Succinic acid optical probe as well as preparation method and application thereof

Abstract

The invention relates to a succinic acid optical probe as well as a preparation method and application thereof. In one aspect, the present invention relates to an optical probe comprising a succinic acid-sensitive polypeptide or functional variant thereof and an optically active polypeptide or functional variant thereof wherein the optically active polypeptide or functional variant thereof is located within the sequence of the succinic acid-sensitive polypeptide or functional variant thereof. The invention also relates to a preparation method of the probe and application of the probe in succinic acid detection.

Description

Technical Field

[0001] The present invention relates to the technical field of optical probes, and in particular to a succinic acid optical probe and a preparation method and application thereof. Background Art

[0002] Succinate is an important natural organic acid. Abnormal succinate metabolism is closely related to a variety of diseases, including cardiovascular disease and tumors. Prag, HA and others found that succinate and the succinate receptor SUCNR1 signaling axis can be used as a metabolite sensing mechanism to regulate the dynamics of leptin in a biological clock-related manner, thereby controlling the energy balance of the organism.

[0003] Existing methods for detecting succinic acid include colorimetry, liquid chromatography-mass spectrometry, high performance liquid chromatography, etc. These detection and analysis methods either require professional analytical instruments or have complicated steps, cannot distinguish succinic acid from xanthine, are prone to human errors, and are only applicable to in vitro detection and cannot monitor the changes in succinic acid concentration in living cells in real time. Therefore, it is urgent to develop new detection methods to achieve simple, rapid, highly specific, real-time localization, quantitative, and high-throughput detection of succinic acid inside and outside cells. Summary of the invention

[0004] The object of the present invention is to provide a probe and method for real-time localization, high throughput, and quantitative detection of succinic acid inside and outside cells. In order to achieve the above-mentioned invention object, the present invention provides the following technical solutions:

[0005] The first aspect of the present invention provides a succinate binding protein variant, which:

[0006] (1) having the sequence shown in SEQ ID NO: 1 and having a mutation at one, two, three, four, five or six of the following positions: S154, F155, Y160, N182, V183, S184, wherein the mutation comprises a modification, substitution or deletion of an amino acid,

[0007] (2) is a truncated variant of (1) having amino acids 65-320, or

[0008] (3) is a sequence having at least 70% sequence identity with the sequence of (1) or (2) and having the mutation described in (1) and retaining the ability to bind to succinate.

[0009] In one or more embodiments, the mutation comprises a mutation at any of the following sites: S154, F155, Y160, N182, V183, S184.

[0010] In one or more embodiments, the mutation comprises a mutation at a site selected from any one of the following groups: (1) F155 and Y160, (2) Y160 and N182, (3) Y160, N182 and V183, (4) Y160, N182 and S184, (5) S154, Y160, N182 and S184;

[0011] In one or more embodiments, S154 is mutated to R. In one or more embodiments, F155 is mutated to T, P, Q, or C. In one or more embodiments, Y160 is mutated to Y or W. In one or more embodiments, N182 is mutated to S. In one or more embodiments, V183 is mutated to Y or W. In one or more embodiments, S184 is mutated to L.

[0012] In one or more embodiments, the mutation comprises a mutation selected from any one of the following groups: (1) F155T and Y160W, (2) F155P and Y160W, (3) F155Q and Y160W, (4) F155C and Y160W, (5) Y160W and N182S, (6) Y160W, N182S and V183Y, (7) Y160W, N182S and V183W, (8) Y160W, N182S and S184L, (9) S154R, Y160W, N182S and S184L.

[0013] Another aspect of the present invention provides a succinic acid optical probe, comprising a succinic acid sensitive polypeptide and an optically active polypeptide, wherein the optically active polypeptide is located within the sequence of the succinic acid sensitive polypeptide.

[0014] In one or more embodiments, the succinate-sensitive polypeptide comprises a succinate-binding protein or a functional variant thereof.In one or more embodiments, the succinate-sensitive polypeptide is derived from Agrobacterium tumefaciens.

[0015] In one or more embodiments, the succinate-sensitive polypeptide has:

[0016] (1) the sequence shown in SEQ ID NO: 1 or a truncated variant thereof having amino acids 65 to 320, or a sequence having at least 70% sequence identity therewith and retaining succinate binding activity,

[0017] (2) the sequence of the succinate binding protein variant described in any embodiment of the first aspect of the present invention, or

[0018] (3) A sequence having at least 70% sequence identity with the sequence described in (2) and having the mutation described in (2) and retaining sensitivity to succinic acid.

[0019] 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 yellow fluorescent protein (such as cpYFP as shown in SEQ ID NO: 2), orange fluorescent protein (such as cpmOrange as shown in SEQ ID NO: 3), red fluorescent protein (such as mKate as shown in SEQ ID NO: 4 or 8, such as mcherry as shown in SEQ ID NO: 5), green fluorescent protein (such as cpGFP as shown in SEQ ID NO: 6), blue fluorescent protein (such as cpBFP as shown in SEQ ID NO: 7), apple red fluorescent protein (such as cpmApple as shown in SEQ ID NO: 9). Preferably, the optically active polypeptide is cpYFP. In one or more embodiments, the fluorescent protein has a sequence as shown in any one of SEQ ID NO: 2-9.

[0020] In one or more embodiments, the fluorescent protein functional variant has: (1) the sequence shown in SEQ ID NO: 2 and has mutations at 1, 2, 3, 4, 5 or 6 of the following positions: M9, S132, Y141, F201, N207, Y245, wherein the mutations include modification, substitution or deletion of amino acids, and the numbering of the amino acids corresponds to the sequence of the fluorescent protein, or (2) a sequence having at least 70% sequence identity with the sequence of (1). In one or more embodiments, the mutations of the fluorescent protein are selected from any one or more of the following: M9T, S132R, Y141N, F201S, N207T and Y245F. In one or more embodiments, the mutations of the fluorescent protein are M9T, S132R, Y141N, F201S, N207T and Y245F.

[0021] In one or more embodiments, the optical probe comprises the succinate binding protein variant described in any embodiment of the first aspect of the present invention and the above-mentioned fluorescent protein functional variant, and comprises a mutation selected from any one of the following groups: (1) F155T and Y160W of succinate binding protein, (2) F155P and Y160W of succinate binding protein, (3) F155Q and Y160W of succinate binding protein, (4) F155C and Y160W of succinate binding protein, (5) Y160W and N182S of succinate binding protein, (6) Y160W, N182S and V183Y of succinate binding protein, (7) Y160W of succinate binding protein. 160W, N182S and V183W of succinate binding protein, (8) Y160W, N182S and S184L of succinate binding protein, (9) S154R, Y160W, N182S and S184L of succinate binding protein, (10) Y160W, N182S and S184L of succinate binding protein, and M9T, S132R, Y141N, F201S, N207T and Y245F of fluorescent protein, (11) S154R, Y160W, N182S and S184L of succinate binding protein, and M9T, S132R, Y141N, F201S, N207T and Y245F of fluorescent protein.

[0022] In one or more embodiments, the optically active polypeptide is located between residues 108-112, 181-185, 196-201 and 148-156 of the succinate-sensitive polypeptide, numbered corresponding to the full length of the succinate-sensitive polypeptide. Preferably, the optically active polypeptide is located at one or more of the following sites of the succinate-sensitive polypeptide: 108 / 109, 108 / 110, 108 / 111, 108 / 112, 109 / 110, 109 / 111, 109 / 112, 110 / 111, 110 / 112, 111 / 112, 181 / 182, 181 / 183, 181 / 184, 181 / 185, 182 / 183, 182 / 184, 182 / 185 5, 183 / 184, 183 / 185, 184 / 185, 196 / 197, 196 / 198, 196 / 199, 196 / 200, 196 / 201, 197 / 198, 197 / 199, 197 / 200, 197 / 201, 198 / 199, 198 / 200, 198 / 201, 199 / 200, 199 / 201, 200 / 201, 148 / 149, 148 / 150, 148 / 151 , 148 / 152, 148 / 153, 148 / 154, 148 / 155, 148 / 156, 149 / 150, 149 / 151, 149 / 152, 149 / 153, 149 / 154, 149 / 155, 149 / 156, 150 / 151, 150 / 152, 150 / 153, 150 / 154, 150 / 155, 150 / 156, 151 / 152, 151 / 153, 151 / 154 , 151 / 155, 151 / 156, 152 / 151, 152 / 152, 152 / 153, 152 / 154, 152 / 155, 152 / 156, 153 / 151, 153 / 152, 153 / 153, 153 / 154, 153 / 155, 153 / 156, 154 / 151, 154 / 152, 154 / 153, 154 / 154, 154 / 155, 154 / 156, 155 / 156. More preferably, the optically active polypeptide is located at any one or more of the following sites of the succinate-sensitive polypeptide: 150 / 152, 150 / 153, 150 / 155, 150 / 156, 151 / 153, 152 / 152, 152 / 153, 152 / 154, 153 / 152, 153 / 153, 153 / 154, 154 / 151, 154 / 152, 154 / 154, 181 / 182.

[0023] In one or more embodiments, the optical probe further comprises one or more linkers flanking the optically active polypeptide. The linker of the present invention can be any amino acid sequence of any length. In one or more embodiments, the optically active polypeptide flank comprises a linker of no more than 5 amino acids, such as a linker of 0, 1, 2, 3, or 4 amino acids. In one or more embodiments, the linker flanking the optically active polypeptide comprises amino acid Y. In one or more embodiments, linker Y is located at the N-terminus and / or C-terminus of the optically active polypeptide. In one or more embodiments, the optical probe is as follows: the first part B1-Y of the succinic acid-sensitive polypeptide-optically active polypeptide A-the second part B2 of the succinic acid-sensitive polypeptide. In one or more embodiments, the optical probe of the present invention does not comprise a linker.

[0024] In one or more embodiments, the optical probes of the present invention further comprise a localization sequence for localizing the probe to, for example, a specific organelle of a cell.

[0025] In one or more embodiments, the optically active polypeptide is a cpYFP or a variant thereof located at any one or more of the following sites of the succinate-sensitive polypeptide: 108 / 110, 108 / 111, 108 / 112, 181 / 182, 181 / 185, 182 / 183, 182 / 185, 183 / 184, 148 / 150, 148 / 153, 148 / 155, 149 / 150, 149 / 151, 149 / 152, 149 / 154, 150 / 152, 150 / 153, 150 / 154, 150 / 155, 150 / 156, 151 / 152, 151 / 153, 151 / 154, 151 / 156, 152 ... 1, 152 / 152, 152 / 153, 152 / 154, 152 / 156, 153 / 152, 153 / 153, 153 / 154, 153 / 156, 154 / 151, 154 / 152, 154 / 153, 154 / 154, 154 / 155, 155 / 156, (preferably 150 / 152, 15 0 / 153, 150 / 155, 150 / 156, 151 / 153, 152 / 152, 152 / 153, 152 / 154, 153 / 152, 153 / 153, 153 / 154, 154 / 151, 154 / 152, 154 / 154, 181 / 182), the succinate-sensitive polypeptide (1) is as shown in SEQ ID NO:1 or is a truncated variant thereof having amino acids 65-320, or (2) is the succinate-binding protein variant described in any embodiment of the first aspect of the present invention. Preferably, the succinate binding protein variant has a mutation selected from any one of the following groups: (1) F155T and Y160W, (2) F155P and Y160W, (3) F155Q and Y160W, (4) F155C and Y160W, (5) Y160W and N182S, (6) Y160W, N182S and V183Y, (7) Y160W, N182S and V183W, (8) Y160W, N182S and S184L, (9) S154R, Y160W, N182S and S184L.

[0026] In one or more embodiments, the cpYFP variant has the sequence shown in SEQ ID NO:2 and has a mutation at 1, 2 or 3, 4, 5 or 6 of the following positions: M9, S132, Y141, F201, N207, Y245, wherein the mutation comprises an amino acid modification, substitution or deletion.

[0027] In one or more embodiments, the mutations of the cpYFP variant are M9T, S132R, Y141N, F201S, N207T, Y245F.

[0028] In one or more embodiments, the optically active polypeptide is a cpGFP located at any one or more of the following sites of the succinate-sensitive polypeptide: 181 / 182, 183 / 185, 184 / 185, 197 / 198, 198 / 200, 198 / 201, 199 / 200, 199 / 201, 200 / 201, 148 / 152, 148 / 154, 148 / 156, 149 / 150, 149 / 151, 149 / 156, 150 / 151, 150 / 152, 150 / 153, 150 / 155, 150 / 156 150 / 153, 151 / 152, 152 / 153, 152 / 154, 151 / 155, 151 / 156, 152 / 152, 152 / 153, 152 / 154, 152 / 155, 152 / 156, 153 / 151, 153 / 153, 153 / 155, 153 / 156, 154 / 151, 154 / 154, 154 / 155, 154 / 156, 155 / 156, (preferably 150 / 153, 151 / 152, 152 / 153, 152 / 154), the succinate-sensitive polypeptide (1) is as shown in SEQ ID NO: 1 or is a truncated variant thereof having amino acids 65-320, or (2) is the succinate-binding protein variant described in any embodiment of the first aspect of the present invention. Preferably, the succinate binding protein variant has a mutation selected from any one of the following groups: (1) F155T and Y160W, (2) F155P and Y160W, (3) F155Q and Y160W, (4) F155C and Y160W, (5) Y160W and N182S, (6) Y160W, N182S and V183Y, (7) Y160W, N182S and V183W, (8) Y160W, N182S and S184L, (9) S154R, Y160W, N182S and S184L.

