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为比较鱼源乳酸菌表达系统口服疫苗在不同免疫程序下诱导鱼免疫应答水平的差异,确定鱼源乳酸菌表达系统口服免疫虹鳟幼鱼的免疫程序。本研究构建了重组表达IPNV VP2-VP3蛋白的鱼源植物乳杆菌L1212,将重组菌pPG612-VP2-VP3/L1212包裹颗粒饲料,口服免疫虹鳟幼鱼。免疫程序分为连续免疫组、免疫1 d后间隔1 d再免疫1 d组、连续免疫4 d后32 d加强免疫1次组和间隔免疫后32 d加强免疫1次组。免疫后进行尾动脉采血,间接ELISA方法检测各组血清抗体效价,免疫接种66 d后,腹腔注射IPNV,计算各组相对免疫保护率。确定颗粒饲料按照1 mL/g比例与108 CFU/mL重组菌pPG612-VP2-VP3/L1212和2.5%海藻酸钠混合,于20℃烘干制备口服疫苗。间隔免疫组血清抗体效价和攻毒保护率均显著高于其他组,并且免疫2次的间隔免疫组的血清抗体效价和攻毒保护率均显著高于免疫1次间隔免疫组。采用重组菌包被颗粒饲料饲喂虹鳟幼鱼,间隔免疫的免疫程序和免疫保护率优于连续免疫的免疫程序,对IPNV的入侵起到保护作用。
In order to compare the difference of the level of fish immune response induced by oral vaccine of Lactobacillus sporogenes expression system under different immunization programs and to determine the immune procedure of orally immunized rainbow trout juvenile with lactic acid bacteria expression system. In this study, we constructed Lactobacillus plantarum L1212 recombinantly expressing VP2-VP3 protein of IPNV and orally immunized rainbow trout juveniles with the recombinant plasmid pPG612-VP2-VP3 / L1212. Immunization procedures were divided into continuous immunization group, 1 d after immunization 1 d after immunization 1 d group after immunization, four days after continuous immunization 32 d enhanced immunization group and 32 d after immunization immunization 1 group. After the immunization, the tail artery blood was drawn and the antibody titer of each group was detected by indirect ELISA. IPNV was injected intraperitoneally after 66 days of immunization, and the relative immune protection rate was calculated. The pellets were determined to be mixed with 108 CFU / mL of recombinant bacteria pPG612-VP2-VP3 / L1212 and 2.5% sodium alginate at a rate of 1 mL / g and dried at 20 ° C. to prepare oral vaccine. Serum antibody titer and challenge protection rate of interval immunization group were significantly higher than those of other groups, and serum antibody titer and challenge protection rate of interval immunization group were significantly higher than that of immunization interval 1 immunization group. The juveniles of rainbow trout fed with recombinant bacteria-coated pellet feed had better protective immunity against IPNV invasion than those immunized with continuous immunization.