CN113501881B

CN113501881B - Fusion proteins

Info

Publication number: CN113501881B
Application number: CN202110661610.6A
Authority: CN
Inventors: 李毓龙; 井淼; 冯杰思; 万金霞; 王欢
Original assignee: Peking University
Current assignee: Peking University
Priority date: 2017-09-27
Filing date: 2017-09-27
Publication date: 2023-07-28
Anticipated expiration: 2037-09-27
Also published as: CN113501881A; CN109553687B; CN109553687A

Abstract

The invention utilizes the characteristic that a G Protein Coupled Receptor (GPCR) senses a specific ligand and generates conformational change, and inserts a circularly rearranged fluorescent protein into a third intracellular loop of the G protein coupled receptor, so that the conformational change of the G protein coupled receptor is converted into a change of an optical signal, and the concentration of the specific ligand is detected by detecting the change of the optical signal, thereby constructing an activated fluorescent probe (GRAB probe) based on the GPCR based on the principle. The invention also discloses a method for detecting the specific ligand by utilizing the GRAB probe.

Description

Fusion proteins

Technical Field

The invention relates to a fluorescent probe based on G protein coupled receptor construction and coded by genes.

Background

Because of the important role of neurotransmitters in the nervous system, many scientists have conducted extensive studies on neurotransmitter properties, synthesis, storage, release and action since the first neurotransmitter acetylcholine was identified to date for nearly 100 years (Valenstein, e.s. the discovery of chemical neuro-domains, brain and cognition 49,73-95 (2002)). However, compared to the now rapidly evolving field of cognitive neurobiology, the technology of detecting neurotransmitters is still limited by low spatial-temporal resolution and cell specificity, which makes our fine characterization of the release and action of transmitters difficult.

Coupled with biochemical assays by microdialysis methods is one of the classical methods of studying neurotransmitter release. The method was originally developed by Bito L in 1966 for detecting the content and dynamic changes of various amino acids in the brain (just, J. B. Quantitative micro analysis of neuro-relays. Journal of Neuroscience Methods, 263-276 (1993)). Understedt and Pycock as precursors in this field have improved and developed microdialysis techniques and applied this technique to detect the presence of a variety of important neurotransmitters such as dopamine in the cerebral nerve loop (Watson, C.J., venton, B.J. & Kennedy, R.T.In vivo measurements of neurotransmitters by microdialysis sample. Analytical chemistry 78,1391-1399 (2006)). Although this method can achieve the objective of detecting neurotransmitters, it is extremely lack of spatial information of transmitter release due to its need to obtain neurotransmitters through dialysis membranes and to isolate and identify specific molecules by biochemical methods, and it is difficult to guarantee complete manifestation of physiological states due to its complicated operation. The now developed microdialysis nano-LC-microdialysis allows for the careful separation and characterization of very small amounts of neurotransmission in tissue by further increasing the resolution of the biochemical detection process, with a minimum of 4nL of required sample volume, which is up to several seconds, however it is also a defect lacking cell-specific detection caused by poor spatial resolution (Olive, m.f., mehmert, K.K.& Hodge, c.w. Microdialysis in the mouse nucleus accumbens: A method for detection of monoamine and amino acid neurotransmitters with simultaneous assessment of locomotor activity.brain Research Protocols 5,16-24 (2000); lee, g.j., park, j.h. & Park, h.k.microdialysis applications in biological research 30,661-668 (2008)).

In addition to biochemical methods, electrochemical techniques developed by chemical redox methods to detect monoamine neurotransmitters such as dopamine, serotonin, etc. are currently one of the more widely used methods for detecting neurotransmitter release. The method can be coupled with electric signals, has better sensitivity and time resolution, so that the release and regulation mechanism of monoamine neurotransmitters such as dopamine, serotonin and the like are known

The detection of neurotransmitters by means of optical imaging has been rapidly developed in recent years due to its high sensitivity and real-time observation. The neural transmitter detection method CNiFERs based on detection cell line construction developed in David Kleinfeld laboratories of san Diego university, U.S.A., detects neurotransmitter release in a specific brain region by implanting modified human HEK293 cells into the brain region (Muller, A., joseph, V., slesinger, P.A., kleinfeld, D.cell-based reporters reveal in vivo dynamics of dopamine and norepinephrine release in murine Cortex.Nature methods 11,1245-1252 (2014); nguyen, Q.T.et al An in vivo biosensor for neurotransmitter release and in situ receptor activity. Nature neuroscience 13,127-132 (2010)). The cell line has a G protein coupled receptor corresponding to a particular neurotransmitter and coupled downstream of the receptor with a fluorescent calcium indicator, thereby converting neurotransmitter binding to the detection of intracellular calcium signals. The method has specific neurotransmitter detection, and plays an important role in detection of epinephrine, dopamine and acetylcholine. Meanwhile, the detection signal is not neurotransmitter binding per se, but is a secondary calcium signal which is subjected to a cascade method downstream, so that the method has higher sensitivity and time resolution of seconds. However, since the principle is to transplant an exogenous cell line into a specific location in the brain, its detection of the level of neurotransmitters by endogenous neural cells, subcellular axons, dendrites, and even individual synapses remains difficult. On the other hand, its complex procedures and possible immunological rejection also limit its widespread use in the field of neurobiology.

The removal of cell-based probes, neurotransmitter probes at the molecular level, has also evolved. Fluorescent probes ACh-snifilt for detecting acetylcholine based on the FRET principle were developed by Kai Johnsson laboratories, university of rocky, switzerland (Alberto Schena, et al Sensing Acetylcholine and anticholinesterase compounds, angewandte Chemie, vol 53, issue 5,1302-1305, dec 13,2013). Although small in size compared to CNiFERs, the construction of ACh-SNIFIT requires expression and collection of proteins and additional modification of chemical fluorophores, which are injected into the corresponding brain region for use, and is not suitable for in vivo, non-invasive detection of neurotransmitters.

The neurotransmitter probe which can be coded by genes and is constructed based on fluorescent protein can not only have cell-specific expression and detection, but also can achieve high signal-to-noise ratio detection by optimizing the fluorescent protein probe. The method realizes noninvasive and in-vivo detection of neurotransmitters, and records neurotransmitter release and actions thereof in brain cognitive nerve loops in real time. The us Janelia research center has been working on the development of a new generation of fluorescent probes, where the Loren log laboratory constructed in 2013 the fluorescent probe iGluSnFR for the important excitatory neurotransmitter glutamate (Marvin, j.s. et al an optimized fluorescent probe for visualizing glutamate neuregransmsision, nature methods 10,162-170 (2013)). The probe converts the conformational change of the binding protein into the change of the brightness of the fluorescent protein by fusing the fluorescent protein with the protein binding glutamic acid in the periplasmic space of the bacterium. The method not only achieves the concentration and release of the cell-specific detection glutamic acid through gene coding, but also enables the probe to have high sensitivity and rapid time resolution based on the principle of conformational change. After the glutamate receptor probe coded by the gene is optimized and purposely expressed on the postsynaptic membrane of a specific neuron, the postsynaptic glutamate release of the neuron can be accurately recorded, neurotransmitter effect is detected at the synaptic level through an imaging method, and important information is provided for the connection between the neurons and the establishment of a neural network. While this approach provides a precedent for the detection of neurotransmitters using genetically encoded fluorescent probes, the use of the metabotropic glutamate binding proteins in bacteria by iGluSnFR makes the probe construction approach difficult to apply simply to the detection of other neurotransmitters, requiring the re-search or design of their binding proteins for different neurotransmitters, while for some key higher biological specific macromolecular neurotransmitters such as peptide neurotransmitters it is difficult to find the corresponding binding proteins in prokaryotes; furthermore, the effective localization of different small molecule binding proteins to the cell membrane for detecting neurotransmitter dynamics may require the help of different localization sequence optimizations or helper proteins, which limit the applicability of this approach. In summary, due to the complexity of the nervous system and dynamic diversity of neurotransmitters, there is now a lack of effective means to detect the dynamic changes of neurotransmitters with space-time specificity, which greatly restricts the development of neurobiology.

Disclosure of Invention

The invention utilizes the characteristic that a G Protein Coupled Receptor (GPCR) senses a specific ligand and generates conformational change, utilizes a circulating rearranged fluorescent protein coupled with the G protein coupled receptor to convert the conformational change of the G protein coupled receptor into a change of an optical signal, detects the concentration of the specific ligand by detecting the change of the optical signal, constructs an activated fluorescent probe (GRAB probe) based on GPCR based on the principle,GPCR Activation Ban ased Sensor, which may also be referred to as a fluorescent probe constructed based on a G protein-coupled receptor) and a method for detecting the specific ligand using a GRAB probe.

Since the receptors of most classical neurotransmitters are ligand-mediated ion channels or G-protein coupled receptors (GPCRs). Thus, the GRAB probes and detection methods of the invention are particularly useful for detecting neurotransmitters by preparing specific G-protein coupled receptors that bind to neurotransmitters, and coupling them directly to the output of a light signal via conformational changes that are activated by the ligand, thereby reflecting the dynamic changes in neurotransmitter concentration.

One aspect of the present invention relates to a fluorescent probe (GRAB probe) constructed based on a G-protein coupled receptor, which is a fusion protein obtained by engineering the G-protein coupled receptor, the engineering comprising inserting a circularly rearranged fluorescent protein in a third intracellular loop between a fifth transmembrane region and a sixth transmembrane region of the G-protein coupled receptor.

The fluorescent probe (i.e., GRAB probe) constructed based on the G protein-coupled receptor can be expressed on a cell membrane. The fluorescent probe constructed based on the G protein-coupled receptor may bind to the specific ligand of the G protein-coupled receptor when contacted therewith, thereby resulting in a detectable change in the fluorescent intensity of the fluorescent probe. The fluorescent probe constructed based on the G protein-coupled receptor can be used to qualitatively detect the binding of the specific ligand of the G protein-coupled receptor or the change of the concentration thereof, or quantitatively analyze the concentration of the specific ligand of the G protein-coupled receptor.

In some embodiments, the engineering comprises truncating the third intracellular loop between the fifth and sixth transmembrane regions of the G protein-coupled receptor and inserting the circularly rearranged fluorescent protein at the truncated position.

In some embodiments, the circularly rearranged fluorescent protein is linked at both ends to a third intracellular loop of a G protein-coupled receptor by a linker peptide, respectively.

In some embodiments, the connecting peptide comprises a flexible amino acid. Preferably, the flexible amino acids include glycine and/or alanine. More preferably, the connecting peptide consists of glycine and alanine.

In some embodiments, the connecting peptide at the N-terminus of the cyclically rearranged fluorescent protein is GG and/or the connecting peptide at the C-terminus of the cyclically rearranged fluorescent protein is GGAAA.

In some embodiments, the cyclically rearranged fluorescent protein is selected from the group consisting of cyclically rearranged green fluorescent protein (cpGFP), cyclically rearranged yellow fluorescent protein (cpYFP), cyclically rearranged red fluorescent protein (cpRFP), cyclically rearranged blue fluorescent protein (cpBFP), cyclically rearranged enhanced green fluorescent protein (cpGFP), and cyclically rearranged enhanced yellow fluorescent protein (cpEYFP).

In some embodiments, the cyclically rearranged fluorescent protein is a cyclically rearranged enhanced green fluorescent protein (cpEGFP). The circularly rearranged enhanced green fluorescent protein is preferably cpEGFP from GCaMP6s, GCaMP6m or G-GECO.

In some embodiments, the circularly rearranged fluorescent protein is a circularly rearranged red fluorescent protein (cpRFP). Circularly rearranged red fluorescent proteins include, but are not limited to cpmApple, cpmCherry, cpmRuby, cpmKate2 and cpfshinonRed, preferably cpmApple. The cpmeAN is preferably a cpmeAN from R-GECO 1.

In some embodiments, the cyclically rearranged fluorescent protein is a cyclically rearranged yellow fluorescent protein (cpYFP). The cyclically rearranged yellow fluorescent protein includes, but is not limited to, cyclically rearranged Venus (cpVenus), cyclically rearranged Citrin (cpCitrine).

In some embodiments, the cyclically rearranged fluorescent protein is a cyclically rearranged infrared fluorescent protein (cp infrared fluorescent protein, cpiRFP).

In some embodiments, the fluorescent probes of the invention may be constructed with a cyclically rearranged luciferase (cp luciferase) in place of the fluorescent protein.

In some embodiments, the G protein-coupled receptor is a G protein-coupled receptor that specifically binds to a specific ligand.

In some embodiments, the specific ligand is a neurotransmitter, including, but not limited to, epinephrine, norepinephrine, acetylcholine, serotonin and/or dopamine. The G-protein coupled receptor is a G-protein coupled receptor that specifically binds neurotransmitters, such as, but not limited to, G-protein coupled receptors that specifically bind epinephrine, norepinephrine, acetylcholine, serotonin, and/or dopamine.

In other embodiments, the specific ligand is a hormone, a metabolic molecule, or a nutritional molecule and the G protein-coupled receptor is a G protein-coupled receptor that specifically binds to a hormone, a metabolic molecule, or a nutritional molecule.

In some embodiments, the specific ligand is a synthetic small molecule or drug that activates a specific receptor, and the G protein-coupled receptor is a G protein-coupled receptor that specifically binds to the synthetic small molecule or drug that activates a specific receptor. Such artificially synthesized small molecules or drugs that activate specific receptors include, but are not limited to, isoprenaline (ISO).

In some embodiments, the G protein-coupled receptor is of human origin.

In some embodiments, the G protein-coupled receptor is of animal origin.

In some embodiments, the fluorescent probe constructed based on a G protein-coupled receptor is a fluorescent probe for detecting epinephrine, wherein the G protein-coupled receptor is a GPCR that specifically binds epinephrine.

In some embodiments, the GPCR that specifically binds epinephrine is a human β2 adrenergic receptor and the fluorescent probe constructed based on a G protein-coupled receptor is a fluorescent probe constructed based on a human β2 adrenergic receptor.

In some embodiments, in a fluorescent probe constructed based on the human β2 adrenergic receptor, the circularly rearranged fluorescent protein is linked to the third intracellular loop of the human β2 adrenergic receptor by a linking peptide at the N-terminus and the C-terminus. In some preferred embodiments, the linking peptides at both ends of the cyclically rearranged fluorescent protein are 1 or 2 amino acids in length and/or 1, 2, 3, 4 or 5 amino acids in carbon-terminal, respectively. In some more preferred embodiments, the linking peptides at both ends of the cyclically rearranged fluorescent protein are each 2 amino acids in length at the nitrogen end and 5 amino acids at the carbon end. In other preferred embodiments, the linking peptides at both ends of the cyclically rearranged fluorescent protein are each 1 amino acid in length at the nitrogen end and 1 amino acid at the carbon end. In some preferred embodiments, the connecting peptides at both ends of the cyclically rearranged fluorescent protein are each GG at the N-terminus and GGAAA at the C-terminus. In other preferred embodiments, the connecting peptides at both ends of the cyclically rearranged fluorescent protein are each GG at the N-terminus and SPSVA at the C-terminus. In other preferred embodiments, the linking peptides at both ends of the cyclically rearranged fluorescent protein are each GG at the N-terminus and APSVA at the C-terminus. In other preferred embodiments, the linking peptides at both ends of the cyclically rearranged fluorescent protein are each G at the N-terminus and G at the C-terminus.

In some embodiments, the circularly rearranged fluorescent protein inserted into the human β2 adrenergic receptor is cpEGFP. In some embodiments, the cpgfp is cpgfp from GCaMP6 s. In other embodiments, the cpgfp is cpgfp from GCaMP6m or GECO 1.2.

In a preferred embodiment, the amino acid sequence of the human β2 adrenergic receptor is:

MGQPGNGSAFLLAPNRSHAPDHDVTQQRDEVWVVGMGIVMSLIVLAIVFGNVLVITAIAKFER LQTVTNYFITSLACADLVMGLAVVPFGAAHILMKMWTFGNFWCEFWTSIDVLCVTASIETLCVIAV DRYFAITSPFKYQSLLTKNKARVIILMVWIVSGLTSFLPIQMHWYRATHQEAINCYANETCCDFFTN QAYAIASSIVSFYVPLVIMVFVYSRVFQEAKRQLQKIDKSEGRFHVQNLSQVEQDGRTGHGLRRSSKFCLKEHKALKTLGIIMGTFTLCWLPFFIVNIVHVIQDNLIRKEVYILLNWIGYVNSGFNPLIYCRSPDF RIAFQELLCLRRSSLKAYGNGYSSNGNTGEQSGYHVEQEKENKLLCEDLPGTEDFVGHQGTVPSDNI DSQGRNCSTNDSLL(SEQ ID NO:1)；

wherein the underlined section is the third intracellular loop.

In some embodiments, the circularly rearranged fluorescent protein is inserted between amino acid 240 and amino acid 241 of the human β2 adrenergic receptor. In some embodiments, the circularly rearranged fluorescent protein is inserted between amino acid 250 and amino acid 251 of the human β2 adrenergic receptor.

In some embodiments, the fluorescent probe constructed based on a G protein-coupled receptor is a fluorescent probe for detecting epinephrine and/or norepinephrine, wherein the G protein-coupled receptor is a GPCR that specifically binds epinephrine and/or norepinephrine.

In some embodiments, the GPCR that specifically binds epinephrine and/or norepinephrine is a human ADRA2A receptor and the fluorescent probe constructed based on G-protein coupled receptors is a fluorescent probe constructed based on human ADRA2A receptor.

In some embodiments, in the fluorescent probe constructed based on the human ADRA2A receptor, the third intracellular loop of the human ADRA2A receptor is truncated and a circularly rearranged fluorescent protein is inserted at the truncated position.

In some preferred embodiments, in fluorescent probes constructed based on the human ADRA2A receptor, the circularly rearranged fluorescent protein is linked to the third intracellular loop of the human ADRA2A receptor via a linking peptide at the N-and C-terminus. In some preferred embodiments, the linking peptides at both ends of the cyclically rearranged fluorescent protein are each 2 amino acids in length at the nitrogen end and 5 amino acids at the carbon end. In some preferred embodiments, the connecting peptides at both ends of the cyclically rearranged fluorescent protein are each GG at the N-terminus and GGAAA at the C-terminus. In some preferred embodiments, the linking peptides at both ends of the cyclic rearranged fluorescent protein are each GG at the N-terminus and TGAAA at the C-terminus.

In some embodiments, the circularly rearranged fluorescent protein inserted into the human ADRA2A receptor is cpEGFP. In some embodiments, the cpgfp is cpgfp from GCaMP6 s. In other embodiments, the cpgfp is cpgfp from GCaMP6m or GECO 1.2.

In some preferred embodiments, the amino acid sequence of the human ADRA2A receptor is:

MFRQEQPLAEGSFAPMGSLQPDAGNASWNGTEAPGGGARATPYSLQVTLTLVCLAGLLMLLT VFGNVLVIIAVFTSRALKAPQNLFLVSLASADILVATLVIPFSLANEVMGYWYFGKAWCEIYLALDV LFCTSSIVHLCAISLDRYWSITQAIEYNLKRTPRRIKAIIITVWVISAVISFPPLISIEKKGGGGGPQPAEP RCEINDQKWYVISSCIGSFFAPCLIMILVYVRIYQIAKRRTRVPPSRRGPDAVAAPPGGTERRPNGLGPERSAGPGGAEAEPLPTQLNGAPG EPAPAGPRDTDALDLEESSSSDHAERPPGPRRPERGPRGKGKARASQVKPGDSLPRRGPGATGIGTPAAGPGEERV GAAKASRWRGRQNREKRFTFVLAVVIGVFVVCW FPFFFTYTLTAVGCSVPRTLFKFFFWFGYCNSSLNPVIYTIFNHDFRRAFKKILCRGDRKRIV(SEQ ID NO:2)；

Wherein the underlined section is the third intracellular loop.

In some preferred embodiments, amino acids 79-138 of the third intracellular loop of the human ADRA2A receptor described above are truncated and a circularly rearranged fluorescent protein is inserted at the truncated position. In other preferred embodiments, amino acids 79-143 of the third intracellular loop of the human ADRA2A receptor described above are truncated and a circularly rearranged fluorescent protein is inserted at the truncated position.

In some embodiments, the fluorescent probe constructed based on a G protein-coupled receptor is a fluorescent probe for detecting acetylcholine, wherein the G protein-coupled receptor is a GPCR that specifically binds to acetylcholine.

In some embodiments, the GPCR that specifically binds epinephrine is a human acetylcholine receptor M3R subtype and the fluorescent probe constructed based on G protein-coupled receptors is a fluorescent probe constructed based on the human acetylcholine receptor M3R subtype.

In some embodiments, in the fluorescent probe constructed based on the human acetylcholine receptor M3R subtype, the third intracellular loop of the human acetylcholine receptor M3R subtype is truncated and a circularly rearranged fluorescent protein is inserted at the truncated position.

In some embodiments, in a fluorescent probe constructed based on the human acetylcholine receptor M3R subtype, the circularly rearranged fluorescent protein is linked to the third intracellular loop of the human acetylcholine receptor M3R subtype by an N-terminal and C-terminal linking peptide. In some embodiments, the connecting peptides at both ends of the cyclically rearranged fluorescent protein are 2 amino acids in length and 5 amino acids in carbon end, respectively. In some preferred embodiments, the connecting peptides at both ends of the cyclically rearranged fluorescent protein are each GG at the N-terminus and GGAAA at the C-terminus. In other preferred embodiments, the linking peptides at both ends of the cyclically rearranged fluorescent protein are each GG at the N-terminus and HGAAA at the C-terminus. In other preferred embodiments, the linking peptides at both ends of the cyclically rearranged fluorescent protein are each GG at the N-terminus and HNAAA at the C-terminus. In other preferred embodiments, the linking peptides at both ends of the cyclically rearranged fluorescent protein are each GG at the N-terminus and HNAK at the C-terminus.

In some embodiments, the circularly rearranged fluorescent protein inserted into the human acetylcholine receptor M3R subtype is cpEGFP. In some embodiments, the cpgfp is cpgfp from GCaMP6 s. In other embodiments, the cpgfp is cpgfp from GCaMP6m or GECO 1.2.

In a preferred embodiment, the amino acid sequence of the human acetylcholine receptor M3R subtype is:

MTLHNNSTTSPLFPNISSSWIHSPSDAGLPPGTVTHFGSYNVSRAAGNFSSPDGTTDDPLGGHT VWQVVFIAFLTGILALVTIIGNILVIVSFKVNKQLKTVNNYFLLSLACADLIIGVISMNLFTTYIIMNR WALGNLACDLWLAIDYVASNASVMNLLVISFDRYFSITRPLTYRAKRTTKRAGVMIGLAWVISFVL WAPAILFWQYFVGKRTVPPGECFIQFLSEPTITFGTAIAAFYMPVTIMTILYWRIYKETEKRTKELAGLQASGTEAETENFVHPTGSSRSCSSY ELQQQSMKRSNRRKYGRCHFWFTTKSWKPSSEQMDQDHSSSDSWNNNDAAASLENSASSDEEDIGSETRAIYSIVL KLPGHSTILNSTKLPSSDNLQVPEEELGMVDLERKADKLQAQKSVDDGGSFPKSFSKLPIQLESAVDTAKTSDVNS SVGKSTATLPLSFKEATLAKRFALKTRSQITKRKRMSLVKEKKAAQTLSAILLAFIITWTPYNIMVLVNTFCDSCIPKTFWNLGYWLCYIN STVNPVCYALCNKTFRTTFKMLLLCQCDKKKRRKQQYQQRQSVIFHKRAPEQAL(SEQ ID NO:3)；

wherein the underlined part is the third intracellular loop (ICL 3), and the ICL3 is amino acids 253-491.

In some embodiments, amino acids 260-490 of the above human acetylcholine receptor M3R subtype are truncated and a circularly rearranged fluorescent protein is inserted at the truncated position. In some embodiments, amino acids 260-491 of the above human acetylcholine receptor M3R subtype are truncated and a circularly rearranged fluorescent protein is inserted at the truncated position.

In some embodiments, the fluorescent probe constructed based on G protein-coupled receptor is a fluorescent probe for detecting 5-hydroxytryptamine, wherein the G protein-coupled receptor is a GPCR that specifically binds 5-hydroxytryptamine and the fluorescent probe constructed based on G protein-coupled receptor is a fluorescent probe constructed based on a GPCR that specifically binds 5-hydroxytryptamine.

In some embodiments, the GPCR that specifically binds 5-hydroxytryptamine is a human HTR2C receptor and the fluorescent probe constructed based on a G protein coupled receptor is a fluorescent probe constructed based on a human HTR2C receptor.

In some embodiments, in the fluorescent probe constructed based on the human HTR2C receptor, the third intracellular loop of the human HTR2C receptor is truncated and a circularly rearranged fluorescent protein is inserted at the truncated position.

In some preferred embodiments, in a fluorescent probe constructed based on the human HTR2C receptor, the cyclically rearranged fluorescent protein is linked to the third intracellular loop of the human HTR2C receptor via a linking peptide at the N-and C-termini. In some preferred embodiments, the connecting peptides at both ends of the cyclically rearranged fluorescent protein are each 2 amino acids in length at the nitrogen end and 5 amino acids at the carbon end. In some preferred embodiments, the connecting peptides at both ends of the cyclically rearranged fluorescent protein are each GG at the N-terminus and GGAAA at the C-terminus. In some preferred embodiments, the connecting peptides at both ends of the circulating rearranged fluorescent protein are each NG at the N-terminus and GFAAA at the C-terminus.

In some embodiments, the circularly rearranged fluorescent protein inserted into the human HTR2C receptor is cpgfp. In some embodiments, the cpgfp is cpgfp from GCaMP6 s. In other embodiments, the cpgfp is cpgfp from GCaMP6m or GECO 1.2.

In some preferred embodiments, the amino acid sequence of the human HTR2C receptor is:

MVNLRNAVHSFLVHLIGLLVWQCDISVSPVAAIVTDIFNTSDGGRFKFPDGVQNWPALSIVIIII MTIGGNILVIMAVSMEKKLHNATNYFLMSLAIADMLVGLLVMPLSLLAILYDYVWPLPRYLCPVWI SLDVLFSTASIMHLCAISLDRYVAIRNPIEHSRFNSRTKAIMKIAIVWAISIGVSVPIPVIGLRDEEKVFV NNTTCVLNDPNFVLIGSFVAFFIPLTIMVITYCLTIYVLRRQALMLLHGHTEEPPGLSLDFLKCCKRNTAEEENSANPNQDQNARRRKKKE RRPRGTMQAINNERKASKVLGIVFFVFLIMWCPFFITNILSVLCE KSCNQKLMEKLLNVFVWIGYVCSGINPLVYTLFNKIYRRAFSNYLRCNYKVEKKPPVRQIPRVAAT ALSGRELNVNIYRHTNEPVIEKASDNEPGIEMQVENLELPVNPSSVVSERISSV(SEQ ID NO:4)；

wherein the underlined section is the third intracellular loop.

In some preferred embodiments, amino acids 16-55 of the third intracellular loop of the human HTR2C receptor are truncated and a circularly rearranged fluorescent protein is inserted at the truncated position. In some preferred embodiments, amino acids 11-60 of the third intracellular loop of the human HTR2C receptor are truncated and a circularly rearranged fluorescent protein is inserted at the truncated position. In some preferred embodiments, amino acids 16-70 of the third intracellular loop of the human HTR2C receptor are truncated and a circularly rearranged fluorescent protein is inserted at the truncated position. In other preferred embodiments, amino acids 15-68 of the third intracellular loop of the human HTR2C receptor are truncated and a circularly rearranged fluorescent protein is inserted at the truncated position.

In some more preferred embodiments, amino acids 15-68 of the third intracellular loop of the human HTR2C receptor described above are truncated and a circularly rearranged fluorescent protein is inserted at the truncated position, and leucine L at position 13 of the third intracellular loop is mutated to phenylalanine F.

In some embodiments, the fluorescent probe constructed based on a G protein-coupled receptor is a fluorescent probe for detecting dopamine, wherein the G protein-coupled receptor is a GPCR that specifically binds dopamine.

In some embodiments, the GPCR that specifically binds dopamine is a human DRD2 receptor and the fluorescent probe constructed based on a G protein coupled receptor is a fluorescent probe constructed based on a human DRD2 receptor.

In some embodiments, in the fluorescent probe constructed based on the human DRD2 receptor, the third intracellular loop of the human DRD2 receptor is truncated and a circularly rearranged fluorescent protein is inserted at the truncated position.

In some preferred embodiments, in a fluorescent probe constructed based on the human DRD2 receptor, the circularly rearranged fluorescent protein is linked to the third intracellular loop of the human DRD2 receptor via a linking peptide at the N-terminus and the C-terminus. In some preferred embodiments, the linking peptides at both ends of the cyclically rearranged fluorescent protein are each 2 amino acids in length at the nitrogen end and 5 amino acids in length at the carbon end. In some preferred embodiments, the connecting peptides at both ends of the circulating rearranged fluorescent protein are each GG at the N-terminus and GGAAA at the C-terminus.

In some embodiments, the circularly rearranged fluorescent protein inserted into the human DRD2 receptor is cpgfp. In some embodiments, the cpgfp is cpgfp from GCaMP6 s. In other embodiments, the cpgfp is cpgfp from GCaMP6m or GECO 1.2.

In some preferred embodiments, the amino acid sequence of the human DRD2 receptor is:

MDPLNLSWYDDDLERQNWSRPFNGSDGKADRPHYNYYATLLTLLIAVIVFGNVLVCMAVSRE KALQTTTNYLIVSLAVADLLVATLVMPWVVYLEVVGEWKFSRIHCDIFVTLDVMMCTASILNLCAI SIDRYTAVAMPMLYNTRYSSKRRVTVMISIVWVLSFTISCPLLFGLNNADQNECIIANPAFVVYSSIV SFYVPFIVTLLVYIKIYIVLR RRRKRVNTKRSSRAFRAHLRAPLKGNCTHPEDMKLCTVIMKSNGSFPVNRRRVEAARRAQELEMEMLSSTSPPERT RYSPIPPSHHQLTLPDPSHHGLHSTPDSPAKPEKNGHAKDHPKIAKIFEIQTMPNGKTRTSLKTMSRRKLSQQKEK KATQMLAIVLGVFIICWLPFFITHILNIHCD CNIPPVLYSAFTWLGYVNSAVNPIIYTTFNIEFRKAFLKILHC(SEQ ID NO:5)；

wherein the underlined section is the third intracellular loop.

In some preferred embodiments, amino acids 253-357 of the human DRD2 receptor are truncated and a circularly rearranged fluorescent protein is inserted at the truncated position. In some preferred embodiments, amino acids 254-360 of the human DRD2 receptor are truncated and a circularly rearranged fluorescent protein is inserted at the truncated position.

In some preferred embodiments, in a fluorescent probe constructed based on the human DRD2 receptor, the circularly rearranged fluorescent protein is linked to the third intracellular loop of the human DRD2 receptor via a linking peptide at the N-terminus and the C-terminus. In some preferred embodiments, the linking peptides at both ends of the cyclically rearranged fluorescent protein are 5 amino acids in length and 3 amino acids in carbon end, respectively. In some preferred embodiments, the connecting peptides at both ends of the circulating rearranged fluorescent protein are each PVVSE at the N-terminus and ATR at the C-terminus.

In some embodiments, the circularly rearranged fluorescent protein inserted into the human DRD2 receptor is cpmApple, and in some embodiments, the cpmApple is a cpmApple from R-GECO 1.

wherein the underlined section is the third intracellular loop.

In some preferred embodiments, amino acids 223-349 of the human DRD2 receptor are truncated and a circularly rearranged fluorescent protein is inserted at the truncated position. In some preferred embodiments, amino acids 268-364 of the human DRD2 receptor are truncated and a circularly rearranged fluorescent protein is inserted at the truncated position. In some preferred embodiments, amino acids 224-365 of the human DRD2 receptor are truncated and a circularly rearranged fluorescent protein is inserted at the truncated position.

In some preferred embodiments, in a fluorescent probe constructed based on the human HTR2C receptor, the cyclically rearranged fluorescent protein is linked to the third intracellular loop of the human HTR2C receptor via a linking peptide at the N-and C-termini. In some preferred embodiments, the connecting peptides at both ends of the cyclically rearranged fluorescent protein are 5 amino acids in length and 3 amino acids in carbon end, respectively. In some preferred embodiments, the connecting peptides at both ends of the cyclically rearranged fluorescent protein are each PVVSE at the N-terminus and ATR at the C-terminus.

In some embodiments, the circularly rearranged fluorescent protein inserted into the human HTR2C receptor is cpmAppe, and in some embodiments, the cpmAppe is cpmAppe from R-GECO 1.

MDPLNLSWYDDDLERQNWSRPFNGSDGKADRPHYNYYATLLTLLIAVIVFGNVLVCMAVSRE KALQTTTNYLIVSLAVADLLVATLVMPWVVYLEVVGEWKFSRIHCDIFVTLDVMMCTASILNLCAI SIDRYTAVAMPMLYNTRYSSKRRVTVMISIVWVLSFTISCPLLFGLNNADQNECIIANPAFVVYSSIV SFYVPFIVTLLVYIKIYIVLR RRRKRVNTKRSSRAFRAHLRAPLKGNCTHPEDMKLCTVIMKSNGSFPVNRRRVEAARRAQELEMEMLSSTSPPERT RYSPIPPSHHQLTLPDPSHHGLHSTPDSPAKPEKNGHAKDHPKIAKIFEIQTMPNGKTRTSLKTMSRRKLSQQKEK KATQMLAIVLGVFIICWLPFFITHILNIHCD CNIPPVLYSAFTWLGYVNSAVNPIIYTTFNIEFRKAFLKILHC(SEQ ID NO:4)；

wherein the underlined section is the third intracellular loop.

