Zygmunt M S, Dubray G, Bundle D R, Perry M P
Institut National de la Recherche Agronomique, Station de Pathologie de la Reproduction, Nouzilly, France.
Ann Inst Pasteur Microbiol. 1988 Jul-Aug;139(4):421-33. doi: 10.1016/0769-2609(88)90105-6.
Purification of the Brucella polysaccharide referred to as native hapten (NH) and extracted from cells by the autoclaving procedure, was accomplished by ultrafiltration, followed by repetitive gel filtration using high-performance liquid chromatography on a "TSK-G2000-SW" column. The purified NH was analysed by SDS-PAGE, gas-liquid chromatography mass spectroscopy, and 13C and 1H NMR spectroscopy. NH from B. abortus B19 (NH-A) was shown to have a structure identical to that of A polysaccharide from B. abortus 1119-3, a linear homopolymer of alpha-1,2-linked-4,6-dideoxy-4-formamido-D-mannopyrannosyl residues. The structure of the NH from B. melitensis 16M (NH-M) was identified as a linear homopolysaccharide of the same sugar but composed of a pentasaccharide repeating unit in which four alpha 1,2-linked-4,6-dideoxy-4-formamido-D-mannopyrannosyl residues are linked alpha-1,3 to the last monosaccharide of the sequence. This structure is similar to that determined for the Brucella M polysaccharide from B. melitensis 16M. The discovery in highly purified NH preparations of covalently bound monosaccharides characteristic of lipopolysaccharide inner core regions e.g., quinovosamine, mannose and 3-deoxy-D-manno-octulosonate (KDO), indicates that this polysaccharide is derived from lipopolysaccharides (LPS) by hydrolytic conditions fortuitously generated during the extraction protocol. The antigenically important polysaccharides of Brucella are now established to be either A or M antigens. Polysaccharide B is a cyclic glucan with no structural or serological relationship to A or M polysaccharides, its apparent activity in diagnostic tests of infected cattle results from O polysaccharide contamination. This artefact, previously referred to as NH, results from LPS hydrolysis under the extraction conditions used to prepare polysaccharide B.
通过高压灭菌程序从细胞中提取的布鲁氏菌多糖（称为天然半抗原，NH），经超滤后，再使用“TSK-G2000-SW”柱进行高效液相色谱法的重复凝胶过滤，从而实现纯化。通过十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）、气-液色谱-质谱联用以及碳-13（13C）和氢-1（1H）核磁共振光谱对纯化后的NH进行分析。流产布鲁氏菌B19的NH（NH-A）显示其结构与流产布鲁氏菌1119-3的A多糖相同，是由α-1,2-连接的4,6-二脱氧-4-甲酰胺基-D-甘露吡喃糖基残基组成的线性同聚物。马尔他布鲁氏菌16M的NH（NH-M）结构被鉴定为相同糖类的线性同多糖，但由一个五糖重复单元组成，其中四个α-1,2-连接的4,6-二脱氧-4-甲酰胺基-D-甘露吡喃糖基残基通过α-1,3连接到该序列的最后一个单糖上。此结构与从马尔他布鲁氏菌16M中确定的布鲁氏菌M多糖结构相似。在高度纯化的NH制剂中发现了共价结合的脂多糖内核心区域特征性单糖，例如奎诺糖胺、甘露糖和3-脱氧-D-甘露辛酮酸（KDO），这表明该多糖是在提取过程中偶然产生的水解条件下从脂多糖（LPS）衍生而来。现已确定布鲁氏菌具有抗原重要性的多糖为A或M抗原。多糖B是一种环状葡聚糖，与A或M多糖无结构或血清学关系，其在感染牛的诊断测试中的明显活性是由O多糖污染导致的。这种假象，以前称为NH，是在用于制备多糖B的提取条件下LPS水解的结果。
