Fructose-1,6-bisphosphate optical probe, its preparation method, and applications

Abstract

The present invention relates to a fructose-bisphosphate optical probe. Specifically, the present invention provides a fructose-bisphosphate optical probe comprising a fructose-1,6-bisphosphate-sensitive polypeptide and an optically active polypeptide, wherein the optically active polypeptide is located within the sequence of the fructose-1,6-bisphosphate-sensitive polypeptide, the fructose-1,6-bisphosphate-sensitive polypeptide is a fructose-1,6-bisphosphate-binding protein or a functional mutant thereof, and the optically active polypeptide is a fluorescent protein or a functional mutant thereof. The fluorescent probe of the present invention exhibits large dynamic changes in fluorescence, excellent specificity, and can detect fructose diphosphate both inside and outside cells in a high-throughput and quantitative manner.

Description

Technical Field

[0001] The present invention relates to the technical field of optical probes, and particularly to a fructose-1,6-bisphosphate optical probe, its preparation method and uses.

Background Art

[0002] Fructose-1,6-bisphosphate (FBP) is a compound that naturally exists in cells and is an important intermediate produced by cells in the glycolytic pathway. By regulating the activities of multiple enzymes in sugar metabolism, it exhibits pharmacological effects within cells. Fructose-1,6-bisphosphate is an important metabolite in the glycolytic pathway and gluconeogenesis process. It is produced from fructose-6-phosphate by the catalytic action of phosphofructokinase, and then, by the action of aldolase, dihydroxyacetone phosphate and 3-phosphoglyceraldehyde are produced, and finally, pyruvate is produced and enters the tricarboxylic acid cycle pathway. Furthermore, fructose-1,6-bisphosphate regulates the activities of multiple enzymes in various metabolic pathways.

[0003] Due to its important role in the metabolic pathway, fructose-1,6-bisphosphate is used in the treatment of metabolic-related diseases and respiratory system-related diseases, and excellent therapeutic effects can be obtained. For example, abnormal sugar metabolism in the body causes a decrease in ATP content, leading to a decrease in the oxygen-carrying ability of hemoglobin, causing metabolic disorders and diseases. However, fructose-1,6-bisphosphate promotes intracellular glycolysis and treats related diseases. Regarding respiratory system diseases, fructose-1,6-bisphosphate promotes the decomposition of intracellular phosphofructokinase, thereby increasing the storage content of phosphate in the body, enhancing the relaxation ability of pulmonary smooth muscle, and achieving the repair of damaged cells.

[0004] Currently, common detection methods for fructose-1,6-bisphosphate include chromatography (Ma B et al., European Journal of Pharmacology, 2013, 718(1-3), 524-532), enzymatic methods (Iwamoto S et al., Applied and Environmental Microbiology, 2007, 73(17), 5676-5678), and colorimetric methods (Wang C et al., Journal of biomedical engineering, 2000, 17(3), 363-365). Chromatographic and enzymatic detection methods offer excellent specificity but are expensive (Wu Liangyong et al., Chinese Medical Standards, 2007, 8(4), 63-65); chromatography can rapidly detect the content of fructose-1,6-bisphosphate and can detect even low levels of fructose-1,6-bisphosphate, but is prone to errors during experiments (Wu Yan et al., Journal of Central South Medical Sciences, 2000, (6), 608-609); and colorimetric methods suffer from interference from substances such as fructose and fructose monophosphate (Du Zhenning et al., Chinese Journal of Biochemistry and Pharmacology 1993, (2), 59-62). Furthermore, the above methods are only suitable for in vitro detection and cannot monitor changes in fructose-1,6-bisphosphate concentration in living cells in real time. Therefore, it is crucial to develop a new method that offers excellent specificity, a fast response rate, and real-time quantitative detection of fructose-1,6-bisphosphate in vivo. [Overview of the project] [Problems that the invention aims to solve]

[0005] The object of the present invention is to provide probes and methods for real-time localization, high-throughput, and quantitative detection of fructose-1,6-bisphosphate inside and outside cells. [Means for solving the problem]

[0006] To achieve the above-mentioned objectives of the invention, the present invention provides the following technical solutions: In a first aspect of the present invention, the following fructose-1,6-bisphosphate-binding protein mutants are provided: (1) Sequences having the sequence shown in SEQ ID NO:1 and having mutations including amino acid modifications, substitutions, or deletions at one, two, three, four, five or more sites selected from the following: T148, R175, G176, G177, L178, E180, D181, V182, K183, N184, Q185, F271, (2) A shortened mutant having amino acids between positions 88 and 340 of (1), or (3) A sequence having at least 70% sequence identity with the sequence of (1) or (2), having the mutation described in (1), and retaining the ability to bind to fructose-1,6-bisphosphate.

[0007] In one or more embodiments, the mutation sites are one, two, three, four, or five selected from any of the following groups: (a) T148, R175, G176, G177, L178, D181, V182, K183, N184, Q185, (b) G177, L178, E180, D181, V182, N184, Q185, F271. In one or more embodiments, the mutation includes mutations at sites selected from any of the following groups: (1) G177 and L178, (5) D181 and V182, (6) E180 and D181, (7) N184 and Q185; or the mutation includes mutations at sites selected from any of the following groups: (1) G177, L178, (2) G177, L178 and Q185, (3) G177, L178 and T148, (4) G177, L178 and F271, (5) D181, V182, (6) E180, D181, (7) N184, Q185, (8) N184, Q185, D181, V182, G177, (9) N184, Q185, G177, (10) N184, Q185, D181, V182.

[0008] In one or more embodiments, T148 is mutated to S. In one or more embodiments, G177 is mutated to A, I, L, M, or N, preferably L or M. In one or more embodiments, L178 is mutated to Y, F, or N, preferably N. In one or more embodiments, E180 is mutated to G. In one or more embodiments, D181 is mutated to F, L, or E, preferably E. In one or more embodiments, V182 is mutated to F, L, or G, preferably G. In one or more embodiments, N184 is mutated to V. In one or more embodiments, Q185 is mutated to S, A, T, D, or P. In one or more embodiments, F271 is mutated to Y. In one or more embodiments, the mutations include one or more selected from: G177L, D181E, V182G, N184V, Q185A.

[0009] In one or more embodiments, the mutations include mutations selected from any of the following groups: (1) G177A and L178Y, (2) G177I and L178F, (3) G177L and L178Y, (4) G177M and L178N, (5) G177N and L178Y, (6) G177M, L178N and Q185S, (7) G177M, L178N and Q185A, (8) G177M, L178N and Q185T, (9) G177M, (10) G177, L178 and T148S, (11) G177, L178 and F271Y, (12) D181F and V182F, (13) D181L and V182L, (14) E180G and D181G, (15) N184V and Q185P, (16) N184V, Q185P, D181E, V182G and G177L, (17) N184V, Q185P and G177L, (18) N184V, Q185P, D181E and V182G.

[0010] Another aspect of the present invention provides a fructose-1,6-bisphosphate optical probe comprising a fructose-1,6-bisphosphate-sensitive polypeptide and an optically active polypeptide, wherein the optically active polypeptide is located within the sequence of the fructose-1,6-bisphosphate-sensitive polypeptide. The fructose-1,6-bisphosphate-sensitive polypeptide is divided into a first and second part by the optically active polypeptide. In one or more embodiments, the fructose-1,6-bisphosphate optical probe comprises a fructose-1,6-bisphosphate-sensitive polypeptide B and an optically active polypeptide A, wherein the optically active polypeptide A is located within the sequence of the fructose-1,6-bisphosphate-sensitive polypeptide B, and the fructose-1,6-bisphosphate-sensitive polypeptide B is divided into a first part B1 and a second part B2, forming a probe structure of the formula B1-A-B2.

[0011] In one or more embodiments, the optically active polypeptide is located between residues 174-185 and / or 201-208 of the fructose-1,6-bisphosphate-sensitive polypeptide, where the numbers correspond to the full length of the fructose-1,6-bisphosphate-sensitive polypeptide. Preferably, the optically active polypeptide is located at one or more of the following sites selected from the fructose-1,6-bisphosphate-sensitive polypeptide: 174 / 175, 174 / 176, 174 / 177, 174 / 178, 174 / 179, 174 / 180, 174 / 181, 174 / 182, 174 / 183, 174 / 184, 174 / 185, 155 / 176, 175 / 177, 175 / 178, 175 / 179, 175 / 180, 175 / 181, 175 / 182, 175 / 183, 175 / 184, 175 / 185, 176 / 177, 176 / 178, 176 / 179, 176 / 180, 176 / 181, 176 / 182, 176 / 183, 176 / 184, 176 / 185, 177 / 178, 177 / 179, 177 / 180, 177 / 181, 177 / 182, 177 / 183, 177 / 184, 177 / 185, 178 / 179, 178 / 180, 178 / 181, 178 / 182, 178 / 183, 178 / 184 , 178 / 185, 179 / 180, 179 / 181, 179 / 182, 179 / 183, 179 / 184, 179 / 185, 180 / 181, 180 / 182, 180 / 183, 180 / 184, 180 / 185, 181 / 182, 181 / 182, 181 / 183, 181 / 184, 181 / 185, 182 / 183, 182 / 184, 182 / 185, 183 / 184, 183 / 185, 184 / 185, 201 / 202, 201 / 203, 201 / 204, 20 1 / 205, 201 / 206, 201 / 207, 201 / 208, 202 / 203, 202 / 204, 202 / 205, 202 / 206, 202 / 207, 202 / 208, 203 / 204, 203 / 205, 203 / 206, 203 / 207, 203 / 208, 204 / 205, 204 / 206, 204 / 207, 204 / 208, 205 / 206, 205 / 206, 205 / 207, 205 / 208, 206 / 207, 206 / 208 and / or 207 / 208.More preferably, the optically active polypeptide is located at one or more of the following sites selected from the fructose-1,6-bisphosphate-sensitive polypeptide: 176 / 177, 176 / 178, 177 / 181, 179 / 180, 182 / 184, 182 / 185, 203 / 206, 203 / 207, and 203 / 208.

[0012] In one or more embodiments, the fructose-1,6-bisphosphate-sensitive polypeptide is a fructose-1,6-bisphosphate-binding protein or a functional mutant thereof, wherein the functional mutant of the fructose-1,6-bisphosphate-binding protein has a mutation within the seven amino acids linked to the optically active polypeptide. In one or more embodiments, the fructose-1,6-bisphosphate-sensitive polypeptide has the following: (1) The sequence shown in SEQ ID NO:1 or a truncated mutant having the amino acids between the 88th and 340th positions, or a sequence having at least 70% sequence identity with them and retaining the activity to bind to fructose-1,6-bisphosphate, (2) A sequence of a fructose-1,6-bisphosphate-binding protein mutant described in any one embodiment of the first aspect of this specification, or (3) A sequence having at least 70% sequence identity with the sequence described in (2), having the mutation described in (2), and retaining fructose-1,6-bisphosphate sensitivity. In one or more embodiments, the optically active polypeptide is a fluorescent protein or a functional mutant thereof, wherein the functional mutant of the fluorescent protein has a mutation within three amino acids linked to the optically active polypeptide.

[0013] In one embodiment, the fluorescent protein is selected from yellow fluorescent protein, orange fluorescent protein, red fluorescent protein, green fluorescent protein, blue fluorescent protein, and apple red fluorescent protein. In one embodiment, the fluorescent protein has the sequence shown in one of SEQ ID NO: 2-9. In one or more embodiments, a functional mutant of a fluorescent protein has a mutation in the amino acid at position 1-3, preferably at position 1. Preferably, a functional mutant of a fluorescent protein includes a fluorescent protein in which the amino acid at position 1 is mutated to I or V. In one or more embodiments, a functional mutant of the fluorescent protein has the sequence shown in SEQ ID NO:2 and has a mutation at the Y1 site. Preferably, the mutation is Y1I or Y1V.

[0014] In one embodiment, the optical probe further comprises one or more linkers that flank the optically active polypeptide. The linkers described in the present invention may be any amino acid sequence of any length. In one embodiment, the flanking of the optically active polypeptide comprises a linker of five or fewer amino acids, e.g., a linker of 0, 1, 2, 3, or 4 amino acids. In one embodiment, the linker of the flanking of the optically active polypeptide comprises amino acid Y. In one embodiment, linker Y is located at the N-terminus and / or C-terminus of the optically active polypeptide. In one embodiment, the optical probe is as shown below: first portion B1, Y of the fructose-1,6-bisphosphate-sensitive polypeptide, optically active polypeptide A, and second portion B2 of the fructose-1,6-bisphosphate-sensitive polypeptide. In one embodiment, the optical probe of the present invention does not contain a linker. In one embodiment, the optical probe of the present invention further includes a localization sequence for localizing the probe to, for example, a specific organelle of a cell.

[0015] In one or more embodiments, the fructose-1,6-bisphosphate-sensitive polypeptide is a truncated mutant having amino acids 88-340 of SEQ ID NO:1, and the optically active polypeptide is located at one or more of the following sites selected from the fructose-1,6-bisphosphate-sensitive polypeptide: 176 / 177, 176 / 178, 177 / 181, 179 / 180, 182 / 184, 182 / 185, 203 / 206, 203 / 207, and 203 / 208. Preferably, the optically active polypeptide is as shown in SEQ ID NO:2. In one or more embodiments, the fructose-1,6-bisphosphate-sensitive polypeptide is a truncated mutant having amino acids 88-340 of SEQ ID NO:1, and the optically active polypeptide is located at one or more of the following sites selected from the fructose-1,6-bisphosphate-sensitive polypeptide: 176 / 177, 177 / 181, 179 / 180, or 182 / 184; and the optical probe has one or more mutations selected from the following: T1 of the fructose-1,6-bisphosphate-sensitive polypeptide 48S, G177A, G177I, G177L, G177M, G177N, L178Y, L178F, L178Y, L178N, L178Y, L178N, E180G, D181E, D181F, D181L, D181G, V182F, V182L, V182G, N184V, Q185A, Q185S, Q185A, Q185T, Q185D, Q185P, F271Y; The 1-position amino acid of the optically active polypeptide is mutated to V or I. Preferably, the optically active polypeptide is as shown in SEQ ID NO:2.