In some preferred embodiments, amino acids 241-306 of the human HTR2C receptor are truncated and a circularly rearranged fluorescent protein is inserted at the truncated position. In some preferred embodiments, amino acids 240-309 of the human HTR2C receptor are truncated and a circularly rearranged fluorescent protein is inserted at the truncated position.

In some embodiments, in any of the above-described fluorescent probes constructed based on G protein-coupled receptors, the engineering further comprises ligating a gα protein peptide at the C-terminus of the G protein-coupled receptor. The G.alpha.protein peptide may preferably be linked after the last amino acid at the C-terminus of the G protein-coupled receptor. The G alpha protein peptide segment can be 20 amino acids of the carbon end of any G protein. In some preferred embodiments, the specific sequence of the gα protein peptide fragment is: VFAAVKDTILQLNLKEYNLV (Gαq20, SEQ ID NO: 6). In another preferred embodiment, the specific sequence of the gα protein peptide fragment is: VFNDCRDIIQRMHLRQYELL (Gαs20, SEQ ID NO: 7). In other preferred embodiments, the specific sequences of the gα protein peptide fragment are: VFDAVTDVIIKNNLKDCGLF (Gαi20, SEQ ID NO: 8).

In a preferred embodiment, in any of the fluorescent probes previously described constructed based on the human acetylcholine receptor M3R subtype, the ga protein peptide fragment is linked at the C-terminus of the human acetylcholine receptor M3R subtype. The G.alpha.protein peptide may preferably be linked after the last amino acid at the C-terminus of the M3R subtype of the human acetylcholine receptor. The G alpha protein peptide segment can be 20 amino acids of the carbon end of any G protein. In a preferred embodiment, the specific sequence of the gα protein peptide fragment is: VFAAVKDTILQLNLKEYNLV (Gαq20, SEQ ID NO: 6). In other preferred embodiments, the specific sequences of the gα protein peptide fragment are: VFNDCRDIIQRMHLRQYELL (Gαs20, SEQ ID NO: 7). In other preferred embodiments, the specific sequences of the gα protein peptide fragment are: VFDAVTDVIIKNNLKDCGLF (Gαi20, SEQ ID NO: 8).

In some embodiments, in any of the above-described fluorescent probes constructed based on G-protein coupled receptors, the engineering further comprises inserting a luciferase at the C-terminus of the G-protein coupled receptor, such that light emitted by the luciferase-catalyzed chemical reaction is capable of exciting a cyclically rearranged fluorescent protein in the fluorescent probe.

In some embodiments, the peak of light emitted by the luciferase-catalyzed chemical reaction is close to the wavelength of excitation light of the circulating rearranged fluorescent protein contained in the probe.

In some embodiments, the luciferase is Nanoluc.

In other embodiments, the luciferase is Fluc (firefly luciferase ) or Rluc (renilla luciferase, renilla luciferase).

In some embodiments, in any of the foregoing fluorescent probes constructed based on human HTR2C receptors, the luciferase is inserted into the C-terminus of the fluorescent probe, the luciferase is linked to the C-terminus of the fluorescent probe by its N-and C-terminal linker peptides, and the linker peptides of both the N-and C-termini of the luciferase are GSG.

In some embodiments, the luciferase is inserted between amino acids 582 and 583 of the fluorescent probe GRAB-5-HT2.0 and is linked at both ends to the fluorescent probe GRAB-5-HT2.0 by a linker peptide, wherein the linker peptide at both the N-and C-termini of the luciferase is GSG; wherein the fluorescent probe GRAB-5-HT2.0 is a fluorescent probe obtained by cleaving the 15 th to 68 th positions of the third intracellular loop of the human HTR2C receptor and inserting a cpEGFP (preferably a cpEGFP from GCaMP6 s) into the cleaved position, wherein the N-terminus of the cpEGFP is linked to the human HTR2C receptor through an N-terminal connecting peptide NG and the C-terminus is linked to the human HTR2C receptor through a C-terminal connecting peptide GFAAA. The amino acid sequence of the human HTR2C receptor is:

Wherein the underlined section is the third intracellular loop.

Another aspect of the present invention relates to a method for constructing a GRAB fluorescent probe, comprising completely intercepting a third intracellular loop of a fluorescent probe constructed based on a first G protein-coupled receptor together with a circularly rearranged fluorescent protein inserted therein, replacing the third intracellular loop of a second G protein-coupled receptor, and obtaining a fluorescent probe constructed based on a second G protein-coupled receptor, namely the GRAB fluorescent probe.

In the present invention, in this method of constructing a GRAB probe, the GRAB fluorescent probe finally obtained can be regarded as a fluorescent probe constructed based on a second G protein-coupled receptor.

In some embodiments, the first G-protein coupled receptor and the second G-protein coupled receptor bind the same specific ligand or bind different specific ligands.

The fluorescent probe constructed based on the first G protein-coupled receptor may be any one of the GRAB probes described above.

The specific ligand of the second G-protein coupled receptor may be a neurotransmitter, hormone, metabolic molecule, nutritional molecule or an artificially synthesized small molecule or drug that activates a specific receptor, and the second G-protein coupled receptor may be a G-protein coupled receptor that binds to the neurotransmitter, hormone, metabolic molecule, nutritional molecule or the artificially synthesized small molecule or drug that activates a specific receptor.

The neurotransmitters include, but are not limited to, epinephrine, norepinephrine, acetylcholine, serotonin and/or dopamine.

Such synthetic small molecules or drugs that activate specific receptors include, but are not limited to, isoprenaline (ISO).

The second G protein-coupled receptor may be of human or animal origin.

The GRAB fluorescent probe constructed as described above can be expressed on a cell membrane and can be bound to the specific ligand of the second G protein-coupled receptor when contacted therewith, thereby resulting in a detectable change in the fluorescence intensity of the fluorescent probe. The GRAB fluorescent probe constructed by the above method can be used to qualitatively detect the binding of the specific ligand of the second G protein-coupled receptor or a change in the concentration thereof, or quantitatively analyze the concentration of the specific ligand of the second G protein-coupled receptor.

In a preferred embodiment, the first G protein-coupled receptor is a human β2 adrenergic receptor, the amino acid sequence of which is:

MGQPGNGSAFLLAPNRSHAPDHDVTQQRDEVWVVGMGIVMSLIVLAIVFGNVLVITAIAKFER LQTVTNYFITSLACADLVMGLAVVPFGAAHILMKMWTFGNFWCEFWTSIDVLCVTASIETLCVIAV DRYFAITSPFKYQSLLTKNKARVIILMVWIVSGLTSFLPIQMHWYRATHQEAINCYANETCCDFFTN QAYAIASSIVSFYVPLVIMVFVYSRVFQEAKRQLQKIDKSEGRFHVQNLSQVEQDGRTGHGLRRSSKFCLKEHKALKTLGIIMGTFTLCWLPFFIVNIVHVIQDNLIRKEVYILLNWIGYVNSGFNPLIYCRSPDF RIAFQELLCLRRSSLKAYGNGYSSNGNTGEQSGYHVEQEKENKLLCEDLPGTEDFVGHQGTVPSDNI DSQGRNCSTNDSLL(SEQ ID NO:1)，

wherein the underlined section is the third intracellular loop.

In some embodiments, the circularly rearranged fluorescent protein is linked to a third intracellular loop of a human β2 adrenergic receptor by a linking peptide at the N-terminus and the C-terminus, wherein the linking peptides at both ends of the circularly rearranged fluorescent protein are each GG at the N-terminus and GGAAA at the C-terminus; alternatively, the connecting peptides at two ends of the circularly rearranged fluorescent protein are GG at the N end and SPSVA at the C end respectively; alternatively, the connecting peptides at two ends of the fluorescent protein rearranged circularly are GG at the N end and APSVA at the C end respectively.

In a more preferred embodiment, the second G protein-coupled receptor is the human acetylcholine receptor M3R subtype. In some embodiments, the specific sequences are:

MTLHNNSTTSPLFPNISSSWIHSPSDAGLPPGTVTHFGSYNVSRAAGNFSSPDGTTDDPLGGHTV WQVVFIAFLTGILALVTIIGNILVIVSFKVNKQLKTVNNYFLLSLACADLIIGVISMNLFTTYIIMNRW ALGNLACDLWLAIDYVASNASVMNLLVISFDRYFSITRPLTYRAKRTTKRAGVMIGLAWVISFVLW APAILFWQYFVGKRTVPPGECFIQFLSEPTITFGTAIAAFYMPVTIMTILYWRIYKETEKRTKELAGLQASGTEAETENFVHPTGSSRSCSSY ELQQQSMKRSNRRKYGRCHFWFTTKSWKPSSEQMDQDHSSSDSWNNNDAAASLENSASSDEEDIGSETRAIYSIVL KLPGHSTILNSTKLPSSDNLQVPEEELGMVDLERKADKLQAQKSVDDGGSFPKSFSKLPIQLESAVDTAKTSDVNS SVGKSTATLPLSFKEATLAKRFALKTRSQITKRKRMSLVKEKKAAQTLSAILLAFIITWTPYNIMVLVNTFCDSCIPKTFWNLGYWLCYINST VNPVCYALCNKTFRTTFKMLLL CQCDKKKRRKQQYQQRQSVIFHKRAPEQAL(SEQ ID NO:3)；

wherein the sequence of the underlined section is the third intracellular loop thereof and is replaced.

In other preferred embodiments, the first G protein-coupled receptor is a human HTR2C receptor, which human HTR2C receptor has the amino acid sequence:

wherein the underlined section is the third intracellular loop.

In some preferred embodiments, in a fluorescent probe constructed based on the human HTR2C receptor, the cyclically rearranged fluorescent protein is linked to the third intracellular loop of the human HTR2C receptor by a linking peptide at the N-terminus and the C-terminus, wherein the linking peptide at both ends of the cyclically rearranged fluorescent protein is GG at the N-terminus and GGAAA at the C-terminus, respectively; alternatively, the connecting peptides at both ends of the fluorescent protein rearranged circularly are NG at N-terminal and GFAAA at C-terminal.

In a more preferred embodiment, the second G protein-coupled receptor is a human HTR2B receptor or a human HTR6 receptor.

In some embodiments, the amino acid sequence of the human HTR2B receptor is:

MALSYRVSELQSTIPEHILQSTFVHVISSNWSGLQTESIPEEMKQIVEEQGNKLHWAALLILMVII PTIGGNTLVILAVSLEKKLQYATNYFLMSLAVADLLVGLFVMPIALLTIMFEAMWPLPLVLCPAWLF LDVLFSTASIMHLCAISVDRYIAIKKPIQANQYNSRATAFIKITVVWLISIGIAIPVPIKGIETDVDNPNN ITCVLTKERFGDFMLFGSLAAFFTPLAIMIVTYFLTIHALQKKAYLVKNKPPQRLTWLTVSTVFQRDETPCSSPEKVAMLDGSRKDKAL PNSGDETLMRRTSTIGKKSVQTISNEQRASKVLGIVFFLFLLMWCP FFITNITLVLCDSCNQTTLQMLLEIFVWIGYVSSGVNPLVYTLFNKTFRDAFGRYITCNYRATKSVKT LRKRSSKIYFRNPMAENSKFFKKHGIRNGINPAMYQSPMRLRSSTIQSSSIILLDTLLLTENEGDKTEE RVSYV(SEQ ID NO:9)；

wherein the underlined section is the third intracellular loop.

In some embodiments, the amino acid sequence of the human HTR6 receptor is:

MVPEPGPTANSTPAWGAGPPSAPGGSGWVAAALCVVIALTAAANSLLIALICTQPALRNTSNFF LVSLFTSDLMVGLVVMPPAMLNALYGRWVLARGLCLLWTAFDVMCCSASILNLCLISLDRYLLILS PLRYKLRMTPLRALALVLGAWSLAALASFLPLLLGWHELGHARPPVPGQCRLLASLPFVLVASGLT FFLPSGAICFTYCRILLAARK QAVQVASLTTGMASQASETLQVPRTPRPGVESADSRRLATKHSRKALKASLTLGILLGMFFVTWLPFFVANIVQAVCDCISPGLFDVLTWLGYCNSTMNPIIYPLFMRDFKRA LGRFLPCPRCPRERQASLASPSLRTSHSGPRPGLSLQQVLPLPLPPDSDSDSDAGSGGSSGLRLTAQLL LPGEATQDPPLPTRAAAAVNFFNIDPAEPELRPHPLGIPTN(SEQ ID NO:10)；

wherein the underlined section is the third intracellular loop.

The invention also relates to the GRAB probe constructed and obtained by the method.

Another aspect of the invention relates to polynucleotides encoding the above-described fluorescent probes constructed based on G protein-coupled receptors.

Another aspect of the invention relates to an expression vector comprising the polynucleotide described above.

Another aspect of the invention relates to a host cell comprising the polynucleotide described above or the expression vector described above.

In some embodiments, the host cell is a neuronal cell.

Another aspect of the present invention relates to a method for detecting the presence or absence of a specific ligand of a G protein-coupled receptor in a test sample or a test tissue using any of the above-described fluorescent probes constructed based on G protein-coupled receptors, the method comprising expressing the fluorescent probes constructed based on G protein-coupled receptors on a cell membrane and contacting the expressed fluorescent probes with the test sample or the test tissue, determining the fluorescent signal intensity F0 before and the fluorescent signal intensity F after the contacting of the fluorescent probes constructed based on G protein-coupled receptors with the test sample or the test tissue, and determining the presence or absence of the specific ligand of the G protein-coupled receptor in the test sample or the test tissue based on the change of the fluorescent intensity of F relative to F0.

In some embodiments, the change in fluorescence signal intensity comprises an increase or decrease in fluorescence signal intensity.

Another aspect of the present invention relates to a method for qualitatively detecting a change in concentration of a specific ligand of a G protein-coupled receptor in a sample or tissue to be tested using any one of the above-mentioned fluorescent probes constructed based on the G protein-coupled receptor, the method comprising expressing the fluorescent probe constructed based on the G protein-coupled receptor on a cell membrane and contacting the expressed fluorescent probe with the sample or tissue to be tested containing the specific ligand, measuring a fluorescence signal intensity F1 of the fluorescent probe at a first time point, which is a second time point after the first time point or before the fluorescent probe is contacted with the sample or tissue to be tested, measuring a fluorescence signal intensity F2 of the fluorescent probe at the second time point after the first time point, and determining a change in the concentration of the specific ligand at the second time point relative to the first time point based on the change in the fluorescence signal intensity of F2 relative to F1.

In some embodiments, the change in fluorescence signal intensity comprises an increase, decrease, or no change in fluorescence signal intensity. In some embodiments, the change in concentration of the specific ligand comprises an increase, decrease, or no change in its concentration.

Another aspect of the present invention relates to a method for quantitatively detecting a change in concentration of a specific ligand of a G protein-coupled receptor in a sample or tissue to be tested using any of the above-described fluorescent probes constructed based on the G protein-coupled receptor, the method comprising:

(1) Reacting the fluorescent probe with different samples each comprising a known concentration of the specific ligand, and determining a standard curve of the correspondence of the specific ligand concentration to the fluorescent signal intensity;

(2) And (2) expressing the fluorescent probe on a cell membrane, contacting the expressed fluorescent probe with a sample to be tested or a tissue to be tested containing the specific ligand, measuring the fluorescent signal intensity, and obtaining the concentration of the specific ligand in the sample to be tested or the tissue to be tested according to the measured fluorescent signal intensity and the standard curve obtained in the step (1).

Another aspect of the invention relates to a method for screening a drug, comprising expressing any of the above fluorescent probes constructed based on G protein-coupled receptors on a cell membrane, adding a drug candidate to the cell, measuring the fluorescent signal intensity before and after the drug candidate is added, and determining whether the drug candidate is an agonist of the G protein-coupled receptor based on a change in the fluorescent signal intensity after the drug candidate is added relative to the fluorescent signal intensity before the drug candidate is added.

Another aspect of the invention relates to a method of drug screening comprising:

(1) Expressing any one of the fluorescent probes constructed based on the G protein-coupled receptor on a cell membrane, adding a molecule capable of activating the G protein-coupled receptor into the cell, and measuring the change of the fluorescent signal intensity;

(2) Adding a drug candidate to the cells, and screening the drug candidate capable of reversing the change in fluorescence signal intensity in step (1) as an antagonist capable of blocking the G protein-coupled receptor.

Wherein, if the fluorescence signal intensity increases after adding the molecule capable of activating the G protein-coupled receptor in step (1), the inversion in step (2) means that the fluorescence signal intensity decreases after adding the drug candidate; if the intensity of the fluorescent signal decreases after the addition of the molecule that activates the G protein-coupled receptor in step (1), the reversal in step (2) means that the intensity of the fluorescent signal increases after the addition of the drug candidate.

In another aspect, the invention relates to a method for detecting the distribution of a specific ligand of a G protein coupled receptor in an animal body, which comprises expressing the fluorescent probe constructed based on the G protein coupled receptor in the animal body, measuring the fluorescent signal intensity in the animal body in real time, and determining whether the specific ligand exists in different areas in the animal body, whether the concentration of the specific ligand among the different areas is different and whether the concentration of the same area changes with time according to the existence, the intensity and the change condition of the fluorescent signal intensity of the different areas in the animal body.

In some embodiments, the animal is induced to produce the specific ligand prior to determining the fluorescent signal intensity.

In some embodiments, the specific ligand is induced to be produced by the animal body by odor stimulation or visual stimulation.

In any of the above methods, when measuring the fluorescence signal intensity, the cyclically rearranged fluorescent protein can be caused to generate a fluorescence signal by excitation of an external light source.

In any of the above methods, when a luciferase inserted at the C-terminal end of the G protein-coupled receptor is further included in the fluorescent probe, detection is performed by BRET (bioluminescence resonance energy transfer) in which the circularly rearranged fluorescent protein does not need to be caused to generate a fluorescent signal by excitation of an external light source, but the fluorescent probe is brought into contact with a substrate of the luciferase before measuring the intensity of the fluorescent signal.

By utilizing the fluorescent probe and the method, the high-efficiency and accurate detection of the specific ligand of the G protein coupled receptor can be realized, the time resolution is higher, and the dynamic change of the specific ligand of the G protein coupled receptor in a specific environment can be tracked in real time.

While the following examples illustrate the present invention by way of example of different neurotransmitters, it will be appreciated by those skilled in the art that the fluorescent probes of the present invention take advantage of the structural commonality of the seven transmembrane regions of the G-protein coupled receptor and thus can be used for other ligands of the G-protein coupled receptor, such as hormones, metabolic molecules or nutritional molecules, and are not limited to neurotransmitters.

Drawings

FIG. 1 is a schematic representation of the insertion of a cyclic rearranged fluorescent protein of the β2 adrenergic receptor at different positions (upper panel) and the results of fluorescence imaging (lower panel). Wherein fluorescence imaging was performed using a Nikon confocal microscope A1.

The upper left hand corner of the lower panel indicates the insertion of a fluorescent protein at the amino acid position of the GPCR, its corresponding position on the receptor being indicated by the dash in the upper panel. In the lower panel, the arrow indicates the fusion protein successfully expressed on the cell membrane.

FIG. 2 is a typical reaction of GRAB-EPI 0.1 to saturation concentration (2. Mu.M) ISO. Upon addition of ISO, the receptor undergoes a conformational change resulting in a rapid increase in fluorescence signal with an average magnitude of 6% ΔF/F ₀ . After washing off the ISO with physiological solution, the conformation of the receptor reverts back toIn the inactive state, the corresponding cellular fluorescence values also return to baseline. Wherein the lower graph is a schematic diagram of fluorescence intensity of single cells represented by pseudo color before and after addition of ISO, and obvious reversible change of fluorescence value on cell membranes before and after addition of ISO can be observed.

FIG. 3 shows the construction and performance of GRAB-EPI probes using different circularly rearranged fluorescent proteins. Probes constructed with cyclically rearranged EGFP had better folding and cell membrane transport, whereas probes constructed with cyclically rearranged superfolder GFP could not fold correctly, and in the following figures the image acquisition was taken using an Olympus IX81 inverted fluorescence microscope, where the arrow indicates visible, distinct intracellular fluorescent protein aggregation.

FIG. 4 shows the results obtained by altering the insertion site of a fluorescent protein at the third intracellular loop of the β2 adrenergic receptor. The signal change was found to be about 15% ΔF/F ₀ Shows a sensitive, rapid, reversible optical change to the ligand.

FIG. 5 shows that will beta ₂ Short ICL3 migration of AR to M _1-5 GRAB-ACh probe was generated in R. a: beta ₂ AR and M _1-5 R, wherein the region between TM5 and TM6 is shown, the boundary of the graft is indicated by a black dashed line. b: m is M _1-5 R-β ₂ Fluorescent response of R ICL3-cpEGFP chimera to ACh (100. Mu.M) derived from M alone ₃ The probe of R showed a detectable increase in fluorescence, data were collected by a TECAN fluorescence analyzer (n=6-10 wells/chimera,>100 cells/well). M is M ₁ R,ΔF/F ₀ -2.11±1.58％；M ₂ R,ΔF/F 2.09±1.19％；M ₃ R,ΔF/F ₀ 22.03±0.86％；M ₄ R,ΔF/F ₀ 2.16±1.63％；M ₅ R,ΔF/F ₀ -0.49±0.16％。

FIG. 6 shows the construction of a GRAB-ACh probe to acetylcholine. a: principle of GRAB-ACh probe. b: GRAB-ACh probes based on different muscarinic receptors typically model membrane uptake in HEK293T cells, based on M ₃ The probe of R, named GRAB-ACh 1.0, has good coating properties. c&d: optimization of GRAB-ACh 1.0, random mutagenesis of the connecting peptide sequence of cpEGFP (N-terminalScreening at the end of 2 amino acids, 5 amino acids at the C-terminus, the single residues with best results (C-panels) were further combined (d-panels) to produce a probe designated GRAB-ACh 2.0, ΔF/F ₀ Near 100%. Each data point is an average response of 2-10 cells. e-g: GRAB-ACh 1.0&2.0 response in HEK293T cells. Pseudo-color plots are their peak response to perfusion of 100. Mu.M ACh (e plot), f plot shows quantitative values for e plot experiments, g plot shows GRAB-ACh 1.0&Group data of 2.0 (GRAB-ACh 1.0: ΔF/F) ₀ 24.62±1.51%, n=19 cells; GRAB-ACh 2.0: ΔF/F ₀ 90.12±1.74%, n=29 cells; z= -5.79, p<0.001). h-j: comparison of GRAB-ACh 2.0 with FRET Probe-based muscarinic receptor when 100. Mu.M ACh is perfused, GRAB-ACh 2.0 reacts at peak (ΔF/F) compared to FRET Probe ₀ Both 94.0±3.0% versus Δfret ratio 6.6±0.4%, panel i) and signal to noise ratio (724±9 versus 8.3±1.1, panel j) showed significantly stronger signals (n=10 cells per group). Mann-Whitney rank and nonparametric test, x, p in g plot<0.05； **,p<0.01；***；p<0.001; n.s., no significance. All scales are 10 μm.

FIG. 7 shows the results of an optimized screen for the length of the connecting peptide between fluorescent protein and GPCR. Wherein the ON probe with the highest signal variation is 2-5 of the length of the connecting peptide, and the OFF probe is 1-1 of the length of the connecting peptide. The numbers below each column in the figure represent the nitrogen-terminal peptide length-carbon-terminal peptide length, e.g., 1-3 represents the nitrogen-terminal 1 amino acid and 3 amino acids at the carbon-terminal.

FIG. 8 shows optimization of GRAB-ACh 1.0 by random mutagenesis of the linker peptide. a: the two and five amino acid connecting peptides (left panel) connecting the N and C termini of cpgfp were randomly mutated individually to 20 possible amino acids. 373 variants of 7 residues were tested alone and were tested for their ΔF/F for ACh (100. Mu.M) in HEK293T cells ₀ The reactions were quantitatively detected (right panel). The most effective one to four mutations per residue were selected for the second round of screening. b: sequence information and ΔF/F for each of the 23 candidates in the second round of screening ₀ Reaction in which GRAB-ACh 2.0. DELTA.F/F ₀ Approximately 0.9.

Figure 9 shows that FRET probes based on muscarinic receptors respond poorly to ACh. a: construction based on M as reported previously ₁ R's FRET probe (Markovic, D., et al FRET-based detection of M1 muscarinic acetylcholine receptor activation by orthosteric and allosteric agonists. PloS one 7, e29946 (2012)), wherein CFP is inserted between K361 and K362 of its ICL3, YFP is fused to its C-terminus, and chimeric proteins are poorly membranous. b: ACh (100 μm) induced a slight decrease in fluorescence in YFP channels and an increase in fluorescence in CFP channels (n=average result of 10 cells). c: after perfusion of ACh, FRET ratio (CFP/YFP) of ACh probe showed a moderate increase.

FIG. 10 shows the spectral properties of GRAB probes and their pH sensitivity. GRAB probes constructed based on green fluorescent protein have excitation peaks and emission peaks similar to GFP and are respectively positioned near 490 nanometers and 520 nanometers, and the fluorescence intensity of the GRAB probes also shows sensitivity to the pH value of the solution.

FIG. 11 shows the change in fluorescence signal that the GRAB probe produces in the case of activation by a ligand. When a receptor-specific blocker is added, the same concentration of agonist cannot produce a change in fluorescence signal due to failure to bind to the receptor.

Figure 12 shows that mutation of the domain directed against the GPCR binding ligand can significantly affect the performance of the probe. A: upon mutation of the ligand binding domain to the β2 adrenergic receptor, agonist ISO fails to cause an increase in fluorescence signal. B: after reducing the affinity of the acetylcholine receptor for the ligand by mutation, the acetylcholine fluorescent probe exhibits a reduced affinity for acetylcholine.

FIG. 13 shows that GRAB probes exhibit ligand concentration-dependent fluorescence signal changes. A: the GRAB-EPI 1.0 probe exhibited enhanced fluorescent signal for different concentrations of agonist ISO, similar to the endogenous β2 adrenergic receptor. B: the GRAB-ACh 1.0 probe exhibited a change in fluorescence value for different concentrations of acetylcholine, similar to the endogenous M3-type acetylcholine receptor.

FIG. 14 shows GRAB-ACh 2.0 detection of ACh with sub-second kinetics and micromoleSensitivity of the mole. a: a schematic of a rapid perfusion system in which glass pipettes containing ACh and red rhodamine-6G dye were placed near GRAB-ACh 2.0 expressing cells, with white lines representing the line scans performed. b: in line scan experiments performed on ACh and Tio, perfusion of ACh or Tio resulted in an increase or decrease in fluorescence of GRAB-ACh 2.0 with time constants of 185ms and 696ms, respectively. c: panel b, average on time constant 279.4±32.6ms, n=18, off time constant 762.3 ±74.9ms, n=11. d, d&e: dose-dependent response of GRAB-ACh 2.0 to ACh. pEC of GRAB-ACh 2.0 (-0.7. Mu.M, n=4) ₅₀ = -6.12±0.11M, and WT-M ₃ Kd (0.5-2. Mu.M) for R (JakubI.k, J., bacA kov A, L., el-Fakahany, E.E).&Tucek, S.positive cooperativity of acetylcholine and other agonists with allosteric ligands on muscarinic acetylcholine receptors, molecular pharmacology 52,172-179 (1997)) are very close. AF-DX384, M ₃ Antagonists of R completely block the increase in fluorescence. The units in panel d are μM, the average response of 3 experiments performed using the same HEK293T cells.

FIG. 15 shows a significant decrease in the coupling of GRAB probes to G-protein mediated signaling pathways. After treatment of cells with calcium dye, perfusion experiments with different concentrations of acetylcholine were performed to compare whether there was a difference in calcium signal in cells expressing the GRAB-ACh 1.0 probe and endogenous M3 type acetylcholine receptor. Wherein the lower graph shows the response curve of calcium signal and ligand concentration, and the coupling degree of the calcium signal of the GRAB probe-expressing cell is reduced by about 5 times.

FIG. 16 shows that by utilizing the feature that a G.alpha.protein carbon-terminal peptide can stabilize a GPCR in an activated state but cannot transmit downstream signals, the G.alpha.protein peptide is attached to the end of a GRAB probe, thereby reducing G-protein mediated activation of downstream pathways by competing for endogenous G-protein binding. A: GRAB-ACh 2.0-Gq20 probe fluorescence imaging, the probe was well folded and expressed on the cell membrane. B: GRAB-ACh 2.0-Gq20 exhibited an increase in fluorescence signal with a change in signal of about 70% ΔF/F upon addition of saturated acetylcholine ₀ . C: obtaining cells expressing different probes at different concentrations of neurotransmitters using calcium imaging methodsUnder the condition of calcium signal change, the probe connected with the G alpha peptide segment can be obviously reduced for G protein mediated downstream channel coupling by calculating the Kd value.

FIG. 17 shows the detection of receptor coupling to endocytic signaling pathway by fluorescent probes constructed based on GPCR receptor endocytic principles. A: principle of endocytic probes. B: beta-based ₂ Probe pHluorin-beta constructed by adrenergic receptor ₂ AR exhibits a marked activation of endocytic signaling pathways, i.e. a decrease in cellular fluorescent signal.

FIG. 18 shows that the coupling efficiency of GRAB probes to the arestin-mediated endocytic signal pathway is greatly reduced. A: for the GRAB-EPI 1.0 probe, the fluorescence value on the cell membrane was observed not to change over time with a saturated concentration of agonist ISO for 30 minutes. B: comparing the endocytic signal coupling efficiency of the GRAB-EPI 1.0 probe and the endogenous beta 2 adrenergic receptor, the GRAB probe can be found to almost completely block the coupling of endocytic signal channels, thereby truly reflecting the dynamic change of ligand concentration.

FIG. 19 is a fluorescence imaging of GRAB probes in cultured neurons. A: imaging of GRAB-EPI 1.0 in cortical neurons. B: fluorescence imaging of GRAB-ACh 1.0 in cortical neurons (left panel) and partial enlargement thereof (right panel).

FIG. 20 shows the response of GRAB probes in cultured neurons. A: the GRAB-ACh 1.0 probe showed ligand-specific fluorescence signal elevation in cultured cortical neurons. B: the GRAB-EPI 1.0 probe and the GRAB-ACh 1.0 probe showed ligand concentration-dependent fluorescence reaction in neurons.

FIG. 21 shows the specificity of the response of GRAB probes to specific neurotransmitters. A & B: GRAB-ACh 1.0 produces a reproducible, reversible, specific response only to epinephrine (Epi) and its analogs (ISO), which is absent when the blocker ICI is added. C & D: the GRAB-ACh 1.0 probe produces only reproducible specific responses to acetylcholine, and no fluorescent response to other major neurotransmitters.

FIG. 22 shows GRAB-ACh 1.0 probe specific detection of endogenous acetylcholine release from the Drosophila olfactory system. After administration of isoamyl acetate scent, the probe optical signal at the antennal nerve lobes showed a rapid rise with an amplitude that was dependent on the concentration of scent molecules (upper graph). At the same time, the rise of the fluorescent signal shows olfactory bulb specificity, and in the olfactory bulb projected by the olfactory receptor neuron receiving isoamyl acetate, such as DM2, the signal is larger, while in the olfactory bulb not projected by the neuron, such as DA1, the fluorescent signal is unchanged (lower graph).

FIG. 23 shows the effect of detecting in vivo over-expressed GRAB probes on cellular calcium signaling using the red calcium indicator RGECO. A: to have similar magnitudes in Drosophila expressing RGECO alone and Drosophila co-expressing RGECO and GRAB-ACh 1.0 probes, the odor molecule induced elevation of DM2 olfactory bulb calcium signaling in the antennary nerve lobes. B: is a statistical result of a plurality of fruit flies.

FIG. 24 shows the performance of GRAB-ACh probe on mouse hippocampal acute acquisition brain slices. A: for fluorescent imaging of GRAB-ACh probes in hippocampal neurons under a two-photon microscope, red dye Alexa 594 is used for dyeing and imaging the neurons from left to right, and the images of neurons transferred into GRAB-ACh are overlapped, so that the GRAB probes are uniformly distributed on the cell membranes of the neurons, and the axon positions of the neurons are visibly expressed. B: cells expressing GRAB-ACh exhibited a specific acetylcholine-induced increase in fluorescence compared to non-expressing cells. C: cells expressed as GRAB-ACh probes respond fluorescently to the M receptor agonist acetylcholine, oxo-M, without significant changes in fluorescence intensity to nicotine and physiological solutions (ACSF, artificial cerebrospinal fluid) themselves.

FIG. 25 shows the selection of human norepinephrine receptors for the construction of fluorescent probes. a: chemical structures of norepinephrine NE and epinephrine Epi. b: n-terminal fusion expresses three different expression conditions of noradrenergic receptor ADRA1D, ADRB and ADRA2A of green fluorescent protein pHluorin in mammalian cell HEK 293T. In panel b, the arrows in ADRA1D and ADRB3 indicate cells with worse conditions of coating, and the arrows in ADRA2A indicate cells with better conditions of coating. Scale=50 μm.

