Abstract
Objective
We 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).
Results
For construction of DNA aptamer and NanoLuc conjugate using the catalytic domain of Rep from PCV2. pRep fused to NanoLuc was genetically constructed and expressed in E. 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.
Conclusion
We have demonstrated that DNA-NanoLuc conjugates via the catalytic domain of PCV2 Rep could be applied for DNA aptamer-based sandwich assay system.
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Acknowledgements
This work was supported in part by JSPS KAKENHI Grant Numbers 16K01388 (M.M.), 15K13781 and 6289310J (E.K.).
Supplementary Information
Supplementary Figure 1—The gene of the catalytic domain of PCV2 Rep comprising residues 1–116 (pRep). The amino acid sequence of the pRep was shown as red. The sequence of the pRep optimized for E.coli expression was shown as black (Opt).
Supplementary Figure 2—Immobilization of DNA-protein conjugates on the hydrophobic plate surface.
Supplementary Figure 3—Evaluation of specific binding abilities of Thrombin DNA aptamer conjugated to protein.
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Mie, M., Niimi, T., Mashimo, Y. et al. Construction of DNA-NanoLuc luciferase conjugates for DNA aptamer-based sandwich assay using Rep protein. Biotechnol Lett 41, 357–362 (2019). https://doi.org/10.1007/s10529-018-02641-7
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DOI: https://doi.org/10.1007/s10529-018-02641-7