Purification And Polyclonal Antibody Preparation Of The Aeromonas Hydrophila Natural Flagellin And Recombinant FlaB Protein

Aeromonas hydrophila belongs to Vibrionaceae Aeramonas,which can infect avariety of aquatic and terrestrial animala with different disease outcomes and is apathogenetic strain spread among human beings, beats and fishes.They causefurunculosis and motile aeromonad septicemia in freshwater species;both diseases area serious problem for aquaculture.The human can suffer from diarrhea,food poisoning,and secondary infection because of pathogenic Aeramonas hydrophila,which makes aserious threat to human public health and safety.The flagellum is a special structure ofAeromonas hydrophila.Flagellar motility represents an important advantage forbacteria in moving toward favorable conditions or in avoiding detrimentalenvironments,and it allows flagellated bacteria to successfully compete with othermicroorganisms.In addition,flagella is an important virulence factor,which play acrucial role in adhesion,biofilm formation,and colonization of pathogenic bacteria.In this experiment, we purificate the natural flagellin of Aeromonashydrophila.The bacterial and flagella were identified by the transmission electronmicroscopy.SDS-PAGE showed that the purified flagellin protein strip was located atMr40×103region.Purification of the IgM in serum of the carp immunitied withnatural flagellin.Rabbit anti fish immunoglobulins antibodies labeled with HRP wereprepared.In addition,the flagellar gene flaB of Aeromonas hydrophila was amplifiedby PCR from the Aeromonas hydrophila genome,and cloned into the prokaryoticexpression vector pET30a(+).After being sequenced,the recombinant plasmidpET30a-flaB was transformed into strain BL21(DE3), and FlaB protein was expressedby the recombinant strain after IPTG(0.6mmol/L)induction.By Western-blotanalysis,the His-tagged proteins were confirmed.The molecular weight of the inducedprotein was about38kD as expected.The purificated recombinant protein was inoculated to New Zealand rabbits. Theprepared polyclonal antibody of recombinant protein were determinded for tier byindirect ELISA and for reactogenicity with the natural flagellin of Aeromonashydrophila.Our result showed that the rabbit antibodies raised against the recombinant could recognize the natural flagellin and the antibodies raised against natural flagellincould recognize the recombinant protein,suggesting flagellin FlaB may be animportant protective antigens of Aeromonas hydrophila and has protential for a noveltarget for vaccine development.