[0029] In one or more embodiments, the optically active polypeptide is a cpBFP located at any one or more of the following sites of the succinate sensitive polypeptide: 108 / 109, 108 / 110, 110 / 111, 110 / 112, 181 / 185, 183 / 184, 183 / 185, 197 / 201, 200 / 201, 148 / 150, 148 / 151, 148 / 152, 148 / 153, 149 / 151, 149 / 153, 149 / 155, 150 / 151, 150 / 152, 150 / 155, 151 / 152, 151 / 153, 151 / 154, 151 / 156, 152 / 151, 152 / 152, 152 / 154, 152 / 155, 152 / 156, 153 / 151, 153 / 152, 153 / 153, 153 / 156, 154 / 151, 154 / 152, 155 / 156, (preferably 148 / 152, 148 / 153, 151 / 153, 151 / 154, 152 / 155, 152 / 156, 153 / 151, 154 / 152), the succinate-sensitive polypeptide (1) is as shown in SEQ ID NO: 1 or is a truncated variant thereof having amino acids 65-320, or (2) is the succinate-binding protein variant described in any embodiment of the first aspect of the present invention. Preferably, the succinate binding protein variant has a mutation selected from any one of the following groups: (1) F155T and Y160W, (2) F155P and Y160W, (3) F155Q and Y160W, (4) F155C and Y160W, (5) Y160W and N182S, (6) Y160W, N182S and V183Y, (7) Y160W, N182S and V183W, (8) Y160W, N182S and S184L, (9) S154R, Y160W, N182S and S184L.

[0030] In one or more embodiments, the optically active polypeptide is cpmApple, which is located at any one or more of the following positions of the succinate sensitive polypeptide: 108 / 109, 108 / 110, 110 / 111, 196 / 201, 197 / 201, 199 / 200, 200 / 201, 148 / 149, 148 / 151, 148 / 155, 149 / 152 , 149 / 156, 150 / 155, 150 / 156, 151 / 156, 152 / 154, 152 / 155, 152 / 156, 153 / 151, 153 / 152, 153 / 153, 154 / 152, 154 / 153, 154 / 156, (preferably 152 / 154, 153 / 153), the succinate-sensitive polypeptide (1) is as shown in SEQ ID NO:1 or is a truncated variant thereof having amino acids 65-320, or (2) is the succinate-binding protein variant described in any embodiment of the first aspect of the present invention. Preferably, the succinate binding protein variant has a mutation selected from any one of the following groups: (1) F155T and Y160W, (2) F155P and Y160W, (3) F155Q and Y160W, (4) F155C and Y160W, (5) Y160W and N182S, (6) Y160W, N182S and V183Y, (7) Y160W, N182S and V183W, (8) Y160W, N182S and S184L, (9) S154R, Y160W, N182S and S184L.

[0031] In one or more embodiments, the optical probe comprises any one of the amino acid sequences SEQ ID NO: 10-31 or a variant thereof. In one or more embodiments, the optical probe provided by the present invention comprises 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 NO: 10-31. Preferably, the optical probe provided by the present invention comprises a sequence substantially similar or identical to any one of the amino acid sequences SEQ ID NO: 10-31.

[0032] Another aspect of the present invention provides a fusion polypeptide comprising the optical probe described herein and other polypeptides. In some embodiments, the optical probe described herein further comprises other polypeptides fused thereto. The other polypeptides described herein do not affect the properties of the optical probe. In some embodiments, the other polypeptides are located at the N-terminus and / or C-terminus of the optical probe. In some embodiments, the other polypeptides include polypeptides for locating the optical probe to different organelles or sub-organelles, tags for purification, or tags for immunoblotting. A joint may be provided between the optical probe and other polypeptides in the fusion polypeptide described herein.

[0033] Another aspect of the present invention provides a nucleic acid molecule comprising: (a) a coding sequence of a polypeptide or probe as described in any embodiment of the present invention, or (b) a complementary sequence of (a), or (c) a fragment of (a) or (b). The fragment is a primer.

[0034] In one or more embodiments, the nucleic acid sequence comprises an amino acid sequence encoding any one of SEQ ID NOs: 25-31. Preferably, the nucleic acid sequence comprises any one of nucleotide sequences SEQ ID NOs: 30-31 or a variant thereof. More preferably, the nucleic acid sequence comprises a sequence having 99%, 95%, 90%, 80%, 70% or 50% identity to any one of nucleotide sequences SEQ ID NOs: 30-31; or comprises a nucleotide sequence substantially similar or identical to any one of nucleotide sequences SEQ ID NOs: 30-31.

[0035] The present invention also relates to the complementary sequence of the above-mentioned nucleic acid sequence or its variant, which may comprise the nucleic acid sequence encoding the fragment, analog, derivative, soluble fragment and variant of the optical probe or fusion protein of the present invention or its complementary sequence.

[0036] The present invention also provides a nucleic acid construct comprising the nucleic acid molecule described herein. The nucleic acid sequence encodes the optical probe or fusion polypeptide described in the present invention.

[0037] In one or more embodiments, the nucleic acid construct is a cloning vector, an expression vector, or a recombinant vector.

[0038] In one or more embodiments, the nucleic acid molecule is operably linked to an expression control sequence.

[0039] In some embodiments, the expression vector is selected from a prokaryotic expression vector, a eukaryotic expression vector, and a viral vector.

[0040] The present invention also provides a host cell, wherein the host cell: (1) expresses the optical probe or fusion polypeptide according to any embodiment of the present invention; (2) contains the nucleic acid molecule according to any embodiment of the present invention; or (3) contains the nucleic acid construct according to any embodiment of the present invention. The host cell is preferably Escherichia coli.

[0041] Another aspect of the present invention provides a succinate detection kit, comprising the optical probe or fusion polypeptide or polynucleotide described herein or the optical probe prepared by the method described herein.

[0042] In one or more embodiments, the kit further comprises one or more reagents selected from the group consisting of a buffer, a culture medium, and a succinate standard.

[0043] The present invention provides a method for preparing the optical probe described herein, comprising: providing a host cell expressing the optical probe or fusion polypeptide described herein, culturing the host cell under conditions where the optical probe or fusion polypeptide is expressed, and isolating the optical probe or fusion polypeptide.

[0044] In one or more embodiments, the method for preparing the succinate optical probe or fusion polypeptide described herein comprises the following steps: 1) transferring the expression vector encoding the succinate optical probe described herein into a host cell; 2) culturing the host cell under conditions suitable for the expression of the expression vector; 3) isolating the succinate optical probe.

[0045] The present invention also provides a method for detecting succinic acid in a sample, comprising: contacting the optical probe or fusion polypeptide described herein or the optical probe or fusion polypeptide prepared by the method described herein with the sample, and detecting changes in the optically active polypeptide. The detection can be performed in vivo, in vitro, in subcellular or in situ. The sample is, for example, blood.

[0046] Also provided herein is a method for quantifying succinic acid in a sample, comprising: contacting the optical probe or fusion polypeptide described herein or the optical probe or fusion polypeptide prepared by the method described herein with the sample, detecting changes in the optically active polypeptide, and quantifying succinic acid in the sample based on the changes in the optically active polypeptide.

[0047] The present invention also provides a method for screening compounds (e.g., drugs), comprising: contacting the optical probe or fusion polypeptide described herein or the optical probe or fusion polypeptide prepared by the method described herein with a candidate compound in a system containing succinic acid, detecting changes in the optically active polypeptide, and screening the compound based on the changes in the optically active polypeptide. The method can screen compounds with high throughput.

[0048] In one or more embodiments, a host cell described herein is contacted with a candidate compound in a system containing succinate, and a change in the optics of the optically active polypeptide indicates whether the candidate compound is capable of modulating succinate uptake by the cell.

[0049] Another aspect of the present invention provides a method for localizing the succinic acid inside and / or outside a cell, comprising: contacting a system containing succinic acid with the optical probe or the host cell, and detecting the optical change of the optically active polypeptide.

[0050] In one or more embodiments, the system is a solution system, a cellular system, or a subcellular system.

[0051] Another aspect of the present invention provides the use of the succinic acid optical probe or fusion polypeptide or host cell described herein in detecting succinic acid in a sample, screening compounds or localizing succinic acid inside or outside a cell. In one or more embodiments, the localization is real-time localization.

[0052] Beneficial effects of the present invention: The succinic acid optical probe provided by the present invention is easy to mature, has large dynamic changes in fluorescence, has good specificity, and can be expressed in cells by genetic manipulation methods, and can be used for real-time positioning, high-throughput, and quantitative detection of succinic acid inside and outside cells, eliminating the time-consuming sample processing steps. Experimental results show that the maximum response of the succinic acid optical probe provided by the present application to succinic acid is about 4 times that of the control, and cells can be positioned, qualitatively, and quantitatively detected in subcellular structures such as cytoplasm, mitochondria, cell nuclei, and nuclear exclusion, and high-throughput compound screening and quantitative detection of succinic acid in blood can be performed. BRIEF DESCRIPTION OF THE DRAWINGS

[0053] The present invention will be further described below in conjunction with the accompanying drawings and embodiments.

[0054] Figure 1 is an SDS-PAGE image of the exemplary succinic acid optical probe described in Example 1;

[0055] Figure 2 is a titration curve diagram of the response of the exemplary succinic acid optical probe described in Example 6 to different concentrations of succinic acid;

[0056] Figure 3 is the affinity of the exemplary succinate optical probe described in Example 8 for succinate;

[0057] Figure 4 A bar graph showing the specific detection of four succinate analogs by the exemplary succinate optical probe described in Example 8;

[0058] Figure 5 This is a photograph of the subcellular organelle localization of the exemplary succinate optical probe described in Example 9 in mammalian cells;

[0059] Figure 6 A schematic diagram of dynamically monitoring the succinate concentration in the cytoplasm of mammalian cells using an exemplary succinate optical probe as described in Example 9;

[0060] Figure 7 A dot plot of high-throughput compound screening at the living cell level using the exemplary succinate optical probe described in Example 10;

[0061] Figure 8 A bar graph showing the quantification of succinate in mouse and human blood by the exemplary succinate optical probe described in Example 11; DETAILED DESCRIPTION

[0062] As used herein, the term "about" when giving a value or range means that the value or range is within 20%, within 10%, and within 5% of the given value or range.

[0063] As used herein, the terms "comprising", "including" and equivalent forms thereof include the meanings of "containing" as well as "consisting of", for example, a composition "comprising" X may consist of X alone or may contain other substances, such as X+Y.

[0064] As used herein, the term "succinate-sensitive polypeptide" or "succinate-responsive polypeptide" refers to a polypeptide that responds to succinate, including any response to a chemical, biological, electrical or physiological parameter of the polypeptide associated with the interaction of the sensitive polypeptide. Responses include small changes, for example, changes in the orientation of the amino acids or peptide fragments of the polypeptide and changes in, for example, the primary, secondary or tertiary structure of the polypeptide, including, for example, changes in protonation, electrochemical potential and / or conformation. "Conformation" is the three-dimensional arrangement of the primary, secondary and tertiary structures of a molecule containing side groups in the molecule; when the three-dimensional structure of the molecule changes, the conformation changes. Examples of conformational changes include transitions from α-helix to β-fold or from β-fold to α-helix. It is understood that as long as the fluorescence of the fluorescent protein portion is changed, the detectable change does not need to be a conformational change. The succinate-sensitive polypeptides described herein may also include functional variants thereof. Functional variants of succinate-sensitive polypeptides include, but are not limited to, variants that can interact with succinic acid to undergo the same or similar changes as the parent succinate-sensitive polypeptide.

[0065] The term "optical probe" as used herein refers to a succinate-sensitive polypeptide fused to an optically active polypeptide, wherein the optically active polypeptide (e.g., a fluorescent protein) is operably inserted into the succinate-sensitive polypeptide. The inventors have found that when an optically active polypeptide is fused to a succinate-sensitive polypeptide such as a succinate-binding protein, the conformational changes produced by the succinate-sensitive polypeptide specifically binding to physiological concentrations of succinate will cause conformational changes in the optically active polypeptide (e.g., a fluorescent protein), thereby causing the optical properties of the optically active polypeptide to change. By drawing a standard curve with the help of the fluorescence of the fluorescent protein measured under different succinate concentrations, the presence and / or level of succinate can be detected and analyzed. The succinate-sensitive polypeptide of the present invention includes, but is not limited to, the succinate-binding protein DctBp or a variant having more than 90% homology thereto. The exemplary succinate-binding protein DctBp of the present invention is derived from Agrobacterium tumefaciens. An exemplary DctBp protein is shown in SEQ ID NO: 1, and an exemplary DctBp protein truncated variant is a fragment of SEQ ID NO: 1 comprising amino acids 65 to 320. When describing the optical probe, succinate-sensitive polypeptide or succinate-binding protein of the present invention (e.g., when describing the insertion site or mutation site), the amino acid residue numbers are all referenced to SEQ ID NO: 1.