FIG. 26 shows the development and optimization of norepinephrine fluorescence probes. a: schematic representation of the truncated and inserted circulating rearranged fluorescent protein cpEGFP against the third intracellular loop ICL3 of the ADRA2A receptor. b: the first round of screening resulted in GRAB-NE1.0 with a fluorescent signal change to NE. c: the second round of fine screening of the insertion sites can obtain better fluorescent brightness, and the fluorescent signal of NE is changed to be larger GRAB-NE2.0.d: through a drug perfusion experiment, the fluorescence signal of the NE1.0 and 2.0 versions respectively changes by more than 100% and 200% under the condition of 100 mu M NE, the reaction of the probe is reversible, and the fluorescence intensity is recovered to an initial value after the drug is washed out. e: pseudo-color maps of GRAB-NE1.0 and 2.0. Scale=10 μm.

FIG. 27 shows the continued optimisation of GRAB-NE2.0 on the connecting peptide fragments. a: schematic representation of truncated screening libraries of linked peptide fragments. b: truncated selection of the connecting peptide fragment did not result in a probe with higher fluorescence intensity and greater change in fluorescence signal than GRAB-NE2.0. c: schematic representation of the pool of linked peptide stretch amino acid mutations. d: the third round of screening of the connecting peptide segment results in GRAB-NE2.1 with higher fluorescence intensity and larger change of NE fluorescence signal, which is mutation from glycine of the third connecting amino acid to threonine.

FIG. 28 shows basic characterization of GRAB-NE probes and development of GRAB-NE 2.2. a: drug-specific assay of GRAB-NE 2.0. It has a fluorescent signal change only for neurotransmitters NE and Epi, and is not responsive to saturated concentrations of the beta receptor-specific activator ISO and other neurotransmitters. The addition of both the alpha receptor specific blocker Yohimbine (2 μm) and the ligand binding domain S204A mutation of the ADRA2A receptor inhibited ligand induced NE probe signal changes. b: the ligand concentration dependence curve of GRAB-NE2.0 was obtained by sequential perfusion of 10nM to 100. Mu.M NE, which reaction was also completely inhibited by the addition of 1. Mu.M of the blocker Yohimbine (unit/. Mu.M). c: the introduction of the T373K mutation resulted in GRAB-NE2.2 whose concentration-dependent curve shifted to the left in comparison with GRAB-NE2.1, with an increase in affinity for ligand NE of 10-fold. d: expression of GRAB-NE2.1, GRAB-NE 2.1S 204A, GRAB-NE2.2 in HEK293T cells and membranous conditions. e: the enhanced versions of NE2.1 and NE2.2 of GRAB-NE optimization are improved over the version of GRAB-NE2.0 in both fluorescence intensity and fluorescence response signal. f: GRAB-NE2.2 has a similar concentration-dependent curve for ligands NE and Epi, and the affinity of both ligands is improved. The scale bar is 10 μm.

FIG. 29 shows that the GRBA-NE2.2 probe has rapid reaction kinetics. a: schematic of NPEC group caged NE releasing free NE and NPEC groups upon uv activation. b: using 405nm laser light to photolyze the white area surrounding GRAB-NE2.2, a 20% change in fluorescence signal was observed for GRAB-NE2.2 when photolyzed with 100. Mu.M NPEC-NE, which was inhibited in the presence of 10. Mu.M blocker Yohimbine. c: GRAB-NE2.2 graph of change in fluorescence signal upon simulated photolysis, 100. Mu.M NPEC-NE and addition of 10. Mu.M Yohimbine. Amplifying the time 2000ms around the photolysis time point fits a rate constant of 104ms for the photolysis reaction to cause the fluorescence signal of the GRAB-NE2.2 probe to rise.

FIG. 30 is a depiction of the decoupling of GRAB-NE probe from downstream G protein signaling. a & b: schematic representation of the green fluorescent protein in the NE receptor protein ADRA2A third intracellular loop insertion to uncouple the gαi protein from the GPCR. c & d: concentration dependence curves of NE2.0 on ligand (unit: μM) were not changed after cotransformation of GRAB-NE2.0 with PTX. e: the concentration dependence curve of GRAB-NE2.0 on NE is not changed by adding GTPγS under the treatment of digitonin (a saponin which is used for opening a pore on a cell membrane and allowing exogenously added medicines to enter the cell, especially small molecules (GTPγS and the like) with poor fat solubility and difficulty in crossing the cell membrane) to inhibit the activation cycle of G alpha protein. f. By following the downstream TGF-alpha release assay with GRAB-NE2.0, the receptor proteins ADRA2A and TPA (which directly activate intracellular PLC (downstream of GPCR), which can be used as a positive control in TGF-alpha assays to verify if the system is working properly) at 100 nM NE treatment, it was found that GRAB-NE2.0 activates the downstream signal only 1/3 as strongly as the receptor protein.

FIG. 31 shows that GRAB-NE2.1 has an optical signal change to specific neurotransmitters in cultured neurons. a & b: co-transformed GRAB-NE2.1 and PSD95-mcherry showed that GRAB-NE was distributed more evenly on the neuronal membrane, with a slightly more aggregated cell cytoplasmic fraction (1), but better on the dendritic membrane (arrows 1, 2). There is also a pronounced distribution (e.g., 1, 2 triangles) on the dendritic spine co-located with PSD 95. c: when 100 mu M NE drug is infused, the fluorescence signal changes on the cell membrane and on the dendritic spines are about 200%, and similar to those in mammalian cells, the cell bodies are about 60% in response due to poor upper membrane condition. d: pseudocolor image after drug elution when neuron drug transfected with GRAB-NE2.1 is perfused. e: comparison of fluorescence response signals of cell bodies, cell membranes and dendritic spines. f & g: dependence curve of GRAB-NE2.1 neuronal cell bodies on different concentrations of NE, drug infusion from 10nM to 100. Mu.M, ligand affinity 790nM (unit:. Mu.M). The scale bar is 10 μm.

FIG. 32 shows that neurotransmitter fluorescent probe GRAB-NE2.1 has an optical signal change for specific neurotransmitters in cultured rat cardiomyocytes. a: expression of GRAB-NE2.1 in rat cardiomyocytes and conditions of tunica media. b: GRAB-NE2.1 has a greater than 300% change in fluorescence signal in cardiomyocytes at 100. Mu.M NE drug perfusion, and the response is reversible. c: pseudo-color map of GRAB-NE2.1 response in cardiomyocytes. d & e: the reaction of the probe in myocardial cells also has ligand concentration dependence, and NE with 1nM to 100 mu M is perfused once to obtain a ligand concentration dependence curve of the probe, wherein the affinity is 500nM; the reaction was inhibited with 1. Mu.M of the blocker Yohimbine (unit:. Mu.M); the scale bar is 50. Mu.M.

FIG. 33 shows that GRAB-5-HT2.1 probe shows ligand concentration dependent fluorescence in HEK293T cells, K _d The value was about 131 nM, similar to the affinity of HTR2C receptors under physiological conditions.

Fig. 34 shows: a: the GRAB-5-HT2.1 probe only reacts specifically to serotonin and does not respond fluorescently to other major neurotransmitters such as Gly, epi, ach, etc. B: serotonin and the specific agonist CP809 of HTR2C can cause a change in the fluorescence signal of the GRAB-5-HT2.1 probe, whereas the specific agonist BWT23C83 of HTR2B and the specific agonist CGS12066B of HTR1B cannot cause a change in the probe signal; the specific antagonist RS102221 of HTR2C antagonizes the increase in fluorescence signal of the GRAB-5-HT2.1 probe induced by serotonin, whereas the specific antagonist SB204741 of HTR2B does not antagonize the increase in signal.

FIG. 35 shows the response of a series of serotonin fluorescent probes constructed based on different HTR receptors after addition of saturated concentrations of serotonin.

FIG. 36 shows that GRAB-5-HT2.0 probe specifically detects endogenous serotonin release from the olfactory system of Drosophila. The optical signal of the probe showed a rapid rise after the scent (isoamyl acetate, banana scent) stimulus was given.

FIG. 37 shows the signal change of probe GRAB-GDA3.0 constructed based on DRD2 under treatment with saturated concentration of dopamine.

FIG. 38 shows the pharmacological characterization of GRAB-GDA3.0 in HEK293T cells. GRAB-GDA3.0 is activated only by dopamine and the hDRD2 specific agonist quinpirole (quinpirole) and blocked by the hDRD2 specific antagonist Haloperidol.

FIG. 39 shows the odor-activated GRAB-GDA3.0 (shown as GDA) signal in MB. A: schematic of 2-PT imaging in Drosophila body after odor stimulation. GRAB-GDA3.0 is expressed in dopaminergic neurons (DANs), driven by TH-GAL4, focusing on the Mushroom (MB), which receives dopaminergic enhancement signals. MB beta' lobe is outlined by a dashed line. The scale bar is 25 μm. B: GDA located on the cell membrane of DAN can report dopamine release in the synaptic cleft. C1-C3: pseudo-color imaging of GRAB-GDA3.0 in β' lobe after IA (1% isoamyl acetate,5 seconds) stimulation. The scale bar is 25 μm. D: average time of 3 trials of GRAB-GDA3.0 signal in β' lobe after IA stimulation in one drosophila.

FIG. 40 shows that the odor-activated GRAB-GDA3.0 (shown as GDA) signal in MB is dopamine-specific. a-C: GDA signal in IA-stimulated β' lobe can be blocked by the hDRD 2-specific antagonist halo (10. Mu.M haloperidol). Pseudo-color imaging in a fruit fly before and after halo application. Scale bar 25 μm (panel A); average time of three trials in the same drosophila before and after halo administration (panel B); statistical results showed significant inhibition of GDA by halo (panel C). Error bars represent SEM (n=6). D-F: GDA signal in the stimulated MB.beta. -lobe of IA cannot be blocked by the octopamine receptor antagonist epinastine (10. Mu.M). Pseudo-color imaging in one fruit fly before and after epinastine application. Scale bar 25 μm (D plot); average time of three trials in the same drosophila before and after application of epinastine (E plot); statistical results showed no inhibitory effect of epinastine on GDA (panel F). Error bars represent SEM (n=6). G-J: when DAT-RNAi is expressed in DAN and driven by TH-GAL4, the GDA signal decays τ. DAT is located in the presynaptic membrane of DAN, which releases DA from the gap cycle (G plot). Average time in one WT drosophila and one DAT-deficient drosophila. Fitting results of the decay curves are shown in the H plot; error bars represent SEM (n=6). The WT fruit fly and DAT deficient fruit fly were imaged for false color after odor stimulation. The scale bar is 25 μm (J plot).

FIG. 41 shows the construction of a cpmeAN-based dopamine fluorescence probe. A: ligand-induced reaction (ΔF/F) of variants in a constructed library ₀ ). Perfusion was performed to test the performance of 92 variants, 16 of which showed no fluorescence, 56 did not show ligand-induced responses, 16 showed on-responses, and 5 showed off-responses. The dashed rectangular box represents the candidate with the highest on-and off-reactions. 222-349/267-364 represents the insertion site of cpmAppe into HTR 2C. B: the left plot is the imaging properties of the two candidates selected, the right plot is the corresponding response curve, scale bar 20 μm, and the results are shown as mean ± SEM, red curve n=6 cells, blue curve n=5 cells. C: the left panel shows ligand-induced response (ΔF/F) of variants in a random mutant library of connecting peptides ₀ ). Only the response characteristics of the variants are shown in the figure. The dashed rectangle represents the candidate with the greatest response. The middle panel shows the imaging properties of the best candidate of the fine tuning library. The right plot is the corresponding response curve, scale bar 20 μm, and the results are shown as mean ± SEM, red curve n=5 cells, black curve n=6 cells. D: the right panel shows ligand-induced response (ΔF/F) of variants in a random mutant library of connecting peptides ₀ ) And relative brightness. The library is a mixture of five independent libraries, each independent library being a library directed against one amino acid random mutation. The red dot represents the nature of the starting template, which is the best candidate selected from the fine tuning library. Black dots represent the characteristics of variants of the random mutation library of the connecting peptide. X in the left graph represents random mutant connecting peptide ammoniaThe positions of the amino acids are randomly mutated individually, one by one, in the linker peptide amino acids.

FIG. 42 shows the construction of a cpmeAN-based serotonin fluorescent probe. A: ligand-induced response (Δf/F) of variants in a library constructed from a cpRFP insertion strategy and a trimming strategy ₀ ). The dashed rectangular box represents the candidate with the highest on-and off-reactions. 240-306/239-309 represent the insertion site of cpmeapple into HTR 2C. B: the left plot is the imaging properties of the two candidates selected, the right plot is the corresponding response curve, scale bar 20 μm, and the results are shown as mean ± SEM, red curve n=8 cells, blue curve n=6 cells. C: the right panel shows ligand-induced response (ΔF/F) of variants in a random mutant library of connecting peptides ₀ ) And relative brightness. The library is a mixture of five independent libraries, each independent library being a library directed against one amino acid random mutation. The red dot represents the nature of the starting template, which is the best candidate selected from the fine tuning library. Black dots represent the characteristics of variants of the random mutation library of the connecting peptide. In the left panel, X represents the position of the random mutated connecting peptide amino acids, which are individually randomly mutated one by one.

FIG. 43 is a signal change profile of a serotonin fluorescent probe based on bioluminescence resonance energy transfer. Where R is the ratio of the signal intensity of the 535nm channel to the 450nm channel. Where dR is ΔR, the value of the change in R. Wherein 535nm channel indicates the emission wavelength of the GRAB probe and 450nm channel is the emission wavelength of Nanoluc, the ratio of which is a measure of energy resonance transfer.

FIG. 44 shows that a specific receptor blocker (Tio) can block the response of the acetylcholine probe GRAB-ACh 1.0 to the ligand acetylcholine.

FIG. 45 shows optimized screening of fluorescent probes constructed based on acetylcholine M3R receptors. a & b: randomly selecting one site from 7 sites at the N end and 8 sites at the C end of ICL3, truncating a peptide fragment between the two sites and inserting the cpEGFP; c: selecting a part from screening results on Opera Phenix, and confirming the part by using Confocal perfusion; d: perfusion results of partial mutants.

FIG. 46 shows the optimization of the binding peptide of cpEGFP to the M3R receptor, showing better probe performance when the first amino acid at the C-terminus is histidine His.

FIG. 47 shows GRAB-ACh4.0 obtained by optimized screening of the connecting peptide fragments.

FIG. 48 shows the perfusion results of GRAB-ACh4.0.

FIG. 49 shows that GRAB-ACh4.0 has no significant differences in affinity for its ligand acetylcholine compared to the reported wild-type M3R receptor.

FIG. 50 shows that GRAB-ACh4.0 can be, and is only, activated by ACh to produce a change in fluorescence intensity.

FIG. 51 shows that GRAB-ACh4.0 does not activate downstream Gq-directed signal pathways.

FIG. 52 shows experimental results of drug screening using a cell line expressing GRAB-5HT1.0 probe.

FIG. 53 shows that the C-terminal attachment of different G.alpha.protein peptide fragments to the acetylcholine probe results in a decrease in the ability of the probe to couple to downstream G-protein signaling pathways.

Detailed Description

The principle of the G-protein coupled receptor (GPCR) -activated fluorescent probe (GPCR Activation Based Sensor, also referred to as GRAB probe in the present invention, and the fluorescent probe constructed based on the G-protein coupled receptor) of the present invention is: inserting the cyclically rearranged fluorescent protein into a third intracellular loop of the GPCR, the binding of the ligand to the GPCR inducing a conformational change in the GPCR, thereby causing the conformational change in the cyclically rearranged fluorescent protein, causing a change in the intensity of the fluorescent signal, thereby converting the ligand-induced conformational change in the GPCR into a change in the optical signal.

G Protein Coupled Receptors (GPCRs) are a class of seven transmembrane proteins expressed on the cytoplasmic membrane, the GPCR protein body consists of 7 segments of alpha helical structures spanning the cytoplasmic membrane, the N-terminus and 3 loops are extracellular, and the C-terminus and 3 loops are intracellular. In studies on G protein-coupled receptors, resolution of the crystal structure helped scientists understand the specific mechanism by which it triggered intracellular downstream pathways upon ligand activation. The Masashi Miyano group first performed a crystal structure analysis on classical GPCRs, namely the photoreceptor rhodopsin in vision (Palczewski, K.et al crystal Structure of Rhodopsin: A G Protein-Coupled receptor. Science (New York, N.Y.) 289,739-745 (2000)) 16 by a method of stabilizing receptors using G Protein peptide fragments. In structural comparison to its activated and non-activated state, it was found that GPCRs upon ligand binding induce a series of conformational changes, most notably the outward extension of the fifth and sixth transmembrane regions, exposing a structural pore for carbon-terminal entry of the G protein. Subsequently, by a variety of methods of stabilizing the crystal structure of GPCRs, especially the use of single chain antibodies nanobody, which stabilize the receptor in an activated state, the crystal structure of the β2adrenergic receptor was successfully resolved by the brian kobilka group around 2012 (Rasmussen, S.G.F.et al Crystal structure of the human beta, 5, 383-387 (2007), rasmussen, S.G.et al crystal structure of the β2adrenergic receptor-Gs protein complex.477, 549-555 (2011), cherezov, V.et al, high-Resolution Crystal Structure of an Engineered Human $/betaG Protein Coupled, releaser.Science 318,1258-1265 (2007)), the activation of the β2adrenergic receptor was also accompanied by a significant change in the molecular conformation and a fifth transmembrane domain, similar to rhodopsin. To further demonstrate that this specific conformational change is a conserved activation pattern common to most GPCRs, further a crystal structure analysis was performed against the M-type acetylcholine receptor (Kruse, a.c. et al structure and dynamics of the M3 muscarinic acetylcholine receptor nature 482,552-556 (2012)), the opaid (Huang, w.et al structure insights into micro-opaid receptor activity. Nature 524,315-321 (2015)) receptor, and it was likewise found to have a similar conformational change pattern, thus speculating that this activation pattern might be common to most GPCRs. From the crystal structure analysis of GPCRs, GPCRs themselves can be regarded as specific ligand probes that evolve naturally, reacting as conservative conformational changes to mediate activation of downstream pathways. Depending on the resolution of the crystal structure of the different GPCRs in the active and inactive states, the pattern of conformational changes resulting from ligand binding is quite conserved, being the outward folding of the fifth and sixth transmembrane regions.

As described above, the resolved GPCR crystal structure information shows that the location of the GPCR where conformational change is greatest during activation is the fifth and sixth transmembrane regions, while the third intracellular loop is the region connecting the fifth sixth transmembrane region, which stretches as a segment of the flexible region with conformational change in the transmembrane region. In contrast, in the primary screening, it was also found that insertion of fluorescent protein in the third intracellular loop not only maintained proper folding and membrane transport of the receptor itself, but also had better fluorescent signal changes upon conformational changes of the receptor.

In the invention, the fusion protein is constructed by the cyclic rearranged fluorescent protein and the GPCR, when the concentration of ligand molecules changes, the conformation of the GPCR changes, thereby influencing the environment of the fluorescent protein chromophore to cause the change of the fluorescence intensity, and the change of the fluorescence intensity can be detected in real time by an optical imaging method, so that the change of the fluorescence intensity of the cyclic rearranged fluorescent protein can be used for indicating the concentration change of the ligand (such as an exogenous neurotransmitter). In the present invention, the probe is named as GRAB probe, which is an abbreviation of GPCR Activation Based Sensor. Since most known neurotransmitters have corresponding specific GPCRs, the fusion proteins of the circularly rearranged fluorescent proteins of the invention with the GPCRs can be used as probes for detecting neurotransmitters; in addition, the probes of the invention may also be used to detect ligands for other GPCRs.

In the present invention, a GRAB probe means a fusion protein constructed by inserting a circularly rearranged fluorescent protein at an appropriate position in a third intracellular loop between fifth and sixth transmembrane regions of a GPCR and connecting the circularly rearranged fluorescent protein to the third intracellular loop with a linker peptide at the N-terminus and C-terminus, and which can be used as a probe for detecting a ligand of the GPCR.

The invention provides a fluorescent probe (GRAB probe) constructed based on a G protein-coupled receptor, wherein the fluorescent probe is a fusion protein obtained by modifying the G protein-coupled receptor, and the modification comprises inserting a circularly rearranged fluorescent protein into a third intracellular loop between a fifth transmembrane region and a sixth transmembrane region of the G protein-coupled receptor.

As used herein, a "G protein-coupled receptor (GPCR)" is a large family of proteins that are transmembrane receptors that sense extracellular molecules, activate intracellular signaling pathways and ultimately activate cellular responses. Ligands that bind to and activate these receptors include photoactive compounds, odors, pheromones, hormones and neurotransmitters, and vary in size from small molecules to peptides to large proteins. GPCRs are involved in many diseases and are targets of about half of all modern pharmaceutical drugs. GPCRs can be classified into at least 5 classes based on sequence homology and functional similarity: class a rhodopsin-like, class B secretin-like, class C metabolic/pheromone, class D fungal pheromone, and class E cAMP receptor.

Class a rhodopsin-like receptors include: amine acceptor: acetylcholine, alpha adrenergic receptor, beta adrenergic receptor, dopamine, histamine, 5-hydroxytryptamine, octopamine, and trace amines; peptide receptor: angiotensin, bombesin, bradykinin, C5a anaphylatoxin, fmet-leu-phe, APJ-like substances, interleukin-8, chemokine receptors (C-C chemokines, C-X-C chemokines, B0 n Z0 receptor (CXC 6R), C-X3-C chemokines and XC chemokines), CCK receptor, endothelin receptor, melanocortin receptor, neuropeptide Y receptor, neurotensin receptor, opioid receptor, somatostatin receptor, tachykinin receptor (substance P (NK 1), substance K (NK 2), neuromodulatory peptide K (NK 3), tachykinin-like 1 and tachykinin-like 2), vasopressin-like receptors (vasopressin, oxytocin and conopresin), galanin-like receptors (galanin, aprotinin and GPCR 54), protease-like receptors (e.g., thrombin), peptide & neuropeptides FF, sclerostin II receptor, adrenoceptor (G10D, 37/D receptor and U-like receptor; hormone protein receptor: follicle stimulating hormone, luteinizing hormone-chorionic gonadotrophin, thyrotropin and gonadotrophin; (rhodi) an opsin receptor; nose receptors; prostanoid receptors: prostaglandins, prostacyclins, and thromboxane; nucleotide-like receptors: adenosine and purine receptors; a cannabis receptor; platelet activating factor receptor; gonadotropin releasing hormone receptors; thyrotropin releasing hormone & secretagogue receptor: thyrotropin releasing hormone, growth hormone secretagogues and growth hormone secretagogue-like; melatonin receptors; a viral receptor; a soluble sphingolipid (lysosphingoid) and LPA (EDG) receptor; leukotriene m receptor: leukotriene beta 4 receptor BLT1 and leukotriene m receptor BLT2; and class a orphan/other receptors: platelet ADP & KI01 receptor, SREB, mas protooncogene, RDC1, ORPH, LGR-like (hormone receptor), GPR 45-like, cysteinyl leukotriene, mas related receptor (MRGs) and GP 40-like receptor.

Class B (secretin receptor family) of GPCRs includes polypeptide hormone receptors (calcitonin, corticotropin releasing factor, incretin, glucagon-like peptide-1, -2, growth hormone releasing hormone, parathyroid hormone, PACAP, secretin, vasoactive intestinal peptide, diuretic hormone, EMR1, spider toxin receptor (latophilin)), a molecule thought to mediate intercellular interactions in the plasma membrane (brain-specific angiogenesis inhibitor (BAI)), and a group of drosophila proteins that regulate stress response and longevity (mehurselah-like proteins).

Class C metabotropic glutamate/pheromone receptors include metabotropic glutamate, group I metabotropic glutamate, group II metabotropic glutamate, group III metabotropic glutamate, other metabotropic glutamate, extracellular calcium sensing, putative pheromone receptors, GABA-B receptors (GABA-B receptors are composed of two subunits (B1, B2), are a dimeric protein) and orphan GPRC5 receptors.

GPCRs are involved in a variety of physiological processes including vision, olfaction, behavioral and emotional modulation, modulation of immune system activity and inflammation, autonomic nervous system transmission, cell density sensing, and many others. Inactive G proteins are known to bind to receptors in their inactive state. Once the ligand is recognized, the receptor or subunit thereof is conformational changed and thus mechanically activates the G protein, which detaches from the receptor. The receptor can now activate another G protein, or switch back to its inactive state. The receptor molecules are believed to exist in a conformational balance between active and inactive biophysical states. Binding of the ligand to the receptor may shift the equilibrium to the active receptor state.

G protein-coupled receptors that may be used in the present invention include, but are not limited to, beta 2 adrenergic receptor (ADRB 2), alpha 2A adrenergic receptor (ADRA 2A), acetylcholine receptor subtype M3R (M3 muscarinic acetylcholine receptor, CHRM 3), dopamine D2 receptor (DRD 2), 5-hydroxytryptamine 2C receptor (HTR 2C), 5-hydroxytryptamine 2B receptor (HTR 2B), 5-hydroxytryptamine receptor 6 (HTR 6), the sequences of which are well known to those skilled in the art and the sequences of which may be obtained by a variety of routes, such as well known database queries.

The N-terminus, transmembrane region, intracellular loop and C-terminus of a G protein-coupled receptor can be readily determined by one skilled in the art, for example, based on their amino acid sequence and similarity to the transmembrane region of known G protein-coupled receptors. Various bioinformatics methods can be used to determine the position and structure of the transmembrane region in a protein, for example, BLAST program or CLUSTAL W program can be used for alignment and amino acid sequence comparison as is conventional in the art. Based on alignment with G-protein coupled receptors known to contain a transmembrane region, one skilled in the art can predict the location and structure of the transmembrane region of other GPCRs. There are also a number of procedures available for predicting the location and structure of transmembrane regions in proteins. For example, one or a combination of the following procedures may be used: TMpred, which predicts transmembrane protein fragments; topPred, which predicts the topology of membrane proteins; predor, which predicts secondary structures from single and multiple sequences; TMAP, which predicts the transmembrane region of a protein from a plurality of aligned sequences; and AL0M2, which predicts the transmembrane region from a single sequence. The numbering of the transmembrane region and intracellular loop is relative to the N-terminus of the GPCR, according to standard nomenclature.

The term "cyclically rearranged fluorescent protein (circular permutated FP, cpFP)" as used herein is well known to those skilled in the art and refers to a fluorescent protein formed by ligating the molecular ends of the original fluorescent protein and then cleaving the protein from any site to form new carbon and nitrogen ends. The fluorescent protein itself has its own chromophore center composed of three amino acids, the chemical reaction of which determines the spectral properties and fluorescence intensity of the fluorescent protein, most of the chromophores of the fluorescent protein are located inside the protein and protected by the barrel structure of the surrounding beta-sheet, when the fluorescent protein is fused with the target protein, the environmental change around the chromophores is difficult to be caused by the involvement of the tail end of the fluorescent protein, and the fluorescence intensity of the fluorescent protein is difficult to be changed. The chromophore of the circularly rearranged fluorescent protein is relatively close to the newly formed end, and when the chromophore is connected with the target protein, the conformational change of the target protein can involve the end of the circularly rearranged fluorescent protein, so that the surrounding environment of the chromophore is changed, and the fluorescence intensity of the fluorescent protein is increased or reduced, thereby converting the conformational change of the target protein into the change of the fluorescence intensity of the fluorescent protein, and the fluorescent protein can be detected in real time by an optical imaging method. The cyclic rearranged fluorescent protein was originally derived from green fluorescent protein, its amino acid sequence was very highly homologous to GFP, and Roger Tsien was originally designed and applied in the development of gene-encoded calcium indicators (Baird, g.s., zacharias, D.a. & Tsien, r.y. Cyclic permutation and receptor insertion within green fluorescent proteins.proceedings of the National Academy of Sciences of the United States of America, 11241-11246 (1999)). A variety of cyclically rearranged fluorescent proteins have been constructed for probe construction, which have a high sensitivity to conformational changes in the protein and which are characterized by changes in fluorescence. cpFP usable in the present invention include a cyclic rearranged enhanced green fluorescent protein (circular permutated EGFP, cpgfp) and a cyclic rearranged red fluorescent protein (circular permutated RFP, cpFP). The cpeGFP may be a cpeGFP from GCaMP6s or GCaMP6m (Chen, T. -W.et al. Ultrasensitive fluorescent proteins for imaging neuronal activity. Nature 499,295-300 (2013)), or a cpeGFP from GECO1.2 (Zhao, Y.et al. An Expanded Palette of Genetically Encoded Ca2 +Industors. Science 333, 1888-1891 (2011)). The cprFP may be a cpmAppe, such as from R-GECO1 (Yongxin Zhao, et al, an Expanded Palette of Genetically Encoded Ca2+ indicators, science, 2011). Their sequences are available from NCBI databases or adedge databases. It will be appreciated by those skilled in the art that any other cyclically rearranged fluorescent protein may also be used in the present invention, including, but not limited to, cyclically rearranged green fluorescent protein, red fluorescent protein, infrared fluorescent protein, yellow fluorescent protein, blue fluorescent protein, etc., such as cyclically rearranged green fluorescent protein (cpGFP), cyclically rearranged superfolder GFP, cyclically rearranged mApple (cpmApple), cyclically rearranged mCherry (cpmCherry), cyclically rearranged mKate (cpmKate), cyclically rearranged enhanced green fluorescent protein (cpGFP), cyclically rearranged Venus (cpVenus), cyclically rearranged Citrin (cpCitrine), cyclically rearranged enhanced yellow fluorescent protein (cpEYFP), and cyclically rearranged infrared fluorescent protein (cp infrared fluorescent protein, cpfp, see Daria M Shcherbakova, et al, near-infrared fluorescent proteins for multicolor in vivo imaging, nature methods,2013;Pandey N,et al,Tolerance of a Knotted Near-Infrared fluorescent protein to random circular permutation, biochemistry, 2016), without being limited to the above cpGFP and cpapple. Wherein the excitation wavelength of cpiRFP is longer, thus having better tissue penetration and being less affected by tissue autofluorescence.

In some embodiments of the invention, a circularly rearranged fluorescent protein cpEGFP from GCaMP6s is used, the specific sequence of which is:

NVYIKADKQKNGIKANFHIRHNIEDGGVQLAYHYQQNTPIGDGPVLLPDNHYLSVQSKLSKDP NEKRDHMVLLEFVTAAGITLGMDELYKGGTGGSMVRKGEELFTGVVPILVELDGDVNGHKFSVSG EGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYIQERTI FFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYN(SEQ ID NO:11)。

in some embodiments of the invention, a cyclic rearranged fluorescent protein cpmeapple is used, the specific amino acid sequence of which is:

PVVSERMYPEDGALKSEIKKGLRLKDGGHYAAEVKTTYKAKKPVQLPGAYIVDIKLDIVSHNE DYTIVEQCERAEGRHSTGGMDELYKGGTGGSLVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIE GEGEGRPYEAFQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYIKHPADIPDYFKLSFPEGFRWERV MNFEDGGIIHVNQDSSLQDGVFIYKVKLRGTNFPPDGPVMQKKTMGWEATR(SEQ ID NO:12)。

in addition, in the present invention, a circularly rearranged luciferase (cp luciferase) may be used instead of a fluorescent protein, and the conformational change of the receptor is involved in the folding change of the luciferase, thereby changing the activity of the catalytic substrate thereof, by applying the same principle.

Suitable available cyclically rearranged fluorescent proteins can be readily determined experimentally for a particular G protein-coupled receptor. For example, the suitability of an inserted cyclically rearranged fluorescent protein may be determined by detecting whether the GRAB probe is capable of folding correctly after insertion of the cyclically rearranged fluorescent protein and detecting whether binding of the GRAB probe to its ligand results in a change in the intensity of the fluorescent signal.

The fluorescent probe constructed based on the G protein-coupled receptor (i.e., GRAB probe) of the present invention should be capable of being expressed on a cell membrane. Methods for detecting whether the probe is capable of being expressed on a cell membrane are well known to those skilled in the art, for example, by expressing the probe in a cell (e.g., HEK293T cells) and analyzing the expression morphology of fluorescent proteins in the cell, wherein the proteins expressed on the cell membrane are formed as a thin circle around the outermost periphery of the cell, and the contours of the cell can be obtained by comparing the fluorescent channel with the open field channel, and then analyzing the result. Probes that fail to normally coat are often clustered in cells, which under a microscope are clustered signals within the cell. Quantitative measurements can also be made by expressing another protein that is known to localize to the cell membrane, by calculating the co-localization of the fluorescent probe signal with the protein.

The fluorescent probe constructed based on the G protein-coupled receptor according to the present invention should be capable of binding to the specific ligand of the G protein-coupled receptor when it is contacted therewith, thereby resulting in a detectable change in the fluorescent intensity of the probe. Methods for detecting this are known to those skilled in the art, and for example, the probe may be contacted with a ligand specific for the G protein-coupled receptor, then the fluorescent probe may be detected for a fluorescent response to a particular ligand by performing fluorescence imaging on cells expressing the fluorescent probe, performing continuous photographing recording before and after the addition of the ligand, and analyzing the change in fluorescence intensity recorded before and after the addition of the ligand.

In the present invention, the term "a detectable change in fluorescence intensity" means that the GRAB probe has a fluorescence intensity after binding to the ligandVarying ΔF/F ₀ The absolute value of (c) is 5% or more, 10% or more, 15% or more, 20% or more, 30% or more, 60% or more, 70% or more, 80% or more, 90% or more, 100% or more, 200% or more, 300% or more, 350% or more, 500% or more, or even more. The change may be an increase in fluorescence intensity or a decrease in fluorescence intensity. The greater the change in fluorescence, the more excellent the properties of the probe, and the greater the likelihood of being used for intracellular detection.

