乳酸菌表达与传递猪流行性腹泻病毒保护性抗原靶向融合肽诱导黏膜免疫的研究

The project is designed to build of recombinant Lactobacillus expressing the protective antigens of the porcine epidemic diarrhea based on its pathogenic and immune characteristics. A vaccination strategy was established by using Lactobacillus to deliver PEDV protective antigen(PA) via specific dendritic cell-targeting peptides to dendritic cells (DCs) or the M cell-targeting ligand to M cells, which reside in the periphery and mucosal surfaces, thus directing and regulating acquired immunity. The efficiency of oral delivery of Lactobacillus expressing a PA-DCpep fusion was evaluated in mice and pigs.The expression of the protective antigens of the porcine epidemic diarrhea virus was detected via sodium dodecyl sulfate-polyacrylamide gel electrophoresis, Western blotting, and immuno-fluorescence, and both local mucosal and systemic immune responses against porcine epidemic diarrhea virus were detected in BALB/c mice and Piglets vaccinated with recombinant Lactobacillus expressing PA-DC pep induce protective immunity against porcine epidemic diarrhea virus compared with mice vaccinated with Lactobacillus expressing PA-control peptide or an empty vector. Additionally, serum anti-PA titers, neutralizing PA antibodies, and the levels of IgA-expressing cells were all comparable with immunized orally with the recombinant Lactobacillus expressing Fusion protein. This is new way for the prevention of porcine epidemic diarrhea virus induced diarrhea in pigs, and the development of this strategy for oral delivery of DC-targeted antigens provides a safe and protective vaccine via a bacterial adjuvant that may potentiate mucosal immune responses against pathogens

猪流行性腹泻在我国流行范围广，危害严重。常规灭活疫苗非经口免疫所产生的循环抗体对该病毒的免疫保护效果并不理想。本课题依据猪流行性腹泻病毒肠道黏膜感染与免疫特性，探索以提高肠道黏膜免疫功效进行疾病的科学预防。实验设计以肠道益生性乳酸菌为表达和传递系统进行口服接种，通过菌体表面表达猪流行性腹泻病毒免疫保护性抗原靶向融合肽，将其完整无损传递于肠道黏膜，进一步通过巨噬细胞类靶向肽将抗原物质准确传递于抗原转运和递呈细胞，启动肠道黏膜免疫应答。表达猪流行性腹泻病毒保护性抗原靶向融合肽乳酸菌活菌物质口服免疫模型动物小鼠和本动物猪后，在不同免疫时期测定肠道和肠道外局部黏膜免疫应答、系统免疫应答、体外抗原中和能力及体内免疫保护作用。在此基础上，进一步测定靶向肽定向传递抗原于M细胞或树突状细胞的功能活性，深入探讨表达外源抗原并具有靶向传递作用的重组乳酸菌粘膜免疫保护机制，为猪流行性腹泻免疫预防探索新途径。

猪流行性腹泻在我国流行范围广，危害严重。常规灭活疫苗非经口免疫所产生的循环抗体对该病毒的免疫保护效果并不理想。本课题依据猪流行性腹泻病毒肠道黏膜感染与免疫特性，探索以提高肠道黏膜免疫功效进行疾病的科学预防。实验设计以肠道益生性乳酸菌为表达和传递系统进行口服接种，通过菌体表面表达猪流行性腹泻病毒免疫保护性抗原靶向融合肽，将其完整无损传递于肠道黏膜，进一步通过巨噬细胞类靶向肽将抗原物质准确传递于抗原转运和递呈细胞，启动肠道黏膜免疫应答。表达猪流行性腹泻病毒保护性抗原靶向融合肽乳酸菌活菌物质口服免疫模型动物小鼠和本动物猪后，在不同免疫时期测定肠道和肠道外局部黏膜免疫应答、系统免疫应答、体外抗原中和能力及体内免疫保护作用。在此基础上，进一步测定靶向肽定向传递抗原于M细胞或树突状细胞的功能活性，深入探讨表达外源抗原并具有靶向传递作用的重组乳酸菌粘膜免疫保护机制，为猪流行性腹泻免疫预防探索新途径。