WO2024094002A1 - 一种果糖-1，6-二磷酸光学探针及其制备方法和应用 - Google Patents

一种果糖-1，6-二磷酸光学探针及其制备方法和应用 Download PDF

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WO2024094002A1
WO2024094002A1 PCT/CN2023/128488 CN2023128488W WO2024094002A1 WO 2024094002 A1 WO2024094002 A1 WO 2024094002A1 CN 2023128488 W CN2023128488 W CN 2023128488W WO 2024094002 A1 WO2024094002 A1 WO 2024094002A1
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fructose
bisphosphate
polypeptide
optically active
optical probe
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English (en)
French (fr)
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杨弋
赵玉政
姚静
邹叶君
张莉娟
陈琰
李写
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华东理工大学
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/62—DNA sequences coding for fusion proteins
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/70—Vectors or expression systems specially adapted for E. coli
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/74—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/531—Production of immunochemical test materials
    • G01N33/532—Production of labelled immunochemicals
    • G01N33/533—Production of labelled immunochemicals with fluorescent label
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • C12R2001/185—Escherichia
    • C12R2001/19—Escherichia coli

Definitions

  • the present invention relates to the technical field of optical probes, and in particular to a fructose-1,6-diphosphate optical probe and a preparation method and application thereof.
  • FBP Fructose-1,6-bisphosphate
  • Fructose-1,6-bisphosphate is an important metabolite in the glycolysis pathway and gluconeogenesis process. It is produced by fructose-6-phosphate under the catalysis of phosphofructokinase, and then generates dihydroxyacetone phosphate and 3-phosphoglyceraldehyde under the action of aldolase, and finally generates pyruvate and enters the tricarboxylic acid cycle pathway. In addition, fructose-1,6-bisphosphate regulates the activity of multiple enzymes in multiple metabolic pathways.
  • fructose-1,6-bisphosphate Due to its important role in metabolic pathways, fructose-1,6-bisphosphate is used to treat metabolic-related and respiratory-related diseases, and has achieved good results. For example, when sugar metabolism in the body is abnormal, the ATP content decreases, which in turn leads to a decrease in the oxygen-carrying capacity of hemoglobin, causing metabolic disorders and diseases. Fructose-1,6-bisphosphate can promote the decomposition of sugar in cells and treat related diseases. In terms of respiratory diseases, fructose-1,6-bisphosphate increases the content of the phosphate pool in the body by promoting the decomposition of intracellular phosphofructokinase, thereby increasing the relaxation capacity of lung smooth muscles and repairing damaged cells.
  • fructose-1,6-bisphosphate mainly include chromatography (Ma B et al. European Journal of Pharmacology, 2013, 718 (1-3), 524-532), enzymatic method (Iwamoto S et al. Applied and Environmental Microbiology, 2007, 73 (17), 5676-5678), and colorimetric method (Wang C et al. Journal of biomedical engineering, 2000, 17 (3), 363-365). Chromatographic enzymatic detection has good specificity, but has the disadvantage of high cost (Wu Liangyong et al.
  • Chromatographic method can quickly detect the content of fructose-1,6-diphosphate, and can detect low content of fructose-1,6-diphosphate, but errors are prone to occur in the experimental process (Wu Yan et al. Journal of Central South Medical Science, 2000, (6), 608-609); and the colorimetric method has the problem of interference from substances such as fructose and fructose monophosphate (Du Zhenning et al. Chinese Journal of Biochemical Pharmacy, 1993, (2), 59-62), and the above methods are only suitable for in vitro detection, and cannot monitor the concentration changes of fructose-1,6-diphosphate in living cells in real time. Therefore, it is of great significance to develop a new method with good specificity, fast response speed and real-time quantitative detection of fructose-1,6-diphosphate in vivo.
  • the purpose of the present invention is to provide a probe and method for real-time localization, high-throughput and quantitative detection of fructose-1,6-bisphosphate inside and outside cells.
  • the first aspect of the present invention provides a fructose-1,6-bisphosphate binding protein variant, which:
  • (2) is a truncated variant of (1) having amino acids 88 to 340, or
  • the mutated sites are selected from 1, 2 or 3, 4, or 5 of any one of the following groups: (a) T148, R175, G176, G177, L178, D181, V182, K183, N184, Q185, (b) G177, L178, E180, D181, V182, N184, Q185, F271.
  • the mutation comprises a mutation at a site selected from any one of the following groups: (1) G177 and L178, (5) D181 and V182, (6) E180 and D181, (7) N184 and Q185; or, the mutation comprises a mutation at a site selected from any one 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.
  • 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.
  • F271 is mutated to Y.
  • the mutation comprises one or more selected from the group consisting of: G177L, D181E, V182G, N184V, Q185A.
  • the mutation comprises a mutation selected from any one 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 and Q185D, (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.
  • 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 part and a second part by the optically active polypeptide.
  • 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 to form a probe structure of the type B1-A-B2.
  • the optically active polypeptide is located between residues 174-185 and/or 201-208 of the fructose-1,6-bisphosphate sensitive polypeptide, numbered corresponding to the full length of the fructose-1,6-bisphosphate sensitive polypeptide.
  • the optically active polypeptide is located at any one or more of the following sites of 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, 175/176, 175/177, 175/178, 175/179, 175/180, 175/181, 175/182, 175/183, 175/184, 175/185 75/185, 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, 176/184, 176/185, 177/178, 177/179, 177/180, 177/181, 177/182, 177/183, 176/184,
  • the optically active polypeptide is located at any one or more of 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.
  • the fructose-1,6-bisphosphate-sensitive polypeptide is a fructose-1,6-bisphosphate binding protein or a functional variant thereof, wherein the functional variant of the fructose-1,6-bisphosphate binding protein has a mutation within 7 amino acids at the site where it is connected to the optically active polypeptide.
  • the fructose-1,6-bisphosphate-sensitive polypeptide has:
  • the optically active polypeptide is a fluorescent protein or a functional variant thereof, wherein the functional variant of the fluorescent protein has a mutation within 3 amino acids at the junction with the optically active polypeptide.
  • the fluorescent protein is selected from yellow fluorescent protein, orange fluorescent protein, red fluorescent protein, green fluorescent protein, blue fluorescent protein, apple red fluorescent protein. In one embodiment, the fluorescent protein has a sequence shown in any one of SEQ ID NO: 2-9.
  • the functional variant of the fluorescent protein has a mutation at amino acids 1-3, preferably at position 1.
  • the functional variant of the fluorescent protein comprises a mutation in which the amino acid at position 1 of the fluorescent protein is mutated to I or V.
  • the functional variant of the fluorescent protein has a sequence as shown in SEQ ID NO: 2 and has a mutation at the Y1 position.
  • the mutation is Y1I or Y1V.
  • the optical probe further comprises one or more linkers flanking the optically active polypeptide.
  • the linker of the present invention can be any amino acid sequence of any length.
  • the optically active polypeptide flank comprises a linker of no more than 5 amino acids, such as a linker of 0, 1, 2, 3, or 4 amino acids.
  • the linker flanking the optically active polypeptide comprises amino acid Y.
  • linker Y is located at the N-terminus and/or C-terminus of the optically active polypeptide.
  • the optical probe is as follows: the first part B1, Y, optically active polypeptide A, and the second part B2 of the fructose-1,6-bisphosphate sensitive polypeptide.
  • the optical probe of the present invention does not comprise a linker.
  • the optical probe of the present invention further comprises a localization sequence for localizing the probe to, for example, a specific organelle of a cell.
  • the fructose-1,6-bisphosphate sensitive polypeptide is a truncated variant of SEQ ID NO: 1 having amino acids 88-340, and the optically active polypeptide is located at any one or more of the following positions 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.
  • the optically active polypeptide is as shown in SEQ ID NO:2.
  • the fructose-1,6-bisphosphate sensitive polypeptide is a truncated variant of SEQ ID NO: 1 having amino acids 88-340
  • the optically active polypeptide is located at any one or more of the following sites of the fructose-1,6-bisphosphate sensitive polypeptide: 176/177, 177/181, 179/180 or 182/184
  • the optical probe has one or more mutations selected from the following: T148S, G177A of the fructose-1,6-bisphosphate sensitive polypeptide , 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
  • the fructose-1,6-bisphosphate-sensitive polypeptide is a truncated variant of SEQ ID NO: 1 having amino acids 88-340
  • the optically active polypeptide is located at any one or more of the following positions of the fructose-1,6-bisphosphate-sensitive polypeptide: 176/177, 177/181, 179/180 or 182/184
  • the optical probe has a mutation selected from 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 L178Y of the fructose-1,6-bisphosphate-sensitive polypeptide L178N, (5) G177N and L178Y of fructose-1,6-bisphosphate sensitive poly
  • Another aspect of the present invention provides a fusion polypeptide comprising an optical probe as described herein and other polypeptides.
  • the other polypeptides are located at the N-terminus and/or C-terminus of the optical probe.
  • the other polypeptides include polypeptides that locate the optical probe to different organelles or sub-organelles, tags for purification, or tags for immunoblotting.
  • nucleic acid molecule comprising: (a) a coding sequence of a polypeptide or probe as described in any embodiment of the present invention, or (b) a complementary sequence of (a), or (c) a fragment of (a) or (b).
  • the fragment is a primer.
  • the present invention also relates to variants of the above-mentioned nucleic acid molecules, including nucleic acid sequences encoding fragments, analogs, derivatives, soluble fragments and variants of the optical probe or fusion protein of the present invention or their complementary sequences.
  • nucleic acid construct comprising the nucleic acid molecule described herein.
  • the nucleic acid sequence encodes the optical probe or fusion polypeptide described in the present invention.
  • the nucleic acid construct is a cloning vector, an expression vector, or a recombinant vector.
  • the nucleic acid molecule is operably linked to an expression control sequence.
  • the expression vector is selected from a prokaryotic expression vector, a eukaryotic expression vector, and a viral vector.
  • Another aspect of the present invention provides a host cell, wherein the host cell: (1) expresses the optical probe or fusion polypeptide according to any embodiment of the present invention; (2) comprises the nucleic acid molecule according to any embodiment of the present invention; or (3) comprises the nucleic acid construct according to any embodiment of the present invention.
  • the host cell is preferably Escherichia coli.
  • Another aspect of the present invention provides a fructose-1,6-bisphosphate detection kit, comprising the optical probe or fusion polypeptide or polynucleotide described herein or the optical probe prepared by the method described herein.
  • the kit further comprises one or more reagents selected from the group consisting of a buffer, a culture medium, and a fructose-1,6-bisphosphate standard.
  • Another aspect of the present invention provides a method for preparing the optical probe described herein, comprising: providing a host cell expressing the optical probe or fusion polypeptide described herein, culturing the host cell under conditions where the cell expresses the optical probe, and isolating the optical probe or fusion polypeptide.
  • 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 expression of the expression vector; 3) isolating the fructose-1,6-bisphosphate optical probe.
  • Another aspect of the present invention provides a method for detecting fructose-1,6-bisphosphate in a sample, comprising: contacting the optical probe or fusion polypeptide or host cell described herein with the sample, and detecting changes in the optically active polypeptide.
  • the detection can be performed in vivo, in vitro, in subcellular or in situ.
  • the sample is, for example, blood.
  • the present invention also provides a method for quantifying fructose-1,6-bisphosphate in a sample, comprising: contacting the optical probe or fusion polypeptide or host cell described in the present invention with the sample, detecting the optical change of the optically active polypeptide, and quantifying the fructose-1,6-bisphosphate in the sample based on the optical change of the optically active polypeptide.
  • Another aspect of the present invention provides a method for screening compounds (e.g., drugs), comprising: contacting the optical probe or fusion polypeptide described herein or a host cell with a candidate compound in a system containing fructose-1,6-bisphosphate, detecting the optical change of the optically active polypeptide, and screening the compound based on the optical change of the optically active polypeptide.
  • the method can screen compounds with high throughput.
  • a host cell described herein is contacted with a candidate compound in a system containing fructose-1,6-bisphosphate, and an optical change in the optically active polypeptide indicates whether the candidate compound can modulate the uptake of fructose-1,6-bisphosphate by the cell.
  • Another aspect of the present invention provides a method for localizing the fructose-1,6-bisphosphate inside and/or outside the cell, comprising: contacting a system containing fructose-1,6-bisphosphate with the optical probe or the host cell, and detecting optical changes of the optically active polypeptide.
  • the system is a solution system, a cellular system, or a subcellular system.
  • Another aspect of the present invention provides the use of the fructose-1,6-bisphosphate optical probe or fusion polypeptide or host cell described herein in detecting fructose-1,6-bisphosphate in a sample, screening compounds or fructose-1,6-bisphosphate intracellular/extracellular localization.
  • the localization is real-time localization.
  • the fructose-1,6-bisphosphate optical probe provided by the present invention is easy to mature, has large dynamic changes in fluorescence, good specificity, and can be expressed in cells through genetic manipulation methods. It can detect fructose-1,6-bisphosphate in real time inside and outside cells, with high throughput and quantitative detection, eliminating the time-consuming sample processing steps.
  • FIG1 is an SDS-PAGE image of an exemplary fructose-1,6-bisphosphate optical probe described in Example 2;
  • FIG2 is a table showing the response changes of the exemplary fructose-1,6-bisphosphate optical probe comprising cpYFP and fructose-1,6-bisphosphate binding protein described in Example 2 to fructose-1,6-bisphosphate;
  • FIG3 is a table showing the response changes of the exemplary fructose-1,6-bisphosphate optical probe comprising cpGFP and fructose-1,6-bisphosphate binding protein to fructose-1,6-bisphosphate as described in Example 3;
  • FIG4 is a table showing the response changes of the exemplary fructose-1,6-bisphosphate optical probe comprising cpBFP and fructose-1,6-bisphosphate binding protein described in Example 4 to fructose-1,6-bisphosphate;
  • FIG5 is a graph showing the fluorescence spectral properties of an exemplary fructose-1,6-bisphosphate optical probe as described in Example 6;
  • FIG6 is a titration curve of the exemplary fructose-1,6-bisphosphate optical probe described in Example 6 to different concentrations of fructose-1,6-bisphosphate;
  • FIG7 is a bar graph showing the specific detection of multiple similar substrates in the glycolytic pathway by the exemplary fructose-1,6-bisphosphate optical probe described in Example 6;
  • FIG8 is a photograph of the subcellular organelle localization of the exemplary fructose-1,6-bisphosphate optical probe described in Example 7 in mammalian cells;
  • FIG9 is a schematic diagram of dynamically monitoring the concentration of fructose-1,6-bisphosphate in the cytoplasm of mammalian cells using an exemplary fructose-1,6-bisphosphate optical probe as described in Example 7;
  • FIG10 is a dot plot of high-throughput compound screening at the living cell level using an exemplary fructose-1,6-bisphosphate optical probe as described in Example 8;
  • 11 is a bar graph showing the quantification of fructose-1,6-bisphosphate in human blood by the exemplary fructose-1,6-bisphosphate optical probe described in Example 9.
  • the term "about" when giving a value or range means that the value or range is within 20%, within 10%, and within 5% of the given value or range.
  • compositions “comprising”, “including” and equivalent forms thereof include the meanings of "containing” as well as “consisting of”, for example, a composition “comprising” X may consist of X alone or may contain other substances, such as X+Y.
  • fructose-1,6-bisphosphate-sensitive polypeptide refers to a polypeptide that responds to fructose-1,6-bisphosphate, including any response of a chemical, biological, electrical or physiological parameter of the polypeptide associated with the interaction of the sensitive polypeptide.
  • Responses include small changes, for example, changes in the orientation of the amino acids or peptide fragments of the polypeptide and changes in, for example, the primary, secondary or tertiary structure of the polypeptide, including, for example, changes in protonation, electrochemical potential and/or conformation.
  • Conformation is the three-dimensional arrangement of the primary, secondary and tertiary structures of a molecule containing side groups in the molecule; when the three-dimensional structure of the molecule changes, the conformation changes. Examples of conformational changes include transitions from an alpha-helix to a beta-sheet or from a beta-sheet to an alpha-helix. It will be understood that the detectable change does not need to be a conformational change as long as the fluorescence of the fluorescent protein portion is altered.
  • the fructose-1,6-bisphosphate-sensitive polypeptides described herein may also include functional variants thereof.
  • Functional variants of fructose-1,6-bisphosphate-sensitive polypeptides include, but are not limited to, variants that can interact with fructose-1,6-bisphosphate to cause the same or similar changes as the parent fructose-1,6-bisphosphate-sensitive polypeptide.
  • the fructose-1,6-bisphosphate sensitive polypeptide of the present invention includes but is not limited to the fructose-1,6-bisphosphate binding protein CggR or a variant with more than 90% homology thereto.
  • the exemplary fructose-1,6-bisphosphate binding protein CggR of the present invention is derived from Bacillus subtilis.
  • CggR belongs to the SorC/DeoR family of prokaryotic transcriptional regulatory factors and consists of two domains: a DNA binding domain at the N-terminus and a ligand domain at the C-terminus.
  • Fructose-1,6-bisphosphate binding protein can sense changes in fructose-1,6-bisphosphate concentration, and the spatial conformation of fructose-1,6-bisphosphate binding protein will also change during the dynamic change of fructose-1,6-bisphosphate concentration.
  • An exemplary CggR protein truncation is shown in SEQ ID NO: 1.
  • the amino acid residue numbers are all referred to SEQ ID NO: 1.
  • optical probe refers to a fructose-1,6-bisphosphate sensitive polypeptide fused to an optically active polypeptide.
  • the inventors have found that the conformational changes produced by the fructose-1,6-bisphosphate sensitive polypeptide, such as fructose-1,6-bisphosphate binding protein, specifically binding to physiological concentrations of fructose-1,6-bisphosphate will cause conformational changes in optically active polypeptides (such as fluorescent proteins), thereby causing changes in the optical properties of the optically active polypeptides.
  • optically active polypeptides such as fluorescent proteins
  • an optically active polypeptide e.g., a fluorescent protein
  • a protein-based "optically active polypeptide” is a polypeptide that has the ability to emit fluorescence. Fluorescence is an optical property of an optically active polypeptide that can be used as a means of detecting the responsiveness of the optical probe of the present invention.
  • the term "fluorescence property" refers to the molar extinction coefficient at an appropriate excitation wavelength, the fluorescence quantum efficiency, the shape of the excitation spectrum or the emission spectrum, the excitation wavelength maximum and the emission wavelength maximum, the amplitude of excitation at two different wavelengths, the ratio of the emission amplitude at two different wavelengths, the excited state lifetime, or the fluorescence anisotropy.
  • a measurable difference in any of these properties between the active and inactive states is sufficient for the utility of the fluorescent protein substrate of the present invention in activity assays.
  • the measurable difference can be determined by determining the relative ...
  • the amount of fluorescence at a particular wavelength or the integral of the fluorescence over the emission spectrum can be determined.
  • the protein substrate is selected to have fluorescence properties that are easily distinguished in the unactivated and activated conformational states.
  • the optically active polypeptides described herein may also include functional variants thereof.
  • Functional variants of optically active polypeptides include, but are not limited to, variants that may undergo the same or similar fluorescence property changes as the parent optically active polypeptide.
