Ratiometric fluorescent sensor constructed based on g protein-coupled receptor and its construction method

A ratiometric fluorescent sensor constructed based on a G protein-coupled receptor (GPCR), wherein the ratiometric fluorescent sensor comprises the GPCR, a circularly permuted fluorescent protein, and peptide-linkers;

• the peptide-linkers comprise an N-terminal linker and a C-terminal linker;
• the circularly permuted fluorescent protein is inserted between a fifth transmembrane domain and a sixth transmembrane domain of the GPCR, an N-terminus of the circularly permuted fluorescent protein is connected to the fifth transmembrane domain of the GPCR via the N-terminal linker;
• and a C-terminus of the circularly permuted fluorescent protein is connected to the sixth transmembrane domain of the GPCR via the C-terminal linker;
• the N-terminal linker comprises 5 amino acids, and a third amino acid of the N-terminal linker is mutated;
• the ratiometric fluorescent sensor is capable of being expressed on a cell membrane;
• and the ratiometric fluorescent sensor is capable of binding to a specific ligand of the GPCR when in contact with the specific ligand of the GPCR, thereby causing a detectable change in a fluorescence intensity of the ratiometric fluorescent sensor;
• the GPCR is of human origin;
• the specific ligand is a neurotransmitter;
• the neurotransmitter comprises at least one selected from the group consisting of neurotensin (NTS), somatostatin (SST), pituitary adenylate cyclase-activating polypeptide (PACAP), neuropeptide Y (NPY), dopamine, and serotonin;
• the circularly permuted fluorescent protein is a circularly permuted enhanced green fluorescent protein (cpEGFP);
• when the cpEGFP is connected to the GPCR specifically binding the NTS to obtain a first resulting protein, the first resulting protein has the amino acid sequence as shown in SEQ ID NO:
• 1 or a variant thereof with a glutamic acid substitution at position 127;
• when the cpEGFP is connected to the GPCR specifically binding the SST to obtain a second resulting protein, the second resulting protein has the amino acid sequence as shown in SEQ ID NO:
• 2 or a variant thereof with a glutamic acid substitution at position 127;
• when the cpEGFP is connected to the GPCR specifically binding the PACAP to obtain a third resulting protein, the third resulting protein has the amino acid sequence as shown in SEQ ID NO:
• 3;
• when the cpEGFP is connected to the GPCR specifically binding the NPY to obtain a fourth resulting protein, the fourth resulting protein has the amino acid sequence as shown in SEQ ID NO:
• 4;
• when the cpEGFP is connected to the GPCR specifically binding the dopamine to obtain a fifth resulting protein, the fifth resulting protein has the amino acid sequence as shown in SEQ ID NO:
• 5 or a variant thereof with a valine substitution at position 127;
• when the cpEGFP is connected to the GPCR specifically binding the serotonin to obtain a sixth resulting protein, the sixth resulting protein has the amino acid sequence as shown in SEQ ID NO:
• 6;
• when the cpEGFP is connected to the GPCR specifically binding the NTS, the SST, the PACAP, the NPY, or the serotonin, the sequence of the N-terminal linker before mutation of the third amino acid is LEEGG;
• when the cpEGFP is connected to the GPCR specifically binding the NTS, the SST, or the NPY, the third amino acid of the N-terminal linker is mutated to leucine, and the mutated sequence of the N-terminal linker is LELGG;
• when the cpEGFP is connected to the GPCR specifically binding the PACAP, the third amino acid of the N-terminal linker is mutated to leucine, isoleucine, or methionine, and the mutated sequence of the N-terminal linker is any one of LELGG, LEIGG, or LEMGG;
• when the cpEGFP is connected to the GPCR specifically binding the serotonin, the third amino acid of the N-terminal linker is mutated to leucine or methionine, and the mutated sequence of the N-terminal linker is any one of LELGG or LEMGG;
• when the cpEGFP is connected to the GPCR specifically binding the dopamine, the sequence of the N-terminal linker before mutation of the third amino acid is LNSLI;
• when the cpEGFP is connected to the GPCR specifically binding the dopamine, the third amino acid of the N-terminal linker is mutated to leucine, alanine, or histidine, and the mutated sequence of the N-terminal linker is any one of LNLLI, LNALI, or LNHLI.