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首页> 外文期刊>Infection and immunity >Purification and characterization of an exopolysaccharide of Burkholderia (Pseudomonas) pseudomallei.
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Purification and characterization of an exopolysaccharide of Burkholderia (Pseudomonas) pseudomallei.

机译：伯克霍尔德氏菌（假单胞菌）假小麦芽胞外多糖的纯化和表征。

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Burkholderia pseudomallei (basonym Pseudomonas pseudomallei) is the causative organism of melioidosis, a disease which is recognized as a major public health problem primarily in Southeast Asia and Northern Australia. In this paper, we report on the identification, purification, and characterization of a species-specific exopolysaccharide of B. pseudomallei. After immunization of mice with a B. pseudomallei strain exhibiting mucoid growth characteristics, we isolated an immunoglobulin G1 monoclonal antibody (MAb) (3015) with specificity for a carbohydrate structure as determined by immunoblotting following sodium dodecyl sulfatepolyacrylamide gel electrophoresis. Electron microscopy studies with MAb 3015 revealed reactivity with an exopolysaccharide with a capsule-like appearance in the immunizing strain. All of the mucoid and nonmucoid B. pseudomallei strains tested from geographically different tropical regions were recognized by MAb 3015 in an enzyme-linked immunosorbent assay or immunoblot, indicating that the exopolysaccharide is constitutively expressed among this species. Intensive testing for cross-reactivity including members of all the Pseudomonas rRNA groups showed no cross-reactivity except in the case of the closely related species Burkholderia mallei. A protocol for purification of the exopolysaccharide which is based principally on mechanical separation from the cell surface followed by repetitive ethanol precipitation steps and finally affinity chromatography using MAb 3015 was established. The exopolysaccharide yielded was of high purity. Gel permeation chromatography was performed, and the molecular mass was estimated to be > 150 kDa. Sera from patients with melioidosis were strongly reactive with the purified exopolysaccharide, indicating its in vivo expression and immunogenicity in natural infection. The diagnostic value of the exopolysaccharide and its role in the pathogenesis of disease must still be determined.
机译：Burkholderia pseudomallei（假名Pseudomonas pseudomallei）是类弧菌病的病原体，该病主要在东南亚和北澳大利亚被认为是主要的公共卫生问题。在本文中，我们报告了假单胞菌种特异性胞外多糖的鉴定，纯化和表征。用表现出粘液样生长特征的假苹果芽孢杆菌菌株对小鼠进行免疫后，我们分离出了一种免疫球蛋白G1单克隆抗体（MAb）（3015），该抗体对碳水化合物的结构具有特异性，如十二烷基硫酸钠聚丙烯酰胺凝胶电泳后的免疫印迹法所确定。用MAb 3015进行的电子显微镜研究显示，在免疫菌株中，其与胞外多糖具有胶囊样外观的反应性。 MAb 3015在酶联免疫吸附测定法或免疫印迹法中识别了从地理上不同的热带地区测试的所有粘液状和非粘液状假小芽孢杆菌菌株，表明胞外多糖在该物种中组成性表达。对包括所有假单胞菌rRNA组成员在内的交叉反应性进行的严格测试表明，除了紧密相关的物种Burkholderia Mallei以外，没有交叉反应性。建立了一种纯化胞外多糖的方案，该方案主要基于从细胞表面机械分离，然后重复乙醇沉淀步骤，最后使用MAb 3015进行亲和层析。所产生的胞外多糖具有高纯度。进行凝胶渗透色谱，估计分子量＞ 150kDa。来自类me突病患者的血清与纯化的胞外多糖具有强反应性，表明其在自然感染中的体内表达和免疫原性。胞外多糖的诊断价值及其在疾病发病机理中的作用仍必须确定。

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