  • Linker refers to an amino acid or nucleotide sequence that connects two parts in the polypeptide, protein or nucleic acid of the present invention.
  • the number of amino acids at the amino terminal of the connecting region between the fructose-1,6-bisphosphate sensitive polypeptide and the optically active polypeptide is selected from 0 to 3
  • the number of amino acids at the carboxyl terminal is selected from 0 to 2; when the recombinant optical probe is connected to the functional protein as a basic unit, it can be fused to the amino acid or carboxyl terminal of the recombinant optical probe.
  • the linker sequence can be a short peptide chain composed of one or more flexible amino acids, such as Y.
  • fluorescent protein refers to a protein that emits fluorescence under the irradiation of excitation light. Fluorescent proteins are used as a basic detection method in the field of biological sciences, such as the green fluorescent protein GFP commonly used in the field of biotechnology and the circular rearranged blue fluorescent protein (cpBFP), circular rearranged green fluorescent protein (cpGFP), and circular rearranged yellow fluorescent protein (cpYFP) derived from mutations of the protein; there is also the red fluorescent protein RFP commonly used in the field of technology, and the circular rearranged proteins derived from the protein, such as cpmApple, cpmOrange, cpmKate, etc.
  • GFP green fluorescent protein GFP commonly used in the field of biotechnology
  • cpBFP circular rearranged blue fluorescent protein
  • cpGFP circular rearranged green fluorescent protein
  • cpYFP circular rearranged yellow fluorescent protein
  • cpYFP is shown in SEQ ID NO: 2
  • cpmOrange is shown in SEQ ID NO: 3
  • cpmKate is shown in SEQ ID NO: 4 or 8
  • mCherry is shown in SEQ ID NO: 5
  • cpGFP is shown in SEQ ID NO: 6
  • cpBFP is shown in SEQ ID NO: 7
  • cpmApple is shown in SEQ ID NO: 9.
  • the fluorescent protein in the optical probe also includes functional variants with mutations, including but not limited to fluorescent proteins with mutations in amino acids 1-3 (preferably 1), such as mutations to V or I.
  • the functional variant of cpYFP has a sequence shown in SEQ ID NO: 2 and has a mutation V or I at the Y1 position.
  • the optically active polypeptide is located between residues 174-185 and/or 201-208 of the fructose-1,6-bisphosphate-sensitive polypeptide in the N-C direction, and the numbering corresponds to the full length of the fructose-1,6-bisphosphate-sensitive polypeptide.
  • the optically active polypeptide is located at the following positions of 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, 176/177, 176/178, 176/179, 176/180, 176/181, 176/182, 176/183, 174/184, 174/185 171,176/182,176/183,176/184,176/185,177/178,177/179,177/180,177/181,177/182,177/183,177/184,177/185,178/179,178/180,178/181,178/182,178/183,178/184,178/185,179/180,179/181,179/182,179/183,178/184,178/185,179/180,179/181,179/182,179/183,178/184,178/185,179/180,179/181,179/18
  • the optically active polypeptide is located between the amino acids described by the numbers.
  • the insertion site 147/148 means that the optically active polypeptide is located between the amino acids 147 and 148 of the fructose-1,6-bisphosphate sensitive polypeptide.
  • the two numbers in the position represented by the "X/Y" format are not consecutive integers, it means that the optically active polypeptide replaces the amino acids between the amino acids indicated by the numbers.
  • the insertion site 174/185 means that the optically active polypeptide replaces the amino acids 175-184 of the fructose-1,6-bisphosphate-sensitive polypeptide.
  • the optically active polypeptide is inserted into the following positions 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.
  • 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 variants (e.g., SEQ ID NO: 1 or a fragment thereof comprising amino acids 88-340).
  • the fructose-1,6-bisphosphate sensitive polypeptide in the optical probe is as shown in amino acids 88-340 of SEQ ID NO: 1
  • the optically active polypeptide is as shown in any one of SEQ ID NO: 2-9
  • 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.
  • variants having the same function of the polypeptide or protein but different sequences.
  • variants of polypeptides or proteins may include: homologous sequences, conservative variants, allelic variants, natural mutants, induced mutants. These variants include but are not limited to: deletion, insertion and/or substitution of one or more (usually 1-30, preferably 1-20, more preferably 1-10, and most preferably 1-5) amino acids in the sequence of the polypeptide or protein, and sequences obtained by adding one or more (usually within 20, preferably within 10, and more preferably within 5) amino acids to its carboxyl terminal and/or amino terminal.
  • variants may also include polypeptides or proteins having a sequence identity of at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99% or 100% with the polypeptide or protein.
  • a change in an amino acid residue without changing the overall configuration and function of a polypeptide or protein is a function conservative mutation.
  • amino acids with similar properties often refer to a family of amino acids with similar side chains, which has been clearly defined in the art.
  • amino acids with basic side chains e.g., lysine, arginine, histidine
  • amino acids with acidic side chains e.g., aspartic acid, glutamic acid
  • amino acids with uncharged polar side chains e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine
  • amino acids with non-polar side chains e.g., alanine, valine, leucine, isoleucine, arginine, phenylalanine, methionine, tryptophan
  • amino acids with ⁇ -branched side chains e.g., threonine, valine, isoleucine
  • amino acids with aromatic side chains e.g., tyrosine, phenylalanine, tryptophan, histidine
  • adding one or more amino acids to the amino and/or carboxyl termini generally does not change the function of a polypeptide or protein.
  • Conservative amino acid substitutions for many common known non-genetically encoded amino acids are known in the art.
  • Conservative substitutions for other non-coded amino acids can be determined based on a comparison of their physical properties with the properties of the genetically encoded amino acids.
  • a fructose-1,6-bisphosphate binding protein variant having a mutation at a site selected from the following positions of SEQ ID NO: 1 or a truncated variant thereof exhibits a binding activity different from fructose-1,6-bisphosphate: T148, R175, G176, G177, L178, E180, D181, V182, K183, N184, Q185, F271.
  • the amino acid mutation includes modification, substitution or deletion of an amino acid.
  • a fructose-1,6-bisphosphate binding protein variant having a mutation at one, two or three, four or five sites selected from any one of the following groups of SEQ ID NO: 1 or its truncated variants is more advantageous for use in the optical probe of the present invention to more efficiently detect fructose-1,6-bisphosphate: (a) T148, R175, G176, G177, L178, D181, V182, K183, N184, Q185, (b) G177, L178, E180, D181, V182, N184, Q185, F271.
  • the mutation of the fructose-1,6-bisphosphate binding protein variant includes a mutation at a site selected from any one of the following groups: (1) G177 and L178, (5) D181 and V182, (6) E180 and D181, (7) N184 and Q185; or, the mutation includes a mutation at a site selected from any one of the following groups: (1) G177, L178, (2) G177, L178 and Q185.
  • T148 mutates to S; G177 mutates to A, I, L, M or N, preferably mutates to L or M; L178 mutates to Y, F or N, preferably mutates to N; E180 mutates to G; D181 mutates to F, L or E, preferably mutates to E; V182 mutates to F, L or G, preferably mutates to G; N184 mutates to V; Q185 mutates to S, A, T, D or P, preferably mutates to P; F271 mutates to Y.
  • the mutation for the fructose-1,6-bisphosphate binding protein variant is selected from any 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 Q185A, (9) G177M, L178N and Q185E, (10) G177M, L178N and Q185A, (11) G177M, L178N and Q185E L178N and Q185T, (9) G177M, L178N and Q185D, (10) G177M, L178N and T148S, (11) G177M, L178N and F271Y, (12) D181F and V182F, (13) D181L and V182L
  • the present invention provides fructose-1,6-bisphosphate binding protein variants having these mutations and optical probes comprising such fructose-1,6-bisphosphate binding protein variants as fructose-1,6-bisphosphate sensitive polypeptides. Therefore, in one or more embodiments, the fructose-1,6-bisphosphate sensitive polypeptide in the optical probe is a fructose-1,6-bisphosphate binding protein variant as described in any embodiment of the present invention, and the fluorescent protein in the optical probe is as shown in SEQ ID NO: 2-9 and the first amino acid of the fluorescent protein is mutated to I or V.
  • the fructose-1,6-bisphosphate sensitive polypeptide in the optical probe is as shown in amino acids 88-340 of SEQ ID NO: 1
  • the optically active polypeptide is as shown in SEQ ID NO: 2
  • the optically active polypeptide is located at position 176/177 of the fructose-1,6-bisphosphate sensitive polypeptide
  • the fructose-1,6-bisphosphate sensitive polypeptide has a mutation selected from any one of the following: (1) G177A and L178Y, (2) G177I and L178 F, (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 Q185D, (10) G177M, L178N and T148S, (11) G177M, L178N and F271Y.
  • the amino acid sequence of the optical probe shown in item (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 item (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 item (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 item (9) is shown in SEQ ID NO: 13, and the nucleic acid sequence is shown in SEQ ID NO: 20.
  • the fructose-1,6-bisphosphate-sensitive polypeptide in the optical probe is as shown in amino acids 88-340 of SEQ ID NO: 1
  • the optically active polypeptide is as shown in SEQ ID NO: 2
  • the optically active polypeptide is located at the 177/181 position of the fructose-1,6-bisphosphate-sensitive polypeptide
  • the fructose-1,6-bisphosphate-sensitive polypeptide has a mutation selected from any one of the following: (12) D181F and V182F, (13) D181L and V182L.
  • the fructose-1,6-bisphosphate-sensitive polypeptide in the optical probe is as shown in amino acids 88-340 of SEQ ID NO: 1
  • the optically active polypeptide is as shown in SEQ ID NO: 2
  • the optically active polypeptide is located at the 179/180 position of the fructose-1,6-bisphosphate-sensitive polypeptide
  • the fructose-1,6-bisphosphate-sensitive polypeptide has mutations (14) E180G and D181G.
  • the fructose-1,6-bisphosphate-sensitive polypeptide in the optical probe is as shown in amino acids 88-340 of SEQ ID NO: 1
  • the optically active polypeptide is as shown in SEQ ID NO: 2
  • the optically active polypeptide is located at position 182/184 of the fructose-1,6-bisphosphate-sensitive polypeptide
  • the optical probe has a mutation selected from any one of the following: (15) N184V and Q185P of the fructose-1,6-bisphosphate-sensitive polypeptide, (16) N184V, Q185P, D181E, V182G, G177L of the fructose-1,6-bisphosphate-sensitive polypeptide, and the first amino acid of the optically active polypeptide is mutated to V, (17 ) N184V, Q185P, D181E, V182G, G177L of a fructose-1,6-bisphosphate-sensitive polypeptide, and the first amino acid of an optically active poly
  • amino acid sequence of the optical probe shown in item (16) is shown as SEQ ID NO: 14, and the nucleic acid sequence is shown as SEQ ID NO: 21; the amino acid sequence of the optical probe shown in item (17) is shown as SEQ ID NO: 15, and the nucleic acid sequence is shown as SEQ ID NO: 22; the amino acid sequence of the optical probe shown in item (20) is shown as SEQ ID NO: 16, and the nucleic acid sequence is shown as SEQ ID NO: 23.
  • identity means that two or more sequences or subsequences are identical or that a certain percentage of amino acid residues or nucleotides in a specified region are identical (e.g., 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical) when compared and aligned for maximum correspondence over a comparison window or specified region using methods known in the art, such as sequence comparison algorithms, by manual alignment and visual inspection.
  • BLAST and BLAST 2.0 algorithms are the BLAST and BLAST 2.0 algorithms, see Altschul et al. (1977) Nucleic Acids Res. 25:3389 and Altschul et al. (1990) J. Mol. Biol. 215:403, respectively.
  • fusion proteins promote the expression of recombinant proteins, obtain recombinant proteins that are automatically secreted outside the host cell, or facilitate the purification of recombinant proteins
  • it is often necessary to add some amino acids to the N-terminus, C-terminus or other suitable regions in the protein of the recombinant protein for example, including but not limited to, suitable linker peptides, signal peptides, leader peptides, terminal extensions, glutathione S-transferase (GST), maltose E binding protein, protein A, tags such as 6His or Flag, or proteolytic enzyme sites of factor Xa or thrombin or enterokinase.
  • Functional variants, derivatives or analogs of the polypeptide or protein (e.g., fructose-1,6-bisphosphate binding protein or fluorescent protein) of the present invention may be (i) proteins with one or more conservative or non-conservative amino acid residues (preferably conservative amino acid residues) substituted, and such substituted amino acid residues may or may not be encoded by the genetic code, or (ii) proteins with substitution groups in one or more amino acid residues, or (iii) proteins formed by fusion of a mature protein with another compound (e.g., a compound that prolongs the half-life of the protein, such as polyethylene glycol), or (iv) proteins formed by fusion of an additional amino acid sequence to the protein sequence (e.g., a secretory sequence or a sequence used to purify the protein or a proprotein sequence, or a fusion protein formed with an antigen IgG fragment).
  • proteins with one or more conservative or non-conservative amino acid residues preferably conservative amino acid residues
  • these functional variants, derivatives and analogs belong to the well-known scope of those skilled in the art.
  • the analogs also include analogs with residues different from natural L-amino acids (such as D-amino acids), and analogs with non-natural or synthetic amino acids (such as ⁇ , ⁇ -amino acids).
  • L-amino acids such as D-amino acids
  • non-natural or synthetic amino acids such as ⁇ , ⁇ -amino acids.
  • the fructose-1,6-bisphosphate sensitive polypeptide of the present invention is not limited to the representative proteins, variants, derivatives and analogs listed above. Modifications (usually without changing the primary structure) include: chemical derivatization forms of proteins in vivo or in vitro such as acetylation or carboxylation.
  • Modifications also include glycosylation, such as those produced by glycosylation modification during protein synthesis and processing or in further processing steps. This modification can be accomplished by exposing the protein to a glycosylation enzyme (such as a mammalian glycosylase or deglycosylation enzyme). Modified forms also include sequences with phosphorylated amino acid residues (such as phosphotyrosine, phosphoserine, phosphothreonine). It also includes proteins that have been modified to improve their anti-proteolytic properties or optimize solubility.
  • glycosylation enzyme such as a mammalian glycosylase or deglycosylation enzyme
  • Modified forms also include sequences with phosphorylated amino acid residues (such as phosphotyrosine, phosphoserine, phosphothreonine). It also includes proteins that have been modified to improve their anti-proteolytic properties or optimize solubility.
  • the fusion polypeptide of the present invention comprises the optical probe described herein and other polypeptides.
  • the optical probe described herein further comprises other polypeptides fused thereto.
  • the other polypeptides described herein do not affect the properties of the optical probe.
  • the other polypeptides may be located at the N-terminus and/or C-terminus of the optical probe.
  • the other polypeptides include polypeptides that locate the optical probe to different organelles or sub-organelles, tags for purification, or tags for immunoblotting.
  • a linker may be provided between the optical probe and other polypeptides in the fusion polypeptide described herein.
  • tags for purification or tags for immunoblotting include 6*histidine (6*His), glutathione S-transferase (GST), Flag.
  • the present invention includes nucleic acid molecules encoding the fructose-1,6-bisphosphate sensitive polypeptide or optical probe of the present invention.
  • nucleic acid or “nucleotide” or “polynucleotide” or “nucleic acid sequence” used in the present invention can be in the form of DNA or RNA.
  • DNA forms include cDNA, genomic DNA or artificially synthesized DNA.
  • DNA can be single-stranded or double-stranded.
  • DNA can be a coding strand or a non-coding strand.
  • variant used herein can be a naturally occurring allelic variant or a non-naturally occurring variant.
  • nucleotide variants include degenerate Variants, substitution variants, deletion variants and insertion variants.
  • an allelic variant is an alternative form of a nucleic acid, which may be a substitution, deletion or insertion of one or more nucleotides, but does not substantially change the function of the protein encoded by it.
  • the nucleic acid of the present invention may contain a nucleotide sequence that has a sequence identity of 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% with the nucleic acid sequence.
  • the full-length sequence of the optical probe or fusion protein of the present invention or its fragments can usually be obtained by PCR amplification, artificial synthesis or recombination.
  • the steps and reagents used in conventional PCR, synthesis and recombination are known in the art.
  • mutations can be introduced into the protein sequence of the present invention by methods such as mutation PCR or chemical synthesis.
  • the present invention also relates to nucleic acid constructs comprising 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 a variety of ways to ensure expression of the polypeptide or protein.
  • the nucleic acid constructs can be manipulated prior to insertion into a vector depending on the differences or requirements of the expression vector. Techniques for altering polynucleotide sequences using recombinant DNA methods are known in the art.
  • the nucleic acid construct is a vector.
  • the vector can be a cloning vector, an expression vector, or a homologous recombination vector.
  • the polynucleotides of the present invention can be cloned into many types of vectors, for example, plasmids, phagemids, phage derivatives, animal viruses, and cosmids.
  • Typical expression vectors contain expression control sequences that can be used to regulate the expression of the desired nucleic acid sequence, and are operably connected to the nucleic acid sequence of the present invention or its complementary sequence.
  • expression control sequence used herein refers to an element that can be operably connected to the target gene to regulate the transcription, translation and expression of the target gene, which can be a replication origin, a promoter, a marker gene or a translation control element, including an enhancer, an operator, a terminator, a ribosome binding site, etc. The selection of the expression control sequence depends on the host cell used.
  • operable connection refers to the connection of the target nucleotide sequence with the regulatory sequence in a manner that allows the expression of the nucleotide sequence.
  • Those skilled in the art are familiar with methods that can be used to construct expression vectors containing the fusion protein coding sequence of the present invention and suitable transcription/translation control signals. These methods include in vitro recombinant DNA technology, DNA synthesis technology, in vivo recombination technology, etc.
  • the DNA sequence can be effectively connected to an appropriate promoter in the expression vector to guide mRNA synthesis.
  • promoters include: lac or trp promoters of Escherichia coli; PL promoters of ⁇ phage; eukaryotic promoters include CMV immediate early promoter, HSV thymidine kinase promoter, early and late SV40 promoter, LTR of retrovirus and other known promoters that can control gene expression in prokaryotic or eukaryotic cells or their viruses.
  • the expression vector also includes a ribosome binding site for translation initiation and a transcription termination site.
  • the expression vector may be a commercially available pCDF vector, without other special requirements.
  • the nucleotide sequence encoding the optical probe and the expression vector are double-digested with BamHI and EcoRI, respectively, and then the digestion products of the two are connected to obtain a recombinant expression vector.
  • the present invention does not specifically limit the specific steps and parameters of digestion and connection, and conventional steps and parameters in the art can be used.
  • the vector is transformed into a host cell to produce a protein or peptide including a fusion protein.
  • This transfer process can be performed using conventional techniques known to those skilled in the art such as transformation or transfection.
  • the host cell described in the present invention refers to a cell that can receive and accommodate a recombinant DNA molecule, and is a site for recombinant gene amplification.
  • the ideal recipient cell should meet the two conditions of easy acquisition and proliferation.
  • the "host cell” of the present invention may include prokaryotic cells and eukaryotic cells, specifically including bacterial cells, yeast cells, insect cells and mammalian cells.
