WO2025232721A1 - D-glucose optical probe, and preparation method therefor and use thereof - Google Patents

D-glucose optical probe, and preparation method therefor and use thereof

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Publication number
WO2025232721A1
WO2025232721A1 PCT/CN2025/092836 CN2025092836W WO2025232721A1 WO 2025232721 A1 WO2025232721 A1 WO 2025232721A1 CN 2025092836 W CN2025092836 W CN 2025092836W WO 2025232721 A1 WO2025232721 A1 WO 2025232721A1
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Prior art keywords
glucose
optically active
sensitive
polypeptide
optical probe
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Application number
PCT/CN2025/092836
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French (fr)
Chinese (zh)
Inventor
杨弋
赵玉政
李写
施泽恩
卢世洁
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East China University of Science and Technology
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East China University of Science and Technology
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Publication of WO2025232721A1 publication Critical patent/WO2025232721A1/en
Pending legal-status Critical Current
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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
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • 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
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/54—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving glucose or galactose
    • 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
    • G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
    • G01N21/76—Chemiluminescence; Bioluminescence
    • 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
    • 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
    • G01N33/5308—Immunoassay; Biospecific binding assay; Materials therefor for analytes not provided for elsewhere, e.g. nucleic acids, uric acid, worms, mites
    • 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
    • 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
    • G01N33/582—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances 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
    • G01N33/66—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving blood sugars, e.g. galactose
    • 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
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/60—Fusion polypeptide containing spectroscopic/fluorescent detection, e.g. green fluorescent protein [GFP]

Definitions

  • This invention relates to the field of optical probe technology, and in particular to a D-glucose optical probe, its preparation method, and its application.
  • the present invention provides the following technical solution:
  • (b) is a sequence that has at least 70% sequence identity with the sequence of (a) and has the mutation described in (a) and retains the ability to bind to D-glucose.
  • the mutation includes a mutation at a site selected from any of the following groups:
  • W8 mutates to R, H, E, A, V, L, F, I, M, C, N, G, K, D, or T; in one or more embodiments, W9 mutates to R, H, E, A, V, L, F, I, M, C, P, D, N, G, K, Y, S, or T; in one or more embodiments, A42 mutates to R, E, W, F, M, C, P, N, G, K, Y, or T; in one or more embodiments, H66 mutates to R, E, A, F, M, C, P, Q, or T; in one or more embodiments, K312 mutates to R, A, Q, G, H, or S; in one or more embodiments, V347 mutates to W; in one or more embodiments, H348 mutates to T, G, or S.
  • the mutation comprises a mutation selected from any of the following groups: (1) V347W and H348T, (2) V347W and H348G, (3) V347W and H348S, (4) V347W, H348T and H66R, (5) V347W, H348T and H66E, (6) V347W, H348T and H66A, (7) V347W, H348T and H66F, (8) V347W (9) V347W, H348T and H66C, (10) V347W, H348T and H66P, (11) V347W, H348T and H66Q, (12) V347W, H348T and H66T, (13) V347W, H348T and W9R, (14) V347W, H348T and W9H, (15) V347W, H348T and W9E, (16) V347 W, H348T and W9A, (17) V347W, H3
  • Another aspect of the present invention provides a D-glucose optical probe comprising a D-glucose-sensitive polypeptide and an optically active polypeptide, wherein the optically active polypeptide is located within the sequence of the D-glucose-sensitive polypeptide.
  • the D-glucose-sensitive polypeptide is divided into a first part and a second part by the optically active polypeptide.
  • the optically active polypeptide is located between residues 117-123, 266-288, and/or 340-353 of the D-glucose-sensitive polypeptide, the numbering corresponding to the full length of the D-glucose-sensitive polypeptide.
  • the optically active polypeptide is located at any one or more sites selected from the following groups of the D-glucose-sensitive polypeptide: 266/267, 266/268, 266/269, 266/270, 266/271, 266/272, 266/273, 266/274, 266/275, 266/276, 266/277, 266/278, 266/279, 266/280, 266/281, 266/282, 266/283.
  • the D-glucose-sensitive polypeptide is a D-glucose-binding protein or a functional variant thereof.
  • the D-glucose-sensitive polypeptide has:
  • (3) has at least 70% sequence identity with the sequence described in (2) and has the mutation described in (2) and retains the sequence sensitive to D-glucose.
  • 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 linker to the optically active polypeptide.
  • the fluorescent protein is selected from yellow fluorescent protein, green fluorescent protein, blue fluorescent protein, and apple red fluorescent protein.
  • the fluorescent protein has the sequence shown in any of SEQ ID NO: 2-9.
  • the fluorescent protein has the sequence shown in any of SEQ ID NO: 2, 6, 7, and 9.
  • the functional variant of the fluorescent protein has a mutation at amino acid positions 1-3, preferably position 1.
  • the functional variant of the fluorescent protein includes a mutation in the amino acid position 1 of the fluorescent protein to A or G.
  • a functional variant of the fluorescent protein has the sequence shown in SEQ ID NO: 2 and a mutation at the Y1 site.
  • the mutation is Y1A or Y1G.
  • the fluorescent protein has the sequence shown in SEQ ID NO: 2 or a variant Y1A or Y1G with any of the following mutations at its first amino acid position, and the optically active polypeptide is located at position 348/352 of the D-glucose-sensitive polypeptide.
  • the optical probe further comprises one or more linkers flanking the optically active polypeptide.
  • the linkers of this invention can be any amino acid sequence of any length.
  • the optically active polypeptide flanking the linker comprises a linker of no more than 5 amino acids, for example, linkers 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: a first portion B1 of a D-glucose-sensitive polypeptide, Y, an optically active polypeptide A, and a second portion B2 of a D-glucose-sensitive polypeptide.
  • the optical probe of this invention does not comprise a linker.
  • the optical probe of the present invention further includes a positioning sequence for positioning the probe to a specific organelle, such as a cell.
  • the sequence of the D-glucose-sensitive polypeptide is as shown in SEQ ID NO: 1, and the optically active polypeptide is as shown in any one of SEQ ID NO: 2, 6, 7, and 9, wherein the optically active polypeptide is located at any one or more sites of the D-glucose-sensitive polypeptide selected from the following: 266/267, 266/268, 266/269, 266/270, 266/271, 266/272, 266/273, 266/274, 266...
  • the D-glucose-sensitive polypeptide is as shown in SEQ ID NO: 1
  • the optically active polypeptide is as shown in any one of SEQ ID NO: 2, 6, 7, 9 or a variant thereof having a mutation selected from any one or more of the following at the amino acid position corresponding to the first amino acid of SEQ ID NO: 2: Y1A or Y1G, and the optically active polypeptide is located between residues 117-123, 266-288 and/or 340-353 of the D-glucose-sensitive polypeptide, the numbers corresponding to the full length of the D-glucose-sensitive polypeptide.
  • the optically active polypeptide is located at any one or more sites of the D-glucose-sensitive polypeptide selected from the following: 266/267, 266/268, 266/269, 266/270, 266/271, 266/272, 266/273, 266/274, 266/275, 266/276, 266/277, 266/278, 266/279, 266/280, 266/281, 266/282.
  • the optical probe contains a D-glucose-sensitive polypeptide as shown in SEQ ID NO: 1, and has one or more of the following mutations: W8, W9, A42, H66, K312, V347, H348.
  • the optically active polypeptide is shown in SEQ ID NO: 2, 6, 7, 9, or has a variant containing one or more mutations selected from Y1A or Y1G at the amino acid position corresponding to the first amino acid of SEQ ID NO: 2.
  • optically active polypeptide is located at 266/267, 266/268, 266/269, 266/270, 266/271, 266/272, 266/273, 266/274, 266/275, 266 of the D-glucose-sensitive polypeptide.
  • the mutations in the D-glucose-sensitive peptide include mutations selected from any of the following groups: (1) V347W and H348T, (2) V347W and H348G, (3) V347W and H348S, (4) V347W, H348T and H66R, (5) V347W, H348T and H66E, (6) V347W, H348T and H66A, (7) V347W, H348T and H66F, (8) V347W (9) V347W, H348T and H66C, (10) V347W, H348T and H66P, (11) V347W, H348T and H66Q, (12) V347W, H348T and H66T, (13) V347W, H348T and W9R, (14) V347W, H348T and W9H, (15) V347W, H348T and W9E, (16) V347 W, H348T and
  • the optical probe contains a D-glucose-sensitive polypeptide as shown in SEQ ID NO: 1 and an optically active polypeptide as shown in SEQ ID NO: 2, 6, 7, and 9, wherein the optically active polypeptide is located at position 348/352 of the D-glucose-sensitive polypeptide, and the optical probe has the following mutations: (1) V347W and H348T of the D-glucose-sensitive polypeptide and 1A of the optically active polypeptide; (2) V347W and H348T of the D-glucose-sensitive polypeptide.
  • the optical probe is as shown in SEQ ID NO: 11.
  • a fusion polypeptide comprising the optical probe 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 a localization sequence (e.g., a polypeptide that localizes the optical probe to a different organelle or sub-organelle), a tag for easy purification, or a tag for use in an immunoreaction (e.g., immunoblotting).
  • a linker may be present between the optical probe and the other polypeptides in the fusion polypeptide described herein.
  • the present invention also relates to variants of the aforementioned nucleic acid molecules, including fragments, analogs, derivatives, soluble fragments and variants of the present invention encoding the optical probes or fusion proteins of the present invention, or their complementary sequences.
  • the present invention also provides nucleic acid constructs comprising the nucleic acid molecules described herein.
  • the nucleic acid sequence encodes the optical probe or fusion polypeptide described herein.
  • the nucleic acid construct is a cloning vector, an expression vector, or a recombinant vector.
  • the nucleic acid molecule is operatively linked to an expression control sequence.
  • the kit further comprises one or more reagents selected from the following: buffer, culture medium, D-glucose 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 of cell expression, and isolating the optical probe or fusion polypeptide.
  • the method includes the following steps: 1) incorporating a nucleic acid molecule encoding the D-glucose optical probe described herein into an expression vector; 2) transferring the expression vector into a host cell; 3) culturing the host cell under conditions suitable for expression of the expression vector; and 4) isolating the D-glucose optical probe.
  • Another aspect of the present invention provides a method for detecting D-glucose in a sample, comprising: contacting the sample with the optical probe or fusion peptide or host cell described herein, and detecting changes in the optically active peptide.
  • the detection can be performed in vivo, in vitro, subcellular, or in situ.
  • the sample may be, for example, blood.
  • This article also provides a method for quantifying D-glucose in a sample, comprising: contacting the optical probe or fusion peptide or host cell described herein with the sample, detecting optical changes in the optically active peptide, and quantifying D-glucose in the sample based on the optical changes in the optically active peptide.
  • Another aspect of the present invention provides a method for screening compounds (e.g., drugs), comprising: contacting the optical probe or fusion peptide or host cell described herein with a candidate compound in a system containing D-glucose; detecting optical changes in the optically active peptide; and screening the compound based on the optical changes in the optically active peptide.
  • the method can screen compounds in high throughput.
  • the host cells described herein are contacted with the candidate compound in a system containing D-glucose, and optical changes in the optically active peptide indicate whether the candidate compound can regulate the uptake of D-glucose by the cells.
  • Another aspect of the present invention provides a method for intracellular and/or extracellular localization of the D-glucose, comprising: contacting a system containing D-glucose with the optical probe or the host cell, and detecting optical changes in the optically active peptide.
  • the system is a solution system, a cellular system, or a subcellular system.
  • the beneficial effects of this invention are as follows:
  • the D-glucose optical probe provided by this invention is easy to mature, exhibits large fluorescence dynamics, and has good specificity. Furthermore, it can be expressed in cells through gene manipulation, enabling real-time, high-throughput, and quantitative detection of D-glucose both inside and outside cells, eliminating time-consuming sample processing steps.
  • Experimental results show that the D-glucose optical probe provided by this application achieves a response to D-glucose that is more than 10 times that of the control. It can also perform localization, qualitative, and quantitative detection of D-glucose in subcellular structures such as the cytoplasm, nucleus, cell membrane, and mitochondria, and allows for high-throughput compound screening and quantitative detection of D-glucose in blood.
  • Figure 1 shows an SDS-PAGE image of an exemplary D-glucose optical probe
  • Figure 2 shows the fluorescence spectral properties of an exemplary D-glucose optical probe
  • Figure 3 shows the titration curves of different concentrations of D-glucose using an exemplary D-glucose optical probe
  • Figure 5 shows a photograph of the subcellular organelle localization of an exemplary D-glucose optical probe in mammalian cells
  • Figure 6 is a schematic diagram of the dynamic monitoring of D-glucose concentration in the cytoplasm of mammalian cells using an exemplary D-glucose optical probe.
  • Figure 7 is a dot plot of an exemplary D-glucose optical probe used for high-throughput compound screening at the live cell level
  • Figure 8 is a bar chart showing the quantification of D-glucose in mouse and human blood using an exemplary D-glucose optical probe.
  • compositions “containing” X may consist of only X or may contain other substances, such as X+Y.
  • D-glucose-sensitive peptide refers to a peptide that responds to D-glucose, and the response includes any response to chemical, biological, electrical, or physiological parameters of the peptide in relation to the interaction with the sensitive peptide.
  • Responses include small changes, such as changes in the orientation of amino acids or peptide fragments of the peptide, and changes in the primary, secondary, or tertiary structure of the peptide, 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; a conformational change occurs when the three-dimensional structure of the molecule changes. Examples of conformational changes include a change from an ⁇ -helix to a ⁇ -sheet or vice versa. It is understood that a detectable change need not be a conformational change, as long as the fluorescence of the fluorescent protein moiety is altered.
  • the D-glucose-sensitive peptides described herein may also include their functional variants. Functional variants of D-glucose-sensitive peptides include, but are not limited to, variants that can interact with D-glucose to undergo the same or similar changes as the parental D-glucose-sensitive peptide.
  • the D-glucose-sensitive polypeptides described in this invention include, but are not limited to, the glucose-galactose-binding protein TtGBP derived from the thermophilic bacterium *T. thermophilus*, or variants thereof with more than 90% homology.
  • the D-glucose-binding protein can sense changes in D-glucose concentration, and its spatial conformation also changes during dynamic changes in D-glucose concentration.
  • optical probe refers to a D-glucose-sensitive peptide fused to an optically active peptide.
  • D-glucose-sensitive peptides such as D-glucose-binding proteins
  • optically active peptides e.g., fluorescent proteins
  • an optically active polypeptide e.g., a fluorescent protein
  • a protein-based "optically active polypeptide” is a polypeptide capable of emitting fluorescence. Fluorescence is an optical property of the optically active polypeptide that can be used as a means of detecting the responsiveness of the optical probes of the present invention.
  • fluorescence property refers to the molar extinction coefficient at an appropriate excitation wavelength, fluorescence quantum efficiency, shape of the excitation or emission spectrum, maximum excitation wavelength and maximum emission wavelength, amplitude of excitation at two different wavelengths, ratio of emission amplitudes at two different wavelengths, excited-state lifetime, or fluorescence anisotropy.
  • a measurable difference in any of these properties between active and inactive states is sufficient for the utility of the fluorescent protein substrate of the present invention in activity assays.
  • the measurable difference can be determined by determining the amount of any quantitative fluorescence property, for example, the amount of fluorescence at a specific wavelength or the integral of fluorescence over the emission spectrum.
  • the protein substrate is selected to have fluorescence properties that are easily distinguishable between inactive and activated conformational states.
  • the optically active polypeptides described herein may also include functional variants thereof.
  • Functional variants of optically active peptides include, but are not limited to, variants that can undergo changes in fluorescence properties that are the same as or similar to those of the parent optically active peptide.
  • fluorescent protein refers to a protein that emits fluorescence under excitation light. Fluorescent proteins are fundamental detection methods in the field of bioscience. Examples include the commonly used green fluorescent protein GFP and its cyclically rearranged derivatives such as blue fluorescent protein (cpBFP), green fluorescent protein (cpGFP), and yellow fluorescent protein (cpYFP); and the commonly used red fluorescent protein RFP, and its cyclically rearranged derivatives such as cpmApple, cpmOrange, and cpmKate.
  • cpYFP is shown in SEQ ID NO: 2
  • cpGFP in SEQ ID NO: 6
  • cpBFP in SEQ ID NO: 7
  • cpmApple 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 a mutation at the first amino acid corresponding to SEQ ID NO: 2.
  • the mutation at position 1 is preferably A or G.
  • the functional variant of the fluorescent protein has the sequence shown in any of SEQ ID NO: 2, 6, 7, 9 and has a mutation selected from any group of the following at the first amino acid corresponding to SEQ ID NO: 2: Y1A or Y1G. 1S indicates that the first amino acid is mutated to S, and so on.
  • the optically active polypeptide is located in the N-C direction in the residues 266/267, 266/268, 266/269, 266/270, 266/271, 266/272, 266/273, 266/274, 266/275, 266/276, 266/277, 266/278, 266/279, 266/280, 266/281, 266/282, 266...
  • insertion site 266/267 indicates that the optically active polypeptide is located between amino acids 266 and 267 of the D-glucose-sensitive polypeptide. If the two numbers in the "X/Y" format are not consecutive integers and X is less than Y, it indicates that the optically active polypeptide replaces the amino acids between those numbers. For example, insertion site 266/269 indicates that the optically active polypeptide replaces amino acids 267-268 of the D-glucose-sensitive polypeptide.
  • X is greater than Y in the "X/Y" format, it indicates that the D-glucose-sensitive polypeptide portion located at the N-terminus of the optically active polypeptide terminates at the Xth amino acid in the D-glucose-sensitive polypeptide sequence, while the D-glucose-sensitive polypeptide portion located at the C-terminus of the optically active polypeptide terminates at the Xth amino acid in the D-glucose-sensitive polypeptide sequence.
  • insertion site 346/344 indicates that the N-terminus of the optically active polypeptide is fused with the N-terminal starting amino acid (e.g., any amino acid from position 1 to 394) to amino acid 346 of the D-glucose-sensitive polypeptide sequence, and the C-terminus of the optically active polypeptide is fused with the amino acid from position 344 to the C-terminal ending amino acid (e.g., amino acid 394) of the D-glucose-sensitive polypeptide sequence, with an exemplary structure of: (amino acids 1 to 346 of the D-glucose-sensitive polypeptide sequence) - (optically active polypeptide) - (amino acids 344 to 394 of the D-glucose-sensitive polypeptide sequence); if the two numbers in the site represented in the form of "X/Y" are not consecutive integers and X equals Y, it indicates that the optically active polypeptide is inserted between the amino acids
  • the optically active polypeptide represented by SEQ ID NO: 2, 6, 7, or 9 is located at any one or more of the following sites of the D-glucose-sensitive polypeptide represented by SEQ ID NO: 1: 266/267, 266/268, 266/269, 266/270, 266/271, 266/272, 266/273, 266/274, 266/275, 266/276, 266/277, 266/278, 2 66/279, 266/280, 266/281, 266/282, 266/283, 266/284, 266/285, 266/286, 266/287, 266/288, 267/267, 267/268, 267/269, 267/270, 267/271, 267/272, 267/273, 267/274, 267/275, 267/276, 267/277, 267/278 267/279, 267/280, 267/281, 267/282, 267/283, 267/283, 267
  • the optical probe comprises, from the N-terminus to the C-terminus, residues 1-X of SEQ ID NO: 1, an optically active polypeptide or a variant thereof shown in any one of SEQ ID NO: 2, 6, 7, 9, and residues Y-394 of SEQ ID NO: 1, wherein X and Y are selected from any one of the following groups:
  • X is 266, Y is 267.
  • X is 266, Y is 268.
  • X is 266, Y is 269.
  • (4) X is 266, Y is 270.
  • X is 266, Y is 271.
  • (6) X is 266, Y is 272.
  • (7) X is 266, Y is 273.
  • (8) X is 266, Y is 274.
  • (9) X is 266, Y is 275.
  • Y 280, (125) X is 271, Y is 281, (126) X is 271, Y is 282, (127) X is 271, Y is 283, (128) X is 271, Y is 284, (129) X is 271, Y is 285, (130) X is 271, Y is 286, (131) X is 271, Y is 287, (132) X is 271, Y is 288, (133) X is 272, Y is 267, (134) X is 272, Y is 268, (135) X is 272, Y is 269, (136) X is 272, Y is 270, (137) X is 272, Y is 271, (138) X is 272, Y is 272, (139) X is 272, Y is 273, (140) X is 272, Y is 274, (141) X is 272, Y is 275, (142) X is 272, Y is 276, (143) X is 272, Y is 277, (
  • Y is 278, (189) X is 274, Y is 279, (190) X is 274, Y is 280, (191) X is 274, Y is 281, (192) X is 274, Y is 282, (193) X is 274, Y is 283, (194) X is 274, Y is 284, (195) X is 274, Y is 285, (196) X is 274, Y is 286, (197) X is 274, Y is 287, (198) X is 274, Y is 288, (199) X is 275, Y is 267, (200) X is 275, Y is 268, (201) X is 275, Y is 269, (202) X is 275, Y is 270, (203) X is 275, Y is 271, (204) X is 275, Y is 272, (205) X is 275, Y is 273, (206) X is 275, Y is 274, (207) X is 275, Y is 275, (208)
  • Y is 288, (221) X is 276, Y is 267, (222) X is 276, Y is 268, (223) X is 276, Y is 269, (224) X is 276, Y is 270, (225) X is 276, Y is 271, (226) X is 276, Y is 272, (227) X is 276, Y is 273, (228) X is 276, Y is 274, (229) X is 276, Y is 275, (230) X is 276, Y is 276, (231) X is 276, Y is 277, (232) X is 276, Y is 278, (233) X is 276, Y is 279, (234) X is 276, Y is 280, (235) X is 276, Y is 281, (236) X is 276, Y is 282, (237) X is 276, Y is 283, (238) X is 276, Y is 284, (239) X is 276,
  • Y is 276, (253) X is 277, Y is 277, (254) X is 277, Y is 278, (255) X is 277, Y is 279, (256) X is 277, Y is 280, (257) X is 277, Y is 281, (258) X is 277, Y is 282, (259) X is 277, Y is 283, (260) X is 277, Y is 284, (261) X is 277, Y is 285, (262) X is 277, Y is 286, (263) X is 277, Y is 287, (264) X is 277, Y is 288, (265) X is 278, Y is 267, (266) X is 278, Y is 268, (267) X is 278, Y is 269, (268) X is 278, Y is 270, (269) X is 278, Y is 271, (270) X is 278,
  • Y is 286, (285)X is 278, Y is 287, (286)X is 278, Y is 288, (287)X is 279, Y is 267, (288)X is 279, Y is 268, (289)X is 279, Y is 269, (290)X is 279, Y is 270, (291)X is 279, Y is 271, (292)X is 279, Y is 272, (293)X is 279, Y is 273, (294)X is 279, Y is 274, (295 ...
  • Y is 284, (349) X is 281, Y is 285, (350) X is 281, Y is 286, (351) X is 281, Y is 287, (352) X is 281, Y is 288, (353) X is 282, Y is 267, (354) X is 282, Y is 268, (355) X is 282, Y is 269, (356) X is 282, Y is 270, (357) X is 282, Y is 271, (358) X is 282, Y is 272, (359) X is 282, Y is 273, (360) X is 282, Y is 274, (361) X is 282, Y is 275, (362) X is 282, Y is 276, (363) X is 282, Y is 277, (364) X is 282, Y is 278, (365) X is 282, Y is 279, (366) X is 282, Y is 280, (367) X is 28
  • Y is 272, (381) X is 283, Y is 273, (382) X is 283, Y is 274, (383) X is 283, Y is 275, (384) X is 283, Y is 276, (385) X is 283, Y is 277, (386) X is 283, Y is 278, (387) X is 283, Y is 279, (388) X is 283, Y is 280, (389) X is 283, Y is 281, (390) X is 283, Y is 282, (391) X is 283, Y is 283, (392) X is 283, Y is 284, (393) X is 283, Y is 285, (394) X is 283, Y is 286, (395) X is 283, Y is 287, (396) X is 283, Y is 288, (397) X is 284, Y is 267, (398) X is 284, Y is 268, (399) X is 284,
  • Y is 282, (413) X is 284, Y is 283, (414) X is 284, Y is 284, (415) X is 284, Y is 285, (416) X is 284, Y is 286, (417) X is 284, Y is 287, (418) X is 284, Y is 288, (419) X is 285, Y is 267, (420) X is 285, Y is 268, (421) X is 285, Y is 269, (422) X is 285, Y is 270, (423) (424) X is 285, Y is 271, (425) X is 285, Y is 272, (426) X is 285, Y is 273, (427) X is 285, Y is 274, (428) X is 285, Y is 275, (429) X is 285, Y is 277, (430) X is 285, Y is 278, (431) X is 285, Y is 279, (432) X
  • Y is 270, (445) X is 286, Y is 271, (446) X is 286, Y is 272, (447) X is 286, Y is 273, (448) X is 286, Y is 274, (449) X is 286, Y is 275, (450) X is 286, Y is 276, (451) X is 286, Y is 277, (452) X is 286, Y is 278, (453) X is 286, Y is 279, (454) X is 286, Y is 280, (455) (456) X is 286, Y is 281, (457) X is 286, Y is 282, (458) X is 286, Y is 283, (459) X is 286, Y is 284, (460) X is 286, Y is 286, (461) X is 286, Y is 287, (462) X is 286, Y is 288, (463) X is 287, Y is 267, (464)
  • Y is 347, (541) X is 341, Y is 348, (542) X is 341, Y is 349, (543) X is 341, Y is 350, (544) X is 341, Y is 351, (545) X is 341, Y is 352, (546) X is 341, Y is 353, (547) X is 342, Y is 341, (548) X is 342, Y is 342, (549) X is 342, Y is 343, (550) X is 342, Y is 344, (551) (552) X is 342, Y is 345, (553) X is 342, Y is 346, (554) X is 342, Y is 347, (555) X is 342, Y is 348, (556) X is 342, Y is 349, (557) X is 342, Y is 350, (558) X is 342, Y is 352, (559) X is 342, Y is 353, (560)
  • Y is 353, (573) X is 344, Y is 341, (574) X is 344, Y is 342, (575) X is 344, Y is 343, (576) X is 344, Y is 344, (577) X is 344, Y is 345, (578) X is 344, Y is 346, (579) X is 344, Y is 347, (580) X is 344, Y is 348, (581) X is 344, Y is 349, (582) X is 344, Y is 350, (583) X is 344, Y is 351, (584) X is 344, Y is 352, (585) X is 344, Y is 353, (586) X is 345, Y is 341, (587) X is 345, Y is 342, (588) X is 345, Y is 343, (589) X is 345, Y is 344, (590)
  • Y is 346, (605) X is 346, Y is 347, (606) X is 346, Y is 348, (607) X is 346, Y is 349, (608) X is 346, Y is 350, (609) X is 346, Y is 351, (610) X is 346, Y is 352, (611) X is 346, Y is 353, (612) X is 347, Y is 341, (613) X is 347, Y is 342, (614) X is 347, Y is 343, (615) X is 347, Y is 344, (616) X is 347, Y is 345, (617) X is 347, Y is 346, (618) X is 347, Y is 347, (619) X is 347, Y is 348, (620) X is 347, Y is 349, (621) X is 347, Y is 350, (62
  • Y is 352, (637)X is 348, Y is 353, (638)X is 349, Y is 341, (639)X is 349, Y is 342, (640)X is 349, Y is 343, (641)X is 349, Y is 344, (642)X is 349, Y is 345, (643)X is 349, Y is 346, (644)X is 349, Y is 347, (645)X is 349, Y is 348, (646)X is 349, Y is 349, (647) X is 349, Y is 350, (648) X is 349, Y is 351, (649) X is 349, Y is 352, (650) X is 349, Y is 353, (651) X is 350, Y is 341, (652) X is 350, Y is 342, (653) X is 350, Y is 343, (654) X is 350, Y is 344, (655)
  • Y is 345, (669) X is 351, Y is 346, (670) X is 351, Y is 347, (671) X is 351, Y is 348, (672) X is 351, Y is 349, (673) X is 351, Y is 350, (674) X is 351, Y is 351, (675) X is 351, Y is 352, (676) X is 351, Y is 353, (677) X is 352, Y is 341, (678) X is 352, Y is 342, (679) X is 352, Y is 343, (680) X is 352, Y is 344, (681) X is 352, Y is 345, (682) X is 352, Y is 346, (683) X is 352, Y is 347, (684) X is 352, Y is 348, (685) X is 352, Y is 349, (686) X is 352, Y is 350, (687)
  • response fold refers to the standardized fluorescence ratio. The greater the deviation of the probe's response fold from 1 (whether it increases or decreases), the greater the change in the probe's response to the substrate relative to the control, or the greater its responsiveness.
  • the response fold is calculated by detecting the change in the ratio of fluorescence intensity at 528nm emission from 420nm excitation to fluorescence intensity at 528nm emission from 485nm excitation (Normalized Ratio 420/485), as detailed below:
  • Fluorescence signal values were corrected by subtracting the detection signal values from cells that did not express the probe protein. pH-sensitive interference was eliminated by dividing the probe detection signals from parallel experimental groups by the control detection signals to obtain corrected data.
  • F represents fluorescence intensity.
  • F ⁇ sub> sample ⁇ /sub> represents the total fluorescence intensity of the sample expressing the fluorescent probe
  • F ⁇ sub>BLK ⁇ /sub> represents the background fluorescence intensity of the sample not expressing the fluorescent probe
  • F ⁇ sub> cpYFP ⁇ /sub> represents the fluorescence intensity of the sample used as a pH control.
  • F ⁇ sub>485 ⁇ /sub> represents the fluorescence intensity emitted at 528 nm when the fluorescent protein sample is excited at 485 nm
  • F ⁇ sub>420 ⁇ /sub> represents the fluorescence intensity emitted at 528 nm when the fluorescent protein sample is excited at 420 nm.
  • Ratiosensor represents the fluorescence intensity ratio of the probe
  • Ratio cpYFP represents the fluorescence intensity ratio of the corresponding probe to the pH control fluorescent protein.
  • NormalizedRatio 420/485 is the fold change of the probe or the response multiple. The greater the deviation of NormalizedRatio 485/420 from 1 (whether it increases or decreases), the greater the fold change of the probe or the response multiple.
  • variants include variants that have the same function as the polypeptide or protein but have a different sequence.
  • variants of polypeptides or proteins may include: homologous sequences, conserved variants, allelic variants, natural mutants, and induced mutants.
  • variants include, but are not limited to: deletions, insertions, and/or substitutions of one or more (typically 1-30, preferably 1-20, more preferably 1-10, most preferably 1-5) amino acids in the sequence of the polypeptide or protein, and sequences obtained by adding one or more (typically up to 20, preferably up to 10, more preferably up to 5) amino acids to its carboxyl terminus and/or amino terminus.
  • variants may also comprise polypeptides or proteins with at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100% sequence identity with the polypeptide or protein.
  • amino acids with basic side chains e.g., lysine, D-glucose, 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 nonpolar side chains e.g., alanine, valine, leucine, isoleucine, D-glucose, phenylalanine, methionine, D-glucose
  • amino acids with ⁇ -branched side chains e.g., threonine, valine, isoleucine
  • amino acids with aromatic side chains e.g., tyrosine, phenylalanine, D-glucose, histidine
  • amino acids For example, adding one or more amino acids to the amino terminus and/or carboxyl terminus generally does not alter the function of a polypeptide or protein.
  • conserved amino acid substitutions for many common, known non-genetically encoded amino acids are known in the art.
  • conserveed substitutions for other non-coding amino acids can be determined based on a comparison of their physical properties with those of their genetically encoded amino acids.
  • Connector or “linking region” refers to an amino acid or nucleotide sequence that links two parts in a polypeptide, protein, or nucleic acid of the present invention.
  • the number of amino acids at the amino terminus of the linking region between the D-glucose-sensitive polypeptide and the optically active polypeptide is selected to be 0-3, and the number of amino acids at the carboxyl terminus is selected to be 0-2; when the recombinant optical probe is used as a basic unit to link with a functional protein, it can be fused to the amino acid or carboxyl terminus of the recombinant optical probe.
  • the connector sequence can be a short peptide chain composed of one or more flexible amino acids, such as Y.
  • D-glucose-binding protein variants with mutations at the following sites exhibit different binding activities than D-glucose: W8, W9, E13, A42, H66, D278, K312, V347, and H348 of SEQ ID NO: 1.
  • the amino acid mutations include modifications, substitutions, or deletions of amino acids.
  • the D-glucose-sensitive peptide in the optical probe is a D-glucose-binding protein variant as described in any embodiment herein, and the fluorescent protein in the optical probe is as shown in SEQ ID NO: 2-9 or a functional variant thereof.
  • the fluorescent protein is as shown in SEQ ID NO: 2, 6, 7, 9 or a functional variant thereof.
  • the D-glucose-sensitive polypeptide in the optical probe is shown in SEQ ID NO: 1
  • the optically active polypeptide is shown in SEQ ID NO: 2.
  • the optically active polypeptide is located at positions 266/267, 266/268, 266/269, 266/270, 266/271, 266/272, 266/273, 266/274, 266/275, 266/276, 266/277, and 266/278 of the D-glucose-sensitive polypeptide.
  • the optical probe contains a D-glucose-sensitive polypeptide as shown in SEQ ID NO: 1 and an optically active polypeptide as shown in SEQ ID NO: 2, 6, 7, and 9, wherein the optically active polypeptide is located at position 348/352 of the D-glucose-sensitive polypeptide, and the optical probe has the following mutations: (1) V347W and H348T of the D-glucose-sensitive polypeptide and 1A of the optically active polypeptide; (2) V347W and H348T of the D-glucose-sensitive polypeptide.
  • identity refers to the fact that, when compared and matched for maximum correspondence using methods known in the art, such as sequence comparison algorithms, by manual alignment and visual inspection, two or more sequences or subsequences are identical or have a certain percentage of amino acid residues or nucleotides identical in a specified region (e.g., 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical).
  • the preferred algorithms for determining the percentage of sequence identity and the percentage of sequence similarity are BLAST and BLAST 2.0 algorithms, which can be found in Altschul et al. (1977) Nucleic Acids Res. 25: 3389 and Altschul et al. (1990) J.Mol.Biol. 215: 403, respectively.
  • gene cloning often requires the design of suitable restriction enzyme sites, which inevitably introduces one or more irrelevant residues at the end of the expressed polypeptide or protein, without affecting the activity of the target polypeptide or protein.
  • promote the expression of recombinant proteins obtain recombinant proteins that are automatically secreted outside host cells, or facilitate the purification of recombinant proteins, it is often necessary to add certain amino acids to the N-terminus, C-terminus, or other suitable regions within the recombinant protein.
  • Suitable adaptor peptides include, but are not limited to, suitable adaptor peptides, signal peptides, leader peptides, terminal extensions, glutathione S-transferase (GST), maltose E-binding proteins, protein A, tags such as 6His or Flag, or proteolytic enzyme sites such as factor Xa, thrombin, or enterokinase.
  • the terms “functional fragment,” “derivative,” and “analyte” refer to a protein that substantially retains the same biological function or activity as the original polypeptide or protein (e.g., D-glucose-binding protein or fluorescent protein).
  • analogues also include those having residues different from naturally occurring L-amino acids (such as D-amino acids), and those having non-naturally occurring or synthetic amino acids (such as ⁇ - or ⁇ -amino acids).
  • L-amino acids such as D-amino acids
  • non-naturally occurring or synthetic amino acids such as ⁇ - or ⁇ -amino acids.
  • D-glucose-sensitive polypeptides of the present invention are not limited to the representative proteins, variants, derivatives, and analogues listed above.
  • Modifications (generally without altering the primary structure) include chemically derived forms of proteins, such as acetylation or carboxylation, either in vivo or in vitro.
  • Modifications also include glycosylation, such as those resulting from glycosylation modifications during protein synthesis and processing or further processing steps. This modification can be accomplished by exposing the protein to glycosylation enzymes (such as mammalian glycosylation or deglycosylation enzymes). Modifications also include sequences having phosphorylated amino acid residues (such as phosphotyrosine, phosphotyserine, phosphotythreonine). Proteins modified to improve their resistance to proteolytic hydrolysis or optimize their solubility are also included.
  • glycosylation enzymes such as mammalian glycosylation or deglycosylation enzymes.
  • Modifications also include sequences having phosphorylated amino acid residues (such as phosphotyrosine, phosphotyserine, phosphotythreonine). Proteins modified to improve their resistance to proteolytic hydrolysis or optimize their solubility are also included.
  • the fusion polypeptide of this invention comprises the optical probe described herein and other polypeptides.
  • the optical probe described herein further comprises other polypeptides fused thereto. These other polypeptides 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 for targeting the optical probe to different organelles or subcellular organelles, tags for purification, or tags for immunoblotting.
  • a linker may be present between the optical probe and other polypeptides in the fusion polypeptide described herein.
  • the subcellular organelles described herein include the cytoplasm, mitochondria, nucleus, endoplasmic reticulum, cell membrane, Golgi apparatus, lysosomes, and peroxisomes.
  • the tags used for purification or for immunoblotting include 6-histidine (6*His), glutathione S-transferase (GST), and Flag.
  • nucleic acid molecules encoding the D-glucose-sensitive polypeptide or optical probe described herein.
  • nucleic acid can be in DNA or RNA form.
  • DNA form includes 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 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 a substitution of a nucleic acid, which may be a substitution, deletion, or insertion of one or more nucleotides, but does not substantially alter the function of the protein it encodes.
  • the nucleic acid of this invention may comprise a nucleotide sequence with at least about 50%, at least about 60%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100% sequence identity with the nucleic acid sequence described herein.
  • nucleic acid fragments that hybridize with the above-described sequences.
  • a "nucleic acid fragment” contains at least 15 nucleotides in length, preferably at least 30 nucleotides, more preferably at least 50 nucleotides, and most preferably at least 100 nucleotides or more.
  • the nucleic acid fragments can be used in nucleic acid amplification techniques (such as PCR).
  • the full-length sequence or fragment of the optical probe or fusion protein of this invention can typically be obtained by PCR amplification, artificial synthesis, or recombinant methods.
  • the steps and reagents used in conventional PCR, synthesis, and recombinant methods are known in the art.
  • mutations can be introduced into the protein sequence of this invention through methods such as mutagenic PCR or chemical synthesis.
  • This invention also relates to nucleic acid constructs containing the polynucleotides described herein, and one or more regulatory sequences operatively linked to these sequences.
  • the polynucleotides described herein can be manipulated in various ways to ensure the expression of the polypeptide or protein.
  • the nucleic acid constructs can be manipulated before insertion into a vector, depending on the expression vector or requirements. 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, such as plasmids, phage particles, phage derivatives, animal viruses, and entrapments.
  • Typical expression vectors contain expression control sequences that can be used to regulate the expression of a desired nucleic acid sequence, operatively linked to the nucleic acid sequence described herein or its complement.
  • expression control sequence refers to an element that can be operatively linked to the target gene to regulate the transcription, translation, and expression of the target gene. This can be an origin of replication, promoter, marker gene, or translation control element, including enhancers, operons, terminators, ribosome binding sites, etc. The choice of expression control sequence depends on the host cell used.
  • "operative linking” refers to the linking of the target nucleotide sequence to the regulatory sequence in a manner that allows the nucleotide sequence to be expressed.
  • the expression vector also includes a ribosome binding site for translation initiation and a transcription terminator.
  • the expression vector may be a commercially available pCDF vector, with no other special requirements.
  • the host cell is preferably a variety of cells conducive to gene product expression or fermentation production, such cells being 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*, animal cells such as CHO, COS, HEK293, HeLa cells, or Bowes melanoma cells, etc.
  • the exemplary host cell used in the embodiments of this invention is *Escherichia coli* strain BL21-DE3. Those skilled in the art will understand how to select appropriate vectors, promoters, enhancers, and host cells.
  • This invention involves transforming an expression vector into host cells, followed by amplification and expression culture of the host cells to isolate the D-glucose optical probe.
  • the host cell amplification and expression culture can be performed using conventional methods.
  • the culture medium can be any conventional medium. Culture is carried out under conditions suitable for host cell growth.
  • the optical probe is expressed intracellularly, on the cell membrane, or secreted extracellularly.
  • the recombinant protein can be separated or purified using various separation methods based on its physical, chemical, and other properties. This invention does not specifically limit the method for separating the D-glucose fluorescent protein; conventional methods for separating fusion proteins in the art can be used.
  • optical probe is separated using His-tagged affinity chromatography.
  • This invention also provides the application of the D-glucose optical probe in real-time localization, quantitative detection, and high-throughput compound screening of D-glucose.
  • the D-glucose optical probe is preferably linked to a signal peptide at different sites within the cell and transferred into the cell. Real-time localization of D-glucose is achieved by detecting the intensity of fluorescence signals within the cell; quantitative detection of D-glucose is then performed by combining a standard D-glucose titration curve with changes in fluorescence signals. Changes in fluorescence signals are displayed, for example, by a standardized fluorescence signal ratio.
  • this ratio is the ratio of the sample's 485 nm fluorescence signal to its 420 nm fluorescence signal to the corresponding ratio of the control.
  • the D-glucose standard titration curve of this invention is plotted based on the fluorescence signals obtained by the D-glucose optical probe at different concentrations of D-glucose.
  • the D-glucose optical probe of this invention is directly transferred into the cell, eliminating the need for time-consuming sample processing during real-time localization and quantitative detection of D-glucose, thus improving accuracy.
  • the D-glucose optical probe of this invention when used for high-throughput compound screening, involves adding different compounds to cell culture medium and measuring changes in D-glucose content to screen for compounds that affect these changes.
  • the application of the D-glucose optical probe described in this invention for real-time localization, quantitative detection, and high-throughput compound screening is not for diagnostic or therapeutic purposes and does not involve the diagnosis or treatment of diseases.
  • This invention also provides a detection kit comprising the optical probes, nucleic acid molecules, nucleic acid constructs, and/or cells described herein.
  • the kit further comprises other reagents required for the detection of D-glucose.
  • these other reagents are well known in the art, such as buffers, cell culture media, and D-glucose standards.
  • Exemplary buffers include, for example, 100 mM HEPES and 100 mM NaCl, pH 7.4.
  • the pCDF-cpYFP and pCDF-ttGBP plasmids used in the examples were constructed by the Protein Laboratory of East China University of Science and Technology, and the pCDF plasmid vectors were purchased from Invitrogen. All primers used for PCR were synthesized, purified, and identified correctly by mass spectrometry by Shanghai Jierui Biotechnology Co., Ltd. and BGI Genomics. The expression plasmids constructed in the examples were all sequenced, and the sequencing was performed by BGI Genomics and J. Lee Sequencing.
  • the Taq DNA polymerase used in each example was purchased from Dongsheng Biotechnology, the pfu DNA polymerase was purchased from Tiangen Biotech (Beijing) Co., Ltd., and the primeSTAR DNA polymerase was purchased from TaKaRa.
  • the corresponding polymerase buffer and dNTPs were included with the purchase of the three polymerases. Restriction endonucleases such as BamHI, BglII, HindIII, NdeI, XhoI, EcoRI, and SpeI, T4 ligase, and T4 phosphorylase (T4PNK) were purchased from Fermentas, and the corresponding buffers were included with the purchase.
  • the Lip2000 transfection kit was purchased from Invitrogen.
  • D-glucose and other compounds were purchased from Sigma-Aldrich. Unless otherwise stated, inorganic salts and other chemical reagents were purchased from Sigma-Aldrich. HEPES salt, ampicillin (Amp), and puromycin were purchased from Amersco. The 96-well detection blackboard and the 384-well fluorescence detection blackboard were purchased from Grenier.
  • the DNA purification kits used in these examples were purchased from BBI (Canada), and the general plasmid extraction kits were purchased from Tiangen Biotech (Beijing) Co., Ltd.
  • the cloned strain Mach1 was purchased from Invitrogen. Nickel affinity chromatography columns and desalting column packing materials were both from GE Healthcare.
  • the main instruments used in the examples include: Biotek Synergy2 multi-functional microplate reader (Bio-Tek, USA), X-15R high-speed refrigerated centrifuge (Beckman, USA), Microfuge 22R benchtop high-speed refrigerated centrifuge (Beckman, USA), PCR amplifier (Biometra, Germany), ultrasonic disruptor (Ningbo Xinzhi Co., Ltd.), nucleic acid electrophoresis apparatus (Shenneng Bocai Co., Ltd.), fluorescence spectrophotometer (Varian, USA), CO2 incubator (SANYO), and inverted fluorescence microscope (Nikon, Japan).
  • the PCR amplification reaction system 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 (10 mM) 1 ⁇ L, sterile ultrapure water ( ddH2O ) 41.5-42 ⁇ L, total volume 50 ⁇ L.
  • the PCR amplification program is as follows: denaturation at 95°C 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 (600 bp/min)), extension at 72°C for 10 minutes.
  • the long-fragment amplification used in this invention is mainly a reverse PCR amplification vector, a technique used in the following examples to obtain site-directed mutagenesis.
  • Reverse PCR primers are designed at the mutation site, with one primer containing the mutated nucleotide sequence at its 5' end.
  • the amplified product then contains the corresponding mutation site.
  • the long-fragment amplification PCR reaction system is as follows: template sequence (10 pg-1 ng) 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 (2.5 mM) 4 ⁇ L, sterile ultrapure water ( ddH2O ) 33.5 ⁇ L, total volume 50 ⁇ L.
  • the PCR amplification program is as follows: denaturation at 95°C for 5 minutes, 30 cycles (98°C for 10 seconds, 50-68°C for 5-15 seconds, 72°C for a certain time (1000bp/min)), extension at 72°C for 10 minutes; or denaturation at 95°C for 5 minutes, 30 cycles (98°C for 10 seconds, 68°C for a certain time (1000bp/min)), extension at 72°C for 10 minutes.
  • the double enzyme digestion system for the plasmid vector is as follows: 20 ⁇ L plasmid vector (approximately 1.5 ⁇ g), 5 ⁇ L 10 ⁇ buffer, 11-2 ⁇ L restriction endonuclease, 21-2 ⁇ L restriction endonuclease, and bring the total volume to 50 ⁇ L with sterile ultrapure water. Reaction conditions: 37°C, 1-7 hours.
  • Plasmids or genomes extracted from microorganisms contain phosphate groups at their ends, while PCR products do not. Therefore, a phosphate addition reaction is required at the 5' end of the PCR product. Only DNA molecules with phosphate groups at their ends can undergo ligation.
  • the phosphorylation reaction system is as follows: 5-8 ⁇ L of PCR product DNA sequence, 1 ⁇ L of 10 ⁇ T4 ligase buffer, 1 ⁇ L of T4 polynucleotide kinase (T4 PNK), and 0-3 ⁇ L of sterile ultrapure water, for a total volume of 10 ⁇ L. The reaction conditions are 37°C for 30 minutes to 2 hours, followed by inactivation at 72°C for 20 minutes.
  • the principle of this method is that the blunt-end product obtained by PCR is phosphorylated at the 5' end of a DNA fragment using T4 PNK, and then ligated with a linearized vector using PEG4000 and T4 DNA ligase to obtain a recombinant plasmid.
  • the homologous recombination ligation system is as follows: 4 ⁇ L of T4 PNK-treated DNA fragment, 4 ⁇ L of linearized vector fragment, 1 ⁇ L of PEG4000, 1 ⁇ L of 10 ⁇ T4 ligase buffer, and 1 ⁇ L of T4 DNA ligase, for a total of 10 ⁇ L.
  • the reaction conditions are 22°C for 30 minutes.
  • DNA fragments digested by restriction endonucleases typically produce prominent sticky ends, which can then be ligated to vector fragments containing sequence-complementary sticky ends to form recombinant plasmids.
  • the ligation reaction system is as follows: 1-7 ⁇ L of the digested PCR product DNA fragment, 0.5-7 ⁇ L of the digested plasmid, 1 ⁇ L of 10 ⁇ T4 ligase buffer, 1 ⁇ L of T4 DNA ligase, and sterile ultrapure water to a total volume of 10 ⁇ L.
  • the reaction conditions are 16°C for 4-8 hours.
  • the 5' phosphorylated DNA fragment was ligated to the 3' and 5' ends of the linearized vector via a self-circularization ligation reaction to obtain a recombinant plasmid.
  • the self-circularization ligation reaction system was as follows: 10 ⁇ L phosphorylation reaction mixture, 0.5 ⁇ L T4 ligase (5 U/ ⁇ L), total volume 10.5 ⁇ L. Reaction conditions: 16°C, 4–16 hours.
  • Resuspension buffer CaCl2 (100mM), MgCl2 (70mM), NaAc (40mM)
  • Storage buffer 0.5 mL DMSO, 1.9 mL 80% glycerol, 1 mL 10 ⁇ CaCl2 (1 M), 1 mL 10 ⁇ MgCl2 (700 mM), 1 mL 10 ⁇ NaAc (400 mM), 4.6 mL ddH2O
  • Example 2 Expression and detection of cpYFP optical probes at different insertion sites
  • a DNA fragment of cpYFP was generated using PCR. Simultaneously, a homologous sequence from the cpYFP terminal was introduced at the 5' end using primers. PCR amplification produced a linearized pCDF-D-glucose-binding protein vector, whose 5' and 3' ends contained sequences completely identical to those at the two ends of cpYFP (15-20 bp).
  • the linearized pCDF-TtGBP and cpYFP fragments underwent homologous recombination using HieffCloneEnzyme.
  • the product was transformed into DH5 ⁇ , and the transformed DH5 ⁇ was plated on LB agar plates (streptomycin 100 ⁇ g/mL) and incubated overnight at 37°C. Positive clones identified by PCR were subjected to plasmid extraction and sequencing. Sequencing was performed by J. Lee Sequencing.
  • D-glucose response screening was performed using the supernatant of fragmented *E. coli* expressing the TtGBP-cpYFP fusion protein.
  • the detection signal of the fusion fluorescent protein containing 100 mM D-glucose was divided by the detection signal of the fusion fluorescent protein without D-glucose.
  • Table 1 the detection results showed that optical probes expressing the TtGBP-cpYFP fusion protein in the fragmented supernatant exhibited a D-glucose response greater than 1.2 times: 273/279, 273/284, 350/342, 348/343, 349/343, 348/344, 349/344, 349/345, 348/349, 349/349, and 3...
  • optical probes 48/350, 349/350, 346/351, 347/351, 347/352, 348/352, and 350/353. Seven probes showed a response to D-glucose exceeding 1.25 times: 273/279, 348/343, 348/344, 349/345, 349/349, 349/350, and 347/352.
  • Example 3 Expression and detection of cpGFP optical probes at different insertion sites
  • cpYFP was replaced with cpGFP to construct a D-glucose green fluorescent protein probe.
  • Table 2 the detection results showed that optical probes expressing the TtGBP-cpGFP fusion protein responded to D-glucose more than 1.3 times to optical probes at sites 348/344, 349/345, 349/349, 349/350, and 347/352, or the corresponding amino acid sites of its family proteins, were inserted.
  • Example 4 Expression and detection of cpBFP optical probes at different insertion sites
  • cpYFP was replaced with cpBFP to construct a D-glucose blue fluorescent protein probe.
  • Table 3 the detection results showed that optical probes expressing the TtGBP-cpBFP fusion protein responded to D-glucose more than 1.3 times to optical probes with insertion sites at 348/343, 348/344, 349/345, and 349/349 or corresponding amino acid sites of its family proteins were inserted.
  • Example 5 Expression and detection of cpmApple optical probes at different insertion sites
  • cpYFP was replaced with cpmApple to construct a D-glucose red fluorescent protein probe.
  • Table 4 the detection results show that optical probes expressing TtGBP-cpmApple fusion protein respond more than 1.3 times to D-glucose by replacing cpYFP with cpmApple.
  • Optical probes with insertion sites at 349/345 and 349/349 sites or corresponding amino acid sites of its family proteins are also included.
  • glucose detection was performed using a concentration gradient (0–100 mM), and the change in the ratio of fluorescence intensity at 420 nm excitation and 528 nm emission to fluorescence intensity at 485 nm excitation and 528 nm emission was detected.
  • Example 7 Expression and detection of mutated cpYFP optical probes
  • Optical probe mutants were constructed based on TtGBP-348/352-cpYFP.
  • the plasmid pCDF-TtGBP-348/352-cpYFP was linearized by PCR, with primers containing the base sequences of the desired mutation sites. Homologous recombination was performed on the obtained PCR product to obtain mutant plasmids for 12 sites: V347, H348, H66, D278, W9, E13, W8, A42, K312 of the D-glucose-sensitive polypeptide, and Y1 of the optically active polypeptide. Sequencing was performed by J. Lee Sequencing. As an example, the amino acid sequence of 348/352-TtGBP-V347W/H348S/H66C-cpYFP-Y1A is shown in SEQ ID NO: 11.
  • the successfully constructed mutant plasmid was transformed into BL21(DE3) to induce expression.
  • the supernatant from the fragmented *E. coli* expressing the probe protein was used for response screening to D-glucose and other non-specific substrates.
  • the detection signal of the fusion fluorescent protein containing D-glucose or other non-specific substrates was divided by the detection signal of the fusion fluorescent protein without D-glucose. The results are shown in Table 5. The detection results show that the optical probes with a response to D-glucose greater than 3-fold and good specificity are listed below.
  • the D-glucose optical probes described in Table 5 of Example 7 were used to detect D-glucose at concentration gradients (0–100 mM). After treating the probes for 10 minutes, the change in the ratio of fluorescence intensity at 420 nm excitation and 528 nm emission was measured to be that at 485 nm excitation and 528 nm emission. The probe titration results are shown in Figure 3, indicating that different mutants have different affinities for D-glucose.
  • the D-glucose probes in Table 5 were subjected to specificity testing. Reactivity was measured against D-glucose structural analogs, glucose-1-phosphate, sorbitol, mannitol, sodium lactate, sucrose, pyruvate, ribose, fructose, oxaloacetic acid, ATP, glucose-6-phosphate, citric acid, D-glucose, isocitrate, sodium gluconate, NAD+, NADH, malic acid, NADPH, arabinose, PEP, lactose, maltose, 2-deoxyglucose, and galactose. The results showed good specificity, as shown in Figure 4.
  • Example 9 Subcellular organelle localization of optical probes and performance of optical probes within subcellular organelles
  • different localization signal peptides are fused with the optical probe 348/352-TtGBP-V347W/H348S/H66C-cpYFP-Y1A to localize the optical probe to different organelles.
  • the D-glucose optical probes by fusing with different specific localization signal peptides, can localize to subcellular organelles including the cytoplasm, outer membrane, nucleus, endoplasmic reticulum, mitochondria, and nuclear exclusion. Fluorescence was observed in all different subcellular structures, and the distribution and intensity of the fluorescence varied.
  • HEK293 cells were transfected with a cytoplasmic optical probe plasmid for 36 hours. After washing with PBS, the cells were placed in HBSS solution, and the changes in the fluorescence intensity ratio at 420 nm excitation and 528 nm emission (485 nm excitation and 528 nm emission) were detected over a 30-minute period. The results are shown in Figure 6. After adding 5 mM D-glucose, the detection was continued for another 30 minutes. The 420/485 ratio gradually increased in the D-glucose-added samples, reaching a maximum of 1.8 times the initial value, while the 420/485 ratio remained unchanged in the control group without Oxalate.
  • Example 10 High-throughput compound screening in living cells based on optical probes
  • HeLa cells with cytoplasmic expression of 348/352-TtGBP-V347W/H348S/H66C-cpYFP-Y1A for high-throughput compound screening.
  • Transfected 293 cells were washed with PBS, treated with HBSS solution (without D-glucose) for 1 hour, and then treated with 10 ⁇ M of the compound for 1 hour. D-glucose was added to each sample. The ratio of fluorescence intensity at 420 nm excitation to 528 nm emission and the ratio of fluorescence intensity at 485 nm excitation to 528 nm emission were recorded using a microplate reader. Samples without any compound treatment were used as controls for standardization. The results are shown in Figure 7. Of the 2000 compounds used, the vast majority had minimal effect on D-glucose uptake by cells. Five compounds increased the cells' ability to take up D-glucose, while nine compounds significantly reduced it.
  • Example 11 Quantitative Detection of D-Glucose in Blood Using an Optical Probe
  • purified Kd 48.33 ⁇ M 348/352-TtGBP-V347W/H348S-cpYFP-Y1A was used to analyze D-glucose in the blood supernatant of mice and humans.
  • the D-glucose optical probe provided by the present invention has a relatively small protein molecular weight and is easy to mature. It exhibits large fluorescence dynamic changes, good specificity, and can be expressed in cells through gene manipulation. It can locate and quantify D-glucose in and out of cells in real time and can also perform high-throughput compound screening.