[0066] Protein-based "optically active polypeptides" are polypeptides that have the ability to emit fluorescence. Fluorescence is an optical property of optically active polypeptides that can be used as a means of detecting the responsiveness of the optical probes of the present invention. As used herein, the term "fluorescence property" refers to the molar extinction coefficient at an appropriate excitation wavelength, the fluorescence quantum efficiency, the shape of the excitation spectrum or the emission spectrum, the excitation wavelength maximum and the emission wavelength maximum, the amplitude of two different wavelength excitations, the emission amplitude ratio of two different wavelengths, the excited state lifetime or the fluorescence anisotropy. The measurable difference in any of these properties between the active and inactive states is sufficient for the utility of the fluorescent protein substrate of the present 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 specific wavelength or the integral of fluorescence on the emission spectrum. Preferably, the protein substrate is selected to have fluorescent properties that are easily distinguished in the unactivated and activated conformational states. The optically active polypeptides described herein may also include functional variants thereof. Functional variants of optically active polypeptides include, but are not limited to, variants that can undergo the same or similar fluorescent property changes as the parent optically active polypeptide.

[0067] Herein, "response multiple" is the normalized fluorescence ratio. The more the probe response multiple deviates from 1 (whether it increases or decreases), the greater the change multiple or response ability of the probe to the substrate relative to the control. For example, the embodiment of the present application detects the change in the ratio of the fluorescence intensity at 528nm emission when excited by 420nm and the fluorescence intensity at 528nm emission when excited by 485nm (Normalized Ratio 485 / 420 ) to calculate the response multiple, as follows:

[0068] The fluorescence signal value was corrected by deducting the detection signal value of cells that did not express the probe protein. The probe detection signal in the parallel experimental group was divided by the control detection signal to eliminate pH-sensitive interference and obtain the correction data.

[0069] F=F sample =F BLK

[0070]

[0071]

[0072]

[0073]

[0074] F represents the fluorescence intensity. sample Represents the total fluorescence intensity of the sample expressing the fluorescent probe, F BLKrepresents the background fluorescence intensity of the sample without expressing the fluorescent probe, F cpYFP represents the fluorescence intensity of the sample used as a pH control. 485 The fluorescence intensity of the fluorescent protein sample is excited at 485 nm and emitted at 528 nm, F 420 It represents the fluorescence intensity of the fluorescent protein sample when excited at 420nm and emitted at 528nm. sensor Represents the fluorescence intensity ratio of the probes, Ratio cpYFP Normalized Ratio represents the fluorescence intensity ratio of the corresponding probe to the pH control fluorescent protein. 485 / 420 Normalized Ratio is the multiple of the probe’s change or response. 485 / 420 The greater the deviation from 1 (whether it becomes larger or smaller), the greater the change fold or response fold of the probe.

[0075] "Linker" or "connector region" refers to an amino acid or nucleotide sequence that connects two parts in the polypeptide, protein or nucleic acid of the present invention. For example, the number of amino acids at the amino terminus of the connecting region between the succinic acid-sensitive polypeptide and the optically active polypeptide in the present invention is 0-3, and the number of amino acids at the carboxyl terminus is 0-2; when the recombinant optical probe is connected to the functional protein as a basic unit, it can be fused to the amino acid or carboxyl terminus of the recombinant optical probe. The linker sequence can be a short peptide chain composed of one or more flexible amino acids, such as Y.

[0076] The terms "chromophore", "fluorophore" and "fluorescent protein" used herein are synonymous with proteins that emit fluorescence under excitation light. Fluorescent proteins are used as basic detection methods in the field of biological sciences, such as the green fluorescent protein GFP commonly used in the field of biotechnology and the cyclic rearranged blue fluorescent protein (cpBFP), cyclic rearranged green fluorescent protein (cpGFP), cyclic rearranged yellow fluorescent protein (cpYFP) derived from mutations of the protein; and the red fluorescent protein RFP commonly used in the field of technology, and the cyclic rearranged proteins derived from the protein, such as cpmApple, cpmOrange, cpmKate, etc. The sequence of an exemplary fluorescent protein is shown in any one of SEQ ID NO: 2-9.

[0077] The succinate optical probe of the present invention comprises a succinate sensitive polypeptide B, such as a succinate binding protein or a variant thereof, and an optically active polypeptide A, such as a fluorescent protein. The optically active polypeptide A is inserted into the succinate sensitive polypeptide B, and B is divided into a first part B1 and a second part B2, forming a probe structure of the type B1-A-B2; the interaction between the succinate sensitive polypeptide B and succinic acid causes the optical signal of the optically active polypeptide A to become stronger.

[0078] In the optical probe of the present invention, the optically active polypeptide can be located at any position of the succinate-sensitive polypeptide. In one or more embodiments, the optically active polypeptide is located in the following regions of the succinate-sensitive polypeptide in the NC direction: amino acid residues 108-112, 181-185, 196-201 and 148-156 regions. Exemplarily, the optically active polypeptide is located at 108 / 109, 108 / 110, 108 / 111, 108 / 112, 109 / 110, 109 / 111, 109 / 112, 110 / 111, 110 / 112, 111 / 112, 181 / 182, 181 / 183, 181 / 184, 181 / 185, 182 / 183, 182 / 184, 182 / 185, 183 / 184,183 / 185,184 / 185,196 / 197,196 / 198,196 / 199,196 / 200,196 / 201,197 / 198,197 / 199,197 / 200,197 / 201,198 / 199,198 / 200,198 / 201,199 / 200,199 / 201,200 / 201,148 / 149,148 / 150,148 / 151,148 / 152 8 / 152, 148 / 153, 148 / 154, 148 / 155, 148 / 156, 149 / 150, 149 / 151, 149 / 152, 149 / 153, 149 / 154, 149 / 155, 149 / 156, 150 / 151, 150 / 152, 150 / 153, 150 / 154, 150 / 155, 150 / 156, 151 / 152, 151 / 153, 151 / 154, 1 151 / 155,151 / 156,152 / 151,152 / 152,152 / 153,152 / 154,152 / 155,152 / 156,153 / 151,153 / 152,153 / 153,153 / 154,153 / 155,153 / 156,154 / 151,154 / 152,154 / 153,154 / 154,154 / 155,154 / 156,155 / 156.

[0079] Herein, in the site expressed in the form of "X / Y", both ends of the optically active polypeptide respectively have partial succinic acid-sensitive polypeptides, wherein the N-terminus of the optically active polypeptide is from the N-terminal starting amino acid (e.g., any amino acid from position 1 to position 65) to the X-th amino acid of the succinic acid-sensitive polypeptide sequence, and the C-terminus of the optically active polypeptide is from the Y-th amino acid to the C-terminal last amino acid (e.g., any amino acid from position Y to amino acid 320) of the succinic acid-sensitive polypeptide sequence. Among them, if the two numbers in the site represented by the form of "X / Y" are consecutive integers, it means that the optically active polypeptide is located between the amino acids described by the numbers, for example, the insertion site 152 / 153 indicates that the optically active polypeptide is located between amino acids 152 and 153 of the succinic acid-sensitive polypeptide; if the two numbers in the site represented by the form of "X / Y" are not consecutive integers and X is less than Y, it means that the optically active polypeptide replaces the amino acids between the amino acids indicated by the numbers, for example, the insertion site 150 / 153 indicates that the optically active polypeptide replaces amino acids 151-152 of the succinic acid-sensitive polypeptide; if X in the site represented by the form of "X / Y" is greater than or equal to Y, it means that the succinic acid-sensitive polypeptide portion located at the N-terminus of the optically active polypeptide is replaced to the succinic acid-sensitive polypeptide. The acid-sensitive polypeptide sequence ends at the Xth amino acid, and the succinic acid-sensitive polypeptide portion located at the C-terminus of the optically active polypeptide starts from the Yth amino acid of the succinic acid-sensitive polypeptide sequence; for example, the insertion site 131 / 126 indicates that the N-terminus of the optically active polypeptide is fused with the N-terminal starting amino acid (e.g., any amino acid from the 1st to the 654th amino acid) to the 131st amino acid of the succinic acid-sensitive polypeptide sequence, and the C-terminus of the optically active polypeptide is fused with the 126th amino acid to the C-terminal last amino acid (e.g., the 320th amino acid) of the succinic acid-sensitive polypeptide sequence, and its exemplary structure is: (amino acids from positions 65 to 131 of the succinic acid-sensitive polypeptide sequence)-(optically active polypeptide)-(amino acids from positions 126 to 320 of the succinic acid-sensitive polypeptide sequence).

[0080] In one or more embodiments, the optical probe comprises, from N-terminus to C-terminus, residues 65-X of SEQ ID NO: 1, an optically active polypeptide or a variant thereof as shown in any one of SEQ ID NO: 2-9, and residues Y-320 of SEQ ID NO: 1, wherein X and Y are selected from any of the following groups:

[0081] (1) X is 108, Y is 109,

[0082] (2) X is 108, Y is 110,

[0083] (3) X is 108, Y is 111,

[0084] (4) X is 108, Y is 112,

[0085] (5) X is 109, Y is 110,

[0086] (6) X is 109, Y is 111,

[0087] (7) X is 109, Y is 112,

[0088] (8) X is 110, Y is 111,

[0089] (9) X is 110, Y is 112,

[0090] (10) X is 111, Y is 112,

[0091] (11) X is 181, Y is 182,

[0092] (12) X is 181, Y is 183,

[0093] (13) X is 181, Y is 184,

[0094] (14) X is 181, Y is 185,

[0095] (15)X is 182,Y is 183,

[0096] (16) X is 182, Y is 184,

[0097] (17) X is 182, Y is 185,

[0098] (18) X is 183, Y is 184,

[0099] (19) X is 183, Y is 185,

[0100] (20)X is 184,Y is 185,

[0101] (21) X is 196, Y is 197,

[0102] (22)X is 196,Y is 198,

[0103] (23) X is 196, Y is 199,

[0104] (24)X is 196,Y is 200,

[0105] (25)X is 196,Y is 201,

[0106] (26)X is 197,Y is 198,

[0107] (27)X is 197,Y is 199,

[0108] (28)X is 197,Y is 200,

[0109] (29)X is 197,Y is 201,

[0110] (30)X is 198,Y is 199,

[0111] (31)X is 198,Y is 200,

[0112] (32)X is 198,Y is 201,

[0113] (33)X is 199,Y is 200,

[0114] (34)X is 199,Y is 201,

[0115] (35)X is 200,Y is 201,

[0116] (36)X is 148,Y is 149,

[0117] (37)X is 148,Y is 150,

[0118] (38)X is 148,Y is 151,

[0119] (39)X is 148,Y is 152,

[0120] (40)X is 148,Y is 153,

[0121] (41)X is 148,Y is 154,

[0122] (42)X is 148,Y is 155,

[0123] (43)X is 148,Y is 156,

[0124] (44)X is 149,Y is 150,

[0125] (45)X is 149,Y is 151,

[0126] (46)X is 149,Y is 152,

[0127] (47)X is 149,Y is 153,

[0128] (48)X is 149,Y is 154,

[0129] (49)X is 149,Y is 155,

[0130] (50)X is 149,Y is 156,

[0131] (51)X is 150,Y is 151,

[0132] (52)X is 150,Y is 152,

[0133] (53)X is 150,Y is 153,

[0134] (54)X is 150,Y is 154,

[0135] (55)X is 150,Y is 155,

[0136] (56)X is 150,Y is 156,

[0137] (57)X is 151,Y is 152,

[0138] (58)X is 151,Y is 153,

[0139] (59)X is 151,Y is 154,

[0140] (60)X is 151,Y is 155,

[0141] (61)X is 151,Y is 156,

[0142] (62)X is 152,Y is 151,

[0143] (63)X is 152,Y is 152,

[0144] (64)X is 152,Y is 153,

[0145] (65)X is 152,Y is 154,

[0146] (66)X is 152,Y is 155,

[0147] (67)X is 152,Y is 156,

[0148] (68)X is 153,Y is 151,

[0149] (69)X is 153,Y is 152,

[0150] (70)X is 153,Y is 153,

[0151] (71)X is 153,Y is 154,

[0152] (72)X is 153,Y is 155,

[0153] (73)X is 153,Y is 156,

[0154] (74)X is 154,Y is 151,

[0155] (75)X is 154,Y is 152,

[0156] (76)X is 154,Y is 153,

[0157] (77)X is 154,Y is 154,

[0158] (78)X is 154,Y is 155,

[0159] (79)X is 154,Y is 156,

[0160] (80)X is 155,Y is 156.

