关键词: 迟缓爱德华氏菌, 口服疫苗, 肠溶微球, 免疫保护效应

Abstract: Seawater factory farming is a modern high-tech fishery with the advantage of water and energy saving. However, the diseases of the cultured fish happened frequently for the high density and intensive farming model and has caused great economic loss to the culture enterprises. Vaccination is an effective way to control the disease of cultured fish. Compared with injection and soaking, oral vaccine has the advantages of convenient inoculation, no injury to the body and good compliance of the receptor, which has become a hot spot of research. Sodium alginate is a natural macromolecule polysaccharide from the ocean. It is nontoxic, biocompatible and degradable. The degradation products can be fully absorbed by the organism. The use of sodium alginate as the carrier material to prepare the microsphere vaccine can increase the viscosity and prolong the release time of the drug. Sodium alginate is feasible to prepare the oral vaccine for fishing as a carrier for its low price, uncomplicated technology and convenient source. The study developed a new method to generate fish oral vaccine of Edwardsiella tarda by emulsfication with sodium alginate as the carrier. The results showed that the optimum preparation process of oral vaccine was 3%（m/v）sodium alginate solution, 4%（m/v）calcium chloride solution, 1∶2 ratios of water and oil, 1% Span80 emulgator, 1 200 r/min stirring rate. The embedding rate was 91.04%. The mean grain size of the microballon was（16.51±11.87）µm. It is necessary to prepare the microsphere vaccine suitable for the intestinal targeted drug delivery, and the artificial simulated gastric and intestinal juice were used to analyze the tolerance of vaccine microspheres when treated by them in vitro. The results showed that the releasing rate of the microballon were 0.8% and 98.1% after dealing with simulated gastric and intestinal juice for 16 hours, respectively. Escherichia coli expressing green fluorescent protein emulsficated with sodium alginate was orally administrated to Cynoglossus semilaevis Gunther to detect the tolerance of vaccine microspheres in gastric and intestinal juice in vivo. It showed that the microspheres do not dilate or disintegrate in the acid stomach, or disintegrate in the neutral environment of the intestine. The above results indicated that the vaccine can endure the acid environment of the stomach, reach the hindgut of the fish and be absorbed by the villus, which is suitable for drug targeting. The results of the immunprotection experiment showed that the highest serum titer was 1∶322 after oral vaccinatione for 14 days, the highest leukocyte phagocytosis rate was 27.06% after oral vaccination for 14 days, and the highest phagocytosis index was 1.65 after oral vaccination for 14 days. All fishes were challenged with Edwardsiella tarda after oral vaccination for 28 days, the mortality rate of control group and vaccine group were 58.82% and 13.33% respectively, and the immune protective rate was 77.33%. The present study proposed a new fish oral vaccine preparation technology. The oral vaccine could activate the fish immune response and prevent fish disease.

Key words: Edwardsiella tarda, oral vaccine, enteric microsphere, immunoprotective effect