Zhang W W, Charest H, Ghedin E, Matlashewski G
Institute of Parasitology, McGill University, Quebec, Canada.
Mol Biochem Parasitol. 1996 Jun;78(1-2):79-90. doi: 10.1016/s0166-6851(96)02612-6.
Leishmania protozoa must adapt rapidly to widely different environments and thus exist as promastigotes in their sandfly host and as amastigotes in their mammalian host. Promastigote differentiation into amastigotes is accompanied by both morphological and biological changes. The molecular mechanisms regulating the differentiation and survival of the different life cycle stages are poorly understood. We have therefore undertaken to identify and characterize amastigote-specific genes and their corresponding products based on the rationale that such products may be involved in the survival in the mammalian host. Previous studies in our laboratory have revealed that the A2 gene family-derived transcripts are abundant in L. donovani amastigotes but are barely detectable in promastigotes. In the present study, we have raised polyclonal and monoclonal antibodies against a recombinant A2 protein synthesized in Escherichia coli. These antibodies have been used to identify a family of A2 proteins ranging from 45 kDa to about 100 kDa which are specifically detected in L. donovani cells when they are cultured in 37 degrees C, and pH 4.5 (conditions which mimic the macrophage phagolysosome) but not in promastigotes cultured at 26 degrees C and pH 7.4. A2 protein therefore represents a unique amastigote-specific protein marker for L. donovani. It is also demonstrated that it was possible to overexpress the A2 protein specifically in amastigote-like cells using a plasmid construct containing the A2 coding and non-coding sequences. These advances set the foundation for defining the biological function of the A2 protein and other genes when specifically expressed in amastigotes.
利什曼原虫必须迅速适应广泛不同的环境，因此在其沙蝇宿主中以前鞭毛体形式存在，而在其哺乳动物宿主中以无鞭毛体形式存在。前鞭毛体向无鞭毛体的分化伴随着形态和生物学变化。调节不同生命周期阶段分化和存活的分子机制尚不清楚。因此，我们基于这样的理由开展研究，即此类产物可能参与在哺乳动物宿主中的存活，从而鉴定和表征无鞭毛体特异性基因及其相应产物。我们实验室之前的研究表明，源自A2基因家族的转录本在杜氏利什曼原虫无鞭毛体中丰富，但在前鞭毛体中几乎检测不到。在本研究中，我们制备了针对在大肠杆菌中合成的重组A2蛋白的多克隆和单克隆抗体。这些抗体已用于鉴定一个A2蛋白家族，其分子量范围从45 kDa到约100 kDa，当杜氏利什曼原虫细胞在37摄氏度和pH 4.5（模拟巨噬细胞吞噬溶酶体的条件）下培养时能被特异性检测到，而在26摄氏度和pH 7.4条件下培养的前鞭毛体中则检测不到。因此，A2蛋白是杜氏利什曼原虫独特的无鞭毛体特异性蛋白标志物。还证明了使用包含A2编码和非编码序列的质粒构建体可以在无鞭毛体样细胞中特异性过表达A2蛋白。这些进展为确定A2蛋白和其他基因在无鞭毛体中特异性表达时的生物学功能奠定了基础。