[0161] Preferably, the optically active polypeptide is located at 150 / 152, 150 / 153, 150 / 155, 150 / 156, 151 / 153, 152 / 152, 152 / 153, 152 / 154, 153 / 152, 153 / 153, 153 / 154, 154 / 151, 154 / 152, 154 / 154, 181 / 182 of the amino acid sequence of the succinate binding protein as shown in SEQ ID NO: 10-24.

[0162] When referring to a certain polypeptide or protein, the term "variant" or "mutant" used in the present invention includes variants having the same function of the polypeptide or protein but different sequences. Variants of polypeptides or proteins may include: homologous sequences, conservative variants, allelic variants, natural mutants, induced mutants. These variants include but are not limited to: deletion, insertion and / or substitution of one or more (usually 1-30, preferably 1-20, more preferably 1-10, and most preferably 1-5) amino acids in the sequence of the polypeptide or protein, and sequences obtained by adding one or more (usually within 20, preferably within 10, and more preferably within 5) amino acids to its carboxyl terminal and / or amino terminal. Without wishing to be limited by theory, changes in amino acid residues without changing the overall configuration and function of the polypeptide or protein are functional conservative mutations. For example, in the art, when amino acids with similar or similar properties are substituted, the function of the polypeptide or protein is generally not changed. In the art, amino acids with similar properties often refer to amino acid families with similar side chains, which have been clearly defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), amino acids with acidic side chains (e.g., aspartic acid, glutamic acid), amino acids with uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), amino acids with non-polar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, succinic acid), amino acids with β-branched side chains (e.g., threonine, valine, isoleucine), and amino acids with aromatic side chains (e.g., tyrosine, phenylalanine, succinic acid, histidine). For another example, adding one or more amino acids to the amino and / or carboxyl termini generally does not change the function of a polypeptide or protein. Conservative amino acid substitutions for many common known non-genetically encoded amino acids are known in the art. Conservative substitutions for other non-encoded amino acids can be determined based on a comparison of their physical properties with the properties of the genetically encoded amino acids. It is well known to those skilled in the art that in gene cloning operations, it is often necessary to design suitable restriction sites, which will inevitably introduce one or more irrelevant residues at the end of the expressed polypeptide or protein, but this does not affect the activity of the target polypeptide or protein. For example, in order to construct fusion proteins, promote the expression of recombinant proteins, obtain recombinant proteins that are automatically secreted outside the host cell, or facilitate the purification of recombinant proteins, it is often necessary to add some amino acids to the N-terminus, C-terminus or other suitable regions within the protein of the recombinant protein, for example, including but not limited to, suitable linker peptides, signal peptides, leader peptides, terminal extensions, glutathione S-transferase (GST), maltose E binding protein, protein A, tags such as 6His or Flag, or proteolytic enzyme sites of factor Xa or thrombin or enterokinase. Variants of polypeptides or proteins may include: homologous sequences, conservative variants, allelic variants, natural mutants, and induced mutants.These variants may also comprise a polypeptide or protein having a sequence identity of 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% with the polypeptide or protein. Exemplary DctBp protein truncated variants are fragments of SEQ ID NO:1 comprising amino acids 65-320, which retain the binding function of DctBp protein to succinic acid and do not affect the changes in optical properties of the inserted optically active polypeptide in response to succinic acid binding.

[0163] The optical probe of the present invention may include a succinate-sensitive polypeptide with a mutation. A succinate-binding protein variant with a mutation at a site selected from the following of SEQ ID NO: 1 or its truncated variant exhibits a binding activity different from succinate: S154, F155, Y160, N182, V183, S184. The amino acid mutation includes modification, substitution or deletion of an amino acid. In a preferred embodiment, the mutation of the succinate-binding protein variant includes a mutation at a site selected from any of the following groups: (1) F155 and Y160, (2) Y160 and N182, (3) Y160, N182 and V183, (4) Y160, N182 and S184, (5) S154, Y160, N182 and S184.

[0164] Wherein, as an example in the embodiments, in SEQ ID NO: 1 or a truncated variant thereof, S154 is mutated to R. In one or more embodiments, F155 is mutated to T, P, Q or C. In one or more embodiments, Y160 is mutated to Y or W. In one or more embodiments, N182 is mutated to S. In one or more embodiments, V183 is mutated to Y or W. In one or more embodiments, S184 is mutated to L.

[0165] In one or more embodiments, the mutation comprises a mutation selected from any one of the following groups: (1) F155T and Y160W, (2) F155P and Y160W, (3) F155Q and Y160W, (4) F155C and Y160W, (5) Y160W and N182S, (6) Y160W, N182S and V183Y, (7) Y160W, N182S and V183W, (8) Y160W, N182S and S184L, (9) S154R, Y160W, N182S and S184L. The present invention provides succinate binding protein variants having these mutations and optical probes comprising such succinate binding protein variants as succinate sensitive polypeptides.

[0166] The optical probe of the present invention may include an optically active polypeptide with a mutation. In some embodiments, the mutated optically active polypeptide has a sequence as shown in SEQ ID NO: 2 and has a mutation at one, two, three, four, five or six of the following sites: M9, S132, Y141, F201, N207, Y245, wherein the mutation includes a modification, substitution or deletion of an amino acid, and in one or more embodiments, the mutation includes a mutation at any of the following sites: M9, S132, Y141, F201, N207, Y245. Specifically, the mutation is M9T, S132R, Y141N, F201S, N207T and Y245F;

[0167] In one or more embodiments, the optical probe comprises any one of the amino acid sequences SEQ ID NO: 10-31 or a variant thereof. In one or more embodiments, the optical probe provided by the present invention comprises a sequence having 35%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99% sequence identity with any one of the amino acid sequences SEQ ID NO: 10-31. In a preferred embodiment, the optical probe provided by the present invention comprises a sequence substantially similar or identical to any one of the amino acid sequences SEQ ID NO: 10-31. Preferably, the optical probe has a sequence as shown in SEQ ID NO: 25-31; more preferably, the optical probe has a sequence as shown in SEQ ID NO: 29-31.

[0168] In some specific embodiments, the succinate-sensitive polypeptide in the optical probe is as shown in amino acids 65-320 of SEQ ID NO:1, the optically active polypeptide is as shown in SEQ ID NO:2, the optically active polypeptide is located at position 150 / 153 of the succinate-sensitive polypeptide, and the succinate-sensitive polypeptide has a mutation selected from any one of the following: (1) F155T and Y160W, (2) F155P and Y160W, (3) F155Q and Y160W, (4) F155C and Y160W, (5) Y160W and N182S, (6) Y160W, N182S and V183Y, (7) Y160W, N182S and V183W, (8) Y160W, N182S and S184L, (9) S154R, Y160W, N182S and S184L; or the optical probe has a mutation selected from any one of the following: (10) Y160W, N182S and S184L of a succinate-sensitive polypeptide, and M9T, S132R, Y141N, F201S, N207T and Y245F of an optically active polypeptide; (11) S154R, Y160W, N182S and S184L of a succinate-sensitive polypeptide, and M9T, S132R, Y141N, F201S, N207T and Y245F of an optically active polypeptide.

[0169] Illustratively, the amino acid sequence of the optical probe shown in items (1)-(4) is shown in SEQ ID NO:25; the amino acid sequence of the optical probe shown in item (5) is shown in SEQ ID NO:26; the amino acid sequence of the optical probe shown in items (6)-(7) is shown in SEQ ID NO:27; the amino acid sequence of the optical probe shown in item (8) is shown in SEQ ID NO:28; the amino acid sequence of the optical probe shown in item (9) is shown in SEQ ID NO:29; the amino acid sequence of the optical probe shown in item (10) is shown in SEQ ID NO:30; the amino acid sequence of the optical probe shown in item (11) is shown in SEQ ID NO:31.

[0170] In the context of 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 identical or wherein a certain percentage of amino acid residues or nucleotides are identical over a specified region (e.g., 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical) when compared and aligned for maximum correspondence over a comparison window or specified region using methods known in the art, such as sequence comparison algorithms, by manual alignment and visual inspection. For example, preferred algorithms suitable for determining percent sequence identity and percent sequence similarity are the BLAST and BLAST 2.0 algorithms, as described in Altschul et al. (1977) Nucleic Acids Res. 25:3389 and Altschul et al. (1990) J. Mol. Biol. 215:403, respectively.

[0171] It is well known to those skilled in the art that in gene cloning operations, it is often necessary to design suitable restriction sites, which will inevitably introduce one or more irrelevant residues at the end of the expressed polypeptide or protein, but this does not affect the activity of the target polypeptide or protein. For example, in order to construct fusion proteins, promote the expression of recombinant proteins, obtain recombinant proteins that are automatically secreted outside the host cell, or facilitate the purification of recombinant proteins, it is often necessary to add some amino acids to the N-terminus, C-terminus or other suitable regions in the protein of the recombinant protein, for example, including but not limited to, suitable linker peptides, signal peptides, leader peptides, terminal extensions, glutathione S-transferase (GST), maltose E binding protein, protein A, tags such as 6His or Flag, or proteolytic enzyme sites of factor Xa or thrombin or enterokinase.

[0172] The terms "functional variant", "derivative" and "analog" as used herein refer to proteins that substantially retain the same biological function or activity as the original polypeptide or protein (e.g., DctBp protein or fluorescent protein). Functional variants, derivatives or analogs of the polypeptide or protein (e.g., DctBp protein or fluorescent protein) of the present invention may be (i) proteins in which one or more conservative or non-conservative amino acid residues (preferably conservative amino acid residues) are substituted, and such substituted amino acid residues may or may not be encoded by the genetic code, or (ii) proteins having substitution groups in one or more amino acid residues, or (iii) proteins 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) proteins formed by fusion of an additional amino acid sequence to this protein sequence (e.g., a secretory sequence or a sequence or proprotein sequence used to purify the protein, or a fusion protein formed with an antigen IgG fragment). According to the teachings herein, these functional variants, derivatives and analogs belong to the well-known scope of those skilled in the art.

[0173] The difference between the analog and the original polypeptide or protein can be a difference in the amino acid sequence, or a difference in the form of modification that does not affect the sequence, or both. These proteins include natural or induced genetic variants. Induced variants can be obtained by various techniques, such as random mutagenesis by radiation or exposure to mutagens, and can also be obtained by site-directed mutagenesis or other known molecular biological techniques.

[0174] The analogs also include analogs with residues different from natural L-amino acids (such as D-amino acids), and analogs with non-natural or synthetic amino acids (such as β, γ-amino acids). It should be understood that the succinic acid sensitive polypeptide of the present invention is not limited to the representative proteins, variants, derivatives and analogs listed above. Modifications (usually without changing the primary structure) include: chemical derivatization forms of proteins in vivo or in vitro such as acetylation or carboxylation. Modifications also include glycosylation, such as those produced by glycosylation modification during protein synthesis and processing or in further processing steps. This modification can be accomplished by exposing the protein to an enzyme that performs glycosylation (such as a mammalian glycosylase or deglycosylation enzyme). Modified forms also include sequences with phosphorylated amino acid residues (such as phosphotyrosine, phosphoserine, phosphothreonine). It also includes proteins that have been modified to improve their anti-proteolytic properties or optimize solubility properties.

[0175] The fusion polypeptide of the present invention comprises the optical probe described herein and other polypeptides. In some embodiments, the optical probe described herein further comprises other polypeptides fused thereto. The other polypeptides described herein do not affect the properties of the optical probe. The other polypeptides may be located at the N-terminus and / or C-terminus of the optical probe. In some embodiments, the other polypeptides include polypeptides that locate the optical probe to different organelles or sub-organelles, tags for purification, or tags for immunoblotting. A linker may be provided between the optical probe and other polypeptides in the fusion polypeptide described herein.

[0176] The subcellular organelles described herein include cytoplasm, mitochondria, nucleus, endoplasmic reticulum, cell membrane, Golgi apparatus, lysosome and peroxisome, etc. In some embodiments, tags for purification or tags for immunoblotting include 6*histidine (6*His), glutathione S-transferase (GST), Flag.

[0177] The present invention also provides a method for preparing the above-mentioned succinic acid optical probe, comprising the following steps: 1) incorporating the nucleic acid sequence encoding the succinic acid optical probe described herein into an expression vector; 2) transferring the expression vector into a host cell; 2) culturing the host cell under conditions suitable for the expression of the expression vector; 3) isolating the succinic acid optical probe.

