The Study Of Four Flagellins Of Vibrio Anguillarum: Expression, Purification Of The Flagenllins In Escherichia Coli And Their Immune Application Research

With the rapid development of science and technology, more and more advancedbreeding techniques and means are used in aquaculture. But on the other hand, thefrequent fish diseases seriously restrict the development of aquaculture because it’sstart rapidly and hard to control. The pathogenic bacteria especially the vibrio havecaused severe economic loss, and increasingly become the focus of attention.Vibrio anguillarum is a marine pathogen of Vibrios which cause vibriosis inclose to50species of fish, including cultured and wild fish, mollusks, and crustaceans,in marine, brackish, and fresh water. More and more reports indicate that theflagenllum-mediated motility is required for optimal bacterial virulence in manyspecies of Vibrio, including Vibrio anguillarum.The genome of V. anguillarum contains five flagellin genes, i.e. flaA, flaB, flaC,flaD, and flaE. In this study, we analyzed the vaccine potential and adjuvanticity ofFlaA, FlaB, FlaD, and FlaE in a model of Japanese flounder (Paralichthys olivaceus).For this purpose, recombinant FlaA, FlaB, FlaD, and FlaE were expressed in andpurified from Escherichia coli (BL21).In vivo immunogenicity analysis showed that antibodies against rFlaA, rFlaB,rFlaD, and rFlaE were detected in rat antiserum raised against live V. anguillarum,with the highest antibody level being that against rFlaB, about2-4times higher thanrFlaA, rFlaD, or rFlaE. When administered into flounder via intraperitoneal injection,rFlaA, rFlaD, and rFlaE induced comparable relative percent survival (RPS) rates,which were significantly lower than that induced by rFlaB; rFlaB75%，rFlaA53.9%,rFlaD55.7%，rFlaE49.9%. Specific serum antibodies were induced by all flagellins,however, the antibody level induced by rFlaB was significantly higher than thoseinduced by other three flagellins about more than two times. Compared to sera from fish vaccinated with rFlaA, rFlaD, and rFlaE, serum from fish vaccinated with rFlaBsignificantly reduced the infectivity of V. anguillarum against host cells.To examine the potential adjuvant effect of the flagellins, flounder wereimmunized with rEsa1, a D15-like surface antigen that induces protective immunityas a subunit vaccine, in the presence or absence of rFlaA, rFlaB, rFlaD, and rFlaErespectively. The results showed that rFlaE, but not other three flagellins, significantlyincreased the RPS of rEsa1about20%. Compared to fish vaccinated with rEsa1, fishvaccinated with rEsa1plus rFlaE exhibited a significantly higher level of serumantibodies and enhanced expression of the genes involved in innate and adaptiveimmunity, such as IL-β、TNF、IgM、MHCⅠα、CD8α、CD40、TLR5.Taken together, these results indicate that FlaA, FlaB, FlaD, and FlaE havedifferent immunological properties and, as a result, differ in vaccine and adjuvantpotentials.