CN121591850A - Novel Acetyl-CoA Optical Probe, Its Preparation Method and Application - Google Patents

Abstract

This invention relates to an acetyl-CoA probe, its preparation method, and its application. Specifically, this invention provides an acetyl-CoA optical probe comprising an acetyl-CoA-sensitive polypeptide and an optically active polypeptide, wherein the optically active polypeptide is located within the sequence of the acetyl-CoA-sensitive polypeptide.

Description

Novel acetyl-CoA optical probe and preparation method and application thereof

Technical Field

The invention relates to the technical field of optical probes, in particular to a novel acetyl-CoA optical probe, a preparation method and application thereof.

Background

Acetyl-coa plays a critical role in the cell, as a key intermediate in metabolic processes, and plays a central role in a variety of biological functions. It is not only involved in the energy metabolism of cells, such as tricarboxylic acid cycle and oxidative phosphorylation processes, providing energy to cells, but also an important precursor for the synthesis of fatty acids, cholesterol, and other bioactive molecules. In addition, acetyl-coa also plays a regulatory role in the field of epigenetic science, regulating gene expression by acetylation of histones. Notably, the altered levels of acetyl-coa are closely linked to a variety of metabolic diseases, including tumors, obesity, and neurodegenerative diseases.

Currently, methods mainly used for detecting acetyl-CoA are GC-MS, LC-MS, HPLC, NMR, enzyme-linked reaction and the like. The conventional chemical analysis method is to directly or indirectly measure the total acetyl-CoA of cells, and has complex operation process, and in addition, the method cannot meet the real-time dynamic in-vivo monitoring of the level of the acetyl-CoA in living cells and even in organelles. In recent years, researchers have also developed acetyl-CoA fluorescent probes based on different principles. For example, the detection method of acetyl-CoA is developed by using bioluminescence resonance energy transfer technology, and the visual and quantitative detection of the level of living cells is realized by using bacteria PanZ protein and cpGFP to design an acetyl-CoA fluorescent protein probe. The currently developed acetyl-CoA protein probes solve some problems to a certain extent, but still have the problems of unsuitable affinity, small response amplitude, incapability of being applied to mammalian cells and the like. Therefore, development of a new detection method is needed to realize the simple, convenient, rapid and high-specificity real-time, positioning, quantitative and high-throughput detection of acetyl-CoA in and out cells.

Disclosure of Invention

The invention aims to provide a probe and a method for detecting acetyl-CoA in real time in a cell and outside the cell in a high-throughput and quantitative manner.

In order to achieve the above object, the present invention provides the following technical solutions:

The first aspect of the invention provides an acetyl-coa binding protein variant which:

(1) Having the sequence shown in SEQ ID NO. 1 and having a mutation at 1,2, 3 or more sites selected from the group consisting of I19, R21, T77, K85, G87, P133, P134, said mutation comprising a modification, substitution or deletion of an amino acid,

(2) Is a sequence having at least 70% sequence identity to the sequence of (1) and having the mutation of (1) and retaining the ability to bind acetyl-CoA.

In one or more embodiments, the mutation comprises a mutation at a site selected from any of (1) P133, P134, and I19, (2) P133, P134, and R21, (3) P133, P134, and T77, (4) P133, P134, and K85, (5) P133, P134, and G87.

In one or more embodiments, I19 is mutated to L, in one or more embodiments R21 is mutated to M or D, in one or more embodiments T77 is mutated to V, in one or more embodiments K85 is mutated to N, in one or more embodiments G87 is mutated to A, in one or more embodiments P133 is mutated to Y, and in one or more embodiments P134 is mutated to D.

In one or more embodiments, the mutation comprises a mutation selected from any one of (1) P133Y, P D and I19L, (2) P133Y, P D and R21M, (3) P133Y, P D and R21D, (4) P133Y, P D and T77V, (5) P133Y, P134D and K85N, (6) P133Y, P D and G87A.

In a second aspect, the invention provides an optical probe comprising an acetyl-coa sensitive polypeptide and an optically active polypeptide, wherein the optically active polypeptide is located within the sequence of the acetyl-coa sensitive polypeptide. The acetyl-coa sensitive polypeptide is separated into a first portion and a second portion by the optically active polypeptide.

In one or more embodiments, the acetyl-CoA optical probe includes an acetyl-CoA sensitive polypeptide B and an optically active polypeptide A, wherein the optically active polypeptide A is located within the sequence of the acetyl-CoA sensitive polypeptide B, and the acetyl-CoA sensitive polypeptide B is divided into a first portion B1 and a second portion B2, forming a probe structure of the formula B1-A-B2.

In one or more embodiments, the optically active polypeptide is located at one or more ：20/21,20/22,20/23,20/24,20/25,20/26,20/27,21/22,21/23,21/24,21/25,21/26,21/27,22/23,22/24,22/25,22/26,22/27,23/24,23/25,23/26,23/27,37/38,37/39,38/39,46/47,46/48,47/48,59/60,59/61,59/62,59/63,59/64,59/65,59/66,59/67,59/68,59/69,60/61,60/62,60/63,60/64,60/65,60/66,60/67,60/68,60/69,61/62,61/63,61/64,61/65,61/66,61/67,61/68,61/69,62/63,62/64,62/65,62/66,62/67,62/68,62/69,63/64,63/65,63/66,63/67,63/68,63/69,64/65,64/66,64/67,64/68,64/69,65/66,65/67,65/68,65/69,66/67,66/68,66/69,67/68,67/69,78/79,78/80,78/81,78/82,78/83,78/84,79/80,79/81,79/82,79/83,79/84,80/81,80/82,80/83,80/84,81/82,81/83,81/84,82/83,82/84,83/84,102/103,102/104,103/104,112/113,122/123,132/133,132/134,132/135,132/136,133/134,133/135,133/136,134/135,134/136 and 135/136 of the acetyl-coa sensitive polypeptide selected from the group consisting of. Numbering corresponds to the full length of the acetyl-coa sensitive polypeptide.

In one or more embodiments, the acetyl-coa sensitive polypeptide is an acetyl-coa binding protein or a functional variant thereof.

In one or more embodiments, the acetyl-coa sensitive polypeptide has:

(1) The sequence shown in SEQ ID No. 1, or a sequence which has at least 70% sequence identity thereto and retains binding activity to acetyl-CoA,

(2) The sequence of an acetyl-CoA binding protein variant as described in any of the embodiments of the first aspect herein, or

(3) Sequences having at least 70% sequence identity to the sequence of (2) and having the mutation of (2) and retaining sensitivity to acetyl-CoA.

In one or more embodiments, the optically active polypeptide is a fluorescent protein or a functional variant thereof. In one or more embodiments, the fluorescent protein is selected from the group consisting of yellow fluorescent protein (cpYFP as shown in SEQ ID NO: 2), orange fluorescent protein (cpmOrange as shown in SEQ ID NO: 3), red fluorescent protein (mKate as shown in SEQ ID NO:4 or 8, mcherry as shown in SEQ ID NO: 5), green fluorescent protein (cpGFP as shown in SEQ ID NO: 6), blue fluorescent protein (cpBFP as shown in SEQ ID NO: 7), apple red fluorescent protein (cpmApple as shown in SEQ ID NO: 9). Preferably, the optically active polypeptide is cpYFP, cpGFP, cpBFP or cpmApple. More preferably, the optically active polypeptide is cpYFP. In one or more embodiments, the fluorescent protein has a sequence as set forth in any one of SEQ ID NOs 2-9, preferably the fluorescent protein has a sequence as set forth in any one of SEQ ID NOs 2, 6, 7, 9.

In one or more embodiments, the optically active polypeptide has (1) a sequence as set forth in any one of SEQ ID NO:2-9, (2) a sequence as set forth in SEQ ID NO:2 and has a mutation at the Y1 site, including a modification, substitution or deletion of an amino acid, preferably the mutation is Y1V, or (3) a variant sequence having at least 70% sequence identity to (1) or (2) and retaining fluorescent protein function.

In one or more embodiments, the sequence of the acetyl-CoA sensitive polypeptide is shown in SEQ ID NO.1, the optically active polypeptide is shown in any one of SEQ ID NO. 2, 6, 7, 9, and the optically active polypeptide is located at any one or more of the following positions of the acetyl-CoA sensitive polypeptide ：20/21,20/22,20/23,20/24,20/25,20/26,20/27,21/22,21/23,21/24,21/25,21/26,21/27,22/23,22/24,22/25,22/26,22/27,23/24,23/25,23/26,23/27,37/38,37/39,38/39,46/47,46/48,47/48,59/60,59/61,59/62,59/63,59/64,59/65,59/66,59/67,59/68,59/69,60/61,60/62,60/63,60/64,60/65,60/66,60/67,60/68,60/69,61/62,61/63,61/64,61/65,61/66,61/67,61/68,61/69,62/63,62/64,62/65,62/66,62/67,62/68,62/69,63/64,63/65,63/66,63/67,63/68,63/69,64/65,64/66,64/67,64/68,64/69,65/66,65/67,65/68,65/69,66/67,66/68,66/69,67/68,67/69,78/79,78/80,78/81,78/82,78/83,78/84,79/80,79/81,79/82,79/83,79/84,80/81,80/82,80/83,80/84,81/82,81/83,81/84,82/83,82/84,83/84,102/103,102/104,103/104,112/113,122/123,132/133,247920

132/134,132/135,132/136,133/134,133/135,133/136,134/135,134/136 And 135/136. Numbering corresponds to the full length of the acetyl-coa sensitive polypeptide.

In one or more embodiments, the acetyl-CoA sensitive polypeptide is as shown in SEQ ID NO. 1, the optically active polypeptide is as shown in any of SEQ ID NO. 2, 6, 7, 9 or a variant thereof having a mutation at an amino acid corresponding to amino acid 1 of SEQ ID NO. 2 of Y1V, the optically active polypeptide being located at any one or more ：20/21,20/22,20/23,20/24,20/25,20/26,20/27,21/22,21/23,21/24,21/25,21/26,21/27,22/23,22/24,22/25,22/26,22/27,23/24,23/25,23/26,23/27,37/38,37/39,38/39,46/47,46/48,47/48,59/60,59/61,59/62,59/63,59/64,59/65,59/66,59/67,59/68,59/69,60/61,60/62,60/63,60/64,60/65,60/66,60/67,60/68,60/69,61/62,61/63,61/64,61/65,61/66,61/67,61/68,61/69,62/63,62/64,62/65,62/66,62/67,62/68,62/69,63/64,63/65,63/66,63/67,63/68,63/69,64/65,64/66,64/67,64/68,64/69,65/66,65/67,65/68,65/69,66/67,66/68,66/69,67/68,67/69,78/79,78/80,78/81,78/82,78/83,78/84,79/80,79/81,79/82,79/83,79/84,80/81,80/82,80/83,80/84,81/82,81/83,81/84,82/83,82/84,83/84,102/103,102/104,103/104,112/113,122/123,132/133,132/134,132/135,132/136,133/134,133/135,133/136,134/135,134/136 and 135/136 of the sites of the acetyl-CoA sensitive polypeptide. Numbering corresponds to the full length of the acetyl-coa sensitive polypeptide.

In one or more embodiments, the optical probe has an acetyl-CoA sensitive polypeptide as shown in SEQ ID NO.1 and has one or more mutations of I19, R21, T77, K85, G87, P133, P134, an optically active polypeptide as shown in SEQ ID NO.2, 6, 7,9 or a variant thereof having a mutation of Y1V at an amino acid corresponding to amino acid 1 of SEQ ID NO.2, the optically active polypeptide being located at 20/21,20/22,20/23,20/24,20/25,20/26,20/27,21/22,21/23,21/24,21/25,21/26,21/27,22/23,22/24,22/25,22/26,22/27,23/24,23/25,23/26,23/27,37/38,37/39,38/39,46/47,46/48,47/48,59/60,59/61,59/62,59/63,59/64,59/65,59/66,59/67,59/68,59/69,60/61,60/62,60/63,60/64,60/65,60/66,60/67,60/68,60/69,61/62,61/63,61/64,61/65,61/66,61/67,61/68,61/69,62/63,62/64,62/65,62/66,62/67,62/68,62/69,63/64,63/65,63/66,63/67,63/68,63/69,64/65,64/66,64/67,64/68,64/69,65/66,65/67,65/68,65/69,66/67,66/68,66/69,67/68,67/69,78/79,78/80,78/81,78/82,78/83,78/84,79/80,79/81,79/82,79/83,79/84,80/81,80/82,80/83,80/84,81/82,81/83,81/84,82/83,82/84,83/84,102/103,102/104,103/104,112/113,122/123,132/133,132/134,132/135,132/136,133/134,133/135,133/136,134/135,134/136 and 135/136 of the acetyl-CoA sensitive polypeptide. Numbering corresponds to the full length of the acetyl-coa sensitive polypeptide. Preferably, the mutation of the acetyl-CoA sensitive polypeptide comprises a mutation selected from any one of (1) P133Y, P D and I19L, (2) P133Y, P D and R21M, (3) P133Y, P D and R21D, (4) P133Y, P D and T77V, (5) P133Y, P134D and K85N, and (6) P133Y, P134D and G87A.

