两歧双歧杆菌S17新的寡肽ABC转运系统及其调控机制的研究

Bifidobacteria are the predominant commensal bacteria in the intestinal microflora and exert various health benefits in humans. The mechanism of oligopeptide translocation of bifidobacterium can reveal its adaptation to the human gastrointestinal tract. By proteomics, transcriptics, and bioinformation anaslysis, the novel ABC translocation protein specific to oligopeptides showed higher levels of expression in cells grown on oligopeptide as sole nitrogen source; In the present study, we will construct the mutants by gene kncked-out, and investigate the comparative proteomics and comparative transcriptics of these mutants grown in different substances, then confirm and appreciate the function of interesting genes. At the same time, regulatory mechanism and adhesive mechanism of this novel ABC transporter system possibly for oligopeptide will be investigated by using these mutants and wild type strains in adhesion to IECs. In addition, oligopeptide binding specificity assay by equilibrium dialysis, ITC and CE will be carried out for selecting the specific to oligopeptide. All of the above results will propose that the uptake of oligopeptide into the cell may be conducted by a specific transport system, which include four enzymes: BBIF_0636, BBIF_0635, BBIF_0633 and BBIF_0634. At last, the mechanism of protein-protein interaction will be confirmed by GST-pull down assay and immunoprecipitation experiment, and define their phosphorylation sites. These findings were very interest in further investigating the mechanism of oligopeptide translocation and oligopeptide uptake and utilization.

双歧杆菌寡肽的吸收和转运机制可以揭示其在肠道中的适应性。本课题组利用家兔肠培养模型的蛋白质组学、转录组学和生物信息学分析，首次发现在两歧双歧杆菌中存在寡肽诱导的寡肽转运相关蛋白。拟通过基因敲除构建转运蛋白缺失突变体和回复突变体，进行不同寡肽发酵的研究以确定和验证它们的基因功能；研究突变株及野生株与宿主IECs相互作用确定转运蛋白的转录调控机制和粘附机制；进一步采用透析平衡试验、ITC试验和CE试验它们与底物结合的特异性，最终得到寡肽ABC转运系统的四个酶：BBIF_0636、BBIF_0635、BBIF_0633和BBIF_0634。然后采用GST沉降实验、免疫共沉淀实验等进行蛋白质-蛋白质相互作用的体内体外实验，研究寡肽ABC转运系统蛋白两两是否存在特异性相互作用；确定寡肽转运蛋白两两间相互作用的功能结构域，和转运蛋白的磷酸化作用及作用位点，从而揭示双歧杆菌寡肽转运的分子机制。

双歧杆菌寡肽的吸收和转运机制可以揭示其在肠道中的适应性。本课题组通过对两歧双歧杆菌S17的基因组分析，对基因功能、蛋白质分泌系统和基因表达调控因子的生物信息学分析和蛋白结构域的预测，确定了受寡肽诱导的寡肽转运相关蛋白（BBIF_0633-BBIF_0636）。通过比较蛋白质组学、转录组学分析等进一步确定和验证转运蛋白的基因功能。完成了两歧双歧杆菌B.bifidum S17的全基因组测序精细图及不同氮源培养中iTRAQ比较蛋白质组研究；完成了RNA-seq鉴定两歧双歧杆菌B. bifidum S17在体内体外环境中差异表达基因。进一步确定了可能的寡肽诱导的肽转运系统的四个蛋白，获得了受寡肽诱导的肽转运系统的四个新型蛋白质；完成了寡肽转运系统的寡肽结合蛋白BopA（BBIF_0636）的功能研究；进行了两歧双歧杆菌菌株中寡肽转运系统中bopA基因的检测及表达分析；完成了BopA表面蛋白抑制剂处理前后两歧双歧杆菌S17的粘附作用研究；纯化的BopA-His蛋白与B.bifidum S17体外竞争性黏附作用的研究。完成了N-端带FLAG标签的重组质粒的构建，完成了免疫共沉淀试验。分析了双歧杆菌寡肽转运的分子机制。完成了寡肽转运系统转运蛋白两两间相互作用的功能结构域的确定，建立双歧杆菌寡肽ABC转运的分子模型。