  • the host cell is preferably a variety of cells that are conducive to the expression or fermentation production of gene products, and such cells are well known and commonly used in the art. Specifically, it can be bacterial cells of Escherichia coli, Streptomyces, Salmonella typhimurium, fungal cells such as yeast, plant cells, insect cells of Drosophila S2 or Sf9, CHO, COS, HEK293, HeLa cells, or animal cells of Bowes melanoma cells, etc.
  • the exemplary host cell used in the embodiments of the present invention is the Escherichia coli BL21-DE3 strain. Those of ordinary skill in the art are aware of how to select appropriate vectors, promoters, enhancers and host cells.
  • the method of transferring to the host cell described in the present invention is a conventional method in the art, including calcium phosphate or calcium chloride coprecipitation, DEAE-mannan-mediated transfection, lipofection, natural competence, chemically mediated transfer or electroporation.
  • the host is a prokaryotic organism such as Escherichia coli
  • the method is preferably a CaCl2 method or a MgCl2 method, and the steps used are well known in the art.
  • the host cell is a eukaryotic cell, the following DNA transfection methods can be selected: calcium phosphate coprecipitation method, conventional mechanical methods such as microinjection, electroporation, liposome packaging, etc.
  • the host cell into which the expression vector is transferred is cultured for amplification and expression to separate and obtain the fructose-1,6-bisphosphate optical probe.
  • the host cell amplification and expression culture can be carried out by conventional methods.
  • the culture medium used in the culture can be various conventional culture media.
  • the culture is carried out under conditions suitable for the growth of the host cell.
  • the optical probe is expressed in the cell, on the cell membrane, or secreted outside the cell.
  • the recombinant protein can be separated or purified by various separation methods using its physical, chemical and other properties.
  • the present invention does not specifically limit the method for separating the fructose-1,6-bisphosphate fluorescent protein, and the conventional separation method of the fusion protein in the art can be used.
  • optical probe is separated using affinity chromatography using a His tag.
  • the present invention also provides the use of the fructose-1,6-bisphosphate optical probe in real-time localization, quantitative detection and high-throughput compound screening of fructose-1,6-bisphosphate.
  • the fructose-1,6-bisphosphate optical probe is preferably connected to a signal peptide in different parts of the cell and transferred into the cell.
  • the real-time localization of fructose-1,6-bisphosphate is performed by detecting the strength of the fluorescence signal in the cell; the corresponding fructose-1,6-bisphosphate is quantitatively detected by combining the change of the fluorescence signal with the fructose-1,6-bisphosphate standard drop curve.
  • the change of the fluorescence signal is displayed by, for example, a standardized fluorescence signal ratio.
  • the ratio is the ratio of the ratio of the sample's 485-nanometer fluorescence signal to the 420-nanometer fluorescence signal to the corresponding ratio of the control.
  • the fructose-1,6-bisphosphate standard drop curve of the present invention is drawn based on the fluorescence signal of the fructose-1,6-bisphosphate optical probe under different concentrations of fructose-1,6-bisphosphate.
  • the fructose-1,6-bisphosphate optical probe of the present invention is directly transferred into cells, and in the process of real-time positioning and quantitative detection of fructose-1,6-bisphosphate, time-consuming sample processing is not required, and it is more accurate.
  • the fructose-1,6-bisphosphate optical probe of the present invention is used for high-throughput compound screening, different compounds are added to the cell culture fluid, and the change of fructose-1,6-bisphosphate content is measured, so as to screen out compounds that affect the change of fructose-1,6-bisphosphate content.
  • fructose-1,6-bisphosphate optical probe described in the present invention in real-time positioning, quantitative detection and high-throughput compound screening of fructose-1,6-bisphosphate is for non-diagnostic and therapeutic purposes, and does not involve the diagnosis and treatment of diseases.
  • the present invention also provides a detection kit comprising the optical probe, nucleic acid molecule, nucleic acid construct and/or cell described herein.
  • the kit also contains other reagents required for detecting fructose-1,6-bisphosphate.
  • the other reagents are well known in the art, such as buffer, cell culture medium, fructose-1,6-bisphosphate standard.
  • Exemplary buffers are, for example, 100 mM HEPES and 100 mM NaCl, pH 7.4.
  • a fructose-1,6-bisphosphate binding protein variant which:
  • (2) is a truncated variant of (1) having amino acids 88 to 340, or
  • the mutated sites are selected from 1, 2, 3, 4 or 5 of any one of the following groups: (a) T148, R175, G176, G177, L178, D181, V182, K183, N184, Q185, (b) G177, L178, E180, D181, V182, N184, Q185, F271,
  • the mutation comprises a mutation at a site selected from any one of the following groups: (1) G177 and L178, (5) D181 and V182, (6) E180 and D181, (7) N184 and Q185; or, the mutation comprises a mutation at a site selected from any one of the following groups: (1) G177, L178, (2) G177, L178 and Q185, (3) G17 7. 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,
  • T148 mutates to S; G177 mutates to A, I, L, M or N, preferably mutates to L or M; L178 mutates to Y, F or N, preferably mutates to N; E180 mutates to G; D181 mutates to F, L or E, preferably mutates to E; V182 mutates to F, L or G, preferably mutates to G; N184 mutates to V; Q185 mutates to S, A, T, D or P, preferably mutates to P; F271 mutates to Y,
  • the mutation comprises a mutation selected from any one 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 and Q185D, (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.
  • 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 variant thereof, and the optically active polypeptide is a fluorescent protein or a functional variant thereof; wherein the functional variant of the fructose-1,6-bisphosphate binding protein has a mutation within 7 amino acids at the connection with the optically active polypeptide, and the functional variant of the fluorescent protein has a mutation within 3 amino acids at the connection with the optically active polypeptide.
  • Item 3 An optical probe as described in Item 1, wherein the fructose-1,6-bisphosphate binding protein has the sequence shown in SEQ ID NO: 1 or a truncation thereof having amino acids 88-340, or a sequence having at least 70% sequence identity therewith and retaining sensitivity to fructose-1,6-bisphosphate.
  • Item 4 The optical probe of Item 1, wherein the optically active polypeptide is located between residues 174-185 and/or 201-208 of the fructose-1,6-bisphosphate sensitive polypeptide,
  • the optically active polypeptide is located at any one or more of the following sites of 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, 175/176, 175/177, 175/178, 175/179, 175/180, 174/181, 174/182, 174/183, 174/184, 174/185, 176/177, 176/178, 176/1 79, 176/180, 176/181, 176/182, 176/183, 176/184, 176/185, 177/178, 177/179, 177/180, 177/181, 177/182, 177/183, 176/184, 176/185, 177/178, 177/179, 177/180, 177/181, 177/182, 177/183, 177/184, 177
  • the functional variant of fructose-1,6-bisphosphate binding protein is as described in claim 1;
  • the fluorescent protein is selected from yellow fluorescent protein, orange fluorescent protein, red fluorescent protein, green fluorescent protein, blue fluorescent protein, apple red fluorescent protein, and the functional variant of the fluorescent protein has a mutation at the amino acid at position 1-3; more preferably, the fluorescent protein has a sequence shown in any one of SEQ ID NOs: 2-9, and the functional variant of the fluorescent protein has a mutation at the amino acid at position 1,
  • the functional variant of the fluorescent protein comprises a mutation in which the first amino acid of the fluorescent protein is mutated to I or V,
  • the fructose-1,6-bisphosphate-sensitive polypeptide is a truncated variant of SEQ ID NO: 1 having amino acids 88-340, and the optically active polypeptide is located at any one or more of the following sites of the fructose-1,6-bisphosphate-sensitive polypeptide selected from: 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 a sequence shown in any one of SEQ ID NO: 10-18.
  • the fructose-1,6-bisphosphate-sensitive polypeptide is a truncated variant of SEQ ID NO: 1 having amino acids 88-340
  • the optically active polypeptide is located at any one or more of the following sites of the fructose-1,6-bisphosphate-sensitive polypeptide: 176/177, 177/181, 179/180 or 182/184
  • the optical probe has one or more mutations selected from the following: T148S, 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 first amino acid of the optically active polypeptide is mutated to V or I
  • the optical probe has a mutation selected from any one of the following groups: (1) G177A and L178Y of a fructose-1,6-bisphosphate-sensitive polypeptide, (2) G177I and L178F of a fructose-1,6-bisphosphate-sensitive polypeptide, (3) G177L and L178Y of a fructose-1,6-bisphosphate-sensitive polypeptide, (4) G177M and L178N of a fructose-1,6-bisphosphate-sensitive polypeptide, (5) G177N and L178Y of a fructose-1,6-bisphosphate-sensitive polypeptide, (6) G177M, L178N and Q185S, (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 fructos
  • a nucleic acid molecule comprising:
  • the nucleic acid construct is a cloning vector, an expression vector or a recombinant vector.
  • Item 7 A host cell, wherein:
  • a detection kit comprising:
  • the detection kit optionally further comprises other reagents required for detecting fructose-1,6-bisphosphate using an optical probe.
  • the detection kit further comprises one or more reagents selected from the following: a buffer, a culture medium, and a fructose-1,6-bisphosphate standard.
  • Item 9 A method for preparing the optical probe described in any one of Items 2-4, comprising: providing the host cell described in Item 7, culturing the host cell under conditions where the optical probe is expressed, and isolating the optical probe.
  • Detecting fructose-1,6-bisphosphate in a sample comprises the steps of: contacting the optical probe or host cell with the sample, detecting an optical change of an optically active polypeptide, and detecting fructose-1,6-bisphosphate in the sample according to the optical change of the optically active polypeptide.
  • the screening compound comprises the steps of: contacting the optical probe or host cell with a candidate compound in a system containing fructose-1,6-bisphosphate, detecting an optical change of an optically active polypeptide, and screening the compound according to the optical change of the optically active polypeptide; preferably, the screening compound comprises the steps of: contacting the host cell 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 regulates the cell's uptake of fructose-1,6-bisphosphate,
  • the intracellular/extracellular localization of fructose-1,6-bisphosphate comprises the steps of 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.
  • the system is a solution system, a cell system or a subcellular system.
  • fructose-1,6-bisphosphate optical probe provided by the present invention is described in detail below in conjunction with the examples, but they should not be construed as limiting the scope of protection of the present invention.
  • the pCDF-cpYFP and pCDF-fructose-1,6-bisphosphate-binding protein plasmids used in the examples were constructed by the Protein Laboratory of East China University of Science and Technology, and the pCDF plasmid vector was purchased from Invitrogen. All primers used for PCR were synthesized, purified and identified by mass spectrometry by Shanghai Jierui Bioengineering Technology Co., Ltd. and BGI. The expression plasmids constructed in the examples were sequenced, and the sequence determination was completed by BGI and Jie Li Sequencing.
  • the Taq DNA polymerase used in each example was purchased from Dongsheng Bio, pfu DNA polymerase was purchased from Tiangen Biochemical Technology (Beijing) Co., Ltd., and primeSTAR DNA polymerase was purchased from TaKaRa.
  • the three polymerases were purchased with corresponding polymerase buffers and dNTPs.
  • BamHI, BglII, HindIII, NdeI, XhoI, EcoRI, SpeI and other restriction endonucleases, T4 ligase, T4 phosphorylase (T4 PNK) were purchased from Fermentas, and the corresponding buffers were provided when purchased.
  • Transfection reagent Lip2000Kit was purchased from Invitrogen.
  • Fructose-1,6-bisphosphate and other compounds were purchased from Sigma. Unless otherwise stated, chemical reagents such as inorganic salts were purchased from Sigma-Aldrich. HEPES salt, ampicillin (Amp) and puromycin were purchased from Ameresco. 96-well black detection plate and 384-well fluorescence detection plate were purchased from Grenier.
  • the DNA purification kit used in the examples was purchased from BBI (Canada), and the common plasmid extraction kit was purchased from Tiangen Biochemical Technology (Beijing) Co., Ltd.
  • the cloned strain Mach1 was purchased from Invitrogen.
  • the nickel column affinity chromatography column and desalting column fillers were both from GE healthcare.
  • the main instruments used in the examples include: Biotek Synergy 2 multifunctional microplate reader (Bio-Tek, USA), X-15R high-speed refrigerated centrifuge (Beckman, USA), Microfuge22R desktop high-speed refrigerated centrifuge (Beckman, USA), PCR amplification instrument (Biometra, Germany), ultrasonic disruptor (Ningbo Xinzhi Company), nucleic acid electrophoresis instrument (Shenneng Gaming Company), fluorescence spectrophotometer (Varian, USA), CO2 constant temperature cell culture incubator (SANYO), and inverted fluorescence microscope (Nikon, Japan).
  • Target fragment amplification PCR 1.
  • This method is mainly used for gene fragment amplification and colony PCR identification of positive clones.
  • the reaction system of PCR amplification is as follows: template sequence 0.5-1 ⁇ L, forward primer (25 ⁇ M) 0.5 ⁇ L, reverse primer (25 ⁇ M) 0.5 ⁇ L, 10 ⁇ pfu buffer 5 ⁇ L, pfu DNA polymerase 0.5 ⁇ L, dNTP (10mM) 1 ⁇ L, sterilized ultrapure water (ddH2O) 41.5-42 ⁇ L, total volume 50 ⁇ L.
  • the PCR amplification procedure is as follows: 95°C denaturation for 2-10 minutes, 30 cycles (94-96°C for 30-45 seconds, 50-65°C for 30-45 seconds, 72°C for a certain time (600bp/min)), 72°C extension for 10 minutes.
  • the long-fragment amplification used in the present invention is mainly a reverse PCR amplification vector, which is a technique used to obtain site-directed mutations in the following embodiments.
  • Reverse PCR primers are designed at the mutation site, and the 5' end of one of the primers contains the mutated nucleotide sequence.
  • the amplified product contains the corresponding mutation site.
  • the long-fragment amplification PCR reaction system is as follows: template sequence (10pg-1ng) 1 ⁇ L, forward primer (25 ⁇ M) 0.5 ⁇ L, reverse primer (25 ⁇ M) 0.5 ⁇ L, 5 ⁇ PrimerSTAR buffer 10 ⁇ L, PrimerSTAR DNA polymerase 0.5 ⁇ L, dNTP (2.5mM) 4 ⁇ L, sterile ultrapure water (ddH2O) 33.5 ⁇ L, total volume 50 ⁇ L.
  • the PCR amplification program was as follows: denaturation at 95°C for 5 min, 30 cycles (98°C for 10 s, 50-68°C for 5-15 s, 72°C for a certain time (1000 bp/min)), and extension at 72°C for 10 min; or denaturation at 95°C for 5 min, 30 cycles (98°C for 10 s, 68°C for a certain time (1000 bp/min)), and extension at 72°C for 10 min.
  • the system for double enzyme digestion of plasmid vector is as follows: 20 ⁇ L of plasmid vector (about 1.5 ⁇ g), 5 ⁇ L of 10 ⁇ buffer, 11-2 ⁇ L of restriction enzyme, 21-2 ⁇ L of restriction enzyme, and the total volume is made up to 50 ⁇ L with sterile ultrapure water.
  • the reaction conditions are 37°C and 1-7 hours.
  • the ends of plasmids or genomes extracted from microorganisms contain phosphate groups, but PCR products do not. Therefore, the 5' end base of the PCR product needs to be subjected to a phosphate group addition reaction. Only DNA molecules with phosphate groups at the end can undergo a ligation reaction.
  • the phosphorylation reaction system is as follows: 5-8 ⁇ L of PCR product fragment DNA sequence, 1 ⁇ L of 10 ⁇ T4 ligase buffer, 1 ⁇ L of T4 polynucleotide kinase (T4 PNK), 0-3 ⁇ L of sterile ultrapure water, and a total volume of 10 ⁇ L.
  • the reaction conditions are 37°C, and inactivation at 72°C for 20 minutes after 30 minutes-2 hours.
  • connection methods between different fragments and vectors are different. Three connection methods are used in the present invention:
  • the principle of this method is that the flat-end product obtained by PCR is phosphorylated at the 5' end of the DNA fragment under the action of T4 PNK, and then connected with the linearized vector under the action of PEG4000 and T4 DNA ligase to obtain a recombinant plasmid.
  • the homologous recombination connection system is as follows: 4 ⁇ L of DNA fragment treated with T4 PNK, 4 ⁇ L of linearized vector fragment, 1 ⁇ L of PEG4000, 1 ⁇ L of 10 ⁇ T4 ligase buffer, 1 ⁇ L of T4 DNA ligase, a total of 10 ⁇ L.
  • the reaction conditions are 22°C, 30 minutes.
  • DNA fragments cut by restriction endonucleases usually produce protruding sticky ends, so they can be connected to vector fragments containing complementary sticky ends to form recombinant plasmids.
  • the ligation reaction system is as follows: 1-7 ⁇ L of PCR product DNA after restriction digestion, 0.5-7 ⁇ L of plasmid after restriction digestion, 1 ⁇ L of 10 ⁇ T4 ligase buffer, 1 ⁇ L of T4 DNA ligase, and sterile ultrapure water to a total volume of 10 ⁇ L. Reaction conditions 16°C, 4-8 hours.
  • the 5'-end phosphorylated DNA fragment was connected to the 3' and 5' ends of the linearized vector by self-circularization ligation to obtain a recombinant plasmid.
  • the self-circularization ligation reaction system is as follows: phosphorylation reaction system 10 ⁇ L, T4 ligase (5U/ ⁇ L) 0.5 ⁇ L, total volume 10.5 ⁇ L. Reaction conditions 16°C, 4-16 hours.
  • Resuspension buffer CaCl 2 (100mM), MgCl 2 (70mM), NaAc (40mM)
  • Storage buffer 0.5 mL DMSO, 1.9 mL 80% glycerol, 1 mL 10 ⁇ CaCl 2 (1M), 1 mL 10 ⁇ MgCl 2 (700 mM), 1 mL 10 ⁇ NaAc (400 mM), 4.6 mL ddH 2 O
  • the supernatant was purified by a self-assembled nickel column affinity chromatography to obtain the protein.
  • the protein after nickel column affinity chromatography was then passed through a self-assembled desalting column to obtain the protein dissolved in 100 mM HEPES buffer (pH 7.4).
  • the probe was diluted into a protein solution with a final concentration of 0.2-5 ⁇ M using assay buffer (100 mM HEPES, 100 mM NaCl, pH 7.4).
  • Fructose-1,6-bisphosphate was prepared into a stock solution with a final concentration of 50 mM using assay buffer (100 mM HEPES, 100 mM NaCl, pH 7.4).
  • the fructose-1,6-bisphosphate optical probe plasmid based on pCDNA3.1+ was transfected into HEK293 cells using the transfection reagent Lipofectamine2000 (Invitrogen) and cultured in a cell culture incubator at 37°C and 5% CO 2. Fluorescence detection was performed after the exogenous gene was fully expressed for 24 to 36 hours.
  • the attached HEK293 cells were rinsed three times with PBS and placed in HBSS solution for detection by fluorescence microscopy and microplate reader.
  • the CggR truncated gene in the Bacillus subtilis gene was amplified by PCR.
  • the PCR product was recovered after gel electrophoresis and digested with HindIII and XhoI.
  • the pCDF vector was double-digested at the same time.
  • T4 DNA ligase the product was used to transform DH5 ⁇ .