[0016] In one or more embodiments, the fructose-1,6-bisphosphate-sensitive polypeptide is a truncated mutant having amino acids at positions 88-340 of SEQ ID NO:1, and the optically active polypeptide is located at one or more of the following sites selected from the fructose-1,6-bisphosphate-sensitive polypeptide: 176 / 177, 177 / 181, 179 / 180, or 182 / 184; the optical probe has mutations represented by one of the following groups: (1) G177A and L178Y of the fructose-1,6-bisphosphate-sensitive polypeptide, (2) fructose-1,6-bisphosphate-sensitive (3) Fructose-1,6-bisphosphate-sensitive polypeptides G177I and L178F, (4) Fructose-1,6-bisphosphate-sensitive polypeptides G177L and L178Y, (5) Fructose-1,6-bisphosphate-sensitive polypeptides G177N and L178Y, (6) Fructose-1,6-bisphosphate-sensitive polypeptides G177M, L178N and Q185S, (7) Fructose-1,6-bisphosphate-sensitive polypeptides G177M, L178N and Q 185A, (8) G177M, L178N and Q185T of fructose-1,6-bisphosphate-sensitive polypeptides, (9) G177M, L178N and Q185D of fructose-1,6-bisphosphate-sensitive polypeptides, (10) G177M, L178N and T148S of fructose-1,6-bisphosphate-sensitive polypeptides, (11) G177M, L178N and F271Y of fructose-1,6-bisphosphate-sensitive polypeptides, (12) D181F and V of fructose-1,6-bisphosphate-sensitive polypeptides 182F, (13) D181L and V182L of fructose-1,6-bisphosphate-sensitive polypeptides, (14) E180G and D181G of fructose-1,6-bisphosphate-sensitive polypeptides, (15) N184V and Q185P of fructose-1,6-bisphosphate-sensitive polypeptides, (16) N184V, Q185P, D181E, V182G, G177L of fructose-1,6-bisphosphate-sensitive polypeptides, and mutations to V at the 1st amino acid of optically active polypeptides, (17) fructose-1,(18) Mutations to the 1st amino acid of the fructose-1,6-bisphosphate-sensitive polypeptide N184V, Q185P, D181E, V182G, G177L, and optically active polypeptides to I; (19) Mutations to the 1st amino acid of the fructose-1,6-bisphosphate-sensitive polypeptide N184V, Q185P, and G177L, and optically active polypeptides to V; (20) Mutations to the 1st amino acid of the fructose-1,6-bisphosphate-sensitive polypeptide N184V, Q185P, and G177L, and optically active polypeptides to I; (20) Mutations to the 1st amino acid of the fructose-1,6-bisphosphate-sensitive polypeptide N184V, Q185P, D181E, and V182G, and optically active polypeptides to V. Preferably, the optically active polypeptide is as shown in SEQ ID NO:2.

[0017] Another aspect of the present invention also provides a fusion polypeptide comprising the optical probe described herein and other polypeptides. In some embodiments, the other polypeptide is located at the N-terminus and / or C-terminus of the optical probe. In some embodiments, the other polypeptide includes polypeptides that localize the optical probe to various organelles or intracellular organelles, tags for purification, or tags for immunoblotting. Another aspect of the present invention provides a nucleic acid molecule comprising: (a) a coding sequence of a polypeptide or probe described in any one embodiment herein, or (b) a complementary sequence of (a), or (c) a fragment of (a) or (b). The fragment is a primer. The present invention further relates to mutants of the nucleic acid molecules and includes a nucleic acid sequence or complementary sequence encoding a mutant with a fragment, analog, derivative, soluble fragment, and mutant of the optical probe or fusion protein of the present invention.

[0018] Another aspect of the present invention further provides a nucleic acid construct comprising a nucleic acid molecule described herein, wherein the nucleic acid sequence encodes an optical probe or fusion polypeptide described in the present invention. In one or more embodiments, the nucleic acid construct is a cloning vector, an expression vector or a recombinant vector. In one or more embodiments, the nucleic acid molecule is operably linked to an expression control sequence. In some embodiments, the expression vector is selected from a prokaryotic expression vector, a eukaryotic expression vector and a viral vector.

[0019] Another aspect of the present invention further provides a host cell, which (1) expresses an optical probe or a fusion polypeptide described in any one of the embodiments of the present invention; (2) contains a nucleic acid molecule described in any one of the embodiments of the present invention; or (3) contains a nucleic acid construct described in any one of the embodiments of the present invention. The host cell is preferably Escherichia coli. Another aspect of the present invention further provides a kit for detecting fructose-1,6-bisphosphate, which kit contains an optical probe described herein or a fusion polypeptide or a polynucleotide or an optical probe prepared by the method described herein. In one or more embodiments, the kit further contains one or more reagents selected from the following: buffer, medium, fructose-1,6-bisphosphate standard.

[0020] Another aspect of the present invention provides a method for preparing an optical probe described herein, which method includes: providing a host cell that expresses an optical probe or a fusion polypeptide described herein, culturing the host cell under conditions in which the cell expresses, and isolating the optical probe or the fusion polypeptide. In one or more embodiments, the method includes the following steps: 1) incorporating a nucleic acid molecule encoding the fructose-1,6-bisphosphate 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 fructose-1,6-bisphosphate optical probe.

[0021] Another aspect of the present invention further provides a method for detecting fructose-1,6-bisphosphate in a sample, the method including: contacting the optical probe or fusion polypeptide or host cell described herein with the sample; and detecting a change in the optically active polypeptide. The detection may be performed in vivo, in vitro, intracellularly, or in situ. The sample is, for example, blood. Another aspect of the present specification further provides a method for quantifying fructose-1,6-bisphosphate in a sample, the method including: contacting the optical probe or fusion polypeptide or host cell described herein with the sample; detecting an optical change in the optically active polypeptide; and quantifying the fructose-1,6-bisphosphate in the sample based on the optical change in the optically active peptide. Another aspect of the present invention further provides a method for screening a compound (for example, a pharmaceutical), the method including: contacting the optical probe or fusion polypeptide or host cell described herein with a candidate compound in a system containing fructose-1,6-bisphosphate; detecting an optical change in the optically active polypeptide; and screening the candidate compound based on the optical change in the optically active peptide. By the above method, high-throughput screening of compounds becomes possible.

[0022] In one or more embodiments, a host cell described herein is brought into contact with a candidate compound in a system containing fructose-1,6-bisphosphate, and the optical change of the optically active polypeptide indicates whether the candidate compound can regulate the intracellular uptake of fructose-1,6-bisphosphate. Another aspect of the present invention further provides a method for localizing fructose-1,6-bisphosphate intracellularly and / or extracellularly, the method comprising: contacting a system comprising fructose-1,6-bisphosphate with the optical probe or the host cell and detecting the optical change of the optically active polypeptide. In one or more embodiments, the system is a solution system, a cell system, or an intracellular system. Another aspect of the present invention further provides applications of the fructose-1,6-bisphosphate optical probes or fusion polypeptides or host cells described herein in the detection of fructose-1,6-bisphosphate in a sample, screening of compounds, or intracellular and / or extracellular localization of fructose-1,6-bisphosphate. In one or more embodiments, the localization is real-time localization.

[0023] Beneficial Effects of the Invention: The fructose-1,6-bisphosphate optical probe provided by the present invention is easy to mature, exhibits large dynamic changes in fluorescence, has excellent specificity, can be expressed in cells using genetic engineering methods, can localize fructose-1,6-bisphosphate in real time both inside and outside cells, can be detected quantitatively and through high throughput, and eliminates time-consuming sample processing procedures. Experimental results show that the fructose-1,6-bisphosphate optical probe provided in this application achieves a maximum response to fructose-1,6-bisphosphate that is more than 12 times that of the control, can be detected qualitatively and quantitatively and locally within intracellular structures such as the cytoplasm, mitochondria, nucleus, endoplasmic reticulum, lysosomes, and Golgi apparatus, can screen compounds through high throughput, and can quantitatively detect fructose-1,6-bisphosphate in blood. [Brief explanation of the drawing]

[0024] The present invention will be further described below based on the drawings and examples. [Figure 1] Figure 1 is an SDS-PAGE image of an exemplary fructose-1,6-bisphosphate optical probe described in Example 2; [Figure 2] Figure 2 is a table showing the change in response to fructose-1,6-bisphosphate for exemplary fructose-1,6-bisphosphate optical probes containing cpYFP and fructose-1,6-bisphosphate-binding protein as described in Example 2; [Figure 3] Figure 3 is a table showing the change in response to fructose-1,6-bisphosphate for exemplary fructose-1,6-bisphosphate optical probes containing cpGFP and fructose-1,6-bisphosphate-binding protein as described in Example 3; [Figure 4] Figure 4 is a table showing the change in response to fructose-1,6-bisphosphate for exemplary fructose-1,6-bisphosphate optical probes containing cpBFP and fructose-1,6-bisphosphate-binding protein as described in Example 4; [Figure 5] Figure 5 is a characteristic image of the fluorescence spectrum of the exemplary fructose-1,6-bisphosphate optical probe described in Example 6; [Figure 6] Figure 6 shows the titration curves of the exemplary fructose-1,6-bisphosphate optical probe described in Example 6 for different concentrations of fructose-1,6-bisphosphate; [Figure 7] Figure 7 is a bar graph showing the specific detection of multiple analogous substrates in the glycolysis pathway by the exemplary fructose-1,6-bisphosphate optical probe described in Example 6; [Figure 8] Figure 8 is a photograph of the localization of the exemplary fructose-1,6-bisphosphate optical probe described in Example 7 to intracellular organelles in mammalian cells; [Figure 9]Figure 9 is a schematic diagram illustrating the dynamic monitoring of fructose-1,6-bisphosphate concentration in the cytoplasm of mammalian cells using the exemplary fructose-1,6-bisphosphate optical probe described in Example 7; [Figure 10] Figure 10 is a dot plot showing high-throughput compound screening at the live cell level using the exemplary fructose-1,6-bisphosphate optical probe described in Example 8; [Figure 11] Figure 11 is a bar graph showing the quantitative determination of fructose-1,6-bisphosphate in human blood using the exemplary fructose-1,6-bisphosphate optical probe described in Example 9. [Modes for carrying out the invention]

[0025] When referring to a number or range, the term "about" as used herein means that the number or range is within 20%, 10%, and 5% of the number or range indicated. As used herein, the terms “contains,” “having,” and their synonyms include the meanings of “containing” and “consisting of.” For example, a composition “contains” X may consist only of X, or it may also contain other substances, such as X + Y.

[0026] As used herein, the term “fructose-1,6-bisphosphate-sensitive polypeptide” refers to a polypeptide that responds to fructose-1,6-bisphosphate, where the response refers to any response of the polypeptide’s chemical, biological, electrical, or physiological parameters related to the interaction of the sensitive polypeptide. The response may include small changes, such as changes in the orientation of amino acids or peptide fragments of the polypeptide, or changes in the polypeptide’s primary, secondary, or tertiary structure (including protonation, electrochemical potential, and / or conformation). “Conformation” refers to the three-dimensional arrangement of the primary, secondary, and tertiary structures of a molecule, including its side chain groups; a change in the three-dimensional structure of a molecule also changes its conformation. Examples of conformational changes include transitions from α-helix to β-sheet or from β-sheet to α-helix. It should be understood that a detectable change does not necessarily have to be a conformational change, as long as the fluorescence of the fluorescent protein moiety changes. The fructose-1,6-bisphosphate-sensitive polypeptides described herein also include their functional mutants. Functional mutants of fructose-1,6-bisphosphate-sensitive polypeptides include, but are not limited to, mutants that interact with fructose-1,6-bisphosphate and can induce changes identical or similar to those of the parent fructose-1,6-bisphosphate-sensitive polypeptide.

[0027] The fructose-1,6-bisphosphate-sensitive polypeptides described in the present invention include, but are not limited to, the fructose-1,6-bisphosphate-binding protein CggR or mutants having 90% or more homology thereto. The exemplary fructose-1,6-bisphosphate-binding protein CggR described in the present invention is derived from Bacillus subtilis. CggR belongs to the SorC / DeoR family of prokaryotic transcription factors and consists of two domains: an N-terminal DNA-binding domain and a C-terminal ligand domain. The fructose-1,6-bisphosphate-binding protein can sense changes in fructose-1,6-bisphosphate concentration, and its spatial conformation also changes during the process of dynamic changes in fructose-1,6-bisphosphate concentration. An exemplary CggR protein truncated form is shown as SEQ ID NO: 1. When describing the optical probe of the present invention (for example, when describing an insertion site or mutation site), all amino acid residue numbers refer to SEQ ID NO:1. As used herein, the term "optical probe" refers to a fructose-1,6-bisphosphate-sensitive polypeptide that fuses with an optically active polypeptide. The inventors have discovered that the conformational changes caused by the specific binding of a fructose-1,6-bisphosphate-sensitive polypeptide, such as a fructose-1,6-bisphosphate-binding protein, to physiological concentrations of fructose-1,6-bisphosphate induce conformational changes in the optically active polypeptide (e.g., a fluorescent protein), thereby causing changes in the optical properties of the optically active polypeptide. The presence and / or level of fructose-1,6-bisphosphate can be detected and analyzed by plotting a standard curve using the fluorescence of the fluorescent protein measured at different fructose-1,6-bisphosphate concentrations.

[0028] In the optical probes of the present invention, an optically active polypeptide (e.g., a fluorescent protein) is operably inserted into a fructose-1,6-bisphosphate-sensitive polypeptide. An "optically active polypeptide" by protein is a polypeptide that possesses the ability to emit fluorescence. Fluorescence is one of the optical properties of an optically active polypeptide and 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 a suitable excitation wavelength, fluorescence quantum efficiency, shape of the excitation or emission spectrum, maximum excitation wavelength and maximum emission wavelength, excitation amplitude at two different wavelengths, ratio of emission amplitude at two different wavelengths, excited state lifetime, or fluorescence anisotropy. A measurable difference in any one of these properties between the active and inactive states is sufficient to use the fluorescent protein substrate of the present invention in an activity assay. The measurable difference can be determined by determining a quantitative amount of the fluorescence property, such as the amount of fluorescence at a particular wavelength or the integral of fluorescence across the entire emission spectrum. Preferably, the protein substrate is selected to have fluorescence properties that are easily distinguishable between the inactivated and activated conformational states. The optically active polypeptides described herein may further include functional mutants thereof. Functional mutants of optically active polypeptides include, but are not limited to, mutants that can induce the same or similar changes in fluorescence properties as the parent optically active polypeptide.

