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目的获得高纯度、性状均一的可溶蛋白肠道病毒71型(Enterovirus 71,EV71)2C。方法截取2C蛋白的6个包含ATPase/解旋酶等核心结构域的基因片段,分别使用含有His标签和MBP促溶标签的表达载体在大肠埃希菌中进行异源表达,分子筛层析纯化2C蛋白,SDS-PAGE电泳检测表达产物和纯化蛋白。结果成功将EV71 2C蛋白的6个片段进行了克隆表达并纯化,获得了高纯度、性状稳定的可溶蛋白MBP-2C91aa-256aa和MBP-2C118aa-256aa。SDS-PAGE检测2C蛋白纯度达97%以上,分子质量与预期结果一致。纯化的2C蛋白浓度为8.8mg/ml。结论91aa-256aa和118aa-256aa片段的MBP-2C截短蛋白比包含其他片段的截短蛋白性状稳定、均一,MBP标签提高了2C的6个截短蛋白的可溶性。高纯度可溶蛋白的制备为其晶体结构和功能研究奠定了基础。
Objective To obtain Enteroviral 71 (EV71) 2C with high purity and homogeneity. Methods Six cDNA fragments containing the core domain of ATPase / helicase were intercepted from the 2C protein and expressed in Escherichia coli using the expression vector containing His tag and MBP promoter respectively. The recombinant protein was purified by molecular sieve chromatography Proteins and SDS-PAGE were used to detect the expression products and purified proteins. Results Six fragments of EV71 2C protein were successfully cloned and purified. The soluble proteins MBP-2C91aa-256aa and MBP-2C118aa-256aa with high purity and stability were obtained. The purity of 2C protein detected by SDS-PAGE was more than 97%, the molecular mass was consistent with the expected result. The purified 2C protein concentration was 8.8 mg / ml. Conclusion The MBP-2C truncated proteins of 91aa-256aa and 118aa-256aa fragments are stable and homogeneous than the truncated proteins containing other fragments. The MBP-tag increases the solubility of 6 truncated proteins of 2C. The preparation of high purity soluble protein laid the foundation for the study of its crystal structure and function.