In one or more embodiments, the optical probe has a mutation shown by (1) P133Y, P134D, I L of the acetyl-CoA sensitive polypeptide and Y1V of the optically active polypeptide, (2) P133Y, P134D, R M of the acetyl-CoA sensitive polypeptide and Y1V of the optically active polypeptide, (3) P133Y, P134D, R D of the acetyl-CoA sensitive polypeptide and Y1V of the optically active polypeptide, (4) P133Y, P134D, T V of the acetyl-CoA sensitive polypeptide and Y1V of the optically active polypeptide, (5) P133Y, P134 3285N of the acetyl-CoA sensitive polypeptide and Y1V of the optically active polypeptide, (6) P133Y, P134D, G A of the acetyl-CoA sensitive polypeptide and Y1V of the optically active polypeptide.

In one or more embodiments, the optical probe is as set forth in any one of SEQ ID NOS: 11-16.

In one or more embodiments, the optical probe further comprises one or more linkers flanking the optically active polypeptide. The linker of the invention may be any amino acid sequence of any length. In one or more embodiments, the optically active polypeptide is flanked by linkers of no more than 5 amino acids, e.g., 0,1, 2, 3, 4 amino acids. In one or more embodiments, the linker flanking the optically active polypeptide comprises amino acid Y. In one or more embodiments, the linker Y is located at the N-terminus and/or the C-terminus of the optically active polypeptide. In one or more embodiments, the optical probe is shown as a first portion B1 of an acetyl-CoA sensitive polypeptide, a first linker Y1, an optically active polypeptide A, a second linker Y2, a second portion B2 of an acetyl-CoA sensitive polypeptide. In one embodiment, the optical probe of the present invention does not comprise a linker.

In one or more embodiments, the optical probes of the invention further comprise a localization sequence for localizing the probe to a specific organelle, e.g., a cell. Preferred organelles are subcellular organelles, more preferably cytoplasm, nucleus, mitochondria, and the like.

In one or more embodiments, the optically active polypeptide is cpYFP, which is located at any one or more of the following sites of the acetyl-CoA sensitive polypeptide selected from 22/25,46/48,59/69,60/68,62/66,133/135,134/135 and 134/136. The acetyl-coa sensitive polypeptide (1) is as shown in SEQ ID No. 1, or (2) is an acetyl-coa binding protein variant as described in any embodiment of the first aspect herein;

in one or more embodiments, the optically active polypeptide is cpGFP, which is located at any one or more of the sites selected from the group consisting of 22/25,22/26,46/48,59/69,60/67,62/67,133/135 and 134/136 of the acetyl-CoA sensitive polypeptide. The acetyl-coa sensitive polypeptide (1) is as shown in SEQ ID No. 1, or (2) is an acetyl-coa binding protein variant as described in any embodiment of the first aspect herein;

In one or more embodiments, the optically active polypeptide is cpBFP, which is located at any one or more of the following positions selected from the group consisting of 22/26,38/39,62/67,133/136 and 134/136 of the acetyl-CoA sensitive polypeptide. The acetyl-coa sensitive polypeptide (1) is as shown in SEQ ID No. 1, or (2) is an acetyl-coa binding protein variant as described in any embodiment of the first aspect herein;

In one or more embodiments, the optically active polypeptide is cpmApple, which is located at any one or more of the following positions selected from the group consisting of 46/47,59/69,60/67,62/66,133/135 and 134/136 of the acetyl-CoA sensitive polypeptide. The acetyl-CoA sensitive polypeptide (1) is as shown in SEQ ID NO. 1, or (2) is an acetyl-CoA binding protein variant as described in any of the embodiments of the first aspect of the document [ supplementary details, corresponding to specific examples 2-5 ]

In another aspect, the invention also provides fusion polypeptides comprising an optical probe as described in any of the embodiments herein and other polypeptides including a localization sequence, a tag for facilitating purification, or a tag for an immune response. In some embodiments, the optical probes described herein further comprise additional polypeptides fused thereto. Other polypeptides described herein do not affect the properties of the optical probe. In some embodiments, the other polypeptide is located at the N-terminus and/or the C-terminus of the optical probe. In some embodiments, other polypeptides include a localization sequence (e.g., a polypeptide that localizes an optical probe to a different organelle or subcellular organelle), a tag that facilitates purification, or a tag for an immune response (e.g., immunoblotting). The fusion polypeptides described herein may have a linker between the optical probe and the other polypeptides.

In another aspect, the invention provides a nucleic acid molecule comprising (1) a coding sequence for an acetyl-CoA binding protein variant, optical probe or fusion polypeptide as described in any of the embodiments herein, or (2) a complement of (1), or (3) a fragment of (1) or (2). The fragments are primers.

The invention also relates to variants of the above nucleic acid molecules, including nucleic acid sequences encoding variants, fragments of optical probes or fusion polypeptides, analogs, derivatives, soluble fragments and variants of the proteins of the invention or their complements.

The invention also provides nucleic acid constructs comprising the nucleic acid molecules described herein. The nucleic acid sequence encodes a protein variant, optical probe or fusion polypeptide according to any of the embodiments of the invention.

In one or more embodiments, the nucleic acid construct is a cloning vector, an expression vector, or a recombinant vector.

In one or more embodiments, the nucleic acid molecule is operably linked to an expression control sequence.

In some embodiments, the expression vector is selected from the group consisting of a prokaryotic expression vector, a eukaryotic expression vector, and a viral vector.

In another aspect, the invention provides a host cell comprising (1) an optical probe or fusion polypeptide according to any of the embodiments of the invention, comprising (2) a nucleic acid molecule according to any of the embodiments of the invention, or comprising (3) a nucleic acid construct according to any of the embodiments of the invention. The host cell is preferably E.coli.

In another aspect, the invention provides a detection kit comprising an optical probe or fusion polypeptide or nucleic acid molecule or nucleic acid construct or host cell as described herein. The detection kit optionally further comprises other reagents required for detection of acetyl-coa using optical probes.

In one or more embodiments, the assay kit further comprises one or more reagents selected from the group consisting of buffers, media, acetyl-CoA standards.

The present invention provides methods of making an optical probe described herein comprising providing a host cell expressing an optical probe or fusion polypeptide described herein, culturing the host cell under conditions in which the cell is expressed, and isolating the optical probe or fusion polypeptide.

In one or more embodiments, the method comprises the steps of 1) incorporating a nucleic acid molecule encoding an optical probe or fusion polypeptide described herein into an expression vector, 2) transferring the expression vector into a host cell, 2) culturing the host cell under conditions suitable for expression of the expression vector, 3) isolating the optical probe or fusion polypeptide.

In another aspect, the invention also provides a method of detecting acetyl-CoA in a sample comprising contacting an optical probe or fusion polypeptide or host cell as described herein with the sample, and detecting a change in the optically active polypeptide. The detection may be performed in vivo, in vitro, subcellular or in situ. Such as blood.

In another aspect, provided herein is a method of quantifying acetyl-CoA in a sample comprising contacting an optical probe or fusion polypeptide or host cell as described herein with the sample, detecting an optical change in the optically active polypeptide, and quantifying acetyl-CoA in the sample based on the optical change in the optically active polypeptide.

In another aspect, the invention also provides a method of screening for a compound (e.g., a drug) comprising contacting an optical probe or fusion polypeptide or host cell described herein with a candidate compound in a system comprising acetyl-CoA, detecting an optical change in an optically active polypeptide, and screening the compound for an optical change in the optically active polypeptide. The method can screen compounds with high throughput.

In one or more embodiments, the host cells described herein are contacted with a candidate compound in an acetyl-coa containing system, and an optical change in the optically active polypeptide is indicative of whether the candidate compound is capable of modulating cellular uptake of acetyl-coa.

In a further aspect the invention provides a method for intracellular and/or extracellular localization of acetyl-CoA comprising contacting an acetyl-CoA containing system with said optical probe or said host cell and detecting an optical change in an optically active polypeptide.

In one or more embodiments, the system is a solution system, a cell system, a subcellular system.

In a further aspect the invention provides the use of an acetyl-CoA optical probe or fusion polypeptide or host cell as described herein for detecting acetyl-CoA, for screening compounds or for intracellular and/or extracellular localization of acetyl-CoA in a sample. In one or more embodiments, the positioning is real-time positioning.

In a further aspect the invention provides the use of an acetyl-coa optical probe or fusion polypeptide or polynucleotide or nucleic acid construct as described herein in the preparation of a kit for detecting acetyl-coa, a screening compound or intracellular and/or extracellular localization of acetyl-coa in a sample.

Drawings

FIG. 1 is a SDS-PAGE diagram of an exemplary acetyl-CoA optical probe as described in example 1;

FIG. 2 is a graph of the change in acetyl-CoA response of an exemplary acetyl-CoA optical probe comprising cpYFP and an acetyl-CoA binding protein described in example 2;

FIG. 3 is a graph of the change in acetyl-CoA response of an exemplary acetyl-CoA optical probe comprising cpGFP and an acetyl-CoA binding protein described in example 3;

FIG. 4 is a graph of the change in acetyl-CoA response of an exemplary acetyl-CoA optical probe comprising cpBFP and an acetyl-CoA binding protein described in example 4;

FIG. 5 is a graph of the change in acetyl-CoA response of an exemplary acetyl-CoA optical probe comprising cpmApple and an acetyl-CoA binding protein described in example 5;

FIG. 6 is a graph of fluorescence spectrum properties of an exemplary acetyl-CoA optical probe described in example 7;

FIG. 7 is a titration curve of response of an exemplary acetyl-CoA probe described in example 7 to different concentrations of acetyl-CoA;

FIG. 8 is a bar graph of specific detection of acetyl-CoA analogs by an exemplary acetyl-CoA optical probe as described in example 7.

FIG. 9 is a photograph of subcellular organelle localization of an exemplary acetyl-CoA optical probe in a mammalian cell according to example 8;

FIG. 10 is a schematic representation of dynamic monitoring of the concentration of acetyl-CoA in the cytosol of an exemplary acetyl-CoA optical probe in a mammalian cell as described in example 8;

FIG. 11 is a plot of high throughput compound screening at the living cell level for an exemplary acetyl-CoA optical probe described in example 9;

FIG. 12 is a bar graph of the quantification of acetyl-CoA in mouse and human blood using an exemplary acetyl-CoA optical probe described in example 10.

Detailed Description

When a value or range is given, the term "about" as used herein means that the value or range is within 20%, within 10% and within 5% of the given value or range.

The terms "comprising," including, "and equivalents thereof as used herein include the meaning of" containing "and" consisting of, for example, a composition "comprising" X may consist of X alone or may contain other materials, such as X+Y.

The term "acetyl-CoA sensitive polypeptide" or "acetyl-CoA responsive polypeptide" as used herein refers to a polypeptide that responds to acetyl-CoA, including any response in a chemical, biological, electrical or physiological parameter of the polypeptide that is related to the interaction of the sensitive polypeptide. Responses include small changes, e.g., changes in the orientation of amino acids or peptide fragments of a polypeptide, e.g., changes in the primary, secondary, or tertiary structure of a polypeptide, including, e.g., changes in protonation, electrochemical potential, and/or conformation. A "conformation" is a three-dimensional arrangement of the primary, secondary and tertiary structures of a molecule comprising pendant groups in the molecule, which changes when the three-dimensional structure of the molecule changes. Examples of conformational changes include a transition from an alpha-helix to a beta-sheet or from a beta-sheet to an alpha-helix. It will be appreciated that the detectable change need not be a conformational change, so long as the fluorescence of the fluorescent protein moiety is altered. The acetyl-coa sensitive polypeptides described herein may also include functional variants thereof. Functional variants of an acetyl-coa sensitive polypeptide include, but are not limited to, variants that can interact with acetyl-coa to effect the same or similar changes as a parent acetyl-coa sensitive polypeptide.