[0029] A "linker" or "connection region" refers to an amino acid or nucleotide sequence that connects two parts in the polypeptide, protein, or nucleic acid of the present invention. Exemplarily, in the present invention, the number of amino acids at the amino terminus of the connection region between a fructose-1,6-bisphosphate-sensitive polypeptide and an optically active polypeptide is selected to be 0 to 3, and the number of amino acids at the carboxyl terminus is selected to be 0 to 2; when a recombinant optical probe is attached to a 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 is a short peptide chain composed of one or more flexible amino acids, such as Y. As used herein, the term "fluorescent protein" refers to a protein that emits fluorescence when irradiated with excitation light. Fluorescent proteins are a fundamental detection method in the biological sciences, and examples include green fluorescent protein (GFP), commonly used in biotechnology, and cyclic rearranged blue fluorescent protein (cpBFP), cyclic rearranged green fluorescent protein (cpGFP), and cyclic rearranged yellow fluorescent protein (cpYFP), which are induced by mutations in this protein; also, red fluorescent protein (RFP), commonly used in this field, and cyclic rearranged proteins derived from this protein (cpmApple, cpmOrange, cpmKate, etc.). For example, cpYFP is shown in SEQ ID NO:2, cpmOrange in SEQ ID NO:3, cpmKate in SEQ ID NO:4 or 8, mCherry in SEQ ID NO:5, cpGFP in SEQ ID NO:6, cpBFP in SEQ ID NO:7, and cpmApple in SEQ ID NO:9.

[0030] The fluorescent proteins in the optical probe include, but are not limited to, functional mutants with mutations, including fluorescent proteins with mutations in the amino acids at positions 1-3 (preferably position 1) (e.g., mutations to V or I). Exemplarily, a functional mutant of cpYFP has the sequence shown in SEQ ID NO:2 and has a mutation to V or I at the Y1 site. In the optical probe of the present invention, the optically active polypeptide is located in the NC direction between residues 174-185 and / or 201-208 of the fructose-1,6-bisphosphate-sensitive polypeptide, where the numbers correspond to the full length of the fructose-1,6-bisphosphate-sensitive polypeptide.

[0031] Exemplary, optically active polypeptides are located at the following sites in the amino acid sequence of fructose-1,6-bisphosphate-binding protein: 174 / 175, 174 / 176, 174 / 177, 174 / 178, 174 / 179, 174 / 180, 174 / 181, 174 / 182, 174 / 183, 174 / 184, 174 / 185, 155 / 176, 175 / 177, 175 / 178, 175 / 179, 175 / 180, 175 / 181, 175 / 182, 175 / 183, 175 / 184, 175 / 185, 176 / 177, 176 / 178, 176 / 179, 176 / 180, 176 / 181, 176 / 182, 176 / 183, 176 / 184, 176 / 185, 177 / 178, 177 / 179, 177 / 180, 177 / 181, 177 / 182, 177 / 183, 177 / 184, 177 / 185, 178 / 179, 178 / 180, 178 / 181, 178 / 182, 178 / 183, 178 / 184, 17 8 / 185, 179 / 180, 179 / 181, 179 / 182, 179 / 183, 179 / 184, 179 / 185, 180 / 181, 180 / 182, 180 / 183, 180 / 184, 180 / 185, 181 / 182, 181 / 182, 181 / 183, 181 / 184, 181 / 185, 182 / 183, 182 / 184, 182 / 185, 183 / 184, 183 / 185, 184 / 185, 201 / 202, 201 / 203, 201 / 204, 2 01 / 205, 201 / 206, 201 / 207, 201 / 208, 202 / 203, 202 / 204, 202 / 205, 202 / 206, 202 / 207, 202 / 208, 203 / 204, 203 / 205, 203 / 206, 203 / 207, 203 / 208, 204 / 205, 204 / 206, 204 / 207, 204 / 208, 205 / 206, 205 / 206, 205 / 207, 205 / 208, 206 / 207, 206 / 208, 207 / 208. In this specification, when two numbers in a site expressed in the form "X / Y" are consecutive integers, it means that the optically active polypeptide is located between the amino acids indicated by those numbers. For example, insertion sites 147 / 148 indicate that the optically active polypeptide is located between amino acids 147 and 148 of the fructose-1,6-bisphosphate-sensitive polypeptide.If the two digits of a site represented in the form "X / Y" are not consecutive integers, it means that the optically active polypeptide will substitute an amino acid between the amino acids indicated by those digits. For example, insertion site 174 / 185 indicates that the optically active polypeptide will substitute amino acids 175-184 of the fructose-1,6-bisphosphate-sensitive polypeptide. Preferably, the optically active polypeptide inserts the following sites of the fructose-1,6-bisphosphate-sensitive polypeptide: 176 / 177, 176 / 178, 177 / 181, 179 / 180, 182 / 184, 182 / 185, 203 / 206, 203 / 207, and 203 / 208.

[0032] In exemplary embodiments, the B1-A-B2 optical probe of the present invention may be a probe formed when the fluorescent protein is located at 176 / 177, 176 / 178, 177 / 181, 179 / 180, 182 / 184, 182 / 185, 203 / 206, 203 / 207 and 203 / 208 of CggR or its truncated variant (e.g., SEQ ID NO:1, or a fragment thereof containing amino acids from positions 88 to 340). In a specific embodiment, the fructose-1,6-bisphosphate-sensitive polypeptide in the optical probe is indicated by the amino acids at positions 88-340 of SEQ ID NO:1, the optically active polypeptide is indicated by any one of SEQ ID NO:2-9, and the optically active polypeptide is located at the following sites of the fructose-1,6-bisphosphate-sensitive polypeptide: 176 / 177, 176 / 178, 177 / 181, 179 / 180, 182 / 184, 182 / 185, 203 / 206, 203 / 207, or 203 / 208.

[0033] When referring to a polypeptide or protein, the terms “variant” or “mutant” as used in this invention include mutants that have the same function as the polypeptide or protein but a different sequence. Mutants of polypeptides or proteins may include homologous sequences, conserved mutants, alleles, native mutants, and induced mutants. These mutants include, but are not limited to, sequences obtained by deleting, inserting, and / or substituting one or more (typically 1 to 30, preferably 1 to 20, more preferably 1 to 10, most preferably 1 to 5) amino acids from the sequence of the polypeptide or protein, and adding one or more (typically up to 20, preferably up to 10, more preferably up to 5) amino acids to the carboxyl and / or amino group ends. These mutants may further include polypeptides or proteins having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100% sequence identity with the aforementioned polypeptide or protein. While not intended to be theoretical, changes in amino acid residues that do not alter the overall conformation and function of a polypeptide or protein are called function-conserving mutations. For example, in this art, substitutions with amino acids of similar properties generally do not alter the function of the polypeptide or protein. In this art, amino acids of similar properties are clearly defined as families of amino acids having similar side chains.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 non-loading side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), amino acids with nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, arginine, phenylalanine, methionine, tryptophan), amino acids with β-branched side chains (e.g., threonine, valine, isoleucine), and amino acids with aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Furthermore, the addition of one or more amino acids to, for example, the amino group terminus and / or carboxyl terminus usually does not alter the function of the polypeptide or protein. Common and known conservation amino acid substitutions of non-genetic coding amino acids are well known in this art. Conservative substitutions of other non-coding amino acids can be determined based on a comparison of their physical properties with those of the genetically coded amino acids.

[0034] The inventors have found that fructose-1,6-bisphosphate-binding protein mutants having mutations at sites selected from the following for SEQ ID NO:1 or its shortened mutants exhibit different binding activity from fructose-1,6-bisphosphate: T148, R175, G176, G177, L178, E180, D181, V182, K183, N184, Q185, F271. The aforementioned amino acid mutations include amino acid modifications, substitutions, or deletions. In particular, fructose-1,6-bisphosphate-binding protein mutants having mutations in one, two, three, four, or five sites from any of the following groups, of SEQ ID NO:1 or its shortened mutants, are more advantageous for more efficient detection of fructose-1,6-bisphosphate with the optical probe of the present invention: (a) T148, R175, G176, G177, L178, D181, V182, K183, N184, Q185, (b) G177, L178, E180, D181, V182, N184, Q185, F271. In a preferred embodiment, the mutation in the fructose-1,6-bisphosphate-binding protein mutant includes mutations at sites selected from any of the following groups: (1) G177 and L178, (5) D181 and V182, (6) E180 and D181, (7) N184 and Q185; or the mutation includes mutations at sites selected from any of the following groups: (1 (1) G177, L178, (2) G177, L178 and Q185, (3) G177, L178 and T148, (4) G177, L178 and F271, (5) D181, V182, (6) E180, D181, (7) N184, Q185, (8) N184, Q185, D181, V182, G177, (9) N184, Q185, G177, (10) N184, Q185, D181, V182.

[0035] However, as an example of the examples, in SEQ ID NO:1 or its shortened mutants, T148 is mutated to S; G177 is mutated to A, I, L, M, or N, preferably L or M; L178 is mutated to Y, F, or N, preferably N; E180 is mutated to G; D181 is mutated to F, L, or E, preferably E; V182 is mutated to F, L, or G, preferably G; N184 is mutated to V; Q185 is mutated to S, A, T, D, or P, preferably P; F271 is mutated to Y.

[0036] In a preferred embodiment, the mutation of the fructose-1,6-bisphosphate-binding protein mutant (SEQ ID NO:1 or its truncated mutant) is selected from one of the following: (1) G177A and L178Y, (2) G177I and L178F, (3) G177L and L178Y, (4) G177M and L178N, (5) G177N and L178Y, (6) G177M, L178N and Q185S, (7) G177M, L178N and Q185A, (8) G177M, L178N and Q185T, (9) G177M, L178N and Q1 85D, (10)G177M, L178N and T148S, (11)G177M, L178N and F271Y, (12)D181F and V182F, (13)D181L and V182L, (14)E180G and D181G, (15)N184V and Q185P, (16)N184V, Q185P, D181E, V182G and G177L, (17)N184V, Q185P and G177L, (18)N184V, Q185P, D181E and V182G.

[0037] The present invention provides fructose-1,6-bisphosphate-binding protein mutants having these mutations, and optical probes containing such fructose-1,6-bisphosphate-binding protein mutants as fructose-1,6-bisphosphate-sensitive polypeptides. Accordingly, in one or more embodiments, the fructose-1,6-bisphosphate-sensitive polypeptide in the optical probe is a fructose-1,6-bisphosphate-binding protein mutant described in any one embodiment herein, the fluorescent protein in the optical probe is indicated by SEQ ID NO: 2-9, and the 1-position amino acid of the fluorescent protein is mutated to I or V. In some specific embodiments, the fructose-1,6-bisphosphate-sensitive polypeptide in the optical probe is shown at amino acids 88-340 of SEQ ID NO:1, the optically active polypeptide is shown at SEQ ID NO:2, the optically active polypeptide is located at the 176 / 177 site of the fructose-1,6-bisphosphate-sensitive polypeptide, and the fructose-1,6-bisphosphate-sensitive polypeptide has a mutation shown in one of the following selected from: (1) G177A and L178Y, (2) G177I and L178F, (3) G177L and L17 8Y, (4) G177M and L178N, (5) G177N and L178Y, (6) G177M, L178N and Q185S, (7) G177M, L178N and Q185A, (8) G177M, L178N and Q185T, (9) G177M, L178N and Q185D, (10) G177M, L178N and T148S, (11) G177M, L178N and F271Y. For example, the amino acid sequence of the optical probe shown in (4) is shown in SEQ ID NO:10, and the nucleic acid sequence is shown in SEQ ID NO:17; the amino acid sequence of the optical probe shown in (7) is shown in SEQ ID NO:11, and the nucleic acid sequence is shown in SEQ ID NO:18; the amino acid sequence of the optical probe shown in (8) is shown in SEQ ID NO:12, and the nucleic acid sequence is shown in SEQ ID NO:19; the amino acid sequence of the optical probe shown in (9) is shown in SEQ ID NO:13, and the nucleic acid sequence is shown in SEQ ID NO:20.

[0038] In some specific embodiments, the fructose-1,6-bisphosphate-sensitive polypeptide in the optical probe is shown at amino acids 88-340 of SEQ ID NO:1, the optically active polypeptide is shown at SEQ ID NO:2, the optically active polypeptide is located at site 177 / 181 of the fructose-1,6-bisphosphate-sensitive polypeptide, and the fructose-1,6-bisphosphate-sensitive polypeptide has a mutation shown in one of the following selected from: (12) D181F and V182F, (13) D181L and V182L. In some specific embodiments, the fructose-1,6-bisphosphate-sensitive polypeptide in the optical probe is shown at amino acids 88-340 of SEQ ID NO:1, the optically active polypeptide is shown at SEQ ID NO:2, the optically active polypeptide is located at the 179 / 180 site of the fructose-1,6-bisphosphate-sensitive polypeptide, and the fructose-1,6-bisphosphate-sensitive polypeptide has mutations (14)E180G and D181G.

[0039] In some specific embodiments, the fructose-1,6-bisphosphate-sensitive polypeptide in the optical probe is shown at amino acids 88-340 of SEQ ID NO:1, the optically active polypeptide is shown at SEQ ID NO:2, the optically active polypeptide is located at the 182 / 184 site of the fructose-1,6-bisphosphate-sensitive polypeptide, and the optical probe has a mutation shown in one of the following selected from: (15) N184V and Q185P of the fructose-1,6-bisphosphate-sensitive polypeptide, (16) mutations to N184V, Q185P, D181E, V182G, G177L of the fructose-1,6-bisphosphate-sensitive polypeptide, and V at the 1st amino acid of the optically active polypeptide, (17) N184V, Q185P, D181E of the fructose-1,6-bisphosphate-sensitive polypeptide, (18) Mutations to the 1st amino acid of the optically active polypeptide V182G, G177L, and (19) Mutations to the 1st amino acid of the optically active polypeptide N184V, Q185P, and G177L, and (20) Mutations to the 1st amino acid of the optically active polypeptide N184V, Q185P, and G177L, and (20) Mutations to the 1st amino acid of the optically active polypeptide N184V, Q185P, D181E, and V182G, and (20) Mutations to the 1st amino acid of the optically active polypeptide N184V, Q185P, D181E, and V182G, and (20) Mutations to the 1st amino acid of the optically active polypeptide For example, the amino acid sequence of the optical probe shown in (16) is shown in SEQ ID NO:14, and the nucleic acid sequence is shown in SEQ ID NO:21; the amino acid sequence of the optical probe shown in (17) is shown in SEQ ID NO:15, and the nucleic acid sequence is shown in SEQ ID NO:22; the amino acid sequence of the optical probe shown in (20) is shown in SEQ ID NO:16, and the nucleic acid sequence is shown in SEQ ID NO:23.

[0040] In two or more polypeptide or nucleic acid molecular sequences, the term “identity” or “percent identity” means that two or more sequences or subsequences are identical when compared by manual alignment and visual inspection using methods known in the Art, such as sequence comparison algorithms, across a comparison window or specified region, and aligned to obtain the greatest possible correspondence, or that a certain percentage of amino acid residues or nucleotides are identical in a particular region (e.g., 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical). For example, preferred algorithms suitable for determining sequence identity percentages and sequence similarity percentages are the BLAST and BLAST2.0 algorithms, for which refer to Altschul et al. (1977) Nucleic Acids Res. 25:3389 and Altschul et al. (1990) J.Mol. Biol. 215:403, respectively.

