Wang Hua-min, Lynn Soong
Department of Microbiology & Immunology, Hainan Medical College, Haikou, China.
Zhongguo Ji Sheng Chong Xue Yu Ji Sheng Chong Bing Za Zhi. 2006 Aug;24(4):266-8.
To detect the expression level of stage-specific genes in Leishmania promastigotes and amastigotes.
Total RNAs were isolated from Leishmania amazonensis stationary promastigotes and three sources of amastigotes: freshly obtained from mouse skin lesions, infected J774.G8 macrophages, and transformed from the cultured promastigotes. mRNAs were conversely transcribed into cDNA with SuperScripII reverse transcriptase and oligo dT primers. The polymerase chain reaction (PCR) was used to amplify the specific fragments of amastigote-specific nuclease (P-4) and promastigote-specific membrane glycoprotein (GP-46). PCR products were analyzed in 1.5% agarose gel.
A P-4-specific band (273 bp) was observed in all three types of amastigotes with similar density, but it was almost undetectable in promastigotes. In contract, a GP-46-specific band (325 bp) was expressed at a higher level in promastigotes than in all three types of amastigotes.
Promastigote-derived amastigotes express high level of P-4-specific gene and can be used as a source of amastigotes for biochemical and immunological studies.
检测利什曼原虫前鞭毛体和无鞭毛体阶段特异性基因的表达水平。
从亚马逊利什曼原虫静止期前鞭毛体和三种无鞭毛体来源中分离总RNA：从小鼠皮肤病变中新鲜获取的、感染J774.G8巨噬细胞的以及由培养的前鞭毛体转化而来的。使用SuperScripII逆转录酶和寡聚dT引物将mRNA逆转录为cDNA。采用聚合酶链反应（PCR）扩增无鞭毛体特异性核酸酶（P-4）和前鞭毛体特异性膜糖蛋白（GP-46）的特异性片段。PCR产物在1.5%琼脂糖凝胶中进行分析。
在所有三种类型的无鞭毛体中均观察到一条P-4特异性条带（273 bp），密度相似，但在前鞭毛体中几乎检测不到。相反，前鞭毛体中GP-46特异性条带（325 bp）的表达水平高于所有三种类型的无鞭毛体。
由前鞭毛体衍生而来的无鞭毛体表达高水平的P-4特异性基因，可作为生化和免疫学研究的无鞭毛体来源。
