RT-PCR检测亚马孙利什曼原虫P-4和GP-46基因表达  

Detection of P-4 and GP-46 Expression in Leishmania amazonensis Amastigotes and Promastigotes by RT-PCR

作　　者：王华民[1] Soong Lynn 

机构地区：[1]海南医学院微生物学与免疫学教研室,海口571101 [2]Dept of Microbiology and Immunology,University of Texas Medical Branch,Galveston,TX.,USA

出　　处：《中国寄生虫学与寄生虫病杂志》2006年第4期266-268,共3页Chinese Journal of Parasitology and Parasitic Diseases

摘　　要：目的证明亚马孙利什曼原虫前鞭毛体和无鞭毛体的基因表达水平。方法用RNA分离试剂盒,分别提取3种不同来源的无鞭毛体(由小鼠模型皮损组织获得的无鞭毛体、由前鞭毛体培养转化而来的无鞭毛体,以及来自J774.G8巨噬细胞株的无鞭毛体)的总RNA,以及前鞭毛体总RNA,然后用SuperScripⅡ逆转录聚合酶将其逆转录为cD-NA,再经PCR扩增无鞭毛体特异核酸酶(P-4)和前鞭毛体特异膜糖蛋白(GP-46)的特异片段,经1.5%琼脂糖凝胶电泳分析。结果3种不同来源的无鞭毛体均观察到P-4特异性条带(273bp),且密度相似,但在前鞭毛体中未观察到;在前鞭毛体中观察到高表达的GP-46特异性条带(325bp),但在3种无鞭毛体中弱表达。结论由前鞭毛体转化的无鞭毛体能高水平表达亚马孙利什曼原虫无鞭毛体P-4特异基因,可为其生物化学及免疫学研究提供无鞭毛体来源。Objective To detect the expression level of stage-specific genes in Leishmania promastigotes and amastigotes. Methods 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. Results 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. Conclusion 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.

关 键 词：亚马孙利什曼原虫 无鞭毛体 前鞭毛体 阶段特异性蛋白 

分 类 号：R382.22[医药卫生—医学寄生虫学]