[0041] Gene cloning operations often require the design of appropriate restriction sites, which inevitably introduce one or more irrelevant residues to the terminus of the expressed polypeptide or protein, but this does not affect the activity of the target polypeptide or protein, as is well known to those skilled in the art. For example, to construct fusion proteins, promote the expression of recombinant proteins, obtain recombinant proteins that are automatically secreted outside host cells, or facilitate the purification of recombinant proteins, it is often necessary to add several amino acids to the N-terminus, C-terminus, or other appropriate region within a recombinant protein, including, but not limited to, appropriate linker peptides, signal peptides, leader peptides, terminus elongators, glutathione S-transferase (GST), maltose E-binding proteins, protein A, tags such as 6His or Flag, or protease sites of factor Xa or thrombin or enterokinase.

[0042] As used herein, the terms “functional fragment,” “derivative,” and “analog” refer to a protein that substantially retains the same biological function or activity as the original polypeptide or protein (e.g., fructose-1,6-bisphosphate-binding protein or fluorescent protein). Functional mutants, derivatives, or analogs of the polypeptide or protein of the present invention (e.g., fructose-1,6-bisphosphate-binding protein or fluorescent protein) may be (i) a protein in which one or more conserved or non-conserved amino acid residues (preferably conserved amino acid residues) are substituted, such substituted amino acid residues may or may not be encoded in the genetic code; or (ii) a protein having substituents on one or more amino acid residues; or (iii) a protein formed by the fusion of a mature protein with another compound (e.g., a compound that extends the half-life of a protein, e.g., polyethylene glycol); or (iv) a protein formed by the fusion of an additional amino acid sequence with this protein sequence (e.g., a secretory sequence, or a sequence or protoprotein sequence for purifying this protein, or a fusion protein formed with an antigen IgG fragment). As taught herein, these functional mutants, derivatives, and analogs are well known to those skilled in the art. These analogs include those having residues different from the natural L-amino acids (e.g., D-amino acids) and those having unnaturally occurring or synthesized amino acids (e.g., β, γ-amino acids). It should be understood that the fructose-1,6-bisphosphate-sensitive polypeptides of the present invention are not limited to the representative proteins, mutants, derivatives, and analogs listed above. Modifications (generally those that do not alter the primary structure) include in vivo or in vitro chemical derivation forms of proteins, such as acetylation or carboxylation. Modifications also include glycosylation, for example, proteins produced by glycosylation modification during or in further processing steps of the protein. This modification can be achieved by exposure to enzymes that glycosylate proteins (e.g., mammalian glycosylation or deglycosylation enzymes).Modifications include sequences containing phosphorylated amino acid residues (e.g., tyrosine phosphate, serine phosphate, threonine phosphate). This also includes proteins whose modification improves their anti-protein hydrolysis performance or optimizes their solubility.

[0043] The fusion polypeptide of the present invention comprises an optical probe described herein and other polypeptides. In some embodiments, the optical probe described herein further comprises other polypeptides that fuse with it. 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 localize the optical probe to various organelles or intracellular organelles, tags for purification, or tags for immunoblotting. A linker may be present between the optical probe and the other polypeptide in the fusion polypeptide described herein. The intracellular organelles described herein include the cytoplasm, mitochondria, nucleus, endoplasmic reticulum, cell membrane, Golgi apparatus, lysosomes, and peroxisomes. In some embodiments, the tag for purification or the tag for immunoblotting comprises hexahistidine (6*His), glutathione S-transferase (GST), and Flag.

[0044] The present invention comprises nucleic acid molecules encoding a fructose-1,6-bisphosphate-sensitive polypeptide or optical probe as described in the present invention. The terms “nucleic acid,” “nucleotide,” “polynucleotide,” or “nucleic acid sequence” as used in the present invention may be in DNA form or RNA form. DNA form includes cDNA, genomic DNA, or artificially synthesized DNA. DNA may be single-stranded or double-stranded. DNA may consist of coding and non-coding strands. When referring to nucleic acids, the term “mutant” as used herein may be a naturally occurring allelic mutant or a non-naturally occurring mutant. These nucleotide mutants include degenerate mutants, substitution mutants, deletion mutants, and insertion mutants. As known in the art, an allelic mutant may be a substitution, deletion, or insertion of one or more nucleotides, but is a form of nucleic acid substitution that does not substantially alter the function of the encoded mutant protein. The nucleic acids of the present invention may include nucleotide sequences having at least about 50%, at least about 60%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100% sequence identity with the nucleic acid sequence. The present invention also relates to nucleic acid fragments that hybridize to the sequence. As used herein, the length of a “nucleic acid fragment” includes at least 15 nucleotides, preferably at least 30 nucleotides, more preferably at least 50 nucleotides, and most preferably at least 100 nucleotides or more. The nucleic acid fragments can be used in nucleic acid amplification techniques (such as PCR).

[0045] The full-length sequences or fragments of the optical probe or fusion protein of the present invention can typically be obtained by PCR amplification, artificial synthesis, or recombination. The procedures and reagents used in common PCR, synthesis, and recombination methods are known in the art. Mutations may also be introduced into the protein sequences of the present invention by methods such as mutagenic PCR or chemical synthesis. The present invention also relates to nucleic acid constructs, which comprise the polynucleotides described herein and one or more regulatory sequences operably linked to these sequences. The polynucleotides of the present invention can be manipulated in various ways to ensure the expression of polypeptides or proteins. Depending on the expression vector or requirements, the nucleic acid constructs may be manipulated before insertion into the vector. Techniques for altering polynucleotide sequences using recombinant DNA methods are known in the art.

[0046] In one embodiment, the nucleic acid construct is a vector. The vector may be a cloning vector, an expression vector, or a cosmogenic recombinant vector. The polynucleotides of the present invention can be cloned into many types of vectors, such as plasmids, phagemids, phage derivatives, animal viruses, and cosmids. A typical expression vector includes an expression regulatory sequence that can be used to control the expression of a desired nucleic acid sequence and is operably ligated to the nucleic acid sequence or its complementary sequence of the present invention. As used herein, “expression regulatory sequence” refers to an element that controls the transcription, translation, and expression of the target gene and is operably ligated to the target gene, and may be an origin of replication, promoter, marker gene, or translational regulatory element (including enhancers, operators, terminators, ribosome binding sites, etc.), and the choice of expression regulatory sequence depends on the host cell used. In recombinant expression vectors, “operably ligated” means that the nucleotide sequence of interest is ligated to the regulatory sequence in a manner that enables the expression of the nucleotide sequence. Those skilled in the art will know well how expression vectors containing the coding sequence of the fusion protein of the present invention and appropriate transcription / translation regulatory signals can be constructed. These methods include in vitro recombinant DNA techniques, DNA synthesis techniques, in vivo recombinant techniques, etc. The DNA sequence can be operably ligated to an appropriate promoter in the expression vector to guide mRNA synthesis. Representative examples of these promoters include the E. coli lac or trp promoter, the λ phage PL promoter, and eukaryotic promoters such as the CMV immediate early promoter, the HSV thymidine kinase promoter, the early and late SV 40 promoters, the reverse transcription virus LTR, and other known promoters that can control gene expression in prokaryotic or eukaryotic cells or their viruses. The expression vector further includes a ribosome binding site for translation initiation and a transcription terminator. In one embodiment, the expression vector may be a commercially available pCDF vector, and there are no other special requirements. Exemplarily, the nucleotide sequence encoding the optical probe and the expression vector are double digested with BamHI and EcoRI, respectively, and then the digest products are ligated to obtain a recombinant expression vector. The present invention is not particularly limited in terms of specific procedures and parameters for enzymatic cleavage and ligation, and conventional procedures and parameters in the art may be used.

[0047] After obtaining a recombinant expression vector, the vector is used to transform host cells to produce a protein or peptide containing the fusion protein. Such an importation process can be carried out by conventional techniques such as transformation or transfection, which are well known to those skilled in the art. The host cells described in this invention refer to cells that can receive and accommodate recombinant DNA molecules and serve as the site for amplification of recombinant genes. Ideal recipient cells must satisfy two conditions: availability and ease of proliferation. The “host cells” of this invention include prokaryotic and eukaryotic cells, and specifically include bacterial cells, yeast cells, insect cells, and mammalian cells. The host cells are preferably cells that are favorable for gene product expression or fermentation production, and such cells are well known in the art and commonly used. Specifically, examples include bacterial cells such as Escherichia coli, Streptomyces, and Salmonella tiphyllum; fungal cells such as yeast; plant cells; insect cells such as Drosophila S2 or Sf9; and animal cells such as CHO, COS, HEK293, HeLa cells, and Bose melanoma cells. The exemplary host cell used in the embodiments of the present invention is the Escherichia coli BL21-DE3 strain. Those skilled in the art will understand how to select appropriate vectors, promoters, enhancers, and host cells.

[0048] The methods for introducing DNA into host cells described in this invention are conventional methods in the art and include calcium phosphate or calcium chloride coprecipitation, DEAE-mannan-mediated transfection, lipofection, innate competence, chemical transfer, or electroporation. When the host is a prokaryote such as Escherichia coli, the above methods are preferably CaCl2 or MgCl2 methods, and the procedures 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. This invention involves introducing an expression vector into host cells, amplifying, expressing, and culturing the host cells into which the expression vector has been introduced, and then isolating the fructose-1,6-bisphosphate optical probe. The amplification, expression, and culturing of the host cells can be done using conventional methods. Depending on the type of host cell used, the culture medium may be a variety of common media. The cells are cultured under conditions suitable for host cell growth.

[0049] In the present invention, the optical probe is expressed intracellularly, on the cell membrane, or secreted extracellularly. If necessary, the recombinant protein may be isolated or purified by various isolation methods utilizing its physical, chemical, or other properties. In the present invention, the method for isolating the fructose-1,6-bisphosphate fluorescent protein is not particularly limited and any conventional isolation method for fusion proteins in the art may be used. These methods are well known to those skilled in the art and include, but are not limited to, conventional refolding, salting-out, centrifugation, osmotic cell disruption, sonication, ultracentrifugation, molecular sieve chromatography, adsorption chromatography, ion exchange chromatography, high-performance liquid chromatography (HPLC), and other liquid chromatography techniques and combinations thereof. In one embodiment, His-tagged affinity chromatography is used for the isolation of the optical probe.

[0050] The present invention further provides applications of the fructose-1,6-bisphosphate optical probe in real-time localization, quantitative detection, and high-throughput compound screening of fructose-1,6-bisphosphate. In one aspect, the fructose-1,6-bisphosphate optical probe performs real-time localization of fructose-1,6-bisphosphate by preferably attaching it to a signal peptide at different sites in a cell, introducing it into the cell, and detecting the intensity of the fluorescence signal in the cell; quantitative detection of the corresponding fructose-1,6-bisphosphate is performed by combining a standard drop curve of fructose-1,6-bisphosphate with the change in fluorescence signal. The change in fluorescence signal is indicated, for example, by a normalized fluorescence signal ratio, and in embodiments relating to cpYFP, ​​the ratio is the ratio between the ratio of the 485 nm fluorescence signal to the 420 nm fluorescence signal of the sample and the corresponding ratio of the control. The fructose-1,6-bisphosphate standard drop curve described in this invention is plotted based on the fluorescence signal of the fructose-1,6-bisphosphate optical probe under different fructose-1,6-bisphosphate concentrations. The fructose-1,6-bisphosphate optical probe described in this invention is directly introduced into cells and is more accurate in the process of real-time localization and quantitative detection of fructose-1,6-bisphosphate without requiring time-consuming sample processing. When the fructose-1,6-bisphosphate optical probe of this invention is used for high-throughput compound screening, different compounds can be added to cell culture medium, the change in fructose-1,6-bisphosphate content can be measured, and compounds that affect the change in fructose-1,6-bisphosphate content can be screened. The application of the fructose-1,6-bisphosphate optical probe described in this invention to real-time localization, quantitative detection of fructose-1,6-bisphosphate and high-throughput compound screening is for non-diagnostic and therapeutic purposes and does not include the diagnosis or treatment of disease.

[0051] The present invention further provides a detection kit comprising an optical probe, nucleic acid molecule, nucleic acid construct and / or cell as described herein. The kit further comprises other reagents necessary for the detection of fructose-1,6-bisphosphate. These other reagents are well known in the art and include, for example, buffers, cell culture media and fructose-1,6-bisphosphate standards. An example buffer is, for example, 100 mM HEPES and 100 mM NaCl, pH 7.4. In this specification, concentrations, content, percentages, and other numerical values ​​may all be expressed in range form. This range form is used solely for convenience and conciseness, and it should be understood that it should be interpreted flexibly to include not only the values ​​explicitly stated at the upper and lower limits of the range, but also all individual values ​​or sub-ranges included within the range.

[0052] (Several specific embodiments) Item 1, (1) Having the sequence shown in SEQ ID NO:1 and having mutations including amino acid modifications, substitutions, or deletions at one, two, three, four, five or more sites selected from the following: T148, R175, G176, G177, L178, E180, D181, V182, K183, N184, Q185, F271, (2) A shortened mutant having amino acids at positions 88-340 of (1), or (3) A sequence having at least 70% sequence identity with the sequence of (1) or (2), having the mutation described in (1), and retaining the ability to bind to fructose-1,6-bisphosphate; A fructose-1,6-bisphosphate-binding protein mutant characterized by the following: Preferably, the mutation sites are one, two, three, four, or five selected from any of the following groups: (a) T148, R175, G176, G177, L178, D181, V182, K183, N184, Q185, (b) G177, L178, E180, D181, V182, N184, Q185, F271, More preferably, the mutations include mutations at sites selected from any of the following groups: (1) G177 and L178, (5) D181 and V182, (6) E180 and D181, (7) N184 and Q185; or, the mutations include mutations at sites selected from any of the following groups: (1) G177, L178, (2) G 177, L178 and Q185, (3) G177, L178 and T148, (4) G177, L178 and F271, (5) D181, V182, (6) E180, D181, (7) N184, Q185, (8) N184, Q185, D181, V182, G177, (9) N184, Q185, G177, (10) N184, Q185, D181, V182, More preferably, T148 is mutated to S; G177 is mutated to A, I, L, M, or N, preferably to L or M; L178 is mutated to Y, F, or N, preferably to N; E180 is mutated to G; D181 is mutated to F, L, or E, preferably to E; V182 is mutated to F, L, or G, preferably to G; N184 is mutated to V; Q185 is mutated to S, A, T, D, or P, preferably to P; F271 is mutated to Y. More preferably, the mutations include mutations selected from any of the following groups: (1) G177A and L178Y, (2) G177I and L178F, (3) G177L and L178Y, (4) G177M and L178N, (5) G177N and L178Y, (6) G177M, L178N and Q185S, (7) G177M, L178N and Q185A, (8) G177M, L178N and Q185T, (9) G177M, L178N (10) G177, L178 and T148S, (11) G177, L178 and F271Y, (12) D181F and V182F, (13) D181L and V182L, (14) E180G and D181G, (15) N184V and Q185P, (16) N184V, Q185P, D181E, V182G and G177L, (17) N184V, Q185P and G177L, (18) N184V, Q185P, D181E and V182G.

