热聚体Thermosome蛋白的纯化与组装体表征

Characterization of Thermosome Protein Assembly and Purification

目的表达纯化热聚体Thermosome蛋白,探讨Thermosome蛋白组装体基本特性与细胞相容性.方法将Thermosome原核表达质粒转化大肠杆菌BL21感受态细胞,诱导表达Thermosome的α和β亚基蛋白,利用凝胶过滤层析法纯化目的蛋白;利用动态光散射和透射电子显微镜分析Thermosome蛋白组装体的粒径和形貌特征;通过MTT法检测Thermosome蛋白组装体的细胞相容性.结果SDS-PAGE结果显示,Thermosome的α和β亚基以可溶性蛋白在BL21大肠杆菌中表达,凝胶过滤层析得到了较高纯度的α和β亚基蛋白;动态光散射结果显示Thermosome蛋白组装体尺寸为20.2 nm,组装体在透射电镜下呈现中空球形结构;MTT结果显示Thermosome纳米粒处理后细胞存活率接近100%;绿色荧光标记的纳米粒能够被细胞摄取进入细胞内.结论本研究成功表达纯化Thermosome的α和β亚基蛋白,得到平均粒径在20.2 nm的中空笼状自组装纳米颗粒;Thermosome蛋白纳米颗粒具有良好生物相容性,能够被肿瘤细胞高效摄取.

Objective To express and purify Thermosome protein,and explore the property and compatibility of Thermosome protein assembly.Method Thermosome expression vector was transformed into E.coli(BL21)competent cells to expressαandβsubunit protein.Both subunit proteins were purified by gel filtration chromatography.The size distribution and morphology of Thermosome protein assembly were determined by dynamic light scattering(DLS)and transmission electron microscope(TEM),respectively.The cell compatibility of Thermosome protein assembly was examined via MTT assay.Results SDS-PAGE results showed that soluableαandβsubunit protein were detected in E.coli cells.We preparedαsubunit andβsubunit of Thermosome protein with satisfied purity by gel filtration chromatography.DLS analysis indicated that the average diameter of Thermosome assembly was 20.2 nm.TEM suggested the hollow nanostructure of assembly.MTT assay demon-strated that the cells treated with Thermosome protein assembly grow well.Thermosome nanoparticle-labeled with green fluorescence could be uptake by CT26 cells.Conclusion We successfully purifiedαsubunit andβsubunit of Thermosome protein.The protein assembly has a mean diameter of 20.2 nm and hollow sphere nano-structure.Thermosome protein nanoparticle has good biocompatibility and efficient cellular uptake.