• 主题
高级搜索  >
历史搜索 清空历史
首页> 外文期刊>Infection and immunity >Detection of Anionic Antimicrobial Peptides in Ovine Bronchoalveolar Lavage Fluid and Respiratory Epithelium
【24h】

Detection of Anionic Antimicrobial Peptides in Ovine Bronchoalveolar Lavage Fluid and Respiratory Epithelium

机译：绵羊支气管肺泡灌洗液和呼吸道上皮中的阴离子抗菌肽的检测

获取原文
           
页面导航
  • 摘要
  • 著录项
  • 相似文献
  • 相关主题

摘要

Three small antimicrobial anionic peptides (AP) were originally isolated from an ovine pulmonary surfactant. However, their presence in bronchoalveolar lavage (BAL) fluid and tissues of the respiratory tract is unknown. In this study, we made affinity-purified rabbit polyclonal and mouse monoclonal antibodies to synthetic H-DDDDDDD-OH. Antibody specificity was assessed by a competitive enzyme-linked immunosorbent assay (ELISA), and the exact epitope binding sites were determined with analog peptides synthesized on derivatized cellulose. These antibodies were used to detect AP in BAL fluid by ELISA and in respiratory tissues by Western blot analysis and immunocytochemistry. BAL fluid from 25 sheep contained 0.83 ± 0.33 mM AP (mean ± standard deviation; range, 0.10 to 1.59 mM) and was antimicrobial. The presence of AP in BAL fluid was confirmed by reverse-phase high-pressure liquid chromatography fractionation followed by matrix-assisted laser desorption/ionization (MALDI) mass spectrometry on those fractions which were positive by competitive ELISA and demonstrated antimicrobial activity. In Western blots, polyclonal antibody PAB96-1 and monoclonal antibody 1G9-1C2 (5.0 μg/ml) detected four bands in solubilized turbinate and tracheal epithelial cells (53.7, 31.2, 28.0, and 25.7 kDa) and five bands in lung homogenates (53.5, 37.1, 31.2, 28.0, and 25.7 kDa). Only a single band was seen in solubilized liver and small-intestine homogenates, and no bands were seen in blots containing BAL fluid, albumin, or kidney or spleen homogenates. In pulmonary-tissue sections, both antibodies PAB96-1 and 1G9-1C2 identified accumulated protein in the apical cytoplasm of the bronchial and bronchiolar epithelia, in the cytoplasm of pulmonary endothelial cells, and in an occasional alveolar macrophage. As a first step in identifying a candidate AP precursor gene(s), degenerate oligonucleotides representing all possible coding combinations for H-GADDDDD-OH and H-DDDDDDD-OH were synthesized and used to probe Southern blots of sheep genomic DNA. Following low-stringency washes and a 2-day exposure, strongly hybridizing bands could be identified. One degenerate oligonucleotide, SH87, was used as a hybridization probe to screen a sheep phage genomic library. Two independent phage contained the H-GADDDDD-OH coding sequence as part of a larger predicted protein. AP may originate as part of an intracellular precursor protein, with multistep processing leading to the release of the heptapeptide into mucosal secretions. There it may interact with other innate pulmonary defenses to prevent microbial infection.
机译：最初从绵羊肺表面活性剂中分离出三种小的抗菌阴离子肽（AP）。但是，它们在支气管肺泡灌洗液（BAL）和呼吸道组织中的存在是未知的。在这项研究中，我们针对合成的H-DDDDDDD-OH制备了亲和纯化的兔多克隆和小鼠单克隆抗体。通过竞争性酶联免疫吸附测定（ELISA）评估抗体的特异性，并使用衍生化纤维素上合成的类似肽确定确切的表位结合位点。这些抗体用于通过ELISA检测BAL液中的AP，以及通过Western blot分析和免疫细胞化学检测呼吸道组织中的AP。来自25只绵羊的BAL液含有0.83±0.33 mM AP（平均值±标准偏差;范围为0.10至1.59 mM），并且具有抗菌作用。 BAL液中AP的存在是通过反相高压液相色谱分级分离，然后通过竞争性ELISA呈阳性并显示出抗菌活性的那些级分进行基质辅助激光解吸/电离（MALDI）质谱确认的。在Western印迹法中，多克隆抗体PAB96-1和单克隆抗体1G9-1C2（5.0μg/ ml）在溶解的鼻甲和气管上皮细胞中分别检测到四个条带（53.7、31.2、28.0和25.7 kDa），在肺匀浆中检测到了五个条带（53.5 ，37.1、31.2、28.0和25.7 kDa）。在溶解的肝脏和小肠匀浆中仅观察到一条条带，在含有BAL液，白蛋白，肾脏或脾脏匀浆的印迹中未观察到条带。在肺组织切片中，两种抗体PAB96-1和1G9-1C2均可在支气管和细支气管上皮的顶端细胞质，肺内皮细胞的细胞质以及偶尔的肺泡巨噬细胞中鉴定出积聚的蛋白质。作为鉴定候选AP前体基因的第一步，合成了代表H-GADDDDD-OH和H-DDDDDDD-OH的所有可能编码组合的简并寡核苷酸，并用于探测绵羊基因组DNA的Southern印迹。经过低严格度的洗涤和2天的暴露后，可以确定强烈杂交的条带。一种简并的寡核苷酸SH87用作杂交探针，以筛选绵羊噬菌体基因组文库。两个独立的噬菌体包含H-GADDDDD-OH编码序列，作为较大的预测蛋白的一部分。 AP可能起源于细胞内前体蛋白，经过多步处理导致七肽释放到粘膜分泌物中。在那里它可能与其他先天性肺部防御相互作用，以防止微生物感染。

著录项

相似文献

  • 外文文献
  • 中文文献
  • 专利
获取原文

客服邮箱：kefu@zhangqiaokeyan.com

京公网安备：11010802029741号 ICP备案号：京ICP备15016152号-6 六维联合信息科技 (北京) 有限公司©版权所有

  • 客服微信

  • 服务号