EV713D聚合酶基因体外重组质粒的构建、表达及活性检测

Construction of a recombinant plasmid containing EV71 3D polymerase, its expression, and an assay of its activity in vitro

目的构建编码肠道病毒71型(EV71)3D聚合酶基因的重组质粒,表达和纯化3D聚合酶并检测其活性。方法将编码3D聚合酶的基因片段克隆至pGEX-4T-1原核表达载体,重组质粒转化入大肠埃希菌BL21(DE3),IPTG诱导表达,经谷胱甘肽琼脂糖亲和层析、TEV酶酶切、Ni-NTA柱亲和纯化后获得高纯度3D聚合酶蛋白,放射测量法是体外酶活力测定方法中较常见的一种方法,本研究通过测定插入poly(U)RNA放射性标志UMP的量确定3D聚合酶的活性。结果经酶切、PCR、测序鉴定pGEX-4T-3D重组质粒构建成功,3D聚合酶基因片段大小为1 386 bp。与未诱导相比,经IPTG诱导后带有GST标签的3D聚合酶成功表达,表达产物的相对分子质量约为79×10~3,TEV酶酶切掉GST标签后的相对分子质量约为55×10~3。纯化的3D聚合酶经体外延伸反应后跑变性尿素聚丙烯酰胺凝胶电泳得知,3D聚合酶溶解液DMSO组相比于3D聚合酶强抑制剂EDTA组,在DMSO组中,可以观察到强电泳条带,表明放射性同位素标志程度强,表明反应速率快,即3D聚合酶酶活较好。结论构建了EV71 3D聚合酶基因重组质粒,其表达产物3D聚合酶为以EV71 3D聚合酶为靶点的抗EV71药物筛选奠定基础。

Objective To construct a recombinant plasmid encoding the 3 D polymerase gene of enterovirus 71(EV71)in order to express and purify 3 D polymerase and evaluate its activity.Methods A DNA fragment encoding 3 D polymerase was cloned into a pGEX-4 T-1 prokaryotic expression vector.The recombinant plasmid was transformed into E.coli BL21(DE3),and protein expression was induced with IPTG.The expressed protein was purified using affinity chromatography with glutathione agarose,enzyme digestion with TEV,and affinity chromatography(Ni-NTA).Highly pure 3 D polymerase was obtained,and its functional activity was evaluated by measuring the amount of UMP inserted into poly(U)RNA.Radiometry is a common method for the determination of enzyme activity in vitro.Results Restriction analysis and sequencing verified that the recombinant plasmid pGEX-4 T-3 D was successfully constructed.The 3 D polymerase gene fragment was 1,386 bp in length.After expression was induced with IPTG,3 D polymerase with a GST tag was successfully expressed in comparison to the untreated group.The expressed product was about 79×10~3 in size.When the GST tag was removed with the TEV enzyme,the purified 3 C polymerase was about 55×10~3 in size.An extension reaction in vitro and denatured urea polyacrylamide gel electrophoresis revealed that 3 D polymerase in the presence of the solvent DMSO produced strong electrophoresis bands in comparison to 3 D polymerase in the presence of the strong inhibitor EDTA,indicating that the amount of radioisotopes was high and the reaction rate was fast,that is,3 D polymerase had better enzyme activity.Experimental results indicated that purified 3 D polymerase has polymerase activity.Conclusion The recombinant plasmid pGEX-4 T-3 D was created and 3 D polymerase with a high level of functional activity was successfully prepared.This polymerase can be used not only to study the function and mechanism of action of EV71 and also to screen and design anti-EV71 drugs targeting 3 D polymerase.