An improved procedure for Percoll gradient separation of sporogonial stages in Encephalitozoon cuniculi (Microsporidia)
An improved procedure for Percoll gradient separation of sporogonial stages in Encephalitozoon cuniculi (Microsporidia)
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DOI：
10.1007/s00436-006-0231-y
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发表时间：
2006-06
影响因子：
2
通讯作者：
V. Taupin;G. Méténier;C. Vivarès;G. Prensier
中科院分区：
文献类型：
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作者：
V. Taupin;G. Méténier;C. Vivarès;G. Prensier
小孢子寄生虫的细胞内发育包括增殖期（分生殖期）和随后的分化期（孢子生殖期），导致抗性孢子的释放。孢子发生依次意味着子粒到孢子的转化、孢子分裂成孢子母细胞和孢子发生。我们报告了一种改进兔脑炎孢子虫孢子阶段分离的方法，兔脑炎虫是一种在哺乳动物细胞寄生泡内发育的物种。 E.的上清液感染兔兔的 Madin-Darby 犬肾细胞培养物提供了大量与宿主细胞碎片混合的寄生虫。将该材料在含有 0.05% 皂苷和 0.05% Triton X-100 的磷酸盐缓冲盐水中轻轻匀化，然后通过玻璃棉柱过滤。将滤液在 70% Percoll–0.23 M 蔗糖梯度上离心，得到不同密度的可重复的条带图案。透射电子显微镜显示，收集的四个级分中的三个没有可见污染物。相应的突出细胞阶段是早期孢子母细胞（组分B）、晚期孢子母细胞加未成熟孢子（组分C）和成熟孢子（组分D）。在 30% Percoll–0.23 M 蔗糖梯度上进一步离心最轻的级分 (A)，生成富含孢子子的级分 (A2)。通过二维凝胶电泳对 A2 和 D 部分的蛋白质进行的首次分析表明，所描述的方法可能用于蛋白质组分析。
Intracellular development of microsporidian parasites comprises a proliferative phase (merogony) followed by a differentiation phase (sporogony) leading to the release of resistant spores. Sporogony implies, successively, meront-to-sporont transformation, sporont division into sporoblasts, and sporogenesis. We report a procedure improving the separation of sporogonial stages ofEncephalitozoon cuniculi, a species that develops inside parasitophorous vacuoles of mammalian cells. Supernatants ofE. cuniculi-infected Madin–Darby canine kidney cell cultures provided a large number of parasites mixed with host-cell debris. This material was gently homogenized in phosphate-buffered saline containing 0.05% saponin and 0.05% Triton X-100 then filtered through glass wool columns. Centrifugation of the filtrate on 70% Percoll–0.23 M sucrose gradient gave a reproducible pattern of bands at different densities. Transmission electron microscopy showed that three of the four collected fractions were free of visible contaminants. Corresponding prominent cell stages were early sporoblasts (fraction B), late sporoblasts plus immature spores (fraction C), and mature spores (fraction D). Further centrifugation of the lightest fraction (A) on 30% Percoll–0.23 M sucrose gradient generated a sporont-rich fraction (A2). First analysis of proteins from fractions A2 and D by two-dimensional gel electrophoresis suggested a potential use of the described method for proteomic profiling.