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Abstract

The present invention relates to a D-glucose optical probe, and a preparation method therefor and the use thereof. Specifically, provided is a D-glucose optical probe, the optical probe containing a D-glucose sensitive polypeptide and an optically active polypeptide, wherein the optically active polypeptide is located within the sequence of the D-glucose sensitive polypeptide.

Description

一种D-葡萄糖光学探针及其制备方法和应用A D-glucose optical probe, its preparation method and application 技术领域Technical Field

本发明涉及光学探针技术领域，尤其涉及一种D-葡萄糖光学探针及其制备方法和应用。This invention relates to the field of optical probe technology, and in particular to a D-glucose optical probe, its preparation method, and its application.

背景技术Background Technology

D-葡萄糖(D-Glucose，D-glc)是地球上包括从细菌到人类各种生物已知最重要、最基本的能量来源，也是人脑和神经系统最主要的供能物质。D-葡萄糖在生物学领域具有重要地位，是生物体的必需营养素，不仅是丰富的势能来源，而且是生物合成途径中广泛的代谢中间体的前体。植物亦可利用光合作用产生葡萄糖。D-葡萄糖是大多数组织的主要能量来源，特别是神经系统、红细胞、肾髓质和骨骼肌。这种营养素的调节失败可导致低血糖和高血糖，甚至可能导致死亡。D-glucose (D-glc) is the most important and fundamental energy source known to all living organisms on Earth, from bacteria to humans, and is also the primary energy source for the human brain and nervous system. D-glucose holds a vital position in biology, serving as an essential nutrient for organisms. It is not only a rich source of potential energy but also a precursor to a wide range of metabolic intermediates in biosynthetic pathways. Plants can also produce glucose through photosynthesis. D-glucose is the primary energy source for most tissues, particularly the nervous system, red blood cells, renal medulla, and skeletal muscle. Failure to regulate this nutrient can lead to both hypoglycemia and hyperglycemia, and may even result in death.

目前最常用的检测D-葡萄糖的方法有酶促探针、电化学法、纳米材料探针及基于上述原理开发的多种可穿戴的葡萄糖检测设备，以及基因编码的葡萄糖探针等。其中酶促探针尽管对葡萄糖具有较高的选择性且具备大规模生产的可行性，但制造成本高、重复性低、稳定性差；电化学法难以平衡成本和对葡萄糖的选择性；目前已经开发出来的基因编码的荧光探针特异性有限，对底物的亲和性无法完成体内原位实时监控，因此亟需开发出能够原位监测活细胞内D-葡萄糖动态变化的遗传编码荧光探针。Currently, the most commonly used methods for detecting D-glucose include enzyme-catalyzed probes, electrochemical methods, nanomaterial probes, various wearable glucose detection devices developed based on the above principles, and gene-encoded glucose probes. Among these, while enzyme-catalyzed probes have high selectivity for glucose and are feasible for large-scale production, they suffer from high manufacturing costs, low reproducibility, and poor stability. Electrochemical methods struggle to balance cost and glucose selectivity. Currently developed gene-encoded fluorescent probes have limited specificity, and their affinity for substrates cannot enable in situ real-time monitoring in vivo. Therefore, there is an urgent need to develop genetically encoded fluorescent probes capable of in situ monitoring of the dynamic changes in D-glucose within living cells.

发明内容Summary of the Invention

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D-葡萄糖的探针和方法。The purpose of this invention is to provide probes and methods for real-time, high-throughput, and quantitative detection of D-glucose inside and outside cells.

为了实现上述发明目的，本发明提供以下技术方案：To achieve the above-mentioned objectives, the present invention provides the following technical solution:

本发明第一方面提供一种D-葡萄糖结合蛋白变体，其：The first aspect of this invention provides a variant of a D-glucose-binding protein, wherein:

(a)具有SEQ ID NO：1所示的序列并且在选自以下的1个、2个、3个或更多个位点具有突变：W8、W9、A42、H66、K312、V347、H348，所述突变包括氨基酸的修饰、取代或缺失，(a) Having the sequence shown in SEQ ID NO: 1 and having mutations at one, two, three or more sites selected from the following: W8, W9, A42, H66, K312, V347, H348, said mutations including amino acid modifications, substitutions or deletions.

(b)是与(a)的序列具有至少70％序列相同性并具有(a)所述突变并保留对D-葡萄糖结合能力的序列。(b) is a sequence that has at least 70% sequence identity with the sequence of (a) and has the mutation described in (a) and retains the ability to bind to D-glucose.

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：In one or more embodiments, the mutation includes a mutation at a site selected from any of the following groups:

(1)V347、H348，(2)V347、H348和K312，(3)V347、H348和H66，(4)V347、H348和W9，(5)V347、H348和E13，(6)V347、H348和W8，(7)V347、H348和A42。(1) V347, H348, (2) V347, H348 and K312, (3) V347, H348 and H66, (4) V347, H348 and W9, (5) V347, H348 and E13, (6) V347, H348 and W8, (7) V347, H348 and A42.

在一个或多个实施方案中，W8突变为R、H、E、A、V、L、F、I、M、C、N、G、K、D或T；在一个或多个实施方案中，W9突变为R、H、E、A、V、L、F、I、M、C、P、D、N、G、K、Y、S或T；在一个或多个实施方案中，A42突变为R、E、W、F、M、C、P、N、G、K、Y或T；在一个或多个实施方案中，H66突变为R、E、A、F、M、C、P、Q或T；在一个或多个实施方案中，K312突变为R、A、Q、G、H或S；在一个或多个实施方案中，V347突变为W；在一个或多个实施方案中，H348突变为T、G或S。In one or more embodiments, W8 mutates to R, H, E, A, V, L, F, I, M, C, N, G, K, D, or T; in one or more embodiments, W9 mutates to R, H, E, A, V, L, F, I, M, C, P, D, N, G, K, Y, S, or T; in one or more embodiments, A42 mutates to R, E, W, F, M, C, P, N, G, K, Y, or T; in one or more embodiments, H66 mutates to R, E, A, F, M, C, P, Q, or T; in one or more embodiments, K312 mutates to R, A, Q, G, H, or S; in one or more embodiments, V347 mutates to W; in one or more embodiments, H348 mutates to T, G, or S.

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)V347W和H348T，(2)V347W和H348G，(3)V347W和H348S，(4)V347W、H348T和H66R，(5)V347W、H348T和H66E，(6)V347W、H348T和H66A，(7)V347W、H348T和H66F，(8)V347W、H348T和H66M，(9)V347W、H348T和H66C，(10)V347W、H348T和H66P，(11)V347W、H348T和H66Q，(12)V347W、H348T和H66T，(13)V347W、H348T和W9R，(14)V347W、H348T和W9H，(15)V347W、H348T和W9E，(16)V347W、H348T和W9A，(17)V347W、H348T和W9V，(18)V347W、H348T和W9L，(19)V347W、H348T和W9F，(20)V347W、H348T和W9I，(21)V347W、H348T和W9M，(22)V347W、H348T和W9C，(23)V347W、H348T和W9P，(24)V347W、H348T和W9N，(25)V347W、H348T和W9G，(26)V347W、H348T和W9K，(27)V347W、H348T和W9Y，(28)V347W、H348T和W9S，(29)V347W、H348T和W9T，(30)V347W、H348T和W9D，(31)V347W、H348T和W8R，(32)V347W、H348T和W8H，(33)V347W、H348T和W8E，(34)V347W、H348T和W8A，(35)V347W、H348T和W8V，(36)V347W、H348T和W8L，(37)V347W、H348T和W8F，(38)V347W、H348T和W8I，(39)V347W、H348T和W8M，(40)V347W、H348T和W8C，(41)V347W、H348T和W8N，(42)V347W、H348T和W8G，(43)V347W、H348T和W8K，(44)V347W、H348T和W8T，(45)V347W、H348T和W8D，(46)V347W、H348T和A42R，(47)V347W、H348T和A42E，(48)V347W、H348T和A42W，(49)V347W、H348T和A42F，(50)V347W、H348T和A42M，(51)V347W、H348T和A42C，(52)V347W、H348T和A42P，(53)V347W、H348T和A42N，(54)V347W、H348T和A42G，(55)V347W、H348T和A42K，(56)V347W、H348T和A42Y，(57)V347W、H348T和A42T，(58)V347W、H348T和K312R，(59)V347W、H348T和K312A，(60)V347W、H348T和K312Q，(61)V347W、H348T和K312G，(62)V347W、H348T和K312H，(63)V347W、H348T和K312S。In one or more embodiments, the mutation comprises a mutation selected from any of the following groups: (1) V347W and H348T, (2) V347W and H348G, (3) V347W and H348S, (4) V347W, H348T and H66R, (5) V347W, H348T and H66E, (6) V347W, H348T and H66A, (7) V347W, H348T and H66F, (8) V347W (9) V347W, H348T and H66C, (10) V347W, H348T and H66P, (11) V347W, H348T and H66Q, (12) V347W, H348T and H66T, (13) V347W, H348T and W9R, (14) V347W, H348T and W9H, (15) V347W, H348T and W9E, (16) V347 W, H348T and W9A, (17) V347W, H348T and W9V, (18) V347W, H348T and W9L, (19) V347W, H348T and W9F, (20) V347W, H348T and W9I, (21) V347W, H348T and W9M, (22) V347W, H348T and W9C, (23) V347W, H348T and W9P, (24) V347W, H348T and W9N, (25) V347W, H348T and W9G, (26) V347W, H348T and W9K, (27) V347W, H348T and W9Y, (28) V347W, H348T and W9S, (29) V347W, H348T and W9T, (30) V347W, H348T and W9D, (31) V347W, H348T and W8R, (32) V347W, H34 8T and W8H, (33)V347W, H348T and W8E, (34)V347W, H348T and W8A, (35)V347W, H348T and W8V, (36)V347W, H348T and W8L, (37)V347W, H348T and W8F, (38)V347W, H348T and W8I, (39)V347W, H348T and W8M, (40)V347W, H348T And W8C, (41)V347W, H348T and W8N, (42)V347W, H348T and W8G, (43)V347W, H348T and W8K, (44)V347W, H348T and W8T, (45)V347W, H348T and W8D, (46)V347W, H348T and A42R, (47)V347W, H348T and A42E, (48)V347W, H348T and A42W, (49) V347W, H348T and A42F, (50) V347W, H348T and A42M, (51) V347W, H348T and A42C, (52) V347W, H348T and A42P, (53) V347W, H348T and A42N, (54) V347W, H348T and A42G, (55) V347W, H348T and A42K, (56) V347W, H 348T and A42Y, (57)V347W, H348T and A42T, (58)V347W, H348T and K312R, (59)V347W, H348T and K312A, (60)V347W, H348T and K312Q, (61)V347W, H348T and K312G, (62)V347W, H348T and K312H, (63)V347W, H348T and K312S.

本发明另一方面提供一种D-葡萄糖光学探针，包含D-葡萄糖敏感多肽和光学活性多肽，其中光学活性多肽位于D-葡萄糖敏感多肽的序列内。D-葡萄糖敏感多肽被光学活性多肽分为第一部分和第二部分。Another aspect of the present invention provides a D-glucose optical probe comprising a D-glucose-sensitive polypeptide and an optically active polypeptide, wherein the optically active polypeptide is located within the sequence of the D-glucose-sensitive polypeptide. The D-glucose-sensitive polypeptide is divided into a first part and a second part by the optically active polypeptide.

在一个或多个实施方案中，所述D-葡萄糖光学探针，包括D-葡萄糖敏感多肽B和光学活性多肽A，其中光学活性多肽A位于D-葡萄糖敏感多肽B的序列内，将D-葡萄糖敏感多肽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。In one or more embodiments, the D-glucose optical probe includes a D-glucose-sensitive polypeptide B and an optically active polypeptide A, wherein the optically active polypeptide A is located within the sequence of the D-glucose-sensitive polypeptide B, and the D-glucose-sensitive polypeptide B is divided into a first part B1 and a second part B2 to form a probe structure of type B1-A-B2.

在一个或多个实施方案中，光学活性多肽位于D-葡萄糖敏感多肽的残基117-123、266-288和/或340-353之间，编号对应于D-葡萄糖敏感多肽的全长。优选地，光学活性多肽位于D-葡萄糖敏感多肽的选自下述位点中的任一个或多个：266/267、266/268、266/269、266/270、266/271、266/272、266/273、266/274、266/275、266/276、266/277、266/278、266/279、266/280、266/281、266/282、266/283、266/284、266/285、266/286、266/287、266/288、267/267、267/268、267/269、267/270、267/271、267/272、267/273、267/274、267/275、267/276、267/277、267/278、267/279、267/280、267/281、267/282、267/283、267/284、267/285、267/286、267/287、267/288、268/267、268/268、268/269、268/270、268/271、268/272、268/273、268/274、268/275、268/276、268/277、268/278、268/279、268/280、268/281、268/282、268/283、268/284、268/285、268/286、268/287、268/288、269/267、269/268、269/269、269/270、269/271、269/272、269/273、269/274、269/275、269/276、269/277、269/278、269/279、269/280、269/281、269/282、269/283、269/284、269/285、269/286、269/287、269/288、270/267、270/268、270/269、270/270、270/271、270/272、270/273、270/274、270/275、270/276、270/277、270/278、270/279、270/280、270/281、270/282、270/283、270/284、270/285、270/286、270/287、270/288、271/267、271/268、271/269、271/270、271/271、271/272、271/273、271/274、271/275、271/276、271/277、271/278、271/279、271/280、271/281、271/282、271/283、271/284、271/285、271/286、271/287、271/288、272/267、272/268、272/269、272/270、272/271、272/272、272/273、272/274、272/275、272/276、272/277、272/278、272/279、272/280、272/281、272/282、272/283、272/284、272/285、272/286、272/287、272/288、273/267、273/268、273/269、273/270、273/271、273/272、273/273、273/274、273/275、273/276、273/277、273/278、273/279、273/280、273/281、273/282、273/283、273/284、273/285、273/286、273/287、273/288、274/267、274/268、274/269、274/270、274/271、274/272、274/273、274/274、274/275、274/276、274/277、274/278、274/279、274/280、274/281、274/282、274/283、274/284、274/285、274/286、274/287、274/288、275/267、275/268、275/269、275/270、275/271、275/272、275/273、275/274、275/275、275/276、275/277、275/278、275/279、275/280、275/281、275/282、275/283、275/284、275/285、275/286、275/287、275/288、276/267、276/268、276/269、276/270、276/271、276/272、276/273、276/274、276/275、276/276、276/277、276/278、276/279、276/280、276/281、276/282、276/283、276/284、276/285、276/286、276/287、276/288、277/267、277/268、277/269、277/270、277/271、277/272、277/273、277/274、277/275、277/276、277/277、277/278、277/279、277/280、277/281、277/282、277/283、277/284、277/285、277/286、277/287、277/288、278/267、278/268、278/269、278/270、278/271、278/272、278/273、278/274、278/275、278/276、278/277、278/278、278/279、278/280、278/281、278/282、278/283、278/284、278/285、278/286、278/287、278/288、279/267、279/268、279/269、279/270、279/271、279/272、279/273、279/274、279/275、279/276、279/277、279/278、279/279、279/280、279/281、279/282、279/283、279/284、279/285、279/286、279/287、279/288、280/267、280/268、280/269、280/270、280/271、280/272、280/273、280/274、280/275、280/276、280/277、280/278、280/279、280/280、280/281、280/282、280/283、280/284、280/285、280/286、280/287、280/288、281/267、281/268、281/269、281/270、281/271、281/272、281/273、281/274、281/275、281/276、281/277、281/278、281/279、281/280、281/281、281/282、281/283、281/284、281/285、281/286、281/287、281/288、282/267、282/268、282/269、282/270、282/271、282/272、282/273、282/274、282/275、282/276、282/277、282/278、282/279、282/280、282/281、282/282、282/283、282/284、282/285、282/286、282/287、282/288、283/267、283/268、283/269、283/270、283/271、283/272、283/273、283/274、283/275、283/276、283/277、283/278、283/279、283/280、283/281、283/282、283/283、283/284、283/285、283/286、283/287、283/288、284/267、284/268、284/269、284/270、284/271、284/272、284/273、284/274、284/275、284/276、284/277、284/278、284/279、284/280、284/281、284/282、284/283、284/284、284/285、284/286、284/287、284/288、285/267、285/268、285/269、285/270、285/271、285/272、285/273、285/274、285/275、285/276、285/277、285/278、285/279、285/280、285/281、285/282、285/283、285/284、285/285、285/286、285/287、285/288、286/267、286/268、286/269、286/270、286/271、286/272、286/273、286/274、286/275、286/276、286/277、286/278、286/279、286/280、286/281、286/282、286/283、286/284、286/285、286/286、286/287、286/288、287/267、287/268、287/269、287/270、287/271、287/272、287/273、287/274、287/275、287/276、287/277、287/278、287/279、287/280、287/281、287/282、287/283、287/284、287/285、287/286、287/287、287/288，117/118、117/119、117/120、117/121、117/122、117/123、118/118、118/119、118/120、118/121、118/122、118/123、119/118、119/119、119/120、119/121、119/122、119/123、120/118、120/119、120/120、120/121、120/122、120/123、121/118、121/119、121/120、121/121、121/122、121/123、122/118、122/119、122/120、122/121、122/122、122/123，340/341、340/342、340/343、340/344、340/345、340/346、340/347、340/348、340/349、340/350、340/351、340/352、340/353、341/341、341/342、341/343、341/344、341/345、341/346、341/347、341/348、341/349、341/350、341/351、341/352、341/353、342/341、342/342、342/343、342/344、342/345、342/346、342/347、342/348、342/349、342/350、342/351、342/352、342/353、343/341、343/342、343/343、343/344、343/345、343/346、343/347、343/348、343/349、343/350、343/351、343/352、343/353、344/341、344/342、344/343、344/344、344/345、344/346、344/347、344/348、344/349、344/350、344/351、344/352、344/353、345/341、345/342、345/343、345/344、345/345、345/346、345/347、345/348、345/349、345/350、345/351、345/352、345/353、346/341、346/342、346/343、346/344、346/345、346/346、346/347、346/348、346/349、346/350、346/351、346/352、346/353、347/341、347/342、347/343、347/344、347/345、347/346、347/347、347/348、347/349、347/350、347/351、347/352、347/353、348/341、348/342、348/343、348/344、348/345、348/346、348/347、348/348、348/349、348/350、348/351、348/352、348/353、349/341、349/342、349/343、349/344、349/345、349/346、349/347、349/348、349/349、349/350、349/351、349/352、349/353、350/341、350/342、350/343、350/344、350/345、350/346、350/347、350/348、350/349、350/350、350/351、350/352、350/353、351/341、351/342、351/343、351/344、351/345、351/346、351/347、351/348、351/349、351/350、351/351、351/352、351/353、352/341、352/342、352/343、352/344、352/345、352/346、352/347、352/348、352/349、352/350、352/351、352/352和352/353。In one or more embodiments, the optically active polypeptide is located between residues 117-123, 266-288, and/or 340-353 of the D-glucose-sensitive polypeptide, the numbering corresponding to the full length of the D-glucose-sensitive polypeptide. Preferably, the optically active polypeptide is located at any one or more sites selected from the following groups of the D-glucose-sensitive polypeptide: 266/267, 266/268, 266/269, 266/270, 266/271, 266/272, 266/273, 266/274, 266/275, 266/276, 266/277, 266/278, 266/279, 266/280, 266/281, 266/282, 266/283. 266/284, 266/285, 266/286, 266/287, 266/288, 267/267, 267/268, 267/269, 267/270, 267/271, 267/272, 267/273, 267/274, 267/275, 267/276, 267/277, 267/278, 267/279, 267/280, 267/281, 267/282, 267/2 83, 267/284, 267/285, 267/286, 267/287, 267/288, 268/267, 268/268, 268/269, 268/270, 268/271, 268/272, 268/273, 268/274, 268/275, 268/276, 268/277, 268/278, 268/279, 268/280, 268/281, 268/282, 26 8/283, 268/284, 268/285, 268/286, 268/287, 268/288, 269/267, 269/268, 269/269, 269/270, 269/271, 269/272, 269/273, 269/274, 269/275, 269/276, 269/277, 269/278, 269/279, 269/280, 269/281, 269/282, 269/283, 269/284, 269/285, 269/286, 269/287, 269/288, 270/267, 270/268, 270/269, 270/270, 270/271, 270/272, 270/273, 270/274, 270/275, 270/276, 270/277, 270/278, 270/279, 270/280, 270/281, 270/2 82, 270/283, 270/284, 270/285, 270/286, 270/287, 270/288, 271/267, 271/268, 271/269, 271/270, 271/271, 271/272, 271/273, 271/274, 271/275, 271/276, 271/277, 271/278, 271/279, 271/280, 271/281, 271 /282, 271/283, 271/284, 271/285, 271/286, 271/287, 271/288, 272/267, 272/268, 272/269, 272/270, 272/271, 272/272, 272/273, 272/274, 272/275, 272/276, 272/277, 272/278, 272/279, 272/280, 272/281 272/282, 272/283, 272/284, 272/285, 272/286, 272/287, 272/288, 273/267, 273/268, 273/269, 273/270, 273/271, 273/272, 273/273, 273/274, 273/275, 273/276, 273/277, 273/278, 273/279, 273/280, 273/28 1, 273/282, 273/283, 273/284, 273/285, 273/286, 273/287, 273/288, 274/267, 274/268, 274/269, 274/270, 274/271, 274/272, 274/273, 274/274, 274/275, 274/276, 274/277, 274/278, 274/279, 274/280, 274 /281, 274/282, 274/283, 274/284, 274/285, 274/286, 274/287, 274/288, 275/267, 275/268, 275/269, 275/270, 275/271, 275/272, 275/273, 275/274, 275/275, 275/276, 275/277, 275/278, 275/279, 275/280, 275/281, 275/282, 275/283, 275/284, 275/285, 275/286, 275/287, 275/288, 276/267, 276/268, 276/269, 276/270, 276/271, 276/272, 276/273, 276/274, 276/275, 276/276, 276/277, 276/278, 276/279, 276/2 80, 276/281, 276/282, 276/283, 276/284, 276/285, 276/286, 276/287, 276/288, 277/267, 277/268, 277/269, 277/270, 277/271, 277/272, 277/273, 277/274, 277/275, 277/276, 277/277, 277/278, 277/279, 277 /280, 277/281, 277/282, 277/283, 277/284, 277/285, 277/286, 277/287, 277/288, 278/267, 278/268, 278/269, 278/270, 278/271, 278/272, 278/273, 278/274, 278/275, 278/276, 278/277, 278/278, 278/279 278/280, 278/281, 278/282, 278/283, 278/284, 278/285, 278/286, 278/287, 278/288, 279/267, 279/268, 279/269, 279/270, 279/271, 279/272, 279/273, 279/274, 279/275, 279/276, 279/277, 279/278, 279/27 9, 279/280, 279/281, 279/282, 279/283, 279/284, 279/285, 279/286, 279/287, 279/288, 280/267, 280/268, 280/269, 280/270, 280/271, 280/272, 280/273, 280/274, 280/275, 280/276, 280/277, 280/278, 280 /279, 280/280, 280/281, 280/282, 280/283, 280/284, 280/285, 280/286, 280/287, 280/288, 281/267, 281/268, 281/269, 281/270, 281/271, 281/272, 281/273, 281/274, 281/275, 281/276, 281/277, 281/278, 2 81/279, 281/280, 281/281, 281/282, 281/283, 281/284, 281/285, 281/286, 281/287, 281/288, 282/267, 282/268, 282/269, 282/270, 282/271, 282/272, 282/273, 282/274, 282/275, 282/276, 282/277, 282/27 8, 282/279, 282/280, 282/281, 282/282, 282/283, 282/284, 282/285, 282/286, 282/287, 282/288, 283/267, 283/268, 283/269, 283/270, 283/271, 283/272, 283/273, 283/274, 283/275, 283/276, 283/277, 283 /278, 283/279, 283/280, 283/281, 283/282, 283/283, 283/284, 283/285, 283/286, 283/287, 283/288, 284/267, 284/268, 284/269, 284/270, 284/271, 284/272, 284/273, 284/274, 284/275, 284/276, 284/277 284/278, 284/279, 284/280, 284/281, 284/282, 284/283, 284/284, 284/285, 284/286, 284/287, 284/288, 285/267, 285/268, 285/269, 285/270, 285/271, 285/272, 285/273, 285/274, 285/275, 285/276, 285/27 7, 285/278, 285/279, 285/280, 285/281, 285/282, 285/283, 285/284, 285/285, 285/286, 285/287, 285/288, 286/267, 286/268, 286/269, 286/270, 286/271, 286/272, 286/273, 286/274, 286/275, 286/276, 286 /277, 286/278, 286/279, 286/280, 286/281, 286/282, 286/283, 286/284, 286/285, 286/286, 286/287, 286/288, 287/267, 287/268, 287/269, 287/270, 287/271, 287/272, 287/273, 287/274, 287/275, 287/276, 2 87/277, 287/278, 287/279, 287/280, 287/281, 287/282, 287/283, 287/284, 287/285, 287/286, 287/287, 287/288, 117/118, 117/119, 117/120, 117/121, 117/122, 117/123, 118/118, 118/119, 118/120, 118/12 1, 118/122, 118/123, 119/118, 119/119, 119/120, 119/121, 119/122, 119/123, 120/118, 120/119, 120/120, 120/121, 120/122, 120/123, 121/118, 121/119, 121/120, 121/121, 121/122, 121/123, 122/118, 122/ 119, 122/120, 122/121, 122/122, 122/123, 340/341, 340/342, 340/343, 340/344, 340/345, 340/346, 340/347, 340/348, 340/349, 340/350, 340/351, 340/352, 340/353, 341/341, 341/342, 341/343, 341/344, 3 41/345, 341/346, 341/347, 341/348, 341/349, 341/350, 341/351, 341/352, 341/353, 342/341, 342/342, 342/343, 342/344, 342/345, 342/346, 342/347, 342/348, 342/349, 342/350, 342/351, 342/352, 342/353 343/341, 343/342, 343/343, 343/344, 343/345, 343/346, 343/347, 343/348, 343/349, 343/350, 343/351, 343/352, 343/353, 344/341, 344/342, 344/343, 344/344, 344/345, 344/346, 344/347, 344/348, 344/ 349, 344/350, 344/351, 344/352, 344/353, 345/341, 345/342, 345/343, 345/344, 345/345, 345/346, 345/347, 345/348, 345/349, 345/350, 345/351, 345/352, 345/353, 346/341, 346/342, 346/343, 346/344, 34 6/345, 346/346, 346/347, 346/348, 346/349, 346/350, 346/351, 346/352, 346/353, 347/341, 347/342, 347/343, 347/344, 347/345, 347/346, 347/347, 347/348, 347/349, 347/350, 347/351, 347/352, 347/353 348/341, 348/342, 348/343, 348/344, 348/345, 348/346, 348/347, 348/348, 348/349, 348/350, 348/351, 348/352, 348/353, 349/341, 349/342, 349/343, 349/344, 349/345, 349/346, 349/347, 349/348, 349/3 49, 349/350, 349/351, 349/352, 349/353, 350/341, 350/342, 350/343, 350/344, 350/345, 350/346, 350/347, 350/348, 350/349, 350/350, 350/351, 350/352, 350/353, 351/341, 351/342, 351/343, 351/344, 35 1/345, 351/346, 351/347, 351/348, 351/349, 351/350, 351/351, 351/352, 351/353, 352/341, 352/342, 352/343, 352/344, 352/345, 352/346, 352/347, 352/348, 352/349, 352/350, 352/351, 352/352 and 352/353.

在一个或多个实施方案中，所述D-葡萄糖敏感多肽是D-葡萄糖结合蛋白或其功能变体。In one or more embodiments, the D-glucose-sensitive polypeptide is a D-glucose-binding protein or a functional variant thereof.

在一个或多个实施方案中，D-葡萄糖敏感多肽具有：In one or more embodiments, the D-glucose-sensitive polypeptide has:

(1)SEQ ID NO：1所示的序列，或与它们有至少70％序列相同性并保留对D-葡萄糖结合活性的序列，(1) The sequence shown in SEQ ID NO: 1, or a sequence that has at least 70% sequence identity with them and retains D-glucose binding activity,

(2)本文第一方面任一实施方案所述的D-葡萄糖结合蛋白变体的序列，或(2) The sequence of the D-glucose-binding protein variant described in any embodiment of the first aspect of this document, or

(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D-葡萄糖敏感性的序列。(3) has at least 70% sequence identity with the sequence described in (2) and has the mutation described in (2) and retains the sequence sensitive to D-glucose.

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。In one or more embodiments, 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 linker to the optically active polypeptide.

在一个实施方式中，荧光蛋白选自黄色荧光蛋白、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。优选地，所述荧光蛋白具有SEQ ID NO：2、6、7、9中任一所示的序列。In one embodiment, the fluorescent protein is selected from yellow fluorescent protein, green fluorescent protein, blue fluorescent protein, and apple red fluorescent protein. In one embodiment, the fluorescent protein has the sequence shown in any of SEQ ID NO: 2-9. Preferably, the fluorescent protein has the sequence shown in any of SEQ ID NO: 2, 6, 7, and 9.

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A或G的突变。In one or more embodiments, the functional variant of the fluorescent protein has a mutation at amino acid positions 1-3, preferably position 1. Preferably, the functional variant of the fluorescent protein includes a mutation in the amino acid position 1 of the fluorescent protein to A or G.

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Y1A或Y1G。In one or more embodiments, a functional variant of the fluorescent protein has the sequence shown in SEQ ID NO: 2 and a mutation at the Y1 site. Preferably, the mutation is Y1A or Y1G.

在一个或多个实施方案中，荧光蛋白具有SEQ ID NO：2所示的序列或是其第1位氨基酸处具有以下任一组所示突变的变体Y1A或Y1G，光学活性多肽位于D-葡萄糖敏感多肽的348/352位点。In one or more embodiments, the fluorescent protein has the sequence shown in SEQ ID NO: 2 or a variant Y1A or Y1G with any of the following mutations at its first amino acid position, and the optically active polypeptide is located at position 348/352 of the D-glucose-sensitive polypeptide.

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：D-葡萄糖敏感多肽的第一部分B1、Y、光学活性多肽A、D-葡萄糖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。In one embodiment, the optical probe further comprises one or more linkers flanking the optically active polypeptide. The linkers of this invention can be any amino acid sequence of any length. In one embodiment, the optically active polypeptide flanking the linker comprises a linker of no more than 5 amino acids, for example, linkers of 0, 1, 2, 3, or 4 amino acids. In one embodiment, the linker flanking the optically active polypeptide comprises amino acid Y. In one embodiment, linker Y is located at the N-terminus and/or C-terminus of the optically active polypeptide. In one embodiment, the optical probe is as follows: a first portion B1 of a D-glucose-sensitive polypeptide, Y, an optically active polypeptide A, and a second portion B2 of a D-glucose-sensitive polypeptide. In one embodiment, the optical probe of this invention does not comprise a linker.

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。In one embodiment, the optical probe of the present invention further includes a positioning sequence for positioning the probe to a specific organelle, such as a cell.

