Rib蛋白在罗伊氏乳杆菌肠道黏附中的功能研究

Lactobacillus reuteri is one of the most potential probiotics in food and dietary supplement industry.It is a member of the normal microbial community of the digestive tract of mammals such as humans,pigs, hamsters, mice, rats, dogs, sheep, cattle, and different birds，which makes it an excellent model for basic studies on ecological and evolutionary mechanisms of a vertebrate gut symbiont.Because this bacteria has strong attachment capability to the gut epithelial cells and can stay in the intestinal tract for up to 2 weeks, it is commonly used as a probiotic to maintain the balance of the gut microbial flora and stimulate the intestinal immune system.The cell surface proteins were shown by previous studies to play very important role in the bacterial adhesion and binding to the gut epithelial cells. Rib protein is a functional unknown large protein found in L. reuteri.The coding gene was cloned by our group. Anaylses of the deduced amino acid sequence showed that this protein contains 1786 amino acid residues, and it's molecular weight is about 190 kDa.Secondary structure prediction with software showed that this protein has a N terminal YSIRK type signal peptide and a C terminal LPXTG sorting signal motif,which indicated that it is mainly distributs on the bacterial cell surface.The BLAST analyses showed that N terminal part of this Rib protein has no specific homology. However there are 8 tandem repeat regains in the C termial part, which was more likely found in a cell surface protein of group B streptococcal bacteria(Streptococcus agalactiae),Rib protein.Although studies showed that it has immune protective effect to the infection of group B streptococcal bacteria,the biological function of the Rib protein is unknown yet. Our hypotheses is this Rib protein has specific interaction with the host factor(s),and plays important role in the adhesion and stay in the host intestinal tract.To prove this hypotheses,our study will consentrate on the biological function and the role of this newly founded Rib protein in L. reuteri attachment to the host gut epithelial cells, using biochemical and molecular biological methods such as gene mutation,protein recombination, in vitro functional analyses and animal test, etc.The functional study of this Rib protein will provide academic basis for further study and application of this bacteria.

罗伊氏乳杆菌是在食品,保健品领域最具潜力的益生菌之一，它在动物肠粘膜上皮细胞表面黏附能力很强。目前对益生菌在肠道内黏附机制的研究很少，研究表明细菌表面蛋白在其肠道黏附与定植中具有重要作用。Rib蛋白是在罗伊氏乳杆菌中发现的功能未知大分子蛋白。本课题组克隆了该蛋白的基因并对推测的氨基酸序列进行了分析，发现该蛋白包含1786个氨基酸残基，分子量为约190 kDa。对其结构的分析发现该蛋白主要分布在细胞表面。其N末端为一未知功能区,C末端含有 8个衔接重复的区域。此重复区域存在于B类链球菌的细菌表面蛋白Rib中，然而这种蛋白的生物学功能还不清楚。本研究推测它具有对宿主的特异性识别作用，并且在细菌的黏附与定植中具有重要作用。为证明该推断，本研究将通过基因突变,蛋白质重组，体外功能检测和动物实验等方法，研究该蛋白的生物学功能及其在细菌黏附和定植中的作用，为该益生菌的进一步研究和应用开发提供理论基础。

Rib蛋白是在罗伊氏乳杆菌中发现的功能未知大分子蛋白。研究表明罗伊氏乳杆菌具有很高的保健功能并具有很强的在肠道定植的能力，为研究其在肠道内黏附机制，本研究推测细菌表面蛋白Rib具有对宿主的特异性识别作用，并且在细菌的黏附与定植中具有重要作用。本课题通过蛋白的异源表达，抗体制备，免疫印迹，突变体的构建，基因组序列分析等方法与技术，对罗伊氏乳杆菌Rib蛋白在肠道黏附中的功能进行了研究。首先通过部分Rib蛋白在大肠杆菌中表达制备了重组Rib蛋白及其特异性抗体，进而通过免疫印迹的方法证实Rib蛋白在罗伊氏乳杆菌中的亚细胞分布为细胞壁上。然而使用免疫电镜却没有发现突变体细胞壁与野生型菌株细胞壁上的明显区别。在罗伊氏乳杆菌中构建rib基因突变体并对其肠道黏附能力进行了研究。小鼠肠上皮细胞黏附的研究发现Rib突变体罗伊氏乳杆菌对细胞的黏附力比野生型菌大大降低。同样，动物实验表明Rib突变体菌株在小鼠肠道中停留的时间大大降低。通过重组Rib蛋白与gut mucin和collagen I 的体外吸附实验表明Rib蛋白与mucin 相互作用较明显，而与collagen I的相互作用不明显。用重组的Rib蛋白对小肠上皮细胞蛋白进行蛋白质pull down发现Rib蛋白与细胞表面一个50kDa和25kDa蛋白可能具有特异性相互作用。使用Rib抗体对不同乳酸菌检测发现Rib类似蛋白在乳酸菌种分布较普遍，预示这可能相似的生物学功能。综合上述研究结果，本课题研究表明Rib蛋白参与了罗伊氏乳杆菌在肠道中黏附过程，这一结论为肠道益生菌与宿主的相互关系研究与应用提供了启示。项目资助发表论文3篇，待发表论文2篇。培养硕士生6名，其中3名已毕业，3名在读。项目投入经费50万元，支出43.868375万元，各项支出基本与预算相符,剩余经费6.131625万元，剩余经费计划用于本项目研究后续支出。