表达猪腹泻冠状病毒保护性抗原转基因乳酸菌诱导黏膜免疫的研究

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.

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。

猪腹泻冠状病毒引起的猪病毒性腹泻对养猪业危害严重。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，设计构建表达病毒保护性抗原的重组乳酸菌，利用该菌肠道黏附和传递表达病毒保护性抗原诱导黏膜免疫应答。本项目主要成果有：构建并测试了温度敏感辅助质粒及主质粒的双质粒基因打靶系统，该系统电转化到乳酸菌内，利用不同的温度，实现目的基因的敲除，同时可定向插入外源基因，实现乳酸菌的基因缺失和外源基因的插入；以乳酸菌基因打靶系统构建了表达猪腹泻冠状病毒保护性抗原的转基因乳酸菌，该重组乳酸菌不携带任何抗性选择标记，是真正意义的食品安全级活菌载体。利用乳酸菌质粒载体系统，构建了组成型表达猪腹泻冠状病毒免疫保护性抗原重组乳酸菌；以重组乳酸菌口服免疫小鼠可诱导机体产生肠道局部黏膜免疫、系统体液免疫和免疫保护作用；重组乳酸菌口服免疫怀孕母猪，生产的仔猪在其体内和母猪初乳中可检测到抗腹泻病毒的特异性抗体存在，仔猪攻毒试验结果显示，免疫母猪所产的仔猪无论是在减轻腹泻的严重性以及降低体内病毒的载量都有一定作用，说明仔猪可以获得被动免疫保护作用；通过乳酸菌菌体和上清液对猪源树突状细胞的作用，阐明了乳酸杆菌具有调节动物体肠道先天性免疫功能。乳酸菌菌体和其培养上清液在调节机体功能方面展示出不同的作用。培养上清具有抗炎作用，而乳酸菌本身具有促炎作用；并且具有刺激MoDCs，促进T细胞分化为Th1/Th2/Treg细胞，在Th1/Th2/Treg型细胞之间对改善平衡方面起着重要作用，而其上清液刺激的MoDCs主要将T细胞定向分化为Th2/Treg。本项研究成果为冠状病毒引起的猪腹泻免疫预防探索了新途径；建立的乳酸菌无痕打靶系统，为乳酸菌基因组表达其他异源功能性蛋白或免疫保护性抗原奠定基础。