丙型肝炎病毒N′-核心蛋白RNA结合区的定位  

Mapping of RNA binding region in HCV core protein

作　　者：关德玉[1] 何长清 武英[1] 黄敦武 路立业[1] 刘碧坚 

机构地区：[1]沈阳军区第202医院传染科,沈阳110003

出　　处：《中华微生物学和免疫学杂志》2002年第4期415-418,共4页Chinese Journal of Microbiology and Immunology

基　　金：军队回国人员科研启动基金资助项目 (H980 0 1)

摘　　要：目的　对丙型肝炎病毒核心蛋白的RNA结合区进行详细定位 ,为制备缺失RNA结合活性突变体做准备。方法　构建GST融合丙型肝炎病毒核心蛋白不同区域片段表达质粒 ,在大肠杆菌中表达相应蛋白并用glutathionesepharose 4B进行纯化 ,利用二次电泳法检测RNA的结合活性 ,利用时相差电泳法比较不同区域RNA结合活性的强弱。结果　核心蛋白第 1～ 132位氨基酸 (AA)之间的片段得到了良好的表达和纯化 ,表达的蛋白经Westernblot得到确认。二次电泳后的放射自显影结果显示 ,第 1～ 10AA、19～ 45AA、80～ 132AA片段无RNA结合活性 ,而含有 10～ 16AA ,45～ 80AA的片段可结合单链和双链RNA。 1～ 2 9AA与 38～ 90AA 2个片段对RNA的结合强度基本相同。结论　丙型肝炎核心蛋白中存在 2个RNA结合区 ,分别定位于 10～ 16AA及 45～ 80AA。 2个结合区都具有较强的RNA结合活性。制备RNA结合活性缺陷突变体须同时对 2个结合区进行突变。Objective To map RNA binding region in hepatitis C (HCV) core protein and provide technical data for establishing mutant core that lacks RNA binding activity. Methods Truncated HCV core proteins were expressed in E.coli and purified by glutathione Sepharose 4B. The protein-RNA complex was detected by autoradiography. Results Truncated core fragments between 1-132 AA were well expressed and purified. Among them 1-10 AA, 19-45 AA and 80-132 AA failed to bind RNA but fragments of 10-16 AA and 45-80 AA did. Time course electrophoreses showed that 1-29 AA and 38-90 AA had similar RNA binding affinity. Conclusions There were two RNA binding regions in HCV core protein mapped as 10-16 AA and 45-80 AA. The two regions bound to RNA with similar affinity suggested that both of them should be mutated when constructing mutant core that lacks RNA binding activity.

关 键 词：丙型肝炎 丙型肝炎病毒 核心蛋白 RNA结合活性 蛋白突变体 

分 类 号：R346[医药卫生—基础医学]