EV-A71病毒3D聚合酶突变体的可溶性高效表达纯化及热稳定性分析Soluble High Efficiency Expression, Purification and Thermal Stability Analysis of EV-A71 3D Polymerase Mutants
秦博;赵蓉;崔胜;
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
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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