  • the transformed DH5 ⁇ was coated on LB plates (streptomycin 100ug/mL) and cultured at 37°C overnight.
  • the grown DH5 ⁇ transformants were subjected to plasmid extraction and PCR identification. After the positive plasmid was sequenced correctly, the subsequent plasmid construction was carried out.
  • Example 2 Expression and detection of cpYFP optical probes at different insertion sites
  • pCDF-CggR 88-340
  • the following sites were selected for insertion of cpYFP according to the crystal structure of fructose-1,6-bisphosphate binding protein to obtain the corresponding pCDF-CggR (88-340)-cpYFP plasmids: 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, 177/180,176/181,176/182,176/183,176/184,176/185, 177/178, 177/179, 177/
  • PCR was used to generate the DNA fragment of cpYFP, and the homologous sequence of the cpYFP terminal was introduced through the 5' end of the primer.
  • PCR amplification produced the pCDF-fructose-1,6-bisphosphate binding protein linearized vector, whose 5' and 3' ends respectively carried completely consistent sequences corresponding to the two ends of cpYFP (15bp ⁇ 20bp).
  • the linearized pCDF-CggR (88-340) and cpYFP fragments were homologously recombined under the action of Hieff Clone Enzyme.
  • the product was transformed into DH5 ⁇ , and the transformed DH5 ⁇ was coated on LB plates (streptomycin 100ug/mL) and cultured at 37°C overnight.
  • the positive clones identified by PCR were sequenced after extracting the plasmid. Sequencing was completed by Jie Li Sequencing Company.
  • the recombinant plasmid was transformed into BL21 (DE3) to induce expression and purify the protein.
  • the size of the protein was around 57Kda by SDS-PAGE electrophoresis. This size is consistent with the size of the CggR (88-340) -cpYFP fusion protein containing the His-tag purification tag expressed by pCDF-CggR (88-340) -cpYFP. The results are shown in Figure 1.
  • the supernatant of E. coli expressing CggR(88-340)-cpYFP fusion protein was used to screen for fructose-1,6-bisphosphate response, and the detection signal of the fusion fluorescent protein containing 1mM 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.
  • the detection results show that the optical probes with a response to fructose-1,6-bisphosphate of more than 1.4 times are inserted at 176/177, 176/178, 177/181, 179/180, 182/184, 182/185, 203/206, 203/207 and 203/208 or the corresponding amino acid sites of their family proteins.
  • the screening results are shown in the following table:
  • Example 3 Expression and detection of cpGFP optical probes at different insertion sites
  • cpYFP was replaced with cpGFP to construct a fructose-1,6-bisphosphate green fluorescent protein fluorescent probe.
  • the detection results show that the optical probes that respond to fructose-1,6-bisphosphate more than 1.4 times include optical probes inserted at sites 176/177, 176/178, 177/181, 182/184, 182/185, 203/204, 203/205, 203/206, 203/207 and 203/208 or the corresponding amino acid sites of their family proteins.
  • Example 4 Expression and detection of cpBFP optical probes at different insertion sites
  • cpYFP was replaced with cpBFP to construct a fructose-1,6-bisphosphate blue fluorescent protein fluorescent probe.
  • the detection results show that the optical probes that respond to fructose-1,6-bisphosphate more than 1.5 times are inserted at 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 their family proteins.
  • Optical probe mutants were constructed based on 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-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-203/208(88-340)-CPYFP.
  • the three purified fructose-1,6-bisphosphate optical probes numbered 4, 15, and 17 in Example 5 were treated with 0 mM and 5 mM fructose-1,6-bisphosphate for 10 minutes, respectively, and then the fluorescence spectra were detected using a fluorescence spectrophotometer.
  • Excitation spectrum measurement The excitation spectrum was recorded with an excitation range of 370nm to 510nm and an emission wavelength of 530nm, and read every 5nm. The results showed that the probe had two excitation peaks at about 410 and 490nm.
  • the excitation wavelengths were fixed at 420 nm and 460 nm, and the emission spectra at 470-600 nm and 490-600 nm were recorded, with a reading every 5 nm.
  • the excitation and emission spectra are shown in FIG5 .
  • the twenty fructose-1,6-bisphosphate optical probes numbered 1-20 in the purified Example 5 were subjected to fructose-1,6-bisphosphate detection with a concentration gradient (0-5 mM). After the purified probes were treated for 10 minutes, the changes in the fluorescence intensity at 528 nm emission under 420 nm excitation and the ratio of the fluorescence intensity at 528 nm emission under 485 nm excitation were detected.
  • the Kd (binding constant) of the twenty fructose-1,6-bisphosphate optical probes are 37 ⁇ M, 25 ⁇ M, 73 ⁇ M, 11 ⁇ M, 38 ⁇ M, 12 ⁇ M, 36 ⁇ M, 22 ⁇ M, 32 ⁇ M, 74 ⁇ M, 12 ⁇ M, 5.7 ⁇ M, 6.2 ⁇ M, 3.3 ⁇ M, 360 ⁇ M, 240 ⁇ M, 769 ⁇ M, 1524 ⁇ M, 117 ⁇ M, 148 ⁇ M and 92 ⁇ M, respectively.
  • the twenty fructose-1,6-bisphosphate optical probes numbered 1-20 in the purified Example 5 were subjected to reactivity detection with eight similar substrates. The results showed that they had good specificity, as shown in FIG7 .
  • Example 7 Subcellular organelle localization of optical probes and performance of optical probes within subcellular organelles
  • the fructose-1,6-bisphosphate optical probe can be localized to subcellular organelles including cytoplasm, nucleus, mitochondria, and nuclear exclusion by fusing with different specific localization signal peptides. Fluorescence is shown in different subcellular structures, and the distribution and intensity of fluorescence are different.
  • H1299 cells were transfected with the cytoplasm-expressed optical probe plasmid for 36 hours, they were rinsed with PBS and placed in HBSS solution containing 0 mM 3-BrPA and 0.5 mM 3-BrPA, and the changes in the ratio of the fluorescence intensity at 528 nm emission at 420 nm excitation and 528 nm emission at 485 nm excitation were detected over a 30-min period.
  • the results are shown in Figure 9.
  • the ratio of 420/485 of the sample with 3-BrPA added gradually increased, reaching up to 3.7 times the initial value, while the 420/485 of the control group without 3-BrPA added remained unchanged at 0.63.
  • Example 8 High-throughput compound screening based on optical probes in living cells
  • HeLa cells expressing 182/184 (CggR: N184V/Q185P/D181E/V182GCPYFP: Y1V) in the cytoplasm for high-throughput compound screening.
  • the transfected HeLa cells were rinsed with PBS, placed in HBSS solution (without fructose-1,6-bisphosphate) for 1 hour, and then treated with 10 ⁇ M compounds for 1 hour.
  • Various compounds were added to each sample. The changes in the ratio of fluorescence intensity at 420nm excitation and 528nm emission and fluorescence intensity at 485nm excitation and 528nm emission were recorded using an ELISA instrument. Samples not treated with any compounds were used as controls for standardization. The results are shown in Figure 10. Among the 1000 compounds used, most of the compounds had little effect on the concentration of fructose-1,6-bisphosphate in cells. 38 compounds can increase the concentration of fructose-1,6-bisphosphate in cells, and 13 compounds can significantly reduce the concentration of fructose-1,6-bisphosphate in cells.
  • Example 9 Quantitative detection of fructose-1,6-bisphosphate in blood using an optical probe
  • fructose-1,6-bisphosphate in the blood supernatants of mice and humans was analyzed using purified 179/180 (CggR: E180G/D181G) with a Kd of approximately 1 ⁇ M.
  • the fructose-1,6-bisphosphate optical probe provided by the present invention has a relatively small protein molecular weight and is easy to mature, has a large dynamic change in fluorescence, has good specificity, can be expressed in cells through genetic manipulation methods, can locate and quantitatively detect fructose-1,6-bisphosphate in real time inside and outside cells, and can perform high-throughput compound screening.

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Abstract

本发明涉及果糖二磷酸光学探针。具体地，本发明提供一种果糖二磷酸光学探针，其包含果糖-1，6-二磷酸敏感多肽和光学活性多肽，其中，光学活性多肽位于果糖-1，6-二磷酸敏感多肽的序列内，所述果糖-1，6-二磷酸敏感多肽是果糖-1，6-二磷酸结合蛋白或其功能变体，所述光学活性多肽是荧光蛋白或其功能变体。本发明的荧光探针荧光动态变化大，特异性好，可在细胞内外高通量、定量检测果糖二磷酸。

Description

一种果糖-1，6-二磷酸光学探针及其制备方法和应用 技术领域
本发明涉及光学探针技术领域，尤其涉及一种果糖-1，6-二磷酸光学探针及其制备方法和应用。
背景技术
果糖-1，6-二磷酸(FBP)是细胞内天然存在的一种化合物，是细胞在糖酵解途径中产生的一个重要中间产物，在细胞中通过调节糖代谢中若干酶的活性产生药理作用。
果糖-1，6-二磷酸是糖酵解途径、糖异生过程中的重要代谢物，由6-磷酸果糖在磷酸果糖激酶的催化下产生，之后在醛缩酶的作用下生成磷酸二羟丙酮和3-磷酸甘油醛，最终生成丙酮酸进入三羧酸循环途径。除此之外，果糖-1，6-二磷酸调控多种代谢途径中多种酶的活性。
由于其在代谢途径中的重要作用，果糖-1，6-二磷酸被用于治疗代谢相关、呼吸系统相关疾病，并取得了良好的疗效。比如生物体内糖代谢异常时引起ATP的含量的降低进而导致血红蛋白的携氧能力下降，造成代谢紊乱产生疾病，而果糖-1，6-二磷酸能促使细胞体内糖的分解，治疗相关疾病；在呼吸系统疾病方面，果糖-1，6-二磷酸通过促进细胞内磷酸果糖激酶的分解来提升生物体内磷酸池的含量进而提升肺部平滑肌的舒张能力，实现受损细胞的修复。