The "GRAB probe" described in the present invention shows a change in fluorescence intensity ΔF/F after binding to a ligand ₀ "refers to the relative change in fluorescence intensity of the GRAB probe after binding to the ligand relative to that before binding to the ligand, wherein F ₀ Refers to the average fluorescence value before the GRAB probe binds to the ligand, and ΔF refers to the average fluorescence value F after the GRAB probe binds to the ligand and the average fluorescence value F before the GRAB probe binds to the ligand ₀ Is (Δf=f-F) ₀ ). In the present invention, Δf may also be referred to as dF.

As used herein, a "ligand" or "specific ligand" of a G protein-coupled receptor is used interchangeably and refers to a molecule capable of binding to and activating (or inhibiting) the G protein-coupled receptor, including photoactive compounds, odors, pheromones, hormones and neurotransmitters. The binding of the G protein-coupled receptor to its ligand is highly specific, with the ligand binding only to a specific receptor, which also binds only to a specific ligand structure. The specificity of binding of a G protein-coupled receptor to its ligand means that the binding affinity of the G protein-coupled receptor to the ligand is significantly higher than to one or more other molecules. "significantly higher than" in "may mean having a statistical significance. Ligands to which different G protein-coupled receptors can bind, or G protein-coupled receptors to which different ligands can bind, are well known to those skilled in the art.

The ligand described in the present invention may be a natural ligand or a synthetic ligand. A natural ligand refers to a molecule that naturally occurs in vivo and binds to a G protein-coupled receptor in vivo. An artificially synthesized ligand refers to a molecule which does not naturally exist in the body and is combined with a G protein-coupled receptor in the body, and the artificially synthesized ligand can be an analogue of the natural ligand, can be an agonist or an antagonist of the G protein-coupled receptor, and can be used as a potential drug for activating or inhibiting the G protein-coupled receptor.

In some embodiments of the invention, in the GRAB probe, a third intracellular loop between the fifth and sixth transmembrane regions of the G protein coupled receptor is truncated and a circularly rearranged fluorescent protein is inserted at the truncated position.

By "truncated" is meant that a portion of the sequence is deleted. "truncated and insertion of a cyclically rearranged fluorescent protein at a truncated position" refers to the replacement of a deleted partial sequence with a cyclically rearranged fluorescent protein.

In some embodiments of the invention, the circularly rearranged fluorescent protein is linked at both ends to a third intracellular loop of a G protein-coupled receptor via a linker peptide, respectively.

The terms "linker peptide" or "linker peptide fragment" as used herein are used interchangeably and refer to a short peptide of a fluorescent protein that links to the third intracellular loop of a G protein-coupled receptor and is rearranged in circulation. In the present invention, since the circularly rearranged fluorescent protein is inserted into the third intracellular loop of the G protein-coupled receptor, "connecting peptide" as described herein includes an N-terminal connecting peptide located at the N-terminal of the circularly rearranged fluorescent protein and a C-terminal connecting peptide located at the C-terminal of the circularly rearranged fluorescent protein. In the present invention, the function of the linker peptide is to assist in the proper folding of the fusion protein while bridging the transfer between the conformational change of the receptor and the change in brightness of the fluorescent protein. The connecting peptide used should therefore be one which can perform the function described. The selection of the linker peptide can be determined by various methods known in the art to detect whether the GRAB probe is properly folded and whether binding of the GRAB probe to its ligand results in a change in fluorescence signal intensity. When the circularly permuted fluorescent protein is inserted into a third intracellular loop of the G protein coupled receptor, an N-terminus of the circularly permuted fluorescent protein may be linked to the third intracellular loop through an N-terminal linker peptide, and a C-terminus of the circularly permuted fluorescent protein may be linked to the third intracellular loop through a C-terminal linker peptide. In the present invention, it is allowed to express a connecting peptide fragment used in a probe in such a manner that "N-terminal connecting peptide-C-terminal connecting peptide".

In the present invention, the connecting peptide may include or consist of a flexible amino acid. The "flexible amino acids" are typically amino acids with smaller side chains that do not affect the conformation of the fusion protein. The flexible amino acids of the present invention may include glycine and alanine.

In the present invention, the amino acids constituting the connecting peptide include, but are not limited to, flexible amino acids, but may include other amino acids, and a person skilled in the art can verify whether or not a connecting peptide composed of different amino acids is feasible by an appropriate means.

In some embodiments of the invention, the specific ligand is a neurotransmitter, including, but not limited to, epinephrine, norepinephrine, acetylcholine, serotonin, and/or dopamine. The G protein-coupled receptor is a G protein-coupled receptor that specifically binds neurotransmitters, such as, but not limited to, G protein-coupled receptors that specifically bind epinephrine, norepinephrine, acetylcholine, serotonin, and/or dopamine.

There are two main classes of adrenergic receptors, one class being the alpha receptor (e.g., the human ADRA2A receptor), and the affinities for epinephrine and norepinephrine are similar. Another broad class is the beta receptors (e.g., human beta 2 adrenergic receptors), which have a higher affinity for epinephrine and about 10-100 times lower affinity for norepinephrine. In vivo, epinephrine mediates mainly in peripheral cardiovascular system and norepinephrine mainly in brain.

In some embodiments of the invention, a G.alpha.protein peptide is further coupled at the C-terminus of the fluorescent probe, which can successfully compete for endogenous G proteins to significantly reduce G protein signaling pathway coupling, so that GRAB probes are free from disturbances that result in significant cell signaling systems when expressed intracellularly.

As used herein, a "G.alpha.protein peptide" refers to the 20 amino acids at the carbon end of the G protein, which belongs to the alpha subunit of the G protein. The alpha subunit of G proteins includes three major classes: αs, αi, αq. The "gαq, gαs, gαi" and "Gq, gs, gi" described herein are used interchangeably. The G alpha protein peptide segment can be 20 amino acids of the carbon end of any G protein. In some preferred embodiments, the gα protein peptide fragment may have the following sequence: VFAAVKDTILQLNLKEYNLV (Gαq20, SEQ ID NO: 6). In other preferred embodiments, the gα protein peptide fragment may have the following sequence: VFNDCRDIIQRMHLRQYELL (Gαs20, SEQ ID NO: 7). In other preferred embodiments, the gα protein peptide fragment may have the following sequence: VFDAVTDVIIKNNLKDCGLF (Gαi20, SEQ ID NO: 8).

In some embodiments of the invention, a luciferase is further inserted at the C-terminus of the fluorescent probe. When the ligand is combined with the GRAB probe, the structure of the receptor is changed, the structural change can change the space distance and the relative position of the luciferase at the C end and the fluorescent protein in the circular rearrangement of the third intracellular ring, and the resonance energy transfer efficiency between the luciferase and the fluorescent protein is changed, so that the fluorescent signal of the fluorescent protein is changed, and the fluorescent probe can image under the condition of no external excitation light.

The term "luciferase" as used herein refers to an enzyme capable of oxidizing luciferin (a naturally occurring fluorophore) to emit light energy. A number of different luciferases and luciferin/luciferase systems are well known to those skilled in the art. Luciferases useful in the present invention include, but are not limited to, nanoluc, firefly luciferase (firefly luciferase, FLuc), renilla luciferase (Renilla luciferase, RLuc).

Luciferases useful in the present invention are those that catalyze substrates that fluoresce at wavelengths close to the excitation wavelength of the fluorescent protein that is cyclically rearranged in the GRAB probe of the present invention. For different cyclically rearranged fluorescent proteins, different luciferases may be used if their excitation wavelengths are different. For example, the excitation wavelength of the cpEGFP in the present invention is 488nm, so when cpEGFP is used in the GRAB probe, luciferases that can be used include renilla luciferase, which has coelenterazine as a substrate and emits light at 480 nm; also included are Gaussia luciferases which use coelenterazine as a substrate to emit light at 470 nm.

In analysis of the crystal structure of GPCRs before and after the activation process, it is hypothesized that conformational changes of GPCRs may be split into two steps. Wherein the first step is a conformational change in the transmembrane region of the receptor (fifth and sixth transmembrane regions) resulting from ligand binding and the second step is a conformational change in the transmembrane region of the receptor involving the opening of the intracellular loop to expose the binding region of the G protein. For different receptors, it triggers different degrees of conformational change of the transmembrane region due to specific ligands (Kruse, A.C.et al. Activation and allosteric modulation of a muscarinic acetylcholine receptor. Nature 504,101-106 (2013); wacker, D.et al. Structural features for functional selectivity at serotonin receptors. Science (New York, N.Y.) 340,615-619 (2013)). However, since endogenous G proteins are only of a few types, conformational changes in their intracellular loops are likely to have a large degree of homology (Rasmussen, S.G.F.et al crystal structure of the β adrenergic receptor-Gs protein complex. Nature 477,549-555 (2011); kruse, A.C.et al Structure and dynamics of the M3 muscarinic acetylcholine receptor. Nature 482,552-556 (2012); huang, W.et al structure insights into micro-optical receptor activity 524,315-321 (2015)). Thus, in the present invention, conservation of conformational changes in the intracellular loops may be exploited to replace the corresponding intracellular loops of other receptors with the intracellular loops of GPCR receptors that have been successful in eliciting a change in the brightness of fluorescent proteins, while leaving the regions of the receptor that bind the ligand exogenously unchanged. By constructing chimeric receptors, on one hand, the good coupling of the receptors and fluorescent proteins can be utilized to expand the feelings of different neurotransmitters by the probes, and on the other hand, the different receptors are combined with ligands to cause different degrees of conformational change, so that the chimeric receptors can be used as natural screening libraries to possibly improve the signal to noise ratio of the probes.

Accordingly, the present invention also provides a method of constructing a GRAB probe, comprising completely cleaving a third intracellular loop of a fluorescent probe constructed based on a first G protein-coupled receptor together with a circularly rearranged fluorescent protein inserted therein, replacing the third intracellular loop of a second G protein-coupled receptor, and obtaining a fluorescent probe constructed based on the second G protein-coupled receptor.

As previously described, the N-terminus, transmembrane region, intracellular loop and C-terminus of a G protein coupled receptor can be readily determined by one skilled in the art.

In some embodiments, the first G-protein coupled receptor and the second G-protein coupled receptor may bind to the same specific ligand or to different specific ligands.

The invention also relates to polynucleotides encoding the GRAB probes of the invention, expression vectors comprising the polynucleotides, and host cells comprising the polynucleotides or expression vectors.

The term "expression vector" refers to an expression vector capable of expressing a protein of interest in a suitable host cell, and is a genetic construct comprising operably linked essential regulatory elements that allow for the expression of the inserted gene. Preferably, the recombinant vector is constructed to carry a coding polynucleotide encoding a GRAB probe of the invention or a fragment thereof. The recombinant vector may be transformed or transfected into a host cell.

The expression vector of the present invention can also be obtained by ligating (inserting) the polynucleotide of the present invention into an appropriate vector. The vector into which the gene of the present invention is to be inserted is not particularly limited as long as it can replicate in a host. For example, plasmid vectors, phage vectors, virus vectors, and the like can be used. Specifically, a commercially available expression vector such as a pDisplay vector, which is available from invitrogen corporation, and animal viruses such as retrovirus, adenovirus and vaccinia virus and insect viruses such as baculovirus may be used, and the plasmid capable of being used in the present invention is not limited to the examples.

In order to operably link the polynucleotide of the present invention to a vector, the vector of the present invention may contain cis-elements such as an enhancer, a splicing signal, a Poly A (Poly A) addition signal (Poly A addition signal), a selection marker, and a ribosome binding sequence in addition to a promoter and the polynucleotide of the present invention.

The structured support may be transformed (orTransfected) into a host cell. The transformation may be performed using any method. In general, the conversion process has the following several methods: caCl (CaCl) ₂ A precipitation method; electroporation; a calcium phosphate precipitation method; a protoplast fusion method; carbonization baby fiber-mediated transformation method; agrobacterium (Agrobacterium) -mediated transformation; PEG-mediated transformation; dextran sulfate, cationic liposome (Iipofectamine), and drying/inhibiting transformation methods. The gene encoding the GRAB probe of the invention can be introduced into a host cell by the vector as described above and transfection using the vector.

The host cell used in the present invention is not particularly limited as long as it can express the GRAB probe of the present invention. In a preferred embodiment, the host cell is HEK293T. In other preferred embodiments, the host cell is a neuronal cell.

The invention also relates to a method for detecting whether the specific ligand of the G protein coupled receptor exists in a sample to be detected or a tissue to be detected by utilizing the GRAB fluorescent probe, a method for qualitatively detecting the concentration change of the specific ligand of the G protein coupled receptor in the sample to be detected or the tissue to be detected by utilizing the GRAB fluorescent probe, a method for quantitatively detecting the concentration change of the specific ligand of the G protein coupled receptor in the sample to be detected or the tissue to be detected by utilizing the GRAB fluorescent probe, a drug screening method and a method for detecting the distribution of the specific ligand of the G protein coupled receptor in an animal body. In these detection methods, it is necessary to measure a change in fluorescence signal intensity, thereby obtaining a detection result or a screening result.

It will be appreciated that the detection method of the present invention is to determine whether a ligand or agonist is present, and whether the ligand or agonist concentration is varied, by a change in the fluorescence intensity of the fluorescent probe. Wherein the change in fluorescence intensity may be an increase or decrease in fluorescence intensity. It should be readily ascertainable by one skilled in the art, given the benefit of this disclosure, how to determine whether a ligand or agonist is present, whether there is a change in ligand or agonist concentration, based on an increase or decrease in fluorescence intensity. For example, if the obtained fluorescent probe is an ON probe, i.e., a probe whose fluorescent signal is enhanced after addition of a ligand, then the presence of a ligand or an agonist, or an increase in the concentration of a ligand or an agonist can be judged as the fluorescent intensity increases during detection; when the fluorescence intensity is unchanged, it can be judged that no ligand or agonist is present, or the concentration of ligand or agonist is unchanged; when the fluorescence intensity is reduced, it can be judged that no ligand or agonist is present, or that the concentration of ligand or agonist is reduced. If the obtained fluorescent probe is an OFF probe, i.e., a probe whose fluorescent signal is reduced after addition of a ligand, then the presence of the ligand or agonist, or an increase in the concentration of the ligand or agonist can be judged when the fluorescent intensity is reduced during the detection; when the fluorescence intensity is unchanged, it can be judged that no ligand or agonist is present, or the concentration of ligand or agonist is unchanged; when the fluorescence intensity increases, it can be judged that no ligand or agonist is present, or that the concentration of ligand or agonist is reduced.

As used herein, "change in fluorescence signal intensity" may refer to a change in fluorescence signal intensity ΔF/F ₀ A change of 5% or more, 10% or more, 15% or more, 20% or more, 30% or more, 60% or more, 70% or more, 80% or more, 90% or more, 100% or more, 200% or more, 300% or more, 350% or more, or 500% or more. The change may be an increase in fluorescence intensity or a decrease in fluorescence intensity.

The "change in fluorescence signal intensity ΔF/F" described in the present invention ₀ "may refer to the relative fluorescence intensity change after change relative to before change, where F ₀ Mean fluorescence value before change, ΔF means mean fluorescence value after change F and mean fluorescence value before change F ₀ Is (Δf=f-F) ₀ )。

The "test sample" as described herein may include samples outside of a living organism, including but not limited to cell cultures or extracts thereof; a biopsy material obtained from a mammal or an extract thereof; and blood, saliva, urine, feces, semen, tears, or other bodily fluids or extracts thereof. The detection of the sample to be tested may be performed in vitro.

The "tissue to be measured" as described herein may include any tissue within an organism, including but not limited to heart tissue, brain tissue, and the like. The detection of the tissue to be examined may be performed in vivo.

In the present invention, human muscarinic acetylcholine receptor M ₃ The R subtype is also known as the M3R subtype of the human acetylcholine receptor, M3R type receptor, M3R or M ₃ R, CHRM3, chrm3, etc.

In the present invention, serotonin is also referred to as 5-hydroxytryptamine.

The method of any of the embodiments of the invention may be performed in vitro or in vivo.

The method of any of the embodiments of the invention may be non-therapeutic.

It will be appreciated that the fluorescent probes of the invention may incorporate a cyclically rearranged fluorescent protein at a different position within the third intracellular loop of the GPCR, and that the two ends of the incorporated fluorescent probe may be linked to the third intracellular loop of the GPCR by different linking peptides, and that the cyclically rearranged fluorescent protein used may be a variety of different cyclically rearranged fluorescent proteins. Thus, in the present invention, different cyclically rearranged fluorescent proteins, different insertion sites on the third intracellular loop, and different connecting peptides may be combined with each other, and the resulting various combinations are within the scope of the present invention.

Furthermore, it is to be understood that in the present invention, when referring to a value or range, the term "about" is used to mean within 20%, within 10%, or within 5% of the given value or range.

Abbreviations used in the present invention include:

GPCR G protein coupled receptor

EGFP enhanced green fluorescent protein

GFP green fluorescent protein

YFP yellow fluorescent protein

RFP red fluorescent protein

CFP blue fluorescent protein

cp cyclic rearrangement (when abbreviated as fluorescent protein thereafter, e.g., cpEGFP is a cyclic rearranged enhanced green fluorescent protein)

Epi epinephrine

NE norepinephrine

ISO isoprenaline

Ach acetylcholine

ICI or ICI118,551 beta 2 adrenergic receptor specific blocker

Tio (tiotropium bromide) tiotropium bromide

AF-DX384 or AF-DX muscarinic acetylcholine receptor antagonists

5-HT 5-hydroxytryptamine

GABA gamma-aminobutyric acid

DA dopamine

Gly glycine

Glu glutamic acid

ACSF artificial cerebrospinal fluid

PTX pertussis toxin

DAN dopaminergic neurons

MB mushroom

Example 1 materials and methods

1. Molecular cloning for GRAB probe construction and mutation screening

In the present invention, all molecular clones were cloned by the method of Gibson assembly (Gibson, D.G.et al. Enzymic assembly of DNA molecules up to several hundred k ilobs. Nature methods 6,343-345 (2009)), i.e.recombinant ligation of homologous fragments was achieved by sequence complementation. Efficient splicing between sequences is achieved using homologous complementary sequences of about 30 bases designed to fit on the primers. All recombining correct clones were sequenced at the equipment center of the college of university of Beijing student's life sciences.

The GRAB probe expression vector used was a pDISPLAY vector from Invitrogen corporation. The GPCR gene part is amplified in full-length human genome cDNA (hORFeome database 8.1.1), firstly transferred to a final vector with att sequence constructed by a pDISPLAY vector by a Gateway cloning method, and then inserted into a specific position of a receptor by a Gibson assembly method. The different fluorescent proteins used in the optimization of the GRAB probes were amplified in their corresponding fusion proteins, with G-GECO (see Yongxin Zhao, et al, an Expanded Palette of Genetically Encoded Ca2+ indicators, science, 2011) being provided by Robert Campbell teaching laboratories, ASAP1 (see Francois St-Pierre, et al, high-fidelity optical reporting of neuronal electrical activity with an ultrafast fluorescent voltage sensor, nature Neuroscience, 2014) being provided by Michael Lin teaching laboratories, and GCaMP6 (Chen TW, et al, ultrasensitive fluorescent peoteins for imaging neuronal activity, aperture, 2013) being obtained by laboratory self-mutagenesis from GCaMP5 according to literature. In the mutation screening process of the probe, a mutation introducing method is to introduce random base combinations into specific primers so as to construct a site-directed mutation library.

GPCR gene sequences can be obtained from NCBI database and Addgene database, and the web addresses are as follows:

NCBI:https://www.ncbi.nlm.nih.gov/

Addgene:https://www.addgene.org/

the construction method of the chimeric probe comprises the steps of amplifying a fragment of a specific receptor which does not contain a third intracellular loop and a third intracellular loop fragment of the GRAB probe through PCR amplification, and then performing sequence splicing through a Gibson assembly method to realize the construction of the chimeric probe. For different GPCRs, the sequence prediction of the third intracellular loop was based on UNIPROT database.

The fluorescent probe molecule based on receptor endocytosis is constructed by adopting a pDISPLAY carrier, specifically, the pHluorin gene is connected at the nitrogen end of the GPCR gene by a Gibson assembly method, and the pHluorin gene is connected by a short peptide segment of 3 amino acids (GGA) between the pHluorin gene and the pHluorin gene, so that the molecule is ensured to be folded correctly. To further enhance the coupling of the GPCR to the endocytic pathway, the last 29 amino acids (amino acids 343-371) of the human AVPR2 gene were fused at the end of the carbon end of the GPCR. This moiety has been shown to have high affinity for β -arestin and thus may enhance the coupling of GPCRs to endocytic signaling pathways.

The plasmid of the GRAB probe transgenic Drosophila was constructed by cloning the full-length GRAB probe into the Drosophila expression vector pUAST vector, which contains UAS sequences that can be regulated by the transcription factor Gal 4. After a large amount of plasmid is extracted from the GRAB probe Drosophila carrier, drosophila embryo injection and transgenic Drosophila screening are carried out by Fungene biotechnology company.

2. Cell culture and transfection

The selection and optimization of the GRAB probe was performed in the HEK293T cell line. HEK293T was cultured in DMEM (Gibco) medium with 10% fbs (north homobiotech) and in a 37 ℃ incubator with 5% carbon dioxide. According to the growth condition and density of the cells, the steps of carrying out cell passage once every two days and reserving one fourth of the passage are adopted for cell culture. When carrying out plasmid transfection and imaging experiments on HEK293T cells, the cells are firstly subjected to pancreatin digestion in 24-well plate holes on imaging circular slides and then are uniformly paved on the slides. In order to ensure uniformity of cells, the cells in the well plate are homogenized by a horizontal shaking method after the cells are inoculated. Plasmid transfection was performed 8-12 hours after passage of cells to the well plate, ensuring that cells were tightly adhered to the bottom of the slide and stretched. For HEK293T cell lines, PEI mediated plasmid transfection methods were used, specifically DNA and PEI were mixed in DMEM solution at a ratio of DNA: pei=1:4, and after resting for 15 minutes at room temperature, added to the cell solution to be transfected. After 4 hours of cell transfection, the cells were changed with DMEM solution containing 5% fbs and PEI was washed off to ensure good cell status. Expression and imaging of the GRAB probe was performed at about 36 hours after transfection.

The primary culture of rat neurons adopts a new born Sprague-Dawley rat, after the skin of the rat is cleaned by alcohol, the head of the rat is dissected by a surgical instrument, the vascular membrane on the surface of the cortex is carefully removed after the brain is taken out, and the cortex tissue is sheared and then placed in a 0.25% pancreatin solution to be digested for 10 minutes in a 37-DEG constant temperature incubator. After digestion, digestion was stopped with DMEM solution containing 5% fbs and the cells were further disrupted by slow pipetting ten times with a pipette. After standing for 5 minutes, the upper solution was aspirated, the sediment containing the tissue fragments was removed, and the solution was centrifuged at 1000rpm for 5 minutesAnd (5) a heart. The supernatant was then discarded, neurons were resuspended in neurobasal+b27 solution used to culture neurons, and density calculations were performed using a cell counter plate. After calculation of cell density, the cell density was calculated according to 0.5-1x10 ⁶ Cells/ml were density diluted and seeded onto polylysine (sigma) -plated slides. Primary neurons were cultured in neurobasal+b27 solution and half-changed every two days. The primary cultured neurons were transfected 6-8 days after dissection, using the calcium phosphate transfection method. After 1.5 hours from the time of cell transfection, the solution was observed by a microscope for the generation of small and uniform calcium phosphate precipitates, and the solution was changed with an HBS solution having a pH of 6.8. After HBS washing, neurons were re-placed in neurobasal+b27 medium for culture until imaging experiments were performed 48 hours later.

3. Fluorescence imaging of cells and drug perfusion

After introducing a specific GRAB probe DNA into cells by transfection, characterization was performed by fluorescence imaging in combination with perfusion experiments. Imaging experiments on HEK293T cells fluorescence imaging was performed using an Olympus IX81 inverted microscope, using a 40 XNA: 1.35 oil mirror and excitation light filters of 475/28 and emission light filters of 515 LP. The optical signal was collected by Zyla sCMOS DG-152V-C1e FI camera (Andor) using the Lambda DG-4 of Sutter Instuments company as the fluorescent light source. The exposure time was set at 50ms, with an acquisition frequency of imaging every 5 seconds. The whole imaging system realizes overall control through micromanager software.

Imaging experiments of neurons employed an inverted Nikon laser scanning confocal fiberscope, which is a microscope based on an inverted Ti-E microscope and an A1Si spectral detection confocal system. Imaging was performed using a 40 XNA 1.35 oil mirror and 488 laser. The microscope body, PMT and image acquisition and processing system of the laser scanning confocal microscope are controlled by NIS element software.

Response detection of GRAB probe to ligand (. DELTA.F/F) ₀ ) The method is carried out by adopting a drug perfusion method. Cells were placed in standard physiological solutions, the solution formulation was:

NaCl	150mM
		KCl	4mM
MgCl ₂	2mM
		CaCl ₂	2mM
HEPES	10mM
		Glucose	10mM

After the pH value of the physiological solution is corrected to be about 7.4, the small molecular medicine is diluted by partial solution, and the solution with the corresponding concentration of the small molecular ligand is prepared. Isoprenaline ISO, ICI118,551 and AF-DX in small molecules of the drug are purchased from sigma company, acetylcholine is purchased from solabio company, tiotropium Bromide is purchased from Yu Dexin good company. If not explicitly stated, the ISO perfusion concentration of isoprenaline is 2 μm and the acetylcholine perfusion concentration is 100 μm.

The perfusion system is arranged on a microscope and comprises a solution introducing system, a multi-way valve, an imaging workbench and a liquid suction pump, wherein the solution introducing system is made of an injection needle tube. In the perfusion process, an imaging workbench is arranged above an objective lens of an inverted microscope, a slide inoculated with cells is placed in the workbench, and perfusion experiments of different medicines are carried out by controlling the switches of different pipelines. In order to ensure the solution level in the table, the perfusion rate is set to be about one drop per second. After the cells are treated by the medicine each time, the cells are washed by adopting physiological solution for more than five minutes, so that the experiment after no residual medicine affects is ensured. After each experiment is finished, the perfusion pipeline and the workbench are washed three times by 75% ethanol, so that residual medicines and impurities are ensured to be sufficiently cleaned.

Aiming at the detection of GRAB probe dynamics, a local spray irrigation drug system is adopted for experiments. The experiment was performed using Olympus front-facing micro-mirror BX51, using 40xNA: imaging was performed with a 0.80 water mirror and image acquisition was performed with a 710M camera (DVC). The medicine spray irrigation system is controlled by ROE-200 of Sutter instruments company, and the position of the medicine spray irrigation needle is controlled by MPS-1 operation rod. In the dynamic characterization experiments of the probe, imaging was performed using a frequency of 50HZ with a resolution of 768x484 pixels and a 2x2 binning.

4. Detecting probe performance using a fluorogenic microplate reader

For the GRAB probe, measurements were made using a fluoroenzyme-labeled instrument for corresponding curves of different neurotransmitter concentrations. The enzyme-labeled instrument is a Safire2 full spectrum scanner of the company TECAN. Cells were first plated on average in 96-well plates pre-treated with polylysine and transfected using the PEI method. Before measuring fluorescent signals, firstly, the physiological solution is used for changing the liquid of the cells, and the interference of the culture medium on the fluorescent signal acquisition is removed. 480 nanometers is adopted as excitation wavelength, 520 nanometers is adopted as emission wavelength, and fluorescence values of cells in physiological solution and after specific drugs are added are respectively read. The experiment adopts the addition of a small amount of drug solution with 100 times of final concentration so as to avoid the change of fluorescence signals caused by the change of the liquid level of the solution. When detection screening is performed on different probes, 6 hole cells are repeated for each probe, and the average value is taken to reduce fluorescence change caused by noise.

5. Imaging of two-photon living fruit fly

Drosophila is fed in a 25-DEG incubator by adopting a standard culture medium, and after the UAS-GRAB transgenic Drosophila is hybridized with a GH146-Gal4 strain, the Drosophila which shows a stronger fluorescent signal is selected for odor treatment experiments. Adult Drosophila to be tested was transferred to a new culture tube 0-2 days after emergence and left at room temperature for 8-12 days. Before the imaging process, the living drosophila is first fixed in a small dish, and then the eye tip square skull part is removed by operation, and part of the brain is exposed. Adipose tissue and air sac in the vicinity of the imaged antennal nerve lobes are surgically removed to prevent interference with the fluorescent signal. In order to further reduce the reduction of imaging quality caused by the movement of the drosophila in the imaging process, the muscles below the balance side of the drosophila are sheared by using surgical forceps. The Drosophila brain is in precooled physiological solution during the whole dissection and imaging process, and the formulation is as follows.

NaCl	108mM
		KCl	5mM
HEPES	5mM
		Trehalose	5mM
Sucrose	5mM
		NaHCO ₃	26mM
NaH ₂ PO ₄	1mM
		CaCl ₂	2mM
MgCl ₂	2mM

Drosophila imaging experiments were performed using an Olympus two-photon microscope. Specifically included are the olympus BX61 WI microscope, 25xNA: 1.05, and a Ti-sapphire mode-locked laser performs two-photon excitation. For imaging experiments with GRAB probes, the wavelength of the emitted light was set to 950 nm to successfully excite the fluorescent protein to produce fluorescence. The odor molecule Isoamyl Acetate (IA) used to stimulate drosophila was purchased from sigma corporation and diluted in mineral oil in a 1:100 ratio, and was further diluted in the experiment by 5-40 times concentration by mixing with the air stream. The air stream mixed with the odor molecules was applied to a position about 1 cm away from the tentacle of the drosophila through a hollow space about 1 cm wide on the laboratory bench, and experiments were performed by controlling the air stream to carry out different concentrations of odor molecules. Each round of imaging process was 17.8 seconds, with between 5 and 10 seconds providing specific odor molecules. After the odor function imaging experiment, the imaging area is scanned layer by adopting high resolution to obtain the distribution information of the olfactory bulb, and the distribution of the olfactory bulb at the antennal nerve leaves is identified according to the reported literature.

6. Image data processing

Fluorescence imaging data were processed using ImageJ software. For fluorescence expression of the GRAB probe in HEK293T cell lines and neurons, whole cell bodies were selected as data-treated regions. For live Drosophila imaging, fluorescence acquisition images at the same Z axis were analyzed by imageJ software. The change of the fluorescence signal is indicated by the relative change, the fluorescence signal is firstly subtracted from the background area without probe expression, thereby obtaining the real expression of the fluorescence protein intensity, and then the fluorescence value F after adding the medicine and the average fluorescence value F before adding are calculated ₀ Obtaining the relative fluorescence change value delta F/F= (F-F) ₀ )/F ₀ As a fluorescent response of the probe to the particular drug. ΔF/F ₀ The change over time was in turn plotted in the Origin 8.6 software.

7. Statistical testing

In the present invention, the data pattern shown in the graph is the mean ± mean standard error.

The materials and methods described in this example are applicable to examples 2-7 below, unless specifically stated otherwise. In other embodiments, materials and methods not explicitly mentioned are employed, unless contradicted by materials and methods described in these embodiments.

EXAMPLE 2 construction of epinephrine-specific and acetylcholine-specific fluorescent probes

1. Insertion of fluorescent proteins into different sites on the beta 2adrenergic receptor to find fusion with correct localization Proteins

A specific fluorescent probe for epinephrine was constructed based on human β2adrenergic receptor (Rasmussen, S.G.F.et al crystal structure of the β2adrenergic receptor-Gs protein complex. Nature 477,549-555 (2011); cherezov, V.et al high-Resolution Crystal Structure of an Engineered Human $\ betaG Protein Coupled receptor. Science 318,1258-1265 (2007)), the fluorescent protein was a circularly rearranged fluorescent protein cpEGFP used in G-GECO.

The sequence of the human β2adrenergic receptor is referenced by NCBI gene ID:154, the links are https:// www.ncbi.nlm.nih.gov/gene/154, and the specific amino acid sequences are:

MGQPGNGSAFLLAPNRSHAPDHDVTQQRDEVWVVGMGIVMSLIVLAIVFGNVLVITAIAKFER LQTVTNYFITSLACADLVMGLAVVPFGAAHILMKMWTFGNFWCEFWTSIDVLCVTASIETLCVIAVD RYFAITSPFKYQSLLTKNKARVIILMVWIVSGLTSFLPIQMHWYRATHQEAINCYANETCCDFFTNQA YAIASSIVSFYVPLVIMVFVYSRVFQEAKRQLQKIDKSEGRFHVQNLSQVEQDGRTGHGLRRSSKFCLKEHKALKTLGIIMGTFTLCWLPFFIVNIVHVIQDNLIRKEVYILLNWIGYVNSGFNPLIYCRSPDFRIAF QELLCLRRSSLKAYGNGYSSNGNTGEQSGYHVEQEKENKLLCEDLPGTEDFVGHQGTVPSDNIDSQ GRNCSTNDSLL(SEQ ID NO:1)。

wherein the underlined section is the third intracellular loop.

