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[目的]原核表达纯化获得可溶性α-OGG1,ACO2蛋白并初步鉴定其相互作用.[方法]将α-OGG1,ACO2基因构建到pGEX-4T-2-TEV和pET-28a-TEV表达载体上,转化至BL21(DE3)大肠杆菌.IPTG诱导表达融合蛋白,通过Ni柱和GST柱亲和层析法纯化目标融合蛋白;采用SDS-PAGE检测表达产物;采用GST-Pulldown方法鉴定蛋白与蛋白的相互作用;采用双质粒共转化,表达纯化α-OGG1,ACO2蛋白复合体.[结果]成功克隆并构建了α-OGG1及其N-半段、C-半段基因和ACO2基因的原核表达载体,并转化至大肠杆菌表达.通过Ni柱和GST柱亲和层析法得到可溶性表达的6×His_α-OGG1(1-326)和GST_ACO2(29-780)蛋白,SDS-PAGE检测结果表明2个蛋白条带均与理论大小相符.GSTPulldown实验结果证实α-OGG1与ACO2直接相互作用.通过共转化、表达纯化得到了α-OGG1与ACO2的复合物.[结论]采用原核表达和亲和层析纯化得到了可溶性6×His_α-OGG1,GST_ACO2融合蛋白及两者的复合体,并初步证实α-OGG1与ACO2存在直接相互作用.
[Objective] The prokaryotic expression system was used to obtain soluble α-OGG1 and ACO2 protein and preliminary identification of their interaction. [Methods] The α-OGG1 and ACO2 genes were cloned into pGEX-4T-2-TEV and pET-28a- Then transformed into BL21 (DE3) E.coli.IPTG induced the expression of the fusion protein, purified the target fusion protein by Ni column and GST column affinity chromatography, detected the expression product by SDS-PAGE and identified the protein-protein interaction by GST-Pulldown OGG1 and ACO2 protein complexes were purified by double-plasmid co-transformation. [Results] The prokaryotic expression vector of α-OGG1 and its N-half, C-half and ACO2 genes were successfully cloned and constructed. And transformed into E.coli.The soluble proteins of 6 × His_α-OGG1 (1-326) and GST_ACO2 (29-780) were obtained by affinity chromatography on Ni column and GST column.The results of SDS-PAGE showed that the two proteins The results of GSTPulldown experiment showed that α-OGG1 interacted directly with ACO2, and the complex of α-OGG1 and ACO2 was obtained by co-transformation and purification. [Conclusion] The prokaryotic expression system was purified by affinity chromatography Soluble 6 × His_α-OGG1, GST_ACO2 fusion protein and both Body, and confirm the presence of the initial direct interaction with α-OGG1 ACO2.