目前常见果糖-1，6-二磷酸的检测方法主要有色谱法(Ma B等.European Journal of Pharmacology，2013，718(1-3)，524-532)、酶法(Iwamoto S等.Applied and Environmental Microbiology，2007，73(17)，5676-5678)、和显色法(Wang C等.Journal ofbiomedical engineering，2000，17(3)，363-365)等。色谱法酶法检测具有较好的特异性，但具有成本较高的缺点(伍良涌等.中国药品标准，2007，8(4)，63-65)；色谱法能快速的检测果糖-1，6-二磷酸的含量，能检测较低含量的果糖-1，6-二磷酸，但其实验过程中容易出现误差(吴燕等.中南医学科学杂志，2000，(6)，608-609)；而显色法则存在果糖和单磷酸果糖等物质的干扰问题(杜振宁等.中国生化药物杂志，1993，(2)，59-62)，并且上述几种方法只适合体外检测，无法实时监控活细胞的果糖-1，6-二磷酸浓度变化。因此，开发新的特异性好响应速度快且可以对体内果糖-1，6-二磷酸实时定量检测的方法具有重要的意义。
发明内容
本发明的目的在于提供在细胞内、外实时定位、高通量、定量检测果糖-1，6-二磷酸的探针和方法。
为了实现上述发明目的，本发明提供以下技术方案：
本发明第一方面提供一种果糖-1，6-二磷酸结合蛋白变体，其：
(1)具有SEQ ID NO：1所示的序列并且在选自以下的1个、2个或3个、4个、5个或更多个位点具有突变：T148、R175、G176、G177、L178、E180、D181、V182、K183、N184、Q185、F271，所述突变包括氨基酸的修饰、取代或缺失，
(2)是(1)的具有第88-340位氨基酸的截短变体，或
(3)是与(1)或(2)的序列具有至少70％序列相同性并具有(1)所述突变并保留对果糖-1，6-二磷酸结合能力的序列。
在一个或多个实施方案中，所述突变的位点选自以下任一组中的1个、2个或3个、4个、5个：(a)T148、R175、G176、G177、L178、D181、V182、K183、N184、Q185，(b)G177、L178、E180、D181、V182、N184、Q185、F271。
在一个或多个实施方案中，所述突变包括选自以下任一组的位点处的突变：(1)G177和L178，(5)D181和V182，(6)E180和D181，(7)N184和Q185；或者，所述突变包含选自以下任一组的位点处的突变：(1)G177、L178，(2)G177、L178和Q185，(3)G177、L178和T148，(4)G177、L178和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。
在一个或多个实施方案中，T148突变为S。在一个或多个实施方案中，G177突变为A、I、L、M或N，优选突变为L或M。在一个或多个实施方案中，L178突变为Y、F或N，优选突变为N。在一个或多个实施方案中，E180突变为G。在一个或多个实施方案中，D181突变为F、L或E，优选突变为E。在一个或多个实施方案中，V182突变为F、L或G，优选突变为G。在一个或多个实施方案中，N184突变为V。在一个或多个实施方案中，Q185突变为S、A、T、D或P。在一个或多个实施方案中，F271突变为Y。
在一个或多个实施方案中，所述突变包含选自以下的一个或多个：G177L、D181E、V182G、N184V、Q185A。
在一个或多个实施方案中，所述突变包含选自以下任一组的突变：(1)G177A和L178Y，(2)G177I和L178F，(3)G177L和L178Y，(4)G177M和 L178N，(5)G177N和L178Y，(6)G177M、L178N和Q185S，(7)G177M、L178N和Q185A，(8)G177M、L178N和Q185T，(9)G177M、L178N和Q185D，(10)G177、L178和T148S，(11)G177、L178和F271Y，(12)D181F和V182F，(13)D181L和V182L，(14)E180G和D181G，(15)N184V和Q185P，(16)N184V、Q185P、D181E、V182G和G177L，(17)N184V、Q185P和G177L，(18)N184V、Q185P、D181E和V182G。
本发明另一方面提供一种果糖-1，6-二磷酸光学探针，包含果糖-1，6-二磷酸敏感多肽和光学活性多肽，其中光学活性多肽位于果糖-1，6-二磷酸敏感多肽的序列内。果糖-1，6-二磷酸敏感多肽被光学活性多肽分为第一部分和第二部分。
在一个或多个实施方案中，所述果糖-1，6-二磷酸光学探针，包括果糖-1，6-二磷酸敏感多肽B和光学活性多肽A，其中光学活性多肽A位于果糖-1，6-二磷酸敏感多肽B的序列内，将果糖-1，6-二磷酸敏感多肽B分为第一部分B1和第二部分B2，形成B1-A-B2式的探针结构。
在一个或多个实施方案中，光学活性多肽位于果糖-1，6-二磷酸敏感多肽的残基174-185和/或201-208之间，编号对应于果糖-1，6-二磷酸敏感多肽的全长。优选地，光学活性多肽位于果糖-1，6-二磷酸敏感多肽的选自下述位点中的任一个或多个：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和/或207/208。更优选地，光学活性多肽位于果糖-1，6-二磷酸敏感多肽的选自下述位点中的任一个或多个：176/177，176/178，177/181，179/180，182/184，182/185，203/206，203/207和203/208。
在一个或多个实施方案中，所述果糖-1，6-二磷酸敏感多肽是果糖-1，6-二磷酸结合蛋白或其功能变体，其中，果糖-1，6-二磷酸结合蛋白的功能变体在与光学活性多肽连接处的7个氨基酸内具有突变。
在一个或多个实施方案中，果糖-1，6-二磷酸敏感多肽具有：
(1)SEQ ID NO：1所示的序列或其具有第88-340位氨基酸的截短变体，或与它们有至少70％序列相同性并保留对果糖-1，6-二磷酸结合活性的序列，
(2)本文第一方面任一实施方案所述的果糖-1，6-二磷酸结合蛋白变体的序列，或
(3)与(2)所述序列具有至少70％序列相同性并具有(2)所述突变并保留对果糖-1，6-二磷酸敏感性的序列。
在一个或多个实施方案中，所述光学活性多肽是荧光蛋白或其功能变体，其中，荧光蛋白的功能变体在与光学活性多肽连接处的3个氨基酸内具有突变。
在一个实施方式中，荧光蛋白选自黄色荧光蛋白、橘黄色荧光蛋白、红色荧光蛋白、绿色荧光蛋白、蓝色荧光蛋白、苹果红荧光蛋白。在一个实施方式中，荧光蛋白具有SEQ ID NO：2-9中任一所示的序列。
在一个或多个实施方案中，荧光蛋白的功能变体在第1-3位，优选第1位，的氨基酸处具有突变。优选地，荧光蛋白的功能变体包括荧光蛋白的第1位氨基酸突变为I或V的突变。
在一个或多个实施方案中，荧光蛋白的功能变体具有SEQ ID NO：2所示的序列并在Y1位点具有突变。优选地，所述突变为Y1I或Y1V。
在一个实施方式中，光学探针还包含侧接所述光学活性多肽的一个或多个接头。本发明所述接头可以是任何长度的任何氨基酸序列。在一个实施方式中，光学活性多肽侧翼包含不超过5个氨基酸的接头，例如0、1、2、3、4个氨基酸的接头。在一个实施方式中，光学活性多肽侧翼的接头包含氨基酸Y。在一个实施方式中，接头Y位于光学活性多肽的N端和/或C端。在一个实施方式中，光学探针如下所示：果糖-1，6-二磷酸敏感多肽的第一部分B1、Y、光学活性多肽A、果糖-1，6-二磷酸敏感多肽的第二部分B2。在一个实施方式中，本发明光学探针不包含接头。
在一个实施方式中，本发明光学探针还包含定位序列，用于将探针定位到例如细胞的特定细胞器。
在一个或多个实施方案中，果糖-1，6-二磷酸敏感多肽是SEQ ID NO：1的具有第88-340位氨基酸的截短变体，并且光学活性多肽位于果糖-1，6-二磷酸敏感多肽的选自下述位点中的任一个或多个：176/177，176/178，177/181，179/180，182/184， 182/185，203/206，203/207和203/208。优选地，光学活性多肽如SEQ ID NO：2所示。
在一个或多个实施方案中，果糖-1，6-二磷酸敏感多肽是SEQ ID NO：1的具有第88-340位氨基酸的截短变体，光学活性多肽位于果糖-1，6-二磷酸敏感多肽的选自下述位点中的任一个或多个：176/177，177/181，179/180或182/184，并且所述光学探针具有选自以下的一个或多个突变：果糖-1，6-二磷酸敏感多肽的T148S、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，光学活性多肽的第1位氨基酸突变为V或I。优选地，光学活性多肽如SEQ ID NO：2所示。
在一个或多个实施方案中，果糖-1，6-二磷酸敏感多肽是SEQ ID NO：1的具有第88-340位氨基酸的截短变体，光学活性多肽位于果糖-1，6-二磷酸敏感多肽的选自下述位点中的任一个或多个：176/177，177/181，179/180或182/184，所述光学探针具有选自以下任一组所示的突变：(1)果糖-1，6-二磷酸敏感多肽的G177A和L178Y，(2)果糖-1，6-二磷酸敏感多肽的G177I和L178F，(3)果糖-1，6-二磷酸敏感多肽的G177L和L178Y，(4)果糖-1，6-二磷酸敏感多肽的G177M和L178N，(5)果糖-1，6-二磷酸敏感多肽的G177N和L178Y，(6)果糖-1，6-二磷酸敏感多肽的G177M、L178N和Q185S，(7)果糖-1，6-二磷酸敏感多肽的G177M、L178N和Q185A，(8)果糖-1，6-二磷酸敏感多肽的G177M、L178N和Q185T，(9)果糖-1，6-二磷酸敏感多肽的G177M、L178N和Q185D，(10)果糖-1，6-二磷酸敏感多肽的G177M、L178N和T148S，(11)果糖-1，6-二磷酸敏感多肽的G177M、L178N和F271Y，(12)果糖-1，6-二磷酸敏感多肽的D181F和V182F，(13)果糖-1，6-二磷酸敏感多肽的D181L和V182L，(14)果糖-1，6-二磷酸敏感多肽的E180G和D181G，(15)果糖-1，6-二磷酸敏感多肽的N184V和Q185P，(16)果糖-1，6-二磷酸敏感多肽的N184V、Q185P、D181E、V182G、G177L，和光学活性多肽的第1位氨基酸突变为V，(17)果糖-1，6-二磷酸敏感多肽的N184V、Q185P、D181E、V182G、G177L，和光学活性多肽的第1位氨基酸突变为I，(18)果糖-1，6-二磷酸敏感多肽的N184V、Q185P和G177L，和光学活性多肽的第1位氨基酸突变为V，(19)果糖-1，6-二磷酸敏感多肽的N184V、Q185P和G177L，和光学活性多肽的第1位氨基酸突变为I，(20)果糖-1，6-二磷酸敏感多肽的N184V、Q185P、D181E和V182G，和光学活性多肽的第1位氨基酸突变为V。优选地，光学活性多肽如SEQ ID NO：2所示。
本发明另一方面还提供融合多肽，包含本文所述光学探针和其它多肽。在一些实施方式中，其他多肽位于所述光学探针的N端和/或C端。在一些实施方式中，其他多肽包括将光学探针定位到不同细胞器或亚细胞器的多肽、用于纯化的标签或者用于免疫印迹的标签。
本发明另一方面还提供核酸分子，其包含：(a)本文任一实施方案所述的多肽或探针的编码序列，或(b)(a)的互补序列，或(c)(a)或(b)的片段。所述片段是引物。
本发明还涉及上述核酸分子的变体，包括编码本发明光学探针或融合蛋白的片段、类似物、衍生物、可溶性片段和变体的核酸序列或其互补序列。
本发明另一方面还提供包含本文所述核酸分子的核酸构建物。该核酸序列编码本发明所述光学探针或融合多肽。
在一个或多个实施方案中，所述核酸构建物是克隆载体、表达载体或重组载体。
在一个或多个实施方案中，所述核酸分子与表达控制序列操作性连接。
在一些实施方案中，表达载体选自原核表达载体、真核表达载体和病毒载体。
本发明另一方面还提供一种宿主细胞，所述宿主细胞：(1)表达本发明任一实施方案所述的光学探针或融合多肽；(2)包含本发明任一实施方案所述的核酸分子；或(3)包含本发明任一实施方案所述的核酸构建物。所述宿主细胞优选大肠杆菌。
本发明另一方面还提供果糖-1，6-二磷酸检测试剂盒，包括本文所述光学探针或融合多肽或多核苷酸或如本文所述方法制备的光学探针。
在一个或多个实施方案中，所述试剂盒还包含选自以下的一种或多种试剂：缓冲液、培养基、果糖-1，6-二磷酸标准品。
本发明另一方面提供制备本文所述光学探针的方法，包括：提供表达本文所述光学探针或融合多肽的宿主细胞，在所述细胞表达的条件下培养所述宿主细胞，和分离光学探针或融合多肽。
在一个或多个实施方案中，所述方法包括以下步骤：1)将编码本文所述果糖-1，6-二磷酸光学探针的核酸分子纳入表达载体；2)将表达载体转移到宿主细胞中；2)在适合所述表达载体表达的条件下培养所述宿主细胞，3)分离果糖-1，6-二磷酸光学探针。
本发明另一方面还提供检测样品中果糖-1，6-二磷酸的方法，包括：使本文所述光学探针或融合多肽或宿主细胞与样品接触，和检测光学活性多肽的变化。所述检测可以在体内、体外、亚细胞或原位进行。所述样品例如血液。
本文另一方面还提供定量样品中果糖-1，6-二磷酸的方法，包括：使本文所述光学探针或融合多肽或宿主细胞与样品接触，检测光学活性多肽的光学变化，和根据光学活性多肽的光学变化定量样品中的果糖-1，6-二磷酸。
本发明另一方面还提供筛选化合物(例如药物)的方法，包括：在含果糖-1，6-二磷酸的体系中使本文所述光学探针或融合多肽或宿主细胞与候选化合物接触，检测光学活性多肽的光学变化，和根据光学活性多肽的光学变化筛选化合物。所述方法可以高通量地筛选化合物。
在一个或多个实施方案中，在含果糖-1，6-二磷酸的体系中使本文所述宿主细胞与候选化合物接触，并且光学活性多肽的光学变化指示所述候选化合物是否能调节细胞对果糖-1，6-二磷酸的摄取。
本发明另一方面还提供对所述果糖-1，6-二磷酸进行细胞内和/或外定位的方法，包括：将含果糖-1，6-二磷酸的体系与所述光学探针或所述宿主细胞接触，和检测光学活性多肽的光学变化。
在一个或多个实施方案中，所述体系是溶液体系、细胞体系、亚细胞体系。
本发明另一方面还提供本文所述果糖-1，6-二磷酸光学探针或融合多肽或宿主细胞在检测样品中的果糖-1，6-二磷酸、筛选化合物或果糖-1，6-二磷酸细胞内/外定位中的应用。在一个或多个实施方案中，所述定位是实时定位。
本发明的有益效果：本发明提供的果糖-1，6-二磷酸光学探针易于成熟，荧光动态变化大，特异性好，并且能够通过基因操作的方法在细胞中表达，可在细胞内外实时定位、高通量、定量检测果糖-1，6-二磷酸，省去了耗时的处理样品步骤。实验效果表明本申请所提供的果糖-1，6-二磷酸光学探针对果糖-1，6-二磷酸的最高响应达到对照的12倍以上，并且可以在细胞浆、线粒体、细胞核、内质网、溶酶体和高尔 基体等亚细胞结构中对细胞进行定位、定性、定量检测，并且可以进行高通量的化合物筛选以及血液中果糖-1，6-二磷酸定量检测。
附图说明
下面结合附图和实施例对本发明作进一步说明。
图1为实施例2所述的示例性果糖-1，6-二磷酸光学探针的SDS-PAGE图；
图2为实施例2所述的示例性的包含cpYFP和果糖-1，6-二磷酸结合蛋白的果糖-1，6-二磷酸光学探针对果糖-1，6-二磷酸响应变化表；
图3为实施例3所述的示例性的包含cpGFP和果糖-1，6-二磷酸结合蛋白的果糖-1，6-二磷酸光学探针对果糖-1，6-二磷酸响应变化表；
图4为实施例4所述的示例性的包含cpBFP和果糖-1，6-二磷酸结合蛋白的果糖-1，6-二磷酸光学探针对果糖-1，6-二磷酸响应变化表；
图5为实施例6所述的示例性果糖-1，6-二磷酸光学探针的荧光光谱性质图；
图6为实施例6所述的示例性的果糖-1，6-二磷酸光学探针对不同浓度果糖-1，6-二磷酸的滴定曲线；
图7为实施例6所述的示例性的果糖-1，6-二磷酸光学探针对糖酵解途径中的多种相似底物的特异性检测的柱状图；
图8为实施例7所述的示例性果糖-1，6-二磷酸光学探针在哺乳动物细胞中的亚细胞器定位照片；
图9为实施例7所述的对示例性果糖-1，6-二磷酸光学探针在哺乳动物细胞中对胞浆中的果糖-1，6-二磷酸浓度进行动态监测的示意图；
图10为实施例8所述的示例性果糖-1，6-二磷酸光学探针在活细胞水平进行高通量化合物筛选的点图；
图11为实施例9所述的示例性果糖-1，6-二磷酸光学探针对人血液中的果糖-1，6-二磷酸进行定量的柱状图。
具体实施方式
在给出数值或范围时，本文所用术语“约”指该数值或范围在给定数值或范围的20％以内、10％以内和5％以内。
本文所用术语“包含”、“包括”和其等同形式包括“含有”以及“由......组成”的含义，例如“包含”X的组合物可仅由X组成或可含有其它物质，例如X+Y。
本文所用术语“果糖-1，6-二磷酸敏感多肽”指对果糖-1，6-二磷酸产生响应的多肽，所述响应包括与敏感多肽的相互作用相关的多肽的化学，生物学，电学或生理学参数的任何响应。响应包括小的变化，例如，多肽的氨基酸或肽片段的方向的变化以及例如多肽的一级，二级或三级结构的变化，包括例如质子化，电化学势和/或构象的变化。“构象”是分子中包含侧基的分子的一级，二级和三级结构的三维排列；当分子的三维结构发生变化时，构象发生变化。构象变化的实例包括从α-螺旋转变为β-折叠或从β-折叠转变为α-螺旋。可以理解的是，只要荧光蛋白部分的荧光被改变，可检测到的改变不需要是构象改变。本文所述果糖-1，6-二磷酸敏感多肽还可包括其功能变体。果糖-1，6-二磷酸敏感多肽的功能变体包括但不限于可以与果糖-1，6-二磷酸相互作用从而发生与亲本果糖-1，6-二磷酸敏感多肽相同或相似变化的变体。
本发明所述果糖-1，6-二磷酸敏感多肽包括但不限于果糖-1，6-二磷酸结合蛋白CggR或与其有90％以上同源性的变体。本发明所述示例性果糖-1，6-二磷酸结合蛋白CggR来源于枯草芽孢杆菌Bacillus subtilis。CggR属于原核生物转录调控因子SorC/DeoR家族，由N端的DNA结合域和C端的配体结构域两个结构域组成。果糖-1，6-二磷酸结合蛋白可以感应果糖-1，6-二磷酸浓度的变化，在果糖-1，6-二磷酸浓度动态变化的过程中果糖-1，6-二磷酸结合蛋白的空间构象也会发生改变。示例性CggR蛋白截短体如SEQ ID NO：1所示。当描述本发明光学探针时(例如描述插入位点或突变位点时)，提及氨基酸残基编号均参考SEQ ID NO：1。
本文所用术语“光学探针”是指与光学活性多肽融合的果糖-1，6-二磷酸敏感多肽。发明人发现，果糖-1，6-二磷酸敏感多肽例如果糖-1，6-二磷酸结合蛋白专一性地对生理浓度的果糖-1，6-二磷酸结合后所产生的构象变化会引起光学活性多肽(例如荧光蛋白)的构象变化，进而导致光学活性多肽的光学性质发生改变。借助不同果糖-1，6-二磷酸浓度下测定的荧光蛋白的荧光绘制标准曲线，可以检测并分析果糖-1，6-二磷酸的存在和/或水平。
在本发明的光学探针中，光学活性多肽(例如荧光蛋白)可操作地插入果糖-1，6-二磷酸敏感多肽中。基于蛋白质的“光学活性多肽”是具有发射荧光能力的多肽。荧光是光学活性多肽的一种光学性质，其可用作检测本发明的光学探针的响应性的手段。如本文所用，术语“荧光性质”是指适当激发波长下的摩尔消光系数，荧光量子效率，激发光谱或发射光谱的形状，激发波长最大值和发射波长最大值，两个不同波长激发的振幅，两个不同波长的发射振幅比，激发态寿命或荧光各向异性。活性和无活性状态之间的这些性质中的任何一个的可测量的差异足以用于本发明的荧光蛋白底物在活性测定中的效用。可测量的差异可通过确定任何定量荧光性质的 量来确定，例如，特定波长处的荧光量或荧光在发射光谱上的积分。优选地，选择蛋白质底物以具有在未激活和活化的构象状态下容易区分的荧光特性。本文所述光学活性多肽还可包括其功能变体。光学活性多肽的功能变体包括但不限于可以发生与亲本光学活性多肽相同或相似荧光性质变化的变体。
“接头”或“连接区”指在本发明多肽、蛋白质或核酸中连接两个部分的氨基酸或核苷酸序列。示例性地，本发明中果糖-1，6-二磷酸敏感多肽与光学活性多肽的连接区氨基端的氨基酸数目选择的是0-3个，羧基端的氨基酸数目选择的是0-2个；当重组光学探针作为基本单元与功能蛋白连接时，可以融合在重组光学探针的氨基酸或羧基端。接头序列可为一个或多个柔性氨基酸组成的短肽链，如Y。