The cpgfp used in this example is the cpgfp in GCaMP6s, and the specific sequence is:

NVYIKADKQKNGIKANFHIRHNIEDGGVQLAYHYQQNTPIGDGPVLLPDNHYLSVQSKLSKDP NEKRDHMVLLEFVTAAGITLGMDELYKGGTGGSMVSKGEELFTGVVPILVELDGDVNGHKFSVSGE GEGDATYGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYIQERTIFF KDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYN(SEQ ID NO:11)。

the cyclic rearranged fluorescent protein insertion site was initially selected as each peptide loop starting from the nitrogen to the carbon end of the human β2adrenergic receptor. After obtaining the fusion protein expression vector inserted at different sites, it was transfected into HEK293T cell line and observed for good fluorescence intensity and membrane distribution. After insertion of fluorescent protein at some sites, the fluorescent protein fused to the cell is in net distribution due to incorrect folding and transportation, and may be in misfolded protein residing in endoplasmic reticulum, as shown in fig. 1, two fusion proteins with good fluorescent intensity and cell membrane fluorescent distribution are obtained by screening, namely fusion protein inserted after 140 th amino acid of the second intracellular loop of the receptor (140 th amino acid of the whole receptor) and fusion protein inserted after 240 th intracellular loop of the receptor (240 th amino acid of the whole receptor).

2. Detection of correctly folded probes by fluorescence imaging in combination with drug perfusion may be receptive to neurotransmitter production Optical signal variation

After finding a fusion protein that can be localized on the cell membrane, it is further examined whether it can convert the conformational change of the receptor during activation into a change in fluorescence intensity. For two fusion proteins obtained by the above screening with good fluorescence intensity and cell membrane fluorescence distribution, HEK293T cells transfected with them were perfused with solutions containing the β2 adrenergic specific agonist isoprenaline ISO, respectively, and observed before agonist additionWhether there is a change in fluorescence intensity. One of them (insertion of fluorescent protein after 240 th position of receptor) showed a small but reversible increase in fluorescence upon addition of 2. Mu.M ISO, with an average change of about 6% (ΔF/F) ₀ ) (FIG. 2), the fluorescent probe was designated GRAB-EPI 0.1, which has the ability to detect epinephrine and its analogs.

3. Optimizing the insertion of a circulating rearranged fluorescent protein and fluorescent protein into the third intracellular loop of a human adrenergic receptor Site(s)

The cyclically rearranged fluorescent protein cpEGFP (Chen, t.—w.et al ultrasensitive fluorescent proteins for imaging neuronal activity. Nature 499,295-300 (2013) (GCaMP 6s, GCaMP6 m), zhao, y.et al Expanded Palette of Genetically Encoded Ca2 +indicators. Science 333,1888-1891 (2011) (GECO 1.2), the sequences of which are available from NCBI database or addgene database, GECO1.2 is one version of G-GECO, GCaMP6s/f/m is three different sub-versions of GCaMP 6), and cyclically rearranged superfolder GFP (p-Pierre, f.et al, hi-fidelity optical reporting of neuronal electrical activity with an ultrafast fluorescent voltage sensor. Nature 8817), used in voltage-sensitive fluorescent probe ASAP1, and St-250 amino acids inserted at position 2019 to the adrenaline receptor after 250. HEK293T cells were transfected with the constructed fusion protein expression vector, as shown in FIG. 3, fluorescent probes constructed using different cpEGFP could all be successfully folded and transported onto the cell membrane, and exhibited similar fluorescence intensity changes under treatment with the same concentration (2. Mu.M) of agonist ISO. However, fluorescent probes constructed using cyclically rearranged superfolder GFP have difficulty in correctly folding and transporting membranes, which are manifested as aggregation in intracellular structures such as the endoplasmic reticulum, as shown by the arrows in fig. 3, which results in their inability to exhibit a change in fluorescent signal upon ligand binding. After comprehensively considering the brightness of fluorescent protein, the folding of the probe, the membrane expression and the signal change of the probe, the circularly rearranged cpEGFP from GCaMP6s was selected as the fluorescent protein used in the GRAB probe.

The cyclic rearranged cpgfp from GCaMP6s was inserted at a different insertion site in the third intracellular loop of the human β2 adrenergic receptor. After cloning and expression of the respectively obtained fusion proteins in HEK293T cells, perfusion treatment was performed with the same concentration (2 μm) of agonist ISO and its fluorescence change before and after addition of agonist was observed by fluorescence imaging. As shown in FIG. 4, the increase in fluorescence of the probe obtained after insertion of the fluorescent protein into the 250 amino acid position of the receptor was more remarkable, and it was possible to reach 15% ΔF/F under ISO treatment at the same concentration (2. Mu.M) ₀ The probe was designated GRAB-EPI 1.0.

4. Construction of acetylcholine fluorescent probes by methods of constructing chimeric receptors

Based on the successfully constructed adrenergic fluorescent probe GRAB-EPI 1.0 (beta. In the upper panel of FIG. 5) ₂ AR), the third intracellular loop thereof was excised along with the inserted circulating rearranged fluorescent protein (fig. 5), and the human muscarinic acetylcholine receptor (M _1-5 R) a corresponding third intracellular loop (ICL 3) moiety to allow insertion of conformationally sensitive cpEGFP into the human muscarinic acetylcholine receptor (M) _1-5 In the third intracellular loop of all five subtypes of R) (FIG. 6 a), a fluorescent probe M of the corresponding neurotransmitter is constructed _1-5 R-β ₂ R ICL3-cpEGFP chimera.

Wherein:

M ₁ r sequence refers to NCBI gene ID 1128;

M ₂ r sequence refers to NCBI gene ID 1129;

M ₃ r sequence refers to NCBI gene ID 1131;

M ₄ r sequence refers to NCBI gene ID 1132;

M ₅ the sequence of R refers to NCBI gene ID 1133.

The specific sequence of interception:

RVFQEAKRQLQKIDKSEGRFHVQNLSQVEQGGNVYIKADKQKNGIKANFHIRHNIEDGGVQLAYHYQQN TPIGDGPVLLPDNHYLSVQSKLSKDPNEKRDHMVLLEFVTAAGITLGMDELYKGGTGGSMVSKGEELFTGVVPILVE LDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYIQ ERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNGGAAADGRTGHGLRRSSKFCLKEHKALKT

wherein the underlined section is the inserted fluorescent protein sequence. Italics are connecting peptide segments.

To test M _1-5 R-β ₂ Whether R ICL3-cpEGFP chimeras are capable of detecting ACh, expressing them in HEK293T cells, wherein M is expressed ₃ R-β ₂ Cells of R ICL3-cpEGFP showed good membrane expression of the chimera, and increased fluorescence response (. DELTA.F/F) upon perfusion of ACh (100. Mu.M) ₀ ) (-30%) (fig. 5b, fig. 6b-f, indicated by arrows). These results indicate that they are derived from M ₃ The probe for R can detect ACh, which is designated GRAB-ACh 1.0.

M ₃ The specific sequence of the R receptor is as follows:

MTLHNNSTTSPLFPNISSSWIHSPSDAGLPPGTVTHFGSYNVSRAAGNFSSPDGTTDDPLGGHT VWQVVFIAFLTGILALVTIIGNILVIVSFKVNKQLKTVNNYFLLSLACADLIIGVISMNLFTTYIIMNR WALGNLACDLWLAIDYVASNASVMNLLVISFDRYFSITRPLTYRAKRTTKRAGVMIGLAWVISFVL WAPAILFWQYFVGKRTVPPGECFIQFLSEPTITFGTAIAAFYMPVTIMTILYWRIYKETEKRTKELAGLQASGTEAETENFVHPTGSSRSCSSY ELQQQSMKRSNRRKYGRCHFWFTTKSWKPSSEQMDQDHSSSDSWNNNDAAASLENSASSDEEDIGSETRAIYSIVL KLPGHSTILNSTKLPSSDNLQVPEEELGMVDLERKADKLQAQKSVDDGGSFPKSFSKLPIQLESAVDTAKTSDVNS SVGKSTATLPLSFKEATLAKRFALKTRSQITKRKRMSLVKEKKAAQTLSAILLAFIITWTPYNIMVLVNTFCDSCIPKTFWNLGYWLCYIN STVNPVCYALCNKTFRTTFKMLLLCQCDKKKRRKQQYQQRQSVIFHKRAPEQAL(SEQ ID NO:3)。

wherein the underlined part is the third intracellular loop (ICL 3), which ICL3 is defined with reference to the uniprot database as amino acids 253-491.

5. Optimizing the linking peptide between a cyclically rearranged fluorescent protein and a GPCR

The cyclic rearranged fluorescent protein cpEGFP and the third intracellular loop of the receptor are provided with a connecting peptide segment, wherein the connecting peptide segment is a manually added peptide segment consisting of a few amino acids, which can help the fusion protein to fold correctly and plays a bridge role in transferring the conformational change of the receptor and the brightness change of the fluorescent protein. According to the design principle of the cyclic rearrangement fluorescent protein, the position of the connecting peptide segment replaces the 145 th asparagine of the original fluorescent protein and has close interaction with a chromophore (Baird, G.S., zacharias, D.a. & Tsien, R.Y. Circular permutation and receptor insertion within green fluorescent proteins.proceedings of the National Academy of Sciences of the United States of America 96,11241-11246 (1999)).

In the previous step, in the construction of the adrenergic fluorescent probe, flexible amino acids (glycine, alanine) are used to form short connecting peptide segments to assist in the proper folding of the fusion protein. The peptide fragment has a length of 2 amino acids GG at the nitrogen end and 5 amino acids GGAAA at the carbon end. The linker peptide was cleaved from GRAB-EPI 1.0 together with ICL3 and grafted into an acetylcholine fluorescent probe.

The linker peptide was optimized based on GRAB-ACh 1.0. First, screening was performed for the length of peptide fragments based on flexible amino acids (glycine, alanine). The specific strategy is to change the peptide length of the nitrogen end and the carbon end to 0-5 amino acids respectively, and randomly combine the nitrogen end and the carbon end to obtain all possible arrangements. Fusion protein expression vectors containing various arrangements were expressed in HEK293T cells and perfusion experiments were performed, as shown in fig. 7, the number of amino acids at the nitrogen end had a decisive effect on whether the probe was fluorescent rising or falling after ligand addition, whereas the number of amino acids at the carbon end affected specific signal changes of the probe. Specifically, the probes can be classified into two types according to the change of fluorescence of the probes after ligand addition, one type is an ON probe with enhanced fluorescence signal after ligand addition, and the other type is an OFF probe with reduced fluorescence. According to the coupling principle of the fluorescent probe and the GPCR, the chromophore of the fluorescent protein in the ON probe is supposed to be in an exposed state before the ligand is added, so that the chromophore is attacked by water molecules to be quenched, and the fluorescent protein has lower fluorescence intensity; after ligand addition causes conformational change of the receptor, the intracellular loop is involved to initiate refolding of the fluorescent protein, thus protecting the chromophore well and enhancing its fluorescence emission intensity. Accordingly, the mechanism of the OFF probe may be reversed. In the screening results, ON probes were found only in probes with a nitrogen-terminal peptide length of 2 amino acids, whereas probes with a nitrogen-terminal peptide length of 1, 3, 4, 5 amino acids all appeared as OFF probes. For signal changes of the probe, the longer the carbon-end peptide fragment amino acid in the ON probe, the higher the signal change, while the shorter the carbon-end peptide fragment amino acid in the OFF probe, the higher the signal change. Combining the length screening of the connecting peptide fragments of the nitrogen end and the carbon end, the best combination of the peptide fragment lengths of the ON probe of 2-5 (namely, the N end is GG and the C end is GGAAA) is identified, and the best combination of the length of the OFF probe of 1-1 (namely, the N end is G and the C end is G).

Next, a combination of 2 to 5 in the length of the connecting peptide fragment was immobilized, and a probe having a larger signal variation was obtained by changing the amino acid type of the probe sequence. Site-directed mutagenesis was used to generate a library of 723 random point mutations on the 2-and 5-amino acid connecting peptides at the N-and C-termini of the cpEGFP of GRAB-ACh 1.0 (FIGS. 6C and 8). These mutants were then expressed in HEK293T cells, respectively, and screened for a large response to ACh perfusion (ΔF/F) ₀ ) Is a candidate for (a). Screening from them to identify the best ΔF/F ₀ (. About.70%) of the variant, designated GRAB-ACh 1.5 (the linker peptide sequence was N-terminal GG, carbon-terminal SPSVA) (FIG. 6 d). A second round of site-directed mutagenesis and selection was then performed using a combination of optimal connecting peptide residues (fig. 6c and fig. 8). After screening 23 combinatorial variants, it was identified that there was a maximum Δf/F at ACh perfusion ₀ An added variant, designated GRAB-ACh 2.0 (FIGS. 6c and 8), which uses GG-APSVA as the connecting peptide. Further analysis showed that GRAB-ACh 2.0 retained excellent expression and membrane uptake properties (FIG. 6 e), with an extended dynamic range (2.5 fold expansion) compared to GRAB-ACh 1.0 (FIG. 6F, g), and increased peak signaling response compared to FRET-based probes (GRAB-ACh 2.0: ΔF/F) ₀ =94.0±3.0%, with the FRET-based probe Δratio=6.6±0.4%, GRAB-ACh 2.0 being up to 20 times higher, the signal-to-noise ratio (SNR) is increased (up to 60 times) compared to the conventional FRET-based ACh probe (Markovic, d., et al FRET-based detection of M1 muscarinic acetylcholine receptor activation by orthosteric and allosteric agnists. Plos one 7, e29946 (2012)) (fig. 6i-j and fig. 9).

The whole ICL3 of GRAB-ACh 1.5 and the whole ICL3 of GRAB-ACh 2.0 were transplanted into GRAB-EPI 1.0 together with the cpEGFP and the connecting peptide therein, respectively, and instead of the third intracellular loop portion thereof, GRAB-EPI 1.1 and GRAB-EPI 2.0 were obtained, respectively, and their expression vectors were transfected into HEK293T cells, and a drug perfusion experiment was performed, in which the fluorescence of the probe increased by about 60% after the addition of agonist ISO (2. Mu.M) to GRAB-EPI 1.1 and about 70% after the addition of agonist ISO (2. Mu.M) to GRAB-EPI 2.0 was performed.

Spectral properties of fluorescent probes and their pH sensitivity were measured using HEK293T cells expressing GRAB-EPI 1.0, with a major excitation peak at around 490 nm and an emission peak at around 520 nm. In the case of triton treatment leading to cell membrane permeabilization, the different pH values of the external solution lead to a change in the brightness of the fluorescent protein with a pKa of about 7.0 (FIG. 10).

Example 3GRAB probes with ligand binding optical signal changes that result in receptor conformational change specificities

To determine that the fluorescence change of the probe was receptor activation specific, HEK293T cells expressing GRAB-EPI 1.0 and GRAB-ACh 1.0 in example 2 were treated with receptor specific blockers, respectively, and observed for the ability to eliminate ligand-induced fluorescence signal changes in the presence of the blocker. For single cells, experiments were first performed with the corresponding ligand, after an increase in signal value was observed, the ligand was then completely washed away, and the cells were again perfused with a mixed solution of blocking agent and ligand. The blocking agent is selected as follows: ICI against β2 adrenergic receptor (Rasmussen, s.g.f.et al crystal structure of the human beta, 2 adrenoceptor-coupled receptor. Nature 450,383-387 (2007)), tiotropium bromide (Tiotropium Bromide, tio) against M3 acetylcholine receptor (Wood, m.d.et al functional comparison of muscarinic partial agonists at muscarinic receptor subtypes hM1, hM2, hM3, hM4 and hM5 using microphysiometry. British journal of pharmacology, 1620-1624 (1999)). As a result, it was found that agonist ISO or ligand Ach did not cause an increase in fluorescence intensity in the presence of the blocker (fig. 11), which revealed that the blocker specifically blocked the receptor fluorescent probe from binding to each other with the agonist ISO or ligand Ach, so that the receptor fluorescent probe could not be activated, and thus there was no conformational change occurring after activation and no change in fluorescence intensity.

Mutation experiments were performed on the binding sites of the receptor and ligand to further verify that the fluorescence change of the probe was receptor activation specific. With respect to the beta 2 adrenergic receptor, amino acids 113 and 114 of the binding site between the receptor and the ligand in GRAB-EPI 1.0 are mutated, and HEK293T cells expressing the mutated probe are observed to respond to fluorescence of an agonist ISO. These two mutations have been shown to significantly reduce the affinity of the receptor and ligand (Del Carmine, R.et al. Mutations inducing divergent shifts of constitutive activity reveal different modes of binding among catecholamine analogues to the beta (2) -adonengic receptor. British journal of pharmacology, 1715-1722 (2002)). Cells expressing the mutant probe did not have any increase in fluorescence intensity under the action of agonist ISO. For M ₃ Type acetylcholine receptor, mutation at amino acid 506 of the ligand binding domain in GRAB-ACh 1.0 (Y506F), and HEK293T cells expressing the mutant probe were known to reduce ligand binding capacity by about ten times in fluorescence response to ligand acetylcholine (Wood, M.D.et al functional comparison of muscarinic partial agonists at muscarinic receptor subtypes hM1, hM2, hM3, hM4 and hM5 using microphysiometer.British journal of pharmacology, 1620-1624 (1999)). In the cells expressing the mutant acetylcholine probes, a decrease in affinity of acetylcholine to the probes of about 10-fold was observed, and the Kd value was decreased from 1. Mu.M to 10. Mu.M. (FIG. 12)

The above experiments show that the fluorescence signal change of the GRAB probe after ligand addition is indeed due to the conformational change that occurs after receptor activation, which involves a change in microenvironment of the cyclically rearranged fluorescent protein, resulting in an increase in fluorescence value.

Example 4GRAB probe has a ligand concentration dependent optical response

Ligand experiments were performed at different concentrations for the adrenergic probe GRAB-EPI 1.0 and the acetylcholine probe GRAB-ACh 1.0 (FIG. 13) using HEK293T cells expressing GRAB-EPI 1.0 and GRAB-ACh 1.0, respectively. It was found that it can exhibit concentration-dependent changes in fluorescence signal over a wide range of neurotransmitter concentration changes, with a curve conforming to the hill distribution. By calculating the Kd values of the curve and comparing the Kd values of the receptor for the ligand in the literature (Wacker, d.et al structure features for functional selectivity at serotonin receptors.science (New York, n.y.) 340,615-619 (2013), gainetdinov, r.r., premont, r.t., bohn, l.m., lefkowitz, R.J. & Caron, m.g. localization of G protein-coupled receptors and neuronal functions, annual review of neuroscience 27,107-144 (2004)), it was found that the GRAB probe did not alter the affinity of the receptor for the specific ligand. Ligand concentration dependent response curves indicate that the GRAB probe can sensitively and quantitatively detect different concentrations of the neural transmitter signal under physiological conditions.

This experiment demonstrates that a GRAB neurotransmitter probe based on receptor construction can not only qualitatively report the binding and concentration changes of neurotransmitters, but also quantitatively analyze the absolute concentration of neurotransmitters in a specific area.

Example 5 sub-second order kinetics and micromolar sensitivity of GRAB-ACh Probe detection

High-speed line scanning of membrane surface fluorescence signals of HEK293T cells expressing GRAB-ACh 2.0 delivered agonists or antagonists with a fast local perfusion system (-2,000 Hz/line) confocal imaging (FIG. 14 a-b.) local ACh perfusion elicited a rapid increase in fluorescence density of GRAB-ACh 2.0 expressing cells fitted with a single exponential function with a time constant of 280+ -32 ms (FIG. 14b-c, left panel.) local perfusion tiotropium bromide (Tio), muscarinic antagonists (AF-DX 384) (Casarosa, P., et al, preclonical evaluation of long-acting muscarinic antagonists: comparison of tiotropium and investigational drugs.the Journal of pharmacology and experimental therapeutics, 660-668 (2009)), reduced fluorescence signals with a slower time constant of 762+ -75 ms in GRAB-ACh 2.0 expressing cells perfusing 100. Mu.M ACh (FIG. 14b-c, right panel).

To determine the sensitivity of the sensor, fluorescence intensities of HEK293T cells expressing GRAB-ACh 2.0 were measured by perfusing solutions containing different concentrations of ACh (FIG. 14 d). The ACh concentration was increased from 10nM to 100. Mu.M and the fluorescence intensity of GRAB-ACh 2.0 expressing cells was gradually increased in relation to the concentration response, fitted with Boltzmann equation, EC ₅₀ About 0.7. Mu.M (FIG. 14 e), with wild type M ₃ R (WT-M ₃ R)(Jakubik,J.,Bacakova,L.,El-Fakahany,E.E.&Tucek, S.positive cooperativity of acetylcholine and other agonists with allosteric ligands on muscarinic acetylcholine receptors, molecular pharmacology 52,172-179 (1997)).

EXAMPLE 6 uncoupling of GRAB probes from downstream Signal channels

In this example, it was verified whether overexpression of fluorescent probes based on important signaling molecule GPCRs in cells would cause unwanted signaling pathway activation. In response to this problem, experiments were performed on two major signaling pathways known for GPCRs, namely the G protein-mediated signaling pathway and the arestin-mediated endocytosis signaling pathway, respectively (gainetdiov, r.r., premont, r.t., bohn, l.m., lefkowitz, r.j. & Caron, m.g. localization of G protein-coupled receptors and neuronal functions.annu review of neuroscience, 107-144 (2004)), to observe the ability of the GRAB probe to couple to downstream pathways of GPCRs.

For the G protein mediated signaling pathway, downstream calcium signaling was detected by calcium imaging using the GRAB-ACh 1.0 probe constructed in example 2 to characterize G protein mediated signaling pathway sensitivity. As the GRAB probe occupies the green light spectrum, red calcium dye Cal590 is adopted, HEK293T cells expressing GRAB-ACh 1.0 are treated by acetylcholine with different concentrations, and Kd values are calculated by obtaining a reaction curve of calcium signals and ligand concentration, so that whether the sensitivity is obviously different is further compared. The experimental results show (FIG. 15) that compared to endogenous M3 type acetylcholine receptors (shown in FIG. 15 as WT-CHRM3 or WT-M ₃ R, the natural M3 type acetylcholine receptor not inserted into cpEGFP corresponding to GRAB-ACh 1.0), GRAB-ACh 1.0 probe has reduced sensitivity to G protein-mediated signaling pathway, and its Kd value decreases about 5-fold.

The method of replacing G protein with G alpha protein peptide fragment commonly used in GPCR crystal structure analysis is used as reference. The G.alpha.protein peptide is 20 amino acids at the carbon end of the G Protein, and in the crystal structure, the G.alpha.protein carbon end plays an important role in inserting the intracellular loop of the GPCR after activation and stabilizing the GPCR in the activated state (Palczewski, K.et al crystal Structure of Rhodopsin: A G Protein-Coupled receptor.science (New York, N.Y.) 289,739-745 (2000)). Since the carbon-terminal peptide of the G.alpha.protein can replace the G protein to stabilize the GPCR in an activated state, and cannot trigger a downstream signal by itself, it is hypothesized whether the carbon-terminal peptide of the G.alpha.protein can be artificially coupled at the end of the carbon end of the GRAB probe, thereby causing the carbon-terminal peptide of the G.alpha.protein to compete with endogenous G protein for the intracellular loop position of the GRAB probe and further reducing the G protein-mediated activation of the downstream signal.

Taking an acetylcholine probe as an example, the last amino acid at the carbon end of the GRAB-ACh 2.0 probe constructed in example 2 is connected with 20 amino acids at the carbon end of the G alpha q protein (the specific sequence is VFAAVKDTILQLNLKEYNLV), whether the probe still has acetylcholine-induced fluorescence rise or not is observed, and meanwhile, a calcium imaging method is adopted to observe whether the signal transmission of a downstream G protein channel can be reduced or not. The probe with the Gαprotein peptide is named GRAB-ACh 2.0-Gq20, wherein Gq20 represents the carbon end 20 amino acids of the connected G αq protein. From the results (FIG. 16), it was found that GRAB-ACh 2.0-Gq20 still had good cell membrane localization and fluorescence intensity, and exhibited a significant increase in fluorescence signal under treatment with the ligand acetylcholine, with an average signal change of 70% ΔF/F ₀ Slightly lower than GRAB-ACh 2.0 (90%). Using the calcium imaging method described above, a response curve of calcium signal was obtained for different concentrations of acetylcholine, which showed a significant decrease in calcium signal coupling. The ability to couple calcium signals is reduced by about 10-fold compared to probes that attach G.alpha.peptide fragments, and 50-fold compared to endogenous M3R-type receptors (i.e., CHRM 3). This indicates that the GRAB probe was successful after its end fused to the G.alpha.peptide fragment Competing for endogenous G proteins significantly reduces the coupling of G protein signaling pathways so that the GRAB probe is free from disturbances that result in significant cell signaling systems when expressed intracellularly.

In addition, on the basis of the GRAB-Ach 2.0 probe constructed in example 2, 20 amino acids (Gq 20: VFAAVKDTILQLNLKEYNLV (SEQ ID NO: 6), gs20: VFNDCRDIIQRMHLRQYELL (SEQ ID NO: 7), gi20: VFDAVTDVIIKNNLKDCGLF (SEQ ID NO: 8)) derived from the carbon end of different G.alpha.proteins were respectively ligated after the last amino acid at the C-terminus of the probe, and after detection of the magnitude of calcium signal at different acetylcholine concentrations, the ability of these acetylcholine probes to couple to the downstream G protein signaling pathway was examined (see FIG. 53 for the results, where chrm3 is the expression of unmodified wild-type acetylcholine receptor M3R). As can be seen from fig. 53, these acetylcholine probes have reduced ability to couple to downstream G protein signaling pathways due to competition of the gα protein peptide with the endogenous G protein.

In addition to the G protein-mediated downstream pathway, another important downstream pathway of GPCRs is the protein-mediated signaling pathway associated with receptor endocytosis, such as arestin. In order to more stably detect extracellular neurotransmitter dynamics, the ideal probe should not be regulated by the endocytic system, so that the actual change in the concentration of the exogenous neurotransmitter can be stabilized. To address this problem, it was first tested whether the GRAB probe would couple to the arestin signaling pathway resulting in endocytosis of the receptor probe. It is further speculated that if the GRAB probe can couple to endocytic signaling pathways and cause endocytosis of the receptor, it should appear as a decrease in fluorescent signal on the cell membrane. If the fluorescent signal on the cell membrane does not show a significant change under prolonged (more than five minutes) ligand treatment, it is likely that the coupling of the probe to the endocytic signal pathway is disrupted. First, a fluorescent probe molecule based on receptor endocytosis (GPCR fused to pH sensitive fluorescent protein) was constructed (see example 1 for specific construction methods, in native. Beta. ₂ Adrenergic receptor (beta) ₂ AR) gene is linked to pHluorin gene by Gibson assembly method, and is linked by short peptide of 3 amino acids (GGA), and fused with human AVPR2 gene at the end of its carbon endThe last 29 amino acids (343-371 amino acids) to obtain pHluorin-beta ₂ AR), demonstrating that adrenergic receptors stably activate endocytic signaling pathways, embodied as pHluorin-beta ₂ The fluorescence intensity of AR showed a significant decrease after a period of time following the addition of agonist ISO (figure 17).

Since the adrenergic receptor can stably activate endocytic signal pathway, a long-time ligand treatment experiment is carried out on the adrenergic probe GRAB-EPI 1.0 constructed based on the adrenergic receptor, and whether the fluorescence intensity of the cell membrane shows obvious fluorescence decrease along with the increase of the treatment time of an agonist ISO is observed. A plot of the fluorescence values of the probe at 30 minutes of agonist ISO treatment was obtained, and it was found that the fluorescence value of GRAB-EPI 1.0 did not change significantly over time, maintaining similar fluorescence intensity over time for the agonist treatment, washing off the agonist after 30 minutes, and the fluorescence signal returned to the basal value, indicating that this fluorescence change was a reversible receptor activation response (fig. 18). From this, it is thought that the coupling efficiency of the GRAB probe to the signal pathway of endocytosis mediated by arestin is greatly reduced, probably because the fluorescent protein in the GRAB probe occupies the binding site of the protein such as arestin, and thus it is difficult to couple the signal pathway. For the probe itself, the reduced signal coupling can better ensure that the fluorescence change is truly reflected by the change of the concentration of the exogenous ligand.

EXAMPLE 7 use of neurotransmitter fluorescent probes

1. Neurotransmitter fluorescent probes respond to specific neurotransmitters in cultured neurons

The adrenergic probe GRAB-EPI 1.0 and the acetylcholine probe GRAB-ACh 1.0 were expressed in cultured neurons, respectively, and their expression in the system and response to specific neurotransmitters were examined.

Primary cultured rat cortical neurons were calcium phosphate transfected and neurons were imaged approximately 48 hours later. Neurons expressing neurotransmitter fluorescent probes were observed to be normal in morphology with a good and extended axon dendritic network. Fluorescent probes constructed based on the receptors are uniformly expressed on the cell membrane of neurons, and the expression of the probes can be clearly seen on different structures of neurons, such as synaptic spines (fig. 19).

The optical response of neurons expressing neurotransmitter probes was observed by perfusion of neurotransmitter solutions. Neurotransmitter-specific optical signals are recorded, which have the characteristic of being fast and stable, with good reproducibility in different neurons. Further, the receptor was stabilized in an inactive state using a specific receptor blocker (Tio), in which case neurotransmitters failed to trigger activation of the receptor, nor did the corresponding optical signal undergo ligand-induced changes (fig. 44). In order to demonstrate that neurotransmitter probes expressed in neurons still have a sensitive response to neurotransmitters at different concentrations, the ligand concentration dependence curves of the probe optical signals are shown in FIG. 20, which in cultured cell lines, conform to the Hill equation in analogy with the values reported in the literature (Wood, M.D. et al functional comparison of muscarinic partial agonists at muscarinic receptor subtypes hM, hM2, hM3, hM4 and hM5 using microphysiometry.British journal of pharmacology, 1620-1624 (1999); hoffmann, C., leitz, M.R., obedorf-Maass, S., lohse, M.J. & Klotz, K.N. compartive pharmacology of human. Beta. -adrenergic receptor subtypes-Characterization of stably transfected receptors in CHO cels, naunyn-Schmieedeg's archives of pharmacology, 151-159 (2004)).

Neurons expressing the same neurotransmitter probe are sequentially treated with saturated concentrations of different neurotransmitters. As a result, it was found that only neurotransmitters for which the probe should detect can trigger reproducible optical signal responses, while other major neurotransmitters cannot trigger any changes in optical signals even at high concentrations (fig. 21). The results demonstrate that fluorescent probes of these neurotransmitters are capable of specifically detecting the corresponding neurotransmitters without being affected by changes in the concentration of other neurotransmitters.

2. Method for detecting release of acetylcholine in drosophila olfactory system by using two-photon imaging method

The central nervous system of Drosophila has acetylcholine as the primary excitatory neurotransmitter involved in information transmission. In its olfactory system, upon receipt of activation of an odorant molecule, a olfactory receptor neuron transmits sensory information to a second level olfactory neuron, namely, an anticornal lobe (Ng, m.et al transmission of olfactory information between three populations of neurons in the antennal lobe of the fly. Neuron 36,463-474 (2002)). Classical calcium imaging methods observe the transmission of olfactory information by expressing a calcium indicator in the antennal nerve lobes, whereas calcium signals are not molecular specific in nature as second messengers within cells, and do not reflect what neurotransmitters play a role in the transmission of information in particular (Wang, j.w., wong, a.m., flores, j., vosshall, l.b. & Axel, r.two-photon calcium imaging reveals an odor-evoked map of activity in the fly brain. Cell 112, 271-282 (2003)). The embodiment adopts the acetylcholine probe coded by the developed gene, and specifically expresses the acetylcholine probe at the lobe of the touch horn nerve, namely after the synapse of the olfactory receptor neuron, so as to detect the acetylcholine released by the olfactory receptor neuron in receiving the olfactory information. Through a drosophila embryo injection combined genetic screening method, GRAB-ACh 1.0 is transferred into drosophila, a UAS-GRAB-ACh 1.0 transgenic drosophila is constructed, after hybridization with a GH146-Gal4 (Ruta, V.et al A dimorphic pheromone circuit in Drosophila from sensory input to descending output. Nature 468,686-690 (2010)) strain, GRAB-ACh 1.0 probes are specifically expressed at antennal nerve leaves, and release of endogenous acetylcholine in the synaptic network under odor stimulation is observed through two-photon imaging.

Specific fluorescence increases were observed at specific sites at the antennal nerve lobes following odor treatment with Isoamyl Acetate (IA). To verify that this reaction is odor-specific induced acetylcholine release, by administering different concentrations of odor molecules, it was observed that there was no change in fluorescence signal when the odor molecule concentration was zero, and that the fluorescence reaction also exhibited a concentration dependence as the odor molecule concentration gradually increased (figure 22 upper graph), indicating that this reaction was indeed neurotransmitter release by odor molecules.

The antennal nerve leaves can be divided into different regions corresponding to different olfactory bulbs which have specific activation patterns for specific olfactory molecules as they receive projections of different olfactory neurons. It was found experimentally that isoamyl acetate can specifically trigger the fluorescent signal changes at the olfactory bulb DM2, DM3, DL1 with the greatest amplitude of change at DM2, consistent with the results obtained in the previous literature using calcium imaging. Correspondingly, isoamyl acetate failed to elicit an increase in optical signal at the DA1 olfactory bulb, consistent with previous reports that DA1 was primarily responsible for activation of sex hormone odorants (Wang, J.W., wong, A.M., flares, J., vosshall, L.B. & Axel, R.two-photon calcium imaging reveals an odor-evoked map of activity in the fly brain.cell 112, 271-282 (2003); couto, A., alenius, M. & Dickson, B.J., molecular, anatomic, and functional organization of the Drosophila olfactory system.current Biology 15,1535-1547 (2005)) (FIG. 22, bottom panel).

