The Optimum Culture Conditions And Preparation Of Immunostimulating Complexes(ISCOMs) For Aeromonas Hydrophila β-hemA Gene Production Expressed In E.coli

Aeromonas hydrophila (Ah) is an important pathagen to fresh water fishes, which frequently cause septicemia in cultured eel and lead to heavy loses every year. Vaccination against Ah become an important issue for aquaculture. Except the inactivated vaccine to Aeromonas hydrophila had been developed and applied onto clinical, the subunit and genetic engineering vaccines also underdeveloping.Hemolysin is one of the most virulent factors and important protective antigen of Ah. The hemolysin gene has been cloned and translated into E.coli, which can provide technological base for preparing the subunit vaccine against Ah.An orthogonal design was carried out to test the optimum culture conditions including temperature, culture medium and time for the recombinant bacteria. The results indicate that the optimizations are TSB medium, 37 ℃ and 30h, respectively. Further experiment showed that the hemolysis activity and the hemolysin amount produced under the following condition : 100mL TSB ( appending 0.05% yeast lixivium and 1.8ug /mL ZnCl2) and shaking at 37℃ for 30 hs were up to 17.44hu/ug and 3258ug, respectively, which were much more higher than that in LB (14hu/jug ,1260ug), or nutritional broth (8.22 hu/ug, 1199u g). Among the three factors , the culture medium was the most important factor which affected the gene production, and the temperature ranking in the second; in turns of hemolysis activity, the temperature is the most important influencing factor and followed by the time. The growth curve at the best culture condition displayed that the recombinant bacteria E.coli DH5a(PCB) could reach the log phase at 6 h after it was inoculated into TSB which prewarmed to 37℃.A method for preparing p-HemA-ISCOMs(Immune Stimulating Complexes),in which the expressed 0 -hemolysin of Ah was extracted by ammonia sulphate gradient precipitation and pretreated by acidization pH(2.5) to reveal hydrophobic regions and then used for constructing the complexes with Quil A was reported in this paper. The β-HemA- ISCOMs was a cagelike structurewith a diameter of 30-40nm under negative contrast electron microscopyThe eels were inoculated with b-HemA-ISCOMs,b-HemA-CFA and b-HemA-PBS. The results showed that the eel immunized with p-HemA-ISCOMs generated strong immunity against the challenge. The protection rate to the homologous challenge with TPS-30 strain was up to 100%, meanwhile, the protection rates of the eels immunized with b-HemA-CFA and p-HemA-PBS were 87.5%, 50%, respectively. Verified the cross protection ability by challenging the animal with a heterologous Ah strain named WQ, the p-HemA-ISCOMs immunized group was 100% survived from the challenge and the p-HemA-PBS immunized group was 33% only. Lymphocyte proliferation assay demonstrated tha the T cell proliferation index in β-HemA-ISCOMs injected eel was obviously higher than that in β-HemA-PBS immunized eel.