秦博;赵蓉;崔胜;

• 1:中国医学科学院病原生物学研究所

摘要(Abstract)：

RNA依赖的RNA聚合酶是EV-A71病毒进行药物设计的药物靶点蛋白之一，本研究通过已知小核糖核酸病毒科聚合酶同源结构的序列比对，设计了三个针对EV-A71 3D聚合酶，有助于提高核苷酸类似物药物结合效率的突变体，以实验室原有野生型EV-A71 3D聚合酶质粒为模板，通过PCR扩增、野生型质粒的消化分解及转化的方式，构建突变体质粒的阳性克隆，并转化入大肠杆菌3016感受态细胞，通过低温过夜诱导，超声破碎富集的菌泥，将上清经过Ni柱亲和纯化、High trap Q离子交换层析纯化及Superdex 200 10/300凝胶过滤层析三个步骤的梯度纯化，获得了高纯度高产量的EV-A71 3D聚合酶突变体重组蛋白，并通过荧光定量的方法，采用荧光定量PCR仪对最终获得的稳定表达突变体重组蛋白，进行不同溶液条件的荧光热稳定性分析，得到了该重组蛋白最佳的蛋白溶解条件。为深入研究设计以EV-A71 3D作为药物靶点进行核苷酸类似物药物设计提供了技术基础。

关键词(KeyWords)： EV-A71病毒;3D聚合酶;药物靶点;荧光定量;热稳定性分析

Abstract:

Keywords:

基金项目(Foundation): 国家自然科学基金(项目号：82072291)，题目：基于小RNA病毒2C解旋酶核心结构特征的抗病毒策略;国家自然科学基金(项目号：81772207)，题目：肠道病毒解旋酶在囊泡膜上组装六聚体及催化双链RNA解旋结构基础~~

作者(Authors): 秦博;赵蓉;崔胜;

DOI: 10.13242/j.cnki.bingduxuebao.004136

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