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

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。

RNA-dependent RNA polymerase is a target protein for drug design against enterovirus(EV)A71.Using sequence alignment of the homologs of polymerases of known oligonucleotide families,we designed three mutants that target EV-A71 3D polymerase,and which help improve the drug-binding efficiency of nucleotide analogs. We used EV-A71 of a wild-type 3D polymerase plasmid as a template. By polymerase chain reaction(PCR)amplification,digestion,decomposition and transformation of wild-type plasmids,we constructed a positive clone of mutant plasmids. Then,we transformed it into Escherichia coli 3016-receptive cells. Cryogenic induction overnight,ultrasonic crushing and enrichment of slime were done. Purification using a Ni column and then high-trap Q ion-exchange chromatography were undertaken. Gel-filtration chromatography led to high-purity and high-yield EV-A71 3D polymerase mutant recombinant proteins. Real-time reverse transcription-quantitative PCR was done to determine stable expression of mutant proteins. Thermal-stability analyses of different solution conditions enabled the optimum conditions of protein dissolution to be obtained. This study provides a technical basis for further research of the design of nucleotide analogs that target EV-A71 3D polymerase.