PURIFICATION AND PARTIAL CHARACTERIZATION OF THE MAJOR OUTER-MEMBRANE PROTEIN OF CHLAMYDIA-TRACHOMATIS
PURIFICATION AND PARTIAL CHARACTERIZATION OF THE MAJOR OUTER-MEMBRANE PROTEIN OF CHLAMYDIA-TRACHOMATIS
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DOI：
10.1128/iai.31.3.1161-1176.1981
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发表时间：
1981-01-01
影响因子：
3.1
通讯作者：
SCHACHTER, J
中科院分区：
文献类型：
--
作者：
CALDWELL, HD;KROMHOUT, J;SCHACHTER, J
对沙眼衣原体血清型 C、E 和 L2 的原体 (EB) 进行外源放射性碘标记，并通过十二烷基硫酸钠-聚丙烯酰胺凝胶电泳 (SDS-PAGE) 比较这些血清型的全细胞裂解物。多肽谱的放射自显影鉴定出一种主要表面蛋白，其表观亚基分子量为 39,500，这是每种沙眼衣原体血清型所共有的。通过对每次去污剂处理后获得的可溶性和不溶性组分进行 SDS-PAGE 分析，研究了非离子 (Triton X-100)、偶极离子 (Zwittergent TM-314)、温和（脱氧胆酸钠和 N-月桂酰肌氨酸钠）和强阴离子 (SDS) 去污剂从 L2 血清型完整 EB 中提取该蛋白质的能力。只有 SDS 才能轻松地从完整的 EB 中提取该蛋白质。肌氨酰处理选择性地溶解了大多数其他 EB 蛋白，留下了与肌氨酰不溶部分相关的 39,500 道尔顿蛋白。对不溶于肌氨酰的 EB 颗粒的超微结构研究表明，它由空的 EB 颗粒组成，具有明显完整的外膜。没有发现肽聚糖样细胞壁的结构证据。从形态上看，这些衣原体外膜复合物 (COMC) 类似于完整的衣原体 EB 外膜。通过在60℃下用2％SDS处理从COMC中定量提取39,500道尔顿的外膜蛋白。 C. 该蛋白占 COMC 相关蛋白总量的 61%，其提取导致 COMC 膜结构和形态的伴随损失。将从 SDS 处理的 COMC 中获得的可溶性提取物吸附到羟基磷灰石柱上，并用线性磷酸钠梯度洗脱。 39,500 道尔顿的蛋白质以单峰形式从柱中洗脱，磷酸盐浓度约为0.3 M。通过 SDS-PAGE 检测，洗脱的蛋白质几乎是均质的，并且似乎不含碳水化合物、糖脂和核酸污染。针对 L2 血清型 39,500 道尔顿蛋白制备的超免疫小鼠抗血清通过 EB 间接免疫荧光与沙眼衣原体血清型 Ba、E、D、K、L1、L2 和 L3 发生反应，但未能与沙眼衣原体小鼠肺炎株或沙眼衣原体肺炎血清型 A、B、C、F、G、H、I 和 J 反应。鹦鹉热猫肺炎、豚鼠包涵体结膜炎或 6BC 株。因此，39,500道尔顿的主要外膜蛋白是沙眼衣原体生物体的血清群抗原。
Elementary bodies (EB) of C. trachomatis serotypes C, E and L2 were extrinsically radioiodinated and whole-cell lysates of these serotypes were compared by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Autoradiography of the polypeptide profiles identified a major surface protein with an apparent subunit MW of 39,500 that was common to each C. trachomatis serotype. The abilities of nonionic (Triton X-100), dipolar ionic (Zwittergent TM-314), mild (sodium deoxycholate and sodium N-lauroyl sarcosine) and strongly anionic (SDS) detergents to extract this protein from intact EB of the L2 serotype were investigated by SDS-PAGE analysis of the soluble and insoluble fractions obtained after each detergent treatment. Only SDS readily extracted this protein from intact EB. Sarkosyl treatment selectively solubilized the majority of other EB proteins, leaving the 39,500-dalton protein associated with the Sarkosyl-insoluble fraction. Ultrastructural studies of the Sarkosyl-insoluble EB pellet showed it to consist of empty EB particles possessing an apparently intact outer membrane. No structural evidence for a peptidoglycanlike cell wall was found. Morphologically these chlamydial outer membrane complexes (COMC) resembled intact chlamydial EB outer membranes. The 39,500-dalton outer membrane protein was quantitatively extracted from COMC by treating them with 2% SDS at 60.degree. C. This protein accounted for 61% of the total COMC-associated protein and its extraction resulted in a concomitant loss of the COMC membrane structure and morphology. The soluble extract obtained from SDS-treated COMC was adsorbed to a hydroxylapatite column and eluted with a linear sodium phosphate gradient. The 39,500-dalton protein was eluted from the column as a single peak at a phosphate concentration of .apprx. 0.3 M. The eluted protein was nearly homogeneous by SDS-PAGE and appeared free of contaminating carbohydrate, glycolipid and nucleic acid. Hyperimmune mouse antiserum prepared against the 39,500-dalton protein from serotype L2 reacted with C. trachomatis serotypes Ba, E, D, K, L1, L2 and L3 by indirect immunofluorescence with EB but failed to react with serotypes A, B, C, F, G, H, I and J with the C. trachomatis mouse pneumonitis strain or with the C. psittaci feline pneumonitis, guinea pig inclusion conjunctivitis or 6BC strains. Thus, the 39,500-dalton major outer membrane protein is a serogroup antigen of C. trachomatis organisms.