[0178] The term "nucleic acid" or "nucleotide" used in the present invention can be in the form of DNA or RNA. DNA forms include cDNA, genomic DNA or artificially synthesized DNA. DNA can be single-stranded or double-stranded. DNA can be a coding strand or a non-coding strand. When referring to nucleic acids, the term "variant" used herein can be a naturally occurring allelic variant or a non-naturally occurring variant. These nucleotide variants include degenerate variants, substitution variants, deletion variants and insertion variants. As known in the art, an allelic variant is an alternative form of a nucleic acid, which may be a substitution, deletion or insertion of one or more nucleotides, but will not substantially change the function of the protein encoded by it. The nucleic acid of the present invention may contain a nucleotide sequence having a sequence identity of 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% with the nucleic acid sequence. The present invention also relates to nucleic acid fragments hybridized with the above-mentioned sequences. As used herein, a "nucleic acid fragment" has a length of at least 15 nucleotides, preferably at least 30 nucleotides, more preferably at least 50 nucleotides, and most preferably at least 100 nucleotides. Nucleic acid fragments can be used in nucleic acid amplification techniques (such as PCR).

[0179] The full-length sequence or fragments of the optical probe or fusion protein of the present invention can usually be obtained by PCR amplification, artificial synthesis or recombination. For PCR amplification, primers can be designed according to the nucleotide sequence disclosed in the present invention, and a commercially available cDNA library or a cDNA library prepared by conventional methods known to those skilled in the art can be used as a template to amplify the relevant sequence. When the nucleotide sequence is greater than 2500 bp, 2 to 6 PCR amplifications are preferably performed, and then the fragments amplified in each time are spliced ​​together in the correct order. The present invention does not specifically limit the PCR amplification procedure and system, and the conventional PCR amplification procedure and system in the art can be used. Recombination methods can also be used to obtain the relevant sequence in large quantities. This is usually cloned into a vector, then transferred into cells, and then separated and purified from the host cells after proliferation by conventional methods to obtain the relevant polypeptide or protein. In addition, artificial synthesis methods can also be used to synthesize the relevant sequence, especially when the fragment length is short. In the present invention, when the nucleotide sequence of the optical probe is less than 2500 bp, an artificial synthesis method can be used for synthesis. The artificial synthesis method is a conventional DNA artificial synthesis method in the art, and there are no other special requirements. Usually, by synthesizing a plurality of small fragments first and then connecting them, a very long sequence can be obtained. At present, the DNA sequence encoding the protein of the present invention (or its functional variant, derivative or analog) can be obtained completely by chemical synthesis. The DNA sequence can then be introduced into various existing DNA molecules (such as vectors) and cells known in the art. Mutations can be introduced into the protein sequence of the present invention by methods such as mutation PCR or chemical synthesis.

[0180] After obtaining the nucleotide sequence encoding the optical probe, the present invention incorporates the nucleotide sequence encoding the optical probe into an expression vector to obtain a recombinant expression vector. The terms "expression vector" and "recombinant vector" used herein are interchangeable and refer to prokaryotic or eukaryotic vectors well known in the art, such as bacterial plasmids, bacteriophages, yeast plasmids, plant cell viruses, mammalian cell viruses such as adenoviruses, retroviruses or other vectors, which can replicate and stably express in the host. An important feature of these recombinant vectors is that they usually contain expression control sequences. The term "expression control sequence" used herein refers to an element that can be operably connected to the target gene to regulate the transcription, translation and expression of the target gene, which can be a replication origin, a promoter, a marker gene or a translation control element, including an enhancer, an operator, a terminator, a ribosome binding site, etc. The selection of the expression control sequence depends on the host cell used. Recombinant vectors applicable to the present invention include but are not limited to bacterial plasmids. In the recombinant expression vector, "operable connection" refers to the connection of the target nucleotide sequence with the regulatory sequence in a manner that allows the expression of the nucleotide sequence. Those skilled in the art are familiar with methods that can be used to construct expression vectors containing the coding sequence of the fusion protein of the present invention and appropriate transcription / translation control signals. These methods include in vitro recombinant DNA technology, DNA synthesis technology, in vivo recombination technology, etc. The DNA sequence can be effectively connected to an appropriate promoter in an expression vector to guide mRNA synthesis. Representative examples of these promoters include: lac or trp promoters of Escherichia coli; λ phage PL promoter; eukaryotic promoters include CMV immediate early promoter, HSV thymidine kinase promoter, early and late SV40 promoter, LTR of retrovirus and other known promoters that can control gene expression in prokaryotic or eukaryotic cells or their viruses. The expression vector also includes a ribosome binding site and a transcription terminator for translation initiation. In one or more embodiments, the expression vector can use a commercially available pCDF vector without other special requirements. Exemplarily, HindIII and XhoI are used to perform double enzyme digestion on the nucleotide sequence encoding the optical probe and the expression vector, respectively, and then the enzyme digestion products of the two are connected to obtain a recombinant expression vector. The present invention does not specifically limit the specific steps and parameters of enzyme digestion and connection, and the conventional steps and parameters in the art can be used.

[0181] After obtaining the recombinant expression vector, the vector is transformed into a host cell to produce a protein or peptide including a fusion protein. This transfer process can be carried out using conventional techniques known to those skilled in the art such as transformation or transfection. The host cell of the present invention refers to a cell that can receive and accommodate a recombinant DNA molecule, and is the site of recombinant gene amplification. The ideal recipient cell should meet the two conditions of easy acquisition and proliferation. The "host cell" of the present invention may include prokaryotic cells and eukaryotic cells, specifically including bacterial cells, yeast cells, insect cells and mammalian cells. Specifically, it can be Escherichia coli, Streptomyces, bacterial cells of Salmonella typhimurium, fungal cells such as yeast, plant cells, insect cells of Drosophila S2 or Sf9, CHO, COS, HEK293, HEK293 cells, or animal cells of Bowes melanoma cells, etc., including but not limited to those host cells mentioned above. The host cell is preferably a variety of cells that are conducive to gene product expression or fermentation production, and such cells are well known and commonly used in the art. The exemplary host cell used in the embodiment of the present invention is the Escherichia coli BL21-DE3 strain. Those skilled in the art will appreciate how to select appropriate vectors, promoters, enhancers and host cells.

[0182] The method of transferring to the host cell described in the present invention is a conventional method in the art, including calcium phosphate or calcium chloride coprecipitation, DEAE-mannan-mediated transfection, lipofection, natural competence, chemical-mediated transfer or electroporation. When the host is a prokaryotic organism such as Escherichia coli, the method is preferably treated with CaCl2 or MgCl2, and the steps used are well known in the art. When the host cell is a eukaryotic cell, the following DNA transfection methods can be selected: calcium phosphate coprecipitation, conventional mechanical methods such as microinjection, electroporation, liposome packaging, etc.

[0183] After the expression vector is transferred into the host cell, the host cell into which the expression vector is transferred is cultured for amplification and expression to separate and obtain the succinic acid optical probe. The host cell amplification and expression culture can be carried out by conventional methods. Depending on the type of host cell used, the culture medium used in the culture can be various conventional culture media. The culture is carried out under conditions suitable for the growth of the host cell.

[0184] In the present invention, the optical probe is expressed in the cell, on the cell membrane, or secreted outside the cell. If necessary, the recombinant protein can be separated or purified by various separation methods using its physical, chemical and other properties. The present invention does not specifically limit the method for separating the succinate fluorescent protein, and the separation method of the fusion protein conventional in the art can be used. These methods are well known to those skilled in the art, including but not limited to: conventional renaturation treatment, salting-out method, centrifugation, osmotic breaking, ultrasonic treatment, 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 or more embodiments, the optical probe is separated using affinity chromatography using a His tag.

[0185] The present invention also provides the application of the succinic acid optical probe in real-time localization, quantitative detection and high-throughput compound screening of succinic acid. In one aspect, the succinic acid optical probe is preferably connected to the signal peptide in different parts of the cell, transferred into the cell, and the real-time localization of succinic acid is performed by detecting the strength of the fluorescent signal in the cell; the corresponding succinic acid is quantitatively detected by the succinic acid standard drop curve. The succinic acid standard drop curve of the present invention is drawn according to the fluorescent signal of the succinic acid optical probe under different concentrations of succinic acid. The succinic acid optical probe of the present invention is directly transferred into the cell, and in the process of real-time localization and quantitative detection of succinic acid, no time-consuming sample processing process is required, which is more accurate. When the succinic acid optical probe of the present invention performs high-throughput compound screening, different compounds are added to the cell culture medium, and the change of succinic acid content is determined, so as to screen out compounds that affect the change of succinic acid content. The application of the succinic acid optical probe in real-time localization, quantitative detection and high-throughput compound screening of succinic acid described in the present invention is non-diagnostic and therapeutic purposes, and does not involve the diagnosis and treatment of diseases.

[0186] In this article, concentration, content, percentage and other numerical values ​​can be expressed in the form of a range. It should also be understood that the use of this range format is only for convenience and brevity, and should be flexibly interpreted as including the numerical values ​​explicitly mentioned at the upper and lower limits of the range, and all individual numerical values ​​or sub-ranges included in the range should also be included.

[0187] Example

[0188] The succinic acid optical probe provided by the present invention is described in detail below in conjunction with the examples, but they should not be construed as limiting the protection scope of the present invention.

[0189] I. Experimental Materials and Reagents

[0190] In the embodiments, conventional genetic engineering molecular biology cloning methods, cell culture and imaging methods are mainly used. These methods are well known to ordinary technicians in the field, for example: "Molecular Biology Laboratory Reference Manual" by Jane Roskams et al., "Molecular Cloning Laboratory Guide" (3rd edition, August 2002, Science Press, Beijing) by J. Sambrook, DW Russell, translated by Huang Peitang et al.; "Animal Cell Culture: Basic Technical Guide" (5th edition) by Frasheny et al., translated by Zhang Jingbo, Xu Cunshuan et al.; "Concise Cell Biology Laboratory Guide" by JS Bonifacion, M. Dassault et al., translated by Zhang Jingbo et al.

[0191] The pCDF-cpYFP, ​​pCDF-succinic acid binding protein plasmid used in the embodiment was constructed by the Protein Laboratory of East China University of Science and Technology, and the pCDF plasmid vector was purchased from Invitrogen. All primers used for PCR were synthesized, purified and identified correctly by mass spectrometry by Shanghai Jierui Bioengineering Technology Co., Ltd. and Huada Gene. The expression plasmids constructed in the embodiment were sequenced, and the sequence determination was completed by Huada Gene and Jie Li Sequencing Company. The TaqDNA polymerase used in each embodiment was purchased from Dongsheng Biology, pfu DNA polymerase was purchased from Tiangen Biochemical Technology (Beijing) Co., Ltd., and primeSTARDNA polymerase was purchased from TaKaRa. The three polymerases were purchased with corresponding polymerase buffer and dNTPs. Restriction enzymes such as BamHI, BglII, HindIII, NdeI, XhoI, EcoRI, SpeI, T4 ligase, and T4 phosphorylase (T4PNK) were purchased from Fermentas, and corresponding buffers were provided when purchased. Transfection reagent Lip2000 Kit was purchased from Invitrogen. Compounds such as succinic acid were purchased from Sigma. Unless otherwise stated, chemical reagents such as inorganic salts were purchased from Sigma-Aldrich. HEPES salt, ampicillin (Amp) and puromycin were purchased from Ameresco. 96-well black plate and 384-well fluorescence black plate were purchased from Grenier.

[0192] The DNA purification kit used in the examples was purchased from BBI (Canada), and the common plasmid extraction kit was purchased from Tiangen Biochemical Technology (Beijing) Co., Ltd. The cloned strain Mach1 was purchased from Invitrogen. The nickel column affinity chromatography column and desalting column fillers were both from GE healthcare.

[0193] The main instruments used in the embodiments include: Biotek Synergy 2 multifunctional microplate reader (Bio-Tek, USA), X-15R high-speed refrigerated centrifuge (Beckman, USA), Microfuge22R desktop high-speed refrigerated centrifuge (Beckman, USA), PCR amplification instrument (Biometra, Germany), ultrasonic disruptor (Ningbo Xinzhi Company), nucleic acid electrophoresis instrument (Shenneng Gaming Company), fluorescence spectrophotometer (Varian, USA), CO2 constant temperature cell culture incubator (SANYO), inverted fluorescence microscope (Nikon, Japan).

[0194] II. Molecular Biology Methods and Cell Experimental Methods

[0195] II.1 Polymerase chain reaction (PCR):

[0196] 1. Target fragment amplification PCR:

[0197] This method is mainly used for gene fragment amplification and colony PCR identification of positive clones. The reaction system of PCR amplification is as follows: template sequence 0.5-1μL, forward primer (25μM) 0.5μL, reverse primer (25μM) 0.5μL, 10×pfu buffer 5μL, pfu DNA polymerase 0.5μL, dNTP (10mM) 1μL, sterilized ultrapure water (ddH2O) 41.5-42μL, total volume 50μL. The PCR amplification procedure is as follows: 95℃ denaturation for 2-10 minutes, 30 cycles (94-96℃ for 30-45 seconds, 50-65℃ for 30-45 seconds, 72℃ for a certain time (600bp / min)), 72℃ extension for 10 minutes.

