创伤弧菌微球口服疫苗的制备及其免疫效果研究

Investigation on the immune effect of Vibrio vulnificus inactivated vaccine in fish

为了研究创伤弧菌灭活疫苗的免疫效果,本研究将实验室保存的创伤弧菌菌株EPL0201经0.3%甲醛灭活,采用海藻酸钠包裹灭活菌液制备了创伤弧菌微球疫苗,通过口服途径免疫健康的珍珠龙胆石斑鱼(Pearl gentian grouper),在免疫后第28天用1×107cfu/mL创伤弧菌腹腔注射珍珠龙胆石斑鱼,检测了免疫后珍珠龙胆石斑鱼部分免疫相关基因的表达趋势和攻毒后的免疫保护率。创伤弧菌微球口服疫苗的粒径为(34.55±5.33)μm,包覆率为90.5%。测其安全性良好。实时定量PCR(quantitative real-time PCR,q RT-PCR)结果显示,与对照组相比,各免疫基因先后出现不同的表达变化。TLR信号通路上的基因TLR1、TLR2、TLR3、TLR5和M y D88及炎性细胞因子IL-1β、IL-12p40和TNF-α在肾脏、脾脏、肝脏和肠组织中都参与免疫应答。TGF-β只在脾脏中有表达。免疫基因在各个组织中的表达变化不同,其中,表达量最高的是IL-1β在脾脏中,最高值为对照组的72.37倍。同一种免疫基因在鱼体不同器官和组织中的峰值表达量时间不同,同一组织中不同免疫基因的峰值表达量也不相同,说明各组织中的基因表达变化有差异性。攻毒试验结果显示,免疫保护率为61.3%。本研究结果显示创伤弧菌微球口服疫苗能够引起免疫基因参与免疫应答反应,对珍珠龙胆石斑鱼具有免疫保护作用,也为微球口服疫苗防治创伤弧菌引起的疾病提供试验数据支持。

In order to study the immune effect of inactivated Vibrio vulnificus vaccine,this study prepared Vibrio vulnificus microsphere vaccine by inactivating the Vibrio vulnificus strain EPL0201 stored in the laboratory with 0.3%formaldehyde and wrapping the inactivated bacteria solution with sodium alginate.Healthy pearl gentian grouper was immunized by oral route,and was injected intraperitoneally with 1×107 cfu/mL Vibrio vulnificus to challenge the pearl gentian grouper.The expression trend of some immune related genes of the pearl gentian grouper after immunization and the immune protection rate after challenge were detected.The results showed that the particle size of Vibrio vulnificus microsphere oral vaccine was(34.55±5.33)μm.The coverage rate was 90.5%.Its safety was good.Quantitative real-time PCR(QRT-PCR)results showed that,compared with the control group,each immune gene had different expression changes successively.They participated in immune response in kidney,spleen,liver and intestine which involved the genes TLR1,TLR2,TLR3,TLR5,and Myd88 in the TLR signaling pathway and inflammatory cytokines IL-1β,IL-12P40 and TNF-α.The expression of immune genes in different tissues was different,and the highest expression level of IL-1βwas in spleen,and the highest value was 72.37 times that of the control group.The peak of peak expression of the same immune gene in different organs and tissues of fish was different,and the peak expression of different immune genes in the same tissue was also different,which indicated that the gene expression changes in different tissues were different.The results of challenge experiment showed that the immune protection rate was 61.3%.The results of this study showed that Vibrio vulnificus oral vaccine can induce immune genes to participate in the immune response,and has immune protection effect on pearl Gentian grouper.It also provides experimental data support for the prevention and treatment of diseases caused by Vibrio vulnificus with microsphere oral vaccine.