[0053] Item 2, a fructose-1,6-bisphosphate optical probe comprising a fructose-1,6-bisphosphate-sensitive polypeptide and an optically active polypeptide, wherein the optically active polypeptide is located within the sequence of the fructose-1,6-bisphosphate-sensitive polypeptide, the fructose-1,6-bisphosphate-sensitive polypeptide is a fructose-1,6-bisphosphate-binding protein or a functional mutant thereof, and the optically active polypeptide is a fluorescent protein or a functional mutant thereof; wherein the functional mutant of the fructose-1,6-bisphosphate-binding protein has a mutation within 7 amino acids linked to the optically active polypeptide, and the functional mutant of the fluorescent protein has a mutation within 3 amino acids linked to the optically active polypeptide.

[0054] Item 3. The optical probe described in Item 1, characterized in that the fructose-1,6-bisphosphate-binding protein has the sequence shown in SEQ ID NO:1 or a shortened form having amino acids between positions 88 and 340, or a sequence having at least 70% sequence identity with those and retaining fructose-1,6-bisphosphate sensitivity.

[0055] Item 4. The optical probe described in Item 1, characterized in that the optically active polypeptide is located between residues 174-185 and / or 201-208 of the fructose-1,6-bisphosphate-sensitive polypeptide. Preferably, the optically active polypeptide is located at one or more of the following sites selected from the fructose-1,6-bisphosphate-sensitive polypeptide: 174 / 175, 174 / 176, 174 / 177, 174 / 178, 174 / 179, 174 / 180, 174 / 181, 174 / 182, 174 / 183, 174 / 184, 174 / 185, 155 / 176, 175 / 177, 175 / 178, 175 / 179, 175 / 180, 175 / 181, 175 / 182, 175 / 183, 175 / 184, 175 / 185, 176 / 177, 176 / 178, 176 / 179, 176 / 180, 176 / 181, 176 / 182, 176 / 183, 176 / 184, 176 / 185, 177 / 178, 177 / 179, 177 / 180, 177 / 181, 177 / 182, 177 / 183, 177 / 184, 177 / 185, 178 / 179, 178 / 180, 178 / 181, 178 / 182, 178 / 183, 178 / 184, 178 / 185, 179 / 180, 179 / 181, 179 / 182, 179 / 183, 179 / 184, 179 / 185, 180 / 181, 180 / 182, 180 / 183, 180 / 184, 180 / 185, 181 / 182, 181 / 182, 181 / 183, 181 / 184, 181 / 185, 182 / 183, 182 / 184, 182 / 185, 183 / 184, 183 / 185, 184 / 185, 201 / 202, 201 / 203, 201 / 204, 201 / 205, 201 / 206, 201 / 207, 201 / 208, 202 / 203, 202 / 204, 202 / 205, 202 / 206, 202 / 207, 202 / 208, 203 / 204, 203 / 205, 203 / 206, 203 / 207, 203 / 208, 204 / 205, 204 / 206, 204 / 207, 204 / 208, 205 / 206, 205 / 206, 205 / 207, 205 / 208, 206 / 207, 206 / 208 and / or 207 / 208; more preferably, the optically active polypeptide is located at one or more of the following sites selected from the fructose-1,6-bisphosphate-sensitive polypeptide: 176 / 177, 176 / 178, 177 / 181, 179 / 180, 182 / 184, 182 / 185, 203 / 206, 203 / 207 and 203 / 208; Preferably, a functional mutant of the fructose-1,6-bisphosphate-binding protein is one of those described in item 1; Preferably, the fluorescent protein is selected from yellow fluorescent protein, orange fluorescent protein, red fluorescent protein, green fluorescent protein, blue fluorescent protein, and apple red fluorescent protein, and the functional mutant of the fluorescent protein has a mutation in the amino acids at positions 1-3; more preferably, the fluorescent protein has the sequence shown in any one of SEQ ID NO:2-9, and the functional mutant of the fluorescent protein has a mutation in the amino acid at position 1. Preferably, a functional mutant of a fluorescent protein includes one in which the amino acid at position 1 of the fluorescent protein is mutated to I or V. Preferably, the fructose-1,6-bisphosphate-sensitive polypeptide is a truncated mutant having amino acids 88-340 of SEQ ID NO:1, and the optically active polypeptide is located at one or more of the following sites selected from the fructose-1,6-bisphosphate-sensitive polypeptide: 176 / 177, 176 / 178, 177 / 181, 179 / 180, 182 / 184, 182 / 185, 203 / 206, 203 / 207, and 203 / 208; more preferably, the optical probe has the sequence shown in one of SEQ ID NO:10-18; Preferably, the fructose-1,6-bisphosphate-sensitive polypeptide is a truncated mutant having amino acids 88-340 of SEQ ID NO:1, the optically active polypeptide is located at one or more of the following sites selected from the fructose-1,6-bisphosphate-sensitive polypeptide: 176 / 177, 177 / 181, 179 / 180, or 182 / 184, and the optical probe has one or more mutations selected from the following: T1 of the fructose-1,6-bisphosphate-sensitive polypeptide 48S, G177A, G177I, G177L, G177M, G177N, L178Y, L178F, L178Y, L178N, L178Y, L178N, E180G, D181E, D181F, D181L, D181G, V182F, V182L, V182G, N184V, Q185A, Q185S, Q185A, Q185T, Q185D, Q185P, F271Y, the 1-position amino acid of optically active polypeptides is mutated to V or I; Preferably, the optical probe has mutations represented in one of the following groups: (1) G177A and L178Y of the fructose-1,6-bisphosphate-sensitive polypeptide, (2) G177I and L178F of the fructose-1,6-bisphosphate-sensitive polypeptide, (3) G177L and L178Y of the fructose-1,6-bisphosphate-sensitive polypeptide, (4) G177M and L178N of the fructose-1,6-bisphosphate-sensitive polypeptide, (5) G177 N and L178Y, (6) G177M, L178N and Q185S of fructose-1,6-bisphosphate-sensitive polypeptide, (7) G177M, L178N and Q185A of fructose-1,6-bisphosphate-sensitive polypeptide, (8) G177M, L178N and Q185T of fructose-1,6-bisphosphate-sensitive polypeptide, (9) G177M, L178N and Q185D of fructose-1,6-bisphosphate-sensitive polypeptide, (10) G177, L178 and T148S of fructose-1,6-bisphosphate-sensitive polypeptide (11) Fructose-1,6-bisphosphate-sensitive polypeptides G177, L178 and F271Y, (12) Fructose-1,6-bisphosphate-sensitive polypeptides D181F and V182F, (13) Fructose-1,6-bisphosphate-sensitive polypeptides D181L and V182L, (14) Fructose-1,6-bisphosphate-sensitive polypeptides E180G and D181G, (15) Fructose-1,6-bisphosphate-sensitive polypeptides N184V and Q185P, (16) Fructose-1,6-bisphosphate-sensitive polypeptides (17) Mutations to the 1st amino acid of the fructose-1,6-bisphosphate-sensitive polypeptide N184V, Q185P, D181E, V182G, G177L, and optically active polypeptides to V, (18) Mutations to the 1st amino acid of the fructose-1,6-bisphosphate-sensitive polypeptide N184V, Q185P and G177L, and optically active polypeptides to I, (19) Fructose-1,(20) Mutations to the 1st amino acid I of the 6-bisphosphate-sensitive polypeptide N184V, Q185P, and G177L, and the optically active polypeptide, (20) Mutations to the 1st amino acid V of the fructose-1,6-bisphosphate-sensitive polypeptide N184V, Q185P, D181E, and V182G, and the optically active polypeptide.

[0056] Item 5, (a) Code sequence of the optical probe as described in any one of items 2-4, (b) Complementary sequence of (a) A nucleic acid molecule characterized by containing [something]. Nucleic acid constructs containing nucleic acid molecules as described in items 6 and 5; Preferably, the nucleic acid construct is a cloning vector, an expression vector, or a recombinant vector. Item 7, (1) Express an optical probe as described in any one of items 2-4; (2) Containing nucleic acid molecules as described in item 5; or (3) Including nucleic acid constructs as described in item 6. A host cell characterized by the following features. Item 8. (1) An optical probe as described in any one of items 2-4; (2) The nucleic acid sequence described in item 5, (3) Nucleic acid constructs as described in item 6, or (4) Host cells as described in item 7, A detection kit characterized by containing; The aforementioned detection kit optionally includes other reagents necessary for detecting fructose-1,6-bisphosphate using an optical probe; Preferably, the detection kit further comprises one or more reagents selected from: buffer, culture medium, and fructose-1,6-bisphosphate standard.

[0057] A method for preparing an optical probe according to any one of items 2-4, comprising providing host cells as described in item 9 and item 7, culturing the host cells under conditions in which the optical probe is expressed, and isolating the optical probe. Item 10, detection of fructose-1,6-bisphosphate in a sample, screening of compounds, or use in host cells of any one of items 2-4, nucleic acid sequences, nucleic acid constructs, or host cells, as described in item 5, item 6, or as described in item 7; Preferably, Detection of fructose-1,6-bisphosphate in a sample involves contacting the sample with the aforementioned optical probe or host cells, detecting the optical change of the optically active polypeptide, and detecting fructose-1,6-bisphosphate in the sample based on the optical change of the optically active peptide; The screening of the aforementioned compounds includes contacting the candidate compound with the optical probe or host cell in a system containing fructose-1,6-bisphosphate, detecting the optical change of the optically active polypeptide, and screening the candidate compound based on the optical change of the optically active peptide; preferably, the screening of the aforementioned compounds includes contacting the candidate compound with the host cell in a system containing fructose-1,6-bisphosphate, and the optical change of the optically active polypeptide indicating whether the candidate compound can regulate the intracellular uptake of fructose-1,6-bisphosphate; The intracellular and / or extracellular localization of fructose-1,6-bisphosphate comprises contacting a system containing fructose-1,6-bisphosphate with the optical probe or the host cell and detecting the optical change of the optically active polypeptide; More preferably, the system is a solution system, a cell system, or an intracellular system. [Examples]

[0058] The fructose-1,6-bisphosphate optical probes provided in the present invention will be described in detail below in conjunction with examples, but these should not be construed as limiting the scope of protection of the present invention. I. Experimental Materials and Reagents The examples mainly utilize conventional genetic engineering molecular biology cloning methods, cell culture methods, and imaging methods, which are well known to those skilled in the art. Examples include "Molecular Biology Laboratory Reference Manual" by Jane Roskams et al.; "Molecular Cloning Laboratory Guide" by J. Sambrook and DWRussell, translated by Huang Peitang et al. (3rd edition, August 2002, Science Press, Beijing); "Animal Cell Culture: Basic Techniques Guide" by Frasier et al., translated by Zhang Jingbo, Xu Cunshuan et al. (5th edition); and "A Concise Cell Biology Laboratory Guide" by JSBonifacion, M. Dassault et al., translated by Zhang Jingbo et al.

[0059] The pCDF-cpYFP and pCDF-fructose-1,6-bisphosphate-binding protein plasmids used in the examples were constructed by the Protein Laboratory at East China University of Science and Technology, and the pCDF plasmid vector was purchased from Invitrogen. All primers used for PCR were synthesized and purified by Shanghai Jierui Bioengineering Technology Co., Ltd. and BGI, and their correctness was verified by mass spectrometry. The expression plasmids constructed in the examples were sequenced by BGI and JIELI Sequencing Company. The TaqDNA polymerase used in each example was purchased from Dongsheng Biotechnology, the pfuDNA polymerase from Tiangen Biochemical Technology (Beijing) Co., Ltd., and the primeSTAR DNA polymerase from TaKaRa. The three polymerases were also provided with their corresponding polymerase buffers and dNTPs at the time of purchase. Restriction enzymes such as BamHI, BglII, HindIII, NdeI, XhoI, EcoRI, and SpeI, as well as T4 ligases and T4 phosphorylase (T4 PNK), were purchased from Fermentas, with corresponding buffers provided at the time of purchase. The Lip2000 transfection reagent kit was purchased from Invitrogen. Compounds such as fructose-1,6-bisphosphate were purchased from Sigma. Unless otherwise specified, chemical reagents such as inorganic salts were purchased from Sigma-Aldrich. HEPES salt, ampicillin (Amp), and puromycin were purchased from Amersco. 96-well detection black plates and 384-well fluorescence detection black plates were purchased from Grenier.

[0060] 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 clone strain Mach1 was purchased from Invitrogen. The nickel affinity chromatography column and desalting column packing material were from GE Healthcare. The main instruments used in the examples include a Biotek Synergy 2 multi-function microplate reader (Bio-Tek, USA), an X-15R high-speed refrigerated centrifuge (Beckman, USA), a Microfuge 22R desktop high-speed refrigerated centrifuge (Beckman, USA), a PCR amplifier (Biometra, Germany), an ultrasonic disruptor (Ningbo Xinzhi Company), a nucleic acid electrophoresis system (Shenneng Bocai Company), a fluorescence spectrophotometer (Varian, USA), a CO2 incubator for cell culture (SANYO), and an inverted fluorescence microscope (Nikon, Japan).

[0061] II. Molecular Biological Techniques and Cellular Experimental Techniques II.1 Polymerase Chain Reaction (PCR): 1. PCR for target fragment amplification: This method was primarily used for the amplification of gene fragments and colony PCR identification of positive clones. The PCR amplification reaction system was as follows: 0.5–1 μL of template sequence, 0.5 μL of forward primer (25 μM), 0.5 μL of reverse primer (25 μM), 5 μL of 10×pfu buffer, 0.5 μL of pfu DNA polymerase, 1 μL of dNTP (10 mM), 41.5–42 μL of sterile ultrapure water (ddH2O), for a total volume of 50 μL. The PCR amplification program consisted of denaturation at 95°C for 2–10 minutes, 30 cycles (30–45 seconds at 94–96°C, 30–45 seconds at 50–65°C, and a constant time at 72°C (600 bp / min)), and extension at 72°C for 10 minutes. 2. PCR for amplifying long fragments (>2500bp): The long fragment amplification used in this invention primarily involves amplifying the vector by reverse PCR, which is the technique used to obtain site-directed mutations in the following examples. A reverse PCR primer is designed at the mutation site, containing the mutated nucleotide sequence at the 5' end of one of the primers. The amplified product contains the corresponding mutation site. The PCR reaction system for long fragment amplification is as follows: 1 μL of template sequence (10 pg-1 ng), 0.5 μL of forward primer (25 μM), 0.5 μL of reverse primer (25 μM), 10 μL of 5 × PrimerSTAR buffer, 0.5 μL of PrimerSTAR DNA polymerase, 4 μL of dNTPs (2.5 mM), 33.5 μL of sterile ultrapure water (ddH2O), for a total volume of 50 μL. The PCR amplification programs were either denaturation at 95°C for 5 minutes, 30 cycles (10 seconds at 98°C, 5-15 seconds at 50-68°C, and a constant time at 72°C (1000 bp / min)), and extension at 72°C for 10 minutes, or denaturation at 95°C for 5 minutes, 30 cycles (10 seconds at 98°C, and a constant time at 68°C (1000 bp / min)), and extension at 72°C for 10 minutes.