[0198] 2. PCR amplification of long fragments (>2500bp):

[0199] The long-fragment amplification used in the present invention is mainly a reverse PCR amplification vector, which is a technique used to obtain site-directed mutations in the following examples. Reverse PCR primers are designed at the mutation site, and the 5' end of one of the primers contains the mutated nucleotide sequence. The amplified product contains the corresponding mutation site. The long-fragment amplification PCR reaction system is as follows: template sequence (10pg-1ng) 1μL, forward primer (25μM) 0.5μL, reverse primer (25μM) 0.5μL, 5×PrimerSTAR buffer 10μL, PrimerSTAR DNA polymerase 0.5μL, dNTP (2.5mM) 4μL, sterile ultrapure water (ddH2O) 33.5μL, total volume 50μL. The PCR amplification program was as follows: denaturation at 95°C for 5 min, 30 cycles (98°C for 10 s, 50-68°C for 5-15 s, 72°C for a certain time (1000 bp / min)), and extension at 72°C for 10 min; or denaturation at 95°C for 5 min, 30 cycles (98°C for 10 s, 68°C for a certain time (1000 bp / min)), and extension at 72°C for 10 min.

[0200] II.2 Endonuclease digestion reaction:

[0201] The system for double enzyme digestion of plasmid vector is as follows: 20 μL of plasmid vector (about 1.5 μg), 5 μL of 10× buffer, 1-2 μL of restriction enzyme 1, 1-2 μL of restriction enzyme 2, and the total volume is made up to 50 μL with sterile ultrapure water. The reaction conditions are 37°C and 1-7 hours.

[0202] II.3 DNA fragment 5' end phosphorylation reaction

[0203] The ends of plasmids or genomes extracted from microorganisms contain phosphate groups, but PCR products do not. Therefore, the 5' end base of the PCR product needs to be subjected to a phosphate group addition reaction. Only DNA molecules with phosphate groups at the end can undergo a ligation reaction. The phosphorylation reaction system is as follows: 5-8μL of PCR product fragment DNA sequence, 1μL of 10×T4 ligase buffer, 1μL of T4 polynucleotide kinase (T4 PNK), 0-3μL of sterile ultrapure water, and a total volume of 10μL. The reaction conditions are 37℃, and inactivation at 72℃ for 20 minutes after 30 minutes-2 hours.

[0204] II.4 Ligation reaction between target fragment and vector

[0205] The connection methods between different fragments and vectors are different. Three connection methods are used in the present invention:

[0206] 1. Blunt-end ligation of blunt-end short fragments and linearized vector

[0207] The principle of this method is that the flat-end product obtained by PCR is phosphorylated at the 5' end of the DNA fragment under the action of T4 PNK, and then connected with the linearized vector under the action of PEG4000 and T4 DNA ligase to obtain a recombinant plasmid. The homologous recombination connection system is as follows: 4μL of DNA fragment treated with T4 PNK, 4μL of linearized vector fragment, 1μL of PEG4000, 1μL of 10×T4 ligase buffer, 1μL of T4 DNA ligase, a total of 10μL. The reaction conditions are 22℃, 30 minutes.

[0208] 2. Ligation of DNA fragments with sticky ends and vector fragments with sticky ends

[0209] DNA fragments cut by restriction endonucleases usually produce protruding sticky ends, so they can be connected to vector fragments containing complementary sticky ends to form recombinant plasmids. The ligation reaction system is as follows: 1-7μL of PCR product DNA after restriction digestion, 0.5-7μL of plasmid after restriction digestion, 1μL of 10×T4 ligase buffer, 1μL of T4DNA ligase, and sterile ultrapure water to a total volume of 10μL. Reaction conditions 16℃, 4-8 hours.

[0210] 3. The ligation reaction of the 5' phosphorylated DNA fragment product after site-directed mutation introduced by inverse PCR and self-circularization

[0211] The 5'-end phosphorylated DNA fragment was connected to the 3' and 5' ends of the linearized vector by self-circularization ligation to obtain a recombinant plasmid. The self-circularization ligation reaction system is as follows: phosphorylation reaction system 10μL, T4 ligase (5U / μL) 0.5μL, total volume 10.5μL. Reaction conditions 16℃, 4-16 hours.

[0212] II.5 Preparation and transformation of competent cells

[0213] Preparation of competent cells:

[0214] 1. Pick a single colony (such as Mach1) and inoculate it into 5 mL LB medium and shake it at 37°C overnight.

[0215] 2. Transfer 0.5-1 mL of overnight culture solution into 50 mL LB medium and culture at 37°C, 220 rpm for 3 to 5 hours until OD 600 Reach 0.5.

[0216] 3. Pre-cool the cells in an ice bath for 2 hours.

[0217] Centrifuge at 4000 rpm for 10 min at 4.4°C.

[0218] 5. Discard the supernatant and resuspend the cells with 5 mL of pre-cooled buffer. After the cells are homogenized, add resuspension buffer to a final volume of 50 mL.

[0219] 6. Ice bath for 45 minutes.

[0220] Centrifuge at 4000 rpm for 10 min at 7.4°C and resuspend the bacteria in 5 mL of ice-cold storage buffer.

[0221] 8. Place 100 μL of bacterial solution in each EP tube and freeze at -80°C or in liquid nitrogen.

[0222] Resuspension buffer: CaCl2 (100mM), MgCl2 (70mM), NaAc (40mM)

[0223] Storage buffer: 0.5 mL DMSO, 1.9 mL 80% glycerol, 1 mL 10×CaCl2 (1 M), 1 mL 10×MgCl2 (700 mM), 1 mL 10×NaAc (400 mM), 4.6 mL ddH2O

[0224] Transformation of competent cells:

[0225] 1. Take 100 μL of competent cells and thaw them in an ice bath.

[0226] 2. Add an appropriate volume of ligation product, gently pipette to mix, and place on ice for 30 minutes. Usually, the volume of ligation product added is less than 1 / 10 of the volume of competent cells.

[0227] 3. Heat shock the bacterial solution in a 42°C water bath for 90 seconds, then quickly transfer to an ice bath for 5 minutes.

[0228] 4. Add 500 μL LB and culture at 37°C on a constant temperature shaker at 200 rpm for 1 hour.

[0229] 5. Centrifuge the bacterial solution at 4000 rpm for 3 minutes, retain 200 μL of supernatant, blow the bacteria evenly, and evenly spread them on the surface of an agar plate containing appropriate antibiotics. Place the plate upside down in a 37°C constant temperature incubator overnight.

[0230] II.6 Protein expression, purification and fluorescence detection

[0231] 1. Transform the expression vector (e.g., the pCDF-based succinate optical probe expression vector) into BL21 (DE3) cells, invert and culture overnight, pick clones from the plate into a 250 ml conical flask, place it in a 37°C shaker, culture at 220 rpm until OD = 0.4-0.8, add 1 / 1000 (v / v) IPTG (1 M), and induce expression at 18°C ​​for 24-36 hours.

[0232] 2. After induction, centrifuge at 4000 rpm for 30 minutes to collect the bacteria, add 50 mM phosphate buffer to resuspend the bacterial precipitate, and ultrasonically disrupt the bacteria until they are clear. Centrifuge at 9600 rpm at 4°C for 20 minutes.

[0233] 3. The centrifuged supernatant was purified by a self-assembled nickel column affinity chromatography column to obtain the protein, and the protein after the nickel column affinity chromatography was then passed through a self-assembled desalting column to obtain the protein dissolved in 100 mM HEPES buffer (pH 7.4).

[0234] 4. After the purified protein was identified by SDS-PAGE, the probe was diluted into a protein solution with a final concentration of 0.2-5 μM using an assay buffer (100 mM HEPES, 100 mM NaCl, pH 7.4). Succinic acid was prepared into a stock solution with a final concentration of 50 mM using an assay buffer (100 mM HEPES, 100 mM NaCl, pH 7.4).

[0235] 5. Take 100 μl of 1 μM protein solution, incubate at 37°C for 10 minutes, add succinic acid for titration, and measure the fluorescence intensity of the protein at 528 nm after 420 nm light excitation and 528 nm after 485 nm light excitation. The fluorescence excitation and emission of the sample are measured using a multifunctional fluorescence microplate reader.

[0236] 6. Take 100 μl of 1 μM protein solution, incubate at 37°C for 10 minutes, add succinic acid, and measure the absorption spectrum and fluorescence spectrum of the protein. The absorption spectrum and fluorescence spectrum of the sample are measured by a spectrophotometer and a fluorescence spectrophotometer.

[0237] II.7 Transfection of mammalian cells and fluorescence detection

[0238] 1. The pCDNA3.1+-based succinic acid optical probe plasmid was transfected into HEK293 cells using the transfection reagent Lipofectamine 2000 (Invitrogen) and cultured in a cell culture incubator at 37°C and 5% CO2. Fluorescence detection was performed after the exogenous gene was fully expressed for 24 to 36 hours.

[0239] 2. After the induction of expression was completed, the attached HEK293 cells were rinsed three times with PBS and placed in HBSS solution for detection by fluorescence microscopy and microplate reader.

[0240] Example 1: Succinate Binding Protein Particles

[0241] The DctBp (65-320) gene in the Escherichia coli gene was amplified by PCR. The PCR product was recovered after gel electrophoresis and digested with BamHI and XhoI. The pCDF vector was double-digested at the same time. After ligation with T4 DNA ligase, the product was used to transform DH5α. The transformed DH5α was coated on an LB plate (streptomycin 100 μg / mL) and cultured at 37°C overnight. The grown DH5α transformants were subjected to plasmid extraction and PCR identification. The positive plasmid was sequenced correctly for subsequent plasmid construction.

[0242] Example 2: Expression and detection of cpYFP optical probes at different insertion sites

[0243] In this example, the following sites were selected based on pCDF-DctBp to insert cpYFP to obtain the corresponding pCDF-DctBp-cpYFP plasmids: 108 / 109, 108 / 110, 108 / 111, 108 / 112, 109 / 110, 109 / 111, 109 / 112, 110 / 111, 110 / 112, 111 / 112, 181 / 182, 181 / 183, 181 / 184, 181 / 185, 182 / 1 83, 182 / 184, 182 / 185, 183 / 184, 183 / 185, 184 / 185, 196 / 197, 196 / 198, 196 / 199, 196 / 200, 196 / 201, 197 / 198, 197 / 199, 197 / 200, 197 / 201, 198 / 199, 198 / 200, 198 / 201, 199 / 200, 199 / 201, 200 / 201, 148 / 149, 148 / 150, 148 / 151, 148 / 152, 148 / 153, 148 / 154, 148 / 155, 148 / 156, 149 / 150, 149 / 151, 149 / 152, 149 / 153, 149 / 154, 149 / 155, 149 / 156, 150 / 151, 150 / 152, 150 / 153, 150 / 154, 150 / 155, 150 / 156, 151 / 152, 151 / 153, 1 151 / 154, 151 / 155, 151 / 156, 152 / 151, 152 / 152, 152 / 153, 152 / 154, 152 / 155, 152 / 156, 153 / 151, 153 / 152, 153 / 153, 153 / 154, 153 / 155, 153 / 156, 154 / 151, 154 / 152, 154 / 153, 154 / 154, 154 / 155, 154 / 156, 155 / 156.

[0244] The DNA fragment of cpYFP and the linearized vector pCDF-DctBp were amplified by PCR technology, and the 5' and 3' ends of the vector respectively carried sequences that were completely consistent with the two ends of cpYFP (15bp to 20bp). The linearized pCDF-DctBp and cpYFP fragments were homologously recombined under the action of Hieff Clone Enzyme. The product was transformed into DH5α, and the transformed DH5α was spread on LB plates (streptomycin 100ug / mL) and cultured at 37℃ overnight. The positive clones identified by PCR were sequenced after plasmid extraction. Sequencing was completed by Jerry Sequencing Company.

[0245] After the sequencing was correct, the recombinant plasmid was transformed into BL21 (DE3) to induce expression and purify the protein. The size of the protein was around 57Kda by SDS-PAGE electrophoresis. This size is consistent with the size of the DctBp-cpYFP fusion protein containing the His-tag purification tag expressed by pCDF-DctBp-cpYFP. The results are as follows Figure 1 shown.

[0246] The supernatant of E. coli expressing DctBp-cpYFP fusion protein was used for succinic acid response screening, and the detection signal of the fusion fluorescent protein containing succinic acid was divided by the detection signal of the fusion fluorescent protein without succinic acid. The results are shown in Table 1. The detection results show:

[0247] Optical probes with responses to succinate greater than 1.1-fold or less than 0.9-fold are: 108 / 110, 108 / 111, 108 / 112, 181 / 182, 181 / 185, 182 / 183, 182 / 185, 183 / 184, 148 / 150, 148 / 153, 148 / 155, 149 / 150, 149 / 151, 149 / 152, 149 / 154, 150 / 152, 150 / 153, 150 / 154 、150 / 155、150 / 156、151 / 152、151 / 153、151 / 154、151 / 156、152 / 151、152 / 152、152 / 153、152 / 154、152 / 156、153 / 152、153 / 153、153 / 154、153 / 156、154 / 151、154 / 152、154 / 153、154 / 154、154 / 155、155 / 156;

[0248] There are 15 optical probes that respond to succinate more than 2-fold, at positions 150 / 152, 150 / 153, 150 / 155, 150 / 156, 151 / 153, 152 / 152, 152 / 153, 152 / 154, 153 / 152, 153 / 153, 153 / 154, 154 / 151, 154 / 152, 154 / 154, and 181 / 182.

