转基因乳酸菌表面展示猪轮状病毒保护性抗原及其诱导黏膜免疫的研究

项目名称： 转基因乳酸菌表面展示猪轮状病毒保护性抗原及其诱导黏膜免疫的研究

项目编号： No.31272594

项目类型： 面上项目

立项/批准年度： 2013

项目学科： 农业科学

项目作者： 李一经

作者单位： 东北农业大学

项目金额： 81万元

中文摘要： 猪轮状病毒所致猪腹泻在我国危害严重。本课题综合分析其致病与免疫特点及乳酸菌多种肠道益生功能，设计稳定表达猪轮状病毒保护性抗原，并能持久刺激黏膜免疫系统的转基因乳酸菌：利用构建的温度敏感型辅助质粒及主质粒构成的双质粒整合型基因打靶系统，定向敲除乳酸菌非必需upp和xgprt基因，在upp插入黏膜佐剂LTB；在xgprt位点插入猪轮状病毒保护性抗原VP4和VP7基因片段，通过温度敏感及氟尿嘧啶筛选，获得具有免疫增强作用的表面锚定表达猪轮状病毒保护性抗原转基因乳酸杆菌。该菌不携带任何选择标记，达到真正意义的食品安全级活菌载体。以此活菌口服免疫动物，测定肠道特异sIgA、血清抗体、抗体中和活性和对猪免疫保护作用，为轮状病毒引起的猪腹泻免疫预防探索新途径；建立的乳酸菌无痕打靶系统，对表达其它异源功能蛋白或其他保护性抗原奠定重要的技术平台。

中文关键词： 猪轮状病毒；转基因乳酸菌；黏膜免疫；免疫保护性抗原；

英文摘要： The project is designed to build of transgenic Lactobacillus expressing the protective antigens of the porcine rotavirus based on its pathogenic and immune characteristics, and using the genetically modified lactic acid bacteria to stimulate the mucosal immune system and induce the specific mucosal immune response of the animals. The two plasmids which consist of the temperature-sensitive helper plasmid and main plasmid are used as a gene targeting system, targeting knockout of lactic acid bacteria of non-essential upp and xgprt of gene. The mucosal adjuvant LTB and protective antigen of porcine rotavirus VP4 and VP7 gene are inserted in the upp and the xgprt sites Respectively.Expression of the protective antigens of the porcine rotavirus was detected via sodium dodecyl sulfate-polyacrylamide gel electrophoresis, Western blotting, and immuno-fluorescence, and both local mucosal and systemic immune responses against rotavirus were detected in BALB/c mice and Piglets immunized orally with the transgenic Lactobacillus expressing VP4-VP7 Fusion protein. This is new way for the prevention of rotavirus-induced diarrhea in pigs, and the targeting system provide an important technology platform for the expression of other heterologous functional proteins or other protective antigen.

英文关键词： Genetically modified lactic acid bacteria；Porcine rotavirus；Mucosal immune；immune protective antigen；