The term "optical probe" as used herein refers to an acetyl-CoA sensitive polypeptide fused to an optically active polypeptide (e.g., a fluorescent protein) operably inserted into an acetyl-CoA sensitive polypeptide (e.g., an acetyl-CoA binding protein). The acetyl-CoA binding protein may sense a change in the concentration of acetyl-CoA, and the spatial conformation of the acetyl-CoA binding protein may change during a dynamic change in the concentration of acetyl-CoA. The inventors have found that when an optically active polypeptide is fused to an acetyl-coa sensitive polypeptide (e.g., an acetyl-coa binding protein), conformational changes that occur upon binding of the acetyl-coa sensitive polypeptide specifically to a physiological concentration of acetyl-coa cause conformational changes in the optically active polypeptide (e.g., a fluorescent protein), which in turn results in a change in the optical properties of the optically active polypeptide. The presence and/or level of acetyl-CoA can be detected and analyzed by plotting a standard curve from the fluorescence of fluorescent proteins measured at different acetyl-CoA concentrations. The acetyl-CoA sensitive polypeptides of the invention include, but are not limited to, acetyl-CoA binding protein GAT or GNATs protein mutants having more than 90% homology thereto. An exemplary GAT protein is shown in SEQ ID NO. 1. The exemplary acetyl-CoA binding protein GAT of the invention is derived from Bacillus licheniformis Bacillus licheniformis, which is capable of sensing changes in acetyl-CoA concentration, and the spatial conformation of the acetyl-CoA binding protein is also altered during dynamic changes in acetyl-CoA concentration. When describing the optical probes of the invention (e.g.when describing insertion sites or mutation sites), reference is made to SEQ ID NO:1 for amino acid residue numbers.

A protein-based "optically active polypeptide" is a polypeptide that has the ability to emit fluorescence. Fluorescence is an optical property of an optically active polypeptide that can be used as a means to detect the responsiveness of an optical probe of the invention. As used herein, the term "fluorescent properties" refers to molar extinction coefficient, fluorescence quantum efficiency, shape of excitation spectrum or emission spectrum, excitation wavelength maximum and emission wavelength maximum, amplitude of excitation at two different wavelengths, emission amplitude ratio at two different wavelengths, excited state lifetime or fluorescence anisotropy at an appropriate excitation wavelength. The measurable difference in any of these properties between active and inactive states is sufficient for the utility of the fluorescent protein substrates of the invention in activity assays. The measurable difference can be determined by determining the amount of any quantitative fluorescent property, for example, the amount of fluorescence at a particular wavelength or the integration of fluorescence over the emission spectrum. Preferably, the protein substrate is selected to have fluorescent properties that are readily distinguishable in the unactivated and activated conformational state. Optically active polypeptides described herein can also include functional variants thereof. Functional variants of an optically active polypeptide include, but are not limited to, variants that can undergo a change in the same or similar fluorescent properties as the parent optically active polypeptide.

Herein, "response fold" is the normalized fluorescence ratio. The more the response fold of the probe deviates from 1 (whether greater or lesser), the greater the fold change in response of the probe to the substrate or response relative to the control. For example, the present embodiment calculates the response times by detecting the change in the Ratio of the fluorescence intensity at 420nm excitation 528nm emission to the fluorescence intensity at 485nm excitation 528nm emission (Normalized Ratio _420/485), as follows:

Correction of the fluorescent signal values was performed by subtracting the detection signal values of cells not expressing the probe protein. Correction data were obtained by dividing the change in the ratio of probe fluorescence intensity in the parallel experimental group by the change in the ratio of control fluorescence intensity to eliminate pH sensitive interference.

F=Fsampte-F_BB_LK

F represents the fluorescence intensity (Fluorescence intensity), fsample represents the total fluorescence intensity of the sample expressing the fluorescent probe, and F _BLK represents the background fluorescence intensity of the sample not expressing the fluorescent probe. F ₄₈₅ represents the fluorescence intensity emitted at 528nm of excitation at 485nm of the fluorescent protein sample, and F ₄₂₀ represents the fluorescence intensity emitted at 528nm of excitation at 420nm of the fluorescent protein sample. Normalized Ratio represents the Ratio of fluorescence intensity of probe response to acetyl-CoA, normalized Ratiocontrol represents the Ratio of fluorescence intensity of pH of fluorescent probe to acetyl-CoA of control probe. pH corrected Normalized Ratio is the fold change or fold response of the probe after pH correction. A pH Normalized Ratio _420/485 that deviates from 1 (whether larger or smaller) indicates a greater fold change or response of the probe.

"Linker" or "junction region" refers to an amino acid or nucleotide sequence that connects two parts in a polypeptide, protein or nucleic acid of the invention. In the present invention, the number of amino acids at the amino terminus of the linker region between the acetyl-CoA sensitive polypeptide and the optically active polypeptide is exemplified by 0 to 3, and the number of amino acids at the carboxy terminus is exemplified by 0 to 2, and when the recombinant optical probe is linked to the functional protein as a basic unit, the amino acid or the carboxy terminus of the recombinant optical probe may be fused. The linker sequence may be a short peptide chain consisting of one or more flexible amino acids, such as Y.

As used herein, the terms "chromophore", "fluorophore" and "fluorescent protein" are synonymous and refer to proteins that fluoresce upon irradiation with excitation light. Fluorescent protein is used as basic detection means in the field of bioscience, such as green fluorescent protein GFP, cyclic rearranged blue fluorescent protein (cpBFP) derived from mutation of the protein, cyclic rearranged green fluorescent protein (cpGFP), cyclic rearranged yellow fluorescent protein (cpYFP) and the like, as well as red fluorescent protein RFP, which is commonly used in the field of technology, and cyclic rearranged proteins derived from the protein, such as cpmApple, cpmOrange, cpmKate and the like. Exemplary fluorescent proteins have the sequence shown in any one of SEQ ID NOS.2-9. Preferably, the sequence of the exemplary fluorescent protein is shown in any one of SEQ ID NOs 2, 6, 7, 9.

Green fluorescent protein GFP was originally extracted from Aequorea victoria (Aequorea Victoria), and consisted of 238 amino acids with a molecular weight of approximately 26kDa. GFP is a unique barrel-like structure formed from 12 beta-sheet chains, in which a chromogenic tripeptide (Ser 65-Tyr66-Gly 67) is entrapped. When in the presence of oxygen, it spontaneously forms the chromophore structure of p-hydroxyphenylmethylene imidazolidinone to fluoresce. GFP fluorescence does not require cofactors and is very stable, a good imaging tool. GFP has two excitation peaks, a main peak at 395nm producing 508nm emission, and excitation light at 475nm at shoulder producing 503nm emission. Exemplary cpGFP is shown in SEQ ID NO. 6.

Yellow fluorescent protein YFP is derived from green fluorescent protein GFP, and the amino acid sequence of the yellow fluorescent protein YFP has the homology of more than 90 percent with GFP, and the YFP is changed from the key to GFP by mutating the 203 th amino acid from threonine to tyrosine (T203Y). The wavelength of the major excitation peak of YFP was red shifted to 514nm and the emission wavelength was changed to 527nm compared to the original AvGFP. On the basis, the 65 th amino acid of YFP is subjected to site-directed mutagenesis (S65T) to obtain fluorescence-enhanced yellow fluorescent protein EYFP. cpYFP is to connect the original N end and C end of GFP through a flexible short peptide chain, to manufacture a new N end and C end at the original GFP near chromophore position, to use the 145 th to 238 th amino acid part as the N end of new protein, and the 1 st to 144 th amino acid part as the C end of new protein, and to connect the two fragments through 5 to 9 flexible short peptide chains. In the present invention, the near chromophore position is preferably at amino acids Y144 and N145, and the short peptide chain with flexibility is preferably VDGGSGGTG or GGSGG. Exemplary cpYFP sequences are shown in SEQ ID NO. 2.

Red fluorescent protein RFP was originally extracted from corals in the ocean, wild RFP was an oligomeric protein that was detrimental to fusion expression by organisms, and then red fluorescent proteins of different colorbands were further derived on the basis of RFP, with mCherry and mKate being the most common. Exemplary cpmKate are shown in SEQ ID NO. 4 or 8. An exemplary mCherry is shown in SEQ ID NO. 5.

In other embodiments, the fluorescent protein may be one or more of blue fluorescent protein cpBFP with the amino acid sequence shown in SEQ ID NO. 7, orange fluorescent protein cpmOrange with the amino acid sequence shown in SEQ ID NO. 3, and apple red fluorescent protein cpmApple with the amino acid sequence shown in SEQ ID NO. 9.

The acetyl-CoA optical probes described herein include acetyl-CoA sensitive polypeptides B, such as acetyl-CoA binding proteins or variants thereof, and optically active polypeptides A, such as fluorescent proteins or variants thereof. The optical active polypeptide A is inserted into the acetyl-CoA sensitive polypeptide B, B is divided into two parts of B1 and B2 to form a probe structure of a formula B1-A-B2, and the interaction of the acetyl-CoA sensitive polypeptide B and the acetyl-CoA leads to the optical signal of the optical active polypeptide A to be strengthened.

In the optical probes of the invention, the optically active polypeptide may be located at any position of the acetyl-CoA sensitive polypeptide. In one embodiment, the optically active polypeptide is located in the N-C direction at any position of the N-C direction of the acetyl-CoA sensitive polypeptide. Illustratively, the optically active polypeptide is located at any one or more ：20/21,20/22,20/23,20/24,20/25,20/26,20/27,21/22,21/23,21/24,21/25,21/26,21/27,22/23,22/24,22/25,22/26,22/27,23/24,23/25,23/26,23/27,37/38,37/39,38/39,46/47,46/48,47/48,59/60,59/61,59/62,59/63,59/64,59/65,59/66,59/67,59/68,59/69,60/61,60/62,60/63,60/64,60/65,60/66,60/67,60/68,60/69,61/62,61/63,61/64,61/65,61/66,61/67,61/68,61/69,62/63,62/64,62/65,62/66,62/67,62/68,62/69,63/64,63/65,63/66,63/67,63/68,63/69,64/65,64/66,64/67,64/68,64/69,65/66,65/67,65/68,65/69,66/67,66/68,66/69,67/68,67/69,78/79,78/80,78/81,78/82,78/83,78/84,79/80,79/81,79/82,79/83,79/84,80/81,80/82,80/83,80/84,81/82,81/83,81/84,82/83,82/84,83/84,102/103,102/104,103/104,112/113,122/123,132/133,132/134,132/135,132/136,133/134,133/135,133/136,134/135,134/136 and/or 135/136 of the following sites selected from the group of acetyl-CoA sensitive polypeptides. In a preferred embodiment, the optical probe is as shown in SEQ ID NO. 10.

In the present context, in the position indicated by the "X/Y" form, the optically active polypeptide has a portion of the acetyl-CoA sensitive polypeptide at each of its two ends, wherein the N-terminus of the optically active polypeptide is the N-terminal starting amino acid (e.g., any one of amino acids 1 to 12) to the X-th amino acid of the acetyl-CoA sensitive polypeptide sequence, and the C-terminus of the optically active polypeptide is the Y-th amino acid to the C-terminal end amino acid (e.g., any one of amino acids Y to 112) of the acetyl-CoA sensitive polypeptide sequence. Wherein, if two numbers in a site represented in the form of "X/Y" are consecutive integers, it is indicated that the optically active polypeptide is located between the amino acids described by the numbers. For example, insertion site 134/135 indicates that the optically active polypeptide is located between amino acids 134 and 135 of the acetyl-CoA sensitive polypeptide. If two numbers in the positions indicated in the form of "X/Y" are not consecutive integers and X is less than Y, then it is indicated that the optically active polypeptide replaces the amino acid between the amino acids indicated by the numbers, e.g., insertion position 133/135 indicates that the optically active polypeptide replaces amino acid 134 of the acetyl-CoA sensitive polypeptide, e.g., insertion position 132/136 indicates that the optically active polypeptide replaces amino acid 133-135 of the acetyl-CoA sensitive polypeptide.