[0249] Table 1

[0250] Insertion site Change multiple Insertion site Change multiple Insertion site Change multiple Insertion site Change multiple Insertion site Change multiple 108 / 109 1.09 182 / 185 1.80 199 / 200 1.02 149 / 155 0.95 152 / 154 3.98 108 / 110 1.10 183 / 184 1.40 199 / 201 1.01 149 / 156 0.95 152 / 155 1.08 108 / 111 1.18 183 / 185 0.99 200 / 201 1.05 150 / 151 1.03 152 / 156 1.10 108 / 112 1.19 184 / 185 0.98 148 / 149 0.95 150 / 152 2.59 153 / 151 1.01 109 / 110 1.03 196 / 197 1.01 148 / 150 1.15 150 / 153 3.32 153 / 152 5.92 109 / 111 1.01 196 / 198 0.99 148 / 151 0.87 150 / 154 1.59 153 / 153 3.65 109 / 112 1.03 196 / 199 0.97 148 / 152 0.96 150 / 155 0.34 153 / 154 3.19 110 / 111 1.05 196 / 200 0.96 148 / 153 1.30 150 / 156 0.20 153 / 155 1.03 110 / 112 1.05 196 / 201 1.09 148 / 154 0.97 151 / 152 1.28 153 / 156 1.18 111 / 112 1.01 197 / 198 0.97 148 / 155 0.88 151 / 153 4.50 154 / 151 6.49 181 / 182 0.45 197 / 199 1.03 148 / 156 1.08 151 / 154 0.74 154 / 152 6.03 181 / 183 0.97 197 / 200 1.02 149 / 150 0.81 151 / 155 0.93 154 / 153 0.70 181 / 184 0.94 197 / 201 0.99 149 / 151 0.56 151 / 156 0.84 154 / 154 6.63 181 / 185 0.81 198 / 199 1.03 149 / 152 0.82 152 / 151 1.28 154 / 155 1.26 182 / 183 0.71 198 / 200 1.05 149 / 153 0.97 152 / 152 2.89 154 / 156 0.91 182 / 184 0.98 198 / 201 1.05 149 / 154 1.14 152 / 153 8.89 155 / 156 1.32 cpYFP 1.00

[0251] Example 3: Expression and detection of cpGFP optical probes at different insertion sites

[0252] According to the method in Example 2, cpYFP was replaced with cpGFP to construct a succinate green fluorescent protein fluorescent probe. As shown in Table 3, the detection results showed:

[0253] Optical probes with responses to succinate greater than 1.1-fold or less than 0.9-fold are: 181 / 182, 183 / 185, 184 / 185, 197 / 198, 198 / 200, 198 / 201, 199 / 200, 199 / 201, 200 / 201, 148 / 152, 148 / 154, 148 / 156, 149 / 150, 149 / 151, 149 / 156, 150 / 151, 150 / 152, 150 / 153 、150 / 155、150 / 156、151 / 152、151 / 153、151 / 154、151 / 155、151 / 156、152 / 152、152 / 153、152 / 154、152 / 155、152 / 156、153 / 151、153 / 153、153 / 155、153 / 156、154 / 151、154 / 154、154 / 155、154 / 156、155 / 156;

[0254] Optical probes that responded to succinate more than 1.5 times were located at positions 150 / 153, 151 / 152, 152 / 153, and 152 / 154.

[0255] Table 2

[0256] Insertion site Change multiple Insertion site Change multiple Insertion site Change multiple Insertion site Change multiple Insertion site Change multiple 108 / 109 0.97 182 / 185 0.97 199 / 200 1.42 149 / 155 0.97 152 / 154 2.21 108 / 110 0.99 183 / 184 0.94 199 / 201 1.29 149 / 156 1.20 152 / 155 1.45 108 / 111 1.00 183 / 185 1.18 200 / 201 1.10 150 / 151 1.24 152 / 156 1.26 108 / 112 1.01 184 / 185 1.14 148 / 149 0.89 150 / 152 1.41 153 / 151 1.39 109 / 110 1.03 196 / 197 1.00 148 / 150 1.04 150 / 153 1.89 153 / 152 1.00 109 / 111 1.01 196 / 198 1.02 148 / 151 1.07 150 / 154 1.09 153 / 153 1.41 109 / 112 1.00 196 / 199 1.00 148 / 152 1.28 150 / 155 1.39 153 / 154 1.04 110 / 111 0.97 196 / 200 0.97 148 / 153 1.04 150 / 156 1.25 153 / 155 1.44 110 / 112 0.99 196 / 201 0.99 148 / 154 1.20 151 / 152 2.01 153 / 156 1.37 111 / 112 1.00 197 / 198 0.89 148 / 155 1.07 151 / 153 1.30 154 / 151 1.29 181 / 182 1.25 197 / 199 0.98 148 / 156 1.25 151 / 154 1.32 154 / 152 1.04 181 / 183 1.09 197 / 200 1.00 149 / 150 0.89 151 / 155 1.39 154 / 153 0.99 181 / 184 1.00 197 / 201 1.04 149 / 151 1.19 151 / 156 1.41 154 / 154 1.89 181 / 185 0.96 198 / 199 1.02 149 / 152 1.08 152 / 151 1.07 154 / 155 0.85 182 / 183 1.08 198 / 200 1.20 149 / 153 1.05 152 / 152 1.24 154 / 156 1.49 182 / 184 0.93 198 / 201 1.31 149 / 154 0.98 152 / 153 2.08 155 / 156 1.28 cpGFP 1.00

[0257] Example 4: Expression and detection of cpBFP optical probes at different insertion sites

[0258] According to the method in Example 2, cpYFP was replaced with cpBFP to construct a succinate blue fluorescent protein fluorescent probe. As shown in Table 3, the detection results showed:

[0259] Optical probes with responses to succinate greater than 1.1 times or less than 0.9 times are: 108 / 109, 108 / 110, 110 / 111, 110 / 112, 181 / 185, 183 / 184, 183 / 185, 197 / 201, 200 / 201, 148 / 150, 148 / 151, 148 / 152, 148 / 153, 149 / 151, 149 / 153, 149 / 155 、150 / 151、150 / 152、150 / 155、151 / 152、151 / 153、151 / 154、151 / 156、152 / 151、152 / 152、152 / 154、152 / 155、152 / 156、153 / 151、153 / 152、153 / 153、153 / 156、154 / 151、154 / 152、155 / 156;

[0260] Optical probes that responded to succinate more than 1.3 times were located at positions 148 / 152, 148 / 153, 151 / 153, 151 / 154, 152 / 155, 152 / 156, 153 / 151, and 154 / 152.

[0261] Table 3

[0262] Insertion site Change multiple Insertion site Change multiple Insertion site Change multiple Insertion site Change multiple Insertion site Change multiple 108 / 109 1.30 182 / 185 1.02 199 / 200 1.07 149 / 155 1.29 152 / 154 1.20 108 / 110 1.23 183 / 184 1.14 199 / 201 0.93 149 / 156 1.02 152 / 155 1.39 108 / 111 1.01 183 / 185 1.29 200 / 201 0.85 150 / 151 1.30 152 / 156 1.44 108 / 112 1.04 184 / 185 1.03 148 / 149 1.03 150 / 152 1.29 153 / 151 2.04 109 / 110 0.98 196 / 197 1.05 148 / 150 1.20 150 / 153 0.96 153 / 152 1.49 109 / 111 0.99 196 / 198 0.99 148 / 151 1.19 150 / 154 0.96 153 / 153 1.24 109 / 112 0.97 196 / 199 0.96 148 / 152 1.50 150 / 155 0.81 153 / 154 1.00 110 / 111 1.21 196 / 200 1.04 148 / 153 1.42 150 / 156 1.02 153 / 155 1.04 110 / 112 1.19 196 / 201 1.07 148 / 154 1.02 151 / 152 1.30 153 / 156 1.18 111 / 112 1.03 197 / 198 0.96 148 / 155 0.98 151 / 153 1.59 154 / 151 1.30 181 / 182 1.00 197 / 199 1.04 148 / 156 1.05 151 / 154 1.83 154 / 152 1.50 181 / 183 1.05 197 / 200 1.08 149 / 150 1.06 151 / 155 0.92 154 / 153 0.92 181 / 184 1.08 197 / 201 1.10 149 / 151 0.89 151 / 156 0.83 154 / 154 1.00 181 / 185 0.89 198 / 199 1.04 149 / 152 0.92 152 / 151 0.81 154 / 155 0.93 182 / 183 0.94 198 / 200 1.06 149 / 153 0.81 152 / 152 0.82 154 / 156 1.03 182 / 184 1.02 198 / 201 0.97 149 / 154 1.04 152 / 153 0.93 155 / 156 1.19 cpBFP 1.00

[0263] Example 5: Expression and detection of cpmApple optical probes at different insertion sites

[0264] According to the method in Example 2, cpYFP was replaced with cpmApple to construct a succinate red fluorescent protein fluorescent probe. As shown in Table 4, the detection results showed:

[0265] The optical probes with responses to succinate exceeding 1.1-fold or less than 0.9-fold are: 108 / 109, 108 / 110, 110 / 111, 196 / 201, 197 / 201, 199 / 200, 200 / 201, 148 / 149, 148 / 151, 148 / 155, 149 / 152, 149 / 156, 150 / 155, 150 / 156, 151 / 156, 152 / 154, 152 / 155, 152 / 156, 153 / 151, 153 / 152, 153 / 153, 154 / 152, 154 / 153, 154 / 156;

[0266] The optical probes that responded to succinate more than 1.2 times were located at positions 152 / 154 and 153 / 153.

[0267] Table 4

[0268] Insertion site Change multiple Insertion site Change multiple Insertion site Change multiple Insertion site Change multiple Insertion site Change multiple 108 / 109 0.89 182 / 185 0.95 199 / 200 1.19 149 / 155 0.93 152 / 154 1.21 108 / 110 0.83 183 / 184 1.03 199 / 201 1.01 149 / 156 0.82 152 / 155 1.17 108 / 111 0.98 183 / 185 1.08 200 / 201 1.17 150 / 151 1.04 152 / 156 1.16 108 / 112 0.93 184 / 185 1.02 148 / 149 1.11 150 / 152 1.02 153 / 151 1.20 109 / 110 1.02 196 / 197 0.97 148 / 150 1.02 150 / 153 1.00 153 / 152 1.10 109 / 111 1.06 196 / 198 0.93 148 / 151 0.86 150 / 154 0.92 153 / 153 1.29 109 / 112 1.08 196 / 199 1.01 148 / 152 0.92 150 / 155 0.85 153 / 154 0.92 110 / 111 1.10 196 / 200 1.04 148 / 153 0.94 150 / 156 0.82 153 / 155 0.95 110 / 112 0.92 196 / 201 1.13 148 / 154 0.97 151 / 152 1.04 153 / 156 1.02 111 / 112 1.02 197 / 198 1.02 148 / 155 0.89 151 / 153 1.01 154 / 151 1.04 181 / 182 1.04 197 / 199 1.07 148 / 156 1.03 151 / 154 1.02 154 / 152 0.85 181 / 183 1.08 197 / 200 1.04 149 / 150 1.02 151 / 155 1.00 154 / 153 0.82 181 / 184 1.00 197 / 201 0.82 149 / 151 1.07 151 / 156 1.12 154 / 154 0.94 181 / 185 0.97 198 / 199 0.92 149 / 152 0.89 152 / 151 1.04 154 / 155 0.97 182 / 183 0.93 198 / 200 0.95 149 / 153 0.92 152 / 152 1.03 154 / 156 1.11 182 / 184 0.91 198 / 201 1.02 149 / 154 1.04 152 / 153 0.98 155 / 156 1.03 cpmApple 1.00

[0269] Example 6: Performance of optical probe

[0270] For the optical probes obtained in Example 2 that respond to succinic acid more than 2 times, that is, the 15 optical probes inserted at sites: 150 / 152, 150 / 153, 150 / 155, 150 / 156, 151 / 153, 152 / 152, 152 / 153, 152 / 154, 153 / 152, 153 / 153, 153 / 154, 154 / 151, 154 / 152, 154 / 154, and 181 / 182, succinic acid detection was carried out in a concentration gradient (0-100mM), and the changes in the fluorescence intensity at 420nm excitation and 528nm emission and the ratio of the fluorescence intensity at 485nm excitation and 528nm emission were detected. The K values ​​of the eight succinate optical probes with insertion sites of 150 / 152, 153 / 152, 153 / 153, 153 / 154, 154 / 151, 154 / 152, 154 / 154, and 181 / 182 were d (binding constant) is too large to be fitted and is not suitable for detection. In addition, the K values ​​of the seven succinate optical probes with insertion sites of 150 / 153, 150 / 155, 150 / 156, 151 / 153, 152 / 152, 152 / 153, and 152 / 154 are d (binding constants) were 0.7mM, 1.9mM, 3.1mM, 0.8mM, 9.3mM, 1.9mM, and 1.5mM, respectively. The results are shown in Figure 2 shown.

