PDF(Pubmed)
Abstract：
Bacterial endotoxins (lipopolysaccharides (LPSs)) are important mediators of inflammatory processes induced by Gram-negative microorganisms. LPSs are the key inducers of septic shock due to a Gram-negative bacterial infection; thus, the structure and functions of LPSs are of specific interest. Often, highly purified bacterial endotoxins must be isolated from small amounts of biological material. Each of the currently available methods for LPS extraction has certain limitations. Herein, we describe a rapid and simple microscale method for extracting LPSs. The method consists of the following steps: ultrasonic destruction of the bacterial material, LPS extraction via heating, LPS purification with organic solvents, and treatment with proteinase K. LPSs that were extracted by using this method contained less than 2-3% protein and 1% total nucleic acid. We also demonstrated the structural integrity of the O-antigen and lipid A via the sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS) methods, respectively. We demonstrated the ability of the extracted LPSs to induce typical secretion of cytokines and chemokines by primary macrophages. Overall, this method may be used to isolate purified LPSs with preserved structures of both the O-antigen and lipid A and unchanged functional activity from small amounts of bacterial biomass.
摘要：
细菌内毒素(脂多糖(LPSs))是革兰氏阴性微生物诱导的炎症过程的重要介质。LPS是由革兰氏阴性细菌感染引起的脓毒性休克的关键诱导剂;因此，LPS的结构和功能是特别感兴趣的。通常,高度纯化的细菌内毒素必须从少量的生物材料中分离出来。目前可用的每种LPS提取方法都有一定的局限性。在这里，我们描述了一种快速简单的微尺度提取LPS的方法。该方法包括以下步骤：超声波破坏细菌材料，通过加热提取LPS，用有机溶剂纯化LPS，用此方法提取的LPS含有少于2-3%的蛋白质和1%的总核酸。我们还通过十二烷基硫酸钠聚丙烯酰胺凝胶电泳（SDS-PAGE）和基质辅助激光解吸电离质谱（MALDI-MS）方法证明了O抗原和脂质A的结构完整性，分别。我们证明了提取的LPS诱导初级巨噬细胞典型分泌细胞因子和趋化因子的能力。总的来说,该方法可用于从少量细菌生物质中分离出具有保留的O抗原和脂质A结构且功能活性不变的纯化的LPS。
