Construction of DNA-NanoLuc luciferase conjugates for DNA aptamer-based sandwich assay using Rep protein
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).
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.
We have demonstrated that DNA-NanoLuc conjugates via the catalytic domain of PCV2 Rep could be applied for DNA aptamer-based sandwich assay system.
KeywordsDNA aptamer DNA–protein conjugate Rep NanoLuc luciferase DNA aptamer-based sandwich assay
This work was supported in part by JSPS KAKENHI Grant Numbers 16K01388 (M.M.), 15K13781 and 6289310J (E.K.).
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.
- Hanai R, Wang JC (1993) The mechanism of sequence-specific DNA cleavage and strand transfer by phi X174 gene A* protein. J Biol Chem 268:23830–23836Google Scholar