Recombinant Aeromonas Veronii Flagellin FlaA Mediate Toll-like Receptor5M Signaling Pathway Of Common Carp

Common carp is the dominant species of freshwater aquaculture in our country andoccupies an important position in the aquaculture industry. With the growing scale offarming and breeding density, Bacterial Infections has become an obvious obstaclerestricting the development of aquaculture. Aeromonas veronii,a ubiquitous pathogenin the aquatic environment, can cause septicemia and ulcerative syndrome offreshwater fishes and cause human diarrhea or food poisoning after infection. Thispathogen poses a serious threat to fish farming and human health.Aeromonas veronii can produce a variety of pathogenic virulence factors, such aslipopolysaccharide, enterotoxin, outer membrane protein and S-layer protein,whichhave indepth study abroad. Flagella is bacterial moving organs, plays an importantrole in competition with other microorganism. However, flagella also plays a key rolein adhesion,biofilm formation and several pathogens colonization. Bacterial flagellaas an important virulence factor can effectively activate the innate immune response.Flagellin as a major component of the bacterial flagellin is TLR5ligand, which cancapable of binding to TLR5of the cell surface by MyD88dependent pathway, quicklyraise IRAKs(IL-1receptor-associated kinase),combine with TRAF6(TNFreceptor-associated factor6), thereby activate nuclear transcription factor NF-κB toinduce of TNF-ɑ, IL-1β and other pro-inflammatory cytokine production, whichmediates the body against invading heterologous pathogens.In this research, Bacteria(CCA1301) was isolated from enlargement liver of deadcarp and proved to be Aeromonas veronii by adopting the physiological andbiochemical methods, drug susceptibility test of antimicrobial analysis, and16SrDNA sequence analysis. Perspective Electron Microscope was used for observingbacteria and flagella form, and natural flagellin was obtained by the method of acidhydrolysis and affinity chromatography. After then, Gene-specific primer of flaA wasdesigned according to Aeromonas gene sequences from GeneBank by MegAlign multiple sequence alignment to conserved regions. The flaA gene sequence of915bpsuccessfully was cloned, and the target gene was successfully connected to theexpression vector pET-30a. Recombinant expression plasmid pET-30a-flaAsuccessfully was constructed and transformed into E.coli BL21(DE3) to induceexpression,which expressed the purpose protein about38kDa with a His-tag. Ni-ionaffinity chromatography was used to purify protein. Flagellin FlaA antiserum wasobtained by immunizing rabbits. Western blot confirmed FlaA antiserum not only canreact with recombinant flagellin, but also react with native flagellin. Our resultshowed that recombinant flagellin FlaA has strong immunogenicity, which will lay thefoundation for further research flagellin antigenicity and immune adjuvant effect.Finally, Aeromonas veronii, native flagellin and recombinant flagellin respectivelystimulated carp, and then we extracted the total RNA of head kidney after12h and4h.We analysted expression level of TLR5M,TRAF6, TNF–α by fluorescencequantitative PCR method.The results showed that compared with the the bacteria,natural, the recombinant flagellin FlaA also can cause higher expression aboutTLR5M, TRAF6, TNF-α in head kidney of carp. Flagellin can adjust carp TLR5Mand related factors in signal pathway, and produce immune response.