发酵黄芪多糖对鸡肠道乳酸菌FGM表面粘附蛋白的表达调控研究

Surface proteins, which are the main material basis of lactic acid bacteria adhering to intestinal host cells and mucous membrane, play a key role in the process of bacterial colonization, and have been a hot area of research for recent years. Our group's research before showed that the astragalus polysaccharide fermented by FGM, lactic acid bacteria can improve extraction ratio and biological activity of the polysaccharides, and the polysaccharides help to improve chicken intestinal micro ecological environment. It is need to make further research whether the fermentation astragalus polysaccharide play a role through regulating the expression of FGM’s surface proteins. This project will screen and identify FGM’s surface proteins by the western blot hybridization technology, the mass spectrometry identification and sequence analysis. The key structure of the protein domain relevant to adhesion action will be analysised by bioinformatics. Using gene recombination technology and immune analysis technology, the impacts of fermented astragalus polysaccharides on the surface proteins adhering to intestinal host cells, inhibiting pathogenic bacteria and positioning in the bacterial surface will be studied. In order to analyze the regulation mechanism of fermented astragalus polysaccharides on expression of FGM’s surface proteins, the regulation of different molecular weight of fermented astragalus polysaccharides in the expression of FGM’s surface proteins will be studied from the transcription and translation level by fluorescent quantitative PCR and western blot, which will provide the scientific basis for the research on the biological synergy mechanism of lactic acid bacteria and astragalus polysaccharides.

表面蛋白是乳酸菌粘附宿主肠道细胞的主要物质基础，在细菌定植及益生功能发挥过程中起关键作用，是近年研究热点。课题组已建立了分离于鸡盲肠的乳酸菌FGM发酵黄芪并提取多糖的技术工艺，前期研究发现该法提取的黄芪多糖在改善鸡肠道微生态环境方面有显著意义。发酵黄芪多糖是否可通过影响乳酸菌FGM表面蛋白的表达调控，发挥其益生作用呢？需深入研究。本项目拟利用细菌表面粘附配体与肠上皮特异性受体相互作用的机理，筛选乳酸菌FGM表面粘附蛋白，通过质谱、测序和生物信息学相结合的方法解析特异性粘附蛋白关键结构域；利用基因重组和免疫荧光等技术，研究发酵黄芪多糖对FGM表面粘附蛋白粘附作用和抑制致病菌功能的影响；利用荧光定量PCR和Western blot等方法，分别在转录和翻译水平分析发酵黄芪多糖对乳酸菌FGM表面粘附蛋白表达的调控作用，阐述乳酸菌与发酵黄芪多糖在动物机体内生物学协同作用机制。

表面蛋白是乳酸菌粘附宿主肠道细胞的主要物质基础，在细菌定植及益生功能发挥过程中起关键作用。因此，研究乳酸菌表面粘附蛋白介导作用，对改善肠道环境，提高机体免疫力以及阐述中药有效成分的生物学作用机制具有重要意义。有鉴于此，本项目开展了乳酸菌FGM益生性研究，明确FGM菌株无溶血性、无耐药性，耐酸、耐胆碱，3.5Mc FGM细菌在Caco-2的黏附率为37.51 ± 3.18 %，可显著抑制E.Coli生长及对Caco-2的黏附率。体内试验表明，FGM可通过保护肠绒毛结构、调节肠道免疫力与抗氧化能力，增强E.Coli感染肉鸡的抵抗力，保护鸡肠道健康。采用碱性氯化锂法提取菌体表面蛋白，利用质谱技术，筛选FGM表面粘附蛋白为Elongation Factor-Tu（EF-Tu，共计1197个bp, 398aa）。本研究建立EF-Tu重组蛋白体外表达系统，获得纯度为90%的His-tag标签目的蛋白，并免疫白兔制备出效价高达128k的多克隆抗体，进一步利用CO-IP、抗体封闭及间接免疫荧光检测技术，明确EF-Tu蛋白参与了FGM粘附肠上皮细胞和抑制、抵御、清除大肠杆菌的生物学作用，其与Caco-2细胞外膜可能作用的靶点为热休克蛋白、ACTB肌动蛋白β及SRY-box转录因子14。通过比较0.315-1.25mg/mL、10KD-100KD的发酵黄芪多糖、黄芪多糖对FGM生长、粘附、抑制E.Coli粘附Caco-2及其对粘附蛋白EF-Tu表达的调控研究，明确发酵黄芪多糖可促进FGM菌株生长和抑菌活性，可通过负向调控EF-Tu基因的表达促进FGM菌株拮抗、预防、清除侵染肠上皮细胞的E.Coli，其中0.315mg/mL、30-50KD的FAPS活性最高。