本文所用术语“荧光蛋白”指在激发光照射下发出荧光的蛋白质。荧光蛋白作为生物科学领域的基础检测手段，例如生物技术领域常用的绿色荧光蛋白GFP及由该蛋白突变衍生出的环状重排的蓝色荧光蛋白(cpBFP)、环状重排的绿色荧光蛋白(cpGFP)、环状重排的黄色荧光蛋白(cpYFP)等；还有本技术领域常用的红色荧光蛋白RFP，及由该蛋白衍生出来的环状重排的蛋白，如cpmApple，cpmOrange，cpmKate等。示例性地，cpYFP如SEQ ID NO：2所示，cpmOrange如SEQ ID NO：3所示，cpmKate如SEQ ID NO：4或8所示，mCherry如SEQ ID NO：5所示，cpGFP如SEQ ID NO：6所示，cpBFP如SEQ ID NO：7所示，cpmApple如SEQ ID NO：9所示。
光学探针中的荧光蛋白也包括具有突变的功能变体，包括但不限于在第1-3位(优选第1位)氨基酸具有突变的荧光蛋白，例如突变为V或I。示例性地，cpYFP的功能变体具有SEQ ID NO：2所示的序列并在Y1位点具有突变V或I。
在本发明的光学探针中，光学活性多肽以N-C方向位于N-C方向的果糖-1，6-二磷酸敏感多肽的残基174-185和/或201-208之间，编号对应于果糖-1，6-二磷酸敏感多肽的全长。
示例性地，光学活性多肽位于果糖-1，6-二磷酸结合蛋白的氨基酸序列的如下位点：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， 181/182，181/182，181/183，181/184，181/185，182/183，182/184，182/185，183/184，183/185，184/185，201/202，201/203，201/204，201/205，201/206，201/207，201/208，202/203，202/204，202/205，202/206，202/207，202/208，203/204，203/205，203/206，203/207，203/208，204/205，204/206，204/207，204/208，205/206，205/206，205/207，205/208，206/207，206/208，207/208。本文中，如果以“X/Y”形式表示的位点中的两个数字是连续的整数，则表示光学活性多肽位于该数字所述的氨基酸之间。例如插入位点147/148表示光学活性多肽位于果糖-1，6-二磷酸敏感多肽的氨基酸147与148之间。如果以“X/Y”形式表示的位点中的两个数字不是连续的整数，则表示光学活性多肽置换该数字所示氨基酸之间的氨基酸。例如插入位点174/185表示光学活性多肽置换果糖-1，6-二磷酸敏感多肽的氨基酸175-184。优选地，光学活性多肽插入果糖-1，6-二磷酸敏感多肽的下述位点：176/177，176/178，177/181，179/180，182/184，182/185，203/206，203/207和203/208。
在示例性实施方式中，本发明B1-A-B2式光学探针可为当荧光蛋白位于CggR或其截短变体(例如SEQ ID NO：1或其包含第88-340位氨基酸的片段)的176/177，176/178，177/181，179/180，182/184，182/185，203/206，203/207和203/208时形成的探针。在具体实施方案中，光学探针中的果糖-1，6-二磷酸敏感多肽如SEQ ID NO：1的第88-340位氨基酸所示，光学活性多肽如SEQ ID NO：2-9中任一项所示，并且光学活性多肽位于果糖-1，6-二磷酸敏感多肽的以下位点处：176/177，176/178，177/181，179/180，182/184，182/185，203/206，203/207或203/208。
提到某多肽或蛋白时，本发明所用术语“变体”或“突变体”包括具有所述多肽或蛋白相同功能、但序列不同的变体。多肽或蛋白的变体可包括：同源序列、保守性变体、等位变体、天然突变体、诱导突变体。这些变体包括但并不限于：在所述多肽或蛋白的序列中缺失、插入和/或取代一个或多个(通常为1-30个，较佳地1-20个，更佳地1-10个，最佳地1-5个)氨基酸，以及在其羧基末端和/或氨基末端添加一个或数个(通常为20个以内，较佳地为10个以内，更佳地为5个以内)氨基酸获得的序列。这些变体还可包含与所述多肽或蛋白的序列相同性为至少约70％、至少约75％、至少约80％、至少约85％、至少约90％、至少约95％、至少约98％、至少约99％或100％的多肽或蛋白。不希望受理论限制，氨基酸残基发生改变而不改变多肽或蛋白质的总体构型和功能，即功能保守突变。例如，在本领域中，用性能相近或相似的氨基酸进行取代时，通常不会改变多肽或蛋白的功能。在本领域中，性能相似的氨基酸往往指具有相似侧链的氨基酸家族，在本领域已有明确定义。这 些家族包括具有碱性侧链的氨基酸(例如赖氨酸、精氨酸、组氨酸)、具有酸性侧链的氨基酸(例如天冬氨酸、谷氨酸)、具有不带电荷的极性侧链的氨基酸(例如甘氨酸、天冬酰胺、谷氨酰胺、丝氨酸、苏氨酸、酪氨酸、半胱氨酸)、具有非极性侧链的氨基酸(例如丙氨酸、缬氨酸、亮氨酸、异亮氨酸、精氨酸、苯丙氨酸、甲硫氨酸、色氨酸)、具有β-分支侧链的氨基酸(例如苏氨酸、缬氨酸、异亮氨酸)和具有芳香侧链的氨基酸(例如酪氨酸、苯丙氨酸、色氨酸、组氨酸)。又比如，在氨基末端和/或羧基末端添加一个或数个氨基酸通常也不会改变多肽或蛋白的功能。对于许多常见已知非遗传性编码氨基酸的保守氨基酸取代本领域已知。其他非编码氨基酸的保守取代可基于其物理性质与遗传上编码的氨基酸的性质的比较来确定。
发明人发现，在SEQ ID NO：1或其截短变体的选自以下的位点具有突变的果糖-1，6-二磷酸结合蛋白变体表现出与果糖-1，6-二磷酸不同的结合活性：T148、R175、G176、G177、L178、E180、D181、V182、K183、N184、Q185、F271。所述氨基酸突变包括氨基酸的修饰、取代或缺失。特别地，在SEQ ID NO：1或其截短变体的选自以下任一组中的1个、2个或3个、4个、5个的位点具有突变的果糖-1，6-二磷酸结合蛋白变体更有利于用于本发明的光学探针以更高效检测果糖-1，6-二磷酸：(a)T148、R175、G176、G177、L178、D181、V182、K183、N184、Q185，(b)G177、L178、E180、D181、V182、N184、Q185、F271。在较佳的实施方案中，果糖-1，6-二磷酸结合蛋白变体的突变包括选自以下任一组的位点处的突变：(1)G177和L178，(5)D181和V182，(6)E180和D181，(7)N184和Q185；或者，所述突变包含选自以下任一组的位点处的突变：(1)G177、L178，(2)G177、L178和Q185，(3)G177、L178和T148，(4)G177、L178和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。
其中，作为实施例中的示例，在SEQ ID NO：1或其截短变体中，T148突变为S；G177突变为A、I、L、M或N，优选突变为L或M；L178突变为Y、F或N，优选突变为N；E180突变为G；D181突变为F、L或E，优选突变为E；V182突变为F、L或G，优选突变为G；N184突变为V；Q185突变为S、A、T、D或P，优选突变为P；F271突变为Y。
在优选实施方案中，针对果糖-1，6-二磷酸结合蛋白变体(SEQ ID NO：1或其截短变体的)的突变选自以下中的任一项：(1)G177A和L178Y，(2)G177I和L178F，(3)G177L和L178Y，(4)G177M和L178N，(5)G177N和L178Y，(6)G177M、L178N和Q185S，(7)G177M、L178N和Q185A，(8)G177M、 L178N和Q185T，(9)G177M、L178N和Q185D，(10)G177M、L178N和T148S，(11)G177M、L178N和F271Y，(12)D181F和V182F，(13)D181L和V182L，(14)E180G和D181G，(15)N184V和Q185P，(16)N184V、Q185P、D181E、V182G和G177L，(17)N184V、Q185P和G177L，(18)N184V、Q185P、D181E和V182G。
本发明提供具有这些突变的果糖-1，6-二磷酸结合蛋白变体以及包含此类果糖-1，6-二磷酸结合蛋白变体作为果糖-1，6-二磷酸敏感多肽的光学探针。因此，在一个或多个实施方案中，光学探针中的果糖-1，6-二磷酸敏感多肽是本文任一实施方案所述的果糖-1，6-二磷酸结合蛋白变体，光学探针中的荧光蛋白如SEQ ID NO：2-9所示并且荧光蛋白的第1位氨基酸突变为I或V。
在一些具体实施方案中，光学探针中的果糖-1，6-二磷酸敏感多肽如SEQ ID NO：1的第88-340位氨基酸所示，光学活性多肽如SEQ ID NO：2所示，光学活性多肽位于果糖-1，6-二磷酸敏感多肽的176/177位点处，并且果糖-1，6-二磷酸敏感多肽具有选自以下中的任一项所示的突变：(1)G177A和L178Y，(2)G177I和L178F，(3)G177L和L178Y，(4)G177M和L178N，(5)G177N和L178Y，(6)G177M、L178N和Q185S，(7)G177M、L178N和Q185A，(8)G177M、L178N和Q185T，(9)G177M、L178N和Q185D，(10)G177M、L178N和T148S，(11)G177M、L178N和F271Y。示例性地，第(4)项所示的光学探针的氨基酸序列如SEQ ID NO：10所示，核酸序列如SEQ ID NO：17所示；第(7)项所示的光学探针的氨基酸序列如SEQ ID NO：11所示，核酸序列如SEQ ID NO：18所示。；第(8)项所示的光学探针的氨基酸序列如SEQ ID NO：12所示，核酸序列如SEQ ID NO：19所示；第(9)项所示的光学探针的氨基酸序列如SEQ ID NO：13所示，核酸序列如SEQ ID NO：20所示。
在一些具体实施方案中，光学探针中的果糖-1，6-二磷酸敏感多肽如SEQ ID NO：1的第88-340位氨基酸所示，光学活性多肽如SEQ ID NO：2所示，光学活性多肽位于果糖-1，6-二磷酸敏感多肽的177/181位点处，并且果糖-1，6-二磷酸敏感多肽具有选自以下中的任一项所示的突变：(12)D181F和V182F，(13)D181L和V182L。
在一些具体实施方案中，光学探针中的果糖-1，6-二磷酸敏感多肽如SEQ ID NO：1的第88-340位氨基酸所示，光学活性多肽如SEQ ID NO：2所示，光学活性多肽位于果糖-1，6-二磷酸敏感多肽的179/180位点处，并且果糖-1，6-二磷酸敏感多肽具有突变(14)E180G和D181G。
在一些具体实施方案中，光学探针中的果糖-1，6-二磷酸敏感多肽如SEQ ID NO：1的第88-340位氨基酸所示，光学活性多肽如SEQ ID NO：2所示，光学活性多肽位于果糖-1，6-二磷酸敏感多肽的182/184位点处，并且光学探针具有选自以下中的任一项所示的突变：(15)果糖-1，6-二磷酸敏感多肽的N184V和Q185P，(16)果糖-1，6-二磷酸敏感多肽的N184V、Q185P、D181E、V182G、G177L，和光学活性多肽的第1位氨基酸突变为V，(17)果糖-1，6-二磷酸敏感多肽的N184V、Q185P、D181E、V182G、G177L，和光学活性多肽的第1位氨基酸突变为I，(18)果糖-1，6-二磷酸敏感多肽的N184V、Q185P和G177L，和光学活性多肽的第1位氨基酸突变为V，(19)果糖-1，6-二磷酸敏感多肽的N184V、Q185P和G177L，和光学活性多肽的第1位氨基酸突变为I，(20)果糖-1，6-二磷酸敏感多肽的N184V、Q185P、D181E和V182G，和光学活性多肽的第1位氨基酸突变为V。示例性地，第(16)项所示的光学探针的氨基酸序列如SEQ ID NO：14所示，核酸序列如SEQ ID NO：21所示；第(17)项所示的光学探针的氨基酸序列如SEQ ID NO：15所示，核酸序列如SEQ ID NO：22所示；第(20)项所示的光学探针的氨基酸序列如SEQ ID NO：16所示，核酸序列如SEQ ID NO：23所示。
在两种或多种多肽或核酸分子序列中，术语“相同性”或“相同性百分数”指在比较窗口或指定区域上，采用本领域已知方法如序列比较算法，通过手工比对和目测检查来比较和比对最大对应性时，两个或多个序列或子序列相同或其中在指定区域有一定百分数的氨基酸残基或核苷酸相同(例如，60％、65％、70％、75％、80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或100％相同)。例如，适合测定序列相同性百分数和序列相似性百分数的优选算法是BLAST和BLAST 2.0算法，分别可参见Altschul等(1977)Nucleic Acids Res.25：3389和Altschul等(1990)J.Mol.Biol.215：403。
本领域技术人员公知，在基因克隆操作中，常常需要设计合适的酶切位点，这势必在所表达的多肽或蛋白末端引入了一个或多个不相干的残基，而这并不影响目的多肽或蛋白的活性。又如为了构建融合蛋白、促进重组蛋白的表达、获得自动分泌到宿主细胞外的重组蛋白、或利于重组蛋白的纯化，常常需要将一些氨基酸添加至重组蛋白的N-末端、C-末端或该蛋白内的其它合适区域内，例如，包括但不限于，适合的接头肽、信号肽、前导肽、末端延伸、谷胱甘肽S-转移酶(GST)、麦芽糖E结合蛋白、蛋白A、如6His或Flag的标签，或Xa因子或凝血酶或肠激酶的蛋白水解酶位点。
本文所用术语“功能片段”、“衍生物”和“类似物”是指基本上保持与原始多肽或蛋白(例如果糖-1，6-二磷酸结合蛋白或荧光蛋白)相同的生物学功能或活性的蛋白。本发明的多肽或蛋白(例如果糖-1，6-二磷酸结合蛋白或荧光蛋白)的功能变体、衍生物或类似物可以是(i)有一个或多个保守或非保守性氨基酸残基(优选保守性氨基酸残基)被取代的蛋白，而这样的取代的氨基酸残基可以是也可以不是由遗传密码编码的，或(ii)在一个或多个氨基酸残基中具有取代基团的蛋白，或(iii)成熟蛋白与另一个化合物(比如延长蛋白半衰期的化合物，例如聚乙二醇)融合所形成的蛋白，或(iv)附加的氨基酸序列融合到此蛋白序列而形成的蛋白(如分泌序列或用来纯化此蛋白的序列或蛋白原序列，或与抗原IgG片段的形成的融合蛋白)。根据本文的教导，这些功能变体、衍生物和类似物属于本领域熟练技术人员公知的范围。所述类似物还包括具有不同于天然L-氨基酸的残基(如D-氨基酸)的类似物，以及具有非天然存在的或合成的氨基酸(如β、γ-氨基酸)的类似物。应理解，本发明的果糖-1，6-二磷酸敏感多肽并不限于上述列举的代表性蛋白、变体、衍生物和类似物。修饰(通常不改变一级结构)形式包括：体内或体外的蛋白的化学衍生形式如乙酰化或羧基化。修饰还包括糖基化，如那些在蛋白的合成和加工中或进一步加工步骤中进行糖基化修饰而产生的蛋白。这种修饰可以通过将蛋白暴露于进行糖基化的酶(如哺乳动物的糖基化酶或去糖基化酶)而完成。修饰形式还包括具有磷酸化氨基酸残基(如磷酸酪氨酸，磷酸丝氨酸，磷酸苏氨酸)的序列。还包括被修饰从而提高了其抗蛋白水解性能或优化了溶解性能的蛋白。
本发明融合多肽包含本文所述光学探针和其它多肽。在一些实施方案中，本文所述光学探针还包含与之融合的其它多肽。本文所述其他多肽不影响光学探针的性质。其他多肽可位于所述光学探针的N端和/或C端。在一些实施方案中，其他多肽包括将光学探针定位到不同细胞器或亚细胞器的多肽、用于纯化的标签或者用于免疫印迹的标签。本文所述融合多肽中的光学探针和其它多肽之间可具有接头。
本文所述亚细胞器包括细胞浆、线粒体、细胞核、内质网、细胞膜、高尔基体、溶酶体和过氧化物酶体等。在一些实施方案中，用于纯化的标签或者用于免疫印迹的标签包括6组氨酸(6*His)、谷胱甘肽硫转移酶(GST)、Flag。
本发明包含编码本发明所述果糖-1，6-二磷酸敏感多肽或光学探针的核酸分子。本发明所用术语“核酸”或“核苷酸”或“多核苷酸”或“核酸序列”可以是DNA形式或RNA形式。DNA形式包括cDNA、基因组DNA或人工合成的DNA。DNA可以是单链的或是双链的。DNA可以是编码链或非编码链。提到核酸时，本文所用术语“变体”可以是天然发生的等位变体或非天然发生的变体。这些核苷酸变体包括简并 变体、取代变体、缺失变体和插入变体。如本领域所知的，等位变体是一个核酸的替换形式，它可能是一个或多个核苷酸的取代、缺失或插入，但不会从实质上改变其编码的蛋白的功能。本发明核酸可包含与所述核酸序列的序列相同性为至少约50％、至少约60％、至少约70％、至少约75％、至少约80％、至少约85％、至少约90％、至少约95％、至少约98％、至少约99％或100％的核苷酸序列。本发明还涉及与上述的序列杂交的核酸片段。如本文所用，“核酸片段”的长度至少含15个核苷酸，较好是至少30个核苷酸，更好是至少50个核苷酸，最好是至少100个核苷酸以上。核酸片段可用于核酸的扩增技术(如PCR)。
本发明光学探针或融合蛋白的全长序列或其片段通常可以用PCR扩增法、人工合成法或重组法获得。本领域知晓常规PCR、合成法、重组法的步骤和所用试剂。此外，可通过突变PCR或化学合成等方法将突变引入本发明蛋白序列中。
本发明也涉及核酸构建物，该核酸构建物含有本文所述的多核苷酸，以及与这些序列操作性连接的一个或多个调控序列。本发明所述的多核苷酸可以多种方式被操作以保证所述多肽或蛋白的表达。在将核酸构建物插入载体之前可根据表达载体的不同或要求而对核酸构建物进行操作。利用重组DNA方法来改变多核苷酸序列的技术是本领域已知的。
在某些实施方案中，所述核酸构建物是载体。载体可以是克隆载体、表达载体、或同源重组载体。本发明的多核苷酸可被克隆入许多类型的载体，例如，质粒、噬菌粒、噬菌体衍生物、动物病毒和粘粒。
典型的表达载体包含可用于调节期望核酸序列表达的表达控制序列，与本发明所述的核酸序列或其互补序列操作性连接。本文所用术语“表达控制序列”指调控目的基因的转录、翻译和表达的可以与目的基因操作性连接的元件，可以是复制起点、启动子、标记基因或翻译控制元件，包括增强子、操纵子、终止子、核糖体结合位点等，表达控制序列的选择取决于所用的宿主细胞。在重组表达载体中，“操作性连接”是指目的的核苷酸序列与调节序列以允许核苷酸序列表达的方式连接。本领域的技术人员熟知能用于构建含本发明融合蛋白编码序列和合适的转录/翻译控制信号的表达载体的方法。这些方法包括体外重组DNA技术、DNA合成技术、体内重组技术等。所述的DNA序列可有效连接到表达载体中的适当启动子上，以指导mRNA合成。这些启动子的代表性例子有：大肠杆菌的lac或trp启动子；λ噬菌体PL启动子；真核启动子包括CMV立即早期启动子、HSV胸苷激酶启动子、早期和晚期SV40启动子、反转录病毒的LTR和其他一些已知的可控制基因在原核或真核细胞或其病毒中表达的启动子。表达载体还包括翻译起始用的核糖体结合位点和转录终 止子。在一个实施方案中，表达载体可采用市售的pCDF载体，无其他特殊要求。示例性地，采用BamHI和EcoRI分别对编码所述光学探针的核苷酸序列和表达载体进行双酶切，然后将二者的酶切产物连接得到重组表达载体。本发明对酶切和连接的具体步骤和参数没有特殊限定，采用本领域常规的步骤和参数即可。
在获得重组表达载体后，将该载体转化到宿主细胞中，以产生包括融合蛋白的蛋白或肽。此种转移过程可用转化或转染等本领域技术人员熟知的常规技术进行。本发明所述的宿主细胞是指能够接收和容纳重组DNA分子的细胞，是重组基因扩增的场所，理想的受体细胞应该满足易于获取和增殖两个条件。本发明的“宿主细胞”可包括原核细胞和真核细胞，具体包括细菌细胞、酵母细胞、昆虫细胞和哺乳动物细胞。所述宿主细胞优选各种利于基因产物表达或发酵生产的细胞，此类细胞已为本领域熟知并常用。具体的可为大肠杆菌，链霉菌属，鼠伤寒沙门氏菌的细菌细胞，真菌细胞如酵母，植物细胞，果蝇S2或Sf9的昆虫细胞，CHO、COS、HEK293、HeLa细胞、或Bowes黑素瘤细胞的动物细胞等。在本发明实施例中所用的示例性宿主细胞为大肠杆菌BL21-DE3菌株。本领域一般技术人员都清楚如何选择适当的载体、启动子、增强子和宿主细胞。
本发明所述的转移到宿主细胞的方法为本领域常规的方法，包括磷酸钙或氯化钙共沉淀、DEAE-甘露聚糖-介导的转染、脂转染、天然感受态、化学介导的转移或电穿孔。当宿主为原核生物如大肠杆菌时，所述方法优选的为CaCl2法或MgCl2法处理，所用的步骤为本领域公知。当宿主细胞是真核细胞时，可选用如下的DNA转染方法：磷酸钙共沉淀法，常规机械方法如显微注射、电穿孔、脂质体包装等。
