表达牛乳铁蛋白肽的重组罗伊氏乳酸杆菌替代抗生素改善断乳仔猪肠道黏膜免疫的分子机制

Lactobacillus reuteri isolated from piglets’ intestinal mucosa was used as the vehicle to produce and delivery bovine LFcin and LFampin peptide. The recombinant Lactobacillus systems expressing peptide was constructed using plasmid vector pPG-PHE containing of constitutive promoter of Peptidoglycan hydrolase, enhancer, secret signal peptide and terminator, the expressed peptides will be purified and analysis the structure and function characteristics, and will be compared with the synthesized peptides LFcin and LFampin. In vitro, in order to analysis the molecular modulate mechanisms of Inflammatory and non-inflammatory response of recombinant strain, porcine intestinal epithelial cells and APCs from the payer’s patches of ilea, which isolated from swine, were co-cultured. Recombinant Lactobacillus reuteri fed 3-week-old weaned piglets, determined the indexes of promote the healthy growth; analysis of intestinal microflora, detection the expression of specific mRNAs of endogenous defense peptides and small intestine mucin, characterize the expression of gene and protein of intestinal tight junction protein; determination the expression of mRNA and protein in intestinal mucosal immune signaling pathway associated molecules. And also, 5-week-old piglets of tested will be challenged with enterotoxic Escherichia coli (ETEC) , analysis of mRNA expression of related cytokines; detection the content of TNF-α, PGE2 and NO; Characterization the level of phosphorylation of signaling pathways molecules in relieving inflammation, determination the gene and protein expression levels of pattern recognition receptors TLR (s) and NOD1 in intestinal epithelial cells, these results will reveal the mechanism of recombinant lactobacillus reuteri improving piglets intestinal immune function, which clarify the influence of recombinant lactobacillus reuteri on piglets intestinal immunization, furthermore as immune-health promoting factor to improve growth performance and productivity in post-weaning pigs.

以仔猪小肠黏膜定植的罗伊氏乳酸杆菌作为共表达牛乳铁蛋白多肽LFcin及LFampin的宿主菌，利用带有组成型肽聚糖水解酶启动子和分泌信号肽的载体pPG-PHE构建重组菌；分析表达多肽的体外抗菌及抗病毒活性机制；以PIEC和猪回肠派伊尔氏结APC的共培养分析重组菌在炎症和非炎症反应状态下的免疫调节机制；重组菌饲喂3周龄断乳仔猪，测定其促进健康生长的指标;分析肠道微生物区系，内源性防御肽和小肠黏蛋白mRNA的表达量，小肠紧密连接蛋白的基因和蛋白表达量；肠道黏膜免疫信号通路相关分子的mRNA和蛋白表达。并以ETEC攻击感染5周龄仔猪，分析相关细胞因子的mRNA表达；检测TNF-α、PGE2和NO的含量；检测缓解炎症反应信号通路分子的磷酸化水平和肠道上皮细胞的模式识别受体TLR(s)和NOD1的基因和蛋白表达水平，揭示重组菌对肠道免疫的影响及阐明其作为免疫健康促进因子改善仔猪肠道免疫功能的机制。

我国是世界第一大生猪生产和猪肉消费国，但是在生猪饲养过程中，早期断奶引起的仔猪腹泻是制约仔猪健康生长的重要因素之一。近年来，一些抗菌肽制剂和益生菌制剂在预防仔猪腹泻的生产应用研究上越来越受到重视，尤其是抗菌肽制剂，由于其不产生耐药性及广谱的抑菌效果而备受青睐，但多数抗菌肽制剂在杀死致病菌的同时对益生菌也有抑制作用。如果能同时高效的利用益生菌和抗菌肽制剂预防仔猪腹泻，那么将会产生事半功倍的效果。本项目从仔猪肠道内分离到的罗伊氏乳酸杆菌（Lactobacillus reuteri，LR）中筛选出定植能力强的菌株作为基因工程宿主菌，选用对益生菌基本没有抑制作用的牛乳铁蛋白肽作为抗菌肽制剂，通过基因工程技术构建可以分泌表达牛乳铁蛋白肽（Lactoferricin-Lactoferrampin，LFCA）的重组罗伊氏乳酸杆菌（LR-LFCA）。并优化重组菌表达多肽条件，检测重组菌表达多肽的抗菌、抗病毒活性，将其在肠道黏膜免疫中发挥作用的机制进行了探究，既利用体外的肠道上皮细胞和抗原提呈细胞共培养模型来研究非炎症和炎症反应状态下的免疫调节机制，又通过动物实验来进行研究，阐明了表达乳铁蛋白肽的在肠道黏膜定植的罗伊氏乳酸杆菌对肠道黏膜免疫非特异性的免疫调节机制。结果表明，该重组菌可以促进仔猪生长并增强仔猪肠黏膜屏障功能，包括提高免疫球蛋白含量，促进肠黏膜上皮组织细胞的发育，增强肠屏障功能的完整性。重组菌提高断奶仔猪抗ETEC K88感染能力的肠黏膜免疫机制可能与其可以通过抑制TLR4、Myd88和MLCK蛋白的表达量，增加紧密连接蛋白ZO-1和Claudin-2的表达，改善肠上皮屏障功能，抑制NF-κB通路、DCs成熟和T细胞活化增殖进而提高仔猪抗炎能力以及激活Nrf2 / HO-1通路提高仔猪抗氧化能力有关。本研究为表达牛乳铁蛋白肽的重组猪源罗伊氏乳酸杆菌作为微生态制剂在生产中的应用提供理论依据，期望其可以成为无抗饲养用的抗生素替代品，为在我国实现健康养殖的绿色安全营养关键技术研发提供技术支撑。