安全级乳酸菌载体系统靶向传递抗原促进虹鳟免疫应答的机制

The cell interaction model and vitro experiment are used to filtl the modified and enhanced green fluorescent protein (EGFP) mode molecules, which having a target binding function on trout intestinal epithelial cells, the antigen presenting cells (CDs) and macrophages. The transfer efficiency of antigen uptake and effect on DCs mature and function in the process are analyzed.The protective antigen (VP2) of IPNV replace EGFP, which is the best targeting modification pattern molecule. Lactobacillus expressing VP2 of IPNV (targeting modified live vector system) is constructed. The effect on intestinal tissue development of juvenile and the non-specific immune function after oral immunization of rainbow trout juveniles are studied. The immune response characteristics, pathological changes after challenge, resistance to infection and other series of indicators are analyzed. At the point of the Lactobacillus conditioning bowel function and targeted delivery of antigens, it clarifies the mechanism to promote earlier, faster, stronger immune response in Juvenile Rainbow Trout. It provide new ideas and theoretical basis for the the early immune and prevention of infectious diseases in fish.

采用细胞互作模型、离体实验等方法筛选对虹鳟肠上皮细胞、抗原呈递细胞(CDs)和巨噬细胞（Mø）具有靶向结合功能的修饰性增强型绿色荧光蛋白（EGFP）模式分子，分析在此过程中对抗原的摄取转运效率、对DCs成熟和功能的影响。用IPNV的保护性抗原VP2基因置换优选的最佳靶向修饰模式分子中的EGFP，构建表达靶向修饰IPNV VP2蛋白的安全级乳酸杆菌活载体系统。口服免疫虹鳟幼鱼研究其对幼鱼肠道组织发育、非特异性免疫功能的影响，分析其所诱导产生的免疫应答的特征，攻毒后病理学变化、抗感染能力等系列指标。从乳酸杆菌调理肠道功能和靶向传递抗原的角度，阐明促进虹鳟幼鱼产生更早、更快、更强免疫应答的机制，为鱼类传染性疾病的早期免疫预防提供新思路和理论依据。

传染性胰腺坏死病毒（Infectious Pancreas Necrosis Virus，IPNV）感染鲑科幼鱼引起以肝胰腺等内脏实质器官出血坏死的急性病毒性疾病。本研究采用细胞互作模型、离体实验等方法筛选对虹鳟肠上皮细胞、抗原呈递细胞和巨噬细胞具有靶向结合功能的分子，分析在对抗原的摄取转运效率。构建表达靶向修饰IPNV VP2蛋白的安全级乳酸杆菌活载体系统，口服免疫虹鳟幼鱼后分析其诱导产生的免疫应答水平。结果显示：（1）获得虹鳟鱼黏膜免疫靶向修饰蛋白AHA1-flaE-CK6-EGFP、AHA1-CK6-EGFP、flaE-CK6-EGFP、AHA1-EGFP、flaE-EGFP、CK6-EGFP、AHA1-flaE-EGFP纯化的重组靶向蛋白；体外实验结果表明嗜水气单胞菌粘附素AHA1和虹鳟趋化因子CK6融合表达的靶向修饰蛋白分别具有较强的黏附能力和趋化能力，可作为虹鳟口服疫苗的候选靶向分子佐剂。（2）在IPNV保护性抗原VP2前插入靶向分子AHA1和CK6后获得重组干酪乳杆菌pPG-2-AHA1-CK6-VP2/L. casei393和pPG-2-CK6-VP2/L. casei. 393诱导后表达目的蛋白；同时，靶向修饰的重组干酪乳杆菌在虹鳟消化道内的定植能力更强，表明了黏附素AHA1在重组干酪乳杆菌中起到了良好的黏附效果。（3）将诱导表达目的蛋白的靶向修饰的重组干酪乳杆菌口服免疫虹鳟幼鱼，均可刺激机体产生体液免疫应答和细胞免疫应答，其中血清抗体和黏膜抗体水平和中和病毒能力2组靶向修饰的重组干酪乳杆菌显著高于对照组，而pPG-2-AHA1-CK6-VP2/L. casei. 393组显著高于pPG-2-CK6-VP2/L. casei. 393组。（4）经靶向修饰后的重组乳杆菌口服虹鳟幼鱼后早期可以诱导细胞因子β-防御素、TNF-1α、Mx、IL-8和MHC-II的高表达，使更多的抗原提呈细胞参与免疫反应，同时能够引起更快、更强的早期免疫应答水平；2种靶向修饰后的重组乳杆菌诱导鱼体产生了更有效的免疫应答，降低IPNV对虹鳟的感染力。由此可见嗜水气单胞菌粘附素AHA1和细胞因子CK6可以促进虹鳟幼鱼产生更早、更快、更强免疫应答的机制，为鱼类传染性疾病的早期免疫预防提供了理论依据。