在一个或多个实施方案中，所述D-葡萄糖敏感多肽的序列如SEQ ID NO：1所示，所述光学活性多肽如SEQ ID NO：2、6、7、9中任一所示，所述光学活性多肽位于D-葡萄糖敏感多肽的选自下述位点中的任一个或多个：266/267、266/268、266/269、266/270、266/271、266/272、266/273、266/274、266/275、266/276、266/277、266/278、266/279、266/280、266/281、266/282、266/283、266/284、266/285、266/286、266/287、266/288、267/267、267/268、267/269、267/270、267/271、267/272、267/273、267/274、267/275、267/276、267/277、267/278、267/279、267/280、267/281、267/282、267/283、267/284、267/285、267/286、267/287、267/288、268/267、268/268、268/269、268/270、268/271、268/272、268/273、268/274、268/275、268/276、268/277、268/278、268/279、268/280、268/281、268/282、268/283、268/284、268/285、268/286、268/287、268/288、269/267、269/268、269/269、269/270、269/271、269/272、269/273、269/274、269/275、269/276、269/277、269/278、269/279、269/280、269/281、269/282、269/283、269/284、269/285、269/286、269/287、269/288、270/267、270/268、270/269、270/270、270/271、270/272、270/273、270/274、270/275、270/276、270/277、270/278、270/279、270/280、270/281、270/282、270/283、270/284、270/285、270/286、270/287、270/288、271/267、271/268、271/269、271/270、271/271、271/272、271/273、271/274、271/275、271/276、271/277、271/278、271/279、271/280、271/281、271/282、271/283、271/284、271/285、271/286、271/287、271/288、272/267、272/268、272/269、272/270、272/271、272/272、272/273、272/274、272/275、272/276、272/277、272/278、272/279、272/280、272/281、272/282、272/283、272/284、272/285、272/286、272/287、272/288、273/267、273/268、273/269、273/270、273/271、273/272、273/273、273/274、273/275、273/276、273/277、273/278、273/279、273/280、273/281、273/282、273/283、273/284、273/285、273/286、273/287、273/288、274/267、274/268、274/269、274/270、274/271、274/272、274/273、274/274、274/275、274/276、274/277、274/278、274/279、274/280、274/281、274/282、274/283、274/284、274/285、274/286、274/287、274/288、275/267、275/268、275/269、275/270、275/271、275/272、275/273、275/274、275/275、275/276、275/277、275/278、275/279、275/280、275/281、275/282、275/283、275/284、275/285、275/286、275/287、275/288、276/267、276/268、276/269、276/270、276/271、276/272、276/273、276/274、276/275、276/276、276/277、276/278、276/279、276/280、276/281、276/282、276/283、276/284、276/285、276/286、276/287、276/288、277/267、277/268、277/269、277/270、277/271、277/272、277/273、277/274、277/275、277/276、277/277、277/278、277/279、277/280、277/281、277/282、277/283、277/284、277/285、277/286、277/287、277/288、278/267、278/268、278/269、278/270、278/271、278/272、278/273、278/274、278/275、278/276、278/277、278/278、278/279、278/280、278/281、278/282、278/283、278/284、278/285、278/286、278/287、278/288、279/267、279/268、279/269、279/270、279/271、279/272、279/273、279/274、279/275、279/276、279/277、279/278、279/279、279/280、279/281、279/282、279/283、279/284、279/285、279/286、279/287、279/288、280/267、280/268、280/269、280/270、280/271、280/272、280/273、280/274、280/275、280/276、280/277、280/278、280/279、280/280、280/281、280/282、280/283、280/284、280/285、280/286、280/287、280/288、281/267、281/268、281/269、281/270、281/271、281/272、281/273、281/274、281/275、281/276、281/277、281/278、281/279、281/280、281/281、281/282、281/283、281/284、281/285、281/286、281/287、281/288、282/267、282/268、282/269、282/270、282/271、282/272、282/273、282/274、282/275、282/276、282/277、282/278、282/279、282/280、282/281、282/282、282/283、282/284、282/285、282/286、282/287、282/288、283/267、283/268、283/269、283/270、283/271、283/272、283/273、283/274、283/275、283/276、283/277、283/278、283/279、283/280、283/281、283/282、283/283、283/284、283/285、283/286、283/287、283/288、284/267、284/268、284/269、284/270、284/271、284/272、284/273、284/274、284/275、284/276、284/277、284/278、284/279、284/280、284/281、284/282、284/283、284/284、284/285、284/286、284/287、284/288、285/267、285/268、285/269、285/270、285/271、285/272、285/273、285/274、285/275、285/276、285/277、285/278、285/279、285/280、285/281、285/282、285/283、285/284、285/285、285/286、285/287、285/288、286/267、286/268、286/269、286/270、286/271、286/272、286/273、286/274、286/275、286/276、286/277、286/278、286/279、286/280、286/281、286/282、286/283、286/284、286/285、286/286、286/287、286/288、287/267、287/268、287/269、287/270、287/271、287/272、287/273、287/274、287/275、287/276、287/277、287/278、287/279、287/280、287/281、287/282、287/283、287/284、287/285、287/286、287/287、287/288，117/118、117/119、117/120、117/121、117/122、117/123、118/118、118/119、118/120、118/121、118/122、118/123、119/118、119/119、119/120、119/121、119/122、119/123、120/118、120/119、120/120、120/121、120/122、120/123、121/118、121/119、121/120、121/121、121/122、121/123、122/118、122/119、122/120、122/121、122/122、122/123，340/341、340/342、340/343、340/344、340/345、340/346、340/347、340/348、340/349、340/350、340/351、340/352、340/353、341/341、341/342、341/343、341/344、341/345、341/346、341/347、341/348、341/349、341/350、341/351、341/352、341/353、342/341、342/342、342/343、342/344、342/345、342/346、342/347、342/348、342/349、342/350、342/351、342/352、342/353、343/341、343/342、343/343、343/344、343/345、343/346、343/347、343/348、343/349、343/350、343/351、343/352、343/353、344/341、344/342、344/343、344/344、344/345、344/346、344/347、344/348、344/349、344/350、344/351、344/352、344/353、345/341、345/342、345/343、345/344、345/345、345/346、345/347、345/348、345/349、345/350、345/351、345/352、345/353、346/341、346/342、346/343、346/344、346/345、346/346、346/347、346/348、346/349、346/350、346/351、346/352、346/353、347/341、347/342、347/343、347/344、347/345、347/346、347/347、347/348、347/349、347/350、347/351、347/352、347/353、348/341、348/342、348/343、348/344、348/345、348/346、348/347、348/348、348/349、348/350、348/351、348/352、348/353、349/341、349/342、349/343、349/344、349/345、349/346、349/347、349/348、349/349、349/350、349/351、349/352、349/353、350/341、350/342、350/343、350/344、350/345、350/346、350/347、350/348、350/349、350/350、350/351、350/352、350/353、351/341、351/342、351/343、351/344、351/345、351/346、351/347、351/348、351/349、351/350、351/351、351/352、351/353、352/341、352/342、352/343、352/344、352/345、352/346、352/347、352/348、352/349、352/350、352/351、352/352和352/353。In one or more embodiments, the sequence of the D-glucose-sensitive polypeptide is as shown in SEQ ID NO: 1, and the optically active polypeptide is as shown in any one of SEQ ID NO: 2, 6, 7, and 9, wherein the optically active polypeptide is located at any one or more sites of the D-glucose-sensitive polypeptide selected from the following: 266/267, 266/268, 266/269, 266/270, 266/271, 266/272, 266/273, 266/274, 266... /275, 266/276, 266/277, 266/278, 266/279, 266/280, 266/281, 266/282, 266/283, 266/284, 266/285, 266/286, 266/287, 266/288, 267/267, 267/268, 267/269, 267/270, 267/271, 267/272, 267/273, 267/274, 267 /275, 267/276, 267/277, 267/278, 267/279, 267/280, 267/281, 267/282, 267/283, 267/284, 267/285, 267/286, 267/287, 267/288, 268/267, 268/268, 268/269, 268/270, 268/271, 268/272, 268/273, 268/274, 26 8/275, 268/276, 268/277, 268/278, 268/279, 268/280, 268/281, 268/282, 268/283, 268/284, 268/285, 268/286, 268/287, 268/288, 269/267, 269/268, 269/269, 269/270, 269/271, 269/272, 269/273, 269/274, 26 9/275, 269/276, 269/277, 269/278, 269/279, 269/280, 269/281, 269/282, 269/283, 269/284, 269/285, 269/286, 269/287, 269/288, 270/267, 270/268, 270/269, 270/270, 270/271, 270/272, 270/273, 270/274, 2 70/275, 270/276, 270/277, 270/278, 270/279, 270/280, 270/281, 270/282, 270/283, 270/284, 270/285, 270/286, 270/287, 270/288, 271/267, 271/268, 271/269, 271/270, 271/271, 271/272, 271/273, 271/274, 2 71/275, 271/276, 271/277, 271/278, 271/279, 271/280, 271/281, 271/282, 271/283, 271/284, 271/285, 271/286, 271/287, 271/288, 272/267, 272/268, 272/269, 272/270, 272/271, 272/272, 272/273, 272/274, 2 72/275, 272/276, 272/277, 272/278, 272/279, 272/280, 272/281, 272/282, 272/283, 272/284, 272/285, 272/286, 272/287, 272/288, 273/267, 273/268, 273/269, 273/270, 273/271, 273/272, 273/273, 273/274, 2 73/275, 273/276, 273/277, 273/278, 273/279, 273/280, 273/281, 273/282, 273/283, 273/284, 273/285, 273/286, 273/287, 273/288, 274/267, 274/268, 274/269, 274/270, 274/271, 274/272, 274/273, 274/274 274/275, 274/276, 274/277, 274/278, 274/279, 274/280, 274/281, 274/282, 274/283, 274/284, 274/285, 274/286, 274/287, 274/288, 275/267, 275/268, 275/269, 275/270, 275/271, 275/272, 275/273, 275/274 275/275, 275/276, 275/277, 275/278, 275/279, 275/280, 275/281, 275/282, 275/283, 275/284, 275/285, 275/286, 275/287, 275/288, 276/267, 276/268, 276/269, 276/270, 276/271, 276/272, 276/273, 276/274 276/275, 276/276, 276/277, 276/278, 276/279, 276/280, 276/281, 276/282, 276/283, 276/284, 276/285, 276/286, 276/287, 276/288, 277/267, 277/268, 277/269, 277/270, 277/271, 277/272, 277/273, 277/274 277/275, 277/276, 277/277, 277/278, 277/279, 277/280, 277/281, 277/282, 277/283, 277/284, 277/285, 277/286, 277/287, 277/288, 278/267, 278/268, 278/269, 278/270, 278/271, 278/272, 278/273, 278/27 4, 278/275, 278/276, 278/277, 278/278, 278/279, 278/280, 278/281, 278/282, 278/283, 278/284, 278/285, 278/286, 278/287, 278/288, 279/267, 279/268, 279/269, 279/270, 279/271, 279/272, 279/273, 279/27 4, 279/275, 279/276, 279/277, 279/278, 279/279, 279/280, 279/281, 279/282, 279/283, 279/284, 279/285, 279/286, 279/287, 279/288, 280/267, 280/268, 280/269, 280/270, 280/271, 280/272, 280/273, 280/27 4, 280/275, 280/276, 280/277, 280/278, 280/279, 280/280, 280/281, 280/282, 280/283, 280/284, 280/285, 280/286, 280/287, 280/288, 281/267, 281/268, 281/269, 281/270, 281/271, 281/272, 281/273, 281/27 4, 281/275, 281/276, 281/277, 281/278, 281/279, 281/280, 281/281, 281/282, 281/283, 281/284, 281/285, 281/286, 281/287, 281/288, 282/267, 282/268, 282/269, 282/270, 282/271, 282/272, 282/273, 282/2 74, 282/275, 282/276, 282/277, 282/278, 282/279, 282/280, 282/281, 282/282, 282/283, 282/284, 282/285, 282/286, 282/287, 282/288, 283/267, 283/268, 283/269, 283/270, 283/271, 283/272, 283/273, 283/2 74, 283/275, 283/276, 283/277, 283/278, 283/279, 283/280, 283/281, 283/282, 283/283, 283/284, 283/285, 283/286, 283/287, 283/288, 284/267, 284/268, 284/269, 284/270, 284/271, 284/272, 284/273, 284/ 274, 284/275, 284/276, 284/277, 284/278, 284/279, 284/280, 284/281, 284/282, 284/283, 284/284, 284/285, 284/286, 284/287, 284/288, 285/267, 285/268, 285/269, 285/270, 285/271, 285/272, 285/273, 285/ 274, 285/275, 285/276, 285/277, 285/278, 285/279, 285/280, 285/281, 285/282, 285/283, 285/284, 285/285, 285/286, 285/287, 285/288, 286/267, 286/268, 286/269, 286/270, 286/271, 286/272, 286/273, 286 /274, 286/275, 286/276, 286/277, 286/278, 286/279, 286/280, 286/281, 286/282, 286/283, 286/284, 286/285, 286/286, 286/287, 286/288, 287/267, 287/268, 287/269, 287/270, 287/271, 287/272, 287/273, 287 /274, 287/275, 287/276, 287/277, 287/278, 287/279, 287/280, 287/281, 287/282, 287/283, 287/284, 287/285, 287/286, 287/287, 287/288, 117/118, 117/119, 117/120, 117/121, 117/122, 117/123, 118/118, 118 /119, 118/120, 118/121, 118/122, 118/123, 119/118, 119/119, 119/120, 119/121, 119/122, 119/123, 120/118, 120/119, 120/120, 120/121, 120/122, 120/123, 121/118, 121/119, 121/120, 121/121, 121/122, 121 /123, 122/118, 122/119, 122/120, 122/121, 122/122, 122/123, 340/341, 340/342, 340/343, 340/344, 340/345, 340/346, 340/347, 340/348, 340/349, 340/350, 340/351, 340/352, 340/353, 341/341, 341/342, 34 1/343, 341/344, 341/345, 341/346, 341/347, 341/348, 341/349, 341/350, 341/351, 341/352, 341/353, 342/341, 342/342, 342/343, 342/344, 342/345, 342/346, 342/347, 342/348, 342/349, 342/350, 342/351, 34 2/352, 342/353, 343/341, 343/342, 343/343, 343/344, 343/345, 343/346, 343/347, 343/348, 343/349, 343/350, 343/351, 343/352, 343/353, 344/341, 344/342, 344/343, 344/344, 344/345, 344/346, 344/347, 3 44/348, 344/349, 344/350, 344/351, 344/352, 344/353, 345/341, 345/342, 345/343, 345/344, 345/345, 345/346, 345/347, 345/348, 345/349, 345/350, 345/351, 345/352, 345/353, 346/341, 346/342, 346/343, 3 46/344, 346/345, 346/346, 346/347, 346/348, 346/349, 346/350, 346/351, 346/352, 346/353, 347/341, 347/342, 347/343, 347/344, 347/345, 347/346, 347/347, 347/348, 347/349, 347/350, 347/351, 347/352, 347/353, 348/341, 348/342, 348/343, 348/344, 348/345, 348/346, 348/347, 348/348, 348/349, 348/350, 348/351, 348/352, 348/353, 349/341, 349/342, 349/343, 349/344, 349/345, 349/346, 349/347, 349/348 349/349, 349/350, 349/351, 349/352, 349/353, 350/341, 350/342, 350/343, 350/344, 350/345, 350/346, 350/347, 350/348, 350/349, 350/350, 350/351, 350/352, 350/353, 351/341, 351/342, 351/343, 351/344, 351/345, 351/346, 351/347, 351/348, 351/349, 351/350, 351/351, 351/352, 351/353, 352/341, 352/342, 352/343, 352/344, 352/345, 352/346, 352/347, 352/348, 352/349, 352/350, 352/351, 352/352 and 352/353.

在一个或多个实施方案中，D-葡萄糖敏感多肽如SEQ ID NO：1所示，光学活性多肽如SEQ ID NO：2、6、7、9中任一所示或是其在对应于SEQ ID NO：2的第1位氨基酸的氨基酸处具有选自以下任一个或多个突变的变体：Y1A或Y1G，光学活性多肽位于D-葡萄糖敏感多肽的残基117-123、266-288和/或340-353之间，编号对应于D-葡萄糖敏感多肽的全长。在一个或多个实施方案中，光学活性多肽位于D-葡萄糖敏感多肽的选自下述位点中的任一个或多个：266/267、266/268、266/269、266/270、266/271、266/272、266/273、266/274、266/275、266/276、266/277、266/278、266/279、266/280、266/281、266/282、266/283、266/284、266/285、266/286、266/287、266/288、267/267、267/268、267/269、267/270、267/271、267/272、267/273、267/274、267/275、267/276、267/277、267/278、267/279、267/280、267/281、267/282、267/283、267/284、267/285、267/286、267/287、267/288、268/267、268/268、268/269、268/270、268/271、268/272、268/273、268/274、268/275、268/276、268/277、268/278、268/279、268/280、268/281、268/282、268/283、268/284、268/285、268/286、268/287、268/288、269/267、269/268、269/269、269/270、269/271、269/272、269/273、269/274、269/275、269/276、269/277、269/278、269/279、269/280、269/281、269/282、269/283、269/284、269/285、269/286、269/287、269/288、270/267、270/268、270/269、270/270、270/271、270/272、270/273、270/274、270/275、270/276、270/277、270/278、270/279、270/280、270/281、270/282、270/283、270/284、270/285、270/286、270/287、270/288、271/267、271/268、271/269、271/270、271/271、271/272、271/273、271/274、271/275、271/276、271/277、271/278、271/279、271/280、271/281、271/282、271/283、271/284、271/285、271/286、271/287、271/288、272/267、272/268、272/269、272/270、272/271、272/272、272/273、272/274、272/275、272/276、272/277、272/278、272/279、272/280、272/281、272/282、272/283、272/284、272/285、272/286、272/287、272/288、273/267、273/268、273/269、273/270、273/271、273/272、273/273、273/274、273/275、273/276、273/277、273/278、273/279、273/280、273/281、273/282、273/283、273/284、273/285、273/286、273/287、273/288、274/267、274/268、274/269、274/270、274/271、274/272、274/273、274/274、274/275、274/276、274/277、274/278、274/279、274/280、274/281、274/282、274/283、274/284、274/285、274/286、274/287、274/288、275/267、275/268、275/269、275/270、275/271、275/272、275/273、275/274、275/275、275/276、275/277、275/278、275/279、275/280、275/281、275/282、275/283、275/284、275/285、275/286、275/287、275/288、276/267、276/268、276/269、276/270、276/271、276/272、276/273、276/274、276/275、276/276、276/277、276/278、276/279、276/280、276/281、276/282、276/283、276/284、276/285、276/286、276/287、276/288、277/267、277/268、277/269、277/270、277/271、277/272、277/273、277/274、277/275、277/276、277/277、277/278、277/279、277/280、277/281、277/282、277/283、277/284、277/285、277/286、277/287、277/288、278/267、278/268、278/269、278/270、278/271、278/272、278/273、278/274、278/275、278/276、278/277、278/278、278/279、278/280、278/281、278/282、278/283、278/284、278/285、278/286、278/287、278/288、279/267、279/268、279/269、279/270、279/271、279/272、279/273、279/274、279/275、279/276、279/277、279/278、279/279、279/280、279/281、279/282、279/283、279/284、279/285、279/286、279/287、279/288、280/267、280/268、280/269、280/270、280/271、280/272、280/273、280/274、280/275、280/276、280/277、280/278、280/279、280/280、280/281、280/282、280/283、280/284、280/285、280/286、280/287、280/288、281/267、281/268、281/269、281/270、281/271、281/272、281/273、281/274、281/275、281/276、281/277、281/278、281/279、281/280、281/281、281/282、281/283、281/284、281/285、281/286、281/287、281/288、282/267、282/268、282/269、282/270、282/271、282/272、282/273、282/274、282/275、282/276、282/277、282/278、282/279、282/280、282/281、282/282、282/283、282/284、282/285、282/286、282/287、282/288、283/267、283/268、283/269、283/270、283/271、283/272、283/273、283/274、283/275、283/276、283/277、283/278、283/279、283/280、283/281、283/282、283/283、283/284、283/285、283/286、283/287、283/288、284/267、284/268、284/269、284/270、284/271、284/272、284/273、284/274、284/275、284/276、284/277、284/278、284/279、284/280、284/281、284/282、284/283、284/284、284/285、284/286、284/287、284/288、285/267、285/268、285/269、285/270、285/271、285/272、285/273、285/274、285/275、285/276、285/277、285/278、285/279、285/280、285/281、285/282、285/283、285/284、285/285、285/286、285/287、285/288、286/267、286/268、286/269、286/270、286/271、286/272、286/273、286/274、286/275、286/276、286/277、286/278、286/279、286/280、286/281、286/282、286/283、286/284、286/285、286/286、286/287、286/288、287/267、287/268、287/269、287/270、287/271、287/272、287/273、287/274、287/275、287/276、287/277、287/278、287/279、287/280、287/281、287/282、287/283、287/284、287/285、287/286、287/287、287/288，117/118、117/119、117/120、117/121、117/122、117/123、118/118、118/119、118/120、118/121、118/122、118/123、119/118、119/119、119/120、119/121、119/122、119/123、120/118、120/119、120/120、120/121、120/122、120/123、121/118、121/119、121/120、121/121、121/122、121/123、122/118、122/119、122/120、122/121、122/122、122/123，340/341、340/342、340/343、340/344、340/345、340/346、340/347、340/348、340/349、340/350、340/351、340/352、340/353、341/341、341/342、341/343、341/344、341/345、341/346、341/347、341/348、341/349、341/350、341/351、341/352、341/353、342/341、342/342、342/343、342/344、342/345、342/346、342/347、342/348、342/349、342/350、342/351、342/352、342/353、343/341、343/342、343/343、343/344、343/345、343/346、343/347、343/348、343/349、343/350、343/351、343/352、343/353、344/341、344/342、344/343、344/344、344/345、344/346、344/347、344/348、344/349、344/350、344/351、344/352、344/353、345/341、345/342、345/343、345/344、345/345、345/346、345/347、345/348、345/349、345/350、345/351、345/352、345/353、346/341、346/342、346/343、346/344、346/345、346/346、346/347、346/348、346/349、346/350、346/351、346/352、346/353、347/341、347/342、347/343、347/344、347/345、347/346、347/347、347/348、347/349、347/350、347/351、347/352、347/353、348/341、348/342、348/343、348/344、348/345、348/346、348/347、348/348、348/349、348/350、348/351、348/352、348/353、349/341、349/342、349/343、349/344、349/345、349/346、349/347、349/348、349/349、349/350、349/351、349/352、349/353、350/341、350/342、350/343、350/344、350/345、350/346、350/347、350/348、350/349、350/350、350/351、350/352、350/353、351/341、351/342、351/343、351/344、351/345、351/346、351/347、351/348、351/349、351/350、351/351、351/352、351/353、352/341、352/342、352/343、352/344、352/345、352/346、352/347、352/348、352/349、352/350、352/351、352/352和352/353。In one or more embodiments, the D-glucose-sensitive polypeptide is as shown in SEQ ID NO: 1, and the optically active polypeptide is as shown in any one of SEQ ID NO: 2, 6, 7, 9 or a variant thereof having a mutation selected from any one or more of the following at the amino acid position corresponding to the first amino acid of SEQ ID NO: 2: Y1A or Y1G, and the optically active polypeptide is located between residues 117-123, 266-288 and/or 340-353 of the D-glucose-sensitive polypeptide, the numbers corresponding to the full length of the D-glucose-sensitive polypeptide. In one or more embodiments, the optically active polypeptide is located at any one or more sites of the D-glucose-sensitive polypeptide selected from the following: 266/267, 266/268, 266/269, 266/270, 266/271, 266/272, 266/273, 266/274, 266/275, 266/276, 266/277, 266/278, 266/279, 266/280, 266/281, 266/282. 266/283, 266/284, 266/285, 266/286, 266/287, 266/288, 267/267, 267/268, 267/269, 267/270, 267/271, 267/272, 267/273, 267/274, 267/275, 267/276, 267/277, 267/278, 267/279, 267/280, 267/281, 267/28 2, 267/283, 267/284, 267/285, 267/286, 267/287, 267/288, 268/267, 268/268, 268/269, 268/270, 268/271, 268/272, 268/273, 268/274, 268/275, 268/276, 268/277, 268/278, 268/279, 268/280, 268/281, 268 /282, 268/283, 268/284, 268/285, 268/286, 268/287, 268/288, 269/267, 269/268, 269/269, 269/270, 269/271, 269/272, 269/273, 269/274, 269/275, 269/276, 269/277, 269/278, 269/279, 269/280, 269/281, 2 69/282, 269/283, 269/284, 269/285, 269/286, 269/287, 269/288, 270/267, 270/268, 270/269, 270/270, 270/271, 270/272, 270/273, 270/274, 270/275, 270/276, 270/277, 270/278, 270/279, 270/280, 270/28 1, 270/282, 270/283, 270/284, 270/285, 270/286, 270/287, 270/288, 271/267, 271/268, 271/269, 271/270, 271/271, 271/272, 271/273, 271/274, 271/275, 271/276, 271/277, 271/278, 271/279, 271/280, 271/ 281, 271/282, 271/283, 271/284, 271/285, 271/286, 271/287, 271/288, 272/267, 272/268, 272/269, 272/270, 272/271, 272/272, 272/273, 272/274, 272/275, 272/276, 272/277, 272/278, 272/279, 272/280, 27 2/281, 272/282, 272/283, 272/284, 272/285, 272/286, 272/287, 272/288, 273/267, 273/268, 273/269, 273/270, 273/271, 273/272, 273/273, 273/274, 273/275, 273/276, 273/277, 273/278, 273/279, 273/280, 273/281, 273/282, 273/283, 273/284, 273/285, 273/286, 273/287, 273/288, 274/267, 274/268, 274/269, 274/270, 274/271, 274/272, 274/273, 274/274, 274/275, 274/276, 274/277, 274/278, 274/279, 274/2 80, 274/281, 274/282, 274/283, 274/284, 274/285, 274/286, 274/287, 274/288, 275/267, 275/268, 275/269, 275/270, 275/271, 275/272, 275/273, 275/274, 275/275, 275/276, 275/277, 275/278, 275/279, 275 /280, 275/281, 275/282, 275/283, 275/284, 275/285, 275/286, 275/287, 275/288, 276/267, 276/268, 276/269, 276/270, 276/271, 276/272, 276/273, 276/274, 276/275, 276/276, 276/277, 276/278, 276/279 276/280, 276/281, 276/282, 276/283, 276/284, 276/285, 276/286, 276/287, 276/288, 277/267, 277/268, 277/269, 277/270, 277/271, 277/272, 277/273, 277/274, 277/275, 277/276, 277/277, 277/278, 277/27 9, 277/280, 277/281, 277/282, 277/283, 277/284, 277/285, 277/286, 277/287, 277/288, 278/267, 278/268, 278/269, 278/270, 278/271, 278/272, 278/273, 278/274, 278/275, 278/276, 278/277, 278/278, 278 /279, 278/280, 278/281, 278/282, 278/283, 278/284, 278/285, 278/286, 278/287, 278/288, 279/267, 279/268, 279/269, 279/270, 279/271, 279/272, 279/273, 279/274, 279/275, 279/276, 279/277, 279/278, 2 79/279, 279/280, 279/281, 279/282, 279/283, 279/284, 279/285, 279/286, 279/287, 279/288, 280/267, 280/268, 280/269, 280/270, 280/271, 280/272, 280/273, 280/274, 280/275, 280/276, 280/277, 280/278 280/279, 280/280, 280/281, 280/282, 280/283, 280/284, 280/285, 280/286, 280/287, 280/288, 281/267, 281/268, 281/269, 281/270, 281/271, 281/272, 281/273, 281/274, 281/275, 281/276, 281/277, 281/2 78, 281/279, 281/280, 281/281, 281/282, 281/283, 281/284, 281/285, 281/286, 281/287, 281/288, 282/267, 282/268, 282/269, 282/270, 282/271, 282/272, 282/273, 282/274, 282/275, 282/276, 282/277, 28 2/278, 282/279, 282/280, 282/281, 282/282, 282/283, 282/284, 282/285, 282/286, 282/287, 282/288, 283/267, 283/268, 283/269, 283/270, 283/271, 283/272, 283/273, 283/274, 283/275, 283/276, 283/277 283/278, 283/279, 283/280, 283/281, 283/282, 283/283, 283/284, 283/285, 283/286, 283/287, 283/288, 284/267, 284/268, 284/269, 284/270, 284/271, 284/272, 284/273, 284/274, 284/275, 284/276, 284/2 77, 284/278, 284/279, 284/280, 284/281, 284/282, 284/283, 284/284, 284/285, 284/286, 284/287, 284/288, 285/267, 285/268, 285/269, 285/270, 285/271, 285/272, 285/273, 285/274, 285/275, 285/276, 285 /277, 285/278, 285/279, 285/280, 285/281, 285/282, 285/283, 285/284, 285/285, 285/286, 285/287, 285/288, 286/267, 286/268, 286/269, 286/270, 286/271, 286/272, 286/273, 286/274, 286/275, 286/276, 286/277, 286/278, 286/279, 286/280, 286/281, 286/282, 286/283, 286/284, 286/285, 286/286, 286/287, 286/288, 287/267, 287/268, 287/269, 287/270, 287/271, 287/272, 287/273, 287/274, 287/275, 287/27 6, 287/277, 287/278, 287/279, 287/280, 287/281, 287/282, 287/283, 287/284, 287/285, 287/286, 287/287, 287/288, 117/118, 117/119, 117/120, 117/121, 117/122, 117/123, 118/118, 118/119, 118/120, 118/ 121, 118/122, 118/123, 119/118, 119/119, 119/120, 119/121, 119/122, 119/123, 120/118, 120/119, 120/120, 120/121, 120/122, 120/123, 121/118, 121/119, 121/120, 121/121, 121/122, 121/123, 122/118, 12 2/119, 122/120, 122/121, 122/122, 122/123, 340/341, 340/342, 340/343, 340/344, 340/345, 340/346, 340/347, 340/348, 340/349, 340/350, 340/351, 340/352, 340/353, 341/341, 341/342, 341/343, 341/344 341/345, 341/346, 341/347, 341/348, 341/349, 341/350, 341/351, 341/352, 341/353, 342/341, 342/342, 342/343, 342/344, 342/345, 342/346, 342/347, 342/348, 342/349, 342/350, 342/351, 342/352, 342/3 53, 343/341, 343/342, 343/343, 343/344, 343/345, 343/346, 343/347, 343/348, 343/349, 343/350, 343/351, 343/352, 343/353, 344/341, 344/342, 344/343, 344/344, 344/345, 344/346, 344/347, 344/348, 344 /349, 344/350, 344/351, 344/352, 344/353, 345/341, 345/342, 345/343, 345/344, 345/345, 345/346, 345/347, 345/348, 345/349, 345/350, 345/351, 345/352, 345/353, 346/341, 346/342, 346/343, 346/344, 3 46/345, 346/346, 346/347, 346/348, 346/349, 346/350, 346/351, 346/352, 346/353, 347/341, 347/342, 347/343, 347/344, 347/345, 347/346, 347/347, 347/348, 347/349, 347/350, 347/351, 347/352, 347/35 3, 348/341, 348/342, 348/343, 348/344, 348/345, 348/346, 348/347, 348/348, 348/349, 348/350, 348/351, 348/352, 348/353, 349/341, 349/342, 349/343, 349/344, 349/345, 349/346, 349/347, 349/348, 349/ 349, 349/350, 349/351, 349/352, 349/353, 350/341, 350/342, 350/343, 350/344, 350/345, 350/346, 350/347, 350/348, 350/349, 350/350, 350/351, 350/352, 350/353, 351/341, 351/342, 351/343, 351/344, 35 1/345, 351/346, 351/347, 351/348, 351/349, 351/350, 351/351, 351/352, 351/353, 352/341, 352/342, 352/343, 352/344, 352/345, 352/346, 352/347, 352/348, 352/349, 352/350, 352/351, 352/352 and 352/353.

在一个或多个实施方案中，所述光学探针中，D-葡萄糖敏感多肽如SEQ ID NO：1所示，并且具有以下的一个或多个突变：W8、W9、A42、H66、K312、V347、H348。光学活性多肽如SEQ ID NO：2、6、7、9所示或是其在对应于SEQ ID NO：2的第1位氨基酸的氨基酸处具有选自以下任一个或多个突变的变体：Y1A或Y1G，光学活性多肽位于D-葡萄糖敏感多肽的266/267、266/268、266/269、266/270、266/271、266/272、266/273、266/274、266/275、266/276、266/277、266/278、266/279、266/280、266/281、266/282、266/283、266/284、266/285、266/286、266/287、266/288、267/267、267/268、267/269、267/270、267/271、267/272、267/273、267/274、267/275、267/276、267/277、267/278、267/279、267/280、267/281、267/282、267/283、267/284、267/285、267/286、267/287、267/288、268/267、268/268、268/269、268/270、268/271、268/272、268/273、268/274、268/275、268/276、268/277、268/278、268/279、268/280、268/281、268/282、268/283、268/284、268/285、268/286、268/287、268/288、269/267、269/268、269/269、269/270、269/271、269/272、269/273、269/274、269/275、269/276、269/277、269/278、269/279、269/280、269/281、269/282、269/283、269/284、269/285、269/286、269/287、269/288、270/267、270/268、270/269、270/270、270/271、270/272、270/273、270/274、270/275、270/276、270/277、270/278、270/279、270/280、270/281、270/282、270/283、270/284、270/285、270/286、270/287、270/288、271/267、271/268、271/269、271/270、271/271、271/272、271/273、271/274、271/275、271/276、271/277、271/278、271/279、271/280、271/281、271/282、271/283、271/284、271/285、271/286、271/287、271/288、272/267、272/268、272/269、272/270、272/271、272/272、272/273、272/274、272/275、272/276、272/277、272/278、272/279、272/280、272/281、272/282、272/283、272/284、272/285、272/286、272/287、272/288、273/267、273/268、273/269、273/270、273/271、273/272、273/273、273/274、273/275、273/276、273/277、273/278、273/279、273/280、273/281、273/282、273/283、273/284、273/285、273/286、273/287、273/288、274/267、274/268、274/269、274/270、274/271、274/272、274/273、274/274、274/275、274/276、274/277、274/278、274/279、274/280、274/281、274/282、274/283、274/284、274/285、274/286、274/287、274/288、275/267、275/268、275/269、275/270、275/271、275/272、275/273、275/274、275/275、275/276、275/277、275/278、275/279、275/280、275/281、275/282、275/283、275/284、275/285、275/286、275/287、275/288、276/267、276/268、276/269、276/270、276/271、276/272、276/273、276/274、276/275、276/276、276/277、276/278、276/279、276/280、276/281、276/282、276/283、276/284、276/285、276/286、276/287、276/288、277/267、277/268、277/269、277/270、277/271、277/272、277/273、277/274、277/275、277/276、277/277、277/278、277/279、277/280、277/281、277/282、277/283、277/284、277/285、277/286、277/287、277/288、278/267、278/268、278/269、278/270、278/271、278/272、278/273、278/274、278/275、278/276、278/277、278/278、278/279、278/280、278/281、278/282、278/283、278/284、278/285、278/286、278/287、278/288、279/267、279/268、279/269、279/270、279/271、279/272、279/273、279/274、279/275、279/276、279/277、279/278、279/279、279/280、279/281、279/282、279/283、279/284、279/285、279/286、279/287、279/288、280/267、280/268、280/269、280/270、280/271、280/272、280/273、280/274、280/275、280/276、280/277、280/278、280/279、280/280、280/281、280/282、280/283、280/284、280/285、280/286、280/287、280/288、281/267、281/268、281/269、281/270、281/271、281/272、281/273、281/274、281/275、281/276、281/277、281/278、281/279、281/280、281/281、281/282、281/283、281/284、281/285、281/286、281/287、281/288、282/267、282/268、282/269、282/270、282/271、282/272、282/273、282/274、282/275、282/276、282/277、282/278、282/279、282/280、282/281、282/282、282/283、282/284、282/285、282/286、282/287、282/288、283/267、283/268、283/269、283/270、283/271、283/272、283/273、283/274、283/275、283/276、283/277、283/278、283/279、283/280、283/281、283/282、283/283、283/284、283/285、283/286、283/287、283/288、284/267、284/268、284/269、284/270、284/271、284/272、284/273、284/274、284/275、284/276、284/277、284/278、284/279、284/280、284/281、284/282、284/283、284/284、284/285、284/286、284/287、284/288、285/267、285/268、285/269、285/270、285/271、285/272、285/273、285/274、285/275、285/276、285/277、285/278、285/279、285/280、285/281、285/282、285/283、285/284、285/285、285/286、285/287、285/288、286/267、286/268、286/269、286/270、286/271、286/272、286/273、286/274、286/275、286/276、286/277、286/278、286/279、286/280、286/281、286/282、286/283、286/284、286/285、286/286、286/287、286/288、287/267、287/268、287/269、287/270、287/271、287/272、287/273、287/274、287/275、287/276、287/277、287/278、287/279、287/280、287/281、287/282、287/283、287/284、287/285、287/286、287/287、287/288，117/118、117/119、117/120、117/121、117/122、117/123、118/118、118/119、118/120、118/121、118/122、118/123、119/118、119/119、119/120、119/121、119/122、119/123、120/118、120/119、120/120、120/121、120/122、120/123、121/118、121/119、121/120、121/121、121/122、121/123、122/118、122/119、122/120、122/121、122/122、122/123，340/341、340/342、340/343、340/344、340/345、340/346、340/347、340/348、340/349、340/350、340/351、340/352、340/353、341/341、341/342、341/343、341/344、341/345、341/346、341/347、341/348、341/349、341/350、341/351、341/352、341/353、342/341、342/342、342/343、342/344、342/345、342/346、342/347、342/348、342/349、342/350、342/351、342/352、342/353、343/341、343/342、343/343、343/344、343/345、343/346、343/347、343/348、343/349、343/350、343/351、343/352、343/353、344/341、344/342、344/343、344/344、344/345、344/346、344/347、344/348、344/349、344/350、344/351、344/352、344/353、345/341、345/342、345/343、345/344、345/345、345/346、345/347、345/348、345/349、345/350、345/351、345/352、345/353、346/341、346/342、346/343、346/344、346/345、346/346、346/347、346/348、346/349、346/350、346/351、346/352、346/353、347/341、347/342、347/343、347/344、347/345、347/346、347/347、347/348、347/349、347/350、347/351、347/352、347/353、348/341、348/342、348/343、348/344、348/345、348/346、348/347、348/348、348/349、348/350、348/351、348/352、348/353、349/341、349/342、349/343、349/344、349/345、349/346、349/347、349/348、349/349、349/350、349/351、349/352、349/353、350/341、350/342、350/343、350/344、350/345、350/346、350/347、350/348、350/349、350/350、350/351、350/352、350/353、351/341、351/342、351/343、351/344、351/345、351/346、351/347、351/348、351/349、351/350、351/351、351/352、351/353、352/341、352/342、352/343、352/344、352/345、352/346、352/347、352/348、352/349、352/350、352/351、352/352和352/353位点。优选地，D-葡萄糖敏感多肽的突变包含选自以下任一组的突变：(1)V347W和H348T，(2)V347W和H348G，(3)V347W和H348S，(4)V347W、H348T和H66R，(5)V347W、H348T和H66E，(6)V347W、H348T和H66A，(7)V347W、H348T和H66F，(8)V347W、H348T和H66M，(9)V347W、H348T和H66C，(10)V347W、H348T和H66P，(11)V347W、H348T和H66Q，(12)V347W、H348T和H66T，(13)V347W、H348T和W9R，(14)V347W、H348T和W9H，(15)V347W、H348T和W9E，(16)V347W、H348T和W9A，(17)V347W、H348T和W9V，(18)V347W、H348T和W9L，(19)V347W、H348T和W9F，(20)V347W、H348T和W9I，(21)V347W、H348T和W9M，(22)V347W、H348T和W9C，(23)V347W、H348T和W9P，(24)V347W、H348T和W9N，(25)V347W、H348T和W9G，(26)V347W、H348T和W9K，(27)V347W、H348T和W9Y，(28)V347W、H348T和W9S，(29)V347W、H348T和W9T，(30)V347W、H348T和W9D，(31)V347W、H348T和W8R，(32)V347W、H348T和W8H，(33)V347W、H348T和W8E，(34)V347W、H348T和W8A，(35)V347W、H348T和W8V，(36)V347W、H348T和W8L，(37)V347W、H348T和W8F，(38)V347W、H348T和W8I，(39)V347W、H348T和W8M，(40)V347W、H348T和W8C，(41)V347W、H348T和W8N，(42)V347W、H348T和W8G，(43)V347W、H348T和W8K，(44)V347W、H348T和W8T，(45)V347W、H348T和W8D，(46)V347W、H348T和A42R，(47)V347W、H348T和A42E，(48)V347W、H348T和A42W，(49)V347W、H348T和A42F，(50)V347W、H348T和A42M，(51)V347W、H348T和A42C，(52)V347W、H348T和A42P，(53)V347W、H348T和A42N，(54)V347W、H348T和A42G，(55)V347W、H348T和A42K，(56)V347W、H348T和A42Y，(57)V347W、H348T和A42T，(58)V347W、H348T和K312R，(59)V347W、H348T和K312A，(60)V347W、H348T和K312Q，(61)V347W、H348T和K312G，(62)V347W、H348T和K312H，(63)V347W、H348T和K312S。In one or more embodiments, the optical probe contains a D-glucose-sensitive polypeptide as shown in SEQ ID NO: 1, and has one or more of the following mutations: W8, W9, A42, H66, K312, V347, H348. The optically active polypeptide is shown in SEQ ID NO: 2, 6, 7, 9, or has a variant containing one or more mutations selected from Y1A or Y1G at the amino acid position corresponding to the first amino acid of SEQ ID NO: 2. The optically active polypeptide is located at 266/267, 266/268, 266/269, 266/270, 266/271, 266/272, 266/273, 266/274, 266/275, 266 of the D-glucose-sensitive polypeptide. /276, 266/277, 266/278, 266/279, 266/280, 266/281, 266/282, 266/283, 266/284, 266/285, 266/286, 266/287, 266/288, 267/267, 267/268, 267/269, 267/270, 267/271, 267/272, 267/273, 267/274, 267/275, 26 7/276, 267/277, 267/278, 267/279, 267/280, 267/281, 267/282, 267/283, 267/284, 267/285, 267/286, 267/287, 267/288, 268/267, 268/268, 268/269, 268/270, 268/271, 268/272, 268/273, 268/274, 268/275, 2 68/276, 268/277, 268/278, 268/279, 268/280, 268/281, 268/282, 268/283, 268/284, 268/285, 268/286, 268/287, 268/288, 269/267, 269/268, 269/269, 269/270, 269/271, 269/272, 269/273, 269/274, 269/275, 2 69/276, 269/277, 269/278, 269/279, 269/280, 269/281, 269/282, 269/283, 269/284, 269/285, 269/286, 269/287, 269/288, 270/267, 270/268, 270/269, 270/270, 270/271, 270/272, 270/273, 270/274, 270/275 270/276, 270/277, 270/278, 270/279, 270/280, 270/281, 270/282, 270/283, 270/284, 270/285, 270/286, 270/287, 270/288, 271/267, 271/268, 271/269, 271/270, 271/271, 271/272, 271/273, 271/274, 271/275 271/276, 271/277, 271/278, 271/279, 271/280, 271/281, 271/282, 271/283, 271/284, 271/285, 271/286, 271/287, 271/288, 272/267, 272/268, 272/269, 272/270, 272/271, 272/272, 272/273, 272/274, 272/275 272/276, 272/277, 272/278, 272/279, 272/280, 272/281, 272/282, 272/283, 272/284, 272/285, 272/286, 272/287, 272/288, 273/267, 273/268, 273/269, 273/270, 273/271, 273/272, 273/273, 273/274, 273/275 273/276, 273/277, 273/278, 273/279, 273/280, 273/281, 273/282, 273/283, 273/284, 273/285, 273/286, 273/287, 273/288, 274/267, 274/268, 274/269, 274/270, 274/271, 274/272, 274/273, 274/274, 274/27 5, 274/276, 274/277, 274/278, 274/279, 274/280, 274/281, 274/282, 274/283, 274/284, 274/285, 274/286, 274/287, 274/288, 275/267, 275/268, 275/269, 275/270, 275/271, 275/272, 275/273, 275/274, 275/27 5, 275/276, 275/277, 275/278, 275/279, 275/280, 275/281, 275/282, 275/283, 275/284, 275/285, 275/286, 275/287, 275/288, 276/267, 276/268, 276/269, 276/270, 276/271, 276/272, 276/273, 276/274, 276/2 75, 276/276, 276/277, 276/278, 276/279, 276/280, 276/281, 276/282, 276/283, 276/284, 276/285, 276/286, 276/287, 276/288, 277/267, 277/268, 277/269, 277/270, 277/271, 277/272, 277/273, 277/274, 277/2 75, 277/276, 277/277, 277/278, 277/279, 277/280, 277/281, 277/282, 277/283, 277/284, 277/285, 277/286, 277/287, 277/288, 278/267, 278/268, 278/269, 278/270, 278/271, 278/272, 278/273, 278/274, 278/ 275, 278/276, 278/277, 278/278, 278/279, 278/280, 278/281, 278/282, 278/283, 278/284, 278/285, 278/286, 278/287, 278/288, 279/267, 279/268, 279/269, 279/270, 279/271, 279/272, 279/273, 279/274, 279/ 275, 279/276, 279/277, 279/278, 279/279, 279/280, 279/281, 279/282, 279/283, 279/284, 279/285, 279/286, 279/287, 279/288, 280/267, 280/268, 280/269, 280/270, 280/271, 280/272, 280/273, 280/274, 280 /275, 280/276, 280/277, 280/278, 280/279, 280/280, 280/281, 280/282, 280/283, 280/284, 280/285, 280/286, 280/287, 280/288, 281/267, 281/268, 281/269, 281/270, 281/271, 281/272, 281/273, 281/274, 281 /275, 281/276, 281/277, 281/278, 281/279, 281/280, 281/281, 281/282, 281/283, 281/284, 281/285, 281/286, 281/287, 281/288, 282/267, 282/268, 282/269, 282/270, 282/271, 282/272, 282/273, 282/274, 28 2/275, 282/276, 282/277, 282/278, 282/279, 282/280, 282/281, 282/282, 282/283, 282/284, 282/285, 282/286, 282/287, 282/288, 283/267, 283/268, 283/269, 283/270, 283/271, 283/272, 283/273, 283/274, 2 83/275, 283/276, 283/277, 283/278, 283/279, 283/280, 283/281, 283/282, 283/283, 283/284, 283/285, 283/286, 283/287, 283/288, 284/267, 284/268, 284/269, 284/270, 284/271, 284/272, 284/273, 284/274 284/275, 284/276, 284/277, 284/278, 284/279, 284/280, 284/281, 284/282, 284/283, 284/284, 284/285, 284/286, 284/287, 284/288, 285/267, 285/268, 285/269, 285/270, 285/271, 285/272, 285/273, 285/274 285/275, 285/276, 285/277, 285/278, 285/279, 285/280, 285/281, 285/282, 285/283, 285/284, 285/285, 285/286, 285/287, 285/288, 286/267, 286/268, 286/269, 286/270, 286/271, 286/272, 286/273, 286/274 286/275, 286/276, 286/277, 286/278, 286/279, 286/280, 286/281, 286/282, 286/283, 286/284, 286/285, 286/286, 286/287, 286/288, 287/267, 287/268, 287/269, 287/270, 287/271, 287/272, 287/273, 287/274 287/275, 287/276, 287/277, 287/278, 287/279, 287/280, 287/281, 287/282, 287/283, 287/284, 287/285, 287/286, 287/287, 287/288, 117/118, 117/119, 117/120, 117/121, 117/122, 117/123, 118/118, 118/11 9, 118/120, 118/121, 118/122, 118/123, 119/118, 119/119, 119/120, 119/121, 119/122, 119/123, 120/118, 120/119, 120/120, 120/121, 120/122, 120/123, 121/118, 121/119, 121/120, 121/121, 121/122, 121/12 3, 122/118, 122/119, 122/120, 122/121, 122/122, 122/123, 340/341, 340/342, 340/343, 340/344, 340/345, 340/346, 340/347, 340/348, 340/349, 340/350, 340/351, 340/352, 340/353, 341/341, 341/342, 341/3 43, 341/344, 341/345, 341/346, 341/347, 341/348, 341/349, 341/350, 341/351, 341/352, 341/353, 342/341, 342/342, 342/343, 342/344, 342/345, 342/346, 342/347, 342/348, 342/349, 342/350, 342/351, 342/3 52, 342/353, 343/341, 343/342, 343/343, 343/344, 343/345, 343/346, 343/347, 343/348, 343/349, 343/350, 343/351, 343/352, 343/353, 344/341, 344/342, 344/343, 344/344, 344/345, 344/346, 344/347, 344/ 348, 344/349, 344/350, 344/351, 344/352, 344/353, 345/341, 345/342, 345/343, 345/344, 345/345, 345/346, 345/347, 345/348, 345/349, 345/350, 345/351, 345/352, 345/353, 346/341, 346/342, 346/343, 346/ 344, 346/345, 346/346, 346/347, 346/348, 346/349, 346/350, 346/351, 346/352, 346/353, 347/341, 347/342, 347/343, 347/344, 347/345, 347/346, 347/347, 347/348, 347/349, 347/350, 347/351, 347/352, 347 /353, 348/341, 348/342, 348/343, 348/344, 348/345, 348/346, 348/347, 348/348, 348/349, 348/350, 348/351, 348/352, 348/353, 349/341, 349/342, 349/343, 349/344, 349/345, 349/346, 349/347, 349/348, 349 /349, 349/350, 349/351, 349/352, 349/353, 350/341, 350/342, 350/343, 350/344, 350/345, 350/346, 350/347, 350/348, 350/349, 350/350, 350/351, 350/352, 350/353, 351/341, 351/342, 351/343, 351/344, 35 Loci 1/345, 351/346, 351/347, 351/348, 351/349, 351/350, 351/351, 351/352, 351/353, 352/341, 352/342, 352/343, 352/344, 352/345, 352/346, 352/347, 352/348, 352/349, 352/350, 352/351, 352/352, and 352/353. Preferably, the mutations in the D-glucose-sensitive peptide include mutations selected from any of the following groups: (1) V347W and H348T, (2) V347W and H348G, (3) V347W and H348S, (4) V347W, H348T and H66R, (5) V347W, H348T and H66E, (6) V347W, H348T and H66A, (7) V347W, H348T and H66F, (8) V347W (9) V347W, H348T and H66C, (10) V347W, H348T and H66P, (11) V347W, H348T and H66Q, (12) V347W, H348T and H66T, (13) V347W, H348T and W9R, (14) V347W, H348T and W9H, (15) V347W, H348T and W9E, (16) V347 W, H348T and W9A, (17) V347W, H348T and W9V, (18) V347W, H348T and W9L, (19) V347W, H348T and W9F, (20) V347W, H348T and W9I, (21) V347W, H348T and W9M, (22) V347W, H348T and W9C, (23) V347W, H348T and W9P, (24) V347W, H348T and W9N, (25) V347W, H348T and W9G, (26) V347W, H348T and W9K, (27) V347W, H348T and W9Y, (28) V347W, H348T and W9S, (29) V347W, H348T and W9T, (30) V347W, H348T and W9D, (31) V347W, H348T and W8R, (32) V347W, H34 8T and W8H, (33)V347W, H348T and W8E, (34)V347W, H348T and W8A, (35)V347W, H348T and W8V, (36)V347W, H348T and W8L, (37)V347W, H348T and W8F, (38)V347W, H348T and W8I, (39)V347W, H348T and W8M, (40)V347W, H348T And W8C, (41)V347W, H348T and W8N, (42)V347W, H348T and W8G, (43)V347W, H348T and W8K, (44)V347W, H348T and W8T, (45)V347W, H348T and W8D, (46)V347W, H348T and A42R, (47)V347W, H348T and A42E, (48)V347W, H348T and A42W, (49) V347W, H348T and A42F, (50) V347W, H348T and A42M, (51) V347W, H348T and A42C, (52) V347W, H348T and A42P, (53) V347W, H348T and A42N, (54) V347W, H348T and A42G, (55) V347W, H348T and A42K, (56) V347W, H 348T and A42Y, (57)V347W, H348T and A42T, (58)V347W, H348T and K312R, (59)V347W, H348T and K312A, (60)V347W, H348T and K312Q, (61)V347W, H348T and K312G, (62)V347W, H348T and K312H, (63)V347W, H348T and K312S.