In one or more embodiments, the optical probe comprises, in order from the N-terminus to the C-terminus, an optically active polypeptide or variant thereof as set forth in any one of SEQ ID NO:1, residues 1-X of SEQ ID NO:2, 6, 7, 9, and residues Y-146 of SEQ ID NO:1, wherein X and Y are selected from any one of (1) X is 20, Y is 21, (2) X is 20, Y is 22, (3) X is 20, Y is 23, (4) X is 20, Y is 24, (5) X is 20, Y is 25, (6) X is 20, Y is 26, (7) X is 20, Y is 27, (8) X is 21, Y is 22, (9) X is 21, Y is 23, (10) X is 21, Y is 24, (11) X is 21, Y is 25, (12) X is 21, Y is 26, (13) X is 21, Y is 27, (14) X is 22, Y is 23,247920

(15) X is 22, Y is 24, (16) X is 22, Y is 25, (17) X is 22, Y is 26, (18) X is 22, Y is 27, (19) X is 23, Y is 24, (20) X is 23, Y is 25, (21) X is 23, Y is 26, (22) X is 23, Y is 27, (23) X is 37, Y is 38, (24) X is 37, Y is 39, (25) X is 38, Y is 39, (26) X is 46, Y is 47, (27) X is 46, Y is 48, (28) X is 47, Y is 48, (29) X is 59, Y is 60, (30) X is 59, Y is 61, (31) X is 59, Y is 62, (32) X is 59, Y is 63, (33) X is 59, Y is 64, (34) X is 59, Y is 65, (35) X is 59, Y is 66, (36) X is 59, Y is 67, (37) X is 59, Y is 68, (38) X is 59, Y is 69, (39) X is 60, Y is 61, (40) X is 60, Y is 62, (41) X is 60, Y is 63, (42) X is 60, Y is 64, (43) X is 60, Y is 65, (44) X is 60, Y is 66, (45) X is 60, Y is 67, (46) X is 60, Y is 68, (47) X is 60, Y is 69, (48) X is 61, Y is 62, (49) X is 61, Y is 63, (50) X is 61, Y is 64, (51) X is 61, Y is 65, (52) X is 61, Y is 66, (53) X is 61, Y is 67, (54) X is 61, Y is 68, (55) X is 61, Y is 69, (56) X is 62, Y is 63, (57) X is 62, Y is 64, (58) X is 62, Y is 65, (59) X is 62, Y is 66, (60) X is 62, Y is 67, (61) X is 62, Y is 68, (62) X is 62, Y is 69, (63) X is 63, Y is 64, (64) X is 63, Y is 65, (65) X is 63, Y is 66, (66) X is 63, Y is 67, (67) X is 63, Y is 68, (68) X is 63, Y is 69, (69) X is 64, Y is 65, (70) X is 64, Y is 66, (71) X is 64, Y is 67, (72) X is 64, Y is 68, (73) X is 64, Y is 69, (74) X is 65, Y is 66, (75) X is 65, Y is 67, (76) X is 65, Y is 68, (77) X is 65, Y is 69, (78) X is 66, Y is 67, (79) X is 66, Y is 68, (80) X is 66, Y is 69, (81) X is 67, Y is 68, (82) X is 67, Y is 69, (83) X is 78, Y is 79, (84) X is 78, Y is 80, (85) X is 78, Y is 81, (86) X is 78, Y is 82, (87) X is 78, Y is 83, (88) X is 78, Y is 84, (89) X is 79, Y is 80, (90) X is 79, Y is 81, (91) X is 79, Y is 82, (92) X is 79, Y is 83, (93) X is 79, Y is 84, (94) X is 80, Y is 81, (95) X is 80, Y is 82, (96) X is 80, Y is 83, (97) X is 80, Y is 84, (98) X is 81, Y is 82, (99) X is 81, Y is 83, (100) X is 81, Y is 84, (101) X is 82, Y is 83, (102) X is 82, Y is 84, (103) X is 83, Y is 84, (104) X is 102, Y is 103, (105) X is 102, Y is 104, (106) X is 103, Y is 104, (107) X is 112, Y is 113, (108) X is 122, Y is 123, (109) X is 132, Y is 133, (110) X is 132, Y is 134, (111) X is 132, Y is 135, (112) X is 132, Y is 136, (113) X is 133, Y is 134, (114) X is 133, Y is 135, (115) X is 133, Y is 116) X is 134, Y is 135, (117) X is 134, Y is 136, (Y is 136, and (110) X is 134, Y is 136. an exemplary optical probe sequence is shown in SEQ ID NO. 10, which is a probe formed by an optically active polypeptide located at 134/135 of an acetyl-CoA sensitive polypeptide. The sequences of the remaining probes of the invention can be readily determined from the descriptions herein.

The term "variant" or "mutant" as used herein in reference to a polypeptide or protein includes variants having the same function but different sequences of the polypeptide or protein. Such variants include, but are not limited to, sequences obtained by deleting, inserting and/or substituting one or more (usually 1 to 30, preferably 1 to 20, more preferably 1 to 10, most preferably 1 to 5) amino acids in the sequence of the polypeptide or protein, and adding one or more (usually within 20, preferably within 10, more preferably within 5) amino acids at the carboxy-terminal and/or amino-terminal end thereof. Variants of a polypeptide or protein may include homologous sequences, conservative variants, allelic variants, natural mutants, induced mutants. These variants may also comprise a polypeptide or protein having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100% sequence identity to the polypeptide or protein. Without wishing to be bound by theory, amino acid residues are changed without changing the overall configuration and function of the polypeptide or protein, i.e., function-conservative mutations. For example, in the art, substitution with amino acids having similar or similar properties typically does not alter the function of the polypeptide or protein. Amino acids of similar properties are often referred to in the art as families of amino acids with similar side chains, which are well defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). As another example, the addition of one or more amino acids at the amino-and/or carboxy-terminus typically does not alter the function of the polypeptide or protein. Conservative amino acid substitutions for many commonly known non-genetically encoded amino acids are known in the art. Conservative substitutions of other non-coding amino acids may be determined based on a comparison of their physical properties with those of the genetically encoded amino acid.

In two or more polypeptide or nucleic acid molecule sequences, the term "identity" or "percent identity" refers to two or more sequences or subsequences that are the same or wherein a percentage of amino acid residues or nucleotides are the same (e.g., 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical) as compared and aligned for maximum correspondence over a comparison window or designated region, using methods known in the art, such as sequence comparison algorithms, by manual alignment and visual inspection. For example, preferred algorithms for determining percent sequence identity and percent sequence similarity are the BLAST and BLAST 2.0 algorithms, see Altschul et al (1977) Nucleic Acids Res.25:3389 and Altschul et al (1990) J.mol.biol.215:403, respectively.

It is well known to those skilled in the art that in gene cloning operations, it is often necessary to design suitable cleavage sites, which tend to introduce one or more unrelated residues at the end of the expressed polypeptide or protein, without affecting the activity of the polypeptide or protein of interest. As another example, to construct a fusion protein, facilitate expression of a recombinant protein, obtain an automatic secretion of a recombinant protein outside a host cell, or facilitate purification of a recombinant protein, it is often desirable to add some amino acid to the N-terminus, C-terminus, or other suitable region within the recombinant protein, including, but not limited to, a suitable linker peptide, signal peptide, leader peptide, terminal extension, glutathione S-transferase (GST), maltose E binding protein, a tag such as 6His or Flag, or factor Xa or a proteolytic enzyme site of thrombin or enterokinase, for example. An exemplary GAT protein is the full-length amino acid sequence shown in SEQ ID NO. 1, which retains the binding function to acetyl-CoA and does not affect the change in optical properties of the inserted optically active polypeptide in response to acetyl-CoA binding.

The optical probes of the invention may comprise an acetyl-CoA sensitive polypeptide having a mutation at a site selected from the group consisting of I19, R21, T77, K85, G87, P133, P134 of SEQ ID NO. 1 or a truncated variant thereof. The amino acid mutation includes modification, substitution or deletion of amino acids. In preferred embodiments, the mutation of the variant acetyl-CoA binding protein comprises a mutation at a site selected from any of (1) P133, P134 and I19, (2) P133, P134 and R21, (3) P133, P134 and T77, (4) P133, P134 and K85, (5) P133, P134 and G87.

Wherein, as an example in the examples, in SEQ ID NO.1 or a truncated variant thereof, I19 is mutated to L. In one or more embodiments, R21 is mutated to M or D. In one or more embodiments, T77 is mutated to V. In one or more embodiments, K85 is mutated to N. In one or more embodiments, G87 is mutated to a. In one or more embodiments, P133 is mutated to Y. In one or more embodiments, P134 is mutated to D.

In one or more embodiments, the mutation comprises a mutation selected from any one of (1) P133Y, P D and I19L, (2) P133Y, P D and R21M, (3) P133Y, P D and R21D, (4) P133Y, P D and T77V, (5) P133Y, P134D and K85N, (6) P133Y, P D and G87A. The present invention provides acetyl-coa binding protein variants having these mutations and optical probes comprising such acetyl-coa binding protein variants as acetyl-coa sensitive polypeptides.

The optical probes of the invention may comprise optically active polypeptides having mutations. In some embodiments, the mutated optically active polypeptide has a mutation at the Y1 position, including a modification, substitution, or deletion of an amino acid, and in one or more embodiments, the mutation is Y1V. In one or more embodiments, the optical probe comprises any one of the amino acid sequences SEQ ID NOs 10-16 or a variant thereof. In one or more embodiments, the optical probes provided herein comprise sequences that are 35%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99% sequence identity to any one of the amino acid sequences SEQ ID NOs 10-16. In a preferred embodiment, the optical probes provided herein comprise a sequence substantially similar or identical to any one of the amino acid sequences SEQ ID NOs 10-16.

In one or more embodiments, the optically active polypeptide has a sequence as set forth in SEQ ID NO. 2 or a variant thereof having a mutation at amino acid 1 at 1V, the optically active polypeptide being located at one or more of positions ：20/21,20/22,20/23,20/24,20/25,20/26,20/27,21/22,21/23,21/24,21/25,21/26,21/27,22/23,22/24,22/25,22/26,22/27,23/24,23/25,23/26,23/27,37/38,37/39,38/39,46/47,46/48,47/48,59/60,59/61,59/62,59/63,59/64,59/65,59/66,59/67,59/68,59/69,60/61,60/62,60/63,60/64,60/65,60/66,60/67,60/68,60/69,61/62,61/63,61/64,61/65,61/66,61/67,61/68,61/69,62/63,62/64,62/65,62/66,62/67,62/68,62/69,63/64,63/65,63/66,63/67,63/68,63/69,64/65,64/66,64/67,64/68,64/69,65/66,65/67,65/68,65/69,66/67,66/68,66/69,67/68,67/69,78/79,78/80,78/81,78/82,78/83,78/84,79/80,79/81,79/82,79/83,79/84,80/81,80/82,80/83,80/84,81/82,81/83,81/84,82/83,82/84,83/84,102/103,102/104,103/104,112/113,122/123,132/133,132/134,132/135,132/136,133/134,133/135,133/136,134/135,134/136 and/or 135/136 of the acetyl-CoA sensitive polypeptide.

In some embodiments, the optically active polypeptide is located at position 134/135 of the acetyl-CoA sensitive polypeptide, the acetyl-CoA sensitive polypeptide having a sequence as shown in SEQ ID NO. 1 or a variant having at least 70% sequence identity thereto and retaining activity for acetyl-CoA binding, the optically active polypeptide having a sequence as shown in any one of SEQ ID NO. 2, 6, 7, 9, and the acetyl-CoA variant having a mutation selected from any one of (1) P133Y, P134D and I19L, (2) P133Y, P134D and R21M, (3) P133Y, P134D and R21D, (4) P133Y, P134D and T77V, (5) P133Y, P134D and K85N, (6) P133Y, P D and G87A and optionally the optically active polypeptide being mutated to (7) Y1V.

Illustratively, the present invention provides an optical probe comprising any one of the amino acid sequences SEQ ID NOS 10-16 or variants thereof. In one embodiment, the invention provides an optical probe comprising a sequence having 35%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99% sequence identity to any one of the amino acid sequences SEQ ID NOS 10-16. In a preferred embodiment, the optical probes provided herein comprise sequences substantially similar or identical to those of amino acid sequences SEQ ID NOS 10-16.

The terms "functional variant", "derivative" and "analog" as used herein refer to a protein that retains substantially the same biological function or activity as the original polypeptide or protein (e.g., GAT protein or fluorescent protein). The functional variant, derivative or analogue of a polypeptide or protein of the invention (e.g. a GAT protein or fluorescent protein) may be (i) a protein having one or more, preferably conservative or non-conservative amino acid residues substituted, which may or may not be encoded by the genetic code, or (ii) a protein having a substituent in one or more amino acid residues, or (iii) a protein formed by fusion of a mature protein with another compound (e.g. a compound that prolongs the half-life of the protein, such as polyethylene glycol), or (iv) a protein formed by fusion of an additional amino acid sequence to the protein sequence (e.g. a secretion sequence or a sequence used to purify the protein or a pro-protein sequence, or fusion protein with the formation of an IgG fragment of an antigen). Such functional variants, derivatives and analogs are within the scope of those skilled in the art, as determined by the teachings herein.

The difference between the analog and the original polypeptide or protein may be a difference in amino acid sequence, a difference in modified form that does not affect the sequence, or both. These proteins include natural or induced genetic variants. Induced variants may be obtained by various techniques, such as random mutagenesis by irradiation or exposure to mutagens, as well as by site-directed mutagenesis or other known molecular biological techniques.

