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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 locate the RNA binding region of hepatitis C virus core protein in preparation for preparation of mutants lacking RNA binding activity. Methods GST fusion protein of different regions of HCV core protein was constructed and expressed in E.coli. The recombinant protein was purified by glutathionesepharose 4B. The binding activity of RNA was detected by double electrophoresis. The strength of activity. Results The fragment of amino acids 1 to 132 of core protein was well expressed and purified. The expressed protein was confirmed by Western blot. The results of autoradiography after secondary electrophoresis showed that there was no RNA binding activity in the first ~ 10AA, 19 ~ 45AA and 80 ~ 132AA fragments, while the fragments containing 10 ~ 16AA and 45 ~ 80AA could bind single-stranded and double-stranded RNA. The binding intensities of the 2 fragments of 1 ~ 2 9AA and 38 ~ 90AA to RNA were basically the same. Conclusion There are two RNA binding regions in the core protein of hepatitis C, which are located at 10 ~ 16AA and 45 ~ 80AA, respectively. Both of the two binding regions have strong RNA binding activity. Preparation of RNA binding activity deficient mutants must be simultaneously mutated 2 binding regions.