjuvant group and FliCN-FaeG group than that in FliC-FaeG group and FliCNV-FaeG group(p<0.01).Moreover,the levels of TNF-? and IL-4 produced by spleen cells of immunized mice were measured by indirect ELISA,and the results indicated that the level of TNF-? in the FliC-FaeG group and the FaeG+Freund's adjuvant group was significantly higher than that in the FliCN-FaeG group(p<0.001)and FliCNV-FaeG group(p<0.001).However,the level of IL-4 secretion among the groups was not statistical difference(p>0.05).The real-time PCR results showed that the interleukin 6(IL-6)and tumor necrosis factor alpha(TNF-?)produced in the FliC-FaeG group and FaeG+Freund's group were significantly higher when compared to those in the FliCN-FaeG group(p<0.01)and FliCNV-FaeG group(p<0.01).In conclusion,our results indicated that the transcription and expression of inflammatory genes are dependented on activating the TLR5 signal pathway by complete flagellin structure in mice3.Fusion protein-induced antibodies significantly inhibited adherence of F4ac+ETEC to piglet small intestinal epithelial cellsF4ac+ETEC is recognized as a major bacterial cause of diarrhea in piglets.F4 fimbriae is the main virulence factors contribute to F4ac+ETEC associated diarrhea.F4 fimbriae attachment to specific receptors at piglet intestinal epithelial cells is the initial and critical step of F4ac+ETEC infection.In order to determine whether the specific anti-FaeG antibody induced by the fusion protein above can effectively block the adhesion of F4ac+ETEC to piglet intestinal epithelial cells,two piglet intestinal epithelial cell lines,IPEC-1 and IPEC-J2 were used as in vitro models.The results showed that all serum samples from the mice immunized with fusion proteins significantly inhibited the F4ac+ETEC adhesion to these two cell lines.The serum samples from mice immunized with FliCN-FaeG group and FaeG+Freund adjuvant showed significantly better inhibition efficiency than the serum from mice immunized with FliC-FaeG and FliCNV-FaeG(p<0.01),However,mouse serum samples from either the FliCN-FaeG and the FaeG+Freund's groups(p>0.05),or FliC-FaeG and FliCNV-FaeG groups(p>0.05)showed no significant differences in inhibition against adherence to IPEC-1 and IPEC-J2 cells from F4ac+ETEC strains.Our results indicated that the FliCN-FaeG fusion protein would be priority used as a candidate protein for development of vaccines against F4+ETEC infection in the future.