[0062] II.2 Endonuclease digestion reaction: The system for dual enzymatic digestion of plasmid vectors is as follows: 20 μL (approximately 1.5 μg) of plasmid vector, 5 μL of 10× buffer, 11-2 μL of restriction enzyme, 21-2 μL of restriction enzyme, and a total volume of 50 μL with sterile ultrapure water. Reaction conditions: 37°C, 1-7 hours. II.3 Phosphorylation of the 5' End of DNA Fragments Plasmids and genomes extracted from microorganisms contain phosphate groups at their ends, but PCR products do not. Therefore, a phosphate group addition reaction is necessary at the 5' end of the PCR product, and only DNA molecules with phosphate groups at their ends can undergo the ligation reaction. The phosphorylation reaction system is as follows: 5-8 μL of PCR product fragment DNA sequence, 1 μL of 10×T4 ligase buffer, 1 μL of T4 polynucleotide kinase (T4 PNK), 0-3 μL of sterile ultrapure water, for a total volume of 10 μL. The reaction conditions were 37°C for 30 minutes to 2 hours, followed by inactivation at 72°C for 20 minutes.

[0063] II.4 Ligation reaction between target fragment and vector The ligation methods between different fragments and vectors vary, and in this invention, the following three ligation methods were used. 1. Blunt-end ligation of blunt-end short-chain fragments and linearization vectors The principle of this method is to obtain recombinant plasmids by phosphorylating the blunt-end products obtained by PCR at the 5' end of the DNA fragment under the action of T4 PNK, and then ligating them with a linearization vector under the action of PEG4000 and T4 DNA ligase. The homologous recombination ligation system is as follows: 4 μL of DNA fragment treated with T4 PNK, 4 μL of linearization vector fragment, 1 μL of PEG4000, 1 μL of 10×T4 ligase buffer, 1 μL of T4 DNA ligase, for a total of 10 μL. Reaction conditions: 22°C, 30 minutes. 2. Ligation of sticky-end DNA fragments and sticky-end vector fragments DNA fragments cleaved by restriction enzymes typically generate protruding sticky ends, which can then be ligated with a vector fragment containing sticky ends with a complementary sequence to form a recombinant plasmid. The ligation reaction system is as follows: 1-7 μL of PCR product fragment DNA after restriction digestion, 0.5-7 μL of plasmid after restriction digestion, 1 μL of 10×T4 ligase buffer, 1 μL of T4 DNA ligase, and a total volume of 10 μL with sterile ultrapure water. Reaction conditions: 16°C, 4-8 hours. 3. Ligation reaction of autocirculation of 5' phosphorylated DNA fragment products into which site-directed mutations have been introduced by reverse PCR. A recombinant plasmid was obtained by ligating the 3' and 5' ends of a linearization vector to a 5'-terminus phosphorylated DNA fragment via an auto-circularization ligation reaction. The auto-circularization ligation reaction system was as follows: 10 μL phosphorylation system, 0.5 μL of T4 ligase (5 U / μL), for a total volume of 10.5 μL. Reaction conditions: 16°C, 4-16 hours.

[0064] II.5 Preparation and Transformation of Competent Cells Preparation of competent cells: 1. A single colony (such as Mach1) was collected, inoculated into 5 mL of LB medium, and shaken overnight at 37°C. 2. 0.5-1 mL of the bacterial suspension cultured overnight was transferred to 50 mL of LB medium and incubated at 37°C and 220 rpm for 3-5 hours until the OD600 reached 0.5. 3. The cells were pre-cooled in an ice bath for 2 hours. 4. The mixture was centrifuged at 4°C and 4000 rpm for 10 minutes. 5. Discard the supernatant, resuspend the cells in 5 mL of pre-cooled buffer, homogenize the cells, and then add resuspension buffer to bring the final volume to 50 mL. 6. I took an ice bath for 45 minutes. 7. The bacteria were centrifuged at 4°C and 4000 rpm for 10 minutes, and then resuspended in 5 mL of ice pre-cooled storage buffer. 8. 100 μL of bacterial solution was placed in each EP tube and frozen at -80°C or in liquid nitrogen. Resuspension buffer: CaCl2 (100mM), MgCl2 (70mM), NaAc (40mM) Storage buffer: 0.5 mL DMSO, 1.9 mL 80% glycerin, 1 mL 10 × CaCl2 (1M), 1 mL 10 × MgCl2 (700 mM), 1 mL 10 × NaAc (400 mM), 4.6 mL ddH2O

[0065] Transformation of competent cells: 1. 100 μL of competent cells were thawed in an ice bath. 2. An appropriate volume of ligation product was added, gently blown until uniform, and then the cell was frozen in an ice bath for 30 minutes. Typically, the volume of the added ligation product is less than 1 / 10 of the competent cell volume. 3. The bacterial solution was subjected to a heat shock treatment in a 42°C water bath for 90 seconds, and then immediately transferred to an ice bath and left for 5 minutes. 4. Add 500 μL LB and culture in a constant temperature shaker at 37°C and 200 rpm for 1 hour. 5. The bacterial suspension was centrifuged at 4000 rpm for 3 minutes, leaving 200 μL of supernatant. The bacteria were then uniformly mixed in, and the supernatant was spread evenly onto the surface of an agar plate containing the appropriate antibiotic. The plate was inverted and incubated overnight in a 37°C constant temperature incubator.

[0066] II.6 Protein Expression, Purification, and Fluorescence Detection 1. BL21(DE3) cells were transformed with an expression vector (e.g., a pCDF-based fructose-1,6-bisphosphate optical probe expression vector), inverted, and cultured overnight. Clones were then transferred from the plate to a 250 ml Erlenmeyer flask, placed in a 37°C shaker, and cultured at 220 rpm until OD=0.4-0.8. 1 / 1000 (v / v) IPTG (1M) was added, and expression was induced at 18°C ​​for 24-36 hours. 2. Once expression induction was complete, the cells were collected by centrifugation at 4000 rpm for 30 minutes, resuspended in 50 mM phosphate buffer, and sonicated until the bacteria became clear. The cells were then centrifuged at 9600 rpm at 4°C for 20 minutes. 3. The supernatant was centrifuged and the protein was purified by passing it through a self-made nickel column affinity chromatography column. The nickel column affinity chromatography-purified protein was then passed through a self-made desalting column to obtain the protein dissolved in 100 mM HEPES buffer (pH 7.4). 4. After identifying the purified proteins by SDS-PAGE, the probes were diluted in a protein solution with a final concentration of 0.2–5 μM using the measurement buffer (100 mM HEPES, 100 mM NaCl, pH 7.4). A stock solution of fructose-1,6-bisphosphate was prepared with the measurement buffer (100 mM HEPES, 100 mM NaCl, pH 7.4) to a final concentration of 50 mM. 5. 100 μl of 1 μM protein solution was taken, incubated at 37°C for 10 minutes, and titrated with fructose-1,6-bisphosphate. The fluorescence intensity of the protein emitted at 528 nm after 420 nm photoexcitation and the fluorescence intensity of the protein emitted at 528 nm after 485 nm photoexcitation were measured. Fluorescence excitation and emission measurements of the samples were completed using a multifunctional fluorescence microplate reader. 6. 100 μl of 1 μM protein solution was taken, incubated at 37°C for 10 minutes, and fructose-1,6-bisphosphate was added. The absorption and fluorescence spectra of the protein were measured. The absorption and fluorescence spectra of the samples were measured using a spectrophotometer and a fluorescence spectrophotometer.

[0067] II.7 Transfection of Mammalian Cells and Fluorescence Detection 1. A fructose-1,6-bisphosphate optical probe plasmid based on pCDNA3.1+ was transfected into HEK293 cells using the transfection reagent Lipofectamine2000 (Invitrogen), and the cells were cultured in a cell culture incubator at 37°C and 5% CO2. Fluorescence detection was performed 24–36 hours after the exogenous gene had fully expressed. 2. Once expression induction was complete, the adhered HEK293 cells were rinsed three times with PBS, placed in HBSS solution, and detected using a fluorescence microscope and a microplate reader, respectively. [Examples]

[0068] Fructose-1,6-bisphosphate-binding protein plasmid The CggR truncated gene within the Bacillus subtilis gene was amplified by PCR, the PCR product was recovered after gel electrophoresis, and digested with HindIII and XhoI, while the pCDF vector was simultaneously double-digested correspondingly. After ligating with T4 DNA ligase, DH5α was transformed using the product, and the transformed DH5α was spread on an LB plate (streptomycin 100 ug / mL) and cultured overnight at 37°C. Plasmids of the grown DH5α transformants were extracted and identified by PCR. After the positive plasmid was verified by sequencing, the next plasmid construction was carried out. [Examples]

[0069] Expression and detection of cpYFP optical probes at different insertion sites In this example, based on pCDF-CggR(88-340), the following sites were selected to insert cpYFP according to the crystal structure of the fructose-1,6-bisphosphate-binding protein, and the corresponding pCDF-CggR(88-340)-cpYFP plasmids were obtained: 174 / 175, 174 / 176, 174 / 177, 174 / 178, 174 / 179, 174 / 180, 174 / 181, 174 / 182, 174 / 183, 174 / 184, 174 / 185, 155 / 176, 175 / 1 77, 175 / 178, 175 / 179, 175 / 180, 175 / 181, 175 / 182, 175 / 183, 175 / 184, 175 / 185, 176 / 177, 176 / 178, 176 / 179, 176 / 180, 176 / 181, 176 / 182, 176 / 183, 176 / 184, 176 / 185, 177 / 178, 177 / 179, 177 / 180, 177 / 181, 177 / 182, 177 / 183, 177 / 184, 177 / 185, 178 / 179, 178 / 180, 178 / 1 81, 178 / 182, 178 / 183, 178 / 184, 178 / 185, 179 / 180, 179 / 181, 179 / 182, 179 / 183, 179 / 184, 179 / 185, 180 / 181, 180 / 182, 180 / 183, 180 / 184, 180 / 185, 181 / 182, 181 / 182, 181 / 183, 181 / 184, 181 / 185, 182 / 183, 182 / 184, 182 / 185, 183 / 184, 183 / 185, 184 / 185, 201 / 202, 201 / 2 03, 201 / 204, 201 / 205, 201 / 206, 201 / 207, 201 / 208, 202 / 203, 202 / 204, 202 / 205, 202 / 206, 202 / 207, 202 / 208, 203 / 204, 203 / 205, 203 / 206, 203 / 207, 203 / 208, 204 / 205, 204 / 206, 204 / 207, 204 / 208, 205 / 206, 205 / 206, 205 / 207, 205 / 208, 206 / 207, 206 / 208 and / or 207 / 208.

[0070] Using PCR, a DNA fragment of cpYFP was generated, and simultaneously, a homologous sequence of the cpYFP terminus was introduced through the 5' end of a primer. PCR amplification produced a linearized pCDF-fructose-1,6-bisphosphate-binding protein vector, which had sequences (15 bp to 20 bp) at its 5' and 3' ends that perfectly matched both ends of cpYFP. Homologous recombination occurred between the linearized pCDF-CggR(88-340) and the cpYFP fragment under the action of Hieff Clone Enzyme. DH5α was transformed with the product, and the transformed DH5α was spread on an LB plate (streptomycin 100 ug / mL) and cultured overnight at 37°C. Plasmids of positive clones identified by PCR were extracted and sequenced. Sequencing was completed by JIELI Sequencing Company. After sequencing verification, the recombinant plasmid was transformed into BL21(DE3) to induce expression, and the protein was purified. SDS-PAGE electrophoresis revealed a protein size of approximately 57 kDa. This size was consistent with the size of the CggR(88-340)-cpYFP fusion protein containing the His-tag purified tag, which is expressed by pCDF-CggR(88-340)-cpYFP. The results are shown in Figure 1.

