斑点叉尾鲖源海豚链球菌Srr蛋白海藻酸钠-壳聚糖口服疫苗的制备及其免疫效果研究

制备海藻酸钠-壳聚糖-海豚链球菌Srr蛋白微球疫苗, 并检测其对斑点叉尾鲙的免疫效果。采用乳化法利用海藻酸钠-壳聚糖包被Srr蛋白, 测定其包封率、载药率及包被蛋白的抗原性; 通过拌饲投喂免疫斑点叉尾鲙, 分为Srr组、Srr微球组、空微球组以及对照组, 间接ELISA法检测免疫后斑点叉尾鲙的血清抗体水平, 试剂盒检测多项血清非特异性指标; 于免疫后第4周利用海豚链球菌攻毒, 计算各组相对保护率, 并通过实时荧光定量PCR检测相关基因的表达量。结果显示, 通过乳化法制得形态为圆形或椭圆形、大小较为均一的微球疫苗, 粒径为(4.26±1.13) μm, 包封率为92.38%, 载药率为19.41%, Western-blot分析表明Srr蛋白微球具有较好的抗原性; Srr微球组的抗体效价峰值出现在第4周, 明显高于其他组, 血清总蛋白、T-SOD以及溶菌酶活力均显著或极显著高于其他实验组, 并获得60%的相对保护率。荧光定量分析结果显示, Srr微球组攻毒后24h和48h各免疫基因表达量均有所上调。Srr蛋白微球疫苗能够提高斑点叉尾鲙抵抗海豚链球菌的能力, 对海豚链球菌起到了一定的预防作用。

STUDY ON THE PREPARATION AND IMMUNE EFFICACY OF A ALGINATE-CHITOSAN ORAL VACCINE OF SRR-PROTEIN FROM STREPTOCOCCUS INIAE

The goal of this study was to prepare the alginate-chitosan microcapsules containing the Srr-protein of Streptococcus iniae, and to determine its immune effect in channel catfish by oral route. The alginate-chitosan-Srr-protein microcapsules were prepared by an emulsification method, and the encapsulation efficiency, drug-loaded rate, and immunogenicity integrity were tested. Healthy fish were divided into 4 groups, named Srr group, Srr-microcapsules group, empty-microcapsules group, and control group to immune, respectively. The serum antibody level was detected by an indirect ELISA method, and serum non-specific indexes were detected by reagent kits. 4 weeks after the immunization, the relative percent survivals (RPSs) of each group were calculated by challenging the fish with S. iniae, and the mRNA levels of immune related genes were analyzed by real-time PCR. The alginate-chitosan-Srr-protein microcapsules showed round or oval in shapes, with a mean diameter of 4.26±1.13 μm, an encapsulation efficiency of 92.38%, and a drug-loaded rate of 19.41%. Western-blot indicated a good immunogenicity of the microcapsules. The peak of antibody titer appeared at the 4th week after the immunization, among which, antibody titer in the Srr-microcapsules group was the highest. The serum total protein, T-SOD and lysozyme activity of Srr-microcapsules group were obviously higher than those of the other groups. The relative percentage of survival in Srr-microcapsules group was 60%. The results of real-time PCR presented that mRNA levels of immune related genes in kidneys and spleens from the Srr group increased more notably than those in the other groups during the 24h to 48h post injection. In conclusion, the alginate-chitosan-Srr-protein microcapsules vaccine was able to protect channel catfish from S. iniae infection.