Construction of DNA-NanoLuc luciferase conjugates for DNA aptamer-based sandwich assay using Rep protein
Construction of DNA-NanoLuc luciferase conjugates for DNA aptamer-based sandwich assay using Rep protein
复制标题
使用 Rep 蛋白构建 DNA-NanoLuc 荧光素酶缀合物，用于基于 DNA 适体的夹心测定
DOI：
10.1007/s10529-018-02641-7
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发表时间：
2019
期刊：
影响因子：
--
通讯作者：
Kobatake E
中科院分区：
文献类型：
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作者：
Mie M;Niimi T;Mashimo Y;Kobatake E
利用猪圆环病毒2型复制起始蛋白（Rep）的催化结构域，构建了一种DNA-NanoLuc荧光素酶（NnaoLuc）偶联物，用于基于DNA适体的夹心检测。基因构建与NanoLuc融合的pRep并在E.杆菌纯化后，融合pRep和NanoLuc的活性进行了评估，并通过融合pRep构建DNA-NanoLuc缀合物。最后，构建的DNA-NanoLuc缀合物被应用于基于DNA适配子的夹心检测。在这里，pRep不仅用于缀合的NanoLuc的检测适体，但也用于固定的捕获适体上platesurfaces.ConclusionWe已经证明，DNA-NanoLuc缀合物通过PCV 2 Rep的催化结构域可以应用于DNA适体为基础的夹心检测系统。
ObjectiveWe developed a DNA-NanoLuc luciferase (NnaoLuc) conjugates for DNA aptamer-based sandwich assay using the catalytic domain of the replication initiator protein derived from porcine circovirus type 2 (pRep).ResultsFor construction of DNA aptamer and NanoLuc conjugate using the catalytic domain of Rep from PCV2. pRep fused to NanoLuc was genetically constructed and expressed inE. coli. After purification, the activities of fused pRep and NanoLuc were evaluated, and DNA-NanoLuc conjugates were constructed via the fused pRep. Finally, constructed DNA-NanoLuc conjugates were applied for use in a DNA aptamer-based sandwich assay. Here, pRep was used not only for conjugation of the NanoLuc to the detection aptamer, but also for immobilization of the capture aptamer on the plate surface.ConclusionWe have demonstrated that DNA-NanoLuc conjugates via the catalytic domain of PCV2 Rep could be applied for DNA aptamer-based sandwich assay system.