The analogs also include analogs having residues other than the natural L-amino acid (e.g., D-amino acids), as well as analogs having non-naturally occurring or synthetic amino acids (e.g., beta, gamma-amino acids). It will be appreciated that the acetyl-coa sensitive polypeptides of the invention are not limited to the representative proteins, variants, derivatives and analogues listed above. Modified (typically without altering the primary structure) forms include chemically derivatized forms of the protein, such as acetylation or carboxylation, in vivo or in vitro. Modifications also include glycosylation, such as those resulting from glycosylation modifications during synthesis and processing of the protein or during further processing steps. Such modification may be accomplished by exposing the protein to an enzyme that performs glycosylation (e.g., mammalian glycosylase or deglycosylase). Modified forms also include sequences having phosphorylated amino acid residues (e.g., phosphotyrosine, phosphoserine, phosphothreonine). Proteins modified to increase their proteolytic resistance or to optimize their solubility properties are also included.

The invention also provides a preparation method of the acetyl-CoA optical probe, which comprises the following steps of 1) incorporating a nucleic acid sequence encoding the acetyl-CoA optical probe into an expression vector, 2) transferring the expression vector into a host cell, 3) culturing the host cell under the condition suitable for the expression of the expression vector, and 4) separating the acetyl-CoA optical probe.

The term "nucleic acid" or "nucleotide" as used herein may be in the form of DNA or RNA. DNA forms include cDNA, genomic DNA, or synthetic DNA. The DNA may be single-stranded or double-stranded. The DNA may be a coding strand or a non-coding strand. The term "variant" as used herein when referring to a nucleic acid may be a naturally occurring allelic variant or a non-naturally occurring variant. Such nucleotide variants include degenerate variants, substitution variants, deletion variants and insertion variants. As known in the art, an allelic variant is a substitution pattern of a nucleic acid, which may be a substitution, deletion, or insertion of one or more nucleotides, without substantially altering the function of the encoded protein. The nucleic acids of the invention may comprise a nucleotide sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100% sequence identity to the nucleic acid sequence. The invention also relates to nucleic acid fragments which hybridize to the sequences described above. As used herein, a "nucleic acid fragment" is at least 15 nucleotides, preferably at least 30 nucleotides, more preferably at least 50 nucleotides, and most preferably at least 100 nucleotides or more in length. The nucleic acid fragments may be used in nucleic acid amplification techniques (e.g., PCR).

The full-length sequence of the optical probe or fusion protein of the present invention or a fragment thereof can be generally obtained by PCR amplification, artificial synthesis or recombinant methods. For the PCR amplification method, primers can be designed according to the nucleotide sequences disclosed in the present invention, and the relevant sequences can be obtained by amplification using a commercially available cDNA library or a cDNA library prepared according to a conventional method known to those skilled in the art as a template. When the nucleotide sequence is larger than 2500bp, the PCR amplification is preferably carried out for 2-6 times, and then the amplified fragments are spliced together according to the correct sequence. The PCR amplification procedure and system are not particularly limited, and conventional PCR amplification procedures and systems in the art can be adopted. The sequences of interest can also be obtained in large quantities by recombinant methods. This is usually done by cloning it into a vector, transferring it into cells, and isolating and purifying the relevant polypeptide or protein from the proliferated host cells by conventional methods. Furthermore, the sequences concerned, in particular fragments of short length, can also be synthesized by artificial synthesis. In the present invention, when the nucleotide sequence of the optical probe is less than 2500bp, the optical probe can be synthesized by adopting an artificial synthesis method. The artificial synthesis method is a conventional DNA artificial synthesis method in the field, and has no other special requirements. In general, fragments of very long sequences are obtained by first synthesizing a plurality of small fragments and then ligating them. At present, it is already possible to obtain the DNA sequences encoding the proteins of the invention (or functional variants, derivatives or analogues thereof) entirely by chemical synthesis. The DNA sequence may then be introduced into a variety of existing DNA molecules (e.g., vectors) and cells known in the art. Mutations can be introduced into the protein sequences of the present invention by mutation PCR or chemical synthesis, etc.

After the nucleotide sequence for coding the optical probe is obtained, the nucleotide sequence for coding the optical probe is incorporated into an expression vector to obtain a recombinant expression vector. The terms "expression vector" and "recombinant vector" are used interchangeably herein to refer to a prokaryotic or eukaryotic vector well known in the art, such as a bacterial plasmid, phage, yeast plasmid, plant cell virus, mammalian cell virus such as adenovirus, retrovirus or other vectors, which are capable of replication and stable expression in a host, an important feature of such recombinant vectors being that they typically contain expression control sequences. The term "expression control sequence" as used herein refers to an element operably linked to a gene of interest that regulates the transcription, translation and expression of the gene of interest, and may be an origin of replication, a promoter, a marker gene or a translational control element, including an enhancer, an operator, a terminator, a ribosome binding site, etc., the choice of expression control sequence being dependent upon the host cell used. Recombinant vectors suitable for use in the present invention include, but are not limited to, bacterial plasmids. In recombinant expression vectors, "operably linked" refers to the attachment of a nucleotide sequence of interest to a regulatory sequence in a manner that allows expression of the nucleotide sequence. Methods for constructing expression vectors comprising the fusion protein coding sequences of the invention and appropriate transcriptional/translational control signals are well known to those skilled in the art. These methods include in vitro recombinant DNA techniques, DNA synthesis techniques, in vivo recombinant techniques, and the like. The DNA sequence may be operably linked to an appropriate promoter in an expression vector to direct mRNA synthesis. Representative examples of such promoters are the lac or trp promoter of E.coli, the lambda phage PL promoter, eukaryotic promoters including the CMV immediate early promoter, the HSV thymidine kinase promoter, the early and late SV40 promoters, the LTR of retroviruses and some other known promoters which control gene expression in prokaryotic or eukaryotic cells or viruses thereof. The expression vector also includes a ribosome binding site for translation initiation and a transcription terminator. In one embodiment, the expression vector may be a commercial pCDF vector, with no other special requirements. Illustratively, the nucleotide sequence encoding the optical probe and the expression vector are double digested with BamHI and XhoI, respectively, and then the digested products of the two are ligated to obtain a recombinant expression vector. The specific steps and parameters of the digestion and the connection are not particularly limited, and the steps and parameters conventional in the art are adopted.

After obtaining the recombinant expression vector, the vector is transformed into a host cell to produce a protein or peptide comprising the fusion protein. Such transfer may be carried out by conventional techniques known to those skilled in the art, such as transformation or transfection. The host cell of the invention is a cell capable of receiving and accommodating recombinant DNA molecules, is a site for amplifying recombinant genes, and ideal recipient cells should satisfy both conditions of easy acquisition and proliferation. "host cells" according to the invention may include prokaryotic and eukaryotic cells, including in particular bacterial cells, yeast cells, insect cells and mammalian cells. Specific examples are bacterial cells of E.coli, streptomyces, salmonella typhimurium, fungal cells such as yeast, plant cells, insect cells of Drosophila S2 or Sf9, animal cells of CHO, COS, HEK293, heLa cells, or Bowes melanoma cells, among others, including but not limited to those host cells described above. The host cell is preferably a variety of cells that facilitate expression or fermentative production of the gene product, such cells being well known and commonly used in the art. An exemplary host cell for use in embodiments of the invention is the E.coli BL21-DE3 strain. It will be clear to a person of ordinary skill in the art how to select appropriate vectors, promoters, enhancers and host cells.

Methods of transferring to host cells described herein are conventional in the art and include calcium phosphate or calcium chloride co-precipitation, DEAE-mannan-mediated transfection, lipofection, natural competence, chemical-mediated transfer, or electroporation. When the host is a prokaryote such as E.coli, the method is preferably a CaCl ₂ method or MgCl ₂ method treatment, using procedures well known in the art. When the host cell is eukaryotic, DNA transfection methods such as calcium phosphate co-precipitation, conventional mechanical methods such as microinjection, electroporation, liposome packaging, etc. may be used.

After the expression vector is transferred into a host cell, the host cell transferred into the expression vector is amplified, expressed and cultured, and the acetyl coenzyme A optical probe is obtained by separation. The host cell amplification expression culture can be carried out by adopting a conventional method. The medium used in the culture may be various conventional media depending on the kind of host cell used. The culture is carried out under conditions suitable for the growth of the host cell.

In the present invention, the optical probe is expressed in a cell, on a cell membrane, or secreted outside the cell. If desired, the recombinant proteins can be isolated or purified by various isolation methods using their physical, chemical and other properties. The method for separating the acetyl-CoA fluorescent protein is not particularly limited, and a fusion protein separation method conventional in the art can be adopted. Such methods are well known to those skilled in the art and include, but are not limited to, conventional renaturation treatment, salting-out methods, centrifugation, osmotic sterilization, sonication, ultracentrifugation, molecular sieve chromatography, adsorption chromatography, ion exchange chromatography, high Performance Liquid Chromatography (HPLC), and other various liquid chromatography techniques and combinations of these methods. In one embodiment, the separation of the optical probe is performed using His-tagged affinity chromatography.

The invention also provides application of the acetyl-CoA optical probe in real-time positioning, quantitative detection and high-flux compound screening of acetyl-CoA. In one aspect, the acetyl-CoA optical probe is preferably connected with signal peptides at different parts of a cell, is transferred into the cell, performs real-time positioning of acetyl-CoA by detecting the intensity of a fluorescent signal in the cell, and performs quantitative detection of corresponding acetyl-CoA by an acetyl-CoA standard dripping curve. The standard drop curve of acetyl-CoA is drawn according to fluorescent signals of the acetyl-CoA optical probe under the condition of different concentrations of acetyl-CoA. The acetyl-CoA optical probe is directly transferred into cells, and a time-consuming sample treatment process is not needed in the real-time positioning and quantitative detection process of the acetyl-CoA, so that the method is more accurate. When the acetyl-CoA optical probe is used for high-flux compound screening, different compounds are added into a cell culture solution, and the change of the acetyl-CoA content is measured, so that the compounds with influence on the change of the acetyl-CoA content are screened. The application of the acetyl-CoA optical probe in the invention in real-time positioning and quantitative detection of acetyl-CoA and high-flux compound screening is non-diagnosis and treatment purposes, and does not relate to diagnosis and treatment of diseases.

Concentrations, amounts, percentages, and other numerical values may be expressed herein in terms of ranges. It is also to be understood that such range format is used merely for convenience and brevity and should be interpreted flexibly to include the numerical values explicitly recited as the limits of the range, as well as to include all the individual numerical values or sub-ranges encompassed within that range.

Examples

The acetyl-CoA optical probes provided by the present invention are described in detail below with reference to examples, but they should not be construed as limiting the scope of the present invention.

I. Experimental materials and reagents

In the examples, the conventional cloning method, cell culture and imaging methods of the genetically engineered molecular biology are mainly used, and these methods are well known to those skilled in the art, for example, jianluo Skems et al, J. Sambrook, D.W. Lassel, huang Peitang et al, ind. Molecular cloning laboratory Manual (third edition, 8 months 2002, published by Sci.11, beijing), fei Leixie ni et al, ind. Basic technical Specification (fifth edition), zhang Jingbo, xu Cunshuan et al, J.S. Bonefferson, M.dar et al, ind. Cell biological laboratory Manual, zhang Jingbo et al. Modifications and variations as would be apparent to one skilled in the art are within the scope of the following claims, as may be made in the practice of the application.

The pCDF-cpYFP, pCDF-acetyl-CoA binding protein-based plasmid used in the examples was constructed by the protein laboratory of the university of Wadong, and the pCDF plasmid vector was purchased from Invitrogen. All primers used for PCR were synthesized, purified and identified by mass spectrometry as correct by Shanghai JieRui Bioengineering Co. The expression plasmids constructed in the examples were all subjected to sequence determination, which was performed by Huada gene company and Jie Li Cexu company. Taq DNA polymerase used in each example was purchased from Dongsheng, pfu DNA polymerase was purchased from Tiangen Biochemical technology (Beijing) Co., ltd, PRIMESTAR DNA polymerase was purchased from TaKaRa Co., ltd, and the corresponding polymerase buffer and dNTPs were added when all three polymerases were purchased. BamHI, bglII, hindIII, ndeI, xhoI, ecoRI, speI, etc., a T4 ligase, a T4 phosphorylase (T4 PNK) are purchased from Fermentas, inc., and corresponding buffers are added thereto. Transfection reagent Lip2000 Kit was purchased from Invitrogen company. Amino acids such as acetyl-CoA were purchased from Sigma. Unless otherwise specified, chemical reagents such as inorganic salts were purchased from Sigma-Aldrich corporation. HEPES salts, ampicillin (Amp) and puromycin were purchased from Ameresco. 96-well assay blackboard, 384 Kong Yingguang assay blackboard was purchased from WHB corporation.