To verify whether overexpression of the GRAB-ACh1.0 probe at the antennal nerve leaf coupled to endogenous G protein signaling pathway and thus affected the calcium signaling of cells, calcium signaling at the antennal nerve leaf was measured directly using the gene-encoded red calcium indicator RGECO (Yongxin Zhao, et al, an Expanded Palette of Genetically Encoded Ca2+ indicators, science, 2011). Drosophila expressing the GRAB-ACh1.0 probe did not show a significant difference in calcium signaling when stimulated with isoamyl acetate odor compared to Drosophila expressing RGECO alone (FIG. 23), suggesting that overexpression of the GRAB probe in vivo did not result in observable disturbance of calcium signaling.

3. Detection of expression of acetylcholine probes in mouse living brain slices using viral expression and two-photon imaging

GRAB probes exhibit good ability to detect specific neurotransmitters in both cultured neurons and in vivo Drosophila, and it is desirable to be able to express fluorescent probes in the brain of mammals to detect the dynamic changes in neurotransmitters in more complex neural networks. The fluorescent expression of GRAB-ACh1.0 probe gene coated by slow virus is detected by local spray irrigation of acetylcholine in mouse hippocampal neurons. The GRAB probe also showed a stable response in the mouse living brain slice, and after acetylcholine addition, it showed a rapid fluorescence rise with an average amplitude of about 10% -15%. Since the GRAB-ACh1.0 acetylcholine probe is constructed based on M3 receptor, the specific agonist Oxo-M of M3 receptor can also induce obvious fluorescence enhancement, while the agonist nicotine of N-type acetylcholine can not induce fluorescence signal change, revealing the specificity of GRAB probe signal (FIG. 24).

Example 8 construction of specific fluorescent probes for epinephrine and/or norepinephrine Using human ADRA2A receptor

1. Materials and methods

Molecular cloning for GRAB probe construction and mutation screening

Here, all molecular clones were subjected to the method of Gibson assembly (Gibson, D.G., et al, enzymatic assembly of DNA molecules up to several hundred Kilobases. Nat Methods,2009.6 (5): p.343-5), i.e., recombinant ligation of homologous fragments was achieved using sequence complementation. Efficient splicing between sequences is achieved using homologous complementary sequences of about 30 bases designed on the primer. All recombining correct clones were sequenced at the equipment center of the college of university of Beijing student's life sciences.

The GRAB probe construct vector is a pDISPLAY vector of the company Invitrogen. The GPCR gene part is amplified in full-field human genome cDNA (hORFeome database 8.1.1), firstly transferred to a final vector with att sequence constructed by a pDISPLAY vector by a Gateway cloning method, and then inserted into a specific position of a receptor by a Gibson assembly method. In the mutation screening process of the probe, a mutation introducing method is to introduce random base combinations into specific primers so as to construct a site-directed mutation library. The remaining clones were constructed in a similar manner.

Cell culture and transfection

HEK293T cells were cultured in 10cm dishes with 10% FBS, 1% PSDMEM (DPF complete medium), incubator temperature 37 ℃, CO ₂ The content is 5%. Liquid is changed or passaged according to the cell growth condition. And pouring out the original culture medium when changing the liquid, and adding 15mL of new culture medium. Passaging is performed at cell densities above 80%. The original medium was first decanted, washed twice with 2ml,0.01m PBS, and the remaining magnesium ions and serum removed. 0.5mL of 0.25% trypsin-EDTA was added and digested at 37℃for 1min. The reaction was terminated with 2mL of medium, the cells were gently blown until they were completely detached from the bottom of the dish and dispersed, and then 2mL of medium was added thereto, and the blowing was uniform. About 1mL of the cell suspension was taken into a new 10cm dish, 14mL of medium was added, gently shaken, and returned to the incubator.

The screening process requires cells to be transferred to either 24-well plates (perfusion) or 96-well plates (opera phenox). Cells were digested with pancreatin according to the passaging method described above and after addition of 4mL of medium to form a uniform cell suspension, appropriate volumes of cell suspension were passaged at 50% density into 24-well plates or 96-well plates with clean imaging zero-number round slides. About 500. Mu.L of medium was added to each well of a 24-well plate, about 100. Mu.L of medium was added to each well of a 96-well plate, and the mixture was mixed and placed in an incubator for culturing.

Cells were attached for 8-12h and transfected. DNA was combined with PEI at 1:3 are evenly mixed in DMEM, incubated for 15-20min at room temperature, and then added into the cell culture solution to be transfected, and evenly mixed. The 24-well plate transfected DNA was approximately 800ng per well, and the 96-well plate was 300ng. After 4 hours of transfection, the liquid is changed, and after 24 hours of transfection, fluorescence observation is carried out.

The primary culture of rat neurons adopts a new born Sprague-Dawley rat, after the skin of the rat is cleaned by alcohol, the head of the rat is dissected by an operation instrument, the vascular membrane on the surface of the cortex is carefully removed after the brain is taken out, and the cortex tissue is sheared and placed in 0.25% pancreatin solution and digested for 10 minutes in a 37 ℃ incubator. After digestion, digestion was stopped with DMEM solution containing 5% fbs and the cells were further disrupted by slow pipetting ten times with a pipette. After standing for 5 minutes, the upper solution was aspirated, the sediment containing the tissue fragments was removed, and centrifugation was performed in a centrifuge at 1000rpm for 5 minutes. The supernatant was then discarded, the neurons were resuspended in Neurobasal+B27 solution used to culture the neurons,and density calculations were performed using a cell counting plate. After calculating the cell density, the cell density was calculated according to 0.5-1X 10 ⁶ The density of cells/ml was diluted and plated onto polylysine (sigma) plated slides. Primary neurons were cultured in neurobasal+b27 solution and half-changed every two days. Transfection of primary cultured neurons was performed 6-8 days after dissection, using the calcium phosphate transfection method. After 1.5 hours from the time of cell transfection, the solution was observed by microscopy for small and uniform calcium phosphate precipitates and was changed with HBS solution having a pH of 6.8. After HBS washing, neurons were re-placed in neurobasal+b27 medium for culture until imaging experiments were performed 48 hours later.

Fluorescence imaging of cells and drug perfusion

The perfusion system is arranged on a microscope and comprises a solution introducing system, an imaging pool and a liquid sucking pump. In the perfusion process, an imaging pool is arranged above an objective lens of an inverted microscope, a slide inoculated with cells is placed in the pool, perfusion experiments of different medicines are carried out by controlling the on-off of different pipelines of a solution introducing system, and the perfusion speed is set to be about one drop per second. In order to ensure the stability of the focal plane, the liquid level is required to be kept unchanged all the time, so that the flow rates of the solutions are required to be adjusted consistently. The liquid suction speed is regulated to maintain the liquid level in the pool to be higher than that of the glass slide.

Before perfusion, a cell region to be detected (ROI) and a background region are manually selected, and then a perfusion experiment is carried out. The exposure time was set to be 50ms and the acquisition frequency was imaged every 5 seconds. The solution used for perfusion was physiological solution 4k (pH adjustment=7.3-7.4), and the drug solution was formulated to the desired concentration with 4 k. The program running time is set to be not more than 5min, after the balance is stabilized for about 60-90s by using 4k, the medicine is infused, and after 60s, the medicine is changed back to 4k for flushing. The excitation light of the green fluorescent protein is 488nm, the excitation light of the red fluorescent protein is 568nm, and the laser intensity is adjusted according to the working state of the laser and the cell expression efficiency.

After the program is finished, an acquired time-fluorescence intensity data table is derived, the ROI is subtracted from the background to obtain a corresponding fluorescence value Ft, and the average value of the fluorescence values before adding the medicine is taken as initial fluorescence F ₀ Calculation ofAnd (3) making a relation curve of the ratio and time, and observing the influence of the addition of the medicine on the fluorescence intensity.

The response detection of the GRAB probe to the ligand is carried out by adopting a drug perfusion method. Cells were placed in standard physiological solutions, the solution formulation was:

NaCl	150mM
		KCl	4mM
MgCl ₂	2mM
		CaCl ₂	2mM
HEPES	10mM
		glucose	10mM

After the pH value of the physiological solution is corrected to be about 7.4, the small molecular medicine is diluted by partial solution, and the solution with the corresponding concentration of the small molecular ligand is prepared.

Use of Opera PhenixTM

Opera Phenix (TM) can confocal image 60 wells at a time in the center of a 96-well plate using a 63-fold water mirror. Before the experiment, the cell culture medium was replaced with 100. Mu.L of physiological solution, placed on a sample holder, and then introduced into the instrument. The appropriate imaging focal plane, excitation wavelength and laser intensity are selected, and all imaging apertures and imaging fields of view in each aperture are selected. Running the program, the instrument will automatically image all selected areas. After the first imaging is completed, the 96-well plate is taken out, the physiological solution in each well is replaced by the physiological solution containing the medicine with the required concentration, and the imaging is performed again.

After the two imaging is completed, using a Harmony software analysis program, membrane regions of each field cell are positioned by using mCherry red fluorescence (CAAX sequences carried by RFPs enable positioning on the membrane), the number of stitchable Regions (ROIs) is counted, the ratio (GRratio) of the cpEGFP to mCherry fluorescence intensity in the stitchable regions is calculated, and finally an analysis result report is derived. Comparing the change of GR ratio before and after drug addition can judge whether the fluorescent probe responds to the drug and the intensity of the response.

The difference between Opera Phenix and perfusion is mainly two-fold. Firstly, due to the limitation of a software data processing program, the Opera Phenix TM cannot automatically deduct background fluorescence; secondly, since the image acquisition is not dynamic continuous, the orifice plate must be taken out in the dosing process, so that the ROIs acquired before and after can be different, the confocal planes can also be different, and the absolute value of the fluorescence change of the single cpgfp can be caused by the measurement changes and cannot be used as a standard for whether the fluorescence change is responsive to the drug. This is also why RFP is introduced. In the invention, through reasonable design, the expression metering ratio of GFP and RFP is certain, namely when the external condition is unchanged, the GR ratio of each cell can be approximately equal (but the absolute intensities of fluorescence are not necessarily equal). RFP reflects changes in ROI and focal plane, etc., but does not respond to the fluorescence intensity of the drug. Therefore, the measurement of GR ratio can be adopted to measure the probe response, the decrease of GR ratio corresponds to the decrease of the fluorescent intensity of the cpEGFP after the drug addition, namely the off probe, and the increase of GR ratio corresponds to the increase of the fluorescent intensity of the cpEGFP after the drug addition, namely the on probe.

Photolytic caged NPEC-NE exploration of neurotransmitter response kinetics

100 μm NPEC-NE was formulated in DMSO and photolysis experiments were performed on a Nikon laser scanning confocal microscope with a light stimulation of 80% of a 405nm laser applied for 76ms of light stimulation in a rectangular pattern of 2x2 pixel (1 pixel=0.62 μm).

Image data processing

Fluorescence imaging data were processed using ImageJ software. For fluorescence expression of GRAB probes in HEK293T cell lines and neurons, whole cell bodies were selected as the data-processing region. The change in fluorescence signal is usually indicated by its relative change, the fluorescence signal is first subtracted from the background region of no probe expression to obtain the true appearance of the fluorescent protein intensity, and then the fluorescence value F after drug addition and the average fluorescence value F before addition are calculated ₀ Obtaining the relative fluorescence change value delta F/F ₀ ＝(F-F ₀ )/F ₀ As fluorescent probes for fluorescent responses to specific drugs. ΔF/F ₀ The change over time was in turn plotted in the Origin 8.6 software. The pseudo color map is completed by Matlab.

Statistical testing

2. Selection of human norepinephrine receptor proteins for construction of fluorescent probes

Three different subtypes of human norepinephrine receptor protein and green fluorescent protein pHluorin are selected for fusion expression respectively, green fluorescence is observed under 488nm laser through a confocal microscope, and the expression condition of the three subtypes of human norepinephrine receptor protein and green fluorescent protein on cell membranes is detected (FIG. 25 b). Wherein the human ADRA2A receptor has good membrane localization and a higher affinity for the ligand. The human ADRA2A receptor was therefore chosen as the basic unit for fluorescent probe construction.

The sequence of human ADRA2A is shown in NCBI gene ID:150, and the amino acid sequence is specifically as follows:

MFRQEQPLAEGSFAPMGSLQPDAGNASWNGTEAPGGGARATPYSLQVTLTLVCLAGLLMLLT VFGNVLVIIAVFTSRALKAPQNLFLVSLASADILVATLVIPFSLANEVMGYWYFGKAWCEIYLALDV LFCTSSIVHLCAISLDRYWSITQAIEYNLKRTPRRIKAIIITVWVISAVISFPPLISIEKKGGGGGPQPAEP RCEINDQKWYVISSCIGSFFAPCLIMILVYVRIYQIAKRRTRVPPSRRGPDAVAAPPGGTERRPNGLGPERSAGPGGAEAEPLPTQLNGAPG EPAPAGPRDTDALDLEESSSSDHAERPPGPRRPERGPRGKGKARASQVKPGDSLPRRGPGATGIGTPAAGPGEERV GAAKASRWRGRQNREKRFTFVLAVVIGVFVVCW FPFFFTYTLTAVGCSVPRTLFKFFFWFGYCNSSLNPVIYTIFNHDFRRAFKKILCRGDRKRIV(SEQ ID NO:2)。

wherein the underlined part is the third intracellular loop, specifically amino acids 218-374, as defined in uniprot database.

3. Truncating and inserting a circularly rearranged fluorescent protein against the third intracellular loop ICL3 of the human ADRA2A receptor CapEGFP obtaining GRAB-NE1.0 with optical signal change for high concentration NE

The third intracellular loop of the human ADRA2A receptor has 157 (reference uniprot database) amino acids. It is hypothesized that the length of the third intracellular loop of the receptor may affect the efficiency of the conformational change itself to a change in fluorescence, and that too long an intracellular loop may buffer the conformational change, thereby making it difficult to affect the inserted fluorescent protein. Therefore, truncated insertion of cpgfp was performed against ICL3 of the human ADRA2A receptor. The cpEGFP used was the cyclic rearranged fluorescent protein cpEGFP used in GCaMP6s as in example 2, and its specific sequence was:

By truncating the amino groups at 157An insertion site was selected every 10 amino acids on ICL3 of the acid, and a total of 14 insertion sites were designed. The sites were divided into two groups near the N-terminus and near the C-terminus in order of site, 7 sites per group, and the insertion sites of the two groups were randomly selected for ICL3 truncation and cpEGFP was inserted between the two sites, thus obtaining a truncated insertion library with 49 possibilities (FIG. 26 a). By expressing them in HEK293T cells, respectively, detecting the saturation concentration of agonist (norrenal hormone NE at a concentration of 100. Mu.M and fluorescence intensity on the cell membrane in the absence of agonist) on the high content imaging system Opera Phenix, respectively, while detecting the red fluorescent protein signal localized on the membrane by CAAX modification under the same promoter as an internal reference ₀ ) The largest clone was designated GRAB-NE1.0 (FIG. 26 b), and sequencing gave the truncated insertion sites on ICL3 at positions 78 and 138 of ICL3 (i.e., positions 79-138 of ICL3 were truncated), i.e., 60 amino acids of ICL3 were truncated. Drug perfusion experiments were performed under a confocal laser microscope with 100. Mu.M norepinephrine, and the GRAB-NE1.0 probe had a change in fluorescence intensity of greater than 100% and this change was reversible (FIGS. 26d, e).

In the present invention, when describing the truncation site, the given numbering corresponds to the C-terminal of the amino acid, so that the truncated position is at the C-terminal of the numbering corresponding amino acid, as in this example, the following examples and the present invention, unless otherwise indicated, are all understood as such.

4. Fine tuning of the insertion site of cpEGFP for truncated human ADRA2A receptor ICL3 resulted in a greater response to NE GRAB-NE2.0 with high optical signal variation

The insertion sites were placed one at every 1 amino acid on either side of the truncated ICL sites 78 and 138, i.e.11 total sites from 73-83 (i.e.inserted after any of the sites 73-83), and 11 total sites from 133-143 (i.e.inserted before any of the sites 133-143), with a total of 121 possibilities. Likewise, they are described in HEK2 respectively93T cells, and through screening (100 mu M NE norepinephrine as medicine), the fluorescence intensity is higher, delta F/F is obtained ₀ The largest clone was designated GRAB-NE2.0 (FIG. 26 c), and sequencing gave the truncated insertion sites of this clone at ICL3 at positions 78 and 143 of ICL3 (i.e., positions 79-143 of ICL3 were truncated). Drug perfusion experiments were performed under a confocal laser microscope with a fluorescence intensity change of greater than 200% for the GRAB-NE2.0 probe under 100. Mu.M norepinephrine (FIGS. 26d, e).

5. Screening for truncated human ADRA2A receptor ICL3 and cpEGFP connecting peptide fragment to obtain basic fluorescence intensity Higher degree GRAB-NE2.1 with higher optical signal variation to NE

After the optimal fluorescent protein insertion site was determined, the linking peptide between fluorescent protein and receptor was optimized on the basis of GRAB-NE2.0 probe.

In the previous step, a short connecting peptide segment formed by flexible amino acids (glycine, alanine) is adopted in the construction process of the norepinephrine specific fluorescent probe based on the human ADRA2A receptor, so that the fusion protein can be folded correctly. The peptide fragment has a length of 2 amino acids GG at the nitrogen end and 5 amino acids GGAAA at the carbon end. Based on this, a screen was performed for the length of the peptide fragment. The specific strategy was to change the 5 amino acids of the sequences on both sides of the cpgfp to 0-5 amino acids, starting with a truncation from the direction away from the cpgfp, and randomly combining the nitrogen and carbon ends, resulting in all possible 25 permutations (fig. 27 a). They were expressed in HEK293T cells, respectively, and by high throughput screening (using 100 μm NE), it was found that modification of the length of the connecting peptide with GRAB-NE2.0 as template did not increase the brightness and response of the fluorescent probe (fig. 27 b).

Based on the above experiments, combinations of 2-5 in length of the connecting peptide were immobilized, and an attempt was made to change the amino acid type to obtain probes with larger signal changes. By using a method of designing random primers, NNB base coding is adopted at the amino acid site to be mutated, so that the possibility of 20 different amino acids is obtained, and the probability of occurrence of a stop codon is reduced as much as possible. A total of 7 screening libraries were constructed for random mutations at the amino acid positions of the different connecting peptide fragments, each screening library having 20 possibilities (FIG. 27 c). When they were expressed in HEK293T cells, respectively, and high-throughput screening was performed, GRAB-NE2.1, which was brighter than GRAB-NE2.0 and more reactive, was obtained, and the connecting peptide sequence of the novel fluorescent probe was GG-TGAAA, which was about 1.5 times that of GRAB-NE2.0 (FIGS. 27d and 28 e).

6. Norepinephrine GRAB probes have optical signaling that leads to receptor conformational change specificities with ligand binding Is converted into a form, and the specificity is consistent with that of the corresponding receptor

Cells expressing GRAB-NE2.1 were treated with ADRA2A receptor specific blocker (Yohimbine, 2 μm), beta 2 adrenergic receptor specific blocker (ICI 118,551,2 μm) and other neurotransmitters, respectively, to see if ligand binding-induced changes in fluorescence signals could also be obtained in these cases. The results show that both norepinephrine NE and epinephrine Epi activate GRAB-NE2.1, while β -type receptor specific activator ISO does not activate the probe, which coincides with ADRA2A receptor binding both NE and Epi, but does not bind ISO, demonstrating that the fluorescent probe of neurotransmitter engineered by GPCR retains the selectivity and specificity of endogenous GPCR for ligand. In addition, the specific blocker Yohimbine of the alpha receptor can inhibit the enhancement of the fluorescent signal of the probe caused by NE. Meanwhile, mutation S204A of the ligand binding pocket of ADRA2A receptor can disrupt the fluorescence signal change of GRAB-NE2.1 (FIGS. 28a, d). These results further demonstrate that GRAB-NE probes have receptor-activation specific fluorescent signal changes. In addition, the addition of other common neurotransmitters treated cells transfected with GRAB-NE2.1, respectively, did not significantly activate GRAB-NE2.1 to obtain a change in fluorescence signal (FIG. 28 a). This suggests that the fluorescence change of the norepinephrine GRAB probe is receptor activation specific,

7. Norepinephrine GRAB probes have ligand concentration dependent optical responses

Using different concentrations of ligand (on the norkidney) from 1nM to 1mMProstaglandin NE, at a concentration of 1Nm to 1 mM) HEK293T cells expressing the GRAB-NE2.1 probe were found to exhibit concentration-dependent fluorescent signal changes over a wide range of neurotransmitter concentration changes, which curve conformed to the boltzmann distribution (fig. 28b, c). By calculation of the EC of the curve ₅₀ The value was 0.9. Mu.M, which is on the same order of magnitude as the Kd value of the ligand-binding receptor in the literature, and it was seen that the norepinephrine GRAB probe did not alter the affinity of the receptor for the particular ligand. Because the affinity of the receptor binding ligand is evolving, neurotransmitter signals can be sensitively transmitted to downstream signals in the cell, and thus neurotransmitter fluorescent probes with sensitivity similar to that of the receptor itself can sensitively and quantitatively detect neurotransmitter signals at different concentrations under physiological conditions.

Introduction of the T373K mutation into the GRAB-NE2.1 probe resulted in a GRAB-NE2.2 probe with a 10-fold increase in ligand affinity (FIGS. 28c, d, e), which, although less than GRAB-NE2.1 in background fluorescence intensity and fluorescence signal change, had a ligand affinity of about 100nM which helped to more sensitively detect neurotransmitter signals. It has an affinity on the order of hundred nM for both norepinephrine NE and epinephrine Epi (see FIG. 28 f). This suggests that mutations in the GPCR and ligand binding region can modulate the affinity of the probe for ligand binding, thereby allowing for fluorescent probes with higher or lower ligand affinity, which on the one hand can cover a broader detection range and on the other hand can allow for detection of neurotransmitter release under single action potential stimulation.

8. The rapid kinetics of the norepinephrine GRAB probe can realize sub-second dynamic detection

By caging neurotransmitters, a region of neurotransmitter can be rapidly activated and released by a photolytic reaction, and then the signal of the probe can be rapidly scanned by a microscope to obtain the time constant required for the change in fluorescence intensity after the probe binds to the ligand. Experiments were performed in HEK293T cells using the caged neurotransmitter NPEC-cased NE (i.e., NPEC-NE) (FIG. 29 a), using a 405nm laser to activate NPEC-cased NE, using GRAB-NE 2.2. When photolysis is carried out with a short-term high-energy 405nm laser, an increase in fluorescence signal after photolysis is visible, which is not observed both after addition of the probe-specific inhibitor Yohimbine and without addition of the caged neurotransmitter NPEC-NE (FIGS. 29b, c). This suggests that the increase in fluorescence signal after photolysis is a result of the specific release of NE by photolysis, which is detected by the GRAB-NE2.2 probe. Fitting the rise of the fluorescence signal using a single exponential growth equation yields a rate constant of the rise of the fluorescence signal of about 100ms. This rate constant is already sufficient to specifically capture the process of chemical synaptic signaling in complex neural networks.

9. Decoupling of norepinephrine GRAB probes from downstream signaling pathways

Experiments were performed on the G protein mediated signaling pathway of the norepinephrine receptor, looking at the ability of the GRAB probe to couple to the downstream pathway of the GPCR to determine if intracellular overexpression would cause unwanted signaling pathway activation.

For G-protein dependent signaling pathways, GRAB-NE receptors are fluorescent probes developed based on ADRA2A receptors, whereas ADRA2A receptors are endogenous to the G.alpha.i protein coupled to cause downstream inhibitory signaling. By inhibiting the coupling of the gαi protein, detecting a change in the affinity of the fluorescent probe for the ligand can be used to determine whether the fluorescent probe requires the coupling of a downstream gαi protein to maintain its activated state (see fig. 30a, b). The inhibition of the coupling of the gαi protein by co-expression of PTX pertussis toxin in the cell (by catalyzing ADP ribosylation of the gαi protein, PTX renders gαi inactive) and addition of gtpγs (which can bind the gαi protein to inhibit dissociation of GTP and thus inhibit activation of the G protein), respectively, resulted in the finding that neither of these methods can alter the affinity of the GRAB-NE2.0 fluorescent probe for the ligand (fig. 30 c-e), and that the fluorescent probe itself has not required the coupling of the G protein to stabilize its active conformation.

Whether ligand activation causes activation of downstream G proteins requires determination by direct signaling intensity. The Gqi chimeric G protein cell line is constructed by a traditional resistance screening method, plasmids expressing the Gqi chimeric protein and the antibiotic resistance protein are transfected into cells, insertion of a cell genome is realized by homologous recombination sequences on the plasmids, and then a stable cell line is obtained by resistance screening (the Gqi chimeric protein acts as receptor activation which can convert GPCR receptor activation coupled with a Gi protein pathway into Gq pathway, namely, the Gq pathway downstream detection method (such as TGF alpha assay) can be used for detection). And then, by means of a TGF alpha blending experiment of a signal path of the Gαq, transferring the signal activation of the Gαi coupling to the blending signal of the TGF alpha caused by the Gαq, so that the intensity of the activation of the downstream G protein can be judged through the intensity of the TGF alpha blending. Under the action of 10 μm NE, the tgfα signal caused by the GRAB-NE2.0 probe was only 1/3 of that of the endogenous ADRA2A receptor (fig. 30 f), and it was seen that the construction of this probe did greatly reduce the coupling of this probe to downstream G proteins, so that the GRAB probe was free from causing significant disruption of the cell signaling system when expressed intracellularly. This is probably due to the fact that insertion of cpEGFP affects the position where the GPCR binds to G protein coupling, and thus G protein coupling cannot be completed. While insertion of cpEGFP mimics to some extent the twisting of the GPCR structure by the coupling of the G protein, helping to stabilize the GPCR in an activated state after binding to the ligand.

10. Norepinephrine GRAB fluorescent probes have optical properties on neurotransmitters specific for the cultured neurons Signal variation

Neurotransmitter fluorescent probe GRAB-NE2.1 constructed based on ADRA2A receptor is expressed in cultured neurons, and its expression under the system and response to specific neurotransmitters are observed. Calcium phosphate transfection of primary cultured rat cortical neurons, imaging and characterization of neurons after about 48 hours, showed normal morphology of neurons expressing GRAB-NE2.1 with a good and extended axonal dendritic network. GRAB-NE2.1 was expressed uniformly on the cell membrane of neurons, with a small amount of aggregation at the cell body site, and the distribution of fluorescent probes on dendritic spines was also observed by cotransfection of PSD95-mcherry at different structures of neurons (FIGS. 31a, b).

By perfusing neurotransmitter solution, optical signal change of neuron expressing neurotransmitter probe is observed and recorded to nerveThe transmitter-specific optical signal (fig. 31c, d) which resulted in small changes in optical signal due to small amounts of cell bodies, but the signal on the cell membrane fraction and synapse was similar to that in HEK293T cells (fig. 31 e). The signal has the characteristics of rapidness and stability, and has good repeatability in different neurons. The ligand concentration dependence curve of the probe optical signal is similar to that in the cultured cell line, accords with Boltzmann equation, and has EC ₅₀ The values were similar to those reported in the literature (FIGS. 31f, g). Ligand-induced changes in optical signals of neurotransmitter probes can be inhibited using a specific receptor blocker Yohimbine (fig. 31 f).

11. Epinephrine/norepinephrine fluorescent probes to specific nerves in cultured rat cardiomyocytes The transmitter has optical signal variation

The GRAB-NE2.1 probe was transfected into primary cultured rat cardiomyocytes by liposome, and its optical signal changes for ligand binding and affinity for ligand in cardiomyocytes were detected by drug perfusion. The results show that the probe has good membrane localization expression in cardiomyocytes and has an optical signal change of more than 300% at 100 μm saturated norepinephrine (fig. 32a, b, c). The affinity of the probe for the ligand in cardiomyocytes was also similar to that determined previously, at different concentrations of agonist (norepinephrine NE), at about 0.5 μm (see fig. 32d, e).

EXAMPLE 9 construction of genetically encoded serotonin fluorescent probe

The materials and methods used in this example are the same as in example 1, unless specifically stated otherwise.

Preliminary screening is carried out on different serotonin receptors, and the human HTR1D and human HTR2C receptors still have better expression and membrane localization after fluorescent protein insertion. Then, taking human HTR2C as an example, the optimal insertion site for the fluorescent protein is selected.

1. Construction of serotonin-specific fluorescent probes Using human HTR2C receptor

By human HTR2C receptor is used as skeleton to construct serotonin specific fluorescent probe, and the method of gradually determining the optimal insertion site of fluorescent protein is adopted. Aiming at the third intracellular loop of the human HTR2C receptor, fluorescent protein is inserted every 5 amino acids as an insertion site, meanwhile, the third intracellular loop is truncated at a specific amino acid position and fluorescent protein is inserted at the truncated position, so as to obtain a probe library. The initially constructed probe library is screened by using a fluorescence confocal microscope and a perfusion system, and in the screening process, the probe with reduced fluorescence intensity after sensing the ligand is called as an OFF probe, and the probe with increased fluorescence intensity is called as an ON probe. After a first round of screening (screening drug was serotonin 5HT, at a concentration of 10 μm), two probes with the highest response were selected from them and sequenced, both probes were found to truncate the third intracellular loop of the human HTR2C receptor to varying degrees, wherein the truncations of one probe occurred at positions 15 to 55 of the third intracellular loop (ICL 3) (i.e., positions 16-55 of ICL3 were truncated, designated 15) _N -55 _C ) The truncation of the other probe occurred at positions 10 to 60 of ICL3 (i.e., positions 11-60 of ICL3 were truncated, designated 10) _N -60 _C )。

Wherein the sequence of the human HTR2C receptor is shown in NCBI gene ID 3358, isoferm a, the amino acid sequence of which is specifically:

MVNLRNAVHSFLVHLIGLLVWQCDISVSPVAAIVTDIFNTSDGGRFKFPDGVQNWPALSIVIIII MTIGGNILVIMAVSMEKKLHNATNYFLMSLAIADMLVGLLVMPLSLLAILYDYVWPLPRYLCPVWI SLDVLFSTASIMHLCAISLDRYVAIRNPIEHSRFNSRTKAIMKIAIVWAISIGVSVPIPVIGLRDEEKVFV NNTTCVLNDPNFVLIGSFVAFFIPLTIMVITYCLTIYVLRRQALMLLHGHTEEPPGLSLDFLKCCKRNTAEEENSANPNQDQNARRRKKKE RRPRGTMQAINNERKASKVLGIVFFVFLIMWCPFFITNILSVLCE KSCNQKLMEKLLNVFVWIGYVCSGINPLVYTLFNKIYRRAFSNYLRCNYKVEKKPPVRQIPRVAAT ALSGRELNVNIYRHTNEPVIEKASDNEPGIEMQVENLELPVNPSSVVSERISSV(SEQ ID NO:4)

wherein the underlined part is the third intracellular loop, specifically positions 236-311, defined with reference to the uniprot database.

The fluorescent protein used therein was a cyclic rearranged cpEGFP, which was the same as in example 2 and was a cyclic rearranged fluorescent protein cpEGFP used in GCaMP6s, and its specific sequence was:

2. optimizing the insertion site of fluorescent protein in the third intracellular loop of HTR2C receptor

The screening strategy was to fix the first 10 or 15 amino acids of the N-terminus of the third intracellular loop, and systematically scan the insertion site of the fluorescent protein at the C-terminus of the intracellular loop (designated 10 _N -X _C Or 15 _N -X _C I.e., amino acids 11-X or amino acids 16-X of ICL3 are truncated); similarly, the insertion site of the fluorescent protein at the N-terminus of the intracellular loop was scanned by fixing the amino acid at the 55 th or 60 th position of the third intracellular loop (designated as X _N -55 _C Or X _N -60 _C I.e., amino acids X+1-55 or amino acids X+1-60 of ICL3 were truncated) and selected. The specific screening method comprises transferring human HTR2C receptor inserted with fluorescent protein at different positions into HEK293T cells, respectively, perfusing with serotonin, and measuring ΔF/F ₀ . At 10 _N -X _C Screening library 10 of (C) _N -60C this combination shows the largest OFF response; at 15 _N -X _C When the combination of the insertion sites of the fluorescent protein becomes 15 in the screening library _N -70 _C (i.e., positions 16-70 of ICL3 were truncated), the probe showed about 20% ON response. Thereafter, the amino acid sequence of the human HTR2C receptor following position 70 of the third intracellular loop was immobilized, and the insertion site of the fluorescent protein at the N-terminus of the third intracellular loop was scanned (designated as X _N -70 _C ) No better probes were found.

Then, more accurate screening is carried out on the insertion sites, the left insertion site is defined as amino acids from 13 th to 17 th of a third intracellular loop, the right insertion site is defined as amino acids from 66 th to 74 th, and the sites are arranged and combined to construct a probe libraryAnd screening is performed. Specific screening method As above, by systematically screening fluorescent protein at human HTR2C receptor insertion site, a serotonin fluorescent probe 14 having 80% ON response was obtained _N -68 _C (i.e., ICL3 has its 15-68 th position truncated) and is named GRAB-5-HT1.0.

