肠道病毒71型2C蛋白的异源表达及纯化  

Heterologous expression and purification of the 2C protein from enterovirus 71

作　　者：刘艳[1,2] 李志会[1] 李鹏[1] 岳盈盈[1] 宋楠楠[1] 秦立增[1] 李冰清[1] 孟红[1] 

机构地区：[1]山东省医学科学院基础医学研究所微生物学研究室,山东济南250000 [2]济南大学,山东省医学科学院生命与科学学院

出　　处：《中国病原生物学杂志》2014年第9期800-803,778,共5页Journal of Pathogen Biology

基　　金：国家自然科学基金项目(No.81000720)

摘　　要：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。Objectives To obtain soluble,highly purified,and homogenous 2Cprotein from enterovirus 71（EV71）.Methods Six gene fragments containing the ATPase/helicase core domain of 2Cprotein were truncated and amplified with PCR.The PCR products were ligated into two different expression vectors containing a His tag or MBP tag in order to heterologously express 2Cproteins.Purification was carried out with sieve chromatography.The expressed products and purified protein samples were examined using SDS-PAGE. Results Six fragments of 2Cproteins were successfully cloned,expressed,and purified,resulting in the soluble proteins MBP-2C91aa-256 aa and MBP-2C118aa-256 aa that were highly pure and stable.SDS-PAGE analysis verified that protein purity was as high as〉97% and indicated that the proteins had a molecular weight of 61 KD and 58 KD.These results agreed with the predicted values.Furthermore,the available concentration of purified 2Cprotein was as high as 8.8mg·ml^-1. Conclusion Two individual truncated MBP-2Cproteins containing 91aa-256 aa and 118aa-256 aa fragments were more stable and homogenous than those containing other fragments in this study.Results indicated that the MBP tag was able to greatly improve the solubility of six truncated proteins.Preparations of highly pure soluble proteins should pave the way for further studies of the crystal structure and related functions with of EV71.

关 键 词：肠道病毒71型(enterovirus 71 EV71) 2C蛋白 异源表达 纯化方法 

分 类 号：R373.2[医药卫生—病原生物学]