The DNA purification kit used in the examples was purchased from BBI (Canada), and the ordinary plasmid minipump kit was purchased from Tiangen Biochemical technology (Beijing) Co. Clone strain Mach1 was purchased from Invitrogen. The nickel column affinity chromatography column and desalting column packing were all from GE HEALTHCARE company.

The main instruments used in the examples include Biotek Synergy 2 multifunctional enzyme-labeled instrument (Bio-Tek Co., ltd., U.S.A.), X-15R high-speed refrigerated centrifuge (Beckman Co., ltd., U.S.A.), microfuge22R bench-type high-speed refrigerated centrifuge (Beckman Co., ltd., U.S.A.), PCR amplification instrument (Biometa, germany), ultrasonoscope (Ningbo Zhi Co., ltd.), nucleic acid electrophoresis instrument (Shencan Bo Co., fluorescent spectrophotometer (Varian Co., ltd.), CO ₂ constant temperature cell incubator (SANYO), inverted fluorescent microscope (Nikon Co., ltd.).

II molecular biology method and cell experiment method

II.1 Polymerase Chain Reaction (PCR):

1. Amplification of the fragment of interest PCR:

The method is mainly used for gene fragment amplification and colony PCR identification of positive clones. The PCR amplification reaction system comprises 0.5-1 mu L of template sequence, 0.5 mu L of forward primer (25 mu M), 0.5 mu L of reverse primer (25 mu M), 5 mu L of 10 Xpfu buffer solution, 0.5 mu L of pfu DNA polymerase, 1 mu L of dNTP (10 mM), 41.5-42 mu L of sterilized ultrapure water (ddH ₂ O) and 50 mu L of total volume. The PCR amplification procedure was as follows, denaturation at 95℃for 2-10 min, 30 cycles (94-96℃for 30-45 seconds, 50-65℃for 30-45 seconds, 72℃for a certain time (600 bp/min)), and extension at 72℃for 10 min.

2. Long fragment (> 2500 bp) amplification PCR:

The long fragment amplification used in the present invention is mainly an inverse PCR amplification vector, a technique for obtaining site-directed mutagenesis in the following examples. Reverse PCR primers were designed at the mutation sites, wherein the 5' end of one primer contained the mutated nucleotide sequence. The amplified product contains the corresponding mutation site. The long fragment amplification PCR reaction was performed with 1. Mu.L of the template sequence (10 pg-1 ng), 0.5. Mu.L of the forward primer (25. Mu.M), 0.5. Mu.L of the reverse primer (25. Mu.M), 10. Mu.L of 5X PRIMERSTAR buffer, 0.5. Mu.L of PRIMERSTAR DNA polymerase, 4. Mu.L of dNTPs (2.5 mM), 33.5. Mu.L of sterilized ultrapure water (ddH ₂ O), and a total volume of 50. Mu.L. The PCR amplification procedure was either 95℃for 5 minutes, 30 cycles (98℃for 10 seconds, 50-68℃for 5-15 seconds, 72℃for a certain time (1000 bp/min)), 72℃for 10 minutes, or 95℃for 5 minutes, 30 cycles (98℃for 10 seconds, 68℃for a certain time (1000 bp/min)), 72℃for 10 minutes.

II.2 endonuclease cleavage reaction:

the plasmid vector was digested simultaneously with 20. Mu.L (about 1.5. Mu.g) of the plasmid vector, 5. Mu.L of 10 Xbuffer solution, 1-2. Mu.L of restriction enzyme, and the total volume was made up to 50. Mu.L with sterilized ultrapure water. The reaction conditions were 37℃for 1-7 hours.

II.3 5' -terminal phosphorylation of DNA fragments

The plasmid or genome extracted from the microorganism contains phosphate groups at the terminal, but the PCR product does not, so that the 5' -terminal base of the PCR product needs to be subjected to phosphate group addition reaction, and only DNA molecules containing phosphate groups at the terminal can undergo ligation reaction. The phosphorylation reaction system was as follows, the DNA sequence of the PCR product fragment was 5-8. Mu.L, 10 XT 4 ligase buffer 1. Mu.L, T4 polynucleotide kinase (T4 PNK) 1. Mu.L, sterilized ultrapure water 0-3. Mu.L, and total volume 10. Mu.L. The reaction conditions were 37℃and after 30 minutes to 2 hours the reaction was inactivated at 72℃for 20 minutes. Wherein T4 PNK is a shorthand for T4 polynucleotide kinase for addition reactions to the 5' -terminal phosphate group of DNA molecules.

II.4 ligation of the fragment of interest and the vector

The ligation methods between different fragments and vectors are different, and three ligation methods are used in the present invention

1. Blunt end ligation of blunt end short fragments and linearized vectors

The principle of the method is that a blunt end product obtained by PCR carries out phosphorylation reaction on the 5' end of a DNA fragment under the action of T4 PNK, and then is connected with a linearized vector under the action of PEG4000 and T4 DNA ligase to obtain a recombinant plasmid. The homologous recombination ligation system was 4. Mu.L of T4 PNK treated DNA fragment, 4. Mu.L of linearized vector fragment, PEG4000 1. Mu.L, 10 XT 4 ligase buffer 1. Mu.L, T4 DNA ligase 1. Mu.L, and a total of 10. Mu.L. The reaction conditions were 22 ℃ for 30 minutes.

2. Ligation of DNA fragments containing cohesive ends and vector fragments containing cohesive ends

DNA fragments cleaved by restriction enzymes will typically produce protruding cohesive ends and thus can be ligated to cohesive end vector fragments containing sequence complementarity to form recombinant plasmids. The ligation reaction system was 1-7. Mu.L of the digested PCR fragment DNA, 0.5-7. Mu.L of the digested plasmid, 1. Mu.L of 10 XT 4 ligase buffer solution, 1. Mu.L of T4DNA ligase, and 10. Mu.L of sterilized ultrapure water were added to the total volume. The reaction conditions are 16 ℃ and 4-8 hours. Wherein the mass ratio of the PCR product fragment to the carrier double enzyme product is approximately between 2:1 and 6:1.

3. Ligation of the product of 5' -phosphorylated DNA fragments by self-cyclization after introduction of site-directed mutagenesis by inverse PCR

The DNA fragment with phosphorylated 5' end is connected with the 3' end and the 5' end of the linearization vector through self cyclization connection reaction to obtain the recombinant plasmid. The self-circularization ligation reaction system was 10. Mu.L of the phosphorylation reaction system, 0.5. Mu.L of T4 ligase (5U/. Mu.L), and a total volume of 10.5. Mu.L. The reaction conditions are 16 ℃ and 4-16 hours.

II.5 preparation and transformation of competent cells

Preparation of competent cells:

1. single colonies (e.g., mach 1) were picked and inoculated into 5mL LB medium, and shaken overnight at 37 ℃.

2. 0.5-1ML of the overnight cultured bacterial liquid is transferred to 50mL of LB culture medium, and cultured for 3 to 5 hours at 37 ℃ and 220rpm until OD ₆₀₀ reaches 0.5.

3. The cells were pre-chilled in an ice bath for 2 hours.

Centrifuge at 4000rpm for 10 min at 4.4 ℃.

5. The supernatant was discarded, and the cells were resuspended in 5mL of pre-chilled buffer, and after homogenization, the resuspension buffer was added to a final volume of 50mL.

6. Ice bath for 45 minutes.

The bacteria were resuspended by centrifugation at 4000rpm at 7.4℃for 10 minutes with 5mL of ice-chilled storage buffer.

8. Mu.L of bacterial liquid was placed in each EP tube and frozen at-80℃or with liquid nitrogen.

Resuspension buffer CaCl ₂(100mM)、MgCl₂ (70 mM), naAc (40 mM)

Storage buffer 0.5mL DMSO, 1.9mL 80% glycerol, 1mL 10 XCaCl ₂(1M)、1mL10×MgCl₂(700mM)、1mL 10×NaAc(400mM)、4.6mL ddH₂ O

Transformation of competent cells:

1. 100. Mu.L of competent cells were thawed on an ice bath.

2. Add the appropriate volume of ligation product, gently blow mix, ice bath for 30 minutes. The ligation product is typically added in a volume of less than 1/10 of the competent cell volume.

3. The bacterial liquid is placed in a 42 ℃ water bath for heat shock for 90 seconds, and is quickly transferred to an ice bath for 5 minutes.

4. Mu.L of LB was added and incubated at 200rpm on a 37℃constant temperature shaker for 1 hour.

5. The bacterial liquid was centrifuged at 4000rpm for 3 minutes, 200. Mu.L of supernatant was left to blow the bacterial cells evenly, and the cells were spread evenly on the surface of an agar plate containing the appropriate antibiotics, and the plate was inverted overnight in a 37℃incubator.

II.6 expression, purification and fluorescence detection of proteins

1. Expression vectors (e.g., pCDF-based acetyl-coa optical probe expression vectors) were transformed into BL21 (DE 3) cells, cultured upside down overnight, cloned into 250ml Erlenmeyer flasks were picked from plates, placed on a 37 ℃ shaker, cultured at 220rpm to od=0.4-0.8, added with 1/1000 (v/v) IPTG (1M), and induced for expression at 18 ℃ for 24-36 hours.

2. After the induction expression was completed, the cells were collected by centrifugation at 4000rpm for 30 minutes, and the cell pellet was resuspended in 50mM phosphate buffer and sonicated until the cells were clarified. Centrifugation was performed at 9600rpm at 4℃for 20 minutes.

3. The supernatant was purified by self-contained nickel column affinity chromatography to obtain protein, and the protein after nickel column affinity chromatography was further passed through self-contained desalting column to obtain protein dissolved in 100mM HEPES buffer (pH 7.4).

4. After SDS-PAGE identification of the purified proteins, the probes were diluted with assay buffer (100mM HEPES,100mM NaCl,pH 7.4) to a final concentration of 0.2-5. Mu.M protein solution. acetyl-CoA was formulated with assay buffer (100mM HEPES,100mM NaCl,pH 7.4) as a stock solution at a final concentration of 50 mM.

5. 100 Μl of 1 μM protein solution was incubated at 37deg.C for 10min, and acetyl CoA titration was added to determine the fluorescence intensity of 528nm emission after 420nm excitation and 528nm emission after 485nm excitation. The fluorescence excitation and emission measurement of the sample are completed by a multifunctional fluorescence enzyme-labeling instrument.

6. 100 Μl of 1 μM protein solution was incubated at 37deg.C for 10min, acetyl CoA was added, and the absorption spectrum and fluorescence spectrum of the protein were measured. The measurement of the absorption spectrum and fluorescence spectrum of the sample is performed by a spectrophotometer and a fluorescence spectrophotometer.

II.7 transfection and fluorescence detection of mammalian cells

1. The pcdna3.1+ based acetyl coa optical probe plasmid was transfected into HeLa by the transfection reagent Lipofectamine2000 (Invitrogen) and incubated in a cell incubator at 37 ℃ with 5% CO ₂. And (4) performing fluorescence detection after the exogenous gene is fully expressed for 24-36 hours.

2. After the induction of expression is completed, the adherent HEK293 cells are washed three times by PBS and placed in HBSS solution for detection by a fluorescence microscope and an enzyme-labeled instrument respectively.

EXAMPLE 1 acetyl-CoA binding protein plasmid

GAT (1-146) gene (SEQ ID NO: 1) in Bacillus licheniformis gene was amplified by PCR, and the PCR product was recovered by gel electrophoresis and digested with BamHI and XhoI, while the pCDF vector was subjected to corresponding double digestion. After ligation with T4 DNA ligase, DH 5. Alpha. Was transformed with the product, and the transformed DH 5. Alpha. Was plated on LB plates (streptomycin 100. Mu.g/mL) and incubated overnight at 37 ℃. The growing DH5 alpha transformants were subjected to plasmid extraction and PCR identification. The positive plasmid is sequenced correctly and then the subsequent plasmid construction is carried out.