3. Optimizing the linking peptide between a cyclically rearranged fluorescent protein and an HTR2C receptor

In the initial connecting peptide, the length of the peptide at the N end is 2 amino acids, and the sequence is GG; the C-terminal peptide fragment is 5 amino acids in length and has the sequence GGAAA. On the basis of GRAB-5-HT1.0, carrying out random mutation on each site of the connecting peptide segment in sequence, screening a probe with excellent performance, fixing the amino acid at the position, and continuing carrying out random mutation on the connecting peptide segment of the next site. It was found that after mutation of glycine G at the first position of the N-terminal linker peptide to asparagine N, the signal of the probe increased 3-fold from 80% to nearly 300%, thus fixing the amino acid at the first position to asparagine. After the screening of all the connecting peptide fragments is completed, when the obtained best serotonin probe senses the saturated concentration of serotonin, the fluorescence signal is increased approximately by 350 percent, and the obtained probe is named GRAB-5-HT2.0, and the connecting peptide is N-terminal NG and carbon-terminal GFAAA.

According to the screening result of the connecting peptide fragment, the first amino acid of the N-terminal connecting peptide fragment is found to have a larger influence on the performance of the probe after being changed, and the same strategy is used for screening the three sites in front of the N-terminal connecting peptide fragment, namely the 12 th, 13 th and 14 th sites of the third intracellular loop of the human HTR2C receptor, in consideration of the interaction between the amino acids. As a result, it was found that the changes in amino acids 12 and 14 had no effect on the performance of the probe, whereas the signal of the probe increased to nearly 500% after the mutation of leucine L at position 13 to phenylalanine F, which was designated GRAB-5-HT2.1.

4. Serotonin probes have ligand concentration dependent optical responses

The GRAB-5-HT2.1 probe was deactivated using different concentrations of serotonin, which was found to exhibit a concentration-dependent increase in fluorescence signal over a large range of serotonin concentration changes (FIG. 33), and the curve conformed to the Hill distribution. By calculating the Kd value of GRAB-5-HT2.1 and comparing it with the Kd values reported in the literature for the HTR2C receptor for 5-HT, it was found that the modification of the human HTR2C receptor did not affect its affinity for self-ligands, since the region of 5-HT binding to the human HTR2C receptor was mainly located in the transmembrane region as well as in the extracellular region of the latter, whereas the third intracellular loop of the human HTR2C receptor was modified in the present invention. The ligand concentration dependent response curve shows that the serotonin probe can sensitively and quantitatively detect the serotonin signals with different concentrations under physiological conditions.

5. Serotonin probes with specific ligand-induced optical signal changes

Treatment of the serotonin probe GRAB-5-HT2.1 expressed in HEK293T cells with saturated concentrations of different neurotransmitters resulted in the finding that only serotonin could trigger a larger change in the fluorescent signal of the probe (FIG. 34A), while other neurotransmitters could not cause a change in the optical signal of the GRAB-5-HT2.1 probe even at high concentrations.

The addition of an HTR2C receptor-specific agonist (CP 809) to the GRAB-5-HT2.1 probe can cause a change in fluorescence signal, whereas the HTR2B receptor-specific agonist (BWT 23C 83) and the HTR1B receptor-specific agonist (CGS 12066B) cannot cause a change in fluorescence signal; the addition of serotonin first to the GRAB-5-HT2.1 probe resulted in an increase in fluorescence signal, followed by the addition of an HTR2C receptor-specific antagonist thereto (RS 102221) which antagonizes the increase in fluorescence signal of the GRAB-5-HT2.1 probe, and the addition of an HTR2B receptor-specific antagonist thereto (SB 204741) which did not antagonize the increase in fluorescence signal of the probe, which was initiated by serotonin (FIG. 34B). This demonstrates that probes constructed using the human HTR2C receptor have receptor subtype specificity.

6. Construction of a series of serotonin fluorescent probes by constructing chimeric receptors

Analysis of the binding sites for the ligands based on the structures of the already resolved human HTR1B and human HTR2B receptors, shows that none of these sites is involved in the third intracellular loop of the HTR receptor, and thus it is contemplated that other serotonin receptor-based fluorescent probes may be constructed using methods for constructing chimeric receptors. By sequence alignment of different receptors for HTR, the original third intracellular loop was replaced with the third intracellular loop of GRAB-5-HT2.1 constructed with human HTR2C receptor, and changes in fluorescence signal were observed by addition of saturated concentrations of 5-HT. Probes constructed with human HTR2B and human HTR6 receptors were found to exhibit better membrane localization with an increase in fluorescence signal upon addition of saturated concentrations of 5-HT (fig. 35).

See NCBI gene ID for HTR2B sequences: 3357, isoport 1, specific sequences are:

MALSYRVSELQSTIPEHILQSTFVHVISSNWSGLQTESIPEEMKQIVEEQGNKLHWAALLILMVII PTIGGNTLVILAVSLEKKLQYATNYFLMSLAVADLLVGLFVMPIALLTIMFEAMWPLPLVLCPAWLF LDVLFSTASIMHLCAISVDRYIAIKKPIQANQYNSRATAFIKITVVWLISIGIAIPVPIKGIETDVDNPNN ITCVLTKERFGDFMLFGSLAAFFTPLAIMIVTYFLTIHALQKKAYLVKNKPPQRLTWLTVSTVFQRDETPCSSPEKVAMLDGSRKDKAL PNSGDETLMRRTSTIGKKSVQTISNEQRASKVLGIVFFLFLLMWCP FFITNITLVLCDSCNQTTLQMLLEIFVWIGYVSSGVNPLVYTLFNKTFRDAFGRYITCNYRATKSVKT LRKRSSKIYFRNPMAENSKFFKKHGIRNGINPAMYQSPMRLRSSTIQSSSIILLDTLLLTENEGDKTEE RVSYV(SEQ ID NO:9)

wherein the underlined part is the third intracellular loop, specifically bits 240-324, referring to the uniprot database.

See NCBI gene ID for HTR6 sequences: 3362, isoport 1, specific sequences are:

MVPEPGPTANSTPAWGAGPPSAPGGSGWVAAALCVVIALTAAANSLLIALICTQPALRNTSNFF LVSLFTSDLMVGLVVMPPAMLNALYGRWVLARGLCLLWTAFDVMCCSASILNLCLISLDRYLLILS PLRYKLRMTPLRALALVLGAWSLAALASFLPLLLGWHELGHARPPVPGQCRLLASLPFVLVASGLT FFLPSGAICFTYCRILLAARK QAVQVASLTTGMASQASETLQVPRTPRPGVESADSRRLATKHSRKALKASLTLGILLGMFFVTWLPFFVANIVQAVCDCISPGLFDVLTWLGYCNSTMNPIIYPLFMRDFKRA LGRFLPCPRCPRERQASLASPSLRTSHSGPRPGLSLQQVLPLPLPPDSDSDSDAGSGGSSGLRLTAQLL LPGEATQDPPLPTRAAAAVNFFNIDPAEPELRPHPLGIPTN(SEQ ID NO:10)

wherein the underlined part is the third intracellular loop, specifically bits 209-265, referring to the uniprot database.

7. Method for detecting release of serotonin in central nervous system of drosophila by using two-photon imaging method

Transgenic drosophila of UAS-GRAB-5-HT was constructed using GRAB-5-HT2.0, and after hybridization with Trh-Gal4 strain, the probe was specifically expressed in serotonergic neurons, and neural activity of serotonergic neurons due to olfactory stimulation upon administration of isoamyl acetate scent stimulation was successfully detected by means of two-photon imaging (FIG. 36).

8. High throughput drug screening using GRAB-5-HT1.0 expressing cell lines

The HEK293T cell line stably expressing the GRAB-5-HT1.0 probe was constructed, a high throughput drug screening platform (a platform based on computer-controlled mechanical arm for experimental manipulation, drug addition, precipitation and fluorescent signal detection using computer control, thus achieving better reproducibility and stability) was used, and a stable signal rise was observed compared to the addition of the solvent (control) group by detecting the change in fluorescence after adding 5-hydroxytryptamine to the cells expressing the GRAB-5-HT1.0 probe (FIG. 52). It can be observed from the figure that the detection method has better repeatability and sensitivity (represented by Z factor, which is a parameter that characterizes whether the system is sufficiently sensitive and stable in the high-throughput screening process, and the formula thereof is shown in fig. 52). Generally, systems suitable for high throughput screening, whose Z factor needs to be greater than 0.4, represent sufficient sensitivity and stability for high throughput drug screening based on methods for constructing stable cell lines with GRAB probes.

EXAMPLE 10 construction of genetically encoded dopamine Probe

1. Construction of genetically encoded dopamine probes

The human dopamine receptor has 5 subtypes in vivo, which are designated as DRD1-DRD5, respectively. When constructing fluorescent probes, firstly, carrying out primary screening on partial receptors, and adopting a method of inserting fluorescent protein into any site of a third intracellular loop of the partial receptors to observe the expression and membranous condition of the partial receptors, thereby obtaining a better candidate receptor, namely human DRD2 receptor. The optimal insertion site for the fluorescent protein was then determined step by step using a strategy similar to that of the adrenergic probe in example 2. Specifically, for the third intracellular loop of the human DRD2 receptor, 15 amino acids each served as an insertion site. Human DRD2 receptor inserted with circulating rearranged fluorescent protein at different positions is expressed in HEK293T cells, and drug perfusion experiments are carried out by using dopamine, so that a plurality of fluorescent probes sensitive to dopamine are finally identified, wherein the fluorescent probe with the largest signal change is obtained by cutting off amino acid 253 to amino acid 357 of the human DRD2 receptor, and inserting the circulating rearranged fluorescent protein at the cut-off position. After the site of conformational change sensitivity was determined, the surrounding amino acid sites were next subjected to further screening, i.e. the nitrogen end was around amino acid position 252 and the carbon end was around amino acid position 357, aligned and combined, and they were expressed in HEK293T cells and examined for drug infusion with dopamine. The best probe obtained was a fluorescent protein with amino acids 254 to 360 truncated and a cyclic rearrangement inserted at the truncated position, which can achieve 110% signal change under saturation concentration of dopamine treatment (FIG. 37), designated GRAB-GDA3.0, with a linker peptide of nitrogen end GG and carbon end GGAAA.

Wherein the sequence of the human DRD2 receptor is found in NCBI gene ID:1813,isoform long, the specific amino acid sequence of which is:

MDPLNLSWYDDDLERQNWSRPFNGSDGKADRPHYNYYATLLTLLIAVIVFGNVLVCMAVSRE KALQTTTNYLIVSLAVADLLVATLVMPWVVYLEVVGEWKFSRIHCDIFVTLDVMMCTASILNLCAI SIDRYTAVAMPMLYNTRYSSKRRVTVMISIVWVLSFTISCPLLFGLNNADQNECIIANPAFVVYSSIV SFYVPFIVTLLVYIKIYIVLR RRRKRVNTKRSSRAFRAHLRAPLKGNCTHPEDMKLCTVIMKSNGSFPVNRRRVEAARRAQELEMEMLSSTSPPERT RYSPIPPSHHQLTLPDPSHHGLHSTPDSPAKPEKNGHAKDHPKIAKIFEIQTMPNGKTRTSLKTMSRRKLSQQKEK KATQMLAIVLGVFIICWLPFFITHILNIHCD CNIPPVLYSAFTWLGYVNSAVNPIIYTTFNIEFRKAFLKILHC(SEQ ID NO:5)

wherein the underlined part is the third intracellular loop, specifically positions 214-373, referring to the uniprot database.

NVYIKADKQKNGIKANFHIRHNIEDGGVQLAYHYQQNTPIGDGPVLLPDNHYLSVQSKLSKDP NEKRDHMVLLEFVTAAGITLGMDELYKGGTGGSMVRKGEELFTGVVPILVELDGDVNGHKFSVSG EGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYIQERTI FFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYN(SEQ ID NO:11)

pharmacological studies have shown that GRAB-GDA3.0 can only be activated by DA and not by other neurotransmitters, and in addition, it can be activated or inhibited by DRD 2-specific agonists (Dopamine) or antagonists (haloperidol), respectively (fig. 38).

2. Odor stimulates GDA signaling in the Mushroom (MB)

Construction of transgenic Drosophila UAS-GRAB-GDA3.0, which expresses the GRAB-GDA3.0 probe in specific cells, driven by the corresponding GAL4 line. First, GRAB-GDA3.0 was expressed in dopaminergic neurons (DAN) in all drosophila, and the odor (isoamyl acetate) -induced response was examined by in vivo two-photon imaging (fig. 39A). GRAB-GDA3.0 expressed in the cell membrane of DAN was essentially able to report Dopamine (DA) release by probes located at presynaptic locations (fig. 39B). After releasing the scent within seconds, a robust GRAB-GDA3.0 signal was observed in the whole Mushroom (MB), especially β' lobe (fig. 39C and D).

3. The odor-activated GDA signal in MB is specific for DA

Pharmacological studies on GRAB-GDA3.0, the different drugs were incubated in solution in which Drosophila was imaged, and found to block its GDA signal completely by the DRD 2-specific antagonist halo (haloperidol) (FIGS. 40A-C), whereas as a control it was not blocked by the octopamine receptor-specific antagonist epistatin (FIGS. 40D-F). These results demonstrate that the GDA signal is specific for DA.

In addition, it was further demonstrated from the viewpoint of gene studies that it was specific for GDA, comparing the rate of GDA signal decay between normal Drosophila and Drosophila with reduced expression of DAT (this experiment is a later-written study of gene level with DAT-RNAi, i.e., in DAN, dopamine transporter (DAT) is located in the presynaptic membrane, releasing DA from the synaptic intermittent cycle. DAT expression was inhibited in DAN using DAT-RNAi (fig. 40G). Theoretically, the decay time of GDA signal in DAT-RNAi Drosophila should be longer than that of WT Drosophila. Indeed, the duration of the odor-induced GDA signal (τ=1.85 s) in DAT-RNAi drosophila was indeed longer than WT drosophila (τ=0.48 s) (fig. 40H-J).

EXAMPLE 11 construction of red-fluorescent dopamine and serotonin probes

1. Materials and methods

Molecular cloningThe GRAB probe plasmid was cloned into a pDISPLAY vector (Invitrogen) with an IgK leader sequence before the coding region and a stop codon before the transmembrane region. The cpmApple gene (cpmApple is a cprFP, RFP is red fluorescent protein red fluorescent protein) was amplified by R-GECO1 (Yongxin Zhao, et al, an Expanded Palette of Genetically Encoded Ca2+indicators, science, 2011) (given by Dr. Robert E. Campbell). Full-length human GPCR cDNA was amplified from the hORFeome database 8.1. All molecular cloning, including site-directed mutagenesis, was performed using Gibson assembly, using primers with 30 base overlap. The correct clones were verified by Sanger sequencing.

CpmApple amino acid sequence:

PVVSERMYPEDGALKSEIKKGLRLKDGGHYAAEVKTTYKAKKPVQLPGAYIVDIKLDIVSHNE DYTIVEQCERAEGRHSTGGMDELYKGGTGGSLVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIE GEGEGRPYEAFQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYIKHPADIPDYFKLSFPEGFRWERV MNFEDGGIIHVNQDSSLQDGVFIYKVKLRGTNFPPDGPVMQKKTMGWEATR(SEQ ID NO:12)

cell culture and transfectionHEK293T cells at 37℃and 5% CO ₂ DMEM supplemented with 10% fbs and penicillin-streptomycin was used. 12-mm glass coverslip with cells placed in 24 well platesAnd (3) upper part. Cortical neurons were cultured as follows. P1 rats were dissected and digested with 0.25% Trypsin-EDTA (Gibco) and then placed on poly-D-lysine coated coverslips at a density of 0.5-1X10 ⁶ Individual cells/ml. At the time of transfection, HEK293T cells were transiently transfected by PEI method at a ratio of 1. Mu.g DNA to 4. Mu.g PEI. The medium was refreshed 4-6h after transfection and imaged 24h later. Cultured neurons were transfected with calcium phosphate method and 1xHBS (pH 6.8) was used to dissolve the pellet after 1.5 h. In vitro neurons were transfected after 7-9d and experiments were performed 48h post transfection.

Fluorescence imaging and perfusion of cultured cellsHEK293T cells and cultured cortical neurons were perfused with standard extracellular Tyrode solution containing (in mM): 150NaCl,4KCl,2MgCl ₂ ,2CaCl ₂ 10HEPES and 10 glucose, pH 7.4. The coverslip was placed in a home-made perfusion chamber and connected to miniature manifold (multi-channel tubing connector) for perfusion. HEK293T cells and neurons were imaged using the Nikon confocal system.

2. Strategies for generating red GRAB probes

The circularly rearranged red fluorescent protein cpm apple is inserted into the third intracellular loop of the GPCR, thereby converting the ligand-induced conformational change of the GPCR into an optical signal. The procedure for generating the red GRAB probe was as follows: in the first step, the optimal insertion site of cpmeapple in GPCR is found, cpmeapple is inserted every 5 amino acids in the whole third intracellular loop, and the cpmeapple is truncated at the specific amino acid site and inserted at the truncation. Single constructs with maximal fluorescent response in HEK293 cells were screened in laminar flow chamber with saturated ligand perfusion. Second, the insertion site is fine-tuned. After the possible insertion sites have been located, the insertion sites are trimmed residue by residue around the optimal reaction site in a similar way as in the first step. Step 3, optimizing the N-terminal and C-terminal connecting peptide sequences of cpmApple, firstly, independently optimizing the N-terminal and C-terminal of the cmpApple connecting peptide by repeated mutation and screening, and then combining the optimal N-terminal and C-terminal connecting peptide sequences for screening. In the screening process, the screening has higher delta F/F at the same time ₀ And is higher thanFluorescent mutants.

3. Construction of red fluorescent dopamine Probe

The procedure of the above strategy was followed to find the optimal insertion site for cmpApple with the greatest ligand-induced response and optimal membrane localization. Human dopamine receptor DRD2 (same receptor as in example 10) was selected to construct a probe and 92 variants in the constructed library were perfused. Of which 16 were non-fluorescent and 56 were non-ligand-induced. Of which 15 show on-responses. Of which 5 show an off-response. Herein, the on-reaction means that the fluorescence signal is enhanced when the cells are perfused with buffer containing a saturated concentration of ligand. The off-reaction indicates that the fluorescence signal decreases when the cells are perfused with buffer containing a saturated concentration of ligand. The results of the on reaction and the off reaction are shown in FIG. 41A. The best on reaction candidate DRD 222-349cmpApple (i.e., the 223-349 bits of DRD were truncated and the cmpApple was inserted at the truncated positions) showed more than 13% on reaction, and the best off reaction candidate DRD 267-364cmpApple (i.e., the 268-364 bits of DRD were truncated and the cmpApple was inserted at the truncated positions) showed more than 22% off reaction. The imaging characteristics and response curves are shown in fig. 41B. Herein, the numbers following the DRD represent the insertion site of the cmpApple. Both candidates showed good membrane localization. The on reaction candidate is used for the next optimization since the on reaction probe generally has a better signal-to-noise ratio in the imaging. After fine tuning the insertion site, the ligand-induced response increased to 32%. The left panel of fig. 41C shows the strongest response. The best on candidate DRD 223-365cmpApple (i.e., the 224-365 positions of DRD were truncated and cmpApple was inserted at the truncated position) showed 32% on-response (FIG. 41C, right panel). The membrane was well positioned (fig. 41C, middle panel). The third step was then used to optimize the linker peptide sequence for DRD 223-365 cmpApple. There are 5 linker amino acids at the N-terminus of cmpApple and three linker amino acids at the C-terminus. Random mutation of the linker peptide amino acids independently from one to the other, some variants showed higher Δf/F ₀ And higher brightness, wherein the peptide is linked to one variantThe initial sequence of the segments is PVVSE (N-terminal), ATR (C-terminal) (FIG. 41D).

4. Construction of red fluorescent serotonin Probe

A red GRAB probe of serotonin was constructed using a strategy similar to that of red fluorescent dopamine probe. Human serotonin receptor HTR2C (sequence identical to example 9) was selected to construct the probe. In libraries constructed by the cpmeapple insertion strategy followed by the fine tuning strategy, HTR2C 240-306 cpmeapple (i.e., HTR2C bits 241-306 were truncated and cmpApple inserted at the truncated position) was obtained with 27% on-reaction, and HTR2C 239-309 cpmeapple (i.e., HTR2C bits 240-309 were truncated and cmpApple inserted at the truncated position) with 21% off-reaction (FIGS. 42A and 42B). The connecting peptide fragments are PVVSE (N terminal) and ATR (C terminal). Random mutations of 5 amino acids of the N-terminal linker peptide of cpmeAN, some variants showed higher ΔF/F ₀ And higher brightness (fig. 42C).

EXAMPLE 12 construction of serotonin BRET Probe

The bioluminescence comes from chemical reaction, compared with fluorescence, the imaging can be realized without the excitation of an external light source, and adverse factors such as tissue autofluorescence, phototoxicity, photobleaching and the like caused by the external excitation light are avoided, so that the imaging method is particularly suitable for living animal imaging, especially deep tissue imaging. Nanoluc is a luciferase with extremely high catalytic activity and luminescence brightness, which uses furimazine (2-furanylmethyl-deoxyoementazine) as a substrate, and the peak value of light emitted by catalytic chemical reaction is 450nm, which is similar to the excitation 488nm of the cpEGFP used by each GRAB probe of the present invention. According to the principle of resonance energy transfer of light, energy transfer can occur when the spatial distance and relative position of the Nanoluc and each GRAB probe of the invention meet the requirements.

Therefore, in this example, the light emitted by Nanoluc is used as the energy donor for the serotonin probe, so that the fluorescent signal of the probe is detected without external excitation light. Such a serotonin probe that can be imaged without external excitation light would be advantageous for studying the function of serotonin-related nerve micro-loops in living animals.

According to the structural change characteristics of the G protein coupled receptor when the G protein coupled receptor is combined with a ligand, selecting a peptide segment of a serotonin receptor HTR2C as an insertion site of Nanoluc. Nanoluc was inserted at various positions of its C-terminus and expressed in HEK293T cells based on GRAB-5-HT2.0 obtained in example 9, and furimazine was added after 24 hours of expression of the probe in the cells. And detecting fluorescent signals by using an enzyme-labeled instrument.

When the ligand serotonin (5-HT) binds to the receptor, the structure of the receptor changes, which changes the spatial distance and relative position of Nanoluc at the C-terminus and the cpEGFP at the third intracellular ring, changing the efficiency of resonance energy transfer between the two, and thus changing the fluorescent signal of the cpEGFP. The probe can be imaged under the condition of no external excitation light, and the change of the fluorescence signal can reflect the binding process of serotonin and a receptor.

By optimizing the insertion position and the linker peptide, a probe version was obtained. In this version, after addition of 10. Mu.M of 5-HT, the probe showed a 6% signal enhancement, the signal change being inhibited by HTR2C antagonists, as shown in FIG. 43. The specific insertion position of the probe was between amino acids 582 and 583 of GRAB-5-HT2.0 obtained in example 9 (i.e., between amino acids 582 and 583 of the entire probe after insertion of the fluorescent protein). The N-terminal and C-terminal connecting peptide of Nanoluc are GSG.

The application mode of the probe comprises the following steps: the probe is expressed in brain regions of living animals by transgenic or viral injection, and the substrate furimazine of Nanoluc is added to the animal's diet, allowing the animal to obtain the substrate by ingestion. After a period of time, the bioluminescence imaging device was used to observe changes in serotonin signals in the brain region of the animal.

EXAMPLE 13 optimized screening of acetylcholine probes

1. Materials and methods

As in example 1.

2. Acetylcholine receptor and cpEGFP

As in example 2, wherein the human acetylcholine receptor M3R subtype is also referred to as M3R receptor or CHRM3 in this example.

3. Truncating ICL3 and inserting cpEGFP

ICL3 was truncated between two random sites on ICL3 of the M3R receptor and cpgfp was inserted at the truncated position (fig. 45 a). Libraries of 7*8 =56 sizes were constructed by primer design (fig. 45 b). Due to the random truncations of ICL3, the screening was scaled up to 200 clones in order to cover as much as possible all possible combinations in the library (fig. 45 c). Using a high throughput screening system, a clone with a signal enhancement of up to 30% was obtained, whose truncations at the M3R receptor are at amino acids 259 and 490 (FIG. 45 d). In FIG. 45, the clone is shown as "259-490".

4. Optimizing the connecting peptide fragment of cpEGFP and M3R receptor

In order to systematically optimize the performance of the acetylcholine probe (mainly the basic fluorescence intensity of the acetylcholine probe and its magnitude of response to the ligand at saturation concentration), on the basis of clone 259-490 obtained in the above step, a random mutation was simultaneously performed on one amino acid of the N-terminal linker peptide and one amino acid of the C-terminal linker peptide (the original linker peptide was GG-GGAAA). The N-terminal connecting peptide has 2 amino acid residue sites, the C-terminal connecting peptide has 5 amino acid residue sites, and the total of the N-terminal connecting peptide and the C-terminal connecting peptide is 2*5 =10 libraries; because each site was randomly mutated to potentially mutate any of the 20 amino acids in humans, each library included 20×20=400 possible amino acid residue combinations (fig. 46a, b). The total 4000 plasmids of the 10 libraries were initially screened using an Opera Phenix high content screening platform, since the Opera Phenix high content screening platform can only screen 60 plasmids at a time, only 100 plasmids were taken from each library for workload.

After screening 1000 plasmids of 10 libraries, it was found that: when the first site of the C-terminal linker between cpEGFP and M3R is Histidine (His, H), the probe has a higher basal fluorescence intensity and a higher response to the ligand at saturation concentration (FIG. 46C), i.e., the linker is GG-HGAAA, and thus the first site of the C-terminal linker is immobilized as H, and then random mutation is performed on the remaining 6 sites one by one (FIG. 46C, d).

After fixing the first site of the C-terminal linker peptide to H, the remaining 6 sites were randomly mutated one by one, and it was found that when the 2 nd site of the C-terminal linker peptide was mutated to N, the probe response to acetylcholine was nearly doubled and the basal fluorescence intensity was slightly increased (FIG. 47 e), the linker peptide sequence was GG-HNAAA, and the probe was designated GRAB-ACh3.0.

Fixing the first and second sites of the C-terminal connecting peptide segment as H, N respectively, and continuing to randomly mutate the rest 5 sites one by one on the basis (figures 47b and e), wherein the round of random mutation discovers: the absence of 6 base pairs, which was not artificially introduced, doubles the reaction size of the acetylcholine probe again (FIG. 47 f) -in libraries with random mutations at the fourth site of the C-terminal connecting peptide, amino acid Q at 491 of the M3R receptor was further truncated due to the unexpected lack of 6 base pairs, and the fourth site of the C-terminal connecting peptide was mutated to Lysine (Lysine, lys, K) (FIG. 47 g). This probe was designated GRAB-ACh4.0. In the probe GRAB-ACh4.0, the 260-491 bits of the M3R receptor are cut off, and the connecting peptide between the cpEGFP and the M3R receptor is N-terminal GG and C-terminal HNAK.

The performance of GRAB-ACh4.0 was verified by perfusion experiments. At Confocal, when saturated concentrations (100. Mu. Mol/L) of ACh were added, the single cell expressed GRAB-ACh4.0 probe response reached more than 250% (FIG. 48 a); whereas in the presence of the antagonist tiotropium bromide (Tiotropium bromide, tio), the single cell expressed probe fluorescence signal enhancement was almost completely masked, indicating that the probe fluorescence signal enhancement (i.e. the reaction upon ACh addition) was entirely caused by ACh (fig. 48 a). The same perfusion was performed on 18 cells and the magnitude of their response to ACh was counted as compared to ACh in the presence of the antagonist, and it can be seen that the average response of these 18 cells was over 250% with most cells being over 200% and some even up to 350% (fig. 48b, c).

5. The binding capacity of GRAB-ACh4.0 to ACh is not significantly different from that of the wild-type M3R receptor

An important property of the acetylcholine probe is its ability to bind to acetylcholine Kd. Only if Kd is within a suitable range, the acetylcholine probe can detect the concentration of acetylcholine in vivo, and if Kd is too great or too small, the concentration of acetylcholine in vivo may already be too high in saturation concentration, or below the detection limit of the acetylcholine probe, and the concentration of acetylcholine cannot be detected quantitatively. The binding capacity of GRAB-ACh4.0 to acetylcholine was measured using an Opera Phenix (FIG. 49). It can be seen that Kd=2.61×10 of acetylcholine probe GRAB-ach4.0 ^-7 And can detect the concentration from 10 ^-9 To 10 ^-5 And mol/L of acetylcholine. From other papers on measurements of acetylcholine concentration and Kd of human acetylcholine receptor, the GRAB-ach4.0 probe has a binding capacity to acetylcholine close to that of reported human acetylcholine M3R receptor (Jakubik, j., bacakova, l., el-Fakahany, E.E.&Tucek, S.positive cooperativity of acetylcholine and other agonists with allosteric ligands on muscarinic acetylcholine receptors. Mol Pharmacol 52,172-179 (1997)), can be used to quantitatively measure acetylcholine concentrations in vivo.

6. GRAB-ACh4.0 has very strong specificity

To verify the specificity of the acetylcholine probe GRAB-ACh4.0, different neurotransmitters were added to HEK293T cells expressing GRAB-ACh4.0 on an OPera PhenixTM and the probe response was examined. It can be seen that the GRAB-ACh4.0 probe has high specificity in HEK293T cells-the reaction of the probe is reduced to almost no after the antagonist Tio is added, indicating that the change of fluorescence intensity of GRAB-ACh4.0 is indeed caused by the binding of acetylcholine; the fluorescence intensity of GRAB-ACh4.0 was also hardly changed when other neurotransmitters were added, indicating that the GRAB-ACh4.0 probe did not bind to other neurotransmitters (FIG. 50). In summary, GRAB-ACh4.0 can be, and is only, activated by ACh, resulting in a change in fluorescence intensity.

7. GRAB-ACh4.0 does not activate downstream Gq-directed signaling pathway

The coupling of GRAB-ACh4.0 to downstream G.alpha.q was examined. First, three stably expressed cell lines were constructed against the gαq cell line: M3R (labeled CHRM3 in FIG. 51), GRAB-ACh4.0, and a null (labeled Gq in FIG. 51) cell line; next, the extent to which the G.alpha.q protein is activated is characterized by the extent of release of TGF.alpha.s. It can be seen that only the stable cell line expressing wild type M3R activated the G.alpha.q-directed signal transduction pathway when a gradient concentration of ACh was applied, while the stable cell line expressing GRAB-ACh4.0 was almost flush with the background cell line, indicating that the GRAB-ACh4.0 probe did not activate the G.alpha.q-directed signal transduction pathway (FIG. 51A). Tissue plasminogen activator (Tissue Plasminogen Activator, TPA) is a serum protease that directly lyses the cell membrane, allowing it to release TGF- α with alkaline phosphatase even in the absence of a downstream signal from gαq, thus taking the TPA-added cells as a positive control. The supernatant reaction after TPA addition was maximal, indicating that neither substrate nor enzyme had problems; after ACh, only the cell line expressing M3R is activated, but the cell line expressing GRAB-ACh4.0 is not activated, which indicates that the GRAB-ACh4.0 probe cannot be coupled with G protein, activate downstream signal channels and disturb normal physiological functions of cells; the antagonist Tio can completely mask the downstream signal caused by M3R, indicating that activation of downstream G protein of M3R is indeed caused by the binding of ACh (fig. 51B).

Claims

1. A fluorescent probe constructed based on a G protein-coupled receptor, wherein the fluorescent probe is a fusion protein obtained by modifying the G protein-coupled receptor, and the modification comprises inserting a circularly rearranged luciferase into a third intracellular loop between a fifth transmembrane region and a sixth transmembrane region of the G protein-coupled receptor; wherein the method comprises the steps of

The G protein-coupled receptor is a G protein-coupled receptor which specifically binds to a specific ligand;

the fluorescent probe constructed based on the G protein coupled receptor can be expressed on a cell membrane; and is also provided with

The fluorescent probe constructed based on the G protein-coupled receptor may be bound to the G protein-coupled receptor when contacted with a specific ligand thereof, thereby causing a detectable change in the fluorescent intensity of the fluorescent probe.

2. The fluorescent probe of claim 1, wherein the engineering comprises truncating a third intracellular loop between a fifth transmembrane region and a sixth transmembrane region of the G protein coupled receptor and inserting a circularly rearranged luciferase at the truncated position.

3. The fluorescent probe of claim 2, wherein the circularly rearranged luciferase is linked at both ends to a third intracellular loop of the G protein-coupled receptor via a linker peptide, respectively.

4. The fluorescent probe of claim 3, wherein the linking peptide comprises a flexible amino acid.

5. The fluorescent probe of claim 4, wherein the flexible amino acid comprises glycine and/or alanine.

6. The fluorescent probe of claim 5, wherein the linker peptide consists of glycine and alanine.

7. The fluorescent probe of claim 6, wherein the cyclically rearranged luciferase N-terminal connecting peptide is GG and/or the cyclically rearranged luciferase C-terminal connecting peptide is GGAAA.

8. The fluorescent probe of claim 1, wherein the specific ligand is a neurotransmitter, hormone, metabolic molecule, nutritional molecule, or an artificially synthesized small molecule or drug that activates a specific receptor; the G protein-coupled receptor is a G protein-coupled receptor that specifically binds to neurotransmitters, hormones, metabolic molecules, nutritional molecules, or synthetic small molecules or drugs that activate a particular receptor.

