Comparative proteome analysis of Leishmania donovani at different stages of transformation from promastigotes to amastigotes
Comparative proteome analysis of Leishmania donovani at different stages of transformation from promastigotes to amastigotes
复制标题
杜氏利什曼原虫前鞭毛体向无鞭毛体转化不同阶段的比较蛋白质组分析
DOI：
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发表时间：
2001
期刊：
影响因子：
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通讯作者：
I. Bruchhaus
中科院分区：
文献类型：
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作者：
Meike Thiel;I. Bruchhaus
抽象的。为了经历其生命周期，利什曼原虫属的原生动物寄生虫必须从前鞭毛体分化为无鞭毛体。这一重大转变的分子基础尚不清楚。研究这种现象的一种方法是分离和表征在生命周期的两个阶段之一或阶段分化期间特异性表达的蛋白质。使用二维凝胶电泳，我们绘制了杜氏利什曼原虫在阶段分化期间的蛋白质组图，以识别阶段特异性蛋白质和调节子。开发了一种提取前鞭毛体和无鞭毛体杜氏乳杆菌细胞蛋白质的方案，该方案与等电聚焦兼容。在银染凝胶上可观察到多达 400 个杜氏乳杆菌蛋白点。细胞的代谢标记用于直接比较蛋白质合成模式与蛋白质水平模式。将在分化阶段的不同天收获的杜氏乳杆菌细胞的银染图像与相应的放射自显影照片进行比较。观察到从前鞭毛体到无鞭毛体的分化阶段蛋白质合成显着减少。染色的蛋白质模式以及放射自显影照片上的蛋白质模式发生了巨大变化，特别是在转化后第 3 天（pH 变化后约 24 小时）。
Abstract. To pass through its life cycle, protozoan parasites of the genus Leishmania have to differentiate from promastigotes to amastigotes. The molecular basis underlying this major transformation is poorly understood. One way to study this phenomenon is to isolate and characterize proteins that are specifically expressed in one of the two stages of the life cycle or during the stage differentiation. Using two-dimensional gel electrophoresis, we mapped the Leishmania donovani proteome during stage differentiation to identify stage-specific proteins and regulons. A protocol for extracting proteins of both promastigote and amastigote L. donovani cells was developed, which is compatible with isoelectric focusing. Up to 400 L. donovani protein spots were visualized on a silver-stained gel. Metabolic labeling of the cells was used to compare directly the protein synthesis pattern with the protein level pattern. The silver-stained images of L. donovani cells harvested on different days of stage differentiation were compared to the corresponding autoradiographs. A marked decrease in protein synthesis during stage differentiation from promastigotes to amastigotes was observed. The stained protein pattern as well as the protein pattern on the autoradiograph changed dramatically, especially after day 3 (about 24 h after pH shift) of transformation.