在一个或多个实施方案中，所述光学探针中，D-葡萄糖敏感多肽如SEQ ID NO：1所示，光学活性多肽如SEQ ID NO：2、6、7、9所示，光学活性多肽位于D-葡萄糖敏感多肽的348/352位点，并且，所述光学探针具有如下所示的突变：(1)D-葡萄糖敏感多肽的V347W、H348T，和光学活性多肽的1A，(2)D-葡萄糖敏感多肽的V347W、H348G，和光学活性多肽的1G，(3)D-葡萄糖敏感多肽的V347W、H348S，和光学活性多肽的1A，(4)D-葡萄糖敏感多肽的V347W、H348S，和光学活性多肽的1G，(5)D-葡萄糖敏感多肽的V347W、H348T，和光学活性多肽的1G，(6)D-葡萄糖敏感多肽的V347W、H348T、H66R，和光学活性多肽的1A，(7)D-葡萄糖敏感多肽的V347W、H348T、H66E，和光学活性多肽的1A，(8)D-葡萄糖敏感多肽的V347W、H348T、H66A，和光学活性多肽的1A，(9)D-葡萄糖敏感多肽的V347W、H348T、H66F，和光学活性多肽的1A，(10)D-葡萄糖敏感多肽的V347W、H348T、H66M，和光学活性多肽的1A，(11)D-葡萄糖敏感多肽的V347W、H348T、H66C，和光学活性多肽的1A，(12)D-葡萄糖敏感多肽的V347W、H348T、H66P，和光学活性多肽的1A，(13)D-葡萄糖敏感多肽的V347W、H348T、H66Q，和光学活性多肽的1A，(14)D-葡萄糖敏感多肽的V347W、H348T、H66T，和光学活性多肽的1A，(15)D-葡萄糖敏感多肽的V347W、H348T、W9R，和光学活性多肽的1A，(16)D-葡萄糖敏感多肽的V347W、H348T、W9H，和光学活性多肽的1A，(17)D-葡萄糖敏感多肽的V347W、H348T、W9E，和光学活性多肽的1A，(18)D-葡萄糖敏感多肽的V347W、H348T、W9A，和光学活性多肽的1A，(19)D-葡萄糖敏感多肽的V347W、H348T、W9V，和光学活性多肽的1A，(20)D-葡萄糖敏感多肽的V347W、H348T、W9L，和光学活性多肽的1A，(21)D-葡萄糖敏感多肽的V347W、H348T、W9F，和光学活性多肽的1A，(22)D-葡萄糖敏感多肽的V347W、H348T、W9I，和光学活性多肽的1A，(23)D-葡萄糖敏感多肽的V347W、H348T、W9M，和光学活性多肽的1A，(24)D-葡萄糖敏感多肽的V347W、H348T、W9C，和光学活性多肽的1A，(25)D-葡萄糖敏感多肽的V347W、H348T、W9P，和光学活性多肽的1A，(26)D-葡萄糖敏感多肽的V347W、H348T、W9N，和光学活性多肽的1A，(27)D-葡萄糖敏感多肽的V347W、H348T、W9G，和光学活性多肽的1A，(28)D-葡萄糖敏感多肽的V347W、H348T、W9K，和光学活性多肽的1A，(29)D-葡萄糖敏感多肽的V347W、H348T、W9Y，和光学活性多肽的1A，(30)D-葡萄糖敏感多肽的V347W、H348T、W9S，和光学活性多肽的1A，(31)D-葡萄糖敏感多肽的V347W、H348T、W9T，和光学活性多肽的1A，(32)D-葡萄糖敏感多肽的V347W、H348T、W9D，和光学活性多肽的1A，(33)D-葡萄糖敏感多肽的V347W、H348T、W8R，和光学活性多肽的1A，(34)D-葡萄糖敏感多肽的V347W、H348T、W8H，和光学活性多肽的1A，(35)D-葡萄糖敏感多肽的V347W、H348T、W8E，和光学活性多肽的1A，(36)D-葡萄糖敏感多肽的V347W、H348T、W8A，和光学活性多肽的1A，(37)D-葡萄糖敏感多肽的V347W、H348T、W8V，和光学活性多肽的1A，(38)D-葡萄糖敏感多肽的V347W、H348T、W8L，和光学活性多肽的1A，(39)D-葡萄糖敏感多肽的V347W、H348T、W8F，和光学活性多肽的1A，(40)D-葡萄糖敏感多肽的V347W、H348T、W8I，和光学活性多肽的1A，(41)D-葡萄糖敏感多肽的V347W、H348T、W8M，和光学活性多肽的1A，(42)D-葡萄糖敏感多肽的V347W、H348T、W8C，和光学活性多肽的1A，(43)D-葡萄糖敏感多肽的V347W、H348T、W8N，和光学活性多肽的1A，(44)D-葡萄糖敏感多肽的V347W、H348T、W8G，和光学活性多肽的1A，(45)D-葡萄糖敏感多肽的V347W、H348T、W8K，和光学活性多肽的1A，(46)D-葡萄糖敏感多肽的V347W、H348T、W8T，和光学活性多肽的1A，(47)D-葡萄糖敏感多肽的V347W、H348T、W8D，和光学活性多肽的1A，(48)D-葡萄糖敏感多肽的V347W、H348T、A42R，和光学活性多肽的1A，(49)D-葡萄糖敏感多肽的V347W、H348T、A42E，和光学活性多肽的1A，(50)D-葡萄糖敏感多肽的V347W、H348T、A42W，和光学活性多肽的1A，(51)D-葡萄糖敏感多肽的V347W、H348T、A42F，和光学活性多肽的1A，(52)D-葡萄糖敏感多肽的V347W、H348T、A42M，和光学活性多肽的1A，(53)D-葡萄糖敏感多肽的V347W、H348T、A42C，和光学活性多肽的1A，(54)D-葡萄糖敏感多肽的V347W、H348T、A42P，和光学活性多肽的1A，(55)D-葡萄糖敏感多肽的V347W、H348T、A42N，和光学活性多肽的1A，(56)D-葡萄糖敏感多肽的V347W、H348T、A42G，和光学活性多肽的1A，(57)D-葡萄糖敏感多肽的V347W、H348T、A42K，和光学活性多肽的1A，(58)D一葡萄糖敏感多肽的V347W、H348T、A42Y，和光学活性多肽的1A，(59)D-葡萄糖敏感多肽的V347W、H348T、A42T，和光学活性多肽的1A，(60)D-葡萄糖敏感多肽的V347W、H348T、K312R，和光学活性多肽的1A，(61)D-葡萄糖敏感多肽的V347W、H348T、K312A，和光学活性多肽的1A，(62)D-葡萄糖敏感多肽的V347W、H348T、K312Q，和光学活性多肽的1A，(63)D-葡萄糖敏感多肽的V347W、H348T、K312G，和光学活性多肽的1A，(64)D-葡萄糖敏感多肽的V347W、H348T、K312H，和光学活性多肽的1A，(65)D-葡萄糖敏感多肽的V347W、H348T、K312S，和光学活性多肽的1A。In one or more embodiments, the optical probe contains a D-glucose-sensitive polypeptide as shown in SEQ ID NO: 1 and an optically active polypeptide as shown in SEQ ID NO: 2, 6, 7, and 9, wherein the optically active polypeptide is located at position 348/352 of the D-glucose-sensitive polypeptide, and the optical probe has the following mutations: (1) V347W and H348T of the D-glucose-sensitive polypeptide and 1A of the optically active polypeptide; (2) V347W and H348T of the D-glucose-sensitive polypeptide. 348G, and 1G of optically active peptide, (3) V347W and H348S of D-glucose-sensitive peptide, and 1A of optically active peptide, (4) V347W and H348S of D-glucose-sensitive peptide, and 1G of optically active peptide, (5) V347W and H348T of D-glucose-sensitive peptide, and 1G of optically active peptide, (6) V347W, H348T, and H66R of D-glucose-sensitive peptide, and 1A of optically active peptide, (7) D-glucose-sensitive (8) D-glucose-sensitive peptides V347W, H348T, H66E, and optically active peptide 1A; (9) D-glucose-sensitive peptides V347W, H348T, H66F, and optically active peptide 1A; (10) D-glucose-sensitive peptides V347W, H348T, H66M, and optically active peptide 1A; (11) D-glucose-sensitive peptides V347W, H348T, H66M, and optically active peptide 1A; (12) D-glucose-sensitive peptides V347W, H348T, H66P, and optically active peptide 1A; (13) D-glucose-sensitive peptides V347W, H348T, H66Q, and optically active peptide 1A; (14) D-glucose-sensitive peptides V347W, H348T, H66T, and optically active peptide 1A; (15) D-glucose-sensitive peptides V347W, H348T, H66Q, and optically active peptide 1A; T, W9R, and optically active peptide 1A, (16) D-glucose-sensitive peptides V347W, H348T, W9H, and optically active peptide 1A, (17) D-glucose-sensitive peptides V347W, H348T, W9E, and optically active peptide 1A, (18) D-glucose-sensitive peptides V347W, H348T, W9A, and optically active peptide 1A, (19) D-glucose-sensitive peptides V347W, H348T, W9V, and optically active peptide 1A, 1A of the sex-sensitive peptide, (20) V347W, H348T, W9L of the D-glucose-sensitive peptide, and 1A of the optically active peptide, (21) V347W, H348T, W9F of the D-glucose-sensitive peptide, and 1A of the optically active peptide, (22) V347W, H348T, W9I of the D-glucose-sensitive peptide, and 1A of the optically active peptide, (23) V347W, H348T, W9M of the D-glucose-sensitive peptide, and 1A of the optically active peptide, (24) (25) D-glucose-sensitive peptides V347W, H348T, W9C, and optically active peptide 1A, (26) D-glucose-sensitive peptides V347W, H348T, W9P, and optically active peptide 1A, (27) D-glucose-sensitive peptides V347W, H348T, W9N, and optically active peptide 1A, (28) D-glucose-sensitive peptides (29) D-glucose-sensitive peptides V347W, H348T, W9Y, and optically active peptide 1A; (30) D-glucose-sensitive peptides V347W, H348T, W9S, and optically active peptide 1A; (31) D-glucose-sensitive peptides V347W, H348T, W9T, and optically active peptide 1A; (32) D-glucose-sensitive peptides V347W, H348T, W9K, and optically active peptide 1A; T, W9D, and optically active peptide 1A, (33) D-glucose-sensitive peptides V347W, H348T, W8R, and optically active peptide 1A, (34) D-glucose-sensitive peptides V347W, H348T, W8H, and optically active peptide 1A, (35) D-glucose-sensitive peptides V347W, H348T, W8E, and optically active peptide 1A, (36) D-glucose-sensitive peptides V347W, H348T, W8A, and optically active peptide 1A, 1A of the sex-sensitive peptide, (37) V347W, H348T, W8V of the D-glucose-sensitive peptide, and 1A of the optically active peptide, (38) V347W, H348T, W8L of the D-glucose-sensitive peptide, and 1A of the optically active peptide, (39) V347W, H348T, W8F of the D-glucose-sensitive peptide, and 1A of the optically active peptide, (40) V347W, H348T, W8I of the D-glucose-sensitive peptide, and 1A of the optically active peptide, (41) (42) D-glucose-sensitive peptides V347W, H348T, W8M, and optically active peptide 1A; (43) D-glucose-sensitive peptides V347W, H348T, W8C, and optically active peptide 1A; (44) D-glucose-sensitive peptides V347W, H348T, W8N, and optically active peptide 1A; (45) D-glucose-sensitive peptides V347W, H348T, W8G, and optically active peptide 1A; V347W, H348T, W8K, and optically active peptide 1A, (46) D-glucose-sensitive peptides V347W, H348T, W8T, and optically active peptide 1A, (47) D-glucose-sensitive peptides V347W, H348T, W8D, and optically active peptide 1A, (48) D-glucose-sensitive peptides V347W, H348T, A42R, and optically active peptide 1A, (49) D-glucose-sensitive peptides V347W, H348T, W8K, and optically active peptide 1A, (41) D-glucose-sensitive peptides V347W, H348T, W8K, and optically active peptide 1A, (42) D-glucose-sensitive peptides V347W, H348T, W8K, and optically active peptide 1A, (43) D-glucose-sensitive peptides V347W, H348T, W8K, and optically active peptide 1A, (4 ...5) D-glucose-sensitive peptides V347W, H348T, W8K, 8T, A42E, and optically active peptide 1A, (50) D-glucose-sensitive peptides V347W, H348T, A42W, and optically active peptide 1A, (51) D-glucose-sensitive peptides V347W, H348T, A42F, and optically active peptide 1A, (52) D-glucose-sensitive peptides V347W, H348T, A42M, and optically active peptide 1A, (53) D-glucose-sensitive peptides V347W, H348T, A42 C, and optically active peptide 1A, (54) D-glucose-sensitive peptides V347W, H348T, A42P, and optically active peptide 1A, (55) D-glucose-sensitive peptides V347W, H348T, A42N, and optically active peptide 1A, (56) D-glucose-sensitive peptides V347W, H348T, A42G, and optically active peptide 1A, (57) D-glucose-sensitive peptides V347W, H348T, A42K, and optically active 1A of D-glucose-sensitive peptides, (58) V347W, H348T, A42Y of D-glucose-sensitive peptides, and 1A of optically active peptides, (59) V347W, H348T, A42T of D-glucose-sensitive peptides, and 1A of optically active peptides, (60) V347W, H348T, K312R of D-glucose-sensitive peptides, and 1A of optically active peptides, (61) V347W, H348T, K312A of D-glucose-sensitive peptides, and 1A of optically active peptides. 1A, (62) D-glucose-sensitive peptides V347W, H348T, K312Q, and optically active peptide 1A, (63) D-glucose-sensitive peptides V347W, H348T, K312G, and optically active peptide 1A, (64) D-glucose-sensitive peptides V347W, H348T, K312H, and optically active peptide 1A, (65) D-glucose-sensitive peptides V347W, H348T, K312S, and optically active peptide 1A.

在一个或多个实施方案中，所述光学探针如SEQ ID NO：11所示。In one or more embodiments, the optical probe is as shown in SEQ ID NO: 11.

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，其他多肽包括定位序列(例如将光学探针定位到不同细胞器或亚细胞器的多肽)、便于纯化的标签或者用于免疫反应(例如免疫印迹)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。Another aspect of the present invention provides a fusion polypeptide comprising the optical probe described herein and other polypeptides. In some embodiments, the other polypeptides are located at the N-terminus and/or C-terminus of the optical probe. In some embodiments, the other polypeptides include a localization sequence (e.g., a polypeptide that localizes the optical probe to a different organelle or sub-organelle), a tag for easy purification, or a tag for use in an immunoreaction (e.g., immunoblotting). A linker may be present between the optical probe and the other polypeptides in the fusion polypeptide described herein.

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

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。The present invention also relates to variants of the aforementioned nucleic acid molecules, including fragments, analogs, derivatives, soluble fragments and variants of the present invention encoding the optical probes or fusion proteins of the present invention, or their complementary sequences.

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。In another aspect, the present invention also provides nucleic acid constructs comprising the nucleic acid molecules described herein. The nucleic acid sequence encodes the optical probe or fusion polypeptide described herein.

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

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

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。In some implementations, the expression vector is selected from prokaryotic expression vectors, eukaryotic expression vectors, and viral vectors.

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。In another aspect, the present invention provides a host cell that: (1) expresses the optical probe or fusion polypeptide described in any embodiment of the present invention; (2) contains the nucleic acid molecule described in any embodiment of the present invention; or (3) contains the nucleic acid construct described in any embodiment of the present invention. The host cell is preferably *Escherichia coli*.

本发明另一方面还提供D-葡萄糖检测试剂盒，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。Another aspect of the present invention provides a D-glucose detection kit, comprising the optical probes described herein or fusion peptides or polynucleotides or optical probes prepared as described herein.

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、D-葡萄糖标准品。In one or more embodiments, the kit further comprises one or more reagents selected from the following: buffer, culture medium, D-glucose standard.

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。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 of cell expression, and isolating the optical probe or fusion polypeptide.

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D-葡萄糖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D-葡萄糖光学探针。In one or more embodiments, the method includes the following steps: 1) incorporating a nucleic acid molecule encoding the D-glucose optical probe described herein into an expression vector; 2) transferring the expression vector into a host cell; 3) culturing the host cell under conditions suitable for expression of the expression vector; and 4) isolating the D-glucose optical probe.

本发明另一方面还提供检测样品中D-葡萄糖的方法，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。Another aspect of the present invention provides a method for detecting D-glucose in a sample, comprising: contacting the sample with the optical probe or fusion peptide or host cell described herein, and detecting changes in the optically active peptide. The detection can be performed in vivo, in vitro, subcellular, or in situ. The sample may be, for example, blood.

本文另一方面还提供定量样品中D-葡萄糖的方法，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D-葡萄糖。This article also provides a method for quantifying D-glucose in a sample, comprising: contacting the optical probe or fusion peptide or host cell described herein with the sample, detecting optical changes in the optically active peptide, and quantifying D-glucose in the sample based on the optical changes in the optically active peptide.

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D-葡萄糖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。Another aspect of the present invention provides a method for screening compounds (e.g., drugs), comprising: contacting the optical probe or fusion peptide or host cell described herein with a candidate compound in a system containing D-glucose; detecting optical changes in the optically active peptide; and screening the compound based on the optical changes in the optically active peptide. The method can screen compounds in high throughput.

在一个或多个实施方案中，在含D-葡萄糖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D-葡萄糖的摄取。In one or more embodiments, the host cells described herein are contacted with the candidate compound in a system containing D-glucose, and optical changes in the optically active peptide indicate whether the candidate compound can regulate the uptake of D-glucose by the cells.

本发明另一方面还提供对所述D-葡萄糖进行细胞内和/或外定位的方法，包括：将含D-葡萄糖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。Another aspect of the present invention provides a method for intracellular and/or extracellular localization of the D-glucose, comprising: contacting a system containing D-glucose with the optical probe or the host cell, and detecting optical changes in the optically active peptide.

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

本发明另一方面还提供本文所述D-葡萄糖光学探针或融合多肽或宿主细胞在检测样品中的D-葡萄糖、筛选化合物或D-葡萄糖的细胞内和/或细胞外中的应用。在一个或多个实施方案中，所述定位是实时定位。Another aspect of the present invention provides the use of the D-glucose optical probes, fusion peptides, or host cells described herein in the intracellular and/or extracellular detection of D-glucose in samples, screening compounds, or D-glucose. In one or more embodiments, the localization is real-time localization.

本发明另一方面还提供本文所述D-葡萄糖光学探针或融合多肽或多核苷酸或核酸构建物或宿主细胞在制备试剂盒中的用途，所述试剂盒用于检测样品中的D-葡萄糖、筛选化合物或D-葡萄糖的细胞内和/或细胞外定位。Another aspect of the present invention provides the use of the D-glucose optical probes or fusion peptides or polynucleotides or nucleic acid constructs or host cells described herein in the preparation of kits for detecting D-glucose in samples, screening compounds or intracellular and/or extracellular localization of D-glucose.

本发明的有益效果：本发明提供的D-葡萄糖光学探针易于成熟，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D-葡萄糖，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D-葡萄糖光学探针对D-葡萄糖的最高响应达到对照的10倍以上，并且可以在细胞浆、细胞核、细胞膜、线粒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D-葡萄糖定量检测。The beneficial effects of this invention are as follows: The D-glucose optical probe provided by this invention is easy to mature, exhibits large fluorescence dynamics, and has good specificity. Furthermore, it can be expressed in cells through gene manipulation, enabling real-time, high-throughput, and quantitative detection of D-glucose both inside and outside cells, eliminating time-consuming sample processing steps. Experimental results show that the D-glucose optical probe provided by this application achieves a response to D-glucose that is more than 10 times that of the control. It can also perform localization, qualitative, and quantitative detection of D-glucose in subcellular structures such as the cytoplasm, nucleus, cell membrane, and mitochondria, and allows for high-throughput compound screening and quantitative detection of D-glucose in blood.

附图说明Attached Figure Description

图1为示例性D-葡萄糖光学探针的SDS-PAGE图；Figure 1 shows an SDS-PAGE image of an exemplary D-glucose optical probe;

图2为示例性D-葡萄糖光学探针的荧光光谱性质图；Figure 2 shows the fluorescence spectral properties of an exemplary D-glucose optical probe;

图3为示例性D-葡萄糖光学探针对不同浓度D-葡萄糖的滴定曲线；Figure 3 shows the titration curves of different concentrations of D-glucose using an exemplary D-glucose optical probe;

图4为示例性D-葡萄糖光学探针对另一底物和糖代谢中间物及相似物的特异性柱状图；Figure 4 is a bar chart showing the specificity of an exemplary D-glucose optical probe for another substrate and sugar metabolism intermediates and similar substances;

图5为示例性D-葡萄糖光学探针在哺乳动物细胞中的亚细胞器定位照片；Figure 5 shows a photograph of the subcellular organelle localization of an exemplary D-glucose optical probe in mammalian cells;

图6为对示例性D-葡萄糖光学探针在哺乳动物细胞中对胞浆中的D-葡萄糖浓度进行动态监测的示意图；Figure 6 is a schematic diagram of the dynamic monitoring of D-glucose concentration in the cytoplasm of mammalian cells using an exemplary D-glucose optical probe.

图7为示例性D-葡萄糖光学探针在活细胞水平进行高通量化合物筛选的点图；Figure 7 is a dot plot of an exemplary D-glucose optical probe used for high-throughput compound screening at the live cell level;

图8为示例性D-葡萄糖光学探针对小鼠和人血液中的D-葡萄糖进行定量的柱状图。Figure 8 is a bar chart showing the quantification of D-glucose in mouse and human blood using an exemplary D-glucose optical probe.

具体实施方式Detailed Implementation

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

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。The terms “comprising,” “including,” and their equivalents as used herein include the meanings of “containing” and “composed of,” for example, a composition “containing” X may consist of only X or may contain other substances, such as X+Y.

本文所用术语“D-葡萄糖敏感多肽”指对D-葡萄糖产生响应的多肽，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D-葡萄糖敏感多肽还可包括其功能变体。D-葡萄糖敏感多肽的功能变体包括但不限于可以与D-葡萄糖相互作用从而发生与亲本D-葡萄糖敏感多肽相同或相似变化的变体。As used herein, the term "D-glucose-sensitive peptide" refers to a peptide that responds to D-glucose, and the response includes any response to chemical, biological, electrical, or physiological parameters of the peptide in relation to the interaction with the sensitive peptide. Responses include small changes, such as changes in the orientation of amino acids or peptide fragments of the peptide, and changes in the primary, secondary, or tertiary structure of the peptide, 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; a conformational change occurs when the three-dimensional structure of the molecule changes. Examples of conformational changes include a change from an α-helix to a β-sheet or vice versa. It is understood that a detectable change need not be a conformational change, as long as the fluorescence of the fluorescent protein moiety is altered. The D-glucose-sensitive peptides described herein may also include their functional variants. Functional variants of D-glucose-sensitive peptides include, but are not limited to, variants that can interact with D-glucose to undergo the same or similar changes as the parental D-glucose-sensitive peptide.

本发明所述D-葡萄糖敏感多肽包括但不限于源自T型嗜热菌T.thermophilus的葡萄糖半乳糖结合蛋白TtGBP或与其有90％以上同源性的变体。D-葡萄糖结合蛋白可以感应D-葡萄糖浓度的变化，在D-葡萄糖浓度动态变化的过程中D-葡萄糖结合蛋白的空间构象也会发生改变。The D-glucose-sensitive polypeptides described in this invention include, but are not limited to, the glucose-galactose-binding protein TtGBP derived from the thermophilic bacterium *T. thermophilus*, or variants thereof with more than 90% homology. The D-glucose-binding protein can sense changes in D-glucose concentration, and its spatial conformation also changes during dynamic changes in D-glucose concentration.

本文所用术语“光学探针”是指与光学活性多肽融合的D-葡萄糖敏感多肽。发明人发现，D-葡萄糖敏感多肽例如D-葡萄糖结合蛋白专一性地对生理浓度的D-葡萄糖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D-葡萄糖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D-葡萄糖的存在和/或水平。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。As used herein, the term "optical probe" refers to a D-glucose-sensitive peptide fused to an optically active peptide. The inventors discovered that conformational changes resulting from the specific binding of D-glucose-sensitive peptides, such as D-glucose-binding proteins, to physiological concentrations of D-glucose induce conformational changes in optically active peptides (e.g., fluorescent proteins), thereby altering the optical properties of the optically active peptides. By plotting standard curves using fluorescence data of fluorescent proteins measured at different D-glucose concentrations, the presence and/or level of D-glucose can be detected and analyzed. When describing the optical probes of this invention (e.g., when describing insertion or mutation sites), all amino acid residue numbers are referenced to SEQ ID NO: 1.

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D-葡萄糖敏感多肽中。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。In the optical probes of the present invention, an optically active polypeptide (e.g., a fluorescent protein) is operatively inserted into a D-glucose-sensitive polypeptide. A protein-based "optically active polypeptide" is a polypeptide capable of emitting fluorescence. Fluorescence is an optical property of the optically active polypeptide that can be used as a means of detecting the responsiveness of the optical probes of the present invention. As used herein, the term "fluorescence property" refers to the molar extinction coefficient at an appropriate excitation wavelength, fluorescence quantum efficiency, shape of the excitation or emission spectrum, maximum excitation wavelength and maximum emission wavelength, amplitude of excitation at two different wavelengths, ratio of emission amplitudes at two different wavelengths, excited-state lifetime, or fluorescence anisotropy. A measurable difference in any of these properties between active and inactive states is sufficient for the utility of the fluorescent protein substrate of the present invention in activity assays. The measurable difference can be determined by determining the amount of any quantitative fluorescence property, for example, the amount of fluorescence at a specific wavelength or the integral of fluorescence over the emission spectrum. Preferably, the protein substrate is selected to have fluorescence properties that are easily distinguishable between inactive and activated conformational states. The optically active polypeptides described herein may also include functional variants thereof. Functional variants of optically active peptides include, but are not limited to, variants that can undergo changes in fluorescence properties that are the same as or similar to those of the parent optically active peptide.

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，cpGFP如SEQ ID NO：6所示，cpBFP如SEQ ID NO：7所示，cpmApple如SEQ ID NO：9所示。As used herein, the term "fluorescent protein" refers to a protein that emits fluorescence under excitation light. Fluorescent proteins are fundamental detection methods in the field of bioscience. Examples include the commonly used green fluorescent protein GFP and its cyclically rearranged derivatives such as blue fluorescent protein (cpBFP), green fluorescent protein (cpGFP), and yellow fluorescent protein (cpYFP); and the commonly used red fluorescent protein RFP, and its cyclically rearranged derivatives such as cpmApple, cpmOrange, and cpmKate. For example, cpYFP is shown in SEQ ID NO: 2, cpGFP in SEQ ID NO: 6, cpBFP in SEQ ID NO: 7, and cpmApple in SEQ ID NO: 9.

光学探针中的荧光蛋白也包括具有突变的功能变体，包括但不限于在对应于SEQ ID NO：2的第1位氨基酸具有突变的荧光蛋白。第1位的突变优选突变为A或G。在一些实施方案中，荧光蛋白的功能变体具有SEQ ID NO：2、6、7、9中任一所示的序列并在对应于SEQ ID NO：2的第1位氨基酸处具有选自以下任一组所示的突变：Y1A或Y1G。1S表示第1位氨基酸突变为S，以此类推。The fluorescent protein in the optical probe also includes functional variants with mutations, including but not limited to fluorescent proteins with a mutation at the first amino acid corresponding to SEQ ID NO: 2. The mutation at position 1 is preferably A or G. In some embodiments, the functional variant of the fluorescent protein has the sequence shown in any of SEQ ID NO: 2, 6, 7, 9 and has a mutation selected from any group of the following at the first amino acid corresponding to SEQ ID NO: 2: Y1A or Y1G. 1S indicates that the first amino acid is mutated to S, and so on.

