BIOCHEMICAL AND MOLECULAR BIOLOGICAL STUDIES

气管、支气管粘液分泌物起到重要的保护作用 在呼吸道的正常运作中起着重要作用。粘液糖蛋白 在很大程度上，粘液分泌物中存在的 分泌物的物理性质。在疾病中 国家，粘蛋白可能已经改变了化学和物理 属性，而这又可能会影响 纤毛上皮的分泌物。虽然，有一个 关于低聚糖侧链的大量信息 呼吸道粘蛋白的结构，包括氨基酸序列 缺乏蛋白质骨架。这项研究的目的 是为了研究分子大小、氨基酸序列和 呼吸道粘蛋白蛋白核心的物理性质。这个 这项提议的具体目标是：a)进一步净化和 对原生的(非还原的)和 犬气管粘液分泌物中的脱糖基粘蛋白 对天然粘蛋白和脱糖基粘蛋白进行物理表征以 测定分子大小、回转半径、聚集 疏水结构域的行为和存在，c)制备 天然和脱糖粘蛋白的免疫学探针 为了检测体外细胞产生的特异性无粘蛋白(S) 犬气管上皮(CTE)信使核糖核酸的翻译 细胞并筛选无粘蛋白(S)的表达产物 构建在lambda gt11载体中的文库和d)分离和 犬气管无粘蛋白(S)基因克隆的鉴定 为了测定上皮细胞中的初级氨基酸 序列和氨基酸序列，信号传输和 正在处理。粘蛋白将使用协议进行提纯 在这个实验室建立的。生化特征将会 包括：碳水化合物、硫酸盐、氨基酸的测定 粘蛋白分子的组成、硫醇和二硫键含量。 分子大小、回转半径和团聚行为 将通过光散射和沉淀法确定 平衡法。定向多克隆抗体和单抗 针对粘蛋白的制备将使用建立的标准 协议。粘蛋白基因的分离、鉴定及检测 克隆和核苷酸测序将使用Well进行 建立了分子生物学技术。《知识》 从拟议的研究中获得的成果最终应该会带来更多 阻塞性肺病的有效治疗策略。

Tracheobronchial mucus secretions play an important protective role in the normal functioning of the airways. Mucus glycoproteins present in the mucus secretions are responsible, to a large extent, for the physical properties of the secretions. In the disease states, the mucins may have altered chemical and physical properties and this in turn may influence the clearance of the secretions by ciliated epithelium. Although, there is a considerable information on the oligosaccharide side chains of airway mucins, the structure, including amino acid sequence of the protein backbone is lacking. The objectives of this research are to investigate the molecular size, amino acid sequence, and physical properties of the protein core of the airway mucins. The specific aims of this proposal are to a) further purify and biochemically characterize the native (non-reduced) and deglycosylated mucins from canine tracheal mucus secretions, b) physically characterize the native and deglycosylated mucins to determine molecular size, radius of gyration, aggregation behavior and presence of hydrophobic domains, c) prepare immunological probes against native and deglycosylated mucins in order to detect specific apomucin(s) generated during in vitro cell free translation of mRNA from canine tracheal epithelial (CTE) cells and to screen the production of apomucin(s) in the cDNA library constructed in the lambda gt11 vector and d) isolate and characterize cDNA clones of apomucin(s) from canine tracheal epithelial cells in order to determine the primary amino acid sequence and amino acid sequences which signal transport and processing. The mucins will be purified using protocols established in this laboratory. Biochemical characterization will include: determination of carbohydrate, sulfate, amino acid composition, thiol and disulfide content of the mucin molecules. The molecular size, radius of gyration and agregation behavior will be determined using light scattering and sedimentation equilibrium methods. Poly- and monoclonal antibodies directed against the mucins will be prepared using standard established protocols. Isolation, characterization, detection of mucin cDNA clones and nucleotide sequencing will be carried out using well established molecular biological techniques. The knowledge gained from the proposed studies should eventually lead to more effective treatment strategies for obstructive lung disease.