[0271] Example 7: Expression and detection of mutant cpYFP optical probes

[0272] Optical probe mutants were constructed based on DctBp-150 / 153-cpYFP. Plasmid pCDF-DctBp-150 / 153-cpYFP was linearized by PCR, and the primers contained the base sequences of the desired mutation sites. The obtained PCR products were homologously recombined to obtain mutant plasmids of 12 sites, including S154, F155, Y160, N182, V183, S184 of succinic acid sensitive polypeptides and M9, S132, Y141, F201, N207, and Y245 of optically active polypeptides, and the sequencing was completed by Jie Li Sequencing Company.

[0273] The successfully constructed mutant plasmid was transformed into BL21 (DE3) to induce expression, and the supernatant of E. coli expressing the probe protein was used to screen the response to succinic acid and other non-specific substrates, and the detection signal of the fusion fluorescent protein containing succinic acid or other non-specific substrates was divided by the detection signal of the fusion fluorescent protein without succinic acid. The results are shown in Table 6, and the detection results show that the optical probe with a response to succinic acid of more than 2 times and good specificity is as follows.

[0274] Table 6

[0275]

[0276] Example 8: Performance of Optical Probe Mutants

[0277] The succinic acid optical probes in Table 6 described in Example 7 were subjected to succinic acid detection at a concentration gradient (0-100 mM). After the probes were treated for 10 minutes, the changes in the fluorescence intensity at 420 nm excitation and 528 nm emission and the fluorescence intensity ratio at 485 nm excitation and 528 nm emission were detected. The probe titration results are shown in Figure 2. Figure 3 As shown, the results indicate that different mutants have different affinities for succinate.

[0278] The succinic acid probes in Table 6 were specifically tested for reactivity with succinic acid structural analogs, fumaric acid, malic acid, α-ketoglutaric acid, citric acid, aspartic acid, pyruvic acid, oxaloacetic acid, and malonic acid. The results showed that the probes had good specificity. Figure 4 shown.

[0279] Example 9: Subcellular organelle localization of optical probes and performance of optical probes within subcellular organelles

[0280] In this example, different localization signal peptides were used to fuse with the optical probe DctBp-S154R / Y160W / N182S / S184L-cpYFP-M9T / S132R / Y141N / F201S / N207 / Y245F 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, then rinsed with PBS, placed in HBSS solution, and fluorescence detection was performed under the FITC channel using an inverted fluorescence microscope. The results are shown in Figure 2. Figure 5 As shown. The succinate optical probe can be localized to the cytoplasm, mitochondria, nucleus, nuclear exclusion, outer membrane, and endoplasmic reticulum by fusing with different specific localization signal peptides. Different subcellular structures show fluorescence, and the distribution and intensity of fluorescence are different.

[0281] After transfection of HEK293 cells with the optical probe plasmid expressed in the cytoplasm for 36 hours, the cells were washed with PBS and placed in HBSS solution. The changes in the fluorescence intensity at 528 nm emission under 420 nm excitation and the fluorescence intensity ratio at 528 nm emission under 485 nm excitation were detected over a 30-min period. The results are shown in Figure 2. Figure 6 10 mM succinic acid was added and the test was continued for 30 minutes. The 485 / 420 of the sample with succinic acid added gradually increased, up to 4 times the initial value, while the 485 / 420 of the control group without succinic acid added remained basically unchanged.

[0282] Example 10: High-throughput compound screening based on optical probes in living cells

[0283] In this example, we used HEK293 cells expressing DctBp-S154R / Y160W / N182S / S184L-cpYFP-M9T / S132R / Y141N / F201S / N207 / Y245F in the cytoplasm for high-throughput compound screening.

[0284] The transfected HEK293 cells were rinsed with PBS, placed in HBSS solution (without succinic acid) for 1 hour, and then treated with 10 μM of the compound for 1 hour. Succinic acid was added dropwise to each sample. The fluorescence intensity at 420 nm excitation and 528 nm emission and the ratio of the fluorescence intensity at 485 nm excitation and 528 nm emission were recorded using an ELISA reader. The sample not treated with any compound was used as a control for standardization. The results are shown in Figure 7 As shown. Among the 2000 compounds used, most of them had little effect on the entry of succinate into cells. Seven compounds could increase the cell's ability to take up succinate, and another four compounds could significantly reduce the cell's uptake of succinate.

[0285] Example 11: Quantitative detection of succinic acid in blood using optical probes

[0286] In this example, succinate in the blood supernatant of purified DctBp-S154R / Y160W / N182S / S184L-cpYFP-M9T / S132R / Y141N / F201S / N207 / Y245F mice and humans was used for analysis.

[0287] After DctBp-S154R / Y160W / N182S / S184L-cpYFP-M9T / S132R / Y141N / F201S / N207 / Y245F was mixed with diluted blood supernatant for 10 minutes, the fluorescence intensity at 420nm excitation and 528nm emission and the ratio of the fluorescence intensity at 485nm excitation and 528nm emission were detected using an ELISA reader. Figure 8As shown, the succinic acid content in mouse blood is about 32 μM, and the succinic acid content in human blood is about 16 μM.

[0288] It can be seen from the above examples that the succinic acid optical probe provided by the present invention has a relatively small protein molecular weight and is easy to mature, has large dynamic fluorescence changes, has good specificity, can be expressed in cells through genetic manipulation methods, can locate and quantitatively detect succinic acid in real time inside and outside cells, and can perform high-throughput compound screening.

[0289] Other Implementations

[0290] This specification describes many embodiments. However, it should be understood that various modifications that those skilled in the art would learn from reading this specification without departing from the concept and scope of the present invention should also be included in the scope of the appended claims.

Claims

1. A succinate binding protein variant, which: (1) having the sequence shown in SEQ ID NO: 1 and having a mutation at one, two, three, four, five or six of the following positions: S154, F155, Y160, N182, V183, S184, wherein the mutation comprises a modification, substitution or deletion of an amino acid, (2) is a truncated variant of (1) having amino acids 65-320, or (3) a sequence having at least 70% sequence identity with the sequence of (1) or (2) and having the mutation described in (1) and retaining the ability to bind to succinate, Preferably, the mutated sites are selected from 1, 2, 3, 4, 5 or 6 of any one of the following groups: S154, F155, Y160, N182, V183, S184; More preferably, the mutation includes a mutation at a site selected from any one of the following groups: (1) F155 and Y160, (2) Y160 and N182, (3) Y160, N182 and V183, (4) Y160, N182 and S184, (5) S154, Y160, N182 and S184; More preferably, the S154 mutates to R; F155 mutates to T, P, Q or C; Y160 mutates to Y or W; N182 mutates to S; V183 mutates to Y or W; S184 mutates to L; More preferably, the mutation comprises a mutation selected from any one of the following groups: (1) F155T and Y160W, (2) F155P and Y160W, (3) F155Q and Y160W, (4) F155C and Y160W, (5) Y160W and N182S, (6) Y160W, N182S and V183Y, (7) Y160W, N182S and V183W, (8) Y160W, N182S and S184L, (9) S154R, Y160W, N182S and S184L.

2. An optical probe comprising a succinate-sensitive polypeptide and an optically active polypeptide, wherein the optically active polypeptide is located between residues 108-112, 181-185, 196-201 or 148-156 of the succinate-sensitive polypeptide, the succinate-sensitive polypeptide is a succinate-binding protein or a functional variant thereof, and the optically active polypeptide is a fluorescent protein or a functional variant thereof, Preferably, the succinate-sensitive polypeptide has: (1) the sequence shown in SEQ ID NO: 1 or a truncated variant thereof having amino acids 65 to 320, or a sequence having at least 70% sequence identity therewith and retaining succinate binding activity, (2) the sequence of the succinate binding protein variant according to claim 1, or (3) a sequence having at least 70% sequence identity with the sequence described in (2) and having the mutation described in (2) and retaining sensitivity to succinic acid, Preferably, the optically active polypeptide has: (a) a sequence shown in any one of SEQ ID NOs: 2-9, (b) the sequence shown in SEQ ID NO: 2 and having a mutation at one, two or three, four, five or six positions selected from the group consisting of: M9, S132, Y141, F201, N207, Y245, wherein the mutation comprises a modification, substitution or deletion of an amino acid; preferably, the mutation is selected from any one or more of the following: M9T, S132R, Y141N, F201S, N207T and Y245F, or (c) A variant sequence having at least 70% sequence identity to (a) or (b) and retaining fluorescent protein function.

3. The optical probe according to claim 2, characterized in that The optical probe comprises (2) the succinate binding protein variant and (b) the optically active polypeptide, and comprises a mutation selected from any one of the following groups: (1) F155T and Y160W of the succinate binding protein, (2) F155P and Y160W of the succinate binding protein, (3) F155Q and Y160W of the succinate binding protein, (4) F155C and Y160W of the succinate binding protein, (5) Y160W and N182S of the succinate binding protein, (6) Y160W, N182S and V183Y of the succinate binding protein, (7) Y160W, N182 S and V183W, (8) Y160W, N182S and S184L of succinate binding protein, (9) S154R, Y160W, N182S and S184L of succinate binding protein, (10) Y160W, N182S and S184L of succinate binding protein, and M9T, S132R, Y141N, F201S, N207T and Y245F of fluorescent protein, (11) S154R, Y160W, N182S and S184L of succinate binding protein, and M9T, S132R, Y141N, F201S, N207T and Y245F of fluorescent protein.

4. The optical probe according to claim 2 or 3, characterized in that: The optically active polypeptide is located at one or more of the following sites of the succinate sensitive polypeptide: 108 / 109, 108 / 110, 108 / 111, 108 / 112, 109 / 110, 109 / 111, 109 / 112, 110 / 111, 110 / 112, 111 / 112, 181 / 182, 181 / 183, 181 / 184, 181 / 185, 182 / 183, 182 / 184, 182 / 185, 1 83 / 184, 183 / 185, 184 / 185, 196 / 197, 196 / 198, 196 / 199, 196 / 200, 196 / 201, 197 / 198, 197 / 199, 197 / 200, 197 / 201, 198 / 199, 198 / 200, 198 / 201, 199 / 200, 199 / 201, 200 / 201, 148 / 149, 148 / 150, 148 / 151, 1 48 / 152, 148 / 153, 148 / 154, 148 / 155, 148 / 156, 149 / 150, 149 / 151, 149 / 152, 149 / 153, 149 / 154, 149 / 155, 149 / 156, 150 / 151, 150 / 152, 150 / 153, 150 / 154, 150 / 155, 150 / 156, 151 / 152, 151 / 153, 151 / 154, 151 / 155, 151 / 156, 152 / 151, 152 / 152, 152 / 153, 152 / 154, 152 / 155, 152 / 156, 153 / 151, 153 / 152, 153 / 153, 153 / 154, 153 / 155, 153 / 156, 154 / 151, 154 / 152, 154 / 153, 154 / 154, 154 / 155, 154 / 156, 155 / 156.

5. The optical probe according to claim 4, characterized in that The optical probe has a sequence shown in any one of SEQ ID NOs: 10-31.

6. A nucleic acid molecule comprising: (a) a coding sequence of the optical probe according to any one of claims 2 to 5, or (b) a complementary sequence of (a).

7. A nucleic acid construct comprising the nucleic acid molecule according to claim 6, Preferably, the nucleic acid construct is a cloning vector, an expression vector or a recombinant vector.

8. A host cell, wherein: (1) An optical probe as described in any one of claims 2 to 5; (2) comprising the nucleic acid molecule of claim 6; and / or (3) comprising the nucleic acid construct according to claim 7.

9. A detection kit comprising: (1) The optical probe according to any one of claims 2 to 5, (2) The nucleic acid molecule according to claim 6, (3) The nucleic acid construct according to claim 7, (4) The host cell according to claim 8, The detection kit optionally further comprises other reagents required for detecting succinic acid using an optical probe. Preferably, the detection kit further comprises one or more reagents selected from the following: a buffer, a culture medium, and a succinic acid standard.

10. A method for preparing the optical probe according to any one of claims 2 to 5, comprising: Cultivating the host cell of claim 8, and isolating the optical probe from the culture.

11. Use of the optical probe according to any one of claims 2 to 5, the nucleic acid molecule according to claim 6, the nucleic acid construct according to claim 7 and / or the host cell according to claim 8 in detecting succinic acid in a sample, screening compounds or the intracellular and / or extracellular localization of succinic acid.

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