本发明在将表达载体转入宿主细胞后，对转入表达载体的宿主细胞进行扩增表达培养，分离得到果糖-1，6-二磷酸光学探针。所述宿主细胞扩增表达培养采用常规的方法即可。根据所用的宿主细胞种类，培养中所用的培养基可以是各种常规培养基。在适于宿主细胞生长的条件下进行培养。
在本发明中，光学探针在细胞内、细胞膜上表达、或分泌到细胞外。如果需要，可利用其物理的、化学的和其它特性通过各种分离方法分离或纯化重组的蛋白。本发明对分离所述果糖-1，6-二磷酸荧光蛋白的方法没有特殊限定，采用本领域常规的融合蛋白的分离方法即可。这些方法是本领域技术人员所熟知的，包括但并不限于：常规的复性处理、盐析方法、离心、渗透破菌、超声处理、超离心、分子筛层析、吸附层析、离子交换层析、高效液相层析(HPLC)和其它各种液相层析技术及这些方法的结合。在一个实施方案中，利用His标签的亲和层析法进行光学探针的分离。
本发明还提供了所述果糖-1，6-二磷酸光学探针在果糖-1，6-二磷酸实时定位、定量检测以及高通量化合物筛选中的应用。在一个方面，所述的果糖-1，6-二磷酸光学探针优选与细胞不同部位的信号肽连接，转入到细胞中，通过检测细胞中荧光信号的强弱，进行果糖-1，6-二磷酸的实时定位；通过果糖-1，6-二磷酸标准滴加曲线结合荧光信号的变化进行相应果糖-1，6-二磷酸的定量检测。荧光信号的变化通过例如标准化后的荧光信号比值展示，在涉及cpYFP的实施方案中，所述比值是样品的485纳米荧光信号与420纳米荧光信号之比与对照的相应之比的比值。本发明所述的果糖-1，6-二磷酸标准滴加曲线是根据果糖-1，6-二磷酸光学探针在不同浓度果糖-1，6-二磷酸的情况下的荧光信号绘制而成。本发明所述果糖-1，6-二磷酸光学探针直接转入细胞中，在果糖-1，6-二磷酸实时定位和定量检测过程中，不需要耗时的样品处理过程，更加准确。本发明果糖-1，6-二磷酸光学探针在进行高通量化合物筛选时，将不同的化合物添加到细胞培养液中，测定果糖-1，6-二磷酸含量的变化，从而筛选出对果糖-1，6-二磷酸含量变化有影响的化合物。在本发明中所述的果糖-1，6-二磷酸光学探针在果糖-1，6-二磷酸实时定位、定量检测以及高通量化合物筛选中的应用，均是非诊断和治疗目的，不涉及疾病的诊断和治疗。
本发明还提供包括本文所述光学探针、核酸分子、核酸构建物和/或细胞的检测试剂盒。所述试剂盒还包含检测果糖-1，6-二磷酸所需的其他试剂。所述其他试剂本领域周知，例如缓冲液、细胞培养基、果糖-1，6-二磷酸标品。示例性缓冲液例如100mM HEPES和100mM NaCl，pH 7.4。
在本文中，浓度、含量、百分数和其它数值均可用范围的形式表示。也应理解，使用这种范围形式只是为了方便和简洁，应该被弹性地解读为包括范围上下限所明确提及的数值，还应包括该范围内包括的所有单个数值或子范围。
一些具体实施方案
项目1、一种果糖-1，6-二磷酸结合蛋白变体，其：
(1)具有SEQ ID NO：1所示的序列并且在选自以下的1个、2个或3个、4个、5个或更多个位点具有突变：T148、R175、G176、G177、L178、E180、D181、V182、K183、N184、Q185、F271，所述突变包括氨基酸的修饰、取代或缺失，
(2)是(1)的具有第88-340位氨基酸的截短变体，或
(3)是与(1)或(2)的序列具有至少70％序列相同性并具有(1)所述突变并保留对果糖-1，6-二磷酸结合能力的序列，
优选地，所述突变的位点选自以下任一组中的1个、2个或3个、4个、5个：(a)T148、R175、G176、G177、L178、D181、V182、K183、N184、Q185，(b)G177、L178、E180、D181、V182、N184、Q185、F271，
更优选地，所述突变包括选自以下任一组的位点处的突变：(1)G177和L178，(5)D181和V182，(6)E180和D181，(7)N184和Q185；或者，所述突变包含选自以下任一组的位点处的突变：(1)G177、L178，(2)G177、L178和Q185，(3)G177、L178和T148，(4)G177、L178和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，
更优选地，T148突变为S；G177突变为A、I、L、M或N，优选突变为L或M；L178突变为Y、F或N，优选突变为N；E180突变为G；D181突变为F、L或E，优选突变为E；V182突变为F、L或G，优选突变为G；N184突变为V；Q185突变为S、A、T、D或P，优选突变为P；F271突变为Y，
更优选地，所述突变包含选自以下任一组的突变：(1)G177A和L178Y，(2)G177I和L178F，(3)G177L和L178Y，(4)G177M和L178N，(5)G177N和L178Y，(6)G177M、L178N和Q185S，(7)G177M、L178N和Q185A，(8)G177M、L178N和Q185T，(9)G177M、L178N和Q185D，(10)G177、L178和T148S，(11)G177、L178和F271Y，(12)D181F和V182F，(13)D181L和V182L，(14)E180G和D181G，(15)N184V和Q185P，(16)N184V、Q185P、D181E、V182G和G177L，(17)N184V、Q185P和G177L，(18)N184V、Q185P、D181E和V182G。
项目2、一种果糖-1，6-二磷酸光学探针，包含果糖-1，6-二磷酸敏感多肽和光学活性多肽，其中，光学活性多肽位于果糖-1，6-二磷酸敏感多肽的序列内，所述果糖-1，6-二磷酸敏感多肽是果糖-1，6-二磷酸结合蛋白或其功能变体，所述光学活性多肽是荧光蛋白或其功能变体；其中，果糖-1，6-二磷酸结合蛋白的功能变体在与光学活性多肽连接处的7个氨基酸内具有突变，荧光蛋白的功能变体在与光学活性多肽连接处的3个氨基酸内具有突变。
项目3.如项目1所述的光学探针，其中，果糖-1，6-二磷酸结合蛋白具有SEQ ID NO：1所示的序列或其具有第88-340位氨基酸的截短体，或与它们有至少70％序列相同性并保留对果糖-1，6-二磷酸敏感性的序列。
项目4.如项目1所述的光学探针，其中，光学活性多肽位于果糖-1，6-二磷酸敏感多肽的残基174-185和/或201-208之间，
优选地，光学活性多肽位于果糖-1，6-二磷酸敏感多肽的选自下述位点中的任一个或多个：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和/或207/208，更优选地，光学活性多肽位于果糖-1，6-二磷酸敏感多肽的选自下述位点中的任一个或多个：176/177，176/178，177/181，179/180，182/184，182/185，203/206，203/207和203/208；
优选地，果糖-1，6-二磷酸结合蛋白的功能变体如权利要求1所述；
优选地，所述荧光蛋白选自黄色荧光蛋白、橘黄色荧光蛋白、红色荧光蛋白、绿色荧光蛋白、蓝色荧光蛋白、苹果红荧光蛋白，荧光蛋白的功能变体在第1-3位的氨基酸处具有突变；更优选地，荧光蛋白具有SEQ ID NO：2-9中任一所示的序列，荧光蛋白的功能变体在第1位的氨基酸处具有突变，
优选地，荧光蛋白的功能变体包括荧光蛋白的第1位氨基酸突变为I或V的突变，
优选地，果糖-1，6-二磷酸敏感多肽是SEQ ID NO：1的具有第88-340位氨基酸的截短变体，并且光学活性多肽位于果糖-1，6-二磷酸敏感多肽的选自下述位点中的任一个或多个：176/177，176/178，177/181，179/180，182/184，182/185，203/206，203/207和203/208；更优选地，光学探针具有SEQ ID NO：10-18中任一项所示序列。
优选地，果糖-1，6-二磷酸敏感多肽是SEQ ID NO：1的具有第88-340位氨基酸的截短变体，光学活性多肽位于果糖-1，6-二磷酸敏感多肽的选自下述位点中的任一个或多个：176/177，177/181，179/180或182/184，并且所述光学探针具有选自以下的一个或多个突变：果糖-1，6-二磷酸敏感多肽的T148S、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，光学活性多肽的第1位氨基酸突变为V或I；
优选地，所述光学探针具有选自以下任一组所示的突变：(1)果糖-1，6-二磷酸敏感多肽的G177A和L178Y，(2)果糖-1，6-二磷酸敏感多肽的G177I和L178F，(3)果糖-1，6-二磷酸敏感多肽的G177L和L178Y，(4)果糖-1，6-二磷酸敏感多肽的G177M和L178N，(5)果糖-1，6-二磷酸敏感多肽的G177N和L178Y，(6)果糖-1，6-二磷酸敏感多肽的G177M、L178N和Q185S，(7)果糖-1，6-二磷酸敏感多肽的G177M、L178N和Q185A，(8)果糖-1，6-二磷酸敏感多肽的G177M、L178N和Q185T，(9)果糖-1，6-二磷酸敏感多肽的G177M、L178N和Q185D，(10)果糖-1，6-二磷酸敏感多肽的G177、L178和T148S，(11)果糖-1，6-二磷酸敏感多肽的G177、L178和F271Y，(12)果糖-1，6-二磷酸敏感多肽的D181F和V182F，(13)果糖-1，6-二磷酸敏感多肽的D181L和V182L，(14)果糖-1，6-二磷酸敏感多肽的E180G和D181G，(15)果糖-1，6-二磷酸敏感多肽的N184V和Q185P，(16)果糖-1，6-二磷酸敏感多肽的N184V、Q185P、D181E、V182G、G177L，和光学活性多肽的第1位氨基酸突变为V，(17)果糖-1，6-二磷酸敏感多肽的N184V、Q185P、D181E、V182G、G177L，和光学活性多肽的第1位氨基酸突变为I，(18)果糖-1，6-二磷酸敏感多肽的N184V、Q185P和G177L，和光学活性多肽的第1位氨基酸突变为V，(19)果糖-1，6-二磷酸敏感多肽的N184V、Q185P和G177L，和光学活性多肽的第1位氨基酸突变为I，(20)果糖-1，6-二磷酸敏感多肽的N184V、Q185P、D181E和V182G，和光学活性多肽的第1位氨基酸突变为V。
项目5、一种核酸分子，其包含：
(a)项目2-4中任一项所述的光学探针的编码序列，
(b)(a)的互补序列。
项目6、包含项目5所述的核酸分子的核酸构建物，
优选地，所述核酸构建物是克隆载体、表达载体或重组载体。
项目7、一种宿主细胞，所述宿主细胞：
(1)表达项目2-4中任一项所述的光学探针；
(2)包含项目5所述的核酸分子；或
(3)包含项目6所述的核酸构建物。
项目8.一种检测试剂盒，其包含：
(1)项目2-4中任一项所述的光学探针，
(2)项目5所述的核酸序列，
(3)项目6所述的核酸构建物，或
(4)项目7所述的宿主细胞，
所述检测试剂盒任选还包含利用光学探针检测果糖-1，6-二磷酸所需的其他试剂，
优选地，所述检测试剂盒还包含选自以下的一种或多种试剂：缓冲液、培养基、果糖-1，6-二磷酸标准品。
项目9、制备项目2-4中任一项所述的光学探针的方法，包括：提供项目7所述的宿主细胞，在所述光学探针表达的条件下培养所述宿主细胞，和分离光学探针。
项目10、项目2-4中任一项所述的光学探针、项目5所述的核酸序列、权利要求6所述的核酸构建物或权利要求7所述的宿主细胞在检测样品中的果糖-1，6-二磷酸、筛选化合物、果糖-1，6-二磷酸细胞内/外定位中的应用，
优选地，
检测样品中的果糖-1，6-二磷酸包括步骤：使所述光学探针或宿主细胞与样品接触，检测光学活性多肽的光学变化，和根据光学活性多肽的光学变化检测样品中的果糖-1，6-二磷酸，
所述筛选化合物包括步骤：在含果糖-1，6-二磷酸的体系中使所述光学探针或宿主细胞与候选化合物接触，检测光学活性多肽的光学变化，和根据光学活性多肽的光学变化筛选化合物；优选地，所述筛选化合物包括步骤：在含果糖-1，6-二磷酸的体系中使所述宿主细胞与候选化合物接触，并且光学活性多肽的光学变化指示所述候选化合物是否调节细胞对果糖-1，6-二磷酸的摄取，
所述果糖-1，6-二磷酸细胞内/外定位包括步骤：将含果糖-1，6-二磷酸的体系与所述光学探针或所述宿主细胞接触，和检测光学活性多肽的光学变化，
更优选地，所述体系是溶液体系、细胞体系或亚细胞体系。
实施例
下面结合实施例对本发明提供的果糖-1，6-二磷酸光学探针进行详细的说明，但是不能把它们理解为对本发明保护范围的限定。
I.实验材料和试剂
实施例中主要采用常规的基因工程分子生物学克隆方法和细胞培养以及成像方法等，这些方法是本领域普通技术人员所熟知的，例如：简·罗斯凯姆斯等的《分子生物学实验参考手册》，J.萨姆布鲁克，D.W.拉塞尔著，黄培堂等译：《分子克隆实验指南》(第三版，2002年8月，科学出版社出版，北京)；费雷谢尼等的《动物 细胞培养：基本技术指南》(第五版)，章静波，徐存拴等译；J.S.博尼费斯农，M.达索等的《精编细胞生物学实验指南》，章静波等译。
实施例中所用的基于pCDF-cpYFP，pCDF-果糖-1，6-二磷酸结合蛋白质粒由华东理工大学蛋白质实验室构建，pCDF质粒载体购自Invitrogen公司。所有用于PCR的引物均由上海捷瑞生物工程技术有限公司和华大基因合成、纯化和经质谱法鉴定正确。实施例中构建的表达质粒都经过序列测定，序列测定由华大基因公司和杰李测序公司完成。各实施例所用的Taq DNA聚合酶购自东盛生物，pfu DNA聚合酶购自天根生化科技(北京)有限公司，primeSTAR DNA聚合酶购自TaKaRa公司，三种聚合酶购买时都附带赠送对应聚合酶缓冲液和dNTPs。BamHI、BglII、HindIII、NdeI、XhoI、EcoRI、SpeI等限制性内切酶、T4连接酶、T4磷酸化酶(T4 PNK)购自Fermentas公司，购买时附带有相对应的缓冲液等。转染试剂Lip2000Kit购于Invitrogen公司。果糖-1，6-二磷酸等化合物均购自Sigma公司。除非特别声明，无机盐类等化学试剂均购自Sigma-Aldrich公司。HEPES盐，氨苄青霉素(Amp)和嘌呤霉素购自Ameresco公司。96孔检测黑板、384孔荧光检测黑板购自Grenier公司。
实施例中所用的DNA纯化试剂盒购自BBI公司(加拿大)，普通质粒小抽试剂盒购自天根生化科技(北京)有限公司。克隆菌株Mach1购自Invitrogen公司。镍柱亲和层析柱和脱盐柱填料均来自GE healthcare公司。
实施例中用到的主要仪器包括：Biotek Synergy 2多功能酶标仪(美国Bio-Tek公司)，X-15R高速冷冻离心机(美国Beckman公司)，Microfuge22R台式高速冷冻离心机(美国Beckman公司)，PCR扩增仪(德国Biometra公司)，超声破碎仪(宁波新芝公司)，核酸电泳仪(申能博彩公司)，荧光分光光度计(美国Varian公司)，CO2恒温细胞培养箱(SANYO)，倒置荧光显微镜(日本尼康公司)。
II.分子生物学方法和细胞实验方法
II.1聚合酶链式反应(PCR)：
1.目的片段扩增PCR：
该方法主要用于基因片段扩增和菌落PCR鉴定阳性克隆。所述PCR扩增的反应体系如下：模板序列0.5-1μL，正向引物(25μM)0.5μL，反向引物(25μM)0.5μL，10×pfu缓冲液5μL，pfu DNA聚合酶0.5μL，dNTP(10mM)1μL，灭菌超纯水(ddH2O)41.5-42μL，总体积50μL。PCR扩增程序如下：95℃变性2-10分钟，30轮循环(94-96℃持续30-45秒，50-65℃持续30-45秒，72℃持续一定时间(600bp/min))，72℃延伸10分钟。
2.长片段(＞2500bp)扩增PCR：
本发明中使用的长片段扩增，主要是反向PCR扩增载体，在下述实施例中用于获得定点突变的一种技术。在变异部位设计反向PCR引物，其中一条引物的5’端包含变异的核苷酸序列。扩增后的产物就含有相应的突变位点。长片段扩增PCR反应体系如下：模板序列(10pg-1ng)1μL，正向引物(25μM)0.5μL，反向引物(25μM)0.5μL，5×PrimerSTAR缓冲液10μL，PrimerSTAR DNA聚合酶0.5μL，dNTP(2.5mM)4μL，灭菌超纯水(ddH2O)33.5μL，总体积50μL。PCR扩增程序如下：95℃变性5分钟，30轮循环(98℃持续10秒，50-68℃持续5-15秒，72℃持续一定时间(1000bp/min))，72℃延伸10分钟；或者95℃变性5分钟，30轮循环(98℃持续10秒，68℃持续一定时间(1000bp/min))，72℃延伸10分钟。
II.2核酸内切酶酶切反应：
对质粒载体进行双酶切的体系如下：质粒载体20μL(约1.5μg)，10×缓冲液5μL，限制性内切酶11-2μL，限制性内切酶21-2μL，用灭菌超纯水补至总体积50μL。反应条件37℃，1-7小时。
II.3 DNA片段5’端磷酸化反应
从微生物中抽提出的质粒或者基因组末端都含有磷酸基团，而PCR产物没有，故需对PCR产物的5’端碱基进行磷酸基团加成反应，只有末端含有磷酸基团DNA分子才能发生连接反应。磷酸化反应体系如下：PCR产物片段DNA序列5-8μL，10×T4连接酶缓冲液1μL，T4多聚核苷酸激酶(T4 PNK)1μL，灭菌超纯水0-3μL，总体积10μL。反应条件37℃，30分钟-2小时后72℃灭活20分钟。
II.4目的片段和载体的连接反应
不同的片段和载体之间的连接方法有所差异，本发明中使用了三种连接方法
1.平末端短片段和线性化载体的平末端连接
该方法的原理是PCR获得的平末端产物在T4 PNK作用下对DNA片段的5’末端进行磷酸化反应后，与线性化的载体在PEG4000和T4 DNA连接酶的作用下连接获得重组质粒。同源重组连接体系如下：T4 PNK处理的DNA片段4μL，线性化载体片段4μL，PEG40001μL，10×T4连接酶缓冲液1μL，T4 DNA连接酶1μL，总计10μL。反应条件22℃，30分钟。
2.含有粘性末端的DNA片段和含有粘性末端载体片段的连接
通过限制性内切酶切割的DNA片段通常会产生突出的粘性末端，因此可以和含有序列互补的粘性末端载体片段连接，形成重组质粒。连接反应体系如下：酶切后的PCR产物片段DNA 1-7μL，酶切后的质粒0.5-7μL，10×T4连接酶缓冲液1μL，T4 DNA连接酶1μL，灭菌超纯水补至总体积10μL。反应条件16℃，4-8小时。
3.反向PCR引入定点突变后5’端磷酸化的DNA片段产物自身环化的连接反应
将5’端磷酸化的DNA片段通过自身环化连接反应将线性化载体的3’端和5’端连接反应得到重组质粒。自身环化连接反应体系如下：磷酸化反应体系10μL，T4连接酶(5U/μL)0.5μL，总体积10.5μL。反应条件16℃，4-16小时。
II.5感受态细胞的制备与转化
感受态细胞的制备：
1.挑取单菌落(如Mach1)接种于5mL LB培养基中，37℃摇床过夜。
2.取0.5-1mL过夜培养的菌液转种到50mL LB培养基中，37℃，220rpm培养3至5小时，直到OD600达到0.5。
3.冰浴预冷细胞2小时。
4. 4℃，4000rpm离心10分钟。
5.弃上清，用5mL预冷的缓冲液重悬细胞，待均匀后再加入重悬缓冲液至终体积为50mL。
6.冰浴45分钟。
7. 4℃ 4000rpm离心10分钟，用5mL冰预冷的储存缓冲液重悬细菌。
8.每个EP管中放100μL菌液，-80℃或液氮冻存。
重悬缓冲液：CaCl2(100mM)、MgCl2(70mM)、NaAc(40mM)
储存缓冲液：0.5mL DMSO、1.9mL 80％甘油、1mL 10×CaCl2(1M)、1mL 10×MgCl2(700mM)、1rmL 10×NaAc(400mM)、4.6mL ddH2O
感受态细胞的转化：
1.取100μL感受态细胞于冰浴上融化。
2.加入适当体积的连接产物，轻轻吹打混匀，冰浴30分钟。通常加入的连接产物的体积少于感受态细胞体积的1/10。
3.将菌液放入42℃水浴中热激90秒，迅速转移至冰浴中放置5分钟。
4.加入500μLLB，于37℃恒温摇床上200rpm培养1小时。
5.将菌液4000rpm离心3分钟，留200μL上清将菌体吹匀，均匀涂布于含适当抗生素的琼脂平板表面，平板于37℃恒温培养箱内倒置过夜。
II.6蛋白质的表达，纯化和荧光检测
1.将表达载体(例如以pCDF为基础的果糖-1，6-二磷酸光学探针表达载体)转化到BL21(DE3)细胞中，倒置培养过夜，从平板上挑取克隆到250ml锥形瓶中，置于37℃摇床，220rpm培养至OD＝0.4-0.8，加入1/1000(v/v)的IPTG(1M)，18℃诱导表达24-36小时。
2.诱导表达完成后，4000rpm，30分钟离心收菌，加入50mM的磷酸盐缓冲液重悬菌体沉淀，超声破碎至菌体澄清。9600rpm，4℃离心20分钟。