[0071] Using the lysated supernatant of E. coli expressing the CggR(88-340)-cpYFP fusion protein, a fructose-1,6-bisphosphate response screening was performed. The detection signal of the fusion fluorescent protein containing 1 mM fructose-1,6-bisphosphate was divided by the detection signal of the fusion fluorescent protein without fructose-1,6-bisphosphate. The results are shown in Figure 2. From the detection results, it was found that optical probes that reacted 1.4 times or more to fructose-1,6-bisphosphate were those inserted at the 176 / 177, 176 / 178, 177 / 181, 179 / 180, 182 / 184, 182 / 185, 203 / 206, 203 / 207 and 203 / 208 sites or the corresponding amino acid sites of the family proteins. The screening results are shown in the following table: [Examples]

[0072] Expression and detection of cpGFP optical probes at different insertion sites Following the method of Example 2, fructose-1,6-bisphosphate green fluorescent protein probes were constructed by replacing cpYFP with cpGFP. As shown in Figure 3, detection results revealed that optical probes that reacted with fructose-1,6-bisphosphate at a ratio of 1.4 or more were those inserted at the 176 / 177, 176 / 178, 177 / 181, 182 / 184, 182 / 185, 203 / 204, 203 / 205, 203 / 206, 203 / 207 and 203 / 208 sites or the corresponding amino acid sites of the family proteins. [Examples]

[0073] Expression and detection of cpBFP optical probes at different insertion sites Following the method of Example 2, a fructose-1,6-bisphosphate blue fluorescent protein probe was constructed by replacing cpYFP with cpBFP. As shown in Figure 4, detection results revealed that optical probes that reacted with fructose-1,6-bisphosphate at a ratio of 1.5 or more were those inserted at the 176 / 177, 176 / 178, 177 / 178, 177 / 181, 178 / 181, 182 / 184, 182 / 185, 203 / 205, 203 / 206, 203 / 207 and 203 / 208 sites or the corresponding amino acid sites of the family proteins. [Examples]

[0074] Expression and detection of CPYFP optical probes in linker mutations PCDF-CggR(88-340)-176 / 177-CPYFP, PCDF-CggR-176 / 178(88-340)-CPYFP, PCDF-CggR(88- 340)-177 / 181-CPYFP, PCDF-CggR(88-340)-179 / 180-CPYFP, PCDF-CggR(88-340)-182 / 184-C Optical probe mutants were constructed based on PYFP, PCDF-CggR(88-340)-182 / 185-CPYFP, PCDF-CggR(88-340)-203 / 206-CPYFP, PCDF-CggR(88-340)-203 / 207-CPYFP, and PCDF-CggR-203 / 208(88-340)-CPYFP. Plasmid PCDF-CggR(88-340)-176 / 177-CPYFP, PCDF-CggR(88-340)-176 / 178-CPYFP, PCDF-CggR(88-340)- 177 / 181-CPYFP, PCDF-CggR(88-340)-178 / 180-CPYFP, PCDF-CggR(88-340)-182 / 184-CPYFP, PCDF-CggR( 88-340)-182 / 185-CPYFP, PCDF-CggR(88-340)-203 / 206-CPYFP, PCDF-CggR(88-340)-203 / 207-CPYFP, and PCDF-CggR(88-340)-203 / 208-CPYFP were linearized by PCR. Random NNK mutations were introduced into the linker in the primers. The resulting PCR products were ligated with phosphorus using PNK, T4 DNA ligase and PEG4000, transformed, and a library was constructed. Detection was performed, and mutants with a response to fructose-1,6-bisphosphate more than twofold were screened. Sequencing was completed by JIELI Sequencing Company. The screened mutants are shown in the following table: [Table 1] [Examples]

[0075] Performance of optical probes and their mutants Exemplary, three purified fructose-1,6-bisphosphate optical probes, numbers 4, 15, and 17 of Example 5, were treated with 0 mM and 5 mM fructose-1,6-bisphosphate for 10 minutes, respectively, and their fluorescence spectra were detected using a fluorescence spectrophotometer. Excitation spectrum measurement: The excitation spectrum was recorded in the excitation range of 370 nm to 510 nm and the emission wavelength of 530 nm, with readings taken every 5 nm. The results showed that the probe had two excitation peaks at approximately 410 nm and 490 nm. Measurement of emission spectra: The excitation wavelengths were fixed at 420 nm and 460 nm, respectively. Emission spectra were recorded at 470-600 nm and 490-600 nm, and read at 5 nm intervals. The excitation and emission spectra are shown in Figure 5. Fructose-1,6-bisphosphate was detected in a concentration gradient (0-5 mM) using 20 purified fructose-1,6-bisphosphate optical probes numbered 1-20 from Example 5. After processing the purified probes for 10 minutes, the change in the ratio of fluorescence intensity at 420 nm excitation and 528 nm emission to fluorescence intensity at 485 nm excitation and 528 nm emission was detected. The results are shown in Figure 6, and the Kd (binding constant) of the 20 fructose-1,6-bisphosphate optical probes is 37 μM, 25 μM, 73 μM, 11 μM, 38 μM, 12 μM, 36 μM, 22 μM, 32 μM, 74 μM, 12 μM, 5.7 μM, 6.2 μM, 3.3 μM, 360 μM, 240 μM, 769 μM, 1524 μM, 117 μM, 148 μM, and 92 μM, respectively. The 20 purified fructose-1,6-bisphosphate optical probes (numbers 1-20) from Example 5 were subjected to reactivity detection with eight similar substrates, and the results showed good specificity, as shown in Figure 7. [Examples]

[0076] Localization of optical probes in intracellular organelles and performance of optical probes within intracellular organelles In this example, different localization signal peptides were fused with optical probes 182 / 184 (CggR:N184V / Q185P / D181E / V182G CPYFP:Y1V, number 20 in Example 5), and the optical probes were localized to various organelles. Optical probe plasmids fused with different localization signal peptides were transfected into HeLa cells for 36 hours, washed with PBS, placed in HBSS solution, and fluorescence detection was performed under FITC channel conditions using an inverted fluorescence microscope. The results are shown in Figure 8. Fructose-1,6-bisphosphate optical probes can be localized to intracellular organelles such as the cytoplasm, nucleus, mitochondria, and nucleoplasm (excluding the nucleolus) by fusing with localization signal peptides of different specificities. Fluorescence was observed in different intracellular structures, and the distribution and intensity of fluorescence differed. H1299 cells were transfected with a cytoplasmic optical probe plasmid for 36 hours, washed with PBS, and placed in HBSS solution containing 0 mM 3-BrPA and 0.5 mM 3-BrPA. The change in the ratio of fluorescence intensity at 420 nm excitation and 528 nm emission to fluorescence intensity at 485 nm excitation and 528 nm emission was measured for 30 minutes. The results are shown in Figure 9. The 420 / 485 ratio in the 3-BrPA-treated samples gradually increased, reaching a maximum of 3.7 times the initial value, while the 420 / 485 ratio in the control group without 3-BrPA treatment remained unchanged at 0.63. [Examples]

[0077] High-throughput compound screening using optical probes in living cells In this example, HeLa cells expressing 182 / 184 (CggR:N184V / Q185P / D181E / V182G CPYFP:Y1V) in the cytoplasm were used for high-throughput compound screening. Transfected HeLa cells were washed with PBS, placed in HBSS solution (without fructose-1,6-bisphosphate) for 1 hour, and then treated with 10 μM of the compound for 1 hour. Various compounds were added dropwise to each sample. Using a microplate reader, the ratio of fluorescence intensity at 420 nm excitation and 528 nm emission to fluorescence intensity at 485 nm excitation and 528 nm emission was recorded. Normalization was performed using untreated samples as a control. The results are shown in Figure 10. Of the 1,000 compounds used, the vast majority had little effect on the intracellular concentration of fructose-1,6-bisphosphate. 38 compounds were able to increase the intracellular concentration of fructose-1,6-bisphosphate, and 13 compounds were able to significantly decrease it. [Examples]

[0078] Quantitative detection of fructose-1,6-bisphosphate in blood using an optical probe. In this example, fructose-1,6-bisphosphate in mouse and human blood supernatants was analyzed using purified 179 / 180 (CggR:E180G / D181G) with a Kd of approximately 1 μM. After mixing 179 / 180 (CggR:E180G / D181G) with diluted blood supernatant for 10 minutes, a microplate reader was used to detect the fluorescence intensity at 420 nm excitation and 528 nm emission, and the ratio of fluorescence intensity at 485 nm excitation and 528 nm emission. The results are shown in Figure 11, and the fructose-1,6-bisphosphate content in the blood of healthy individuals is approximately 6 μM. As can be seen from the above examples, the fructose-1,6-bisphosphate optical probe provided by the present invention is a relatively small protein, easy to mature, exhibits large dynamic changes in fluorescence, has excellent specificity, can be expressed in cells using genetic engineering methods, can localize fructose-1,6-bisphosphate in real time both inside and outside cells, can be detected quantitatively and with high throughput, and can also be used for high-throughput compound screening.

[0079] Other Embodiments Various embodiments have been described herein. However, it should be understood that various modifications that a person skilled in the art can learn by reading this specification without departing from the concept and scope of the present invention should also be included in the appended claims.

[0080] The arrangement of this specification 1>MNQLIQAQKKLLPDLLVVMQKRFEILQYIRLTEPIGRRSLSASLGISERVLRGEVQFLKEQNLVEIKTNGMTLTEEGYSLLSILEDTMKDVLGLTVLEKTLKERLNLRDAIIVSGDSDQSPWVKKELGRAAVACMKKRFSGKNIVAVTGGTTIEAVAEMMTPDSKNREL LFVPARGGLGENVKNQANTICAHMAEKASGTYRLLFVPGQLSQGAYSSIIEEPSVKEVLTTIKSASMLVHGIGEAKTMAERRNTPLEDLKKIDENHAVTEAFGYYFNSDGEVVHKVHSVGMQLDDLDAIPDIIAVAGGSSKAGAIEAYFKKPRNTVLVTDEGAAKKLLRDE 2>YNSDNVYIMADKQKNGIKTNFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSFQSVLSKDPNEKRDHMVLLEFVTAAGITLGMDELYNVDGGSGGTGSKGEELFTGVVPILVELDG DVNGHKFSVSGEGEGDATYGKLTLKLICTTGKLPVPWPTLVTTLGYGLKCFARYPDHMKQHDFFKSAMPYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYN 3>VSERMYPEDGVLKSEIKKGLRLKDGGHYAAEVKTTYKAKKPVQLPGAYIVDIKLDIVSHNEDYTIVEQCERAEGRHPTGGRDELYKGGTGGSLVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEEGEGEGRPYEAFQTAKLKVTKGGPLPFAWDILSPQFTYGSKAYIKHPADIPDYFKLSFPEGFRWERVMNFEDGGIIHVNQDSSLQDGVFIYKVKLRGTNFPPDGPVMQKKTMGWEA 4>MGGRSKKPAKNLKMPGVYYVDRRLERIKEADKETYVEQHEVAVARYCDLPSKLGHKLNGGTGGSMVSKGEELIKENMHMKLYMEGTVNNHHFKCTSEGKPYEGTQTMRIKVVEGGPLPFAFDILATSFMYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGGVLTATQDTSLQDGCLIYNVKIRGVNFPSNGPVMQKKTLGWEASTEMLYPADGGLEGRSDMALKLVGGGHLICNLKTTYRSKK 5>MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEEGEGEGRPYEGTQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFEDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALKGEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERAEGRHSTGGMDELYK 6>NVYIKADKQKNGIKANFKIRHNIEDGGVQLAYHYQQNTPIGDGPVLLPDNHYLSVQSILSKDPNEKRDHMVLLEFVTAAGITLGMDELYKGGTGGSMVSKGEELFTGVVPIQVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICCTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYIQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKIDFKEDGNILGHKLEYN 7>NVYIKADKQKNGIKANFKIRHNIEGGGVQLAYHYQQNTPIGDGPVLLPDNHYLSVQSILSKDPNEKRDHMVLLEFVTAAGITLGMDELYKGGTGGSESMVSKGEELFTGVVPIQVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLSHGVQCFSRYPDHMKQHDFFKSAMPGGYIQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKIDFKEDGNILGHKLEYN 8>MSELITENMHMKLYMEGTVNNHHFKCTSEGEGKPYEGTQTMRIKVVEGGPLPFAFDILATSFMYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGGVLTATQDTSLQDGCLIYNVKIRGVNFPPSNGPVMQKKTLGWEASTEMLYPADGGLEGRADMALKLVGGGHLICNLKTTYRSKKPAKNLKMPGVYYVDRRLIKEADKETYVEQHEVAVARYCDLPSKLGHKLN 9>VSERMYPEDGALKSEIKKGLRLKDGGHYAAEVKTTYKAKKPVQLPGAYIVDIKLDIVSHNEDYTIVEQCERAEGRHSTGGMDELYKGGTGGSLVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEEGEGEGRPYEAFQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYIKHPADIPDYFKLSFPEGFRWERVMNFEDGGIIHVNQDSSLQDGVFIYKVKLRGTNFPPDGPVMQKKTMGWEA 10>SNAKDVLGLTLLEKTLKERLNLKDAIIVSGDSDQSPWVKKEMGRAAVACMKKRFSGKNIVAVTGGTTIEAVAEMMTPDSKNRELLFVPARGYNSDNVYIMADKQKNGIKTNFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSFQSVLSKDPNEKRDHMVLLEFVTAAGITLGMDELYNVDGGSGGTGSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKLICTTGKLPVPWPTLVTTLGYGLKCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNMNGEDVKNQANTICAHMAEKASGTYRLLFVPGQLSQGAYSSIIEEPSVKEVLNTIKSASMLVHGIGEAKTMAQRRNTPLEDLKIDDNDAVTEAFGYYFNADGEVVHKVHSVGMQLDDIDAIPDIIAVAGGSSKAEAIEAYFKKPRNTVLVTDEGAAKKLLRDE 11>SNAKDVLGLTLLEKTLKERLNLKDAIIVSGDSDQSPWVKKEMGRAAVACMKKRFSGKNIVAVTGGTTIEAVAEMMTPDSKNRELLFVPARGYNSDNVYIMADKQKNGIKTNFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSFQSVLSKDPNEKRDHMVLLEFVTAAGITLGMDELYNVDGGSGGTGSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKLICTTGKLPVPWPPLVTTLGYGLKCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNMNGEDVKNAANTICAHMAEKASGTYRLLFVPGQLSQGAYSSIIEEPSVKEVLNTIKSASMLVHGIGEAKTMAQRRNTPLEDLKKIDDNDAVTEAFGYYFNADGEVVHKVHSVGMQLDDIDAIPDIIAVAGGSSKAEAIEAYFKKPRNTVLVTDEGAAKKLLRDE 12>SNAKDVLGLTLLEKTLKERLNLKDAIIVSGDSDQSPWVKKEMGRAAVACMKKRFSGKNIVAVTGGTTIEAVAEMMTPDSKNRELLFVPARGYNSDNVYIMADKQKNGIKTNFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSFQSVLSKDPNEKRDHMVLLEFVTAAGITLGMDELYNVDGGSGGTGSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKLICTTGKLPVPWPPLVTTLGYGLKCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNMNGEDVKNTANTICAHMAEKASGTYRLLFVPGQLSQGAYSSIIEEPSVKEVLNTIKSASMLVHGIGEAKTMAQRRNTPLEDLKKIDDNDAVTEAFGYYFNADGEVVHKVHSVGMQLDDIDAIPDIIAVAGGSSKAEAIEAYFKKPRNTVLVTDEGAAKKLLRDE 13>SNAKDVLGLTLLEKTLKERLNLKDAIIVSGDSDQSPWVKKEMGRAAVACMKKRFSGKNIVAVTGGTTIEAVAEMMTPDSKNRELLFVPARGYNSDNVYIMADKQKNGIKTNFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSFQSVLSKDPNEKRDHMVLLEFVTAAGITLGMDELYNVDGGSGGTGSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKLICTTGKLPVPWPPLVTTLGYGLKCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNMNGEDVKNDANTICAHMAEKASGTYRLLFVPGQLSQGAYSSIIEEPSVKEVLNTIKSASMLVHGIGEAKTMAQRRNTPLEDLKIDDNDAVTEAFGYYFNADGEVVHKVHSVGMQLDDIDAIPDIIAVAGGSSKAEAIEAYFKKPRNTVLVTDEGAAKKLLRDE 14>SNAKDVLGLTLLEKTLKERLNLKDAIIVSGDSDQSPWVKKEMGRAAVACMKKRFSGKNIVAVTGGTTIEAVAEMMTPDSKNRELLFVPARGLLGEEGVNSDNVYIMADKQKNGIKTNFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSFQSVLSKDPNEKRDHMVLLEFVTAAGITLGMDELYNVDGGSGGTGSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKLICTTGKLPVPWPTLVTTLGYGLKCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNVPANTICAHMAEKASGTYRLLFVPGQLSQGAYSSIIEEPSVKEVLNTIKSASMLVHGIGEAKTMAQRRNTPLEDLKIDDNDAVTEAFGYYFNADGEVVHKVHSVGMQLDDIDAIPDIIAVAGGSSKAEAIEAYFKKPRNTVLVTDEGAAKKLLRDE 15>SNAKDVLGLTLLEKTLKERLNLKDAIIVSGDSDQSPWVKKEMGRAAVACMKKRFSGKNIVAVTGGTTIEAVAEMMTPDSKNRELLFVPARGLLGEEGINSDNVYIMADKQKNGIKTNFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSFQSVLSKDPNEKRDHMVLLEFVTAAGITLGMDELYNVDGGSGGTGSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKLICTTGKLPVPWPTLVTTLGYGLKCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNVPANTICAHMAEKASGTYRLLFVPGQLSQGAYSSIIEEPSVKEVLNTIKSASMLVHGIGEAKTMAQRRNTPLEDLKIDDNDAVTEAFGYYFNADGEVVHKVHSVGMQLDDIDAIPDIIAVAGGSSKAEAIEAYFKKPRNTVLVTDEGAAKKLLRDE 16>SNAKDVLGLTLLEKTLKERLNLKDAIIVSGDSDQSPWVKKEMGRAAVACMKKRFSGKNIVAVTGGTTIEAVAEMMTPDSKNRELLFVPARGGLGEEGVNSDNVYIMADKQKNGIKTNFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSFQSVLSKDPNEKRDHMVLLEFVTAAGITLGMDELYNVDGGSGGTGSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKLICTTGKLPVPWPTLVTTLGYGLKCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNVPANTICAHMAEKASGTYRLLFVPGQLSQGAYSSIIEEPSVKEVLNTIKSASMLVHGIGEAKTMAQRRNTPLEDLKKIDDNDAVTEAFGYYFNADGEVVHKVHSVGMQLDDIDAIPDIIAVAGGSSKAEAIEAYFKKPRNTVLVTDEGAAKKLLRDE