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D-葡萄糖敏感多肽的残基266/267、266/268、266/269、266/270、266/271、266/272、266/273、266/274、266/275、266/276、266/277、266/278、266/279、266/280、266/281、266/282、266/283、266/284、266/285、266/286、266/287、266/288、267/267、267/268、267/269、267/270、267/271、267/272、267/273、267/274、267/275、267/276、267/277、267/278、267/279、267/280、267/281、267/282、267/283、267/284、267/285、267/286、267/287、267/288、268/267、268/268、268/269、268/270、268/271、268/272、268/273、268/274、268/275、268/276、268/277、268/278、268/279、268/280、268/281、268/282、268/283、268/284、268/285、268/286、268/287、268/288、269/267、269/268、269/269、269/270、269/271、269/272、269/273、269/274、269/275、269/276、269/277、269/278、269/279、269/280、269/281、269/282、269/283、269/284、269/285、269/286、269/287、269/288、270/267、270/268、270/269、270/270、270/271、270/272、270/273、270/274、270/275、270/276、270/277、270/278、270/279、270/280、270/281、270/282、270/283、270/284、270/285、270/286、270/287、270/288、271/267、271/268、271/269、271/270、271/271、271/272、271/273、271/274、271/275、271/276、271/277、271/278、271/279、271/280、271/281、271/282、271/283、271/284、271/285、271/286、271/287、271/288、272/267、272/268、272/269、272/270、272/271、272/272、272/273、272/274、272/275、272/276、272/277、272/278、272/279、272/280、272/281、272/282、272/283、272/284、272/285、272/286、272/287、272/288、273/267、273/268、273/269、273/270、273/271、273/272、273/273、273/274、273/275、273/276、273/277、273/278、273/279、273/280、273/281、273/282、273/283、273/284、273/285、273/286、273/287、273/288、274/267、274/268、274/269、274/270、274/271、274/272、274/273、274/274、274/275、274/276、274/277、274/278、274/279、274/280、274/281、274/282、274/283、274/284、274/285、274/286、274/287、274/288、275/267、275/268、275/269、275/270、275/271、275/272、275/273、275/274、275/275、275/276、275/277、275/278、275/279、275/280、275/281、275/282、275/283、275/284、275/285、275/286、275/287、275/288、276/267、276/268、276/269、276/270、276/271、276/272、276/273、276/274、276/275、276/276、276/277、276/278、276/279、276/280、276/281、276/282、276/283、276/284、276/285、276/286、276/287、276/288、277/267、277/268、277/269、277/270、277/271、277/272、277/273、277/274、277/275、277/276、277/277、277/278、277/279、277/280、277/281、277/282、277/283、277/284、277/285、277/286、277/287、277/288、278/267、278/268、278/269、278/270、278/271、278/272、278/273、278/274、278/275、278/276、278/277、278/278、278/279、278/280、278/281、278/282、278/283、278/284、278/285、278/286、278/287、278/288、279/267、279/268、279/269、279/270、279/271、279/272、279/273、279/274、279/275、279/276、279/277、279/278、279/279、279/280、279/281、279/282、279/283、279/284、279/285、279/286、279/287、279/288、280/267、280/268、280/269、280/270、280/271、280/272、280/273、280/274、280/275、280/276、280/277、280/278、280/279、280/280、280/281、280/282、280/283、280/284、280/285、280/286、280/287、280/288、281/267、281/268、281/269、281/270、281/271、281/272、281/273、281/274、281/275、281/276、281/277、281/278、281/279、281/280、281/281、281/282、281/283、281/284、281/285、281/286、281/287、281/288、282/267、282/268、282/269、282/270、282/271、282/272、282/273、282/274、282/275、282/276、282/277、282/278、282/279、282/280、282/281、282/282、282/283、282/284、282/285、282/286、282/287、282/288、283/267、283/268、283/269、283/270、283/271、283/272、283/273、283/274、283/275、283/276、283/277、283/278、283/279、283/280、283/281、283/282、283/283、283/284、283/285、283/286、283/287、283/288、284/267、284/268、284/269、284/270、284/271、284/272、284/273、284/274、284/275、284/276、284/277、284/278、284/279、284/280、284/281、284/282、284/283、284/284、284/285、284/286、284/287、284/288、285/267、285/268、285/269、285/270、285/271、285/272、285/273、285/274、285/275、285/276、285/277、285/278、285/279、285/280、285/281、285/282、285/283、285/284、285/285、285/286、285/287、285/288、286/267、286/268、286/269、286/270、286/271、286/272、286/273、286/274、286/275、286/276、286/277、286/278、286/279、286/280、286/281、286/282、286/283、286/284、286/285、286/286、286/287、286/288、287/267、287/268、287/269、287/270、287/271、287/272、287/273、287/274、287/275、287/276、287/277、287/278、287/279、287/280、287/281、287/282、287/283、287/284、287/285、287/286、287/287、287/288，117/118、117/119、117/120、117/121、117/122、117/123、118/118、118/119、118/120、118/121、118/122、118/123、119/118、119/119、119/120、119/121、119/122、119/123、120/118、120/119、120/120、120/121、120/122、120/123、121/118、121/119、121/120、121/121、121/122、121/123、122/118、122/119、122/120、122/121、122/122、122/123，340/341、340/342、340/343、340/344、340/345、340/346、340/347、340/348、340/349、340/350、340/351、340/352、340/353、341/341、341/342、341/343、341/344、341/345、341/346、341/347、341/348、341/349、341/350、341/351、341/352、341/353、342/341、342/342、342/343、342/344、342/345、342/346、342/347、342/348、342/349、342/350、342/351、342/352、342/353、343/341、343/342、343/343、343/344、343/345、343/346、343/347、343/348、343/349、343/350、343/351、343/352、343/353、344/341、344/342、344/343、344/344、344/345、344/346、344/347、344/348、344/349、344/350、344/351、344/352、344/353、345/341、345/342、345/343、345/344、345/345、345/346、345/347、345/348、345/349、345/350、345/351、345/352、345/353、346/341、346/342、346/343、346/344、346/345、346/346、346/347、346/348、346/349、346/350、346/351、346/352、346/353、347/341、347/342、347/343、347/344、347/345、347/346、347/347、347/348、347/349、347/350、347/351、347/352、347/353、348/341、348/342、348/343、348/344、348/345、348/346、348/347、348/348、348/349、348/350、348/351、348/352、348/353、349/341、349/342、349/343、349/344、349/345、349/346、349/347、349/348、349/349、349/350、349/351、349/352、349/353、350/341、350/342、350/343、350/344、350/345、350/346、350/347、350/348、350/349、350/350、350/351、350/352、350/353、351/341、351/342、351/343、351/344、351/345、351/346、351/347、351/348、351/349、351/350、351/351、351/352、351/353、352/341、352/342、352/343、352/344、352/345、352/346、352/347、352/348、352/349、352/350、352/351、352/352和352/353中或置换其中的残基，编号对应于D-葡萄糖敏感多肽的全长。本文中，在“X/Y”形式表示的位点中，光学活性多肽的两端分别具有部分D-葡萄糖敏感多肽，其中光学活性多肽的N端为D-葡萄糖敏感多肽序列的N端起始氨基酸(例如第1位至第266位的任一氨基酸)至第X位氨基酸，光学活性多肽的C端为D-葡萄糖敏感多肽序列的第Y位氨基酸至其C端末尾氨基酸(例如第Y位任一氨基酸至第394位氨基酸)。其中，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266/267表示光学活性多肽位于D-葡萄糖敏感多肽的氨基酸266与267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且X小于Y，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266/269表示光学活性多肽置换D-葡萄糖敏感多肽的氨基酸267-268；如果以“X/Y”形式表示的位点中的X大于Y，则表示位于光学活性多肽的N端的D-葡萄糖敏感多肽部分至D-葡萄糖敏感多肽序列的第X位氨基酸终止，而位于光学活性多肽的C端的D-葡萄糖敏感多肽部分从D-葡萄糖敏感多肽序列的第Y位氨基酸开始；例如插入位点346/344表示光学活性多肽的N端融合有D-葡萄糖敏感多肽序列的N端起始氨基酸(例如第1位至第394位的任一氨基酸)至第346位氨基酸，并且光学活性多肽的C端融合有D-葡萄糖敏感多肽序列的第344位氨基酸至C端末尾氨基酸(例如第394位氨基酸)，其示例性的结构为：(D-葡萄糖敏感多肽序列的第1至346位氨基酸)-(光学活性多肽)-(D-葡萄糖敏感多肽序列的第344至394位氨基酸)；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且X等于Y，则表示光学活性多肽插入该数字所示氨基酸之间的氨基酸，例如插入位点267/267表示光学活性多肽插入肌醇敏感多肽的氨基酸267与268之间。在示例性实施方式中，SEQ ID NO：2、6、7或9所示的光学活性多肽位于SEQ ID NO：1所示的D-葡萄糖敏感多肽的选自下述位点中的任一个或多个：266/267、266/268、266/269、266/270、266/271、266/272、266/273、266/274、266/275、266/276、266/277、266/278、266/279、266/280、266/281、266/282、266/283、266/284、266/285、266/286、266/287、266/288、267/267、267/268、267/269、267/270、267/271、267/272、267/273、267/274、267/275、267/276、267/277、267/278、267/279、267/280、267/281、267/282、267/283、267/284、267/285、267/286、267/287、267/288、268/267、268/268、268/269、268/270、268/271、268/272、268/273、268/274、268/275、268/276、268/277、268/278、268/279、268/280、268/281、268/282、268/283、268/284、268/285、268/286、268/287、268/288、269/267、269/268、269/269、269/270、269/271、269/272、269/273、269/274、269/275、269/276、269/277、269/278、269/279、269/280、269/281、269/282、269/283、269/284、269/285、269/286、269/287、269/288、270/267、270/268、270/269、270/270、270/271、270/272、270/273、270/274、270/275、270/276、270/277、270/278、270/279、270/280、270/281、270/282、270/283、270/284、270/285、270/286、270/287、270/288、271/267、271/268、271/269、271/270、271/271、271/272、271/273、271/274、271/275、271/276、271/277、271/278、271/279、271/280、271/281、271/282、271/283、271/284、271/285、271/286、271/287、271/288、272/267、272/268、272/269、272/270、272/271、272/272、272/273、272/274、272/275、272/276、272/277、272/278、272/279、272/280、272/281、272/282、272/283、272/284、272/285、272/286、272/287、272/288、273/267、273/268、273/269、273/270、273/271、273/272、273/273、273/274、273/275、273/276、273/277、273/278、273/279、273/280、273/281、273/282、273/283、273/284、273/285、273/286、273/287、273/288、274/267、274/268、274/269、274/270、274/271、274/272、274/273、274/274、274/275、274/276、274/277、274/278、274/279、274/280、274/281、274/282、274/283、274/284、274/285、274/286、274/287、274/288、275/267、275/268、275/269、275/270、275/271、275/272、275/273、275/274、275/275、275/276、275/277、275/278、275/279、275/280、275/281、275/282、275/283、275/284、275/285、275/286、275/287、275/288、276/267、276/268、276/269、276/270、276/271、276/272、276/273、276/274、276/275、276/276、276/277、276/278、276/279、276/280、276/281、276/282、276/283、276/284、276/285、276/286、276/287、276/288、277/267、277/268、277/269、277/270、277/271、277/272、277/273、277/274、277/275、277/276、277/277、277/278、277/279、277/280、277/281、277/282、277/283、277/284、277/285、277/286、277/287、277/288、278/267、278/268、278/269、278/270、278/271、278/272、278/273、278/274、278/275、278/276、278/277、278/278、278/279、278/280、278/281、278/282、278/283、278/284、278/285、278/286、278/287、278/288、279/267、279/268、279/269、279/270、279/271、279/272、279/273、279/274、279/275、279/276、279/277、279/278、279/279、279/280、279/281、279/282、279/283、279/284、279/285、279/286、279/287、279/288、280/267、280/268、280/269、280/270、280/271、280/272、280/273、280/274、280/275、280/276、280/277、280/278、280/279、280/280、280/281、280/282、280/283、280/284、280/285、280/286、280/287、280/288、281/267、281/268、281/269、281/270、281/271、281/272、281/273、281/274、281/275、281/276、281/277、281/278、281/279、281/280、281/281、281/282、281/283、281/284、281/285、281/286、281/287、281/288、282/267、282/268、282/269、282/270、282/271、282/272、282/273、282/274、282/275、282/276、282/277、282/278、282/279、282/280、282/281、282/282、282/283、282/284、282/285、282/286、282/287、282/288、283/267、283/268、283/269、283/270、283/271、283/272、283/273、283/274、283/275、283/276、283/277、283/278、283/279、283/280、283/281、283/282、283/283、283/284、283/285、283/286、283/287、283/288、284/267、284/268、284/269、284/270、284/271、284/272、284/273、284/274、284/275、284/276、284/277、284/278、284/279、284/280、284/281、284/282、284/283、284/284、284/285、284/286、284/287、284/288、285/267、285/268、285/269、285/270、285/271、285/272、285/273、285/274、285/275、285/276、285/277、285/278、285/279、285/280、285/281、285/282、285/283、285/284、285/285、285/286、285/287、285/288、286/267、286/268、286/269、286/270、286/271、286/272、286/273、286/274、286/275、286/276、286/277、286/278、286/279、286/280、286/281、286/282、286/283、286/284、286/285、286/286、286/287、286/288、287/267、287/268、287/269、287/270、287/271、287/272、287/273、287/274、287/275、287/276、287/277、287/278、287/279、287/280、287/281、287/282、287/283、287/284、287/285、287/286、287/287、287/288，117/118、117/119、117/120、117/121、117/122、117/123、118/118、118/119、118/120、118/121、118/122、118/123、119/118、119/119、119/120、119/121、119/122、119/123、120/118、120/119、120/120、120/121、120/122、120/123、121/118、121/119、121/120、121/121、121/122、121/123、122/118、122/119、122/120、122/121、122/122、122/123，340/341、340/342、340/343、340/344、340/345、340/346、340/347、340/348、340/349、340/350、340/351、340/352、340/353、341/341、341/342、341/343、341/344、341/345、341/346、341/347、341/348、341/349、341/350、341/351、341/352、341/353、342/341、342/342、342/343、342/344、342/345、342/346、342/347、342/348、342/349、342/350、342/351、342/352、342/353、343/341、343/342、343/343、343/344、343/345、343/346、343/347、343/348、343/349、343/350、343/351、343/352、343/353、344/341、344/342、344/343、344/344、344/345、344/346、344/347、344/348、344/349、344/350、344/351、344/352、344/353、345/341、345/342、345/343、345/344、345/345、345/346、345/347、345/348、345/349、345/350、345/351、345/352、345/353、346/341、346/342、346/343、346/344、346/345、346/346、346/347、346/348、346/349、346/350、346/351、346/352、346/353、347/341、347/342、347/343、347/344、347/345、347/346、347/347、347/348、347/349、347/350、347/351、347/352、347/353、348/341、348/342、348/343、348/344、348/345、348/346、348/347、348/348、348/349、348/350、348/351、348/352、348/353、349/341、349/342、349/343、349/344、349/345、349/346、349/347、349/348、349/349、349/350、349/351、349/352、349/353、350/341、350/342、350/343、350/344、350/345、350/346、350/347、350/348、350/349、350/350、350/351、350/352、350/353、351/341、351/342、351/343、351/344、351/345、351/346、351/347、351/348、351/349、351/350、351/351、351/352、351/353、352/341、352/342、352/343、352/344、352/345、352/346、352/347、352/348、352/349、352/350、352/351、352/352和352/353。In the optical probe of the present invention, the optically active polypeptide is located in the N-C direction in the residues 266/267, 266/268, 266/269, 266/270, 266/271, 266/272, 266/273, 266/274, 266/275, 266/276, 266/277, 266/278, 266/279, 266/280, 266/281, 266/282, 266... /283, 266/284, 266/285, 266/286, 266/287, 266/288, 267/267, 267/268, 267/269, 267/270, 267/271, 267/272, 267/273, 267/274, 267/275, 267/276, 267/277, 267/278, 267/279, 267/280, 267/281, 267/282, 2 67/283, 267/284, 267/285, 267/286, 267/287, 267/288, 268/267, 268/268, 268/269, 268/270, 268/271, 268/272, 268/273, 268/274, 268/275, 268/276, 268/277, 268/278, 268/279, 268/280, 268/281, 268/282 268/283, 268/284, 268/285, 268/286, 268/287, 268/288, 269/267, 269/268, 269/269, 269/270, 269/271, 269/272, 269/273, 269/274, 269/275, 269/276, 269/277, 269/278, 269/279, 269/280, 269/281, 269/28 2, 269/283, 269/284, 269/285, 269/286, 269/287, 269/288, 270/267, 270/268, 270/269, 270/270, 270/271, 270/272, 270/273, 270/274, 270/275, 270/276, 270/277, 270/278, 270/279, 270/280, 270/281, 270/ 282, 270/283, 270/284, 270/285, 270/286, 270/287, 270/288, 271/267, 271/268, 271/269, 271/270, 271/271, 271/272, 271/273, 271/274, 271/275, 271/276, 271/277, 271/278, 271/279, 271/280, 271/281, 271 /282, 271/283, 271/284, 271/285, 271/286, 271/287, 271/288, 272/267, 272/268, 272/269, 272/270, 272/271, 272/272, 272/273, 272/274, 272/275, 272/276, 272/277, 272/278, 272/279, 272/280, 272/281, 2 72/282, 272/283, 272/284, 272/285, 272/286, 272/287, 272/288, 273/267, 273/268, 273/269, 273/270, 273/271, 273/272, 273/273, 273/274, 273/275, 273/276, 273/277, 273/278, 273/279, 273/280, 273/281 273/282, 273/283, 273/284, 273/285, 273/286, 273/287, 273/288, 274/267, 274/268, 274/269, 274/270, 274/271, 274/272, 274/273, 274/274, 274/275, 274/276, 274/277, 274/278, 274/279, 274/280, 274/28 1, 274/282, 274/283, 274/284, 274/285, 274/286, 274/287, 274/288, 275/267, 275/268, 275/269, 275/270, 275/271, 275/272, 275/273, 275/274, 275/275, 275/276, 275/277, 275/278, 275/279, 275/280, 275/2 81, 275/282, 275/283, 275/284, 275/285, 275/286, 275/287, 275/288, 276/267, 276/268, 276/269, 276/270, 276/271, 276/272, 276/273, 276/274, 276/275, 276/276, 276/277, 276/278, 276/279, 276/280, 276 /281, 276/282, 276/283, 276/284, 276/285, 276/286, 276/287, 276/288, 277/267, 277/268, 277/269, 277/270, 277/271, 277/272, 277/273, 277/274, 277/275, 277/276, 277/277, 277/278, 277/279, 277/280, 27 7/281, 277/282, 277/283, 277/284, 277/285, 277/286, 277/287, 277/288, 278/267, 278/268, 278/269, 278/270, 278/271, 278/272, 278/273, 278/274, 278/275, 278/276, 278/277, 278/278, 278/279, 278/280 278/281, 278/282, 278/283, 278/284, 278/285, 278/286, 278/287, 278/288, 279/267, 279/268, 279/269, 279/270, 279/271, 279/272, 279/273, 279/274, 279/275, 279/276, 279/277, 279/278, 279/279, 279/280 279/281, 279/282, 279/283, 279/284, 279/285, 279/286, 279/287, 279/288, 280/267, 280/268, 280/269, 280/270, 280/271, 280/272, 280/273, 280/274, 280/275, 280/276, 280/277, 280/278, 280/279, 280/2 80, 280/281, 280/282, 280/283, 280/284, 280/285, 280/286, 280/287, 280/288, 281/267, 281/268, 281/269, 281/270, 281/271, 281/272, 281/273, 281/274, 281/275, 281/276, 281/277, 281/278, 281/279, 281/ 280, 281/281, 281/282, 281/283, 281/284, 281/285, 281/286, 281/287, 281/288, 282/267, 282/268, 282/269, 282/270, 282/271, 282/272, 282/273, 282/274, 282/275, 282/276, 282/277, 282/278, 282/279, 28 2/280, 282/281, 282/282, 282/283, 282/284, 282/285, 282/286, 282/287, 282/288, 283/267, 283/268, 283/269, 283/270, 283/271, 283/272, 283/273, 283/274, 283/275, 283/276, 283/277, 283/278, 283/279 283/280, 283/281, 283/282, 283/283, 283/284, 283/285, 283/286, 283/287, 283/288, 284/267, 284/268, 284/269, 284/270, 284/271, 284/272, 284/273, 284/274, 284/275, 284/276, 284/277, 284/278, 284/27 9, 284/280, 284/281, 284/282, 284/283, 284/284, 284/285, 284/286, 284/287, 284/288, 285/267, 285/268, 285/269, 285/270, 285/271, 285/272, 285/273, 285/274, 285/275, 285/276, 285/277, 285/278, 285/2 79, 285/280, 285/281, 285/282, 285/283, 285/284, 285/285, 285/286, 285/287, 285/288, 286/267, 286/268, 286/269, 286/270, 286/271, 286/272, 286/273, 286/274, 286/275, 286/276, 286/277, 286/278, 286 /279, 286/280, 286/281, 286/282, 286/283, 286/284, 286/285, 286/286, 286/287, 286/288, 287/267, 287/268, 287/269, 287/270, 287/271, 287/272, 287/273, 287/274, 287/275, 287/276, 287/277, 287/278, 28 7/279, 287/280, 287/281, 287/282, 287/283, 287/284, 287/285, 287/286, 287/287, 287/288, 117/118, 117/119, 117/120, 117/121, 117/122, 117/123, 118/118, 118/119, 118/120, 118/121, 118/122, 118/123 119/118, 119/119, 119/120, 119/121, 119/122, 119/123, 120/118, 120/119, 120/120, 120/121, 120/122, 120/123, 121/118, 121/119, 121/120, 121/121, 121/122, 121/123, 122/118, 122/119, 122/120, 122/121 122/122, 122/123, 340/341, 340/342, 340/343, 340/344, 340/345, 340/346, 340/347, 340/348, 340/349, 340/350, 340/351, 340/352, 340/353, 341/341, 341/342, 341/343, 341/344, 341/345, 341/346, 341/3 47, 341/348, 341/349, 341/350, 341/351, 341/352, 341/353, 342/341, 342/342, 342/343, 342/344, 342/345, 342/346, 342/347, 342/348, 342/349, 342/350, 342/351, 342/352, 342/353, 343/341, 343/342, 343/ 343, 343/344, 343/345, 343/346, 343/347, 343/348, 343/349, 343/350, 343/351, 343/352, 343/353, 344/341, 344/342, 344/343, 344/344, 344/345, 344/346, 344/347, 344/348, 344/349, 344/350, 344/351, 34 4/352, 344/353, 345/341, 345/342, 345/343, 345/344, 345/345, 345/346, 345/347, 345/348, 345/349, 345/350, 345/351, 345/352, 345/353, 346/341, 346/342, 346/343, 346/344, 346/345, 346/346, 346/347, 3 46/348, 346/349, 346/350, 346/351, 346/352, 346/353, 347/341, 347/342, 347/343, 347/344, 347/345, 347/346, 347/347, 347/348, 347/349, 347/350, 347/351, 347/352, 347/353, 348/341, 348/342, 348/343 348/344, 348/345, 348/346, 348/347, 348/348, 348/349, 348/350, 348/351, 348/352, 348/353, 349/341, 349/342, 349/343, 349/344, 349/345, 349/346, 349/347, 349/348, 349/349, 349/350, 349/351, 349/35 2, 349/353, 350/341, 350/342, 350/343, 350/344, 350/345, 350/346, 350/347, 350/348, 350/349, 350/350, 350/351, 350/352, 350/353, 351/341, 351/342, 351/343, 351/344, 351/345, 351/346, 351/347, 351/ The residues in or replaced by 348, 351/349, 351/350, 351/351, 351/352, 351/353, 352/341, 352/342, 352/343, 352/344, 352/345, 352/346, 352/347, 352/348, 352/349, 352/350, 352/351, 352/352, and 352/353 correspond to the full length of the D-glucose-sensitive polypeptide. In this paper, at sites represented in the form of "X/Y", the optically active polypeptide has partial D-glucose-sensitive polypeptides at both ends. The N-terminus of the optically active polypeptide is the N-terminal starting amino acid (e.g., any amino acid from position 1 to position 266) to the Xth amino acid of the D-glucose-sensitive polypeptide sequence, and the C-terminus of the optically active polypeptide is the Yth amino acid to the C-terminal ending amino acid (e.g., any amino acid from position Y to position 394) of the D-glucose-sensitive polypeptide sequence. In this context, if the two numbers in the "X/Y" format are consecutive integers, it indicates that the optically active polypeptide is located between the amino acids represented by those numbers. For example, insertion site 266/267 indicates that the optically active polypeptide is located between amino acids 266 and 267 of the D-glucose-sensitive polypeptide. If the two numbers in the "X/Y" format are not consecutive integers and X is less than Y, it indicates that the optically active polypeptide replaces the amino acids between those numbers. For example, insertion site 266/269 indicates that the optically active polypeptide replaces amino acids 267-268 of the D-glucose-sensitive polypeptide. If X is greater than Y in the "X/Y" format, it indicates that the D-glucose-sensitive polypeptide portion located at the N-terminus of the optically active polypeptide terminates at the Xth amino acid in the D-glucose-sensitive polypeptide sequence, while the D-glucose-sensitive polypeptide portion located at the C-terminus of the optically active polypeptide terminates at the Xth amino acid in the D-glucose-sensitive polypeptide sequence. Starting at amino acid position Y; for example, insertion site 346/344 indicates that the N-terminus of the optically active polypeptide is fused with the N-terminal starting amino acid (e.g., any amino acid from position 1 to 394) to amino acid 346 of the D-glucose-sensitive polypeptide sequence, and the C-terminus of the optically active polypeptide is fused with the amino acid from position 344 to the C-terminal ending amino acid (e.g., amino acid 394) of the D-glucose-sensitive polypeptide sequence, with an exemplary structure of: (amino acids 1 to 346 of the D-glucose-sensitive polypeptide sequence) - (optically active polypeptide) - (amino acids 344 to 394 of the D-glucose-sensitive polypeptide sequence); if the two numbers in the site represented in the form of "X/Y" are not consecutive integers and X equals Y, it indicates that the optically active polypeptide is inserted between the amino acids indicated by that number, for example, insertion site 267/267 indicates that the optically active polypeptide is inserted between amino acids 267 and 268 of the inositol-sensitive polypeptide. In an exemplary embodiment, the optically active polypeptide represented by SEQ ID NO: 2, 6, 7, or 9 is located at any one or more of the following sites of the D-glucose-sensitive polypeptide represented by SEQ ID NO: 1: 266/267, 266/268, 266/269, 266/270, 266/271, 266/272, 266/273, 266/274, 266/275, 266/276, 266/277, 266/278, 2 66/279, 266/280, 266/281, 266/282, 266/283, 266/284, 266/285, 266/286, 266/287, 266/288, 267/267, 267/268, 267/269, 267/270, 267/271, 267/272, 267/273, 267/274, 267/275, 267/276, 267/277, 267/278 267/279, 267/280, 267/281, 267/282, 267/283, 267/284, 267/285, 267/286, 267/287, 267/288, 268/267, 268/268, 268/269, 268/270, 268/271, 268/272, 268/273, 268/274, 268/275, 268/276, 268/277, 268/27 8, 268/279, 268/280, 268/281, 268/282, 268/283, 268/284, 268/285, 268/286, 268/287, 268/288, 269/267, 269/268, 269/269, 269/270, 269/271, 269/272, 269/273, 269/274, 269/275, 269/276, 269/277, 269/2 78, 269/279, 269/280, 269/281, 269/282, 269/283, 269/284, 269/285, 269/286, 269/287, 269/288, 270/267, 270/268, 270/269, 270/270, 270/271, 270/272, 270/273, 270/274, 270/275, 270/276, 270/277, 270 /278, 270/279, 270/280, 270/281, 270/282, 270/283, 270/284, 270/285, 270/286, 270/287, 270/288, 271/267, 271/268, 271/269, 271/270, 271/271, 271/272, 271/273, 271/274, 271/275, 271/276, 271/277, 27 1/278, 271/279, 271/280, 271/281, 271/282, 271/283, 271/284, 271/285, 271/286, 271/287, 271/288, 272/267, 272/268, 272/269, 272/270, 272/271, 272/272, 272/273, 272/274, 272/275, 272/276, 272/277, 2 72/278, 272/279, 272/280, 272/281, 272/282, 272/283, 272/284, 272/285, 272/286, 272/287, 272/288, 273/267, 273/268, 273/269, 273/270, 273/271, 273/272, 273/273, 273/274, 273/275, 273/276, 273/277 273/278, 273/279, 273/280, 273/281, 273/282, 273/283, 273/284, 273/285, 273/286, 273/287, 273/288, 274/267, 274/268, 274/269, 274/270, 274/271, 274/272, 274/273, 274/274, 274/275, 274/276, 274/27 7, 274/278, 274/279, 274/280, 274/281, 274/282, 274/283, 274/284, 274/285, 274/286, 274/287, 274/288, 275/267, 275/268, 275/269, 275/270, 275/271, 275/272, 275/273, 275/274, 275/275, 275/276, 275/2 77, 275/278, 275/279, 275/280, 275/281, 275/282, 275/283, 275/284, 275/285, 275/286, 275/287, 275/288, 276/267, 276/268, 276/269, 276/270, 276/271, 276/272, 276/273, 276/274, 276/275, 276/276, 276 /277, 276/278, 276/279, 276/280, 276/281, 276/282, 276/283, 276/284, 276/285, 276/286, 276/287, 276/288, 277/267, 277/268, 277/269, 277/270, 277/271, 277/272, 277/273, 277/274, 277/275, 277/276, 27 7/277, 277/278, 277/279, 277/280, 277/281, 277/282, 277/283, 277/284, 277/285, 277/286, 277/287, 277/288, 278/267, 278/268, 278/269, 278/270, 278/271, 278/272, 278/273, 278/274, 278/275, 278/276 278/277, 278/278, 278/279, 278/280, 278/281, 278/282, 278/283, 278/284, 278/285, 278/286, 278/287, 278/288, 279/267, 279/268, 279/269, 279/270, 279/271, 279/272, 279/273, 279/274, 279/275, 279/276 279/277, 279/278, 279/279, 279/280, 279/281, 279/282, 279/283, 279/284, 279/285, 279/286, 279/287, 279/288, 280/267, 280/268, 280/269, 280/270, 280/271, 280/272, 280/273, 280/274, 280/275, 280/27 6, 280/277, 280/278, 280/279, 280/280, 280/281, 280/282, 280/283, 280/284, 280/285, 280/286, 280/287, 280/288, 281/267, 281/268, 281/269, 281/270, 281/271, 281/272, 281/273, 281/274, 281/275, 281/2 76, 281/277, 281/278, 281/279, 281/280, 281/281, 281/282, 281/283, 281/284, 281/285, 281/286, 281/287, 281/288, 282/267, 282/268, 282/269, 282/270, 282/271, 282/272, 282/273, 282/274, 282/275, 282 /276, 282/277, 282/278, 282/279, 282/280, 282/281, 282/282, 282/283, 282/284, 282/285, 282/286, 282/287, 282/288, 283/267, 283/268, 283/269, 283/270, 283/271, 283/272, 283/273, 283/274, 283/275, 28 3/276, 283/277, 283/278, 283/279, 283/280, 283/281, 283/282, 283/283, 283/284, 283/285, 283/286, 283/287, 283/288, 284/267, 284/268, 284/269, 284/270, 284/271, 284/272, 284/273, 284/274, 284/275, 284/276, 284/277, 284/278, 284/279, 284/280, 284/281, 284/282, 284/283, 284/284, 284/285, 284/286, 284/287, 284/288, 285/267, 285/268, 285/269, 285/270, 285/271, 285/272, 285/273, 285/274, 285/275 285/276, 285/277, 285/278, 285/279, 285/280, 285/281, 285/282, 285/283, 285/284, 285/285, 285/286, 285/287, 285/288, 286/267, 286/268, 286/269, 286/270, 286/271, 286/272, 286/273, 286/274, 286/2 75, 286/276, 286/277, 286/278, 286/279, 286/280, 286/281, 286/282, 286/283, 286/284, 286/285, 286/286, 286/287, 286/288, 287/267, 287/268, 287/269, 287/270, 287/271, 287/272, 287/273, 287/274, 287/ 275, 287/276, 287/277, 287/278, 287/279, 287/280, 287/281, 287/282, 287/283, 287/284, 287/285, 287/286, 287/287, 287/288, 117/118, 117/119, 117/120, 117/121, 117/122, 117/123, 118/118, 118/119, 118 /120, 118/121, 118/122, 118/123, 119/118, 119/119, 119/120, 119/121, 119/122, 119/123, 120/118, 120/119, 120/120, 120/121, 120/122, 120/123, 121/118, 121/119, 121/120, 121/121, 121/122, 121/123, 12 2/118, 122/119, 122/120, 122/121, 122/122, 122/123, 340/341, 340/342, 340/343, 340/344, 340/345, 340/346, 340/347, 340/348, 340/349, 340/350, 340/351, 340/352, 340/353, 341/341, 341/342, 341/343, 341/344, 341/345, 341/346, 341/347, 341/348, 341/349, 341/350, 341/351, 341/352, 341/353, 342/341, 342/342, 342/343, 342/344, 342/345, 342/346, 342/347, 342/348, 342/349, 342/350, 342/351, 342/352 342/353, 343/341, 343/342, 343/343, 343/344, 343/345, 343/346, 343/347, 343/348, 343/349, 343/350, 343/351, 343/352, 343/353, 344/341, 344/342, 344/343, 344/344, 344/345, 344/346, 344/347, 344/34 8, 344/349, 344/350, 344/351, 344/352, 344/353, 345/341, 345/342, 345/343, 345/344, 345/345, 345/346, 345/347, 345/348, 345/349, 345/350, 345/351, 345/352, 345/353, 346/341, 346/342, 346/343, 346/3 44, 346/345, 346/346, 346/347, 346/348, 346/349, 346/350, 346/351, 346/352, 346/353, 347/341, 347/342, 347/343, 347/344, 347/345, 347/346, 347/347, 347/348, 347/349, 347/350, 347/351, 347/352, 347 /353, 348/341, 348/342, 348/343, 348/344, 348/345, 348/346, 348/347, 348/348, 348/349, 348/350, 348/351, 348/352, 348/353, 349/341, 349/342, 349/343, 349/344, 349/345, 349/346, 349/347, 349/348, 34 9/349, 349/350, 349/351, 349/352, 349/353, 350/341, 350/342, 350/343, 350/344, 350/345, 350/346, 350/347, 350/348, 350/349, 350/350, 350/351, 350/352, 350/353, 351/341, 351/342, 351/343, 351/344, 3 51/345, 351/346, 351/347, 351/348, 351/349, 351/350, 351/351, 351/352, 351/353, 352/341, 352/342, 352/343, 352/344, 352/345, 352/346, 352/347, 352/348, 352/349, 352/350, 352/351, 352/352 and 352/353.

在一个或多个实施方案中，光学探针从N端至C端依次包含，SEQ ID NO：1的第1-X位残基、SEQ ID NO：2、6、7、9中任一项所示的光学活性多肽或其变体、和SEQ ID NO：1的第Y-394位残基，其中，X和Y选自以下任一组：In one or more embodiments, the optical probe comprises, from the N-terminus to the C-terminus, residues 1-X of SEQ ID NO: 1, an optically active polypeptide or a variant thereof shown in any one of SEQ ID NO: 2, 6, 7, 9, and residues Y-394 of SEQ ID NO: 1, wherein X and Y are selected from any one of the following groups:

(1)X是266，Y是267，(2)X是266，Y是268，(3)X是266，Y是269，(4)X是266，Y是270，(5)X是266，Y是271，(6)X是266，Y是272，(7)X是266，Y是273，(8)X是266，Y是274，(9)X是266，Y是275，(10)X是266，Y是276，(11)X是266，Y是277，(12)X是266，Y是278，(13)X是266，Y是279，(14)X是266，Y是280，(15)X是266，Y是281，(16)X是266，Y是282，(17)X是266，Y是283，(18)X是266，Y是284，(19)X是266，Y是285，(20)X是266，Y是286，(21)X是266，Y是287，(22)X是266，Y是288，(23)X是267，Y是267，(24)X是267，Y是268，(25)X是267，Y是269，(26)X是267，Y是270，(27)X是267，Y是271，(28)X是267，Y是272，(29)X是267，Y是273，(30)X是267，Y是274，(31)X是267，Y是275，(32)X是267，Y是276，(33)X是267，Y是277，(34)X是267，Y是278，(35)X是267，Y是279，(36)X是267，Y是280，(37)X是267，Y是281，(38)X是267，Y是282，(39)X是267，Y是283，(40)X是267，Y是284，(41)X是267，Y是285，(42)X是267，Y是286，(43)X是267，Y是287，(44)X是267，Y是288，(45)X是268，Y是267，(46)X是268，Y是268，(47)X是268，Y是269，(48)X是268，Y是270，(49)X是268，Y是271，(50)X是268，Y是272，(51)X是268，Y是273，(52)X是268，Y是274，(53)X是268，Y是275，(54)X是268，Y是276，(55)X是268，Y是277，(56)X是268，Y是278，(57)X是268，Y是279，(58)X是268，Y是280，(59)X是268，Y是281，(60)X是268，Y是282，(61)X是268，Y是283，(62)X是268，Y是284，(63)X是268，Y是285，(64)X是268，Y是286，(65)X是268，Y是287，(66)X是268，Y是288，(67)X是269，Y是267，(68)X是269，Y是268，(69)X是269，Y是269，(70)X是269，Y是270，(71)X是269，Y是271，(72)X是269，Y是272，(73)X是269，Y是273，(74)X是269，Y是274，(75)X是269，Y是275，(76)X是269，Y是276，(77)X是269，Y是277，(78)X是269，Y是278，(79)X是269，Y是279，(80)X是269，Y是280，(81)X是269，Y是281，(82)X是269，Y是282，(83)X是269，Y是283，(84)X是269，Y是284，(85)X是269，Y是285，(86)X是269，Y是286，(87)X是269，Y是287，(88)X是269，Y是288，(89)X是270，Y是267，(90)X是270，Y是268，(91)X是270，Y是269，(92)X是270，Y是270，(93)X是270，Y是271，(94)X是270，Y是272，(95)X是270，Y是273，(96)X是270，Y是274，(97)X是270，Y是275，(98)X是270，Y是276，(99)X是270，Y是277，(100)X是270，Y是278，(101)X是270，Y是279，(102)X是270，Y是280，(103)X是270，Y是281，(104)X是270，Y是282，(105)X是270，Y是283，(106)X是270，Y是284，(107)X是270，Y是285，(108)X是270，Y是286，(109)X是270，Y是287，(110)X是270，Y是288，(111)X是271，Y是267，(112)X是271，Y是268，(113)X是271，Y是269，(114)X是271，Y是270，(115)X是271，Y是271，(116)X是271，Y是272，(117)X是271，Y是273，(118)X是271，Y是274，(119)X是271，Y是275，(120)X是271，Y是276，(121)X是271，Y是277，(122)X是271，Y是278，(123)X是271，Y是279，(124)X是271，Y是280，(125)X是271，Y是281，(126)X是271，Y是282，(127)X是271，Y是283，(128)X是271，Y是284，(129)X是271，Y是285，(130)X是271，Y是286，(131)X是271，Y是287，(132)X是271，Y是288，(133)X是272，Y是267，(134)X是272，Y是268，(135)X是272，Y是269，(136)X是272，Y是270，(137)X是272，Y是271，(138)X是272，Y是272，(139)X是272，Y是273，(140)X是272，Y是274，(141)X是272，Y是275，(142)X是272，Y是276，(143)X是272，Y是277，(144)X是272，Y是278，(145)X是272，Y是279，(146)X是272，Y是280，(147)X是272，Y是281，(148)X是272，Y是282，(149)X是272，Y是283，(150)X是272，Y是284，(151)X是272，Y是285，(152)X是272，Y是286，(153)X是272，Y是287，(154)X是272，Y是288，(155)X是273，Y是267，(156)X是273，Y是268，(157)X是273，Y是269，(158)X是273，Y是270，(159)X是273，Y是271，(160)X是273，Y是272，(161)X是273，Y是273，(162)X是273，Y是274，(163)X是273，Y是275，(164)X是273，Y是276，(165)X是273，Y是277，(166)X是273，Y是278，(167)X是273，Y是279，(168)X是273，Y是280，(169)X是273，Y是281，(170)X是273，Y是282，(171)X是273，Y是283，(172)X是273，Y是284，(173)X是273，Y是285，(174)X是273，Y是286，(175)X是273，Y是287，(176)X是273，Y是288，(177)X是274，Y是267，(178)X是274，Y是268，(179)X是274，Y是269，(180)X是274，Y是270，(181)X是274，Y是271，(182)X是274，Y是272，(183)X是274，Y是273，(184)X是274，Y是274，(185)X是274，Y是275，(186)X是274，Y是276，(187)X是274，Y是277，(188)X是274，Y是278，(189)X是274，Y是279，(190)X是274，Y是280，(191)X是274，Y是281，(192)X是274，Y是282，(193)X是274，Y是283，(194)X是274，Y是284，(195)X是274，Y是285，(196)X是274，Y是286，(197)X是274，Y是287，(198)X是274，Y是288，(199)X是275，Y是267，(200)X是275，Y是268，(201)X是275，Y是269，(202)X是275，Y是270，(203)X是275，Y是271，(204)X是275，Y是272，(205)X是275，Y是273，(206)X是275，Y是274，(207)X是275，Y是275，(208)X是275，Y是276，(209)X是275，Y是277，(210)X是275，Y是278，(211)X是275，Y是279，(212)X是275，Y是280，(213)X是275，Y是281，(214)X是275，Y是282，(215)X是275，Y是283，(216)X是275，Y是284，(217)X是275，Y是285，(218)X是275，Y是286，(219)X是275，Y是287，(220)X是275，Y是288，(221)X是276，Y是267，(222)X是276，Y是268，(223)X是276，Y是269，(224)X是276，Y是270，(225)X是276，Y是271，(226)X是276，Y是272，(227)X是276，Y是273，(228)X是276，Y是274，(229)X是276，Y是275，(230)X是276，Y是276，(231)X是276，Y是277，(232)X是276，Y是278，(233)X是276，Y是279，(234)X是276，Y是280，(235)X是276，Y是281，(236)X是276，Y是282，(237)X是276，Y是283，(238)X是276，Y是284，(239)X是276，Y是285，(240)X是276，Y是286，(241)X是276，Y是287，(242)X是276，Y是288，(243)X是277，Y是267，(244)X是277，Y是268，(245)X是277，Y是269，(246)X是277，Y是270，(247)X是277，Y是271，(248)X是277，Y是272，(249)X是277，Y是273，(250)X是277，Y是274，(251)X是277，Y是275，(252)X是277，Y是276，(253)X是277，Y是277，(254)X是277，Y是278，(255)X是277，Y是279，(256)X是277，Y是280，(257)X是277，Y是281，(258)X是277，Y是282，(259)X是277，Y是283，(260)X是277，Y是284，(261)X是277，Y是285，(262)X是277，Y是286，(263)X是277，Y是287，(264)X是277，Y是288，(265)X是278，Y是267，(266)X是278，Y是268，(267)X是278，Y是269，(268)X是278，Y是270，(269)X是278，Y是271，(270)X是278，Y是272，(271)X是278，Y是273，(272)X是278，Y是274，(273)X是278，Y是275，(274)X是278，Y是276，(275)X是278，Y是277，(276)X是278，Y是278，(277)X是278，Y是279，(278)X是278，Y是280，(279)X是278，Y是281，(280)X是278，Y是282，(281)X是278，Y是283，(282)X是278，Y是284，(283)X是278，Y是285，(284)X是278，Y是286，(285)X是278，Y是287，(286)X是278，Y是288，(287)X是279，Y是267，(288)X是279，Y是268，(289)X是279，Y是269，(290)X是279，Y是270，(291)X是279，Y是271，(292)X是279，Y是272，(293)X是279，Y是273，(294)X是279，Y是274，(295)X是279，Y是275，(296)X是279，Y是276，(297)X是279，Y是277，(298)X是279，Y是278，(299)X是279，Y是279，(300)X是279，Y是280，(301)X是279，Y是281，(302)X是279，Y是282，(303)X是279，Y是283，(304)X是279，Y是284，(305)X是279，Y是285，(306)X是279，Y是286，(307)X是279，Y是287，(308)X是279，Y是288，(309)X是280，Y是267，(310)X是280，Y是268，(311)X是280，Y是269，(312)X是280，Y是270，(313)X是280，Y是271，(314)X是280，Y是272，(315)X是280，Y是273，(316)X是280，Y是274，(317)X是280，Y是275，(318)X是280，Y是276，(319)X是280，Y是277，(320)X是280，Y是278，(321)X是280，Y是279，(322)X是280，Y是280，(323)X是280，Y是281，(324)X是280，Y是282，(325)X是280，Y是283，(326)X是280，Y是284，(327)X是280，Y是285，(328)X是280，Y是286，(329)X是280，Y是287，(330)X是280，Y是288，(331)X是281，Y是267，(332)X是281，Y是268，(333)X是281，Y是269，(334)X是281，Y是270，(335)X是281，Y是271，(336)X是281，Y是272，(337)X是281，Y是273，(338)X是281，Y是274，(339)X是281，Y是275，(340)X是281，Y是276，(341)X是281，Y是277，(342)X是281，Y是278，(343)X是281，Y是279，(344)X是281，Y是280，(345)X是281，Y是281，(346)X是281，Y是282，(347)X是281，Y是283，(348)X是281，Y是284，(349)X是281，Y是285，(350)X是281，Y是286，(351)X是281，Y是287，(352)X是281，Y是288，(353)X是282，Y是267，(354)X是282，Y是268，(355)X是282，Y是269，(356)X是282，Y是270，(357)X是282，Y是271，(358)X是282，Y是272，(359)X是282，Y是273，(360)X是282，Y是274，(361)X是282，Y是275，(362)X是282，Y是276，(363)X是282，Y是277，(364)X是282，Y是278，(365)X是282，Y是279，(366)X是282，Y是280，(367)X是282，Y是281，(368)X是282，Y是282，(369)X是282，Y是283，(370)X是282，Y是284，(371)X是282，Y是285，(372)X是282，Y是286，(373)X是282，Y是287，(374)X是282，Y是288，(375)X是283，Y是267，(376)X是283，Y是268，(377)X是283，Y是269，(378)X是283，Y是270，(379)X是283，Y是271，(380)X是283，Y是272，(381)X是283，Y是273，(382)X是283，Y是274，(383)X是283，Y是275，(384)X是283，Y是276，(385)X是283，Y是277，(386)X是283，Y是278，(387)X是283，Y是279，(388)X是283，Y是280，(389)X是283，Y是281，(390)X是283，Y是282，(391)X是283，Y是283，(392)X是283，Y是284，(393)X是283，Y是285，(394)X是283，Y是286，(395)X是283，Y是287，(396)X是283，Y是288，(397)X是284，Y是267，(398)X是284，Y是268，(399)X是284，Y是269，(400)X是284，Y是270，(401)X是284，Y是271，(402)X是284，Y是272，(403)X是284，Y是273，(404)X是284，Y是274，(405)X是284，Y是275，(406)X是284，Y是276，(407)X是284，Y是277，(408)X是284，Y是278，(409)X是284，Y是279，(410)X是284，Y是280，(411)X是284，Y是281，(412)X是284，Y是282，(413)X是284，Y是283，(414)X是284，Y是284，(415)X是284，Y是285，(416)X是284，Y是286，(417)X是284，Y是287，(418)X是284，Y是288，(419)X是285，Y是267，(420)X是285，Y是268，(421)X是285，Y是269，(422)X是285，Y是270，(423)X是285，Y是271，(424)X是285，Y是272，(425)X是285，Y是273，(426)X是285，Y是274，(427)X是285，Y是275，(428)X是285，Y是276，(429)X是285，Y是277，(430)X是285，Y是278，(431)X是285，Y是279，(432)X是285，Y是280，(433)X是285，Y是281，(434)X是285，Y是282，(435)X是285，Y是283，(436)X是285，Y是284，(437)X是285，Y是285，(438)X是285，Y是286，(439)X是285，Y是287，(440)X是285，Y是288，(441)X是286，Y是267，(442)X是286，Y是268，(443)X是286，Y是269，(444)X是286，Y是270，(445)X是286，Y是271，(446)X是286，Y是272，(447)X是286，Y是273，(448)X是286，Y是274，(449)X是286，Y是275，(450)X是286，Y是276，(451)X是286，Y是277，(452)X是286，Y是278，(453)X是286，Y是279，(454)X是286，Y是280，(455)X是286，Y是281，(456)X是286，Y是282，(457)X是286，Y是283，(458)X是286，Y是284，(459)X是286，Y是285，(460)X是286，Y是286，(461)X是286，Y是287，(462)X是286，Y是288，(463)X是287，Y是267，(464)X是287，Y是268，(465)X是287，Y是269，(466)X是287，Y是270，(467)X是287，Y是271，(468)X是287，Y是272，(469)X是287，Y是273，(470)X是287，Y是274，(471)X是287，Y是275，(472)X是287，Y是276，(473)X是287，Y是277，(474)X是287，Y是278，(475)X是287，Y是279，(476)X是287，Y是280，(477)X是287，Y是281，(478)X是287，Y是282，(479)X是287，Y是283，(480)X是287，Y是284，(481)X是287，Y是285，(482)X是287，Y是286，(483)X是287，Y是287，(484)X是287，Y是288，(485)X是117，Y是118，(486)X是117，Y是119，(487)X是117，Y是120，(488)X是117，Y是121，(489)X是117，Y是122，(490)X是117，Y是123，(491)X是118，Y是118，(492)X是118，Y是119，(493)X是118，Y是120，(494)X是118，Y是121，(495)X是118，Y是122，(496)X是118，Y是123，(497)X是119，Y是118，(498)X是119，Y是119，(499)X是119，Y是120，(500)X是119，Y是121，(501)X是119，Y是122，(502)X是119，Y是123，(503)X是120，Y是118，(504)X是120，Y是119，(505)X是120，Y是120，(506)X是120，Y是121，(507)X是120，Y是122，(508)X是120，Y是123，(509)X是121，Y是118，(510)X是121，Y是119，(511)X是121，Y是120，(512)X是121，Y是121，(513)X是121，Y是122，(514)X是121，Y是123，(515)X是122，Y是118，(516)X是122，Y是119，(517)X是122，Y是120，(518)X是122，Y是121，(519)X是122，Y是122，(520)X是122，Y是123，(521)X是340，Y是341，(522)X是340，Y是342，(523)X是340，Y是343，(524)X是340，Y是344，(525)X是340，Y是345，(526)X是340，Y是346，(527)X是340，Y是347，(528)X是340，Y是348，(529)X是340，Y是349，(530)X是340，Y是350，(531)X是340，Y是351，(532)X是340，Y是352，(533)X是340，Y是353，(534)X是341，Y是341，(535)X是341，Y是342，(536)X是341，Y是343，(537)X是341，Y是344，(538)X是341，Y是345，(539)X是341，Y是346，(540)X是341，Y是347，(541)X是341，Y是348，(542)X是341，Y是349，(543)X是341，Y是350，(544)X是341，Y是351，(545)X是341，Y是352，(546)X是341，Y是353，(547)X是342，Y是341，(548)X是342，Y是342，(549)X是342，Y是343，(550)X是342，Y是344，(551)X是342，Y是345，(552)X是342，Y是346，(553)X是342，Y是347，(554)X是342，Y是348，(555)X是342，Y是349，(556)X是342，Y是350，(557)X是342，Y是351，(558)X是342，Y是352，(559)X是342，Y是353，(560)X是343，Y是341，(561)X是343，Y是342，(562)X是343，Y是343，(563)X是343，Y是344，(564)X是343，Y是345，(565)X是343，Y是346，(566)X是343，Y是347，(567)X是343，Y是348，(568)X是343，Y是349，(569)X是343，Y是350，(570)X是343，Y是351，(571)X是343，Y是352，(572)X是343，Y是353，(573)X是344，Y是341，(574)X是344，Y是342，(575)X是344，Y是343，(576)X是344，Y是344，(577)X是344，Y是345，(578)X是344，Y是346，(579)X是344，Y是347，(580)X是344，Y是348，(581)X是344，Y是349，(582)X是344，Y是350，(583)X是344，Y是351，(584)X是344，Y是352，(585)X是344，Y是353，(586)X是345，Y是341，(587)X是345，Y是342，(588)X是345，Y是343，(589)X是345，Y是344，(590)X是345，Y是345，(591)X是345，Y是346，(592)X是345，Y是347，(593)X是345，Y是348，(594)X是345，Y是349，(595)X是345，Y是350，(596)X是345，Y是351，(597)X是345，Y是352，(598)X是345，Y是353，(599)X是346，Y是341，(600)X是346，Y是342，(601)X是346，Y是343，(602)X是346，Y是344，(603)X是346，Y是345，(604)X是346，Y是346，(605)X是346，Y是347，(606)X是346，Y是348，(607)X是346，Y是349，(608)X是346，Y是350，(609)X是346，Y是351，(610)X是346，Y是352，(611)X是346，Y是353，(612)X是347，Y是341，(613)X是347，Y是342，(614)X是347，Y是343，(615)X是347，Y是344，(616)X是347，Y是345，(617)X是347，Y是346，(618)X是347，Y是347，(619)X是347，Y是348，(620)X是347，Y是349，(621)X是347，Y是350，(622)X是347，Y是351，(623)X是347，Y是352，(624)X是347，Y是353，(625)X是348，Y是341，(626)X是348，Y是342，(627)X是348，Y是343，(628)X是348，Y是344，(629)X是348，Y是345，(630)X是348，Y是346，(631)X是348，Y是347，(632)X是348，Y是348，(633)X是348，Y是349，(634)X是348，Y是350，(635)X是348，Y是351，(636)X是348，Y是352，(637)X是348，Y是353，(638)X是349，Y是341，(639)X是349，Y是342，(640)X是349，Y是343，(641)X是349，Y是344，(642)X是349，Y是345，(643)X是349，Y是346，(644)X是349，Y是347，(645)X是349，Y是348，(646)X是349，Y是349，(647)X是349，Y是350，(648)X是349，Y是351，(649)X是349，Y是352，(650)X是349，Y是353，(651)X是350，Y是341，(652)X是350，Y是342，(653)X是350，Y是343，(654)X是350，Y是344，(655)X是350，Y是345，(656)X是350，Y是346，(657)X是350，Y是347，(658)X是350，Y是348，(659)X是350，Y是349，(660)X是350，Y是350，(661)X是350，Y是351，(662)X是350，Y是352，(663)X是350，Y是353，(664)X是351，Y是341，(665)X是351，Y是342，(666)X是351，Y是343，(667)X是351，Y是344，(668)X是351，Y是345，(669)X是351，Y是346，(670)X是351，Y是347，(671)X是351，Y是348，(672)X是351，Y是349，(673)X是351，Y是350，(674)X是351，Y是351，(675)X是351，Y是352，(676)X是351，Y是353，(677)X是352，Y是341，(678)X是352，Y是342，(679)X是352，Y是343，(680)X是352，Y是344，(681)X是352，Y是345，(682)X是352，Y是346，(683)X是352，Y是347，(684)X是352，Y是348，(685)X是352，Y是349，(686)X是352，Y是350，(687)X是352，Y是351，(688)X是352，Y是352，(689)X是352，Y是353。(1) X is 266, Y is 267. (2) X is 266, Y is 268. (3) X is 266, Y is 269. (4) X is 266, Y is 270. (5) X is 266, Y is 271. (6) X is 266, Y is 272. (7) X is 266, Y is 273. (8) X is 266, Y is 274. (9) X is 266, Y is 275. (10) X is 266, Y is 276. (11) X is 266, Y is 277. (12) X is 266, Y is 277. 8, (13) X is 266, Y is 279, (14) X is 266, Y is 280, (15) X is 266, Y is 281, (16) X is 266, Y is 282, (17) X is 266, Y is 283, (18) X is 266, Y is 284, (19) X is 266, Y is 285, (20) X is 266, Y is 286, (21) X is 266, Y is 287, (22) X is 266, Y is 288, (23) X is 267, Y is 267, (24) (25) X is 267, Y is 268, (26) X is 267, Y is 269, (27) X is 267, Y is 270, (28) X is 267, Y is 271, (29) X is 267, Y is 272, (30) X is 267, Y is 273, (31) X is 267, Y is 274, (32) X is 267, Y is 275, (33) X is 267, Y is 276, (34) X is 267, Y is 278, (35) X is 26 7, Y is 279, (36) X is 267, Y is 280, (37) X is 267, Y is 281, (38) X is 267, Y is 282, (39) X is 267, Y is 283, (40) X is 267, Y is 284, (41) X is 267, Y is 285, (42) X is 267, Y is 286, (43) X is 267, Y is 287, (44) X is 267, Y is 288, (45) X is 268, Y is 267, (46) X is 268, Y is 26 8, (47) X is 268, Y is 269, (48) X is 268, Y is 270, (49) X is 268, Y is 271, (50) X is 268, Y is 272, (51) X is 268, Y is 273, (52) X is 268, Y is 274, (53) X is 268, Y is 275, (54) X is 268, Y is 276, (55) X is 268, Y is 277, (56) X is 268, Y is 278, (57) X is 268, Y is 279, (58) (59) X is 268, Y is 280, (60) X is 268, Y is 281, (61) X is 268, Y is 282, (62) X is 268, Y is 283, (63) X is 268, Y is 284, (64) X is 268, Y is 285, (65) X is 268, Y is 286, (66) X is 268, Y is 287, (67) X is 269, Y is 267, (68) X is 269, Y is 268, (69) X is 26 9, Y is 269, (70) X is 269, Y is 270, (71) X is 269, Y is 271, (72) X is 269, Y is 272, (73) X is 269, Y is 273, (74) X is 269, Y is 274, (75) X is 269, Y is 275, (76) X is 269, Y is 276, (77) X is 269, Y is 277, (78) X is 269, Y is 278, (79) X is 269, Y is 279, (80) X is 269, Y is 2 80, (81) X is 269, Y is 281, (82) X is 269, Y is 282, (83) X is 269, Y is 283, (84) X is 269, Y is 284, (85) X is 269, Y is 285, (86) X is 269, Y is 286, (87) X is 269, Y is 287, (88) X is 269, Y is 288, (89) X is 270, Y is 267, (90) X is 270, Y is 268, (91) X is 270, Y is 269, (92) (93) X is 270, Y is 270, (94) X is 270, Y is 271, (95) X is 270, Y is 272, (96) X is 270, Y is 273, (97) X is 270, Y is 274, (98) X is 270, Y is 275, (99) X is 270, Y is 276, (100) X is 270, Y is 277, (101) X is 270, Y is 278, (102) X is 270, Y is 279, (103) X is 270, Y is 280, (104) X is 270, Y is 280, (105) X is 270, Y is 271, (96) X is 270, Y is 271, (97) X is 270, Y is 272, (98) X is 270, Y is 273, (99) X is 270, Y is 274, (105) X is 270, Y is 275, (106) X is 270, Y is 276, (107) X is 270, Y is 277, (108) X is 270, Y is 279, (109) X is 270, Y is 280, (100) X is 270, Y is 279, (100) X is 270, Y is 280, (100) X is 270, Y is 279, (100) X is 270, Y is 279, (100) X is 270, Y is 280, (100) X is 2 X is 270, Y is 281, (104) X is 270, Y is 282, (105) X is 270, Y is 283, (106) X is 270, Y is 284, (107) X is 270, Y is 285, (108) X is 270, Y is 286, (109) X is 270, Y is 287, (110) X is 270, Y is 288, (111) X is 271, Y is 267, (112) X is 271, Y is 268, (113) X is 271, Y is 269 (114) X is 271, Y is 270; (115) X is 271, Y is 271; (116) X is 271, Y is 272; (117) X is 271, Y is 273; (118) X is 271, Y is 274; (119) X is 271, Y is 275; (120) X is 271, Y is 276; (121) X is 271, Y is 277; (122) X is 271, Y is 278; (123) X is 271, Y is 279; (124) X is 271. Y is 280, (125) X is 271, Y is 281, (126) X is 271, Y is 282, (127) X is 271, Y is 283, (128) X is 271, Y is 284, (129) X is 271, Y is 285, (130) X is 271, Y is 286, (131) X is 271, Y is 287, (132) X is 271, Y is 288, (133) X is 272, Y is 267, (134) X is 272, Y is 268, (135) X is 272, Y is 269, (136) X is 272, Y is 270, (137) X is 272, Y is 271, (138) X is 272, Y is 272, (139) X is 272, Y is 273, (140) X is 272, Y is 274, (141) X is 272, Y is 275, (142) X is 272, Y is 276, (143) X is 272, Y is 277, (144) X is 272, Y is 278, (145) X is 272, Y is 279 (146) X is 272, Y is 280; (147) X is 272, Y is 281; (148) X is 272, Y is 282; (149) X is 272, Y is 283; (150) X is 272, Y is 284; (151) X is 272, Y is 285; (152) X is 272, Y is 286; (153) X is 272, Y is 287; (154) X is 272, Y is 288; (155) X is 273, Y is 267; (156) X is 273. Y is 268, (157) X is 273, Y is 269, (158) X is 273, Y is 270, (159) X is 273, Y is 271, (160) X is 273, Y is 272, (161) X is 273, Y is 273, (162) X is 273, Y is 274, (163) X is 273, Y is 275, (164) X is 273, Y is 276, (165) X is 273, Y is 277, (166) X is 273, Y is 278, (167) X is 273, Y is 279, (168) X is 273, Y is 280, (169) X is 273, Y is 281, (170) X is 273, Y is 282, (171) X is 273, Y is 283, (172) X is 273, Y is 284, (173) X is 273, Y is 285, (174) X is 273, Y is 286, (175) X is 273, Y is 287, (176) X is 273, Y is 288, (177) X is 274, Y is 267 (178) X is 274, Y is 268; (179) X is 274, Y is 269; (180) X is 274, Y is 270; (181) X is 274, Y is 271; (182) X is 274, Y is 272; (183) X is 274, Y is 273; (184) X is 274, Y is 274; (185) X is 274, Y is 275; (186) X is 274, Y is 276; (187) X is 274, Y is 277; (188) X is 274. Y is 278, (189) X is 274, Y is 279, (190) X is 274, Y is 280, (191) X is 274, Y is 281, (192) X is 274, Y is 282, (193) X is 274, Y is 283, (194) X is 274, Y is 284, (195) X is 274, Y is 285, (196) X is 274, Y is 286, (197) X is 274, Y is 287, (198) X is 274, Y is 288, (199) X is 275, Y is 267, (200) X is 275, Y is 268, (201) X is 275, Y is 269, (202) X is 275, Y is 270, (203) X is 275, Y is 271, (204) X is 275, Y is 272, (205) X is 275, Y is 273, (206) X is 275, Y is 274, (207) X is 275, Y is 275, (208) X is 275, Y is 276, (209) X is 275, Y is 277 (210) X is 275, Y is 278; (211) X is 275, Y is 279; (212) X is 275, Y is 280; (213) X is 275, Y is 281; (214) X is 275, Y is 282; (215) X is 275, Y is 283; (216) X is 275, Y is 284; (217) X is 275, Y is 285; (218) X is 275, Y is 286; (219) X is 275, Y is 287; (220) X is 275... Y is 288, (221) X is 276, Y is 267, (222) X is 276, Y is 268, (223) X is 276, Y is 269, (224) X is 276, Y is 270, (225) X is 276, Y is 271, (226) X is 276, Y is 272, (227) X is 276, Y is 273, (228) X is 276, Y is 274, (229) X is 276, Y is 275, (230) X is 276, Y is 276, (231) X is 276, Y is 277, (232) X is 276, Y is 278, (233) X is 276, Y is 279, (234) X is 276, Y is 280, (235) X is 276, Y is 281, (236) X is 276, Y is 282, (237) X is 276, Y is 283, (238) X is 276, Y is 284, (239) X is 276, Y is 285, (240) X is 276, Y is 286, (241) X is 276, Y is 287 (242) X is 276, Y is 288; (243) X is 277, Y is 267; (244) X is 277, Y is 268; (245) X is 277, Y is 269; (246) X is 277, Y is 270; (247) X is 277, Y is 271; (248) X is 277, Y is 272; (249) X is 277, Y is 273; (250) X is 277, Y is 274; (251) X is 277, Y is 275; (252) X is 277. Y is 276, (253) X is 277, Y is 277, (254) X is 277, Y is 278, (255) X is 277, Y is 279, (256) X is 277, Y is 280, (257) X is 277, Y is 281, (258) X is 277, Y is 282, (259) X is 277, Y is 283, (260) X is 277, Y is 284, (261) X is 277, Y is 285, (262) X is 277, Y is 286, (263) X is 277, Y is 287, (264) X is 277, Y is 288, (265) X is 278, Y is 267, (266) X is 278, Y is 268, (267) X is 278, Y is 269, (268) X is 278, Y is 270, (269) X is 278, Y is 271, (270) X is 278, Y is 272, (271) X is 278, Y is 273, (272) X is 278, Y is 274, (273) X is 278, Y is 275 (274) X is 278, Y is 276; (275) X is 278, Y is 277; (276) X is 278, Y is 278; (277) X is 278, Y is 279; (278) X is 278, Y is 280; (279) X is 278, Y is 281; (280) X is 278, Y is 282; (281) X is 278, Y is 283; (282) X is 278, Y is 284; (283) X is 278, Y is 285; (284) X is 278. Y is 286, (285)X is 278, Y is 287, (286)X is 278, Y is 288, (287)X is 279, Y is 267, (288)X is 279, Y is 268, (289)X is 279, Y is 269, (290)X is 279, Y is 270, (291)X is 279, Y is 271, (292)X is 279, Y is 272, (293)X is 279, Y is 273, (294)X is 279, Y is 274, (295 ... (296) X is 279, Y is 275, (297) X is 279, Y is 277, (298) X is 279, Y is 278, (299) X is 279, Y is 279, (300) X is 279, Y is 280, (301) X is 279, Y is 281, (302) X is 279, Y is 282, (303) X is 279, Y is 283, (304) X is 279, Y is 284, (305) X is 279, Y is 285 (306) X is 279, Y is 286; (307) X is 279, Y is 287; (308) X is 279, Y is 288; (309) X is 280, Y is 267; (310) X is 280, Y is 268; (311) X is 280, Y is 269; (312) X is 280, Y is 270; (313) X is 280, Y is 271; (314) X is 280, Y is 272; (315) X is 280, Y is 273; (316) X is 280... (317) X is 280, Y is 275, (318) X is 280, Y is 276, (319) X is 280, Y is 277, (320) X is 280, Y is 278, (321) X is 280, Y is 279, (322) X is 280, Y is 280, (323) X is 280, Y is 281, (324) X is 280, Y is 282, (325) X is 280, Y is 283, (326) X is 280, Y is 284, (327) X is 280, Y is 285, (328) X is 280, Y is 286, (329) X is 280, Y is 287, (330) X is 280, Y is 288, (331) X is 281, Y is 267, (332) X is 281, Y is 268, (333) X is 281, Y is 269, (334) X is 281, Y is 270, (335) X is 281, Y is 271, (336) X is 281, Y is 272, (337) X is 281, Y is 273 (338) X is 281, Y is 274; (339) X is 281, Y is 275; (340) X is 281, Y is 276; (341) X is 281, Y is 277; (342) X is 281, Y is 278; (343) X is 281, Y is 279; (344) X is 281, Y is 280; (345) X is 281, Y is 281; (346) X is 281, Y is 282; (347) X is 281, Y is 283; (348) X is 281... Y is 284, (349) X is 281, Y is 285, (350) X is 281, Y is 286, (351) X is 281, Y is 287, (352) X is 281, Y is 288, (353) X is 282, Y is 267, (354) X is 282, Y is 268, (355) X is 282, Y is 269, (356) X is 282, Y is 270, (357) X is 282, Y is 271, (358) X is 282, Y is 272, (359) X is 282, Y is 273, (360) X is 282, Y is 274, (361) X is 282, Y is 275, (362) X is 282, Y is 276, (363) X is 282, Y is 277, (364) X is 282, Y is 278, (365) X is 282, Y is 279, (366) X is 282, Y is 280, (367) X is 282, Y is 281, (368) X is 282, Y is 282, (369) X is 282, Y is 283 (370) X is 282, Y is 284; (371) X is 282, Y is 285; (372) X is 282, Y is 286; (373) X is 282, Y is 287; (374) X is 282, Y is 288; (375) X is 283, Y is 267; (376) X is 283, Y is 268; (377) X is 283, Y is 269; (378) X is 283, Y is 270; (379) X is 283, Y is 271; (380) X is 283. Y is 272, (381) X is 283, Y is 273, (382) X is 283, Y is 274, (383) X is 283, Y is 275, (384) X is 283, Y is 276, (385) X is 283, Y is 277, (386) X is 283, Y is 278, (387) X is 283, Y is 279, (388) X is 283, Y is 280, (389) X is 283, Y is 281, (390) X is 283, Y is 282, (391) X is 283, Y is 283, (392) X is 283, Y is 284, (393) X is 283, Y is 285, (394) X is 283, Y is 286, (395) X is 283, Y is 287, (396) X is 283, Y is 288, (397) X is 284, Y is 267, (398) X is 284, Y is 268, (399) X is 284, Y is 269, (400) X is 284, Y is 270, (401) X is 284, Y is 271 (402) X is 284, Y is 272; (403) X is 284, Y is 273; (404) X is 284, Y is 274; (405) X is 284, Y is 275; (406) X is 284, Y is 276; (407) X is 284, Y is 277; (408) X is 284, Y is 278; (409) X is 284, Y is 279; (410) X is 284, Y is 280; (411) X is 284, Y is 281; (412) X is 284. Y is 282, (413) X is 284, Y is 283, (414) X is 284, Y is 284, (415) X is 284, Y is 285, (416) X is 284, Y is 286, (417) X is 284, Y is 287, (418) X is 284, Y is 288, (419) X is 285, Y is 267, (420) X is 285, Y is 268, (421) X is 285, Y is 269, (422) X is 285, Y is 270, (423) (424) X is 285, Y is 271, (425) X is 285, Y is 272, (426) X is 285, Y is 273, (427) X is 285, Y is 274, (428) X is 285, Y is 275, (429) X is 285, Y is 277, (430) X is 285, Y is 278, (431) X is 285, Y is 279, (432) X is 285, Y is 280, (433) X is 285, Y is 281 (434) X is 285, Y is 282; (435) X is 285, Y is 283; (436) X is 285, Y is 284; (437) X is 285, Y is 285; (438) X is 285, Y is 286; (439) X is 285, Y is 287; (440) X is 285, Y is 288; (441) X is 286, Y is 267; (442) X is 286, Y is 268; (443) X is 286, Y is 269; (444) X is 286. Y is 270, (445) X is 286, Y is 271, (446) X is 286, Y is 272, (447) X is 286, Y is 273, (448) X is 286, Y is 274, (449) X is 286, Y is 275, (450) X is 286, Y is 276, (451) X is 286, Y is 277, (452) X is 286, Y is 278, (453) X is 286, Y is 279, (454) X is 286, Y is 280, (455) (456) X is 286, Y is 281, (457) X is 286, Y is 282, (458) X is 286, Y is 283, (459) X is 286, Y is 284, (460) X is 286, Y is 286, (461) X is 286, Y is 287, (462) X is 286, Y is 288, (463) X is 287, Y is 267, (464) X is 287, Y is 268, (465) X is 287, Y is 26 9, (466) X is 287, Y is 270, (467) X is 287, Y is 271, (468) X is 287, Y is 272, (469) X is 287, Y is 273, (470) X is 287, Y is 274, (471) X is 287, Y is 275, (472) X is 287, Y is 276, (473) X is 287, Y is 277, (474) X is 287, Y is 278, (475) X is 287, Y is 279, (476) X is 287 Y is 280, (477) X is 287, Y is 281, (478) X is 287, Y is 282, (479) X is 287, Y is 283, (480) X is 287, Y is 284, (481) X is 287, Y is 285, (482) X is 287, Y is 286, (483) X is 287, Y is 287, (484) X is 287, Y is 288, (485) X is 117, Y is 118, (486) X is 117, Y is 119, (487) (488) X is 117, Y is 120, (489) X is 117, Y is 121, (490) X is 117, Y is 122, (491) X is 117, Y is 123, (492) X is 118, Y is 118, (493) X is 118, Y is 120, (494) X is 118, Y is 121, (495) X is 118, Y is 122, (496) X is 118, Y is 123, (497) X is 119, Y is 118 (498) X is 119, Y is 119; (499) X is 119, Y is 120; (500) X is 119, Y is 121; (501) X is 119, Y is 122; (502) X is 119, Y is 123; (503) X is 120, Y is 118; (504) X is 120, Y is 119; (505) X is 120, Y is 120; (506) X is 120, Y is 121; (507) X is 120, Y is 122; (508) X is 120 Y is 123, (509) X is 121, Y is 118, (510) X is 121, Y is 119, (511) X is 121, Y is 120, (512) X is 121, Y is 121, (513) X is 121, Y is 122, (514) X is 121, Y is 123, (515) X is 122, Y is 118, (516) X is 122, Y is 119, (517) X is 122, Y is 120, (518) X is 122, Y is 121, (519) X is 122, Y is 122, (520) X is 122, Y is 123, (521) X is 340, Y is 341, (522) X is 340, Y is 342, (523) X is 340, Y is 343, (524) X is 340, Y is 344, (525) X is 340, Y is 345, (526) X is 340, Y is 346, (527) X is 340, Y is 347, (528) X is 340, Y is 348, (529) X is 340, Y is 349 (530) X is 340, Y is 350; (531) X is 340, Y is 351; (532) X is 340, Y is 352; (533) X is 340, Y is 353; (534) X is 341, Y is 341; (535) X is 341, Y is 342; (536) X is 341, Y is 343; (537) X is 341, Y is 344; (538) X is 341, Y is 345; (539) X is 341, Y is 346; (540) X is 341. Y is 347, (541) X is 341, Y is 348, (542) X is 341, Y is 349, (543) X is 341, Y is 350, (544) X is 341, Y is 351, (545) X is 341, Y is 352, (546) X is 341, Y is 353, (547) X is 342, Y is 341, (548) X is 342, Y is 342, (549) X is 342, Y is 343, (550) X is 342, Y is 344, (551) (552) X is 342, Y is 345, (553) X is 342, Y is 346, (554) X is 342, Y is 347, (555) X is 342, Y is 348, (556) X is 342, Y is 349, (557) X is 342, Y is 350, (558) X is 342, Y is 352, (559) X is 342, Y is 353, (560) X is 343, Y is 341, (561) X is 343, Y is 342 (562) X is 343, Y is 343; (563) X is 343, Y is 344; (564) X is 343, Y is 345; (565) X is 343, Y is 346; (566) X is 343, Y is 347; (567) X is 343, Y is 348; (568) X is 343, Y is 349; (569) X is 343, Y is 350; (570) X is 343, Y is 351; (571) X is 343, Y is 352; (572) X is 343. Y is 353, (573) X is 344, Y is 341, (574) X is 344, Y is 342, (575) X is 344, Y is 343, (576) X is 344, Y is 344, (577) X is 344, Y is 345, (578) X is 344, Y is 346, (579) X is 344, Y is 347, (580) X is 344, Y is 348, (581) X is 344, Y is 349, (582) X is 344, Y is 350, (583) X is 344, Y is 351, (584) X is 344, Y is 352, (585) X is 344, Y is 353, (586) X is 345, Y is 341, (587) X is 345, Y is 342, (588) X is 345, Y is 343, (589) X is 345, Y is 344, (590) X is 345, Y is 345, (591) X is 345, Y is 346, (592) X is 345, Y is 347, (593) X is 345, Y is 348 (594) X is 345, Y is 349; (595) X is 345, Y is 350; (596) X is 345, Y is 351; (597) X is 345, Y is 352; (598) X is 345, Y is 353; (599) X is 346, Y is 341; (600) X is 346, Y is 342; (601) X is 346, Y is 343; (602) X is 346, Y is 344; (603) X is 346, Y is 345; (604) X is 346... Y is 346, (605) X is 346, Y is 347, (606) X is 346, Y is 348, (607) X is 346, Y is 349, (608) X is 346, Y is 350, (609) X is 346, Y is 351, (610) X is 346, Y is 352, (611) X is 346, Y is 353, (612) X is 347, Y is 341, (613) X is 347, Y is 342, (614) X is 347, Y is 343, (615) X is 347, Y is 344, (616) X is 347, Y is 345, (617) X is 347, Y is 346, (618) X is 347, Y is 347, (619) X is 347, Y is 348, (620) X is 347, Y is 349, (621) X is 347, Y is 350, (622) X is 347, Y is 351, (623) X is 347, Y is 352, (624) X is 347, Y is 353, (625) X is 348, Y is 341 (626) X is 348, Y is 342; (627) X is 348, Y is 343; (628) X is 348, Y is 344; (629) X is 348, Y is 345; (630) X is 348, Y is 346; (631) X is 348, Y is 347; (632) X is 348, Y is 348; (633) X is 348, Y is 349; (634) X is 348, Y is 350; (635) X is 348, Y is 351; (636) X is 348. Y is 352, (637)X is 348, Y is 353, (638)X is 349, Y is 341, (639)X is 349, Y is 342, (640)X is 349, Y is 343, (641)X is 349, Y is 344, (642)X is 349, Y is 345, (643)X is 349, Y is 346, (644)X is 349, Y is 347, (645)X is 349, Y is 348, (646)X is 349, Y is 349, (647) X is 349, Y is 350, (648) X is 349, Y is 351, (649) X is 349, Y is 352, (650) X is 349, Y is 353, (651) X is 350, Y is 341, (652) X is 350, Y is 342, (653) X is 350, Y is 343, (654) X is 350, Y is 344, (655) X is 350, Y is 345, (656) X is 350, Y is 346, (657) X is 350, Y is 347 (658) X is 350, Y is 348; (659) X is 350, Y is 349; (660) X is 350, Y is 350; (661) X is 350, Y is 351; (662) X is 350, Y is 352; (663) X is 350, Y is 353; (664) X is 351, Y is 341; (665) X is 351, Y is 342; (666) X is 351, Y is 343; (667) X is 351, Y is 344; (668) X is 351. Y is 345, (669) X is 351, Y is 346, (670) X is 351, Y is 347, (671) X is 351, Y is 348, (672) X is 351, Y is 349, (673) X is 351, Y is 350, (674) X is 351, Y is 351, (675) X is 351, Y is 352, (676) X is 351, Y is 353, (677) X is 352, Y is 341, (678) X is 352, Y is 342, (679) X is 352, Y is 343, (680) X is 352, Y is 344, (681) X is 352, Y is 345, (682) X is 352, Y is 346, (683) X is 352, Y is 347, (684) X is 352, Y is 348, (685) X is 352, Y is 349, (686) X is 352, Y is 350, (687) X is 352, Y is 351, (688) X is 352, Y is 352, (689) X is 352, Y is 353.

本文中，“响应倍数”是标准化后的荧光比值。探针的响应倍数越偏离1(不管是变大或者变小)，探针对底物响应能力相对对照的变化倍数或者响应能力越大。例如，本申请实施例通过检测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(Normalized Ratio 420/485)来计算响应倍数，具体如下：In this article, "response fold" refers to the standardized fluorescence ratio. The greater the deviation of the probe's response fold from 1 (whether it increases or decreases), the greater the change in the probe's response to the substrate relative to the control, or the greater its responsiveness. For example, in this application, the response fold is calculated by detecting the change in the ratio of fluorescence intensity at 528nm emission from 420nm excitation to fluorescence intensity at 528nm emission from 485nm excitation (Normalized Ratio 420/485), as detailed below:

通过扣除未表达探针蛋白细胞的检测信号值来进行荧光信号值的校正。将平行实验组中的探针检测信号与对照检测信号进行相除来消除pH敏感干扰获得校正数据。Fluorescence signal values were corrected by subtracting the detection signal values from cells that did not express the probe protein. pH-sensitive interference was eliminated by dividing the probe detection signals from parallel experimental groups by the control detection signals to obtain corrected data.

F＝Fsample-FBLK
F = F sample - F BLK

F表示荧光强度(Fluorescence intensity)，Fsample表示表达荧光探针样品的总的荧光强度，FBLK表示未表达荧光探针样品的背景荧光强度，FcpYFP表示作为pH对照的样品的荧光强度。F485表示荧光蛋白样品的在485nm处激发528nm处发射的荧光强度，F420表示荧光蛋白样品的在420nm处激发528nm处发射的荧光强度。Ratiosensor表示探针的荧光强度比值，RatiocpYFP表示对应探针的pH对照荧光蛋白的荧光强度比值。NormalizedRatio420/485是探针的变化倍数或者响应的倍。NormalizedRatio485/420越偏离1(不管是变大或者变小)表示探针的变化倍数或者响应倍数越大。F represents fluorescence intensity. F<sub> sample </sub> represents the total fluorescence intensity of the sample expressing the fluorescent probe, F <sub>BLK </sub> represents the background fluorescence intensity of the sample not expressing the fluorescent probe, and F<sub> cpYFP </sub> represents the fluorescence intensity of the sample used as a pH control. F <sub>485 </sub> represents the fluorescence intensity emitted at 528 nm when the fluorescent protein sample is excited at 485 nm, and F <sub>420</sub> represents the fluorescence intensity emitted at 528 nm when the fluorescent protein sample is excited at 420 nm. Ratiosensor represents the fluorescence intensity ratio of the probe, and Ratio cpYFP represents the fluorescence intensity ratio of the corresponding probe to the pH control fluorescent protein. NormalizedRatio 420/485 is the fold change of the probe or the response multiple. The greater the deviation of NormalizedRatio 485/420 from 1 (whether it increases or decreases), the greater the fold change of the probe or the response multiple.

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、D-葡萄糖、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、D-葡萄糖、苯丙氨酸、甲硫氨酸、D-葡萄糖)、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、D-葡萄糖、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。When referring to a polypeptide or protein, the term "variant" or "mutant" as used in this invention includes variants that have the same function as the polypeptide or protein but have a different sequence. Variants of polypeptides or proteins may include: homologous sequences, conserved variants, allelic variants, natural mutants, and induced mutants. These variants include, but are not limited to: deletions, insertions, and/or substitutions of one or more (typically 1-30, preferably 1-20, more preferably 1-10, most preferably 1-5) amino acids in the sequence of the polypeptide or protein, and sequences obtained by adding one or more (typically up to 20, preferably up to 10, more preferably up to 5) amino acids to its carboxyl terminus and/or amino terminus. These variants may also comprise polypeptides or proteins with at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100% sequence identity with the polypeptide or protein. The goal is to avoid being limited by theory, where changes to amino acid residues do not alter the overall conformation and function of the polypeptide or protein—that is, functionally conserved mutations. For example, in this field, substitution with amino acids of similar or identical properties typically does not change the function of the polypeptide or protein. In this field, amino acids with similar properties often refer to families of amino acids with similar side chains, which are well-defined. These families include amino acids with basic side chains (e.g., lysine, D-glucose, 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 nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, D-glucose, phenylalanine, methionine, D-glucose), amino acids with β-branched side chains (e.g., threonine, valine, isoleucine), and amino acids with aromatic side chains (e.g., tyrosine, phenylalanine, D-glucose, histidine). For example, adding one or more amino acids to the amino terminus and/or carboxyl terminus generally does not alter the function of a polypeptide or protein. Conserved amino acid substitutions for many common, known non-genetically encoded amino acids are known in the art. Conserved substitutions for other non-coding amino acids can be determined based on a comparison of their physical properties with those of their genetically encoded amino acids.

“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D-葡萄糖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。"Connector" or "linking region" refers to an amino acid or nucleotide sequence that links two parts in a polypeptide, protein, or nucleic acid of the present invention. Exemplarily, in the present invention, the number of amino acids at the amino terminus of the linking region between the D-glucose-sensitive polypeptide and the optically active polypeptide is selected to be 0-3, and the number of amino acids at the carboxyl terminus is selected to be 0-2; when the recombinant optical probe is used as a basic unit to link with a functional protein, it can be fused to the amino acid or carboxyl terminus of the recombinant optical probe. The connector sequence can be a short peptide chain composed of one or more flexible amino acids, such as Y.

发明人发现，在选自以下的位点具有突变的D-葡萄糖结合蛋白变体表现出与D-葡萄糖不同的结合活性：SEQ ID NO：1的W8、W9、E13、A42、H66、D278、K312、V347、H348。所述氨基酸突变包括氨基酸的修饰、取代或缺失。The inventors have discovered that D-glucose-binding protein variants with mutations at the following sites exhibit different binding activities than D-glucose: W8, W9, E13, A42, H66, D278, K312, V347, and H348 of SEQ ID NO: 1. The amino acid mutations include modifications, substitutions, or deletions of amino acids.

本发明提供具有这些突变的D-葡萄糖结合蛋白变体以及包含此类D-葡萄糖结合蛋白变体作为D-葡萄糖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D-葡萄糖敏感多肽是本文任一实施方案所述的D-葡萄糖结合蛋白变体，光学探针中的荧光蛋白如SEQ ID NO：2-9或其功能变体所示。优选地，所述荧光蛋白如SEQ ID NO：2、6、7、9或其功能变体所示。This invention provides D-glucose-binding protein variants having these mutations and optical probes comprising such D-glucose-binding protein variants as D-glucose-sensitive peptides. Therefore, in one or more embodiments, the D-glucose-sensitive peptide in the optical probe is a D-glucose-binding protein variant as described in any embodiment herein, and the fluorescent protein in the optical probe is as shown in SEQ ID NO: 2-9 or a functional variant thereof. Preferably, the fluorescent protein is as shown in SEQ ID NO: 2, 6, 7, 9 or a functional variant thereof.

在一些具体实施方案中，光学探针中的D-葡萄糖敏感多肽如SEQ ID NO：1所示，光学活性多肽如SEQ ID NO：2所示，光学活性多肽位于D-葡萄糖敏感多肽的266/267、266/268、266/269、266/270、266/271、266/272、266/273、266/274、266/275、266/276、266/277、266/278、266/279、266/280、266/281、266/282、266/283、266/284、266/285、266/286、266/287、266/288、267/267、267/268、267/269、267/270、267/271、267/272、267/273、267/274、267/275、267/276、267/277、267/278、267/279、267/280、267/281、267/282、267/283、267/284、267/285、267/286、267/287、267/288、268/267、268/268、268/269、268/270、268/271、268/272、268/273、268/274、268/275、268/276、268/277、268/278、268/279、268/280、268/281、268/282、268/283、268/284、268/285、268/286、268/287、268/288、269/267、269/268、269/269、269/270、269/271、269/272、269/273、269/274、269/275、269/276、269/277、269/278、269/279、269/280、269/281、269/282、269/283、269/284、269/285、269/286、269/287、269/288、270/267、270/268、270/269、270/270、270/271、270/272、270/273、270/274、270/275、270/276、270/277、270/278、270/279、270/280、270/281、270/282、270/283、270/284、270/285、270/286、270/287、270/288、271/267、271/268、271/269、271/270、271/271、271/272、271/273、271/274、271/275、271/276、271/277、271/278、271/279、271/280、271/281、271/282、271/283、271/284、271/285、271/286、271/287、271/288、272/267、272/268、272/269、272/270、272/271、272/272、272/273、272/274、272/275、272/276、272/277、272/278、272/279、272/280、272/281、272/282、272/283、272/284、272/285、272/286、272/287、272/288、273/267、273/268、273/269、273/270、273/271、273/272、273/273、273/274、273/275、273/276、273/277、273/278、273/279、273/280、273/281、273/282、273/283、273/284、273/285、273/286、273/287、273/288、274/267、274/268、274/269、274/270、274/271、274/272、274/273、274/274、274/275、274/276、274/277、274/278、274/279、274/280、274/281、274/282、274/283、274/284、274/285、274/286、274/287、274/288、275/267、275/268、275/269、275/270、275/271、275/272、275/273、275/274、275/275、275/276、275/277、275/278、275/279、275/280、275/281、275/282、275/283、275/284、275/285、275/286、275/287、275/288、276/267、276/268、276/269、276/270、276/271、276/272、276/273、276/274、276/275、276/276、276/277、276/278、276/279、276/280、276/281、276/282、276/283、276/284、276/285、276/286、276/287、276/288、277/267、277/268、277/269、277/270、277/271、277/272、277/273、277/274、277/275、277/276、277/277、277/278、277/279、277/280、277/281、277/282、277/283、277/284、277/285、277/286、277/287、277/288、278/267、278/268、278/269、278/270、278/271、278/272、278/273、278/274、278/275、278/276、278/277、278/278、278/279、278/280、278/281、278/282、278/283、278/284、278/285、278/286、278/287、278/288、279/267、279/268、279/269、279/270、279/271、279/272、279/273、279/274、279/275、279/276、279/277、279/278、279/279、279/280、279/281、279/282、279/283、279/284、279/285、279/286、279/287、279/288、280/267、280/268、280/269、280/270、280/271、280/272、280/273、280/274、280/275、280/276、280/277、280/278、280/279、280/280、280/281、280/282、280/283、280/284、280/285、280/286、280/287、280/288、281/267、281/268、281/269、281/270、281/271、281/272、281/273、281/274、281/275、281/276、281/277、281/278、281/279、281/280、281/281、281/282、281/283、281/284、281/285、281/286、281/287、281/288、282/267、282/268、282/269、282/270、282/271、282/272、282/273、282/274、282/275、282/276、282/277、282/278、282/279、282/280、282/281、282/282、282/283、282/284、282/285、282/286、282/287、282/288、283/267、283/268、283/269、283/270、283/271、283/272、283/273、283/274、283/275、283/276、283/277、283/278、283/279、283/280、283/281、283/282、283/283、283/284、283/285、283/286、283/287、283/288、284/267、284/268、284/269、284/270、284/271、284/272、284/273、284/274、284/275、284/276、284/277、284/278、284/279、284/280、284/281、284/282、284/283、284/284、284/285、284/286、284/287、284/288、285/267、285/268、285/269、285/270、285/271、285/272、285/273、285/274、285/275、285/276、285/277、285/278、285/279、285/280、285/281、285/282、285/283、285/284、285/285、285/286、285/287、285/288、286/267、286/268、286/269、286/270、286/271、286/272、286/273、286/274、286/275、286/276、286/277、286/278、286/279、286/280、286/281、286/282、286/283、286/284、286/285、286/286、286/287、286/288、287/267、287/268、287/269、287/270、287/271、287/272、287/273、287/274、287/275、287/276、287/277、287/278、287/279、287/280、287/281、287/282、287/283、287/284、287/285、287/286、287/287、287/288，117/118、117/119、117/120、117/121、117/122、117/123、118/118、118/119、118/120、118/121、118/122、118/123、119/118、119/119、119/120、119/121、119/122、119/123、120/118、120/119、120/120、120/121、120/122、120/123、121/118、121/119、121/120、121/121、121/122、121/123、122/118、122/119、122/120、122/121、122/122、122/123，340/341、340/342、340/343、340/344、340/345、340/346、340/347、340/348、340/349、340/350、340/351、340/352、340/353、341/341、341/342、341/343、341/344、341/345、341/346、341/347、341/348、341/349、341/350、341/351、341/352、341/353、342/341、342/342、342/343、342/344、342/345、342/346、342/347、342/348、342/349、342/350、342/351、342/352、342/353、343/341、343/342、343/343、343/344、343/345、343/346、343/347、343/348、343/349、343/350、343/351、343/352、343/353、344/341、344/342、344/343、344/344、344/345、344/346、344/347、344/348、344/349、344/350、344/351、344/352、344/353、345/341、345/342、345/343、345/344、345/345、345/346、345/347、345/348、345/349、345/350、345/351、345/352、345/353、346/341、346/342、346/343、346/344、346/345、346/346、346/347、346/348、346/349、346/350、346/351、346/352、346/353、347/341、347/342、347/343、347/344、347/345、347/346、347/347、347/348、347/349、347/350、347/351、347/352、347/353、348/341、348/342、348/343、348/344、348/345、348/346、348/347、348/348、348/349、348/350、348/351、348/352、348/353、349/341、349/342、349/343、349/344、349/345、349/346、349/347、349/348、349/349、349/350、349/351、349/352、349/353、350/341、350/342、350/343、350/344、350/345、350/346、350/347、350/348、350/349、350/350、350/351、350/352、350/353、351/341、351/342、351/343、351/344、351/345、351/346、351/347、351/348、351/349、351/350、351/351、351/352、351/353、352/341、352/342、352/343、352/344、352/345、352/346、352/347、352/348、352/349、352/350、352/351、352/352和352/353位点，光学探针的突变如表5中任一行所示。In some specific embodiments, the D-glucose-sensitive polypeptide in the optical probe is shown in SEQ ID NO: 1, and the optically active polypeptide is shown in SEQ ID NO: 2. The optically active polypeptide is located at positions 266/267, 266/268, 266/269, 266/270, 266/271, 266/272, 266/273, 266/274, 266/275, 266/276, 266/277, and 266/278 of the D-glucose-sensitive polypeptide. 266/279, 266/280, 266/281, 266/282, 266/283, 266/284, 266/285, 266/286, 266/287, 266/288, 267/267, 267/268, 267/269, 267/270, 267/271, 267/272, 267/273, 267/274, 267/275, 267/276, 267/277, 267/27 8, 267/279, 267/280, 267/281, 267/282, 267/283, 267/284, 267/285, 267/286, 267/287, 267/288, 268/267, 268/268, 268/269, 268/270, 268/271, 268/272, 268/273, 268/274, 268/275, 268/276, 268/277, 268/2 78, 268/279, 268/280, 268/281, 268/282, 268/283, 268/284, 268/285, 268/286, 268/287, 268/288, 269/267, 269/268, 269/269, 269/270, 269/271, 269/272, 269/273, 269/274, 269/275, 269/276, 269/277, 269/2 78, 269/279, 269/280, 269/281, 269/282, 269/283, 269/284, 269/285, 269/286, 269/287, 269/288, 270/267, 270/268, 270/269, 270/270, 270/271, 270/272, 270/273, 270/274, 270/275, 270/276, 270/277, 270/ 278, 270/279, 270/280, 270/281, 270/282, 270/283, 270/284, 270/285, 270/286, 270/287, 270/288, 271/267, 271/268, 271/269, 271/270, 271/271, 271/272, 271/273, 271/274, 271/275, 271/276, 271/277, 271 /278, 271/279, 271/280, 271/281, 271/282, 271/283, 271/284, 271/285, 271/286, 271/287, 271/288, 272/267, 272/268, 272/269, 272/270, 272/271, 272/272, 272/273, 272/274, 272/275, 272/276, 272/277, 27 2/278, 272/279, 272/280, 272/281, 272/282, 272/283, 272/284, 272/285, 272/286, 272/287, 272/288, 273/267, 273/268, 273/269, 273/270, 273/271, 273/272, 273/273, 273/274, 273/275, 273/276, 273/277, 27 3/278, 273/279, 273/280, 273/281, 273/282, 273/283, 273/284, 273/285, 273/286, 273/287, 273/288, 274/267, 274/268, 274/269, 274/270, 274/271, 274/272, 274/273, 274/274, 274/275, 274/276, 274/277, 2 74/278, 274/279, 274/280, 274/281, 274/282, 274/283, 274/284, 274/285, 274/286, 274/287, 274/288, 275/267, 275/268, 275/269, 275/270, 275/271, 275/272, 275/273, 275/274, 275/275, 275/276, 275/277 275/278, 275/279, 275/280, 275/281, 275/282, 275/283, 275/284, 275/285, 275/286, 275/287, 275/288, 276/267, 276/268, 276/269, 276/270, 276/271, 276/272, 276/273, 276/274, 276/275, 276/276, 276/277 276/278, 276/279, 276/280, 276/281, 276/282, 276/283, 276/284, 276/285, 276/286, 276/287, 276/288, 277/267, 277/268, 277/269, 277/270, 277/271, 277/272, 277/273, 277/274, 277/275, 277/276, 277/277 277/278, 277/279, 277/280, 277/281, 277/282, 277/283, 277/284, 277/285, 277/286, 277/287, 277/288, 278/267, 278/268, 278/269, 278/270, 278/271, 278/272, 278/273, 278/274, 278/275, 278/276, 278/27 7, 278/278, 278/279, 278/280, 278/281, 278/282, 278/283, 278/284, 278/285, 278/286, 278/287, 278/288, 279/267, 279/268, 279/269, 279/270, 279/271, 279/272, 279/273, 279/274, 279/275, 279/276, 279/27 7, 279/278, 279/279, 279/280, 279/281, 279/282, 279/283, 279/284, 279/285, 279/286, 279/287, 279/288, 280/267, 280/268, 280/269, 280/270, 280/271, 280/272, 280/273, 280/274, 280/275, 280/276, 280/2 77, 280/278, 280/279, 280/280, 280/281, 280/282, 280/283, 280/284, 280/285, 280/286, 280/287, 280/288, 281/267, 281/268, 281/269, 281/270, 281/271, 281/272, 281/273, 281/274, 281/275, 281/276, 281/2 77, 281/278, 281/279, 281/280, 281/281, 281/282, 281/283, 281/284, 281/285, 281/286, 281/287, 281/288, 282/267, 282/268, 282/269, 282/270, 282/271, 282/272, 282/273, 282/274, 282/275, 282/276, 282/ 277, 282/278, 282/279, 282/280, 282/281, 282/282, 282/283, 282/284, 282/285, 282/286, 282/287, 282/288, 283/267, 283/268, 283/269, 283/270, 283/271, 283/272, 283/273, 283/274, 283/275, 283/276, 283 /277, 283/278, 283/279, 283/280, 283/281, 283/282, 283/283, 283/284, 283/285, 283/286, 283/287, 283/288, 284/267, 284/268, 284/269, 284/270, 284/271, 284/272, 284/273, 284/274, 284/275, 284/276, 28 4/277, 284/278, 284/279, 284/280, 284/281, 284/282, 284/283, 284/284, 284/285, 284/286, 284/287, 284/288, 285/267, 285/268, 285/269, 285/270, 285/271, 285/272, 285/273, 285/274, 285/275, 285/276, 28 5/277, 285/278, 285/279, 285/280, 285/281, 285/282, 285/283, 285/284, 285/285, 285/286, 285/287, 285/288, 286/267, 286/268, 286/269, 286/270, 286/271, 286/272, 286/273, 286/274, 286/275, 286/276, 2 86/277, 286/278, 286/279, 286/280, 286/281, 286/282, 286/283, 286/284, 286/285, 286/286, 286/287, 286/288, 287/267, 287/268, 287/269, 287/270, 287/271, 287/272, 287/273, 287/274, 287/275, 287/276, 2 87/277, 287/278, 287/279, 287/280, 287/281, 287/282, 287/283, 287/284, 287/285, 287/286, 287/287, 287/288, 117/118, 117/119, 117/120, 117/121, 117/122, 117/123, 118/118, 118/119, 118/120, 118/121 118/122, 118/123, 119/118, 119/119, 119/120, 119/121, 119/122, 119/123, 120/118, 120/119, 120/120, 120/121, 120/122, 120/123, 121/118, 121/119, 121/120, 121/121, 121/122, 121/123, 122/118, 122/119 122/120, 122/121, 122/122, 122/123, 340/341, 340/342, 340/343, 340/344, 340/345, 340/346, 340/347, 340/348, 340/349, 340/350, 340/351, 340/352, 340/353, 341/341, 341/342, 341/343, 341/344, 341/345 341/346, 341/347, 341/348, 341/349, 341/350, 341/351, 341/352, 341/353, 342/341, 342/342, 342/343, 342/344, 342/345, 342/346, 342/347, 342/348, 342/349, 342/350, 342/351, 342/352, 342/353, 343/34 1, 343/342, 343/343, 343/344, 343/345, 343/346, 343/347, 343/348, 343/349, 343/350, 343/351, 343/352, 343/353, 344/341, 344/342, 344/343, 344/344, 344/345, 344/346, 344/347, 344/348, 344/349, 344/3 50, 344/351, 344/352, 344/353, 345/341, 345/342, 345/343, 345/344, 345/345, 345/346, 345/347, 345/348, 345/349, 345/350, 345/351, 345/352, 345/353, 346/341, 346/342, 346/343, 346/344, 346/345, 346/3 46, 346/347, 346/348, 346/349, 346/350, 346/351, 346/352, 346/353, 347/341, 347/342, 347/343, 347/344, 347/345, 347/346, 347/347, 347/348, 347/349, 347/350, 347/351, 347/352, 347/353, 348/341, 348/ 342, 348/343, 348/344, 348/345, 348/346, 348/347, 348/348, 348/349, 348/350, 348/351, 348/352, 348/353, 349/341, 349/342, 349/343, 349/344, 349/345, 349/346, 349/347, 349/348, 349/349, 349/350, 349/ 351, 349/352, 349/353, 350/341, 350/342, 350/343, 350/344, 350/345, 350/346, 350/347, 350/348, 350/349, 350/350, 350/351, 350/352, 350/353, 351/341, 351/342, 351/343, 351/344, 351/345, 351/346, 351 The mutations of the optical probe at sites /347, 351/348, 351/349, 351/350, 351/351, 351/352, 351/353, 352/341, 352/342, 352/343, 352/344, 352/345, 352/346, 352/347, 352/348, 352/349, 352/350, 352/351, 352/352, and 352/353 are shown in any row of Table 5.