3.离心上清通过自装的镍柱亲和层析柱纯化获得蛋白，镍柱亲和层析后的蛋白再通过自装的脱盐柱获得溶解在100mM HEPES缓冲液(pH 7.4)中的蛋白。
4.纯化的蛋白经过SDS-PAGE鉴定后，使用测定缓冲液(100mM HEPES，100mM NaCl，pH 7.4)稀释探针成终浓度为0.2-5μM的蛋白溶液。用测定缓冲液(100mM HEPES，100mM NaCl，pH 7.4)将果糖-1，6-二磷酸配制成终浓度为50mM的储液。
5.取100μl 1μM的蛋白溶液，37℃温育10分钟，加入果糖-1，6-二磷酸滴定，测定蛋白的420nm光激发后528nm发射和485nm光激发后528nm发射的荧光强度。对样品的荧光激发、发射测定利用多功能荧光酶标仪完成。
6.取100μl 1μM的蛋白溶液，37℃温育10分钟，加入果糖-1，6-二磷酸，测定蛋白的吸收光谱和荧光光谱。对样品的吸收光谱和荧光光谱的测定通过分光光度计和荧光分光光度计完成。
II.7哺乳动物细胞的转染和荧光检测
1.将pCDNA3.1+为基础的果糖-1，6-二磷酸光学探针质粒通过转染试剂Lipofectamine2000(Invitrogen)转染到HEK293中，置于37℃，5％CO2的细胞培养箱中培养。待外源基因充分表达24～36h后进行荧光检测。
2.诱导表达完成后，将贴壁的HEK293细胞，用PBS冲洗三次，置于HBSS溶液中分别进行荧光显微镜和酶标仪检测。
实施例1：果糖-1，6-二磷酸结合蛋白质粒
通过PCR扩增枯草芽孢杆菌基因中的CggR截短体基因，PCR产物凝胶电泳后回收后用HindIII和XhoI酶切，同时对pCDF载体进行相应的双酶切。用T4 DNA连接酶连接后，用产物转化DH5α，转化的DH5α涂布于LB平板(链霉素100ug/mL)，置于37℃培养过夜。将生长DH5α转化子进行质粒抽提后，进行PCR鉴定。阳性质粒经过测序正确后进行后续的质粒构建。
实施例2：不同插入位点的cpYFP光学探针的表达和检测
本实施例中，以pCDF-CggR(88-340)为基础根据果糖-1，6-二磷酸结合蛋白晶体结构选择了下述位点插入cpYFP，得到相应pCDF-CggR(88-340)-cpYFP质粒：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和/或207/208。
利用PCR产生cpYFP的DNA片段，同时通过引物5’端引入cpYFP末端同源序列，PCR扩增产生pCDF-果糖-1，6-二磷酸结合蛋白线性化载体，其5’和3’最末端分别带有和cpYFP两末端对应的完全一致的序列(15bp～20bp)。将线性化的pCDF-CggR(88-340)和cpYFP片段在Hieff Clone Enzyme的作用下发生同源重组。产物转化DH5α，转化的DH5α涂布于LB平板(链霉素100ug/mL)，置于37℃培养过夜。PCR鉴定的阳性克隆抽质粒后测序。由杰李测序公司完成测序。
经过测序正确后，将重组质粒转化到BL21(DE3)中诱导表达，并纯化蛋白质，通过SDS-PAGE电泳大小在57Kda附近。该大小符合pCDF-CggR(88-340)-cpYFP表达出的含His-tag纯化标签的CggR(88-340)-cpYFP融合蛋白质的大小。结果如图1所示。
用表达CggR(88-340)-cpYFP融合蛋白质的大肠杆菌的破碎上清进行果糖-1，6-二磷酸响应筛选，将含有1mM果糖-1，6-二磷酸的融合荧光蛋白质的检测信号除以无果糖-1，6-二磷酸的融合荧光蛋白质的检测信号。结果如图2所示，检测结果显示对果糖-1，6-二磷酸响应超过1.4倍的光学探针有在176/177，176/178，177/181，179/180，182/184，182/185，203/206，203/207和203/208位点或者其家族蛋白的对应氨基酸位点实施插入的光学探针。筛选结果如下表所示：
实施例3：不同插入位点的cpGFP光学探针的表达和检测
按照实施例2中的方法将cpYFP替换为cpGFP，构建果糖-1，6-二磷酸绿色荧光蛋白荧光探针。如图3所示，检测结果显示对果糖-1，6-二磷酸响应超过1.4倍的光学探针有在176/177，176/178，177/181，182/184，182/185，203/204，203/205，203/206，203/207和203/208位点或者其家族蛋白的对应氨基酸位点实施插入的光学探针。
实施例4：不同插入位点的cpBFP光学探针的表达和检测
按照实施例2中的方法将cpYFP替换为cpBFP，构建果糖-1，6-二磷酸蓝色荧光蛋白荧光探针。如图4所示，检测结果显示对果糖-1，6-二磷酸响应超过1.5倍的光学探针有在176/177，176/178，，177/178，177/181，178/181，182/184，182/185，203/205，203/206，203/207和203/208位点或者其家族蛋白的对应氨基酸位点实施插入的光学探针。
实施例5：linker突变的CPYFP光学探针的表达和检测
在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-CPYFP，PCDF-CggR(88-340)-182/185-CPYFP，PCDF-CggR(88-340)-203/206-CPYFP，PCDF-CggR(88-340)-203/207-CPYFP和PCDF-CggR-203/208(88-340)-CPYFP的基础上构建光学探针突变体。通过PCR线性化质粒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和PCDF-CggR(88-340)-203/208-CPYFP引物中在linker处设为NNK随机突变，对得到的PCR产物在PNK、T4 DNA连接酶和PEG4000的作用下加磷连接，转化建库，并进行检测，筛选得到对果糖-1，6-二磷酸响应大于2倍的突变体，并由杰李测序公司完成测序。筛选得到的突变体如下表所示：
连接体突变体

实施例6.光学探针及其突变体突变体的性能
示例性地，将纯化的实施例5中的编号为4、15、17的三种种果糖-1，6-二磷酸光学探针，分别进行0mM和5mM果糖-1，6-二磷酸处理10分钟后，使用荧光分光光度计进行荧光谱的检测。
对激发光谱的测定：以370nm至510nm的激发范围和530nm的发射波长记录激发光谱，每5nm读取一次。结果显示，探针在约410和490nm处有两个激发峰。
对发射光谱的测定：固定激发波长分别为420nm和460nm，记录470-600nm和490-600nm的发射光谱，每5nm读取一次。激发和发射光谱如图5所示。
对纯化的实施例5中编号为1-20的二十种果糖-1，6-二磷酸光学探针进行浓度梯度(0～5mM)的果糖-1，6-二磷酸检测。对纯化的探针处理10分钟后，检测420nm激发528nm发射处荧光强度和485nm激发528nm发射处荧光强度比值的变化。结果如图6所示，二十种果糖-1，6-二磷酸光学探针的Kd(结合常数)分别为37μM、25μM、73μM、11μM、38μM、12μM、36μM、22μM、32μM、74μM、12μM、5.7μM、6.2μM、3.3μM、360μM、240μM、769μM、1524μM、117μM、148μM和92μM。
将对纯化的实施例5中编号为1-20的二十种果糖-1，6二磷酸光学探针与8种相似底物进行反应性检测，结果表明其具有很好的特异性，如图7所示。
实施例7：光学探针的亚细胞器定位和光学探针在亚细胞器内的性能
本实施例中，使用不同的定位信号肽与光学探针182/184(CggR：N184V/Q185P/D181E/V182G CPYFP：Y1V实施例5中的编号20)融合，将光学探针定位到不同的细胞器中。
用融合不同定位信号肽的光学探针质粒转染HeLa细胞36小时后，使用PBS冲洗，置于HBSS溶液中使用倒置荧光显微镜进行FITC通道下进行荧光检测。结果如图8所示。果糖-1，6二磷酸光学探针通过与不同的特异定位信号肽融合能够定位到包括细胞浆、细胞核、线粒体和核排阻等亚细胞器中。不同的亚细胞结构中都显示有荧光，并且荧光的分布和强度各不相同。
用胞浆表达的光学探针质粒转染H1299细胞36小时后，使用PBS冲洗，置于含有0mM 3-BrPA和0.5mM 3-BrPA的HBSS溶液中，检测30min时间段内420nm激发528nm发射处荧光强度和485nm激发528nm发射处荧光强度比值的变化。结果如图9所示。添加3-BrPA的样品的420/485的比值逐渐增加，最高可以达到初始值的3.7倍，而不添加3-BrPA的对照组的420/485为0.63保持不变。
实施例8：在活细胞中基于光学探针进行高通量化合物筛选
本实施例中，我们使用胞浆表达182/184(CggR：N184V/Q185P/D181E/V182GCPYFP：Y1V)的HeLa细胞进行高通量化合物筛选。
经转染的HeLa细胞使用PBS冲洗，置于HBSS溶液中(无果糖-1，6-二磷酸)处理1小时，然后使用10μM的化合物处理1小时。各样品中分别滴加各种化合物。使用酶标仪记录420nm激发528nm发射处荧光强度和485nm激发528nm发射处荧光强度比值变化。以未用任何化合物处理的样品作为对照进行标准化。结果如图10所示。在使用的1000种化合物中，绝大部分的化合物对细胞内果糖-1，6-二磷酸的浓度影响较小。有38种化合物能够提高细胞细胞内的果糖-1，6-二磷酸的浓度，另外有13种化合物能够明显降低细胞细胞内的果糖-1，6-二磷酸的浓度。
实施例9：光学探针定量检测血液中的果糖-1，6-二磷酸
在本实施中，使用纯化的Kd为1μM左右的179/180(CggR：E180G/D181G)对小鼠和人的血液上清中的果糖-1，6二磷酸进行分析。
将179/180(CggR：E180G/D181G)与稀释的血液上清混合处理10分钟后，使用酶标仪检测420nm激发528nm发射处荧光强度和485nm激发528nm发射处荧光强度比值。结果如11所示，健康人的血液中的果糖-1，6-二磷酸含量在6μM左右。
由以上实施例可知，本发明提供的果糖-1，6-二磷酸光学探针，蛋白分子量相对较小且易于成熟，荧光动态变化大，特异性好，并且能够通过基因操作的方法在细胞中表达，可在细胞内外实时定位、定量检测果糖-1，6-二磷酸；并且能够进行高通量的化合物筛选。
其它实施方式
本说明书描述了许多实施方式。然而应理解，本领域技术人员通过阅读本说明书获知的不背离本发明的构思和范围的各种改进，也应包括在所附权利要求书的范围内。
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Claims (10)

  1. 一种果糖-1，6-二磷酸结合蛋白变体，其：
    (1)具有SEQ ID NO：1所示的序列并且在选自以下的1个、2个或3个、4个、5个或更多个位点具有突变：T148、R175、G176、G177、L178、E180、D181、V182、K183、N184、Q185、F271，所述突变包括氨基酸的修饰、取代或缺失，
    (2)是(1)的具有第88-340位氨基酸的截短变体，或
    (3)是与(1)或(2)的序列具有至少70％序列相同性并具有(1)所述突变并保留对果糖-1，6-二磷酸结合能力的序列，
    优选地，所述突变的位点选自以下任一组中的1个、2个或3个、4个、5个：(a)T148、R175、G176、G177、L178、D181、V182、K183、N184、Q185，(b)G177、L178、E180、D181、V182、N184、Q185、F271，
    更优选地，所述突变包括选自以下任一组的位点处的突变：(1)G177和L178，(5)D181和V182，(6)E180和D181，(7)N184和Q185；或者，所述突变包含选自以下任一组的位点处的突变：(1)G177、L178，(2)G177、L178和Q185，(3)G177、L178和T148，(4)G177、L178和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，
    更优选地，T148突变为S；G177突变为A、I、L、M或N，优选突变为L或M；L178突变为Y、F或N，优选突变为N；E180突变为G；D181突变为F、L或E，优选突变为E；V182突变为F、L或G，优选突变为G；N184突变为V；Q185突变为S、A、T、D或P，优选突变为P；F271突变为Y，
    更优选地，所述突变包含选自以下任一组的突变：(1)G177A和L178Y，(2)G177I和L178F，(3)G177L和L178Y，(4)G177M和L178N，(5)G177N和L178Y，(6)G177M、L178N和Q185S，(7)G177M、L178N和Q185A，(8)G177M、L178N和Q185T，(9)G177M、L178N和Q185D，(10)G177、L178和T148S，(11)G177、L178和F271Y，(12)D181F和V182F，(13)D181L和V182L，(14)E180G和D181G，(15)N184V和Q185P，(16)N184V、Q185P、D181E、V182G 和G177L，(17)N184V、Q185P和G177L，(18)N184V、Q185P、D181E和V182G。
  2. 一种果糖-1，6-二磷酸光学探针，包含果糖-1，6-二磷酸敏感多肽和光学活性多肽，其中，光学活性多肽位于果糖-1，6-二磷酸敏感多肽的序列内，所述果糖-1，6-二磷酸敏感多肽是果糖-1，6-二磷酸结合蛋白或其功能变体，所述光学活性多肽是荧光蛋白或其功能变体；其中，果糖-1，6-二磷酸结合蛋白的功能变体在与光学活性多肽连接处的7个氨基酸内具有突变，荧光蛋白的功能变体在与光学活性多肽连接处的3个氨基酸内具有突变。
  3. 如权利要求1所述的光学探针，其中，果糖-1，6-二磷酸结合蛋白具有SEQ ID NO：1所示的序列或其具有第88-340位氨基酸的截短体，或与它们有至少70％序列相同性并保留对果糖-1，6-二磷酸敏感性的序列。
  4. 如权利要求1所述的光学探针，其中，光学活性多肽位于果糖-1，6-二磷酸敏感多肽的残基174-185和/或201-208之间，
    优选地，光学活性多肽位于果糖-1，6-二磷酸敏感多肽的选自下述位点中的任一个或多个：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和/或207/208，更优选地，光学活性多肽位于果糖-1，6-二磷酸敏感多肽的选自下述位点中的任一个或多个：176/177，176/178，177/181，179/180，182/184，182/185，203/206，203/207和203/208；
    优选地，果糖-1，6-二磷酸结合蛋白的功能变体如权利要求1所述；
    优选地，所述荧光蛋白选自黄色荧光蛋白、橘黄色荧光蛋白、红色荧光蛋白、绿色荧光蛋白、蓝色荧光蛋白、苹果红荧光蛋白，荧光蛋白的功能变 体在第1-3位的氨基酸处具有突变；更优选地，荧光蛋白具有SEQ ID NO：2-9中任一所示的序列，荧光蛋白的功能变体在第1位的氨基酸处具有突变，
    优选地，荧光蛋白的功能变体包括荧光蛋白的第1位氨基酸突变为I或V的突变，
    优选地，果糖-1，6-二磷酸敏感多肽是SEQ ID NO：1的具有第88-340位氨基酸的截短变体，并且光学活性多肽位于果糖-1，6-二磷酸敏感多肽的选自下述位点中的任一个或多个：176/177，176/178，177/181，179/180，182/184，182/185，203/206，203/207和203/208；更优选地，光学探针具有SEQ ID NO：10-18中任一项所示序列。
    优选地，果糖-1，6-二磷酸敏感多肽是SEQ ID NO：1的具有第88-340位氨基酸的截短变体，光学活性多肽位于果糖-1，6-二磷酸敏感多肽的选自下述位点中的任一个或多个：176/177，177/181，179/180或182/184，并且所述光学探针具有选自以下的一个或多个突变：果糖-1，6-二磷酸敏感多肽的T148S、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，光学活性多肽的第1位氨基酸突变为V或I；
    优选地，所述光学探针具有选自以下任一组所示的突变：(1)果糖-1，6-二磷酸敏感多肽的G177A和L178Y，(2)果糖-1，6-二磷酸敏感多肽的G177I和L178F，(3)果糖-1，6-二磷酸敏感多肽的G177L和L178Y，(4)果糖-1，6-二磷酸敏感多肽的G177M和L178N，(5)果糖-1，6-二磷酸敏感多肽的G177N和L178Y，(6)果糖-1，6-二磷酸敏感多肽的G177M、L178N和Q185S，(7)果糖-1，6-二磷酸敏感多肽的G177M、L178N和Q185A，(8)果糖-1，6-二磷酸敏感多肽的G177M、L178N和Q185T，(9)果糖-1，6-二磷酸敏感多肽的G177M、L178N和Q185D，(10)果糖-1，6-二磷酸敏感多肽的G177、L178和T148S，(11)果糖-1，6-二磷酸敏感多肽的G177、L178和F271Y，(12)果糖-1，6-二磷酸敏感多肽的D181F和V182F，(13)果糖-1，6-二磷酸敏感多肽的D181L和V182L，(14)果糖-1，6-二磷酸敏感多肽的E180G和D181G，(15)果糖-1，6-二磷酸敏感多肽的N184V和Q185P，(16)果糖-1，6-二磷酸敏感多肽的N184V、Q185P、D181E、V182G、G177L，和光学活性多肽的第1位氨基酸突变为V，(17)果糖-1，6-二磷酸敏感多肽的N184V、Q185P、D181E、V182G、G177L，和光学活性多肽的第1位氨基酸 突变为I，(18)果糖-1，6-二磷酸敏感多肽的N184V、Q185P和G177L，和光学活性多肽的第1位氨基酸突变为V，(19)果糖-1，6-二磷酸敏感多肽的N184V、Q185P和G177L，和光学活性多肽的第1位氨基酸突变为I，(20)果糖-1，6-二磷酸敏感多肽的N184V、Q185P、D181E和V182G，和光学活性多肽的第1位氨基酸突变为V。
  5. 一种核酸分子，其包含：
    (a)权利要求2-4中任一项所述的光学探针的编码序列，
    (b)(a)的互补序列。
  6. 包含权利要求5所述的核酸分子的核酸构建物，
    优选地，所述核酸构建物是克隆载体、表达载体或重组载体。
  7. 一种宿主细胞，所述宿主细胞：
    (1)表达权利要求2-4中任一项所述的光学探针；
    (2)包含权利要求5所述的核酸分子；或
    (3)包含权利要求6所述的核酸构建物。
  8. 一种检测试剂盒，其包含：
    (1)权利要求2-4中任一项所述的光学探针，
    (2)权利要求5所述的核酸序列，
    (3)权利要求6所述的核酸构建物，或
    (4)权利要求7所述的宿主细胞，
    所述检测试剂盒任选还包含利用光学探针检测果糖-1，6-二磷酸所需的其他试剂，
    优选地，所述检测试剂盒还包含选自以下的一种或多种试剂：缓冲液、培养基、果糖-1，6-二磷酸标准品。
  9. 制备权利要求2-4中任一项所述的光学探针的方法，包括：提供权利要求7所述的宿主细胞，在所述光学探针表达的条件下培养所述宿主细胞，和分离光学探针。
  10. 权利要求2-4中任一项所述的光学探针、权利要求5所述的核酸序列、权利要求6所述的核酸构建物或权利要求7所述的宿主细胞在检测样品中的果糖-1，6-二磷酸、筛选化合物、果糖-1，6-二磷酸细胞内/外定位中的应用，
    优选地，
    检测样品中的果糖-1，6-二磷酸包括步骤：使所述光学探针或宿主细胞与样品接触，检测光学活性多肽的光学变化，和根据光学活性多肽的光学变化检测样品中的果糖-1，6-二磷酸，
    所述筛选化合物包括步骤：在含果糖-1，6-二磷酸的体系中使所述光学探针或宿主细胞与候选化合物接触，检测光学活性多肽的光学变化，和根据光学活性多肽的光学变化筛选化合物；优选地，所述筛选化合物包括步骤：在含果糖-1，6-二磷酸的体系中使所述宿主细胞与候选化合物接触，并且光学活性多肽的光学变化指示所述候选化合物是否调节细胞对果糖-1，6-二磷酸的摄取，
    所述果糖-1，6-二磷酸细胞内/外定位包括步骤：将含果糖-1，6-二磷酸的体系与所述光学探针或所述宿主细胞接触，和检测光学活性多肽的光学变化，
    更优选地，所述体系是溶液体系、细胞体系或亚细胞体系。
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