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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Western blot,分析分离效果。结果含50mmol/L NaCl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Western blot、抗GFP抗体Western blot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100~150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。
Objective To establish an improved method for the isolation of nucleoprotein and cytoplasmic protein of Plasmodium falciparum and to analyze and identify the isolated protein components. Methods Nucleoprotein and cytoplasmic protein were extracted from lysis buffer containing different salt concentrations and different milling times, respectively. Western blot was performed using antibodies against different components of the protein, and the separation effect was analyzed. Results The optimal conditions for the isolation of nucleoprotein and cytoplasmic protein from 100 to 150 lysis buffer containing 50 mmol / L NaCl, Western blotting of antibodies against Plasmodium falciparum aldolase cytoplasmic protein and histone 3 nucleoprotein, anti-GFP Antibody Western blot and live cell immunofluorescence assay showed that the method was effective. Conclusion A rapid and effective method for the isolation of nucleoprotein and cytoplasmic protein of Plasmodium falciparum was established (the concentration of lysate salt was 50mmol / L and 100-150 times of milling). To study the intracellular distribution of Plasmodium falciparum protein and the protein / Protein / nucleic acid interaction provides an important technical means.