猪传染性胃肠炎（TGE）和猪流行性腹泻(PED)是由冠状病毒引起的猪肠道传染病，此类疾病在我国流行广，危害严重。综合分析腹泻冠状病毒肠道黏膜感染与黏膜免疫特点及乳酸菌抗腹泻等多种益生功能，设计构建基因组稳定表达病毒保护性抗原的转基因乳酸菌，利用该菌肠道黏附和传递抗原诱导黏膜免疫应答。以构建的温度敏感辅助质粒及主质粒的双质粒基因打靶系统定向敲除乳酸菌非必需基因upp，构建缺失upp的乳酸杆菌，在此插入PEDV S基因COE片段，并在乳酸菌eno基因终止密码子后插入保护性抗原TGEV S基因D位点，通过温度及氟尿嘧啶筛选，获得具有表面表达猪腹泻冠状病毒保护性抗原转基因乳酸菌。转基因乳酸菌不携带任何抗性选择标记，是食品安全级活菌载体，以此口服免疫动物，测定肠道特异sIgA、血清抗体、抗体中和活性、免疫保护作用，并深入探讨特异性抗原黏膜递呈细胞的表型特征，为冠状病毒引起的猪腹泻免疫预防探索新途径。

The project is designed to build of transgenic Lactobacillus expressing the protective antigens of the porcine diarrhea coronaviruses which contain Transmissible gastroenteritis virus(TGEV)and porcine epidemic diarrhea virus(PEDV) based on their 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 the gene targeting system, targeting knockout of lactic acid bacteria of non-essential upp. On the basis of the upp gene deletion of lactobacillus casei, the protective antigen of PEDV COE gene are inserted in the upp TGED D sites are inserted between the termination codon and terminator of the enolase(eno) gene sites Respectively. Expression of the protective antigens of the porcine diarrhea coronaviruses was detected via sodium dodecyl sulfate-polyacrylamide gel electrophoresis, Western blotting, and immuno-fluorescence, and both local mucosal and systemic immune responses against porcine diarrhea coronaviruses were detected in BALB/c mice and Piglets immunized orally with the transgenic Lactobacillus which expresses the protective antigen of PEDV COE gene and TGEV D sites, and further explore the phenotypes of the dendritic antigen presenting cells existed in blood and intestinal submucosa. This is a new way for the prevention of porcine coronaviruses induced diarrhea in pigs.