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Construction of DNA-NanoLuc luciferase conjugates for DNA aptamer-based sandwich assay using Rep protein.
Mie, Masayasu; Niimi, Takahiro; Mashimo, Yasumasa; Kobatake, Eiry.
Affiliation
  • Mie M; Department of Life Science and Technology, School of Life Science and Technology, Tokyo Institute of Technology, 4259 Nagatsuta, Midori-Ku, Yokohama, 226-8502, Japan. mie.m.aa@m.titech.ac.jp.
  • Niimi T; Department of Life Science and Technology, School of Life Science and Technology, Tokyo Institute of Technology, 4259 Nagatsuta, Midori-Ku, Yokohama, 226-8502, Japan.
  • Mashimo Y; Department of Life Science and Technology, School of Life Science and Technology, Tokyo Institute of Technology, 4259 Nagatsuta, Midori-Ku, Yokohama, 226-8502, Japan.
  • Kobatake E; Department of Life Science and Technology, School of Life Science and Technology, Tokyo Institute of Technology, 4259 Nagatsuta, Midori-Ku, Yokohama, 226-8502, Japan.
Biotechnol Lett ; 41(3): 357-362, 2019 Mar.
Article in En | MEDLINE | ID: mdl-30603832
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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Full text: 1 Collection: 01-internacional Database: MEDLINE Main subject: Staining and Labeling / Viral Proteins / Trans-Activators / DNA Helicases / Luminescent Agents / Aptamers, Nucleotide / Luciferases Language: En Journal: Biotechnol Lett Year: 2019 Document type: Article Affiliation country: Japan Country of publication: Netherlands
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Full text: 1 Collection: 01-internacional Database: MEDLINE Main subject: Staining and Labeling / Viral Proteins / Trans-Activators / DNA Helicases / Luminescent Agents / Aptamers, Nucleotide / Luciferases Language: En Journal: Biotechnol Lett Year: 2019 Document type: Article Affiliation country: Japan Country of publication: Netherlands