在一个或多个实施方案中，所述光学探针中，D-葡萄糖敏感多肽如SEQ ID NO：1所示，光学活性多肽如SEQ ID NO：2、6、7、9所示，光学活性多肽位于D-葡萄糖敏感多肽的348/352位点，并且，所述光学探针具有如下所示的突变：(1)D-葡萄糖敏感多肽的V347W、H348T，和光学活性多肽的1A，(2)D-葡萄糖敏感多肽的V347W、H348G，和光学活性多肽的1G，(3)D-葡萄糖敏感多肽的V347W、H348S，和光学活性多肽的1A，(4)D-葡萄糖敏感多肽的V347W、H348S，和光学活性多肽的1G，(5)D-葡萄糖敏感多肽的V347W、H348T，和光学活性多肽的1G，(6)D-葡萄糖敏感多肽的V347W、H348T、H66R，和光学活性多肽的1A，(7)D-葡萄糖敏感多肽的V347W、H348T、H66E，和光学活性多肽的1A，(8)D-葡萄糖敏感多肽的V347W、H348T、H66A，和光学活性多肽的1A，(9)D-葡萄糖敏感多肽的V347W、H348T、H66F，和光学活性多肽的1A，(10)D-葡萄糖敏感多肽的V347W、H348T、H66M，和光学活性多肽的1A，(11)D-葡萄糖敏感多肽的V347W、H348T、H66C，和光学活性多肽的1A，(12)D-葡萄糖敏感多肽的V347W、H348T、H66P，和光学活性多肽的1A，(13)D-葡萄糖敏感多肽的V347W、H348T、H66Q，和光学活性多肽的1A，(14)D-葡萄糖敏感多肽的V347W、H348T、H66T，和光学活性多肽的1A，(15)D-葡萄糖敏感多肽的V347W、H348T、W9R，和光学活性多肽的1A，(16)D-葡萄糖敏感多肽的V347W、H348T、W9H，和光学活性多肽的1A，(17)D-葡萄糖敏感多肽的V347W、H348T、W9E，和光学活性多肽的1A，(18)D-葡萄糖敏感多肽的V347W、H348T、W9A，和光学活性多肽的1A，(19)D-葡萄糖敏感多肽的V347W、H348T、W9V，和光学活性多肽的1A，(20)D-葡萄糖敏感多肽的V347W、H348T、W9L，和光学活性多肽的1A，(21)D-葡萄糖敏感多肽的V347W、H348T、W9F，和光学活性多肽的1A，(22)D-葡萄糖敏感多肽的V347W、H348T、W9I，和光学活性多肽的1A，(23)D-葡萄糖敏感多肽的V347W、H348T、W9M，和光学活性多肽的1A，(24)D-葡萄糖敏感多肽的V347W、H348T、W9C，和光学活性多肽的1A，(25)D-葡萄糖敏感多肽的V347W、H348T、W9P，和光学活性多肽的1A，(26)D-葡萄糖敏感多肽的V347W、H348T、W9N，和光学活性多肽的1A，(27)D-葡萄糖敏感多肽的V347W、H348T、W9G，和光学活性多肽的1A，(28)D-葡萄糖敏感多肽的V347W、H348T、W9K，和光学活性多肽的1A，(29)D-葡萄糖敏感多肽的V347W、H348T、W9Y，和光学活性多肽的1A，(30)D-葡萄糖敏感多肽的V347W、H348T、W9S，和光学活性多肽的1A，(31)D-葡萄糖敏感多肽的V347W、H348T、W9T，和光学活性多肽的1A，(32)D-葡萄糖敏感多肽的V347W、H348T、W9D，和光学活性多肽的1A，(33)D-葡萄糖敏感多肽的V347W、H348T、W8R，和光学活性多肽的1A，(34)D-葡萄糖敏感多肽的V347W、H348T、W8H，和光学活性多肽的1A，(35)D-葡萄糖敏感多肽的V347W、H348T、W8E，和光学活性多肽的1A，(36)D-葡萄糖敏感多肽的V347W、H348T、W8A，和光学活性多肽的1A，(37)D-葡萄糖敏感多肽的V347W、H348T、W8V，和光学活性多肽的1A，(38)D-葡萄糖敏感多肽的V347W、H348T、W8L，和光学活性多肽的1A，(39)D-葡萄糖敏感多肽的V347W、H348T、W8F，和光学活性多肽的1A，(40)D-葡萄糖敏感多肽的V347W、H348T、W8I，和光学活性多肽的1A，(41)D-葡萄糖敏感多肽的V347W、H348T、W8M，和光学活性多肽的1A，(42)D-葡萄糖敏感多肽的V347W、H348T、W8C，和光学活性多肽的1A，(43)D-葡萄糖敏感多肽的V347W、H348T、W8N，和光学活性多肽的1A，(44)D-葡萄糖敏感多肽的V347W、H348T、W8G，和光学活性多肽的1A，(45)D-葡萄糖敏感多肽的V347W、H348T、W8K，和光学活性多肽的1A，(46)D-葡萄糖敏感多肽的V347W、H348T、W8T，和光学活性多肽的1A，(47)D-葡萄糖敏感多肽的V347W、H348T、W8D，和光学活性多肽的1A，(48)D-葡萄糖敏感多肽的V347W、H348T、A42R，和光学活性多肽的1A，(49)D-葡萄糖敏感多肽的V347W、H348T、A42E，和光学活性多肽的1A，(50)D-葡萄糖敏感多肽的V347W、H348T、A42W，和光学活性多肽的1A，(51)D-葡萄糖敏感多肽的V347W、H348T、A42F，和光学活性多肽的1A，(52)D-葡萄糖敏感多肽的V347W、H348T、A42M，和光学活性多肽的1A，(53)D-葡萄糖敏感多肽的V347W、H348T、A42C，和光学活性多肽的1A，(54)D-葡萄糖敏感多肽的V347W、H348T、A42P，和光学活性多肽的1A，(55)D-葡萄糖敏感多肽的V347W、H348T、A42N，和光学活性多肽的1A，(56)D-葡萄糖敏感多肽的V347W、H348T、A42G，和光学活性多肽的1A，(57)D-葡萄糖敏感多肽的V347W、H348T、A42K，和光学活性多肽的1A，(58)D-葡萄糖敏感多肽的V347W、H348T、A42Y，和光学活性多肽的1A，(59)D-葡萄糖敏感多肽的V347W、H348T、A42T，和光学活性多肽的1A，(60)D-葡萄糖敏感多肽的V347W、H348T、K312R，和光学活性多肽的1A，(61)D-葡萄糖敏感多肽的V347W、H348T、K312A，和光学活性多肽的1A，(62)D-葡萄糖敏感多肽的V347W、H348T、K312Q，和光学活性多肽的1A，(63)D-葡萄糖敏感多肽的V347W、H348T、K312G，和光学活性多肽的1A，(64)D-葡萄糖敏感多肽的V347W、H348T、K312H，和光学活性多肽的1A，(65)D-葡萄糖敏感多肽的V347W、H348T、K312S，和光学活性多肽的1A。In one or more embodiments, the optical probe contains a D-glucose-sensitive polypeptide as shown in SEQ ID NO: 1 and an optically active polypeptide as shown in SEQ ID NO: 2, 6, 7, and 9, wherein the optically active polypeptide is located at position 348/352 of the D-glucose-sensitive polypeptide, and the optical probe has the following mutations: (1) V347W and H348T of the D-glucose-sensitive polypeptide and 1A of the optically active polypeptide; (2) V347W and H348T of the D-glucose-sensitive polypeptide. 348G, and 1G of optically active peptide, (3) V347W and H348S of D-glucose-sensitive peptide, and 1A of optically active peptide, (4) V347W and H348S of D-glucose-sensitive peptide, and 1G of optically active peptide, (5) V347W and H348T of D-glucose-sensitive peptide, and 1G of optically active peptide, (6) V347W, H348T, and H66R of D-glucose-sensitive peptide, and 1A of optically active peptide, (7) D-glucose-sensitive (8) D-glucose-sensitive peptides V347W, H348T, H66E, and optically active peptide 1A; (9) D-glucose-sensitive peptides V347W, H348T, H66F, and optically active peptide 1A; (10) D-glucose-sensitive peptides V347W, H348T, H66M, and optically active peptide 1A; (11) D-glucose-sensitive peptides V347W, H348T, H66M, and optically active peptide 1A; (12) D-glucose-sensitive peptides V347W, H348T, H66P, and optically active peptide 1A; (13) D-glucose-sensitive peptides V347W, H348T, H66Q, and optically active peptide 1A; (14) D-glucose-sensitive peptides V347W, H348T, H66T, and optically active peptide 1A; (15) D-glucose-sensitive peptides V347W, H348T, H66Q, and optically active peptide 1A; T, W9R, and optically active peptide 1A, (16) D-glucose-sensitive peptides V347W, H348T, W9H, and optically active peptide 1A, (17) D-glucose-sensitive peptides V347W, H348T, W9E, and optically active peptide 1A, (18) D-glucose-sensitive peptides V347W, H348T, W9A, and optically active peptide 1A, (19) D-glucose-sensitive peptides V347W, H348T, W9V, and optically active peptide 1A, 1A of the sex-sensitive peptide, (20) V347W, H348T, W9L of the D-glucose-sensitive peptide, and 1A of the optically active peptide, (21) V347W, H348T, W9F of the D-glucose-sensitive peptide, and 1A of the optically active peptide, (22) V347W, H348T, W9I of the D-glucose-sensitive peptide, and 1A of the optically active peptide, (23) V347W, H348T, W9M of the D-glucose-sensitive peptide, and 1A of the optically active peptide, (24) (25) D-glucose-sensitive peptides V347W, H348T, W9C, and optically active peptide 1A, (26) D-glucose-sensitive peptides V347W, H348T, W9P, and optically active peptide 1A, (27) D-glucose-sensitive peptides V347W, H348T, W9N, and optically active peptide 1A, (28) D-glucose-sensitive peptides (29) D-glucose-sensitive peptides V347W, H348T, W9Y, and optically active peptide 1A; (30) D-glucose-sensitive peptides V347W, H348T, W9S, and optically active peptide 1A; (31) D-glucose-sensitive peptides V347W, H348T, W9T, and optically active peptide 1A; (32) D-glucose-sensitive peptides V347W, H348T, W9K, and optically active peptide 1A; T, W9D, and optically active peptide 1A, (33) D-glucose-sensitive peptides V347W, H348T, W8R, and optically active peptide 1A, (34) D-glucose-sensitive peptides V347W, H348T, W8H, and optically active peptide 1A, (35) D-glucose-sensitive peptides V347W, H348T, W8E, and optically active peptide 1A, (36) D-glucose-sensitive peptides V347W, H348T, W8A, and optically active peptide 1A, 1A of the sex-sensitive peptide, (37) V347W, H348T, W8V of the D-glucose-sensitive peptide, and 1A of the optically active peptide, (38) V347W, H348T, W8L of the D-glucose-sensitive peptide, and 1A of the optically active peptide, (39) V347W, H348T, W8F of the D-glucose-sensitive peptide, and 1A of the optically active peptide, (40) V347W, H348T, W8I of the D-glucose-sensitive peptide, and 1A of the optically active peptide, (41) (42) D-glucose-sensitive peptides V347W, H348T, W8M, and optically active peptide 1A; (43) D-glucose-sensitive peptides V347W, H348T, W8C, and optically active peptide 1A; (44) D-glucose-sensitive peptides V347W, H348T, W8N, and optically active peptide 1A; (45) D-glucose-sensitive peptides V347W, H348T, W8G, and optically active peptide 1A; V347W, H348T, W8K, and optically active peptide 1A, (46) D-glucose-sensitive peptides V347W, H348T, W8T, and optically active peptide 1A, (47) D-glucose-sensitive peptides V347W, H348T, W8D, and optically active peptide 1A, (48) D-glucose-sensitive peptides V347W, H348T, A42R, and optically active peptide 1A, (49) D-glucose-sensitive peptides V347W, H348T, W8K, and optically active peptide 1A, (41) D-glucose-sensitive peptides V347W, H348T, W8K, and optically active peptide 1A, (42) D-glucose-sensitive peptides V347W, H348T, W8K, and optically active peptide 1A, (43) D-glucose-sensitive peptides V347W, H348T, W8K, and optically active peptide 1A, (4 ...5) D-glucose-sensitive peptides V347W, H348T, W8K, 8T, A42E, and optically active peptide 1A, (50) D-glucose-sensitive peptides V347W, H348T, A42W, and optically active peptide 1A, (51) D-glucose-sensitive peptides V347W, H348T, A42F, and optically active peptide 1A, (52) D-glucose-sensitive peptides V347W, H348T, A42M, and optically active peptide 1A, (53) D-glucose-sensitive peptides V347W, H348T, A42 C, and optically active peptide 1A, (54) D-glucose-sensitive peptides V347W, H348T, A42P, and optically active peptide 1A, (55) D-glucose-sensitive peptides V347W, H348T, A42N, and optically active peptide 1A, (56) D-glucose-sensitive peptides V347W, H348T, A42G, and optically active peptide 1A, (57) D-glucose-sensitive peptides V347W, H348T, A42K, and optically active 1A of the sex-sensitive peptide, (58) V347W, H348T, A42Y of the D-glucose-sensitive peptide, and 1A of the optically active peptide, (59) V347W, H348T, A42T of the D-glucose-sensitive peptide, and 1A of the optically active peptide, (60) V347W, H348T, K312R of the D-glucose-sensitive peptide, and 1A of the optically active peptide, (61) V347W, H348T, K312A of the D-glucose-sensitive peptide, and 1A of the optically active peptide. 1A, (62) D-glucose-sensitive peptides V347W, H348T, K312Q, and optically active peptide 1A, (63) D-glucose-sensitive peptides V347W, H348T, K312G, and optically active peptide 1A, (64) D-glucose-sensitive peptides V347W, H348T, K312H, and optically active peptide 1A, (65) D-glucose-sensitive peptides V347W, H348T, K312S, and optically active peptide 1A.

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。In two or more polypeptide or nucleic acid molecular sequences, the term "identity" or "percentage of identity" refers to the fact that, when compared and matched for maximum correspondence using methods known in the art, such as sequence comparison algorithms, by manual alignment and visual inspection, two or more sequences or subsequences are identical or have a certain percentage of amino acid residues or nucleotides identical in a specified region (e.g., 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical). For example, the preferred algorithms for determining the percentage of sequence identity and the percentage of sequence similarity are BLAST and BLAST 2.0 algorithms, which can be found in Altschul et al. (1977) Nucleic Acids Res. 25: 3389 and Altschul et al. (1990) J.Mol.Biol. 215: 403, respectively.

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。As is known to those skilled in the art, gene cloning often requires the design of suitable restriction enzyme sites, which inevitably introduces one or more irrelevant residues at the end of the expressed polypeptide or protein, without affecting the activity of the target polypeptide or protein. Similarly, to construct fusion proteins, promote the expression of recombinant proteins, obtain recombinant proteins that are automatically secreted outside host cells, or facilitate the purification of recombinant proteins, it is often necessary to add certain amino acids to the N-terminus, C-terminus, or other suitable regions within the recombinant protein. These include, but are not limited to, suitable adaptor peptides, signal peptides, leader peptides, terminal extensions, glutathione S-transferase (GST), maltose E-binding proteins, protein A, tags such as 6His or Flag, or proteolytic enzyme sites such as factor Xa, thrombin, or enterokinase.

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D-葡萄糖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D-葡萄糖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D-葡萄糖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。As used herein, the terms “functional fragment,” “derivative,” and “analyte” refer to a protein that substantially retains the same biological function or activity as the original polypeptide or protein (e.g., D-glucose-binding protein or fluorescent protein). Functional variants, derivatives, or analogs of the polypeptides or proteins of the present invention (e.g., D-glucose-binding protein or fluorescent protein) may be (i) proteins with one or more conserved or non-conserved amino acid residues (preferably conserved amino acid residues) substituted, such substituted amino acid residues may or may not be encoded by the genetic code; or (ii) proteins having substituent groups in one or more amino acid residues; or (iii) proteins formed by the fusion of a mature protein with another compound (e.g., a compound that extends the protein's half-life, such as polyethylene glycol); or (iv) proteins formed by the fusion of an additional amino acid sequence into the protein sequence (e.g., a secreted sequence or a sequence used to purify the protein or the original protein sequence, or a fusion protein formed with an antigen IgG fragment). Based on the teachings herein, these functional variants, derivatives, and analogs are within the scope well known to those skilled in the art. The analogues also include those having residues different from naturally occurring L-amino acids (such as D-amino acids), and those having non-naturally occurring or synthetic amino acids (such as β- or γ-amino acids). It should be understood that the D-glucose-sensitive polypeptides of the present invention are not limited to the representative proteins, variants, derivatives, and analogues listed above. Modifications (generally without altering the primary structure) include chemically derived forms of proteins, such as acetylation or carboxylation, either in vivo or in vitro. Modifications also include glycosylation, such as those resulting from glycosylation modifications during protein synthesis and processing or further processing steps. This modification can be accomplished by exposing the protein to glycosylation enzymes (such as mammalian glycosylation or deglycosylation enzymes). Modifications also include sequences having phosphorylated amino acid residues (such as phosphotyrosine, phosphotyserine, phosphotythreonine). Proteins modified to improve their resistance to proteolytic hydrolysis or optimize their solubility are also included.

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

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。The subcellular organelles described herein include the cytoplasm, mitochondria, nucleus, endoplasmic reticulum, cell membrane, Golgi apparatus, lysosomes, and peroxisomes. In some embodiments, the tags used for purification or for immunoblotting include 6-histidine (6*His), glutathione S-transferase (GST), and Flag.

本发明包含编码本发明所述D-葡萄糖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)。This invention comprises nucleic acid molecules encoding the D-glucose-sensitive polypeptide or optical probe described herein. The terms "nucleic acid," "nucleotide," "polynucleotide," or "nucleic acid sequence" as used herein can be in DNA or RNA form. DNA form includes cDNA, genomic DNA, or artificially synthesized DNA. DNA can be single-stranded or double-stranded. DNA can be a coding strand or a non-coding strand. When referring to nucleic acids, the term "variant" as used herein can be a naturally occurring allelic variant or a non-naturally occurring variant. These nucleotide variants include degenerate variants, substitution variants, deletion variants, and insertion variants. As is known in the art, an allelic variant is a substitution of a nucleic acid, which may be a substitution, deletion, or insertion of one or more nucleotides, but does not substantially alter the function of the protein it encodes. The nucleic acid of this invention may comprise a nucleotide sequence with at least about 50%, at least about 60%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100% sequence identity with the nucleic acid sequence described herein. This invention also relates to nucleic acid fragments that hybridize with the above-described sequences. As used herein, a "nucleic acid fragment" contains at least 15 nucleotides in length, preferably at least 30 nucleotides, more preferably at least 50 nucleotides, and most preferably at least 100 nucleotides or more. The nucleic acid fragments can be used in nucleic acid amplification techniques (such as PCR).

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。The full-length sequence or fragment of the optical probe or fusion protein of this invention can typically be obtained by PCR amplification, artificial synthesis, or recombinant methods. The steps and reagents used in conventional PCR, synthesis, and recombinant methods are known in the art. Furthermore, mutations can be introduced into the protein sequence of this invention through methods such as mutagenic PCR or chemical synthesis.

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。This invention also relates to nucleic acid constructs containing the polynucleotides described herein, and one or more regulatory sequences operatively linked to these sequences. The polynucleotides described herein can be manipulated in various ways to ensure the expression of the polypeptide or protein. The nucleic acid constructs can be manipulated before insertion into a vector, depending on the expression vector or requirements. Techniques for altering polynucleotide sequences using recombinant DNA methods are known in the art.

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。In some embodiments, 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, such as plasmids, phage particles, phage derivatives, animal viruses, and entrapments.

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。Typical expression vectors contain expression control sequences that can be used to regulate the expression of a desired nucleic acid sequence, operatively linked to the nucleic acid sequence described herein or its complement. As used herein, the term "expression control sequence" refers to an element that can be operatively linked to the target gene to regulate the transcription, translation, and expression of the target gene. This can be an origin of replication, promoter, marker gene, or translation control element, including enhancers, operons, terminators, ribosome binding sites, etc. The choice of expression control sequence depends on the host cell used. In recombinant expression vectors, "operative linking" refers to the linking of the target nucleotide sequence to the regulatory sequence in a manner that allows the nucleotide sequence to be expressed. Those skilled in the art are familiar with methods for constructing expression vectors containing the coding sequence of the fusion protein of this invention and suitable transcription/translation control signals. These methods include in vitro recombinant DNA techniques, DNA synthesis techniques, in vivo recombination techniques, etc. The DNA sequence can be efficiently linked to an appropriate promoter in the expression vector to guide mRNA synthesis. Representative examples of these promoters include: the lac or trp promoter of *E. coli*; the PL promoter of *λ* phage; eukaryotic promoters including the CMV immediate early promoter, the HSV thymidine kinase promoter, early and late SV40 promoters, the LTR of retroviruses, and other known promoters that 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 terminator. In one embodiment, the expression vector may be a commercially available pCDF vector, with no other special requirements. Exemplarily, the nucleotide sequence encoding the optical probe and the expression vector are double-digested with BamHI and EcoRI, respectively, and then the digestion products are ligated to obtain the recombinant expression vector. This invention does not specifically limit the specific steps and parameters of digestion and ligation; conventional steps and parameters in the art can be used.

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。After obtaining the recombinant expression vector, the vector is transformed into a host cell to produce a protein or peptide including a fusion protein. This transfer process can be performed using conventional techniques well known to those skilled in the art, such as transformation or transfection. The host cell described in this invention refers to a cell capable of receiving and accommodating recombinant DNA molecules, serving as the site for recombinant gene amplification. Ideally, the recipient cell should meet the conditions of easy acquisition and proliferation. The "host cell" of this invention can include prokaryotic and eukaryotic cells, specifically including bacterial cells, yeast cells, insect cells, and mammalian cells. The host cell is preferably a variety of cells conducive to gene product expression or fermentation production, such cells being 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*, animal cells such as CHO, COS, HEK293, HeLa cells, or Bowes melanoma cells, etc. The exemplary host cell used in the embodiments of this invention is *Escherichia coli* strain BL21-DE3. Those skilled in the art will understand how to select appropriate vectors, promoters, enhancers, and host cells.

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。The method for transferring DNA to host cells described in this invention is a conventional method in the art, including calcium phosphate or calcium chloride co-precipitation, DEAE-mannan-mediated transfection, lipid transfection, native competent cells, chemically mediated transfer, or electroporation. When the host is a prokaryote such as *Escherichia coli*, the preferred method is the CaCl2 or MgCl2 method, and the steps used are well known in the art. When the host cell is a eukaryotic cell, the following DNA transfection methods can be used: calcium phosphate co-precipitation, conventional mechanical methods such as microinjection, electroporation, liposome packaging, etc.

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D-葡萄糖光学探针。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。This invention involves transforming an expression vector into host cells, followed by amplification and expression culture of the host cells to isolate the D-glucose optical probe. The host cell amplification and expression culture can be performed using conventional methods. Depending on the type of host cells used, the culture medium can be any conventional medium. Culture is carried out under conditions suitable for host cell growth.

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D-葡萄糖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。In this invention, the optical probe is expressed intracellularly, on the cell membrane, or secreted extracellularly. If desired, the recombinant protein can be separated or purified using various separation methods based on its physical, chemical, and other properties. This invention does not specifically limit the method for separating the D-glucose fluorescent protein; conventional methods for separating fusion proteins in the art can be used. These methods are well known to those skilled in the art and include, but are not limited to, conventional refolding, salting out, centrifugation, permeation, sonication, ultracentrifugation, molecular sieve chromatography, adsorption chromatography, ion exchange chromatography, high-performance liquid chromatography (HPLC), and various other liquid chromatography techniques and combinations thereof. In one embodiment, the optical probe is separated using His-tagged affinity chromatography.

本发明还提供了所述D-葡萄糖光学探针在D-葡萄糖实时定位、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D-葡萄糖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D-葡萄糖的实时定位；通过D-葡萄糖标准滴加曲线结合荧光信号的变化进行相应D-葡萄糖的定量检测。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D-葡萄糖标准滴加曲线是根据D-葡萄糖光学探针在不同浓度D-葡萄糖的情况下的荧光信号绘制而成。本发明所述D-葡萄糖光学探针直接转入细胞中，在D-葡萄糖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D-葡萄糖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D-葡萄糖含量的变化，从而筛选出对D-葡萄糖含量变化有影响的化合物。在本发明中所述的D-葡萄糖光学探针在D-葡萄糖实时定位、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。This invention also provides the application of the D-glucose optical probe in real-time localization, quantitative detection, and high-throughput compound screening of D-glucose. In one aspect, the D-glucose optical probe is preferably linked to a signal peptide at different sites within the cell and transferred into the cell. Real-time localization of D-glucose is achieved by detecting the intensity of fluorescence signals within the cell; quantitative detection of D-glucose is then performed by combining a standard D-glucose titration curve with changes in fluorescence signals. Changes in fluorescence signals are displayed, for example, by a standardized fluorescence signal ratio. In embodiments involving cpYFP, this ratio is the ratio of the sample's 485 nm fluorescence signal to its 420 nm fluorescence signal to the corresponding ratio of the control. The D-glucose standard titration curve of this invention is plotted based on the fluorescence signals obtained by the D-glucose optical probe at different concentrations of D-glucose. The D-glucose optical probe of this invention is directly transferred into the cell, eliminating the need for time-consuming sample processing during real-time localization and quantitative detection of D-glucose, thus improving accuracy. The D-glucose optical probe of this invention, when used for high-throughput compound screening, involves adding different compounds to cell culture medium and measuring changes in D-glucose content to screen for compounds that affect these changes. The application of the D-glucose optical probe described in this invention for real-time localization, quantitative detection, and high-throughput compound screening is not for diagnostic or therapeutic purposes and does not involve the diagnosis or treatment of diseases.

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D-葡萄糖所需的其他试剂。所述其他试剂本领域周知，例如缓冲液、细胞培养基、D-葡萄糖标品。示例性缓冲液例如100mM HEPES和100mM NaCl，pH 7.4。This invention also provides a detection kit comprising the optical probes, nucleic acid molecules, nucleic acid constructs, and/or cells described herein. The kit further comprises other reagents required for the detection of D-glucose. These other reagents are well known in the art, such as buffers, cell culture media, and D-glucose standards. Exemplary buffers include, for example, 100 mM HEPES and 100 mM 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。In this document, concentrations, contents, percentages, and other values are expressed in range form. It should also be understood that this range form is used for convenience and brevity only, and should be flexibly interpreted to include the values explicitly mentioned at the upper and lower limits of the range, as well as all individual values or subranges included within that range.

实施例Example

下面结合实施例对本发明提供的D-葡萄糖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。The D-glucose optical probe provided by the present invention will be described in detail below with reference to the embodiments, but these should not be construed as limiting the scope of protection of the present invention.

I.实验材料和试剂I. Experimental Materials and Reagents

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。The embodiments mainly employ conventional genetic engineering molecular biology cloning methods, cell culture, and imaging methods, which are well known to those skilled in the art. Examples include: Jane Rothcams et al.'s "Molecular Biology Laboratory Reference Manual," J. Sambrook and D.W. Russell, translated by Huang Peitang et al. (Molecular Cloning Laboratory Manual, 3rd Edition, August 2002, Science Press, Beijing); Fereschney et al.'s "Animal Cell Culture: A Basic Technical Guide" (5th Edition), translated by Zhang Jingbo and Xu Cunshuan et al.; and J.S. Bonifensenon, M. Dassault et al.'s "A Concise Guide to Cell Biology Laboratory Techniques," translated by Zhang Jingbo et al.

实施例中所用的基于pCDF-cpYFP，pCDF-ttGBP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(T4PNK)购自Fermentas公司，购买时附带有相对应的缓冲液等。转染试剂Lip2000 Kit购于Invitrogen公司。D-葡萄糖等化合物均购自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。The pCDF-cpYFP and pCDF-ttGBP plasmids used in the examples were constructed by the Protein Laboratory of East China University of Science and Technology, and the pCDF plasmid vectors were purchased from Invitrogen. All primers used for PCR were synthesized, purified, and identified correctly by mass spectrometry by Shanghai Jierui Biotechnology Co., Ltd. and BGI Genomics. The expression plasmids constructed in the examples were all sequenced, and the sequencing was performed by BGI Genomics and J. Lee Sequencing. The Taq DNA polymerase used in each example was purchased from Dongsheng Biotechnology, the pfu DNA polymerase was purchased from Tiangen Biotech (Beijing) Co., Ltd., and the primeSTAR DNA polymerase was purchased from TaKaRa. The corresponding polymerase buffer and dNTPs were included with the purchase of the three polymerases. Restriction endonucleases such as BamHI, BglII, HindIII, NdeI, XhoI, EcoRI, and SpeI, T4 ligase, and T4 phosphorylase (T4PNK) were purchased from Fermentas, and the corresponding buffers were included with the purchase. The Lip2000 transfection kit was purchased from Invitrogen. D-glucose and other compounds were purchased from Sigma-Aldrich. Unless otherwise stated, inorganic salts and other chemical reagents were purchased from Sigma-Aldrich. HEPES salt, ampicillin (Amp), and puromycin were purchased from Amersco. The 96-well detection blackboard and the 384-well fluorescence detection blackboard were purchased from Grenier.

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。The DNA purification kits used in these examples were purchased from BBI (Canada), and the general plasmid extraction kits were purchased from Tiangen Biotech (Beijing) Co., Ltd. The cloned strain Mach1 was purchased from Invitrogen. Nickel affinity chromatography columns and desalting column packing materials were both from GE Healthcare.

实施例中用到的主要仪器包括：Biotek Synergy2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)。The main instruments used in the examples include: Biotek Synergy2 multi-functional microplate reader (Bio-Tek, USA), X-15R high-speed refrigerated centrifuge (Beckman, USA), Microfuge 22R benchtop high-speed refrigerated centrifuge (Beckman, USA), PCR amplifier (Biometra, Germany), ultrasonic disruptor (Ningbo Xinzhi Co., Ltd.), nucleic acid electrophoresis apparatus (Shenneng Bocai Co., Ltd.), fluorescence spectrophotometer (Varian, USA), CO2 incubator (SANYO), and inverted fluorescence microscope (Nikon, Japan).

II.分子生物学方法和细胞实验方法II. Molecular Biology Methods and Cellular Experimental Methods

II.1聚合酶链式反应(PCR)：II.1 Polymerase Chain Reaction (PCR):

1.目的片段扩增PCR：1. PCR amplification of the target fragment:

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

2.长片段(>2500bp)扩增PCR：2. Long fragment (>2500bp) amplification 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。The long-fragment amplification used in this invention is mainly a reverse PCR amplification vector, a technique used in the following examples to obtain site-directed mutagenesis. Reverse PCR primers are designed at the mutation site, with one primer containing the mutated nucleotide sequence at its 5' end. The amplified product then contains the corresponding mutation site. The long-fragment amplification PCR reaction system is as follows: template sequence (10 pg-1 ng) 1 μL, forward primer (25 μM) 0.5 μL, reverse primer (25 μM) 0.5 μL, 5×PrimerSTAR buffer 10 μL, PrimerSTAR DNA polymerase 0.5 μL, dNTP (2.5 mM) 4 μL, sterile ultrapure water ( ddH₂O ) 33.5 μL, total volume 50 μL. The PCR amplification program is as follows: denaturation at 95℃ for 5 minutes, 30 cycles (98℃ for 10 seconds, 50-68℃ for 5-15 seconds, 72℃ for a certain time (1000bp/min)), extension at 72℃ for 10 minutes; or denaturation at 95℃ for 5 minutes, 30 cycles (98℃ for 10 seconds, 68℃ for a certain time (1000bp/min)), extension at 72℃ for 10 minutes.

II.2核酸内切酶酶切反应：II.2 Endonuclease digestion reaction:

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。The double enzyme digestion system for the plasmid vector is as follows: 20 μL plasmid vector (approximately 1.5 μg), 5 μL 10× buffer, 11-2 μL restriction endonuclease, 21-2 μL restriction endonuclease, and bring the total volume to 50 μL with sterile ultrapure water. Reaction conditions: 37℃, 1-7 hours.

II.3 DNA片段5’端磷酸化反应II.3 Phosphorylation of DNA fragments at the 5' end

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。Plasmids or genomes extracted from microorganisms contain phosphate groups at their ends, while PCR products do not. Therefore, a phosphate addition reaction is required at the 5' end of the PCR product. Only DNA molecules with phosphate groups at their ends can undergo ligation. The phosphorylation reaction system is as follows: 5-8 μL of PCR product DNA sequence, 1 μL of 10×T4 ligase buffer, 1 μL of T4 polynucleotide kinase (T4 PNK), and 0-3 μL of sterile ultrapure water, for a total volume of 10 μL. The reaction conditions are 37°C for 30 minutes to 2 hours, followed by inactivation at 72°C for 20 minutes.

II.4目的片段和载体的连接反应II.4 Ligation reaction between target fragment and vector

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The methods for connecting different fragments and carriers vary, and this invention uses three connection methods.

1.平末端短片段和线性化载体的平末端连接1. Blunt-end junctions of blunt-ended short fragments and linearized vectors

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，PEG4000 1μL，10×T4连接酶缓冲液1μL，T4DNA连接酶1μL，总计10μL。反应条件22℃，30分钟。The principle of this method is that the blunt-end product obtained by PCR is phosphorylated at the 5' end of a DNA fragment using T4 PNK, and then ligated with a linearized vector using PEG4000 and T4 DNA ligase to obtain a recombinant plasmid. The homologous recombination ligation system is as follows: 4 μL of T4 PNK-treated DNA fragment, 4 μL of linearized vector fragment, 1 μL of PEG4000, 1 μL of 10×T4 ligase buffer, and 1 μL of T4 DNA ligase, for a total of 10 μL. The reaction conditions are 22℃ for 30 minutes.

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2. Ligation of DNA fragments with sticky ends and vector fragments with sticky ends.

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，T4DNA连接酶1μL，灭菌超纯水补至总体积10μL。反应条件16℃，4-8小时。DNA fragments digested by restriction endonucleases typically produce prominent sticky ends, which can then be ligated to vector fragments containing sequence-complementary sticky ends to form recombinant plasmids. The ligation reaction system is as follows: 1-7 μL of the digested PCR product DNA fragment, 0.5-7 μL of the digested plasmid, 1 μL of 10×T4 ligase buffer, 1 μL of T4 DNA ligase, and sterile ultrapure water to a total volume of 10 μL. The reaction conditions are 16°C for 4-8 hours.

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3. Ligation reaction involving the self-circularization of DNA fragment products phosphorylated at the 5' end following site-directed mutagenesis using reverse PCR.

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

II.5感受态细胞的制备与转化II.5 Preparation and Transformation of Competent Cells

感受态细胞的制备：Preparation of competent cells:

1.挑取单菌落(如Mach1)接种于5mLLB培养基中，37℃摇床过夜。1. Pick a single colony (e.g., Mach1) and inoculate it into 5 mL of LB medium. Incubate overnight at 37°C with a shaker.

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。2. Take 0.5-1 mL of the overnight culture and transfer it to 50 mL of LB medium. Incubate at 37°C and 220 rpm for 3 to 5 hours until the OD 600 reaches 0.5.

3.冰浴预冷细胞2小时。3. Pre-cool the cells in an ice bath for 2 hours.

4.4℃，4000rpm离心10分钟。Centrifuge at 4.4℃ and 4000rpm for 10 minutes.

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。5. Discard the supernatant, resuspend the cells in 5 mL of pre-cooled buffer, and add resuspending buffer to a final volume of 50 mL after homogenization.

6.冰浴45分钟。6. Ice bath for 45 minutes.

7.4℃4000rpm离心10分钟，用5mL冰预冷的储存缓冲液重悬细菌。Centrifuge at 7.4℃ and 4000rpm for 10 minutes, then resuspend the bacteria in 5mL of ice-cold storage buffer.

8.每个EP管中放100μL菌液，-80℃或液氮冻存。8. Place 100 μL of bacterial culture in each EP tube and store at -80°C or in liquid nitrogen.

重悬缓冲液：CaCl2(100mM)、MgCl2(70mM)、NaAc(40mM)Resuspension buffer: CaCl₂ (100mM), MgCl₂ (70mM), NaAc (40mM)

储存缓冲液：0.5mL DMSO、1.9mL 80％甘油、1mL 10×CaCl2(1M)、1mL10×MgCl2(700mM)、1mL 10×NaAc(400mM)、4.6mL ddH2OStorage buffer: 0.5 mL DMSO, 1.9 mL 80% glycerol, 1 mL 10× CaCl₂ (1 M), 1 mL 10× MgCl₂ (700 mM), 1 mL 10×NaAc (400 mM), 4.6 mL ddH₂O

感受态细胞的转化：Transformation of competent cells:

1.取100μL感受态细胞于冰浴上融化。1. Take 100 μL of competent cells and thaw them on an ice bath.

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。2. Add an appropriate volume of ligation product, gently mix by pipetting, and incubate on ice for 30 minutes. The volume of ligation product added is usually less than 1/10 of the competent cell volume.

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。3. Place the bacterial solution in a 42°C water bath for 90 seconds to heat shock, then quickly transfer it to an ice bath and place it for 5 minutes.

4.加入500μL LB，于37℃恒温摇床上200rpm培养1小时。4. Add 500 μL LB and incubate at 200 rpm for 1 hour on a constant temperature shaker at 37℃.

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。5. Centrifuge the bacterial culture at 4000 rpm for 3 minutes, and keep 200 μL of supernatant. Spread the bacterial cells evenly on the surface of an agar plate containing appropriate antibiotics. Incubate the plate upside down in a 37°C incubator overnight.

II.6蛋白质的表达，纯化和荧光检测II.6 Protein Expression, Purification, and Fluorescence Detection

1.将表达载体(例如以pCDF为基础的D-葡萄糖光学探针表达载体)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。1. Transform the expression vector (e.g., a pCDF-based D-glucose optical probe expression vector) into BL21(DE3) cells, incubate upside down overnight, pick clones from the plate into 250ml Erlenmeyer flasks, place them on a 37℃ shaker, and incubate at 220rpm until OD = 0.4-0.8. Add 1/1000 (v/v) of IPTG (1M) and induce expression at 18℃ for 24-36 hours.

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。2. After induction of expression, centrifuge at 4000 rpm for 30 minutes to collect the bacteria. Resuspend the bacterial pellet in 50 mM phosphate buffer and sonicate until the bacterial cells are clear. Centrifuge at 9600 rpm at 4°C for 20 minutes.

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。3. The supernatant from centrifugation was purified by a self-assembled nickel affinity chromatography column to obtain the protein. The protein after nickel affinity chromatography was then purified by a self-assembled desalting column to obtain the protein dissolved in 100mM HEPES buffer (pH 7.4).

4.纯化的蛋白经过SDS-PAGE鉴定后，使用测定缓冲液(100mMHEPES，100mMNaCl，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D-葡萄糖配制成终浓度为50mM的储液。4. After SDS-PAGE identification of the purified protein, the probe was diluted with assay buffer (100 mM HEPES, 100 mM NaCl, pH 7.4) to a final concentration of 0.2-5 μM. D-glucose 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).

5.取100μl 1μM的蛋白溶液，37℃温育10分钟，加入D-葡萄糖滴定，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。5. Take 100 μl of 1 μM protein solution, incubate at 37℃ for 10 minutes, add D-glucose titration, and measure the fluorescence intensity of the protein at 528 nm emission after excitation by 420 nm light and at 528 nm emission after excitation by 485 nm light. The fluorescence excitation and emission measurements of the samples were performed using a multifunctional fluorescent microplate reader.

6.取100μl 1μM的蛋白溶液，37℃温育10分钟，加入D-葡萄糖，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。6. Take 100 μl of 1 μM protein solution, incubate at 37℃ for 10 minutes, add D-glucose, and measure the absorption and fluorescence spectra of the protein. The absorption and fluorescence spectra of the samples were measured using a spectrophotometer and a fluorescence spectrophotometer.

II.7哺乳动物细胞的转染和荧光检测II.7 Transfection and Fluorescence Detection of Mammalian Cells

1.将pCDNA3.1+为基础的D-葡萄糖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。1. The pCDNA3.1+-based D-glucose optical probe plasmid was transfected into HEK293 cells using Lipofectamine 2000 (Invitrogen) and cultured in a 37°C, 5% CO2 cell culture incubator. Fluorescence detection was performed 24–36 h after the exogenous gene was fully expressed.

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。2. After the expression was induced, the adherent HEK293 cells were washed three times with PBS and placed in HBSS solution for fluorescence microscopy and microplate reader detection.

实施例1：D-葡萄糖结合蛋白质粒Example 1: D-glucose binding protein particles

通过PCR扩增T型嗜热菌基因中的TtGBP基因(SEQ ID NO：1)，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。The TtGBP gene (SEQ ID NO: 1) from *Thermophilus T* was amplified by PCR. After gel electrophoresis, the PCR product was recovered and digested with HindIII and XhoI enzymes, and the pCDF vector was also double-digested accordingly. The DNA was ligated using T4 DNA ligase, and the product was used to transform DH5α cells. The transformed DH5α cells were plated on LB agar plates (streptomycin 100 μg/mL) and incubated overnight at 37°C. Plasmids were extracted from the grown DH5α transformants and identified by PCR. Positive plasmids, after being correctly sequenced, were used for subsequent plasmid construction.

