嗜水气单胞菌菌蜕的制备及其对银鲫的口服免疫

Generation of Aeromonas hydrophila ghosts and their evaluation as oral vac-cine candidates in Carassius auratus gibelio

摘要:

菌蜕系统是一个自身具有佐剂性质的新型疫苗体系, 不含细胞质内容物但具有细菌的完整表面抗原结构, 可诱导机体的体液、细胞免疫应答及增强黏膜免疫反应。本研究通过将带有裂解基因E的质粒pElysis转化至嗜水气单胞菌J-1株中, 对Ah J-1(pElysis)进行温度诱导, 温度从28℃升至42℃, 每隔15min检测菌液的OD600值, 测定其溶菌动力学, 并做无菌检验, 用扫描电镜观察裂解后的细菌形态, 研究其作为口服疫苗对银鲫的效果。结果显示, 通过温度诱导, 嗜水气单胞菌J-1(pElysis)OD值在诱导30min后开始持续下降, 75min时开始趋于平稳, 到120min溶菌效率达99.99%, 诱导16h后进行无菌检验, 证实其无活菌。扫描电镜观察绝大部分菌体经诱导后形成菌蜕, 细胞两端有溶菌通道。动物试验表明, 用菌蜕口服免疫的银鲫, 在第5周产生较高的凝集抗体, 达到27, 并能维持2周; 而甲醛灭活苗组为26, 维持时间仅一周; 生理盐水对照组效价仅2。攻击试验表明, 菌蜕疫苗组和甲醛灭活疫苗组对嗜水气单胞菌强毒株J-1的攻击均有保护作用, 其相对保护率分别为16/20(78.95%)和12/20(57.9%), 显示菌蜕疫苗比普通灭活疫苗能更有效地激活机体的免疫保护。

Abstract:

The bacterial ghost (BG) system is a novel vaccine delivery system endowed with intrinsic adjuvant properties. Bacterial ghosts are nonliving Gram-negative bacterial cell envelopes devoid of cytoplasmic contents while maintaining their cellular morphology and native surface antigenic structures. They are produced by PhiX174 protein E-mediated lysis of Gram-negative bacteria, and can induce humoral and cellular immune response, including mucosal immune responses. Plasmid pElysis consisting E gene was transformed into AhJ-1. Through shifting the culture temperature from 28℃ to 42℃, A. hydrophila J-1 (pElysis) was induced to lyse and the OD600 value of culture media was measured every 15 minutes during the induction. The lysed bacteria were observed by scanning electron microscopy (SEM). The A. hydrophila ghosts (AHG) used as oral vaccine were also investigated. The OD600 value of A. hydrophila J-1(pElysis) began to decline after 30min of induction, and after 75min of induction, the OD600 value decline speed become slowly. The efficiency of ghost induction in non-lyophilized A.hydrophila was 99.99%, 16 hours post induced, no live bacteria can be detected in culture. Scanning electron microscopy observation proved that most lysed bacteria were emptied. Fish vaccination experiments shows that the antibody evoked highest degree after 5 weeks by oral administration of bacterial ghost vaccine and the agglutination antibody titer reached 27 and continued two weeks, while the agglutination antibody titer of formalin killed vaccine only reached 26 and only maintained one week. After challenged with the parent strain J-1, the survival rate of bacterial ghost vaccinated fish was higher than the control group and formalin killed vac-cine group, the relative percent survival (RPS) was 78.95% (16/20), but the RPS of formalin killed vaccine group was 57.9% (12/20). This suggests that the bacterial ghost vaccine has higher potential to induce protective adaptive immu-nity than normal vaccine.