9. The fluorescent probe of claim 8, wherein the neurotransmitter is epinephrine, norepinephrine, acetylcholine, serotonin and/or dopamine.

10. The fluorescent probe of claim 8, wherein the synthetic small molecule or drug that activates a specific receptor is Isoprenaline (ISO).

11. The fluorescent probe of claim 1, wherein the G protein-coupled receptor is of human or animal origin.

12. The fluorescent probe of claim 1, wherein the fluorescent probe constructed based on G protein-coupled receptors is a fluorescent probe for detecting epinephrine, wherein the G protein-coupled receptors are GPCRs that specifically bind epinephrine.

13. The fluorescent probe of claim 12, wherein the GPCR that specifically binds epinephrine is a human β2 adrenergic receptor and the fluorescent probe constructed based on G-protein coupled receptors is a fluorescent probe constructed based on human β2 adrenergic receptor.

14. The fluorescent probe of claim 13, wherein the circularly rearranged luciferase is linked to a third intracellular loop of the human β2 adrenergic receptor through an N-terminal and a C-terminal linking peptide.

15. The fluorescent probe of claim 14, wherein the linker peptide at both ends of the cyclically rearranged luciferase is 1 or 2 amino acids in length at the nitrogen end and/or 1, 2, 3, 4 or 5 amino acids at the carbon end, respectively.

16. The fluorescent probe of claim 15, wherein the linker peptides at both ends of the cyclically rearranged luciferase are each 2 amino acids in length at the nitrogen end and 5 amino acids at the carbon end.

17. The fluorescent probe of claim 16, wherein the connecting peptides at both ends of the circularly rearranged luciferase are GG at the N-terminus and GGAAA at the C-terminus, respectively, or the connecting peptides at both ends of the circularly rearranged luciferase are GG at the N-terminus and SPSVA at the C-terminus, respectively, or the connecting peptides at both ends of the circularly rearranged luciferase are GG at the N-terminus and APSVA at the C-terminus, respectively.

18. The fluorescent probe of claim 15, wherein the linker peptides at both ends of the cyclically rearranged luciferase are each 1 amino acid in length at the nitrogen end and 1 amino acid at the carbon end.

19. The fluorescent probe of claim 18, wherein the linking peptides at both ends of the circularly rearranged luciferase are each G at the N-terminus and G at the C-terminus.

20. The fluorescent probe of claim 13, wherein the amino acid sequence of the human β2 adrenergic receptor is:

MGQPGNGSAFLLAPNRSHAPDHDVTQQRDEVWVVGMGIVMSLIVLAIVFGNVLVITAIAKFERLQTVTNYFITSLACADLVMGLAVVPFGAAHILMKMWTFGNFWCEFWTSIDVLCVTASIETLCVIAVDRYFAITSPFKYQSLLTKNKARVIILMVWIVSGLTSFLPIQMHWYRATHQEAINCYANETCCDFFTNQAYAIASSIVSFYVPLVIMVFVYSRVFQ EAKRQLQKIDKSEGRFHVQNLSQVEQDGRTGHGLRRSSKFCLKEHKALKTLGIIMGTFTLCWLPFFIVNIVHVIQDNLIRKEVYILLNWIGYVNSGFNPLIYCRSPDFRIAFQELLCLRRSSLKAYGNGYSSNGNTGEQSGYHVEQEKENKLLCEDLPGTEDFVGHQGTVPSDNIDSQGRNCSTNDSLL (SEQ ID NO: 1), wherein the underlined part is the third intracellular loop.

21. The fluorescent probe of claim 20, wherein a circularly rearranged luciferase is inserted between amino acid 240 and amino acid 241 of the human β2 adrenergic receptor; or a circularly rearranged luciferase is inserted between amino acid 250 and amino acid 251 of the human β2 adrenergic receptor.

22. The fluorescent probe of claim 1, wherein the fluorescent probe constructed based on G protein-coupled receptors is a fluorescent probe for detecting epinephrine and/or norepinephrine, wherein the G protein-coupled receptors are GPCRs that specifically bind epinephrine and/or norepinephrine.

23. The fluorescent probe of claim 22, wherein the GPCR that specifically binds epinephrine and/or norepinephrine is a human ADRA2A receptor and the fluorescent probe constructed based on G-protein coupled receptors is a fluorescent probe constructed based on human ADRA2A receptor.

24. The fluorescent probe of claim 23, wherein the third intracellular loop of the human ADRA2A receptor is truncated and a circularly rearranged luciferase is inserted at the truncated position.

25. The fluorescent probe of claim 24, wherein the circularly rearranged luciferase is linked to the third intracellular loop of the human ADRA2A receptor via a linker peptide at the N-terminus and the C-terminus, the linker peptide at both ends of the circularly rearranged luciferase being 2 amino acids at the nitrogen terminus and 5 amino acids at the carbon terminus, respectively.

26. The fluorescent probe of claim 25, wherein the connecting peptides at both ends of the circularly rearranged luciferase are GG at the N-terminus and GGAAA at the C-terminus, respectively, or the connecting peptides at both ends of the circularly rearranged luciferase are GG at the N-terminus and TGAAA at the C-terminus, respectively.

27. The fluorescent probe of claim 23, wherein the amino acid sequence of the human ADRA2A receptor is:

MFRQEQPLAEGSFAPMGSLQPDAGNASWNGTEAPGGGARATPYSLQVTLTLVCLAGLLMLLTVFGNVLVIIAVFTSRALKAPQNLFLVSLASADILVATLVIPFSLANEVMGYWYFGKAWCEIYLALDVLFCTSSIVHLCAISLDRYWSITQAIEYNLKRTPRRIKAIIITVWVISAVISFPPLISIEKKGGGGGPQPAEPRCEINDQKWYVISSCIGSFFAPCLIMILVYVRIYQIAKRRTRVPPSRRGPDAVAAPPGGTERRPNGLGPERSAGPGGAEAEPLPTQLNGAPGEPAPAGP RDTDALDLEESSSSDHAERPPGPRRPERGPRGKGKARASQVKPGDSLPRRGPGATGIGTPAAGPGEERVGAAKASR WRGRQNREKRFTFVLAVVIGVFVVCWFPFFFTYTLTAVGCSVPRTLFKFFFWFGYCNSSLNPVIYTIFNHDFRRAFKKILCRGDRKRIV (SEQ ID NO: 2), wherein the underlined part is the third intracellular loop.

28. The fluorescent probe of claim 27, wherein amino acids 71-130 of the third intracellular loop of the human ADRA2A receptor are truncated and a circularly rearranged luciferase is inserted at the truncated position; alternatively, amino acids 71-135 of the third intracellular loop of the human ADRA2A receptor are truncated and a circularly rearranged luciferase is inserted at the truncated position.

29. The fluorescent probe of claim 1, wherein the fluorescent probe constructed based on G protein-coupled receptors is a fluorescent probe for detecting acetylcholine, wherein the G protein-coupled receptors are GPCRs that specifically bind to acetylcholine.

30. The fluorescent probe of claim 29, wherein the GPCR that specifically binds epinephrine is a human acetylcholine receptor M3R subtype and the fluorescent probe constructed based on G protein coupled receptors is a fluorescent probe constructed based on the human acetylcholine receptor M3R subtype.

31. The fluorescent probe of claim 30, wherein the third intracellular loop of the human acetylcholine receptor M3R subtype is truncated and a circularly rearranged luciferase is inserted at the truncated position.

32. The fluorescent probe of claim 31, wherein the cyclically rearranged luciferase is linked to a third intracellular loop of the M3R subtype of human acetylcholine receptor by N-terminal and C-terminal linking peptides that are each 2 amino acids in length at the nitrogen end and 5 amino acids in length at the carbon end.

33. The fluorescent probe of claim 32, wherein the connecting peptides at both ends of the cyclically rearranged luciferase are respectively N-terminal GG and C-terminal GGAAA, or the connecting peptides at both ends of the cyclically rearranged luciferase are respectively N-terminal GG and C-terminal HGAAA, or the connecting peptides at both ends of the cyclically rearranged luciferase are respectively N-terminal GG and C-terminal HNAAA, or the connecting peptides at both ends of the cyclically rearranged luciferase are respectively N-terminal GG and C-terminal HNAK.

34. The fluorescent probe of claim 30, wherein the amino acid sequence of the human acetylcholine receptor M3R subtype is:

MTLHNNSTTSPLFPNISSSWIHSPSDAGLPPGTVTHFGSYNVSRAAGNFSSPDGTTDDPLGGHTVWQVVFIAFLTGILALVTIIGNILVIVSFKVNKQLKTVNNYFLLSLACADLIIGVISMNLFTTYIIMNRWALGNLACDLWLAIDYVASNASVMNLLVISFDRYFSITRPLTYRAKRTTKRAGVMIGLAWVISFVLWAPAILFWQYFVGKRTVPPGECFIQFLSEPTITFGTAIAAFYMPVTIMTILYWRIYKETEKRTKELAGLQASGTEAETENFVHPTGSSRSCSSYELQQQSM KRSNRRKYGRCHFWFTTKSWKPSSEQMDQDHSSSDSWNNNDAAASLENSASSDEEDIGSETRAIYSIVLKLPGHST ILNSTKLPSSDNLQVPEEELGMVDLERKADKLQAQKSVDDGGSFPKSFSKLPIQLESAVDTAKTSDVNSSVGKSTA TLPLSFKEATLAKRFALKTRSQITKRKRMSLVKEKKAAQTLSAILLAFIITWTPYNIMVLVNTFCDSCIPKTFWNLGYWLCYINSTVNPVCYALCNKTFRTTFKMLLLCQCDKKKRRKQQYQQRQSVIFHKRAPEQAL (SEQ ID NO: 3), wherein the underlined part is the third intracellular loop.

35. The fluorescent probe of claim 34, wherein amino acids 260-490 of the human acetylcholine receptor M3R subtype are truncated and a circularly rearranged luciferase is inserted at the truncated position; or the 260-491 amino acids of the M3R subtype of the human acetylcholine receptor are truncated and a circularly rearranged luciferase is inserted at the truncated position.

36. The fluorescent probe of claim 1, wherein the fluorescent probe constructed based on G protein-coupled receptors is a fluorescent probe for detecting 5-hydroxytryptamine, wherein the G protein-coupled receptors are GPCRs that specifically bind 5-hydroxytryptamine.

37. The fluorescent probe of claim 36, wherein the GPCR that specifically binds 5-hydroxytryptamine is a human HTR2C receptor and the fluorescent probe constructed based on G-protein coupled receptors is a fluorescent probe constructed based on human HTR2C receptor.

38. The fluorescent probe of claim 37, wherein the third intracellular loop of the human HTR2C receptor is truncated and a circularly rearranged luciferase is inserted at the truncated position.

39. The fluorescent probe of claim 38, wherein the circularly rearranged luciferase is linked to the third intracellular loop of the human HTR2C receptor by a linker peptide at the N-terminus and the C-terminus, the linker peptide at both ends of the circularly rearranged luciferase being 2 amino acids at the nitrogen terminus and 5 amino acids at the carbon terminus, respectively.

40. The fluorescent probe according to claim 39, wherein the connecting peptides at both ends of the circularly rearranged luciferase are GG at N-terminus and GGAAA at C-terminus, respectively, or the connecting peptides at both ends of the circularly rearranged luciferase are NG at N-terminus and GFAAA at C-terminus, respectively.

41. The fluorescent probe of claim 37, wherein the amino acid sequence of the human HTR2C receptor is:

MVNLRNAVHSFLVHLIGLLVWQCDISVSPVAAIVTDIFNTSDGGRFKFPDGVQNWPALSIVIIIIMTIGGNILVIMAVSMEKKLHNATNYFLMSLAIADMLVGLLVMPLSLLAILYDYVWPLPRYLCPVWISLDVLFSTASIMHLCAISLDRYVAIRNPIEHSRFNSRTKAIMKIAIVWAISIGVSVPIPVIGLRDEEKVFVNNTTCVLNDPNFVLIGSFVAFFIPLTIMVITYCLTIYVLRRQALMLLHGHTEEPPGLSLDFLKCCKRNTAEEENSANPNQDQNARRRKKKERRPRGTM QAINNERKASKVLGIVFFVFLIMWCPFFITNILSVLCEKSCNQKLMEKLLNVFVWIGYVCSGINPLVYTLFNKIYRRAFSNYLRCNYKVEKKPPVRQIPRVAATALSGRELNVNIYRHTNEPVIEKASDNEPGIEMQVENLELPVNPSSVVSERISSV (SEQ ID NO: 4)，

wherein the underlined section is the third intracellular loop.

42. The fluorescent probe of claim 41, wherein amino acids 16-55 of the third intracellular loop of the human HTR2C receptor are truncated and a circularly rearranged luciferase is inserted at the truncated position; alternatively, amino acids 11-60 of the third intracellular loop of the human HTR2C receptor are truncated and a circularly rearranged luciferase is inserted at the truncated position; alternatively, amino acids 16-70 of the third intracellular loop of the human HTR2C receptor are truncated and a circularly rearranged luciferase is inserted at the truncated position; alternatively, amino acids 15-68 of the third intracellular loop of the human HTR2C receptor are truncated and a circularly rearranged luciferase is inserted at the truncated position; alternatively, amino acids 15-68 of the third intracellular loop of the human HTR2C receptor are truncated and a circularly rearranged luciferase is inserted at the truncated position, and leucine L at position 13 of the third intracellular loop thereof is mutated to phenylalanine F.

43. The fluorescent probe of claim 42, the circularly rearranged luciferase is linked to the third intracellular loop of the human HTR2C receptor by a linker peptide of the N-terminus and the C-terminus, the linker peptides at both ends of the circularly rearranged luciferase being 5 amino acids in length at the nitrogen terminus and 3 amino acids at the carbon terminus, respectively.

44. The fluorescent probe of claim 43, wherein the connecting peptides at both ends of the luciferase which are rearranged circularly are PVVSE at the N-terminal and ATR at the C-terminal, respectively.

45. The fluorescent probe of claim 37, wherein the amino acid sequence of the human HTR2C receptor is:

MDPLNLSWYDDDLERQNWSRPFNGSDGKADRPHYNYYATLLTLLIAVIVFGNVLVCMAVSREKALQTTTNYLIVSLAVADLLVATLVMPWVVYLEVVGEWKFSRIHCDIFVTLDVMMCTASILNLCAISIDRYTAVAMPMLYNTRYSSKRRVTVMISIVWVLSFTISCPLLFGLNNADQNECIIANPAFVVYSSIVSFYVPFIVTLLVYIKIYIVLRRRRKRVN TKRSSRAFRAHLRAPLKGNCTHPEDMKLCTVIMKSNGSFPVNRRRVEAARRAQELEMEMLSSTSPPERTRYSPIPP SHHQLTLPDPSHHGLHSTPDSPAKPEKNGHAKDHPKIAKIFEIQTMPNGKTRTSLKTMSRRKLSQQKEKKATQMLAIVLGVFIICWLPFFITHILNIHCDCNIPPVLYSAFTWLGYVNSAVNPIIYTTFNIEFRKAFLKILHC (SEQ ID NO: 4), wherein the underlined part is the third intracellular loop.

46. The fluorescent probe of claim 45, wherein amino acids 241-306 of the human HTR2C receptor are truncated and a circularly rearranged luciferase is inserted at the truncated position; alternatively, amino acids 240-309 of the human HTR2C receptor are truncated and a circularly rearranged luciferase is inserted at the truncated position.

47. The fluorescent probe of claim 1, wherein the fluorescent probe constructed based on G protein-coupled receptors is a fluorescent probe for detecting dopamine, wherein the G protein-coupled receptors are GPCRs that specifically bind dopamine.

48. The fluorescent probe according to claim 47, wherein the GPCR that specifically binds dopamine is a human DRD2 receptor, and the fluorescent probe constructed based on the G protein coupled receptor is a fluorescent probe constructed based on a human DRD2 receptor.

49. The fluorescent probe of claim 48, wherein the third intracellular loop of the human DRD2 receptor is truncated and a circularly rearranged luciferase is inserted at the truncated position.

50. The fluorescent probe of claim 49, wherein the circularly rearranged luciferase is linked to the third intracellular loop of the human DRD2 receptor via a linker peptide at the N-terminus and the C-terminus, the linker peptide at each end of the circularly rearranged luciferase being 2 amino acids in length at the nitrogen terminus and 5 amino acids in length at the carbon terminus.

51. The fluorescent probe of claim 50, wherein the connecting peptides at both ends of the circularly rearranged luciferase are GG at N-terminal and GGAAA at C-terminal, respectively.

52. The fluorescent probe of claim 48, wherein the amino acid sequence of the human DRD2 receptor is:

MDPLNLSWYDDDLERQNWSRPFNGSDGKADRPHYNYYATLLTLLIAVIVFGNVLVCMAVSREKALQTTTNYLIVSLAVADLLVATLVMPWVVYLEVVGEWKFSRIHCDIFVTLDVMMCTASILNLCAISIDRYTAVAMPMLYNTRYSSKRRVTVMISIVWVLSFTISCPLLFGLNNADQNECIIANPAFVVYSSIVSFYVPFIVTLLVYIKIYIVLRRRRKRVN TKRSSRAFRAHLRAPLKGNCTHPEDMKLCTVIMKSNGSFPVNRRRVEAARRAQELEMEMLSSTSPPERTRYSPIPP SHHQLTLPDPSHHGLHSTPDSPAKPEKNGHAKDHPKIAKIFEIQTMPNGKTRTSLKTMSRRKLSQQKEKKATQMLAIVLGVFIICWLPFFITHILNIHCDCNIPPVLYSAFTWLGYVNSAVNPIIYTTFNIEFRKAFLKILHC (SEQ ID NO: 5), wherein the underlined part is the third intracellular loop.

53. The fluorescent probe of claim 52, wherein amino acids 253-357 of the human DRD2 receptor are truncated and a circularly rearranged luciferase is inserted at the truncated position; alternatively, amino acids 254-360 of the human DRD2 receptor are truncated and a circularly rearranged luciferase is inserted at the truncated position.

54. The fluorescent probe of claim 48, wherein the circularly rearranged luciferase is linked to the third intracellular loop of the human DRD2 receptor via N-terminal and C-terminal linker peptides, the linker peptides at each end of the circularly rearranged luciferase being 5 amino acids in nitrogen terminal and 3 amino acids in carbon terminal, respectively.

55. The fluorescent probe of claim 54, wherein the connecting peptides at both ends of the or the circularly rearranged luciferase are PVVSE at the N-terminal and ATR at the C-terminal, respectively.

56. The fluorescent probe of claim 48, wherein the amino acid sequence of the human DRD2 receptor is:

MDPLNLSWYDDDLERQNWSRPFNGSDGKADRPHYNYYATLLTLLIAVIVFGNVLVCMAVSREKALQTTTNYLIVSLAVADLLVATLVMPWVVYLEVVGEWKFSRIHCDIFVTLDVMMCTASILNLCAISIDRYTAVAMPMLYNTRYSSKRRVTVMISIVWVLSFTISCPLLFGLNNADQNECIIANPAFVVYSSIVSFYVPFIVTLLVYIKIYIVLRRRRKRVN TKRSSRAFRAHLRAPLKGNCTHPEDMKLCTVIMKSNGSFPVNRRRVEAARRAQELEMEMLSSTSPPERTRYSPIPP SHHQLTLPDPSHHGLHSTPDSPAKPEKNGHAKDHPKIAKIFEIQTMPNGKTRTSLKTMSRRKLSQQKEKKATQMLAIVLGVFIICWLPFFITHILNIHCDCNIPPVLYSAFTWLGYVNSAVNPIIYTTFNIEFRKAFLKILHC (SEQ ID NO: 5)，

wherein the underlined section is the third intracellular loop.

57. The fluorescent probe of claim 56, wherein amino acids 223-349 of the human DRD2 receptor are truncated and a circularly rearranged luciferase is inserted at the truncated position; alternatively, amino acids 268-364 of the human DRD2 receptor are truncated and a circularly rearranged luciferase is inserted at the truncated position; alternatively, amino acids 224-365 of the human DRD2 receptor are truncated and a circularly rearranged luciferase is inserted at the truncated position.

58. The method for constructing a GRAB fluorescent probe as set forth in claim 1, comprising completely intercepting a third intracellular loop of the fluorescent probe constructed based on the first G protein-coupled receptor together with a circularly rearranged luciferase inserted therein, and replacing the third intracellular loop of the second G protein-coupled receptor to obtain the fluorescent probe constructed based on the second G protein-coupled receptor, namely the GRAB fluorescent probe;

The GRAB fluorescent probe is capable of being expressed on a cell membrane and capable of binding to the specific ligand of the second G protein-coupled receptor when contacted therewith, thereby resulting in a detectable change in the fluorescent intensity of the fluorescent probe;

wherein the first and second G-protein coupled receptors bind the same specific ligand or bind different specific ligands.

59. The method of claim 58, wherein the fluorescent probe constructed based on the first G protein-coupled receptor is the fluorescent probe of any one of claims 1 to 57.

60. The method of claim 58, wherein the specific ligand of the second G-protein coupled receptor is a neurotransmitter, hormone, metabolic molecule, trophic molecule, or synthetic small molecule or drug for activating a particular receptor, and the second G-protein coupled receptor is a G-protein coupled receptor that binds to the neurotransmitter, hormone, metabolic molecule, trophic molecule, or synthetic small molecule or drug for activating a particular receptor.

61. The method of claim 60, wherein the neurotransmitter is epinephrine, norepinephrine, acetylcholine, serotonin and/or dopamine; wherein the synthetic small molecule or drug that activates a particular receptor is Isoproterenol (ISO).

62. The method of claim 58, wherein the first G protein-coupled receptor is a human β2 adrenergic receptor, and the amino acid sequence of the human β2 adrenergic receptor is:

MGQPGNGSAFLLAPNRSHAPDHDVTQQRDEVWVVGMGIVMSLIVLAIVFGNVLVITAIAKFERLQTVTNYFITSLACADLVMGLAVVPFGAAHILMKMWTFGNFWCEFWTSIDVLCVTASIETLCVIAVDRYFAITSPFKYQSLLTKNKARVIILMVWIVSGLTSFLPIQMHWYRATHQEAINCYANETCCDFFTNQAYAIASSIVSFYVPLVIMVFVYSRVFQ EAKRQLQKIDKSEGRFHVQNLSQVEQDGRTGHGLRRSSKFCLKEHKALKTLGIIMGTFTLCWLPFFIVNIVHVIQDNLIRKEVYILLNWIGYVNSGFNPLIYCRSPDFRIAFQELLCLRRSSLKAYGNGYSSNGNTGEQSGYHVEQEKENKLLCEDLPGTEDFVGHQGTVPSDNIDSQGRNCSTNDSLL (SEQ ID NO: 1)，

wherein the underlined section is the third intracellular loop.

63. The method of claim 62, wherein the circularly rearranged luciferase is interposed between amino acid 240 and amino acid 241 of the human β2 adrenergic receptor or the circularly rearranged luciferase is interposed between amino acid 250 and amino acid 251 of the human β2 adrenergic receptor.

64. The method of claim 62, wherein the circularly rearranged luciferase is coupled to the third intracellular loop of the human β2 adrenergic receptor via N-terminal and C-terminal connecting peptides, wherein the connecting peptides at both ends of the circularly rearranged luciferase are each N-terminal GG and C-terminal GGAAA; alternatively, the connecting peptides at both ends of the circularly rearranged luciferase are GG at the N end and SPSVA at the C end respectively; alternatively, the connecting peptides at both ends of the circularly rearranged luciferase are GG at the N-terminal and APSVA at the C-terminal, respectively.

65. The method of claim 58, wherein said second G protein-coupled receptor is the human acetylcholine receptor M3R subtype.

66. The method of claim 65, wherein the specific sequence of the human acetylcholine receptor M3R subtype is:

MTLHNNSTTSPLFPNISSSWIHSPSDAGLPPGTVTHFGSYNVSRAAGNFSSPDGTTDDPLGGHTVWQVVFIAFLTGILALVTIIGNILVIVSFKVNKQLKTVNNYFLLSLACADLIIGVISMNLFTTYIIMNRWALGNLACDLWLAIDYVASNASVMNLLVISFDRYFSITRPLTYRAKRTTKRAGVMIGLAWVISFVLWAPAILFWQYFVGKRTVPPGECFIQFLSEPTITFGTAIAAFYMPVTIMTILYWRIYKETEKRTKELAGLQASGTEAETENFVHPTGSSRSCSSYELQQQSM KRSNRRKYGRCHFWFTTKSWKPSSEQMDQDHSSSDSWNNNDAAASLENSASSDEEDIGSETRAIYSIVLKLPGHST ILNSTKLPSSDNLQVPEEELGMVDLERKADKLQAQKSVDDGGSFPKSFSKLPIQLESAVDTAKTSDVNSSVGKSTA TLPLSFKEATLAKRFALKTRSQITKRKRMSLVKEKKAAQTLSAILLAFIITWTPYNIMVLVNTFCDSCIPKTFWNLGYWLCYINSTVNPVCYALCNKTFRTTFKMLLLCQCDKKKRRKQQYQQRQSVIFHKRAPEQAL (SEQ ID NO: 3)，

67. The method of claim 58, wherein the first G protein-coupled receptor is a human HTR2C receptor and the amino acid sequence of the human HTR2C receptor is:

wherein the underlined section is the third intracellular loop.

68. The method of claim 67, wherein amino acids 16-55 of the third intracellular loop of the human HTR2C receptor are truncated and a circularly rearranged luciferase is inserted at the truncated position; alternatively, amino acids 11-60 of the third intracellular loop of the human HTR2C receptor are truncated and a circularly rearranged luciferase is inserted at the truncated position; alternatively, amino acids 16-70 of the third intracellular loop of the human HTR2C receptor are truncated and a circularly rearranged luciferase is inserted at the truncated position; alternatively, amino acids 15-68 of the third intracellular loop of the human HTR2C receptor are truncated and a circularly rearranged luciferase is inserted at the truncated position; alternatively, amino acids 15-68 of the third intracellular loop of the human HTR2C receptor are truncated and a circularly rearranged luciferase is inserted at the truncated position, and leucine L at position 13 of the third intracellular loop thereof is mutated to phenylalanine F.

69. The method of claim 67, wherein the circularly rearranged luciferase is coupled to a third intracellular loop of the human HTR2C receptor via a connecting peptide at the N-terminus and the C-terminus, wherein the connecting peptides at both ends of the circularly rearranged luciferase are each GG at the N-terminus and GGAAA at the C-terminus; alternatively, the connecting peptides at both ends of the circularly rearranged luciferase are NG at N-terminal and GFAAA at C-terminal, respectively.

70. The method of claim 67, wherein said second G protein-coupled receptor is a human HTR2B receptor or a human HTR6 receptor.

71. The method of claim 70, wherein the amino acid sequence of the human HTR2B receptor is:

MALSYRVSELQSTIPEHILQSTFVHVISSNWSGLQTESIPEEMKQIVEEQGNKLHWAALLILMVIIPTIGGNTLVILAVSLEKKLQYATNYFLMSLAVADLLVGLFVMPIALLTIMFEAMWPLPLVLCPAWLFLDVLFSTASIMHLCAISVDRYIAIKKPIQANQYNSRATAFIKITVVWLISIGIAIPVPIKGIETDVDNPNNITCVLTKERFGDFMLFGSLAAFFTPLAIMIVTYFLTIHALQKKAYLVKNKPPQRLTWLTVSTVFQRDETPCSSPEKVAMLDGSRKDKALPNSGDETLMRRTSTIGKKSVQTISNEQRASKVLGIVFFLFLLMWCPFFITNITLVLCDSCNQTTLQMLLEIFVWIGYVSSGVNPLVYTLFNKTFRDAFGRYITCNYRATKSVKTLRKRSSKIYFRNPMAENSKFFKKHGIRNGINPAMYQSPMRLRSSTIQSSSIILLDTLLLTENEGDKTEERVSYV (SEQ ID NO: 9)，

wherein the underlined section is the third intracellular loop.

72. The method of claim 70, wherein the amino acid sequence of the human HTR6 receptor is:

MVPEPGPTANSTPAWGAGPPSAPGGSGWVAAALCVVIALTAAANSLLIALICTQPALRNTSNFFLVSLFTSDLMVGLVVMPPAMLNALYGRWVLARGLCLLWTAFDVMCCSASILNLCLISLDRYLLILSPLRYKLRMTPLRALALVLGAWSLAALASFLPLLLGWHELGHARPPVPGQCRLLASLPFVLVASGLTFFLPSGAICFTYCRILLAARKQAVQVAS LTTGMASQASETLQVPRTPRPGVESADSRRLATKHSRKALKASLTLGILLGMFFVTWLPFFVANIVQAVCDCISPGLFDVLTWLGYCNSTMNPIIYPLFMRDFKRALGRFLPCPRCPRERQASLASPSLRTSHSGPRPGLSLQQVLPLPLPPDSDSDSDAGSGGSSGLRLTAQLLLPGEATQDPPLPTRAAAAVNFFNIDPAEPELRPHPLGIPTN (SEQ ID NO: 10)，

wherein the underlined section is the third intracellular loop.

73. The GRAB fluorescent probe obtained by the method of any one of claims 58 to 72.

74. A polynucleotide encoding the fluorescent probe according to any one of claims 1 to 57, 73.

75. An expression vector comprising the polynucleotide of claim 74.

76. A host cell comprising the polynucleotide of claim 74 or the expression vector of claim 75.

77. The host cell of claim 76, wherein said host cell is a neuronal cell.

78. A method for qualitatively detecting a change in concentration of a specific ligand for a G protein-coupled receptor in a test sample or a test tissue using the fluorescent probe of any one of claims 1-57, 73, wherein the fluorescent probe is constructed based on the G protein-coupled receptor, the method comprising:

and (2) allowing the fluorescent probe to be expressed on a cell membrane, contacting the expressed fluorescent probe with a sample to be tested or a tissue to be tested containing the specific ligand, and measuring the fluorescent signal intensity F1 of the fluorescent probe at a first time point, wherein the first time point is before the fluorescent probe is contacted with the sample to be tested or the tissue to be tested or after the fluorescent probe is contacted with the sample to be tested or the tissue to be tested, measuring the fluorescent signal intensity F2 of the fluorescent probe at a second time point after the first time point, and determining the change of the concentration of the specific ligand at the second time point relative to the first time point according to the change of the fluorescent signal intensity of F2 relative to F1.

79. The method of claim 78, wherein the change in fluorescence signal intensity comprises an increase, decrease or no change in fluorescence signal intensity and the change in concentration of the specific ligand comprises an increase, decrease or no change in concentration thereof.

80. A method for quantitatively detecting a change in concentration of a specific ligand for a G protein-coupled receptor in a test sample or a test tissue using the fluorescent probe of any one of claims 1 to 57, 73, wherein the fluorescent probe is constructed based on the G protein-coupled receptor, the method comprising:

(2) And (2) expressing the fluorescent probe on a cell membrane, contacting the expressed fluorescent probe with a sample to be tested or tissue to be tested containing the specific ligand, measuring the fluorescent signal intensity, and obtaining the concentration of the specific ligand in the sample to be tested or tissue to be tested according to the measured fluorescent signal intensity and the standard curve obtained in the step (1).

81. A method of drug screening comprising expressing the fluorescent probe of any one of claims 1-57, 73 on a cell membrane, adding a drug candidate to a cell, determining the fluorescent signal intensity of the drug candidate before and after addition, and determining whether the drug candidate is an agonist of a G protein-coupled receptor based on a change in the fluorescent signal intensity of the drug candidate after addition relative to the fluorescent signal intensity before addition, wherein the fluorescent probe is constructed based on the G protein-coupled receptor.

82. A method of drug screening comprising:

(1) Allowing the fluorescent probe of any one of claims 1-57, 73 to express on a cell membrane, adding a molecule capable of activating a G protein-coupled receptor to the cell, and determining a change in fluorescence signal intensity, wherein the fluorescent probe is constructed based on the G protein-coupled receptor;

(2) Adding a drug candidate to the cells, screening for a drug candidate capable of reversing the change in fluorescence signal intensity in step (1) as an antagonist capable of blocking the G protein-coupled receptor;

wherein if the fluorescence signal intensity increases after the addition of the molecule that activates the G protein-coupled receptor in step (1), the reversal in step (2) means that the fluorescence signal intensity decreases after the addition of the drug candidate; if the fluorescence signal intensity decreases after the addition of the molecule that activates the G protein-coupled receptor in step (1), the reversal in step (2) refers to an increase in fluorescence signal intensity after the addition of the drug candidate.

83. A method for detecting the distribution of a specific ligand of a G protein-coupled receptor in an animal, comprising expressing the fluorescent probe of any one of claims 1-57, 73 in the animal, measuring the fluorescent signal intensity in the animal in real time, and determining whether the specific ligand exists in different regions in the animal, whether the concentration of the different regions differs from each other, and whether the concentration of the same region changes with time according to the presence, intensity, and change of the fluorescent signal intensity of the different regions in the animal, wherein the fluorescent probe is constructed based on the G protein-coupled receptor.

84. The method of claim 83, comprising the step of inducing the production of said specific ligand by the animal prior to determining the intensity of the fluorescent signal.

85. The method of claim 84, wherein the specific ligand is induced in the animal by odor stimulation or visual stimulation.