NanoBRET receptor binding drug screening system based on Cy5 labeled ligand
A drug and receptor technology, applied in the field of NanoBRETG protein-coupled receptor combined with high-throughput drug screening system, can solve the problems of limited wide application, high background value, high cost, etc., and achieve low background signal and high sensitivity. , the effect of accurate drug screening
- Summary
- Abstract
- Description
- Claims
- Application Information
AI Technical Summary
Problems solved by technology
Method used
Image
Examples
Embodiment 1
[0047] Example 1 Design of Sulfo-Cy5 fluorescently labeled ligand
[0048] In this embodiment, the sixth Gly of the endogenous ligand gonadotropin-releasing hormone I (GnRH I) of the gonadotropin-releasing hormone receptor (GnRH-R) is replaced by D-Lys, and in D-Lys 6 The bioluminescent resonance energy transfer group Sulfo-Cy5 was connected to the ε-amino group of the ε-amino group, and the GnRH fluorescently labeled polypeptide [Sulfo-Cy5-D-Lys6]GnRH(pyroGlu-His-Trp- Ser-Tyr-D-Lys(-Sulfo-Cy5)-Leu-Arg-Pro-Gly-NH 2 ), the structural formula is shown in formula I, wherein, pyroGlu is pyroglutamic acid (pyroglutamic acid).
Embodiment 2
[0049] Example 2 Detection of Receptor Binding Activity of Sulfo-Cy5 Fluorescently Labeled Ligand
[0050] Culture HEK293 cells stably expressing rat GnRH receptors. When the cell density reaches above 80%, discard the medium, wash the cells once with PBS, and discard the PBS; add an appropriate amount of trypsin to digest the cells to form a cell suspension, and pipette to form a single Cell suspension for cell counting; add 200 μL of single cell suspension to a black 96-well cell culture plate with a transparent bottom and an opaque surrounding, so that the number of cells in each well is 5×10 4 cells;
[0051] After 24 hours, add 100 μL of different concentrations of Sulfo-Cy5 fluorescently labeled ligand [Sulfo-Cy5-D-Lys6]GnRH to the total binding experiment group, and add 100-fold excess GnRH I to the non-specific group at the same time, adjust to the same volume with PBS, Set up 3 replicate wells at each point, incubate at 4°C for 4h, wash the cells with 4°C PBS buffer ...
Embodiment 3
[0054] Example 3 Design and Construction of SecNanoLuc Labeled Receptor
[0055]The human gonadotropin-releasing hormone receptor (hGnRH-R) is a seven-transmembrane protein, and the SecNanoLuc marker is constructed by linking the secretion signal peptide Sec and the bioluminescence resonance energy transfer donor protein nano-luciferase (NanoLuc) at its N-terminus The hGnRH-R specifically comprises the following steps:
[0056] (1) Prepare the enzyme digestion system shown in Table 1, utilize the restriction endonuclease EcoRI and XbaI to digest the recombinant pcDNA3.1 (+) containing HA-hGnRH-R gene and pNLF1-N.CMV Hygro vector (Promega ), after enzyme digestion, in 1×TAE buffer, 120V electrophoresis for 40min, purified and recovered to obtain HA-hGnRH-R gene and linearized pNLF1-N.CMV Hygro vector;
[0057] Table 1
[0058]
[0059]
[0060] (2) Prepare the ligation system shown in Table 2, incubate at 16°C for 1 h, connect the HA-hGnRH-R gene to the pNLF1-N.CMVHygro...
PUM
Abstract
Description
Claims
Application Information
- R&D Engineer
- R&D Manager
- IP Professional
- Industry Leading Data Capabilities
- Powerful AI technology
- Patent DNA Extraction
Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic, Popular Technical Reports.
© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap|About US| Contact US: help@patsnap.com