A High-Throughput Screening Method for Prey Antagonists Based on Membrane-Bound Proteins and Fluorescence Complementation

Abstract

The invention provides a high-flux prey antagonist screening method based on membrane binding protein and fluorescence complementation, relates to the field of gene engineering, and concretely discloses a novel method for screening a receptor antagonist and application thereof. According to the method, a ligand existing outside a cell and a receptor are expressed to the cytomembrane through a signal peptide fusion expression strategy; a ligand structural domain generating mutual action with the receptor is enabled to be remained at the outer side of the cytomembrane; the ligand is coupled with intracellular effect proteins through transmembrane peptide; the ligand and the receptor take mutual effects at the outer side of the cytomembrane, or the effect proteins coupled in the cells are antagonized; and the effect is converted into a macroscopical biological effect, so that the method is provided for the screening of the secretion type ligand and receptor antagonists.

Description

technical field [0001] The invention relates to the field of genetic engineering, and specifically discloses a new method for screening receptor antagonists and its application. Background technique [0002] Antagonist refers to a class of substances that can bind to receptors but do not have the biological function of activating receptors. Its biological function is opposite to that of agonists. It inhibits downstream physiological responses by blocking signal transduction , is an important class of drugs. Antagonists broadly include small molecular compounds, recombinant proteins, antibodies, etc. that can antagonize protein interactions, among which small molecular compound antagonists are the most widely used. Receptor antagonist screening is of great significance in drug development. For example, CXCR4 antagonists AMD3100 and ALX40-4C can inhibit the tumor growth of some CXCR4-related tumors by antagonizing the interaction between CXCL12 and CXCR4; the endothelial gro...

AI Technical Summary

Problems solved by technology

This type of method needs to purify the active ligand protein, and needs to label the ligand protein. It is difficult to guarantee the activity and the reproducibility is not strong.

[0004] For the screening method of intracellular soluble receptor antagonists, a method based on the traditional yeast two-hybrid method has been proposed, but the receptors and ligands studied by the traditional yeast two-hybrid method exist inside the cell, and the compound must be able to enter the cell effectively. Opportunity for blocking, but this factor is difficult to control in high-throughput screening, so the entire compound library is not fully utilized

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