Example 2 expression and detection of cpYFP optical probes at different insertion sites

In this example, the following site insertions cpYFP were selected based on pCDF-GAT to give the corresponding pCDF-GAT-cpYFP plasmid ：20/21,20/22,20/23,20/24,20/25,20/26,20/27,21/22,21/23,21/24,21/25,21/26,21/27,22/23,22/24,22/25,22/26,22/27,23/24,23/25,23/26,23/27,37/38,37/39,38/39,46/47,46/48,47/48,59/60,59/61,59/62,59/63,59/64,59/65,59/66,59/67,59/68,59/69,60/61,60/62,60/63,60/64,60/65,60/66,60/67,60/68,60/69,61/62,61/63,61/64,61/65,61/66,61/67,61/68,61/69,62/63,62/64,62/65,62/66,62/67,62/68,62/69,63/64,63/65,63/66,63/67,63/68,63/69,64/65,64/66,64/67,64/68,64/69,65/66,65/67,65/68,65/69,66/67,66/68,66/69,67/68,67/69,78/79,78/80,78/81,78/82,78/83,78/84,79/80,79/81,79/82,79/83,79/84,80/81,80/82,80/83,80/84,81/82,81/83,81/84,82/83,82/84,83/84,102/103,247920

102/104,103/104,112/113,122/123,132/133,132/134,132/135,132/136,133/134,133/135,133/136,134/135 (I.e.SEQ ID NO: 10), 134/136 and/or 135/136.

And generating cpYFP DNA fragments by PCR, introducing cpYFP terminal homologous sequences through the 5' end of the primer, and generating the pCDF-GlnK1 linearization vector by PCR amplification, wherein the 5' and 3' terminal ends of the linearization vector respectively have completely identical sequences (15 bp-25 bp) corresponding to the two terminal ends of cpYFP. The linearized pCDF-GAT and cpYFP fragments were subjected to homologous recombination under the action of Hieff Clone Enzyme. The product was transformed into DH 5. Alpha. And the transformed DH 5. Alpha. Was plated on LB plates (streptomycin 50. Mu.g/mL) and incubated overnight at 37 ℃. Positive clones identified by PCR were sequenced after drawing the plasmid. Sequencing was done by the Jie Li Huohua big Gene sequencing company.

After sequencing correctly, the recombinant plasmid was transformed into BL21 (DE 3) to induce expression, and the protein was purified and sized around 44kDa by SDS-PAGE. The size of the recombinant protein corresponds to the size of the GAT-cpYFP fusion protein expressed by pCDF-cpYFP-GAT and containing His-tag purification tag. The results are shown in FIG. 1.

The crushed supernatant of E.coli expressing GAT-cpYFP fusion protein was used for acetyl-CoA response screening, and the detection signal of the fusion fluorescent protein containing 100. Mu.M acetyl-CoA was divided by the detection signal of the fusion fluorescent protein without acetyl-CoA. As a result, as shown in FIG. 2, the detection results showed that the optical probes having greater than 1.3-fold or less than 0.75-fold response to acetyl CoA than the control had optical probes for insertion at positions 22/25,46/48,59/69,60/68,62/66,133/135,134/135 and 134/136.

Example 3 expression and detection of cpGFP optical probes at different insertion sites

An acetyl-CoA green fluorescent protein fluorescent probe was constructed by substituting cpYFP with cpGFP as in example 2. As shown in FIG. 3, the detection results showed that the optical probes having greater than 1.3-fold or less than 0.75-fold response to acetyl CoA than the control had optical probes inserted at positions 22/25,22/26,46/48,59/69,60/67,62/67,133/135 and 134/136.

Example 4 expression and detection of cpBFP optical probes at different insertion sites

An acetyl-CoA blue fluorescent protein fluorescent probe was constructed by substituting cpYFP with cpBFP as in example 2. As shown in FIG. 4, the detection results showed that the optical probes having greater than 1.3-fold or less than 0.75-fold response to acetyl CoA than the control had optical probes performing insertions at positions 22/26,38/39,62/67,133/136 and 134/136.

Example 5 expression and detection of cpmApple optical probes at different insertion sites

An acetyl-CoA red fluorescent protein fluorescent probe was constructed by substituting cpYFP with cpmApple as in example 2. As shown in FIG. 5, the detection results showed that the optical probes having greater than 1.2-fold or less than 0.8-fold response to acetyl CoA than the control had optical probes for insertion at positions 46/47,59/69,60/67,62/66,133/135 and 134/136.

EXAMPLE 6 expression and detection of mutated cpYFP optical probes

The optical probe obtained in example 2 was linearized by inverse PCR based on the optical probe inserted at position 134/135, the sequence of the mutation site was introduced into the primer, homologous recombination was performed on the obtained PCR product under the action of Hieff Clone Enzyme, and a mutation library was established. The recombinant plasmid of the mutant library was transformed into BL21 (DE 3) to induce expression, the crushed supernatant of E.coli expressing the probe protein was used for response screening of acetyl-CoA, and the detection signal of the fusion fluorescent protein containing 100. Mu.M acetyl-CoA was divided by the detection signal of the fusion fluorescent protein without acetyl-CoA. The results are shown in Table 1, and the optical probes showing more than 4-fold response to acetyl-CoA are shown below.

TABLE 1 sequence of mutated optical probes

Numbering device	Insertion site	Mutation	R420/485	Sequence(s)
					I3	134/135	GAT-P133Y&P134D&I19L&cpYFP Y1V	4.12	SEQ ID NO:11
T8	134/135	GAT-P133Y&P134D&R21M&cpYFP Y1V	4.39	SEQ ID NO:12
					T9	134/135	GAT-P133Y&P134D&R21D&cpYFP Y1V	4.13	SEQ ID NO:13
F2	134/135	GAT-P133Y&P134D&T77V&cpYFP Y1V	5.11	SEQ ID NO:14
					C19	134/135	GAT-P133Y&P134D&K85N&cpYFP Y1V	4.02	SEQ ID NO:15
G1	134/135	GAT-P133Y&P134D&G87A&cpYFP Y1V	4.37	SEQ ID NO:16

Example 7 Properties of optical Probe mutants

Illustratively, after subjecting purified acetyl-CoA optical probe GAT-P133Y & P134D & R21M & cpYFP Y V to 0mM and 500 μM acetyl-CoA, respectively, for 10 minutes, detection of fluorescence spectra was performed using a fluorescence spectrophotometer. Determination of the excitation spectrum of 350-505nm was recorded with a fixed emission wavelength of 540nm, read every 1 nm. The results showed that the probe had two excitation peaks at 420mm and 500nm, as shown in FIG. 6 (A). For the measurement of emission spectra, the fixed excitation wavelengths were 420nm and 485nm, respectively, and the emission spectra of 500-600nm were recorded, read every 1 nm. The results showed that the emission peak of the probe at 420nm excitation was 512nm and the emission peak of the probe at 485nm excitation was 519nm. The fluorescence intensity of the probe hardly changed under 420nm excitation after 500 mu M of acetyl CoA was added, and the fluorescence intensity was reduced by 7 times under 485nm excitation without acetyl CoA. As shown in fig. 6 (B) and (C).

The concentration gradient (0-500. Mu.M) of the acetyl-CoA optical probe of Table 1 described in example 6 was used for acetyl-CoA detection. After 10 minutes of probe treatment, the change in the ratio of the fluorescence intensity at 528nm emission from 420nm excitation to the fluorescence intensity at 528nm emission from 485nm excitation was detected. The results of the probe titration are shown in FIG. 7, and indicate that the different mutants have different affinities for acetyl-CoA.

The probes of the examples in Table 1 were specifically tested and respectively tested for reactivity with acetyl-CoA analogues, and the results showed good specificity as shown in FIG. 8.

Example 8 subcellular organelle localization of optical probes and the Performance of optical probes within subcellular organelles

In this example, different localization signal peptides were used to fuse with the optical probe GAT-P133Y & P134D & R21M & cpYFP Y V to localize the optical probe to different organelles. HEK293 cells were transfected with optical probe plasmids fused with different localization signal peptides for 36 hours, rinsed with PBS, placed in HBSS solution and fluorescence detected under FITC channel using an inverted fluorescence microscope. The results are shown in FIG. 9. The acetyl-CoA optical probe can be localized to subcellular organelles including cytoplasm, extracellular membrane, nucleus, endoplasmic reticulum, mitochondria and nuclear exclusion by fusion with different specific localization signal peptides. Fluorescence is shown in different subcellular structures, and the distribution and intensity of fluorescence are different.

HEK293 cells were transfected with cytoplasmic expression optical probe plasmid for 36 hours, rinsed with PBS, placed in a solution with 10. Mu.M or 30. Mu.M BMS-303141 added, and the change in the ratio of fluorescence intensity at 420nm excitation 528nm emission to fluorescence intensity at 485nm excitation 528nm emission was detected over a 30min period. BMS-303141 is an inhibitor of ATP-citrate lyase and can reduce intracellular acetyl-CoA levels. As a result, as shown in FIG. 10, R _420/485 of the sample added with 10. Mu.M BMS-303141 was gradually lowered by 6% at the lowest, and R _420/485 of the sample was gradually lowered by 45% at the lowest when 30. Mu.M BMS-303141 was added and detected for 30 minutes.

Example 9 high throughput Compound screening in living cells based on optical probes

In this example, we used HeLa cells with cytosolic expression of GAT-P133Y & P134D & R21M & cpYFP Y V for high throughput compound screening.

Transfected HeLa cells were rinsed with PBS, placed in HBSS solution (without acetyl-CoA) for 1 hour, and then treated with 10. Mu.M compound for 1 hour. acetyl-CoA was added dropwise to each sample. The change in the ratio of the fluorescence intensity at the 528nm emission of 420nm excitation to the fluorescence intensity at the 528nm emission of 485nm excitation was recorded using a microplate reader. Samples not treated with any compound were normalized as controls. The results are shown in FIG. 11. Of the 2000 compounds used, the vast majority of compounds had minimal effect on acetyl-coa entry into the cells. There are 3 compounds that increase the uptake of acetyl-CoA by cells and 2 compounds that significantly decrease the uptake of acetyl-CoA by cells.

Example 10 quantitative detection of acetyl-CoA in blood by optical Probe

In this example, purified GAT-P133Y & P134D & R21M & cpYFP Y V was used to analyze acetyl-CoA in mouse and human blood supernatants.

After mixing GAT-P133Y & P134D & R21M & cpYFP Y V with diluted blood supernatant for 10 minutes, the ratio of the fluorescence intensity at the 528nm emission at 420nm to the fluorescence intensity at the 528nm emission at 485nm was detected using a microplate reader. As a result, FIG. 12 shows that the content of acetyl-CoA in the blood of the mouse was about 700nM and the content of acetyl-CoA in the blood of the human was about 1.6. Mu.M.

The embodiment shows that the acetyl-CoA optical probe provided by the invention has the advantages of relatively small molecular weight, easiness in maturation, large dynamic change of fluorescence, good specificity, capability of expressing in cells by a gene operation method, capability of positioning and quantitatively detecting the acetyl-CoA in real time inside and outside the cells, and capability of performing high-flux compound screening.

Other embodiments

The present specification describes a number of embodiments. It will be appreciated that various modifications may be made by those skilled in the art from a reading of this specification without departing from the spirit and scope of the invention, and are intended to be included within the scope of the appended claims.

Partial sequence

SEQ ID NO:1GAT(1-146)

MIEVKPINAEDTYEIRHRILRPNQPLEACMYETDLLGGAFHLGGYYRGKLISIASFHKAEHSELEGEEQYQLRGM

ATLEGYREQKAGSTLIRHAEELLRKKGADLLWCNARTSVSGYYEKLGFSEQGEVYDIPPIGPHILMYKKLT

SEQ ID NO:2cpYFP

YNSDNVYIMADKQKNGIKANFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSFQSVLSKDPNEKRDH

MVLLEFVTAAGITLGMDELYNVDGGSGGTGSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL

KLICTTGKLPVPWPTLVTTLGYGLKCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGD

TLVNRIELKGIDFKEDGNILGHKLEYN

SEQ ID NO:3cpmOrange

VSERMYPEDGVLKSEIKKGLRLKDGGHYAAEVKTTYKAKKPVQLPGAYIVDIKLDIVSHNEDYTIVEQCERAEG

RHPTGGRDELYKGGTGGSLVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEAFQTAKLKVTK

GGPLPFAWDILSPQFTYGSKAYIKHPADIPDYFKLSFPEGFRWERVMNFEDGGIIHVNQDSSLQDGVFIYKVKLR

GTNFPPDGPVMQKKTMGWEA

SEQ ID NO:4cpmKate

MGGRSKKPAKNLKMPGVYYVDRRLERIKEADKETYVEQHEVAVARYCDLPSKLGHKLNGGTGGSMVSKGEE

LIKENMHMKLYMEGTVNNHHFKCTSEGEGKPYEGTQTMRIKVVEGGPLPFAFDILATSFMYGSKTFINHTQGIP

DFFKQSFPEGFTWERVTTYEDGGVLTATQDTSLQDGCLIYNVKIRGVNFPSNGPVMQKKTLGWEASTEMLYPA

DGGLEGRSDMALKLVGGGHLICNLKTTYRSKK

SEQ ID NO:5mCherry

MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEGTQTAKLKVTKGGPLPFAWDILSPQFMYG