Claims

1. (1) Having the sequence shown in SEQ ID NO: 1, and having mutations including amino acid modifications, substitutions, or deletions at one, two, three, four, five or more sites selected from the following: T148, R175, G176, G177, L178, E180, D181, V182, K183, N184, Q185, F271, (2) A shortened mutant having amino acids between positions 88 and 340 of (1), or (3) A sequence having at least 70% sequence identity with the sequence of (1) or (2), having the mutation described in (1), and retaining the ability to bind to fructose-1,6-bisphosphate; A fructose-1,6-bisphosphate-binding protein mutant characterized by the following: Preferably, the mutation sites are one, two, three, four, or five selected from any of the following groups: (a) T148, R175, G176, G177, L178, D181, V182, K183, N184, Q185, (b) G177, L178, E180, D181, V182, N184, Q185, F271, More preferably, the mutations include mutations at sites selected from any of the following groups: (1) G177 and L178, (5) D181 and V182, (6) E180 and D181, (7) N184 and Q185; or, the mutations include mutations at sites selected from any of the following groups: (1) G177, L178, (2) G 177, L178 and Q185, (3) G177, L178 and T148, (4) G177, L178 and F271, (5) D181, V182, (6) E180, D181, (7) N184, Q185, (8) N184, Q185, D181, V182, G177, (9) N184, Q185, G177, (10) N184, Q185, D181, V182, More preferably, T148 is mutated to S; G177 is mutated to A, I, L, M, or N, preferably to L or M; L178 is mutated to Y, F, or N, preferably to N; E180 is mutated to G; D181 is mutated to F, L, or E, preferably to E; V182 is mutated to F, L, or G, preferably to G; N184 is mutated to V; Q185 is mutated to S, A, T, D, or P, preferably to P; F271 is mutated to Y. More preferably, the mutations include mutations selected from any of the following groups: (1) G177A and L178Y, (2) G177I and L178F, (3) G177L and L178Y, (4) G177M and L178N, (5) G177N and L178Y, (6) G177M, L178N and Q185S, (7) G177M, L178N and Q185A, (8) G177M, L178N and Q185T, (9) G177M, L178N (10) G177, L178 and T148S, (11) G177, L178 and F271Y, (12) D181F and V182F, (13) D181L and V182L, (14) E180G and D181G, (15) N184V and Q185P, (16) N184V, Q185P, D181E, V182G and G177L, (17) N184V, Q185P and G177L, (18) N184V, Q185P, D181E and V182G.

2. A fructose-1,6-bisphosphate optical probe comprising a fructose-1,6-bisphosphate-sensitive polypeptide and an optically active polypeptide, wherein the optically active polypeptide is located within the sequence of the fructose-1,6-bisphosphate-sensitive polypeptide, the fructose-1,6-bisphosphate-sensitive polypeptide is a fructose-1,6-bisphosphate-binding protein or a functional mutant thereof, and the optically active polypeptide is a fluorescent protein or a functional mutant thereof; wherein the functional mutant of the fructose-1,6-bisphosphate-binding protein has a mutation within seven amino acids linked to the optically active polypeptide, and the functional mutant of the fluorescent protein has a mutation within three amino acids linked to the optically active polypeptide.

3. The optical probe according to claim 1 is characterized in that the fructose-1,6-bisphosphate-binding protein has the sequence shown in SEQ ID NO: 1 or a shortened form having amino acids between the 88th and 340th positions, or a sequence having at least 70% sequence identity with those and retaining fructose-1,6-bisphosphate sensitivity.

4. The optical probe according to claim 1 is characterized in that the optically active polypeptide is located between residues 174-185 and / or 201-208 of the fructose-1,6-bisphosphate-sensitive polypeptide; Preferably, the optically active polypeptide is located at one or more of the following sites selected from the fructose-1,6-bisphosphate-sensitive polypeptide: 174 / 175, 174 / 176, 174 / 177, 174 / 178, 174 / 179, 174 / 180, 174 / 181, 174 / 182, 174 / 183, 174 / 184, 174 / 185, 155 / 176, 175 / 177, 175 / 178, 175 / 179, 175 / 180, 175 / 181, 175 / 182, 175 / 183, 175 / 184, 175 / 185, 176 / 177, 176 / 178, 176 / 179, 176 / 180, 176 / 181, 176 / 182, 176 / 183, 176 / 184, 176 / 185, 177 / 178, 177 / 179, 177 / 180, 177 / 181, 177 / 182, 177 / 183, 177 / 184, 177 / 185, 178 / 179, 178 / 180, 178 / 181, 178 / 182, 178 / 183, 178 / 184, 178 / 185, 179 / 180, 179 / 181, 179 / 182, 179 / 183, 179 / 184, 179 / 185, 180 / 181, 180 / 182, 180 / 183, 180 / 184, 180 / 185, 181 / 182, 181 / 182, 181 / 183, 181 / 184, 181 / 185, 182 / 183, 182 / 184, 182 / 185, 183 / 184, 183 / 185, 184 / 185, 201 / 202, 201 / 203, 201 / 204, 201 / 205, 201 / 206, 201 / 207, 201 / 208, 202 / 203, 202 / 204, 202 / 205, 202 / 206, 202 / 207, 202 / 208, 203 / 204, 203 / 205, 203 / 206, 203 / 207, 203 / 208, 204 / 205, 204 / 206, 204 / 207, 204 / 208, 205 / 206, 205 / 206, 205 / 207, 205 / 208, 206 / 207, 206 / 208 and / or 207 / 208; more preferably, the optically active polypeptide is located at one or more of the following sites selected from the fructose-1,6-bisphosphate-sensitive polypeptide: 176 / 177, 176 / 178, 177 / 181, 179 / 180, 182 / 184, 182 / 185, 203 / 206, 203 / 207 and 203 / 208; Preferably, a functional mutant of the fructose-1,6-bisphosphate-binding protein is as described in claim 1; Preferably, the fluorescent protein is selected from yellow fluorescent protein, orange fluorescent protein, red fluorescent protein, green fluorescent protein, blue fluorescent protein, and apple red fluorescent protein, and the functional mutant of the fluorescent protein has a mutation in the amino acids at positions 1-3; more preferably, the fluorescent protein has the sequence shown in any one of SEQ ID NO: 2-9, and the functional mutant of the fluorescent protein has a mutation in the amino acid at position 1. Preferably, a functional mutant of a fluorescent protein includes one in which the amino acid at position 1 of the fluorescent protein is mutated to I or V. Preferably, the fructose-1,6-bisphosphate-sensitive polypeptide is a truncated mutant having amino acids 88-340 of SEQ ID NO: 1, and the optically active polypeptide is located at one or more of the following sites selected from the fructose-1,6-bisphosphate-sensitive polypeptide: 176 / 177, 176 / 178, 177 / 181, 179 / 180, 182 / 184, 182 / 185, 203 / 206, 203 / 207, and 203 / 208; more preferably, the optical probe has the sequence shown in any one of SEQ ID NO: 10-18; Preferably, the fructose-1,6-bisphosphate-sensitive polypeptide is a truncated mutant having amino acids 88-340 of SEQ ID NO: 1, the optically active polypeptide is located at one or more of the following sites selected from the fructose-1,6-bisphosphate-sensitive polypeptide: 176 / 177, 177 / 181, 179 / 180, or 182 / 184, and the optical probe has one or more mutations selected from the following: T1 of the fructose-1,6-bisphosphate-sensitive polypeptide 48S, G177A, G177I, G177L, G177M, G177N, L178Y, L178F, L178Y, L178N, L178Y, L178N, E180G, D181E, D181F, D181L, D181G, V182F, V182L, V182G, N184V, Q185A, Q185S, Q185A, Q185T, Q185D, Q185P, F271Y, the 1-position amino acid of the optically active polypeptide is mutated to V or I; Preferably, the optical probe has mutations represented in any one of the following groups: (1) G177A and L178Y of the fructose-1,6-bisphosphate-sensitive polypeptide, (2) G177I and L178F of the fructose-1,6-bisphosphate-sensitive polypeptide, (3) G177L and L178Y of the fructose-1,6-bisphosphate-sensitive polypeptide, (4) G177M and L178N of the fructose-1,6-bisphosphate-sensitive polypeptide, (5) G177 N and L178Y, (6) G177M, L178N and Q185S of fructose-1,6-bisphosphate-sensitive polypeptide, (7) G177M, L178N and Q185A of fructose-1,6-bisphosphate-sensitive polypeptide, (8) G177M, L178N and Q185T of fructose-1,6-bisphosphate-sensitive polypeptide, (9) G177M, L178N and Q185D of fructose-1,6-bisphosphate-sensitive polypeptide, (10) G177, L178 and T148S of fructose-1,6-bisphosphate-sensitive polypeptide (11) Fructose-1,6-bisphosphate-sensitive polypeptides G177, L178 and F271Y, (12) Fructose-1,6-bisphosphate-sensitive polypeptides D181F and V182F, (13) Fructose-1,6-bisphosphate-sensitive polypeptides D181L and V182L, (14) Fructose-1,6-bisphosphate-sensitive polypeptides E180G and D181G, (15) Fructose-1,6-bisphosphate-sensitive polypeptides N184V and Q185P, (16) Fructose-1,6-bisphosphate-sensitive polypeptides (17) Mutations to the 1st amino acid of the fructose-1,6-bisphosphate-sensitive polypeptide N184V, Q185P, D181E, V182G, G177L, and optically active polypeptide to V, (18) Mutations to the 1st amino acid of the fructose-1,6-bisphosphate-sensitive polypeptide N184V, Q185P and G177L, and optically active polypeptide to I, (19) Fructose-1,(20) Mutations to the 1st amino acid I of the 6-bisphosphate-sensitive polypeptide N184V, Q185P, and G177L, and the 1st amino acid V of the optically active polypeptide, (20) Mutations to the 1st amino acid V of the fructose-1,6-bisphosphate-sensitive polypeptide N184V, Q185P, D181E, and V182G, and the 1st amino acid V of the optically active polypeptide.

5. (a) Code arrangement of an optical probe as described in any one of claims 2-4, (b) Complementary arrangement of (a) A nucleic acid molecule characterized by containing [something].

6. A nucleic acid construct comprising the nucleic acid molecule described in claim 5; Preferably, the nucleic acid construct is a cloning vector, an expression vector, or a recombinant vector.

7. (1) To produce an optical probe as described in any one of claims 2-4; (2) comprising the nucleic acid molecule described in claim 5; or (3) comprising the nucleic acid construct described in claim 6 A host cell characterized by the following features.

8. (1) An optical probe as described in any one of claims 2-4, (2) The nucleic acid sequence described in claim 5, (3) The nucleic acid construct described in claim 6, or (4) Host cell as described in claim 7, A detection kit characterized by including; The aforementioned detection kit optionally includes other reagents necessary for detecting fructose-1,6-bisphosphate using an optical probe; Preferably, the detection kit further comprises one or more reagents selected from the following: buffer, culture medium, and fructose-1,6-bisphosphate standard.

9. A method for preparing an optical probe according to any one of claims 2-4, comprising providing the host cells described in claim 7, culturing the host cells under conditions in which the optical probe is expressed, and isolating the optical probe.

10. An optical probe according to any one of claims 2-4, a nucleic acid sequence according to claim 5, a nucleic acid construct according to claim 6, or a host cell application according to claim 7 for the detection of fructose-1,6-bisphosphate in a sample, for screening of compounds, or for the intracellular and / or extracellular localization of fructose-1,6-bisphosphate; Preferably, Detection of fructose-1,6-bisphosphate in a sample includes contacting the sample with the optical probe or host cells, detecting the optical change of the optically active polypeptide, and detecting fructose-1,6-bisphosphate in the sample based on the optical change of the optically active peptide; The screening of the aforementioned compounds comprises contacting the candidate compound with the optical probe or host cell in a system containing fructose-1,6-bisphosphate, detecting the optical change of the optically active polypeptide, and screening the candidate compound based on the optical change of the optically active peptide; preferably, the screening of the aforementioned compounds comprises contacting the host cell with the candidate compound in a system containing fructose-1,6-bisphosphate, and the optical change of the optically active polypeptide indicating whether the candidate compound can regulate the intracellular uptake of fructose-1,6-bisphosphate; The intracellular and / or extracellular localization of fructose-1,6-bisphosphate comprises contacting a system containing fructose-1,6-bisphosphate with the optical probe or the host cell and detecting the optical change of the optically active polypeptide; More preferably, the system is a solution system, a cell system, or an intracellular system.

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