Hsp60受体靶向型干酪乳杆菌通过NF-κB通路调节肠道黏膜屏障功能的机制研究

乳酸菌能诱导宿主产生黏膜与系统性免疫反应，但受限于定植能力等原因无法推广应用，因此构建新型的乳酸菌疫苗载体成为免疫学研究热点，而目前的研究主要集中于质粒表达抗原递送平台。本研究拟将乳酸菌与基因编辑技术结合，建立干酪乳杆菌pheS*反向筛选系统，构建基因组表达维氏气单胞菌强、弱毒株黏附蛋白的受体靶向型乳酸菌载体疫苗，其最大特性是在机体内竞争肠道黏附位点并分泌释放抗原。通过体外的抗菌活性、细胞黏附、侵染及易位等指标鉴定新型乳酸菌载体的生物学特性。免疫乌鳢后通过细胞黏附、抗体水平检测及攻毒保护试验评价疫苗的免疫效应，并检测NF-κB通路、上皮细胞通透性、表面Hsp60受体反应及Ig+ B细胞类型转换等指标揭示受体靶向型干酪乳杆菌疫苗在鱼类体内引起免疫反应的机制。本项目的研究成果为揭示工程益生菌调节肠道屏障功能的分子机制研究提供科学依据。

Mucosal and systemic immune responses can be triggered by lactic acid bacteria, however, is restricted by the ability to colonize intestinal surfaces. Therefore, novel bioengineered probiotic is becoming more and more attractive as vaccine delivery. Currently, probiotic vectors are mainly targeting for synthesizing protein antigens based on plasmid expressing platform. In this study, we will construct a pheS* counterselectable system in Lactobacillus casei and develop bioengineered probiotic vaccine genomic expressing adhesion protein of pathogenic and non-pathogenic A. veronii, which combines receptor-targeted engineered probiotics with gene editing technology. The attractive characteristic of receptor-targeted engineered probiotics is to compete for intestinal adhesion sites with pathogens and secrete antigens binding to receptors of epithelial cell. The biological characteristics of engineered L. casei vector will be identified by antimicrobial activity, cell adhesion, invasion, and translocation in vitro. The cell adhesion, antibody level and protection against A. veronii will be evaluated after orally administration with vaccine in northern snakehead model. The immunological mechanism induced by receptor-targeted L. casei in fish will be preliminarily elucidated by NF-κB pathway, epithelial cell permeability, activation of Hsp60 receptor on the surface of epithelial cells, and Ig+ B cell isotype switch. The results of this study will provide scientific basis for clarifying the mechanisms of engineered probiotic regulate intestinal barrier function.