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

Pending Publication Date: 2021-03-30
XIAN JIAOTONG LIVERPOOL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, this method also has some disadvantages, such as high cost, high background value, and intramolecular excitation processes (electron transfer, FRET, photobleaching) and fluorescent external interactions of compounds or proteins can cause quenching of receptors or ligands. Annihilation effect, etc., which limit its wide application

Method used

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  • NanoBRET receptor binding drug screening system based on Cy5 labeled ligand
  • NanoBRET receptor binding drug screening system based on Cy5 labeled ligand
  • NanoBRET receptor binding drug screening system based on Cy5 labeled ligand

Examples

Experimental program
Comparison scheme
Effect test

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...

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Abstract

The invention provides a NanoBRET receptor binding high-throughput drug screening system based on Cy5 and a derivative Sulfo-Cy5 fluorescently labeled ligand thereof. The drug screening system comprises the Cy5 and the derivative Sulfo-Cy5 fluorescently labeled ligand thereof and engineered cells for expressing a NanoLuc fusion G protein coupled receptor. According to the invention, NanoLuc is fused at the N end of the receptor to construct a NanoLuc fusion receptor, and the NanoLuc fusion receptor is paired with Cy5 and derivative Sulfo-Cy5 fluorescently-labeled peptides, proteins or small molecular ligands thereof, so that a cheap and sensitive NanoBRET high-throughput receptor drug screening system is created.

Description

technical field [0001] The invention belongs to the field of biotechnology, and relates to a NanoBRET receptor binding drug screening system based on Cy5 or Sulfo-Cy5 labeled ligands, in particular to NanoBRET G protein-coupled receptor binding based on Cy5 and its derivative Sulfo-Cy5 fluorescent labeled ligands High-throughput drug screening system. Background technique [0002] G protein-coupled receptors (GPCRs) are the largest superfamily of membrane proteins in the human genome, widely distributed in almost all organs and tissues such as the nervous system, endocrine system, cardiovascular system, and immune system, mediating neurotransmitters and The cell signal transduction of hormones is involved in the regulation of cell development, differentiation and normal physiological function activities, and is closely related to the occurrence and development of various diseases. In current clinical medicine, about 40% of drugs act on GPCRs, but only a few GPCRs have corre...

Claims

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Application Information

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IPC IPC(8): C12Q1/02C12Q1/66C12N15/85C12N5/10G01N21/64
CPCG01N33/5044C12Q1/66C12N5/0686C12N15/85C07K14/723C12N9/0069C12Y113/12013G01N21/6428C07K2319/61G01N2500/10C12N2503/02C12N2510/00C12N2800/107G01N2021/6439
Inventor 吕志良沈力孟佳荣荣
Owner XIAN JIAOTONG LIVERPOOL UNIV
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