一种改良的恶性疟原虫核蛋白和胞浆蛋白分离方法的建立  

Establishment of an improved method for nuclear protein and cytoplasmic protein isolation in Plasmodium falciparum

作　　者：韩世通[1] 张青锋[1] 潘卫庆[1] 

机构地区：[1]同济大学传染病与疫苗研究所,上海200092

出　　处：《中国病原生物学杂志》2013年第10期874-877,共4页Journal of Pathogen Biology

基　　金：国家自然科学基金项目(No.31271388);中央高校基本科研业务费专项资金

摘　　要：目的建立一种改良的恶性疟原虫核蛋白和胞浆蛋白分离方法，并对分离的蛋白组分进行分析和鉴定。方法分别用含不同盐浓度的裂解缓冲液和不同研磨次数提取核蛋白和胞浆蛋白，并利用针对不同组分特异性蛋白的抗体进行Westernblot，分析分离效果。结果含50mmol／LNaCl的裂解缓冲液和研磨100～150次为分离核蛋白和胞浆蛋白最佳条件，恶性疟原虫醛缩酶胞浆蛋白和组蛋白3核蛋白的抗体Westernblot、抗GFP抗体Westernblot及活细胞免疫荧光检测均表明该方法效果良好。结论建立了一种快速、有效的恶性疟原虫核蛋白和胞浆蛋白的分离方法（裂解液盐浓度为50mmol／L，研磨100150次），为研究恶性疟原虫蛋白的胞内分布以及蛋白质／蛋白质／核酸相互作用提供了重要的技术手段。Objectives To establish an improved method for the isolation of nuclear protein and cytoplasmic protein in Plasmodium falciparurn by further analysis and to verify the nuclear proteins and cytoplasmic proteins isolated. Meth- ods The effectiveness of protein isolation was determined using lysis buffer with different concentrations of NaC1, differ- ent numbers of strokes in a Dounce homogenizer, and Western blotting with antibodies against specific target proteins. Results Nuclear proteins and cytoplasmic proteins were most effectively isolated using a lysis buffer containing 50 mmol/ L NaC1 and 100--150 strokes in the Dounce homogenizer. This finding was corroborated by Western blotting using anti- bodies against specific target proteins, such as aldolase in the cytoplasm and histone H3 in the nucleus. In addition, the effectiveness of isolation was verified by Western blotting and immunofluorescence using anti-GFP antibody in a transgenic model of P. falciparum. Conclusion A fast and effective method for nuclear protein and cytoplasmic protein isolation in P. falciparum has been established （lysis buffer with 50 mmol/L NaC1 and 100--150 strokes in a Dounce homogeni- zer）. This method can help determine the position of certain proteins in P. falciparum and it can also facilitate the study of protein-protein/protein nucleic acid interaction in pathogens.

关 键 词：疟原虫 恶性 核蛋白 胞浆蛋白 

分 类 号：R382.31[医药卫生—医学寄生虫学]