SKAYVKHPADIPDYLKLSFPEGFKWERVMNFEDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTM

GWEASSERMYPEDGALKGEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQY

ERAEGRHSTGGMDELYK

SEQ ID NO:6cpGFP

NVYIKADKQKNGIKANFKIRHNIEDGGVQLAYHYQQNTPIGDGPVLLPDNHYLSVQSILSKDPNEKRDHMVLLE

FVTAAGITLGMDELYKGGTGGSMVSKGEELFTGVVPIQVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTT

GKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYIQERTIFFKDDGNYKTRAEVKFEGDTLVNRIE

LKGIDFKEDGNILGHKLEYN

SEQ ID NO:7cpBFP

NVYIKADKQKNGIKANFKIRHNIEGGGVQLAYHYQQNTPIGDGPVLLPDNHYLSVQSILSKDPNEKRDHMVLLE

FVTAAGITLGMDELYKGGTGGSESMVSKGEELFTGVVPIQVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICT

TGKLPVPWPTLVTTLSHGVQCFSRYPDHMKQHDFFKSAMPGGYIQERTIFFKDDGNYKTRAEVKFEGDTLVNRI

ELKGIDFKEDGNILGHKLEYN

SEQ ID NO:8mKate

MSELITENMHMKLYMEGTVNNHHFKCTSEGEGKPYEGTQTMRIKVVEGGPLPFAFDILATSFMYGSKTFINHTQ

GIPDFFKQSFPEGFTWERVTTYEDGGVLTATQDTSLQDGCLIYNVKIRGVNFPSNGPVMQKKTLGWEASTEMLY

PADGGLEGRADMALKLVGGGHLICNLKTTYRSKKPAKNLKMPGVYYVDRRLERIKEADKETYVEQHEVAVAR

YCDLPSKLGHKLN

SEQ ID NO:9cpmApple

VSERMYPEDGALKSEIKKGLRLKDGGHYAAEVKTTYKAKKPVQLPGAYIVDIKLDIVSHNEDYTIVEQCERAEG

RHSTGGMDELYKGGTGGSLVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEAFQTAKLKVTK

GGPLPFAWDILSPQFMYGSKAYIKHPADIPDYFKLSFPEGFRWERVMNFEDGGIIHVNQDSSLQDGVFIYKVKLR

GTNFPPDGPVMQKKTMGWEA

SEQ ID NO:10GAT-134/135-cpYFP

MIEVKPINAEDTYEIRHRILRPNQPLEACMYETDLLGGAFHLGGYYRGKLISIASFHKAEHSELEGEEQYQLRGM

ATLEGYREQKAGSTLIRHAEELLRKKGADLLWCNARTSVSGYYEKLGFSEQGEVYDIPPYNSDNVYIMADKQK

NGIKANFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSFQSVLSKDPNEKRDHMVLLEFVTAAGITLG

MDELYNVDGGSGGTGSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKLICTTGKLPVPWPT

LVTTLGYGLKCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKE

DGNILGHKLEYNIGPHILMYKKLT

SEQ ID NO:11GAT-134/135-cpYFP(GAT-P133Y&P134D&I19L&cpYFP Y1V)

MIEVKPINAEDTYEIRHRLLRPNQPLEACMYETDLLGGAFHLGGYYRGKLISIASFHKAEHSELEGEEQYQLRGM

ATLEGYREQKAGSTLIRHAEELLRKKGADLLWCNARTSVSGYYEKLGFSEQGEVYDIYDVNSDNVYIMADKQK

NGIKANFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSFQSVLSKDPNEKRDHMVLLEFVTAAGITLG

MDELYNVDGGSGGTGSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKLICTTGKLPVPWPT

LVTTLGYGLKCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKE

DGNILGHKLEYNIGPHILMYKKLT

SEQ ID NO:12 GAT-134/135-cpYFP(GAT-P133Y&P134D&R21M&cpYFP Y1V)

MIEVKPINAEDTYEIRHRILMPNQPLEACMYETDLLGGAFHLGGYYRGKLISIASFHKAEHSELEGEEQYQLRGM

ATLEGYREQKAGSTLIRHAEELLRKKGADLLWCNARTSVSGYYEKLGFSEQGEVYDIYDVNSDNVYIMADKQK

NGIKANFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSFQSVLSKDPNEKRDHMVLLEFVTAAGITLG

MDELYNVDGGSGGTGSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKLICTTGKLPVPWPT

LVTTLGYGLKCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKE

DGNILGHKLEYNIGPHILMYKKLT

SEQ ID NO:13 GAT-134/135-cpYFP(GAT-P133Y&P134D&R21D&cpYFP Y1V)

MIEVKPINAEDTYEIRHRILDPNQPLEACMYETDLLGGAFHLGGYYRGKLISIASFHKAEHSELEGEEQYQLRGM

ATLEGYREQKAGSTLIRHAEELLRKKGADLLWCNARTSVSGYYEKLGFSEQGEVYDIYDVNSDNVYIMADKQK

NGIKANFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSFQSVLSKDPNEKRDHMVLLEFVTAAGITLG

MDELYNVDGGSGGTGSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKLICTTGKLPVPWPT

LVTTLGYGLKCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKE

DGNILGHKLEYNIGPHILMYKKLT

SEQ ID NO:14 GAT-134/135-cpYFP(GAT-P133Y&P134D&T77V&cpYFP Y1V)

MIEVKPINAEDTYEIRHRILRPNQPLEACMYETDLLGGAFHLGGYYRGKLISIASFHKAEHSELEGEEQYQLRGM

AVLEGYREQKAGSTLIRHAEELLRKKGADLLWCNARTSVSGYYEKLGFSEQGEVYDIYDVNSDNVYIMADKQ

KNGIKANFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSFQSVLSKDPNEKRDHMVLLEFVTAAGITL

GMDELYNVDGGSGGTGSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKLICTTGKLPVPWP

TLVTTLGYGLKCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKE

DGNILGHKLEYNIGPHILMYKKLT

SEQ ID NO:15 GAT-134/135-cpYFP(GAT-P133Y&P134D&K85N&cpYFP Y1V)

MIEVKPINAEDTYEIRHRILRPNQPLEACMYETDLLGGAFHLGGYYRGKLISIASFHKAEHSELEGEEQYQLRGM

ATLEGYREQNAGSTLIRHAEELLRKKGADLLWCNARTSVSGYYEKLGFSEQGEVYDIYDVNSDNVYIMADKQK

NGIKANFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSFQSVLSKDPNEKRDHMVLLEFVTAAGITLG

MDELYNVDGGSGGTGSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKLICTTGKLPVPWPT

LVTTLGYGLKCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKE

DGNILGHKLEYNIGPHILMYKKLT

SEQ ID NO:16 GAT-134/135-cpYFP(GAT-P133Y&P134D&G87A&cpYFP Y1V)

MIEVKPINAEDTYEIRHRILRPNQPLEACMYETDLLGGAFHLGGYYRGKLISIASFHKAEHSELEGEEQYQLRGM

ATLEGYREQKAASTLIRHAEELLRKKGADLLWCNARTSVSGYYEKLGFSEQGEVYDIYDVNSDNVYIMADKQK

NGIKANFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSFQSVLSKDPNEKRDHMVLLEFVTAAGITLG

MDELYNVDGGSGGTGSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKLICTTGKLPVPWPT

LVTTLGYGLKCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKE

DGNILGHKLEYNIGPHILMYKKLT

Claims (10)

1. An acetyl-coa binding protein variant which:

(1) Having the sequence shown in SEQ ID NO. 1 and having mutations at 1,2, 3 or more positions selected from the group consisting of I19, R21, T77, K85, G87, P133 and P134, said mutations comprising amino acid modifications, substitutions or deletions,

(2) Is a sequence having at least 70% sequence identity to the sequence of (1) and having the mutation of (1) and retaining the ability to bind acetyl-CoA,

Preferably, the mutations comprise mutations at a site selected from any one of (a) P133, P134 and I19, (b) P133, P134 and R21, (c) P133, P134 and T77, (d) P133, P134 and K85, (e) P133, P134 and G87;

more preferably, the I19 is mutated to L, the R21 is mutated to M or D, the T77 is mutated to V, the K85 is mutated to N, the G87 is mutated to A, the P133 is mutated to Y, and the P134 is mutated to D;

more preferably, the mutation comprises a mutation selected from any one of (I) P133Y, P D and I19L, (ii) P133Y, P D and R21M, (iii) P133Y, P D and R21D, (iv) P133Y, P D and T77V, (V) P133Y, P D and K85N, (vi) P133Y, P D and G87A.

2. An optical probe comprising an acetyl-CoA sensitive polypeptide and an optically active polypeptide, wherein said acetyl-CoA sensitive polypeptide is (1) a sequence shown in SEQ ID NO. 1, or a sequence having at least 70% sequence identity thereto and retaining activity for acetyl-CoA binding, or (2) a sequence of an acetyl-CoA binding protein variant according to claim 1, or (3) a sequence having at least 70% sequence identity to said sequence and having (2) said mutation and retaining sensitivity to acetyl-CoA, and said optically active polypeptide is a fluorescent protein or variant thereof,

The optically active polypeptide is located at one or more sites ：20/21,20/22,20/23,20/24,20/25,20/26,20/27,21/22,21/23,21/24,21/25,21/26,21/27,22/23,22/24,22/25,22/26,22/27,23/24,23/25,23/26,23/27,37/38,37/39,38/39,46/47,46/48,47/48,59/60,59/61,59/62,59/63,59/64,59/65,59/66,59/67,59/68,59/69,60/61,60/62,60/63,60/64,60/65,60/66,60/67,60/68,60/69,61/62,61/63,61/64,61/65,61/66,61/67,61/68,61/69,62/63,62/64,62/65,62/66,62/67,62/68,62/69,63/64,63/65,63/66,63/67,63/68,63/69,64/65,64/66,64/67,64/68,64/69,65/66,65/67,65/68,65/69,66/67,66/68,66/69,67/68,67/69,78/79,78/80,78/81,78/82,78/83,78/84,79/80,79/81,79/82,79/83,79/84,80/81,80/82,80/83,80/84,81/82,81/83,81/84,82/83,82/84,83/84,102/103,102/104,103/104,112/113,122/123,132/133,132/134,132/135,132/136,133/134,133/135,133/136,134/135,134/136 and/or 135/136 selected from the group consisting of acetyl-CoA sensitive polypeptides,

The variant of the fluorescent protein comprises a mutation at the Y1 site,

Preferably, the optically active polypeptide is selected from any one of cpYFP, cpGFP, cpBFP, cpmApple.

3. The optical probe of claim 2, wherein the fluorescent protein is as shown in any one of SEQ ID NOs 2 to 9, and wherein Y1 in the variant of the fluorescent protein is mutated to V;

Preferably, the fluorescent protein is shown in any one of SEQ ID NOs 2, 6, 7 and 9.

4. A fusion polypeptide comprising the optical probe of claim 2 or 3 and an additional polypeptide comprising a targeting sequence, a tag for ease of purification, or a tag for use in an immune response.

5. A nucleic acid molecule comprising the sequence:

(a) A polynucleotide sequence encoding the optical probe of claim 2 or 3, or the fusion polypeptide of claim 4, or

(B) The complement of (a).

6. A nucleic acid construct comprising the nucleic acid molecule of claim 6,

Preferably, the nucleic acid construct is an expression vector.

7. A host cell, the host cell:

(1) Comprising, expressing or secreting the optical probe of claim 2 or 3 or the fusion polypeptide of claim 4;

(2) Comprising the nucleic acid molecule of claim 5, or

(3) Comprising the nucleic acid construct of claim 6.

8. A test kit comprising:

(1) The optical probe according to claim 2 or 3 or the fusion polypeptide according to claim 4,

(2) The nucleic acid molecule according to claim 5,

(3) The nucleic acid construct of claim 6,

(4) The host cell according to claim 7,

The detection kit optionally further comprises other reagents required for detection of acetyl-coa using an optical probe,

Preferably, the assay kit further comprises one or more reagents selected from the group consisting of buffers, media, acetyl-CoA standards.

9. A method of making the optical probe of claim 2 or 3 or the fusion polypeptide of claim 4, comprising culturing the host cell of claim 7 under conditions in which the optical probe is expressed, and isolating the optical probe from the culture.

10. Use of the optical probe of claim 2 or 3, the fusion polypeptide of claim 4, the nucleic acid molecule of claim 5, the nucleic acid construct of claim 6 and/or the host cell of claim 7 for detecting acetyl-coa, a screening compound or intracellular and/or extracellular localization of acetyl-coa in a sample.