MECHANISM OF PATHOGENESIS IN MYCOPLASMAL PNEUMONIA

该项目旨在提供有关生物化学的新信息。 肺炎支原体感染的毒力基础，特别是 人类原发性非典型肺炎(PAP)。我们长期的这一阶段 研究集中在受体部位的性质上， 支原体附着。我们已经确定了这样一个事实，即 肺成纤维细胞上的受体是一种唾液酸糖蛋白 道尔顿山脉。最近的数据表明氨基葡萄糖是一种主要的 这种分子中的碳水化合物。接下来我们将分离肺炎支原体 用凝集素亲和层析法测定其受体活性。粗制受体的制备 通过Triton X-100增加肺成纤维细胞膜的增溶作用 对小麦胚芽凝集素/琼脂糖凝胶，糖蛋白将被洗脱 N-乙酰氨基葡萄糖。纯化的受体将在之后收集 超滤和浓缩，并用聚丙烯酰胺凝胶进行表征 蛋白质和碳水化合物的电泳法和化学测定法。 纯化的受体糖蛋白的特异性和活性将被 由肺炎支原体对肺附着的阻断程度决定 成纤维细胞体外培养。糖蛋白受体将被用作 在小鼠-小鼠杂交瘤中制备单抗。 免疫球蛋白的特异性和克隆的稳定性将通过 辣根过氧化物酶标记的酶联免疫吸附试验技术。是否可以使用 纯化的受体及其同源抗体将使我们能够确定 寄主细胞膜上受体位置的数量和分布。这个 受体分子将共价偶联到琼脂糖凝胶上，以开发一种 改进的P1蛋白的分离和回收方法，由几个 实验室相当于M的活性结合部位。 肺炎病原体和疫苗抗原的主要候选者。我们也 将决定肺炎支原体受体出现的频率 在不同的细胞类型上，并将其与M。 肺炎杆菌附着在这些细胞上。相反，我们将检查 其他支原体利用该受体作为附着部位的能力。 我们分离和研究肺炎支原体受体的能力及其 特异性抗体，将在分子基础上提供独特的视角 致病性，同时对开发有效的 支原体疾病的治疗和预防。

This project is designed to provide new information on the biochemical basis for virulence in Mycoplasma pneumoniae infections, particularly primary atypical pneumonia (PAP) of humans. This phase of our long-term study is concentrating on the nature of the receptor site to which the mycoplasma attaches. We have already established the fact that the receptor site on lung fibroblasts is a sialoglycoprotein in the 100,000 dalton range. Recent data indicate that glucosamine is a major carbohydrate in this molecule. We next will isolate the M. pneumoniae receptor by using lectin affinity chromatography. Crude receptor prepared by triton X-100 solubilization of lung fibroblast membranes will be added to wheat germ agglutinin/sepharose, and glycoproteins will be eluted with N-acetyl glucosamine. Purified receptor will be collected after ultrafiltration and concentration, and characterized by polyacrylamide gel electrophoresis and chemical assays for protein and carbohydrate. Specificity and activity of purified receptor glycoprotein will be determined by the degree of blocking of M. pneumoniae attachment to lung fibroblast cells in vitro. The glycoprotein receptor will be used as an antigen to prepare monoclonal antibodies in mouse-mouse hybridomas. Specificity of immunoglobulins and stability of clones will be assessed by ELISA techniques with a horseradish peroxidase label. The availability of purified receptor and its homologous antibody will permit us to determine the number and distribution of receptor sites on host cell membranes. The receptor molecules will be covalently coupled to Sepharose to develop an improved isolation and recovery method for the P1 protein, shown by several laboratories to be equivalent to the active binding site of the M. pneumoniae organism and a prime candidate for vaccine antigen. We also will determine the frequency of occurrance for the M. pneumoniae receptor site on various cell types, and correlate it with the ability of M. pneumoniae to attach to those cells. Conversely, we will examine the ability of other mycoplasmas to use this receptor as an attachment site. Our ability to isolate and study the M. pneumoniae receptor, along with its specific antibody, will provide a unique perspective on the molecular basis of pathogenicity, while being of direct value to developing effective therapy and prophylaxis for mycoplasma disease.