BORRELIA BURGDORFERI GLYCOSAMINOGLYCAN-BINDING PROTEINS

莱姆疏螺旋体病是一种慢性多系统疾病，由扁虱引起- 传播细菌伯氏疏螺旋体。鉴于以下事实： 约10%的原发性莱姆疏螺旋体病患者逃脱临床 检测和未经治疗的疾病可能会长期使人衰弱 因此，有必要开发一种莱姆型疏螺旋体病疫苗。 侵袭性微生物附着在宿主细胞上是一个关键的早期 疾病发病机制中的一步。之前的调查人员已经证明 伯氏杆菌在体外附着在哺乳动物细胞上，但 依恋的机制尚未阐明。初步研究 由PI执行的结果表明，强毒伯氏杆菌具有一种 中高亲和力的糖胺聚糖结合配体 表面可能裸露。据推测，伯氏假单胞菌 以承载暴露在细胞表面的蛋白多糖。这项建议旨在 糖胺多聚糖介导的丝裂原附着于哺乳动物的特性 分子水平上的细胞。糖胺多聚糖对血管内皮生长因子的影响 对哺乳动物细胞的丝裂原附着的详细描述将在 使用多种细胞类型和多个 强毒和无毒伯氏杆菌菌株(特定目标#1)。B。 伯氏杆菌糖胺多聚糖结合配体将用肝素纯化 琼脂糖亲和层析，并将经过N-末端和 内部氨基酸序列分析(特定目标2)。单克隆 将产生针对蛋白质的抗体，并将评估 它们抑制硼附着的能力(具体目标2)。这个 糖胺多糖结合配体将被克隆、测序和表达 在大肠杆菌中(特定目标3)。重组伯氏杆菌 糖胺多糖结合配体将通过以下组合进行纯化 肝素琼脂糖亲和层析和免疫亲和层析； 它将在体外附着抑制研究中进行评估，并详细说明 将进行研究以确定抗体识别的表位。 这抑制了依恋(具体目标4)。在未来，它是有计划的 鉴定该蛋白的糖胺多糖结合区，并 纯化的重组蛋白在小鼠体内作为疫苗原的评价 莱姆型疏螺旋体病模型(未来研究方向)

Lyme borreliosis is a chronic multisystem disorder caused by the tick- borne bacterium Borrelia burgdorferi. In light of the facts that approximately 10% of cases of primary Lyme borreliosis escape clinical detection and that untreated disease may have debilitating long term consequences, there is a need to develop a Lyme borreliosis vaccine. Attachment of invasive microorganisms to host cells is a critical early step in disease pathogenesis. Previous investigators have demonstrated that B. burgdorferi attaches to mammalian cells in vitro, but the mechanisms of attachment have not been elucidated. Preliminary studies performed by the PI indicate that virulent B. burgdorferi possess a moderately high affinity glycosaminoglycan binding ligand which is putatively surface exposed. It is hypothesized that B. burgdorferi attach to host cell-surface exposed proteoglycans. This proposal aims to characterize glycosaminoglycan-mediated borrelial attachment to mammalian cells at the molecular level. The effects of glycosaminoglycans on borrelial attachment to mammalian cells will be characterized in detail in attachment-inhibition studies using a variety of cell types and a number of virulent and avirulent B. burgdorferi strains (Specific aim #1). The B. burgdorferi glycosaminoglycan-binding ligand will be purified by heparin agarose affinity chromatography and will be subjected to N-terminal and internal amino acid sequence analysis (Specific aim #2). Monoclonal antibodies will be generated against the protein and will be assessed for their ability to inhibit borrelial attachment (Specific aim #2). The glycosaminoglycan binding ligand will be cloned, sequenced, and expressed in Escherichia coli (Specific aim #3). Recombinant B. burgdorferi glycosaminoglycan binding ligand will be purified by a combination of heparin agarose affinity chromatography and immunoaffinity chromatography; it will be assessed in in vitro attachment-inhibition studies and detailed studies will be undertaken to define the epitope recognized by antibodies which inhibit attachment (Specific aim #4). In the future, it is planned to characterize the glycosaminoglycan binding domain of the protein and to assess the purified recombinant protein as a vaccinogen in the murine model of Lyme borreliosis (future directions).