实施例2：不同插入位点的cpYFP光学探针的表达和检测Example 2: Expression and detection of cpYFP optical probes at different insertion sites

本实施例中，以pCDF-TtGBP为基础选择了下述位点插入cpYFP，得到相应pCDF-TtGBP-cpYFP质粒：266/267、266/268、266/269、266/270、266/271、266/272、266/273、266/274、266/275、266/276、266/277、266/278、266/279、266/280、266/281、266/282、266/283、266/284、266/285、266/286、266/287、266/288、267/267、267/268、267/269、267/270、267/271、267/272、267/273、267/274、267/275、267/276、267/277、267/278、267/279、267/280、267/281、267/282、267/283、267/284、267/285、267/286、267/287、267/288、268/267、268/268、268/269、268/270、268/271、268/272、268/273、268/274、268/275、268/276、268/277、268/278、268/279、268/280、268/281、268/282、268/283、268/284、268/285、268/286、268/287、268/288、269/267、269/268、269/269、269/270、269/271、269/272、269/273、269/274、269/275、269/276、269/277、269/278、269/279、269/280、269/281、269/282、269/283、269/284、269/285、269/286、269/287、269/288、270/267、270/268、270/269、270/270、270/271、270/272、270/273、270/274、270/275、270/276、270/277、270/278、270/279、270/280、270/281、270/282、270/283、270/284、270/285、270/286、270/287、270/288、271/267、271/268、271/269、271/270、271/271、271/272、271/273、271/274、271/275、271/276、271/277、271/278、271/279、271/280、271/281、271/282、271/283、271/284、271/285、271/286、271/287、271/288、272/267、272/268、272/269、272/270、272/271、272/272、272/273、272/274、272/275、272/276、272/277、272/278、272/279、272/280、272/281、272/282、272/283、272/284、272/285、272/286、272/287、272/288、273/267、273/268、273/269、273/270、273/271、273/272、273/273、273/274、273/275、273/276、273/277、273/278、273/279、273/280、273/281、273/282、273/283、273/284、273/285、273/286、273/287、273/288、274/267、274/268、274/269、274/270、274/271、274/272、274/273、274/274、274/275、274/276、274/277、274/278、274/279、274/280、274/281、274/282、274/283、274/284、274/285、274/286、274/287、274/288、275/267、275/268、275/269、275/270、275/271、275/272、275/273、275/274、275/275、275/276、275/277、275/278、275/279、275/280、275/281、275/282、275/283、275/284、275/285、275/286、275/287、275/288、276/267、276/268、276/269、276/270、276/271、276/272、276/273、276/274、276/275、276/276、276/277、276/278、276/279、276/280、276/281、276/282、276/283、276/284、276/285、276/286、276/287、276/288、277/267、277/268、277/269、277/270、277/271、277/272、277/273、277/274、277/275、277/276、277/277、277/278、277/279、277/280、277/281、277/282、277/283、277/284、277/285、277/286、277/287、277/288、278/267、278/268、278/269、278/270、278/271、278/272、278/273、278/274、278/275、278/276、278/277、278/278、278/279、278/280、278/281、278/282、278/283、278/284、278/285、278/286、278/287、278/288、279/267、279/268、279/269、279/270、279/271、279/272、279/273、279/274、279/275、279/276、279/277、279/278、279/279、279/280、279/281、279/282、279/283、279/284、279/285、279/286、279/287、279/288、280/267、280/268、280/269、280/270、280/271、280/272、280/273、280/274、280/275、280/276、280/277、280/278、280/279、280/280、280/281、280/282、280/283、280/284、280/285、280/286、280/287、280/288、281/267、281/268、281/269、281/270、281/271、281/272、281/273、281/274、281/275、281/276、281/277、281/278、281/279、281/280、281/281、281/282、281/283、281/284、281/285、281/286、281/287、281/288、282/267、282/268、282/269、282/270、282/271、282/272、282/273、282/274、282/275、282/276、282/277、282/278、282/279、282/280、282/281、282/282、282/283、282/284、282/285、282/286、282/287、282/288、283/267、283/268、283/269、283/270、283/271、283/272、283/273、283/274、283/275、283/276、283/277、283/278、283/279、283/280、283/281、283/282、283/283、283/284、283/285、283/286、283/287、283/288、284/267、284/268、284/269、284/270、284/271、284/272、284/273、284/274、284/275、284/276、284/277、284/278、284/279、284/280、284/281、284/282、284/283、284/284、284/285、284/286、284/287、284/288、285/267、285/268、285/269、285/270、285/271、285/272、285/273、285/274、285/275、285/276、285/277、285/278、285/279、285/280、285/281、285/282、285/283、285/284、285/285、285/286、285/287、285/288、286/267、286/268、286/269、286/270、286/271、286/272、286/273、286/274、286/275、286/276、286/277、286/278、286/279、286/280、286/281、286/282、286/283、286/284、286/285、286/286、286/287、286/288、287/267、287/268、287/269、287/270、287/271、287/272、287/273、287/274、287/275、287/276、287/277、287/278、287/279、287/280、287/281、287/282、287/283、287/284、287/285、287/286、287/287、287/288，117/118、117/119、117/120、117/121、117/122、117/123、118/118、118/119、118/120、118/121、118/122、118/123、119/118、119/119、119/120、119/121、119/122、119/123、120/118、120/119、120/120、120/121、120/122、120/123、121/118、121/119、121/120、121/121、121/122、121/123、122/118、122/119、122/120、122/121、122/122、122/123，340/341、340/342、340/343、340/344、340/345、340/346、340/347、340/348、340/349、340/350、340/351、340/352、340/353、341/341、341/342、341/343、341/344、341/345、341/346、341/347、341/348、341/349、341/350、341/351、341/352、341/353、342/341、342/342、342/343、342/344、342/345、342/346、342/347、342/348、342/349、342/350、342/351、342/352、342/353、343/341、343/342、343/343、343/344、343/345、343/346、343/347、343/348、343/349、343/350、343/351、343/352、343/353、344/341、344/342、344/343、344/344、344/345、344/346、344/347、344/348、344/349、344/350、344/351、344/352、344/353、345/341、345/342、345/343、345/344、345/345、345/346、345/347、345/348、345/349、345/350、345/351、345/352、345/353、346/341、346/342、346/343、346/344、346/345、346/346、346/347、346/348、346/349、346/350、346/351、346/352、346/353、347/341、347/342、347/343、347/344、347/345、347/346、347/347、347/348、347/349、347/350、347/351、347/352、347/353、348/341、348/342、348/343、348/344、348/345、348/346、348/347、348/348、348/349、348/350、348/351、348/352、348/353、349/341、349/342、349/343、349/344、349/345、349/346、349/347、349/348、349/349、349/350、349/351、349/352、349/353、350/341、350/342、350/343、350/344、350/345、350/346、350/347、350/348、350/349、350/350、350/351、350/352、350/353、351/341、351/342、351/343、351/344、351/345、351/346、351/347、351/348、351/349、351/350、351/351、351/352、351/353、352/341、352/342、352/343、352/344、352/345、352/346、352/347、352/348、352/349、352/350、352/351、352/352和352/353。仅作为示例性展示，348/352-TtGBP-cpYFP的氨基酸序列如SEQ ID NO：10所示。In this embodiment, based on pCDF-TtGBP, the following sites were selected for inserting cpYFP to obtain the corresponding pCDF-TtGBP-cpYFP plasmids: 266/267, 266/268, 266/269, 266/270, 266/271, 266/272, 266/273, 266/274, 266/275, 266/276, 266/277, 266/278, 266/279, 266/280, 266/ 281, 266/282, 266/283, 266/284, 266/285, 266/286, 266/287, 266/288, 267/267, 267/268, 267/269, 267/270, 267/271, 267/272, 267/273, 267/274, 267/275, 267/276, 267/277, 267/278, 267/279, 267/280, 26 7/281, 267/282, 267/283, 267/284, 267/285, 267/286, 267/287, 267/288, 268/267, 268/268, 268/269, 268/270, 268/271, 268/272, 268/273, 268/274, 268/275, 268/276, 268/277, 268/278, 268/279, 268/280, 268/281, 268/282, 268/283, 268/284, 268/285, 268/286, 268/287, 268/288, 269/267, 269/268, 269/269, 269/270, 269/271, 269/272, 269/273, 269/274, 269/275, 269/276, 269/277, 269/278, 269/279, 269/28 0, 269/281, 269/282, 269/283, 269/284, 269/285, 269/286, 269/287, 269/288, 270/267, 270/268, 270/269, 270/270, 270/271, 270/272, 270/273, 270/274, 270/275, 270/276, 270/277, 270/278, 270/279, 270/ 280, 270/281, 270/282, 270/283, 270/284, 270/285, 270/286, 270/287, 270/288, 271/267, 271/268, 271/269, 271/270, 271/271, 271/272, 271/273, 271/274, 271/275, 271/276, 271/277, 271/278, 271/279, 27 1/280, 271/281, 271/282, 271/283, 271/284, 271/285, 271/286, 271/287, 271/288, 272/267, 272/268, 272/269, 272/270, 272/271, 272/272, 272/273, 272/274, 272/275, 272/276, 272/277, 272/278, 272/279 272/280, 272/281, 272/282, 272/283, 272/284, 272/285, 272/286, 272/287, 272/288, 273/267, 273/268, 273/269, 273/270, 273/271, 273/272, 273/273, 273/274, 273/275, 273/276, 273/277, 273/278, 273/27 9, 273/280, 273/281, 273/282, 273/283, 273/284, 273/285, 273/286, 273/287, 273/288, 274/267, 274/268, 274/269, 274/270, 274/271, 274/272, 274/273, 274/274, 274/275, 274/276, 274/277, 274/278, 274/ 279, 274/280, 274/281, 274/282, 274/283, 274/284, 274/285, 274/286, 274/287, 274/288, 275/267, 275/268, 275/269, 275/270, 275/271, 275/272, 275/273, 275/274, 275/275, 275/276, 275/277, 275/278, 27 5/279, 275/280, 275/281, 275/282, 275/283, 275/284, 275/285, 275/286, 275/287, 275/288, 276/267, 276/268, 276/269, 276/270, 276/271, 276/272, 276/273, 276/274, 276/275, 276/276, 276/277, 276/278 276/279, 276/280, 276/281, 276/282, 276/283, 276/284, 276/285, 276/286, 276/287, 276/288, 277/267, 277/268, 277/269, 277/270, 277/271, 277/272, 277/273, 277/274, 277/275, 277/276, 277/277, 277/277 8, 277/279, 277/280, 277/281, 277/282, 277/283, 277/284, 277/285, 277/286, 277/287, 277/288, 278/267, 278/268, 278/269, 278/270, 278/271, 278/272, 278/273, 278/274, 278/275, 278/276, 278/277, 278/ 278, 278/279, 278/280, 278/281, 278/282, 278/283, 278/284, 278/285, 278/286, 278/287, 278/288, 279/267, 279/268, 279/269, 279/270, 279/271, 279/272, 279/273, 279/274, 279/275, 279/276, 279/277, 27 9/278, 279/279, 279/280, 279/281, 279/282, 279/283, 279/284, 279/285, 279/286, 279/287, 279/288, 280/267, 280/268, 280/269, 280/270, 280/271, 280/272, 280/273, 280/274, 280/275, 280/276, 280/277 280/278, 280/279, 280/280, 280/281, 280/282, 280/283, 280/284, 280/285, 280/286, 280/287, 280/288, 281/267, 281/268, 281/269, 281/270, 281/271, 281/272, 281/273, 281/274, 281/275, 281/276, 281/277 281/278, 281/279, 281/280, 281/281, 281/282, 281/283, 281/284, 281/285, 281/286, 281/287, 281/288, 282/267, 282/268, 282/269, 282/270, 282/271, 282/272, 282/273, 282/274, 282/275, 282/276, 282/2 77, 282/278, 282/279, 282/280, 282/281, 282/282, 282/283, 282/284, 282/285, 282/286, 282/287, 282/288, 283/267, 283/268, 283/269, 283/270, 283/271, 283/272, 283/273, 283/274, 283/275, 283/276, 283 /277, 283/278, 283/279, 283/280, 283/281, 283/282, 283/283, 283/284, 283/285, 283/286, 283/287, 283/288, 284/267, 284/268, 284/269, 284/270, 284/271, 284/272, 284/273, 284/274, 284/275, 284/276, 2 84/277, 284/278, 284/279, 284/280, 284/281, 284/282, 284/283, 284/284, 284/285, 284/286, 284/287, 284/288, 285/267, 285/268, 285/269, 285/270, 285/271, 285/272, 285/273, 285/274, 285/275, 285/276 285/277, 285/278, 285/279, 285/280, 285/281, 285/282, 285/283, 285/284, 285/285, 285/286, 285/287, 285/288, 286/267, 286/268, 286/269, 286/270, 286/271, 286/272, 286/273, 286/274, 286/275, 286/2 76, 286/277, 286/278, 286/279, 286/280, 286/281, 286/282, 286/283, 286/284, 286/285, 286/286, 286/287, 286/288, 287/267, 287/268, 287/269, 287/270, 287/271, 287/272, 287/273, 287/274, 287/275, 287 /276, 287/277, 287/278, 287/279, 287/280, 287/281, 287/282, 287/283, 287/284, 287/285, 287/286, 287/287, 287/288, 117/118, 117/119, 117/120, 117/121, 117/122, 117/123, 118/118, 118/119, 118/120, 1 18/121, 118/122, 118/123, 119/118, 119/119, 119/120, 119/121, 119/122, 119/123, 120/118, 120/119, 120/120, 120/121, 120/122, 120/123, 121/118, 121/119, 121/120, 121/121, 121/122, 121/123, 122/118 122/119, 122/120, 122/121, 122/122, 122/123, 340/341, 340/342, 340/343, 340/344, 340/345, 340/346, 340/347, 340/348, 340/349, 340/350, 340/351, 340/352, 340/353, 341/341, 341/342, 341/343, 341/3 44, 341/345, 341/346, 341/347, 341/348, 341/349, 341/350, 341/351, 341/352, 341/353, 342/341, 342/342, 342/343, 342/344, 342/345, 342/346, 342/347, 342/348, 342/349, 342/350, 342/351, 342/352, 342 /353, 343/341, 343/342, 343/343, 343/344, 343/345, 343/346, 343/347, 343/348, 343/349, 343/350, 343/351, 343/352, 343/353, 344/341, 344/342, 344/343, 344/344, 344/345, 344/346, 344/347, 344/348, 3 44/349, 344/350, 344/351, 344/352, 344/353, 345/341, 345/342, 345/343, 345/344, 345/345, 345/346, 345/347, 345/348, 345/349, 345/350, 345/351, 345/352, 345/353, 346/341, 346/342, 346/343, 346/344 346/345, 346/346, 346/347, 346/348, 346/349, 346/350, 346/351, 346/352, 346/353, 347/341, 347/342, 347/343, 347/344, 347/345, 347/346, 347/347, 347/348, 347/349, 347/350, 347/351, 347/352, 347/3 53, 348/341, 348/342, 348/343, 348/344, 348/345, 348/346, 348/347, 348/348, 348/349, 348/350, 348/351, 348/352, 348/353, 349/341, 349/342, 349/343, 349/344, 349/345, 349/346, 349/347, 349/348, 349 /349, 349/350, 349/351, 349/352, 349/353, 350/341, 350/342, 350/343, 350/344, 350/345, 350/346, 350/347, 350/348, 350/349, 350/350, 350/351, 350/352, 350/353, 351/341, 351/342, 351/343, 351/344, 3 51/345, 351/346, 351/347, 351/348, 351/349, 351/350, 351/351, 351/352, 351/353, 352/341, 352/342, 352/343, 352/344, 352/345, 352/346, 352/347, 352/348, 352/349, 352/350, 352/351, 352/352, and 352/353. For illustrative purposes only, the amino acid sequence of 348/352-TtGBP-cpYFP is shown in SEQ ID NO: 10.

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-D-葡萄糖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-TtGBP和cpYFP片段在HieffCloneEnzyme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。A DNA fragment of cpYFP was generated using PCR. Simultaneously, a homologous sequence from the cpYFP terminal was introduced at the 5' end using primers. PCR amplification produced a linearized pCDF-D-glucose-binding protein vector, whose 5' and 3' ends contained sequences completely identical to those at the two ends of cpYFP (15-20 bp). The linearized pCDF-TtGBP and cpYFP fragments underwent homologous recombination using HieffCloneEnzyme. The product was transformed into DH5α, and the transformed DH5α was plated on LB agar plates (streptomycin 100 μg/mL) and incubated overnight at 37°C. Positive clones identified by PCR were subjected to plasmid extraction and sequencing. Sequencing was performed by J. Lee Sequencing.

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70Kda附近。该大小符合pCDF-TtGBP-cpYFP表达出的含His-tag纯化标签的TtGBP-cpYFP融合蛋白质的大小。结果如图1所示。After successful sequencing, the recombinant plasmid was transformed into BL21(DE3) to induce expression, and the protein was purified. SDS-PAGE electrophoresis showed a size around 70 kDa. This size is consistent with the size of the pCDF-TtGBP-cpYFP fusion protein containing the His-tag purified label. The results are shown in Figure 1.

用表达TtGBP-cpYFP融合蛋白质的大肠杆菌的破碎上清进行D-葡萄糖响应筛选，将含有100mMD-葡萄糖的融合荧光蛋白质的检测信号除以无D-葡萄糖的融合荧光蛋白质的检测信号。如表1所示，检测结果显示表达有TtGBP-cpYFP融合蛋白质的破碎上清对D-葡萄糖响应超过1.2倍的光学探针有273/279、273/284、350/342、348/343、349/343、348/344、349/344、349/345、348/349、349/349、348/350、349/350、346/351、347/351、347/352、348/352和350/353；对D-葡萄糖响应超过1.25倍的光学探针有7个，分别是273/279、348/343、348/344、349/345、349/349、349/350和347/352。D-glucose response screening was performed using the supernatant of fragmented *E. coli* expressing the TtGBP-cpYFP fusion protein. The detection signal of the fusion fluorescent protein containing 100 mM D-glucose was divided by the detection signal of the fusion fluorescent protein without D-glucose. As shown in Table 1, the detection results showed that optical probes expressing the TtGBP-cpYFP fusion protein in the fragmented supernatant exhibited a D-glucose response greater than 1.2 times: 273/279, 273/284, 350/342, 348/343, 349/343, 348/344, 349/344, 349/345, 348/349, 349/349, and 3... The following optical probes were selected: 48/350, 349/350, 346/351, 347/351, 347/352, 348/352, and 350/353. Seven probes showed a response to D-glucose exceeding 1.25 times: 273/279, 348/343, 348/344, 349/345, 349/349, 349/350, and 347/352.

表1


Table 1


实施例3：不同插入位点的cpGFP光学探针的表达和检测Example 3: Expression and detection of cpGFP optical probes at different insertion sites

按照实施例2中的方法将cpYFP替换为cpGFP，构建D-葡萄糖绿色荧光蛋白荧光探针。如表2所示，检测结果显示表达有TtGBP-cpGFP融合蛋白质的破碎上清对D-葡萄糖响应超过1.3倍的光学探针有348/344、349/345、349/349、349/350和347/352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。Following the method in Example 2, cpYFP was replaced with cpGFP to construct a D-glucose green fluorescent protein probe. As shown in Table 2, the detection results showed that optical probes expressing the TtGBP-cpGFP fusion protein responded to D-glucose more than 1.3 times to optical probes at sites 348/344, 349/345, 349/349, 349/350, and 347/352, or the corresponding amino acid sites of its family proteins, were inserted.

表2
Table 2

实施例4：不同插入位点的cpBFP光学探针的表达和检测Example 4: Expression and detection of cpBFP optical probes at different insertion sites

按照实施例2中的方法将cpYFP替换为cpBFP，构建D-葡萄糖蓝色荧光蛋白荧光探针。如表3所示，检测结果显示表达有TtGBP-cpBFP融合蛋白质的破碎上清对D-葡萄糖响应超过1.3倍的光学探针有348/343、348/344、349/345和349/349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。Following the method in Example 2, cpYFP was replaced with cpBFP to construct a D-glucose blue fluorescent protein probe. As shown in Table 3, the detection results showed that optical probes expressing the TtGBP-cpBFP fusion protein responded to D-glucose more than 1.3 times to optical probes with insertion sites at 348/343, 348/344, 349/345, and 349/349 or corresponding amino acid sites of its family proteins were inserted.

表3
Table 3

实施例5：不同插入位点的cpmApple光学探针的表达和检测Example 5: Expression and detection of cpmApple optical probes at different insertion sites

按照实施例2中的方法将cpYFP替换为cpmApple，构建D-葡萄糖红色荧光蛋白荧光探针。如表4所示，检测结果显示表达有TtGBP-cpmApple融合蛋白质的破碎上清对D-葡萄糖响应超过1.3倍的光学探针有349/345和349/349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。Following the method in Example 2, cpYFP was replaced with cpmApple to construct a D-glucose red fluorescent protein probe. As shown in Table 4, the detection results show that optical probes expressing TtGBP-cpmApple fusion protein respond more than 1.3 times to D-glucose by replacing cpYFP with cpmApple. Optical probes with insertion sites at 349/345 and 349/349 sites or corresponding amino acid sites of its family proteins are also included.

表4
Table 4

实施例6：光学探针的性能Example 6: Performance of the optical probe

对于实施例2中所得的对D-葡萄糖响应超过1.2倍的光学探针，即在：273/279、273/284、350/342、348/343、349/343、348/344、349/344、349/345、348/349、349/349、348/350、349/350、346/351、347/351、347/352、348/352、350/353位点实施插入的17种光学探针，进行进浓度梯度(0～100mM)的葡萄糖检测，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。其中插入位点为273/279、273/284、350/342、348/343、349/343、348/344、348/349、349/349、348/350、349/350、346/351、347/351、347/352、350/353的14种D-葡萄糖光学探针的Kd(结合常数)过大，无法拟合，不适合检测。除此之外，插入位点为349/344、349/345、348/352的3种D-葡萄糖光学探针的Kd(结合常数)分别为1.2mM、0.2mM和0.008mM。For the 17 optical probes obtained in Example 2 that responded to D-glucose more than 1.2 times, namely those inserted at sites 273/279, 273/284, 350/342, 348/343, 349/343, 348/344, 349/344, 349/345, 348/349, 349/349, 348/350, 349/350, 346/351, 347/351, 347/352, 348/352, and 350/353, glucose detection was performed using a concentration gradient (0–100 mM), and the change in the ratio of fluorescence intensity at 420 nm excitation and 528 nm emission to fluorescence intensity at 485 nm excitation and 528 nm emission was detected. Fourteen D-glucose optical probes with insertion sites of 273/279, 273/284, 350/342, 348/343, 349/343, 348/344, 348/349, 349/349, 348/350, 349/350, 346/351, 347/351, 347/352, and 350/353 had excessively high Kd (binding constant), making them unsuitable for detection due to their inability to be properly fitted. In addition, three D-glucose optical probes with insertion sites of 349/344, 349/345, and 348/352 had Kd (binding constant) values of 1.2 mM, 0.2 mM, and 0.008 mM, respectively.

实施例7：突变的cpYFP光学探针的表达和检测Example 7: Expression and detection of mutated cpYFP optical probes

在TtGBP-348/352-cpYFP的基础上构建光学探针突变体。通过PCR线性化质粒pCDF-TtGBP-348/352-cpYFP，引物中含有所要突变位点的碱基序列，对得到的PCR产物进行同源重组得到D-葡萄糖敏感多肽的V347、H348、H66、D278、W9、E13、W8、A42、K312以及光学活性多肽的Y1这12个位点的突变质粒，并由杰李测序公司完成测序。作为示例性展示，348/352-TtGBP-V347W/H348S/H66C-cpYFP-Y1A的氨基酸序列如SEQ ID NO：11所示。Optical probe mutants were constructed based on TtGBP-348/352-cpYFP. The plasmid pCDF-TtGBP-348/352-cpYFP was linearized by PCR, with primers containing the base sequences of the desired mutation sites. Homologous recombination was performed on the obtained PCR product to obtain mutant plasmids for 12 sites: V347, H348, H66, D278, W9, E13, W8, A42, K312 of the D-glucose-sensitive polypeptide, and Y1 of the optically active polypeptide. Sequencing was performed by J. Lee Sequencing. As an example, the amino acid sequence of 348/352-TtGBP-V347W/H348S/H66C-cpYFP-Y1A is shown in SEQ ID NO: 11.

将构建成功的突变质粒转化到BL21(DE3)中诱导表达，用表达探针蛋白的大肠杆菌的破碎上清进行D-葡萄糖以及其他非特异性底物的响应筛选，将含有D-葡萄糖或其他非特异性底物的融合荧光蛋白质的检测信号除以无D-葡萄糖的融合荧光蛋白质的检测信号。结果表5所示，检测结果显示对D-葡萄糖响应超过3倍且特异性较好的光学探针如下所示。The successfully constructed mutant plasmid was transformed into BL21(DE3) to induce expression. The supernatant from the fragmented *E. coli* expressing the probe protein was used for response screening to D-glucose and other non-specific substrates. The detection signal of the fusion fluorescent protein containing D-glucose or other non-specific substrates was divided by the detection signal of the fusion fluorescent protein without D-glucose. The results are shown in Table 5. The detection results show that the optical probes with a response to D-glucose greater than 3-fold and good specificity are listed below.

表5
Table 5

实施例8：光学探针突变体的性能Example 8: Performance of optical probe mutants

将实施例7所述的表5中的D-葡萄糖光学探针分别进行浓度梯度(0～100mM)的D-葡萄糖检测。对探针处理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。探针滴定结果如图3所示，结果表明不同突变体对D-葡萄糖亲和力不同。The D-glucose optical probes described in Table 5 of Example 7 were used to detect D-glucose at concentration gradients (0–100 mM). After treating the probes for 10 minutes, the change in the ratio of fluorescence intensity at 420 nm excitation and 528 nm emission was measured to be that at 485 nm excitation and 528 nm emission. The probe titration results are shown in Figure 3, indicating that different mutants have different affinities for D-glucose.

对表5中的D-葡萄糖探针进行特异性检测，分别与D-葡萄糖结构类似物，葡萄糖-1-磷酸、山梨醇、甘露醇、乳酸钠、蔗糖、丙酮酸、核糖、果糖、草酰乙酸、ATP、6-磷酸葡萄糖、柠檬酸、D-葡萄糖、异柠檬酸、葡萄糖酸钠、NAD+、NADH、苹果酸、NADPH、阿拉伯糖、PEP、乳糖、麦芽糖、2-脱氧葡萄糖、半乳糖进行反应性检测，结果表明具有良好的特异性，如图4所示。The D-glucose probes in Table 5 were subjected to specificity testing. Reactivity was measured against D-glucose structural analogs, glucose-1-phosphate, sorbitol, mannitol, sodium lactate, sucrose, pyruvate, ribose, fructose, oxaloacetic acid, ATP, glucose-6-phosphate, citric acid, D-glucose, isocitrate, sodium gluconate, NAD+, NADH, malic acid, NADPH, arabinose, PEP, lactose, maltose, 2-deoxyglucose, and galactose. The results showed good specificity, as shown in Figure 4.

实施例9：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Example 9: Subcellular organelle localization of optical probes and performance of optical probes within subcellular organelles

本实施例中，使用不同的定位信号肽与光学探针348/352-TtGBP-V347W/H348S/H66C-cpYFP-Y1A融合，将光学探针定位到不同的细胞器中。In this embodiment, different localization signal peptides are fused with the optical probe 348/352-TtGBP-V347W/H348S/H66C-cpYFP-Y1A to localize the optical probe to different organelles.

用融合不同定位信号肽的光学探针质粒转染293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5所示。D-葡萄糖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、细胞外膜、细胞核、内质网、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。After transfecting 293 cells with optical probe plasmids fused with different localization signal peptides for 36 hours, the cells were washed with PBS and placed in HBSS solution for fluorescence detection using an inverted fluorescence microscope under the FITC channel. The results are shown in Figure 5. The D-glucose optical probes, by fusing with different specific localization signal peptides, can localize to subcellular organelles including the cytoplasm, outer membrane, nucleus, endoplasmic reticulum, mitochondria, and nuclear exclusion. Fluorescence was observed in all different subcellular structures, and the distribution and intensity of the fluorescence varied.

用胞浆表达的光学探针质粒转染HEK293细胞36小时后，使用PBS冲洗，置于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6所示，添加5mM D-葡萄糖，继续检测30分钟。添加D-葡萄糖的样品的420/485逐渐增加，最高可以达到初始值的1.8倍，而不添加Oxalate的对照组的420/485保持不变。HEK293 cells were transfected with a cytoplasmic optical probe plasmid for 36 hours. After washing with PBS, the cells were placed in HBSS solution, and the changes in the fluorescence intensity ratio at 420 nm excitation and 528 nm emission (485 nm excitation and 528 nm emission) were detected over a 30-minute period. The results are shown in Figure 6. After adding 5 mM D-glucose, the detection was continued for another 30 minutes. The 420/485 ratio gradually increased in the D-glucose-added samples, reaching a maximum of 1.8 times the initial value, while the 420/485 ratio remained unchanged in the control group without Oxalate.

实施例10：在活细胞中基于光学探针进行高通量化合物筛选Example 10: High-throughput compound screening in living cells based on optical probes

本实施例中，我们使用胞浆表达的348/352-TtGBP-V347W/H348S/H66C-cpYFP-Y1A的HeLa细胞进行高通量化合物筛选。In this embodiment, we used HeLa cells with cytoplasmic expression of 348/352-TtGBP-V347W/H348S/H66C-cpYFP-Y1A for high-throughput compound screening.

经转染的293细胞使用PBS冲洗，置于HBSS溶液中(无D-葡萄糖)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D-葡萄糖。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7所示。在使用的2000种化合物中，绝大部分的化合物对D-葡萄糖进入细胞影响极小。有5种化合物能够提高细胞对D-葡萄糖的摄取能力，另外有9种化合物能够明显降低细胞对D-葡萄糖的摄取。Transfected 293 cells were washed with PBS, treated with HBSS solution (without D-glucose) for 1 hour, and then treated with 10 μM of the compound for 1 hour. D-glucose was added to each sample. The ratio of fluorescence intensity at 420 nm excitation to 528 nm emission and the ratio of fluorescence intensity at 485 nm excitation to 528 nm emission were recorded using a microplate reader. Samples without any compound treatment were used as controls for standardization. The results are shown in Figure 7. Of the 2000 compounds used, the vast majority had minimal effect on D-glucose uptake by cells. Five compounds increased the cells' ability to take up D-glucose, while nine compounds significantly reduced it.

实施例11：光学探针定量检测血液中的D-葡萄糖Example 11: Quantitative Detection of D-Glucose in Blood Using an Optical Probe

在本实施中，使用纯化的Kd为48.33μM的348/352-TtGBP-V347W/H348S-cpYFP-Y1A对小鼠和人的血液上清中的D-葡萄糖进行分析。In this embodiment, purified Kd 48.33 μM 348/352-TtGBP-V347W/H348S-cpYFP-Y1A was used to analyze D-glucose in the blood supernatant of mice and humans.

将348/352-TtGBP-V347W/H348S-cpYFP-Y1A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。结果如图8所示，小鼠血液中的D-葡萄糖含量在8mM左右，人血液中的D-葡萄糖含量在4mM左右。After mixing 348/352-TtGBP-V347W/H348S-cpYFP-Y1A with diluted blood supernatant and treating for 10 minutes, the ratio of fluorescence intensity at 420nm excitation and 528nm emission to fluorescence intensity at 485nm excitation and 528nm emission was detected using an ELISA reader. The results are shown in Figure 8. The D-glucose content in mouse blood was approximately 8 mM, while the D-glucose content in human blood was approximately 4 mM.

由以上实施例可知，本发明提供的D-葡萄糖光学探针，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D-葡萄糖；并且能够进行高通量的化合物筛选。As can be seen from the above embodiments, the D-glucose optical probe provided by the present invention has a relatively small protein molecular weight and is easy to mature. It exhibits large fluorescence dynamic changes, good specificity, and can be expressed in cells through gene manipulation. It can locate and quantify D-glucose in and out of cells in real time and can also perform high-throughput compound screening.

其它实施方式Other implementation methods

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。This specification describes many embodiments. However, it should be understood that various modifications that may be learned by those skilled in the art upon reading this specification without departing from the spirit and scope of the invention should also be included within the scope of the appended claims.

本文序列

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Claims (10)

一种光学探针，包含D-葡萄糖敏感多肽和光学活性多肽，其中，所述D-葡萄糖敏感多肽是TtGBP蛋白或其变体，所述光学活性多肽是荧光蛋白或其变体，所述光学活性多肽位于D-葡萄糖敏感多肽的选自以下一个或多个位点：266/267、266/268、266/269、266/270、266/271、266/272、266/273、266/274、266/275、266/276、266/277、266/278、266/279、266/280、266/281、266/282、266/283、266/284、266/285、266/286、266/287、266/288、267/267、267/268、267/269、267/270、267/271、267/272、267/273、267/274、267/275、267/276、267/277、267/278、267/279、267/280、267/281、267/282、267/283、267/284、267/285、267/286、267/287、267/288、268/267、268/268、268/269、268/270、268/271、268/272、268/273、268/274、268/275、268/276、268/277、268/278、268/279、268/280、268/281、268/282、268/283、268/284、268/285、268/286、268/287、268/288、269/267、269/268、269/269、269/270、269/271、269/272、269/273、269/274、269/275、269/276、269/277、269/278、269/279、269/280、269/281、269/282、269/283、269/284、269/285、269/286、269/287、269/288、270/267、270/268、270/269、270/270、270/271、270/272、270/273、270/274、270/275、270/276、270/277、270/278、270/279、270/280、270/281、270/282、270/283、270/284、270/285、270/286、270/287、270/288、271/267、271/268、271/269、271/270、271/271、271/272、271/273、271/274、271/275、271/276、271/277、271/278、271/279、271/280、271/281、271/282、271/283、271/284、271/285、271/286、271/287、271/288、272/267、272/268、272/269、272/270、272/271、272/272、272/273、272/274、272/275、272/276、272/277、272/278、272/279、272/280、272/281、272/282、272/283、272/284、272/285、272/286、272/287、272/288、273/267、273/268、273/269、273/270、273/271、273/272、273/273、273/274、273/275、273/276、273/277、273/278、273/279、273/280、273/281、273/282、273/283、273/284、273/285、273/286、273/287、273/288、274/267、274/268、274/269、274/270、274/271、274/272、274/273、274/274、274/275、274/276、274/277、274/278、274/279、274/280、274/281、274/282、274/283、274/284、274/285、274/286、274/287、274/288、275/267、275/268、275/269、275/270、275/271、275/272、275/273、275/274、275/275、275/276、275/277、275/278、275/279、275/280、275/281、275/282、275/283、275/284、275/285、275/286、275/287、275/288、276/267、276/268、276/269、276/270、276/271、276/272、276/273、276/274、276/275、276/276、276/277、276/278、276/279、276/280、276/281、276/282、276/283、276/284、276/285、276/286、276/287、276/288、277/267、277/268、277/269、277/270、277/271、277/272、277/273、277/274、277/275、277/276、277/277、277/278、277/279、277/280、277/281、277/282、277/283、277/284、277/285、277/286、277/287、277/288、278/267、278/268、278/269、278/270、278/271、278/272、278/273、278/274、278/275、278/276、278/277、278/278、278/279、278/280、278/281、278/282、278/283、278/284、278/285、278/286、278/287、278/288、279/267、279/268、279/269、279/270、279/271、279/272、279/273、279/274、279/275、279/276、279/277、279/278、279/279、279/280、279/281、279/282、279/283、279/284、279/285、279/286、279/287、279/288、280/267、280/268、280/269、280/270、280/271、280/272、280/273、280/274、280/275、280/276、280/277、280/278、280/279、280/280、280/281、280/282、280/283、280/284、280/285、280/286、280/287、280/288、281/267、281/268、281/269、281/270、281/271、281/272、281/273、281/274、281/275、281/276、281/277、281/278、281/279、281/280、281/281、281/282、281/283、281/284、281/285、281/286、281/287、281/288、282/267、282/268、282/269、282/270、282/271、282/272、282/273、282/274、282/275、282/276、282/277、282/278、282/279、282/280、282/281、282/282、282/283、282/284、282/285、282/286、282/287、282/288、283/267、283/268、283/269、283/270、283/271、283/272、283/273、283/274、283/275、283/276、283/277、283/278、283/279、283/280、283/281、283/282、283/283、283/284、283/285、283/286、283/287、283/288、284/267、284/268、284/269、284/270、284/271、284/272、284/273、284/274、284/275、284/276、284/277、284/278、284/279、284/280、284/281、284/282、284/283、284/284、284/285、284/286、284/287、284/288、285/267、285/268、285/269、285/270、285/271、285/272、285/273、285/274、285/275、285/276、285/277、285/278、285/279、285/280、285/281、285/282、285/283、285/284、285/285、285/286、285/287、285/288、286/267、286/268、286/269、286/270、286/271、286/272、286/273、286/274、286/275、286/276、286/277、286/278、286/279、286/280、286/281、286/282、286/283、286/284、286/285、286/286、286/287、286/288、287/267、287/268、287/269、287/270、287/271、287/272、287/273、287/274、287/275、287/276、287/277、287/278、287/279、287/280、287/281、287/282、287/283、287/284、287/285、287/286、287/287、287/288，117/118、117/119、117/120、117/121、117/122、117/123、118/118、118/119、118/120、118/121、118/122、118/123、119/118、119/119、119/120、119/121、119/122、119/123、120/118、120/119、120/120、120/121、120/122、120/123、121/118、121/119、121/120、121/121、121/122、121/123、122/118、122/119、122/120、122/121、122/122、122/123，340/341、340/342、340/343、340/344、340/345、340/346、340/347、340/348、340/349、340/350、340/351、340/352、340/353、341/341、341/342、341/343、341/344、341/345、341/346、341/347、341/348、341/349、341/350、341/351、341/352、341/353、342/341、342/342、342/343、342/344、342/345、342/346、342/347、342/348、342/349、342/350、342/351、342/352、342/353、343/341、343/342、343/343、343/344、343/345、343/346、343/347、343/348、343/349、343/350、343/351、343/352、343/353、344/341、344/342、344/343、344/344、344/345、344/346、344/347、344/348、344/349、344/350、344/351、344/352、344/353、345/341、345/342、345/343、345/344、345/345、345/346、345/347、345/348、345/349、345/350、345/351、345/352、345/353、346/341、346/342、346/343、346/344、346/345、346/346、346/347、346/348、346/349、346/350、346/351、346/352、346/353、347/341、347/342、347/343、347/344、347/345、347/346、347/347、347/348、347/349、347/350、347/351、347/352、347/353、348/341、348/342、348/343、348/344、348/345、348/346、348/347、348/348、348/349、348/350、348/351、348/352、348/353、349/341、349/342、349/343、349/344、349/345、349/346、349/347、349/348、349/349、349/350、349/351、349/352、349/353、350/341、350/342、350/343、350/344、350/345、350/346、350/347、350/348、350/349、350/350、350/351、350/352、350/353、351/341、351/342、351/343、351/344、351/345、351/346、351/347、351/348、351/349、351/350、351/351、351/352、351/353、352/341、352/342、352/343、352/344、352/345、352/346、352/347、352/348、352/349、352/350、352/351、352/352和352/353，所述TtGBP蛋白具有SEQ ID NO：1所示的序列或其功能片段，所述TtGBP蛋白的变体包含选自下述位点的突变：W8、W9、A42、H66、K312、V347、H348，所述荧光蛋白的变体包含Y1位点的突变，An optical probe comprising a D-glucose-sensitive polypeptide and an optically active polypeptide, wherein the D-glucose-sensitive polypeptide is a TtGBP protein or a variant thereof, and the optically active polypeptide is a fluorescent protein or a variant thereof, wherein the optically active polypeptide is located at one or more sites selected from the following: 266/267, 266/268, 266/269, 266/270, 266/271, 266/272, 266/273, 266/274, 266/275, 266... /276, 266/277, 266/278, 266/279, 266/280, 266/281, 266/282, 266/283, 266/284, 266/285, 266/286, 266/287, 266/288, 267/267, 267/268, 267/269, 267/270, 267/271, 267/272, 267/273, 267/274, 267/275, 267/27 6, 267/277, 267/278, 267/279, 267/280, 267/281, 267/282, 267/283, 267/284, 267/285, 267/286, 267/287, 267/288, 268/267, 268/268, 268/269, 268/270, 268/271, 268/272, 268/273, 268/274, 268/275, 268/276, 2 68/277, 268/278, 268/279, 268/280, 268/281, 268/282, 268/283, 268/284, 268/285, 268/286, 268/287, 268/288, 269/267, 269/268, 269/269, 269/270, 269/271, 269/272, 269/273, 269/274, 269/275, 269/276, 269/ 277, 269/278, 269/279, 269/280, 269/281, 269/282, 269/283, 269/284, 269/285, 269/286, 269/287, 269/288, 270/267, 270/268, 270/269, 270/270, 270/271, 270/272, 270/273, 270/274, 270/275, 270/276, 270/27 7, 270/278, 270/279, 270/280, 270/281, 270/282, 270/283, 270/284, 270/285, 270/286, 270/287, 270/288, 271/267, 271/268, 271/269, 271/270, 271/271, 271/272, 271/273, 271/274, 271/275, 271/276, 271/277, 2 71/278, 271/279, 271/280, 271/281, 271/282, 271/283, 271/284, 271/285, 271/286, 271/287, 271/288, 272/267, 272/268, 272/269, 272/270, 272/271, 272/272, 272/273, 272/274, 272/275, 272/276, 272/277, 272/ 278, 272/279, 272/280, 272/281, 272/282, 272/283, 272/284, 272/285, 272/286, 272/287, 272/288, 273/267, 273/268, 273/269, 273/270, 273/271, 273/272, 273/273, 273/274, 273/275, 273/276, 273/277, 273/278 273/279, 273/280, 273/281, 273/282, 273/283, 273/284, 273/285, 273/286, 273/287, 273/288, 274/267, 274/268, 274/269, 274/270, 274/271, 274/272, 274/273, 274/274, 274/275, 274/276, 274/277, 274/278, 2 74/279, 274/280, 274/281, 274/282, 274/283, 274/284, 274/285, 274/286, 274/287, 274/288, 275/267, 275/268, 275/269, 275/270, 275/271, 275/272, 275/273, 275/274, 275/275, 275/276, 275/277, 275/278, 275/ 279, 275/280, 275/281, 275/282, 275/283, 275/284, 275/285, 275/286, 275/287, 275/288, 276/267, 276/268, 276/269, 276/270, 276/271, 276/272, 276/273, 276/274, 276/275, 276/276, 276/277, 276/278, 276/279 276/280, 276/281, 276/282, 276/283, 276/284, 276/285, 276/286, 276/287, 276/288, 277/267, 277/268, 277/269, 277/270, 277/271, 277/272, 277/273, 277/274, 277/275, 277/276, 277/277, 277/278, 277/279, 27 7/280, 277/281, 277/282, 277/283, 277/284, 277/285, 277/286, 277/287, 277/288, 278/267, 278/268, 278/269, 278/270, 278/271, 278/272, 278/273, 278/274, 278/275, 278/276, 278/277, 278/278, 278/279, 278/ 280, 278/281, 278/282, 278/283, 278/284, 278/285, 278/286, 278/287, 278/288, 279/267, 279/268, 279/269, 279/270, 279/271, 279/272, 279/273, 279/274, 279/275, 279/276, 279/277, 279/278, 279/279, 279/280 279/281, 279/282, 279/283, 279/284, 279/285, 279/286, 279/287, 279/288, 280/267, 280/268, 280/269, 280/270, 280/271, 280/272, 280/273, 280/274, 280/275, 280/276, 280/277, 280/278, 280/279, 280/280, 28 0/281, 280/282, 280/283, 280/284, 280/285, 280/286, 280/287, 280/288, 281/267, 281/268, 281/269, 281/270, 281/271, 281/272, 281/273, 281/274, 281/275, 281/276, 281/277, 281/278, 281/279, 281/280, 281/2 81, 281/282, 281/283, 281/284, 281/285, 281/286, 281/287, 281/288, 282/267, 282/268, 282/269, 282/270, 282/271, 282/272, 282/273, 282/274, 282/275, 282/276, 282/277, 282/278, 282/279, 282/280, 282/281 282/282, 282/283, 282/284, 282/285, 282/286, 282/287, 282/288, 283/267, 283/268, 283/269, 283/270, 283/271, 283/272, 283/273, 283/274, 283/275, 283/276, 283/277, 283/278, 283/279, 283/280, 283/281, 28 3/282, 283/283, 283/284, 283/285, 283/286, 283/287, 283/288, 284/267, 284/268, 284/269, 284/270, 284/271, 284/272, 284/273, 284/274, 284/275, 284/276, 284/277, 284/278, 284/279, 284/280, 284/281, 284/2 82, 284/283, 284/284, 284/285, 284/286, 284/287, 284/288, 285/267, 285/268, 285/269, 285/270, 285/271, 285/272, 285/273, 285/274, 285/275, 285/276, 285/277, 285/278, 285/279, 285/280, 285/281, 285/282, 285/283, 285/284, 285/285, 285/286, 285/287, 285/288, 286/267, 286/268, 286/269, 286/270, 286/271, 286/272, 286/273, 286/274, 286/275, 286/276, 286/277, 286/278, 286/279, 286/280, 286/281, 286/282, 28 6/283, 286/284, 286/285, 286/286, 286/287, 286/288, 287/267, 287/268, 287/269, 287/270, 287/271, 287/272, 287/273, 287/274, 287/275, 287/276, 287/277, 287/278, 287/279, 287/280, 287/281, 287/282, 287/2 83, 287/284, 287/285, 287/286, 287/287, 287/288, 117/118, 117/119, 117/120, 117/121, 117/122, 117/123, 118/118, 118/119, 118/120, 118/121, 118/122, 118/123, 119/118, 119/119, 119/120, 119/121, 119/122, 119/123, 120/118, 120/119, 120/120, 120/121, 120/122, 120/123, 121/118, 121/119, 121/120, 121/121, 121/122, 121/123, 122/118, 122/119, 122/120, 122/121, 122/122, 122/123, 340/341, 340/342, 340/343, 340 /344, 340/345, 340/346, 340/347, 340/348, 340/349, 340/350, 340/351, 340/352, 340/353, 341/341, 341/342, 341/343, 341/344, 341/345, 341/346, 341/347, 341/348, 341/349, 341/350, 341/351, 341/352, 341/3 53, 342/341, 342/342, 342/343, 342/344, 342/345, 342/346, 342/347, 342/348, 342/349, 342/350, 342/351, 342/352, 342/353, 343/341, 343/342, 343/343, 343/344, 343/345, 343/346, 343/347, 343/348, 343/349, 343/350, 343/351, 343/352, 343/353, 344/341, 344/342, 344/343, 344/344, 344/345, 344/346, 344/347, 344/348, 344/349, 344/350, 344/351, 344/352, 344/353, 345/341, 345/342, 345/343, 345/344, 345/345, 345 /346, 345/347, 345/348, 345/349, 345/350, 345/351, 345/352, 345/353, 346/341, 346/342, 346/343, 346/344, 346/345, 346/346, 346/347, 346/348, 346/349, 346/350, 346/351, 346/352, 346/353, 347/341, 347/34 2, 347/343, 347/344, 347/345, 347/346, 347/347, 347/348, 347/349, 347/350, 347/351, 347/352, 347/353, 348/341, 348/342, 348/343, 348/344, 348/345, 348/346, 348/347, 348/348, 348/349, 348/350, 348/351, 3 48/352, 348/353, 349/341, 349/342, 349/343, 349/344, 349/345, 349/346, 349/347, 349/348, 349/349, 349/350, 349/351, 349/352, 349/353, 350/341, 350/342, 350/343, 350/344, 350/345, 350/346, 350/347, 350/ 348, 350/349, 350/350, 350/351, 350/352, 350/353, 351/341, 351/342, 351/343, 351/344, 351/345, 351/346, 351/347, 351/348, 351/349, 351/350, 351/351, 351/352, 351/353, 352/341, 352/342, 352/343, 352/344 Variants of the TtGBP protein, 352/345, 352/346, 352/347, 352/348, 352/349, 352/350, 352/351, 352/352, and 352/353, have the sequence shown in SEQ ID NO: 1 or a functional fragment thereof. Variants of the TtGBP protein contain mutations selected from the following sites: W8, W9, A42, H66, K312, V347, and H348. Variants of the fluorescent protein contain a mutation at the Y1 site. 优选地，所述光学活性多肽选自以下任一种：cpYFP、cpGFP、cpBFP、cpmApple。Preferably, the optically active polypeptide is selected from any one of the following: cpYFP, cpGFP, cpBFP, and cpmApple. 如权利要求1所述的光学探针，其特征在于，所述荧光蛋白如SEQ ID NO：2-9中任一项所示，所述荧光蛋白的变体中Y1突变为A或G；The optical probe according to claim 1, wherein the fluorescent protein is as shown in any one of SEQ ID NO: 2-9, and the Y1 variant of the fluorescent protein is mutated to A or G; 优选地，所述荧光蛋白如SEQ ID NO：2、6、7、9中任一项所示。Preferably, the fluorescent protein is as shown in any one of SEQ ID NO: 2, 6, 7, 9. 如权利要求1所述的光学探针，其特征在于，The optical probe as described in claim 1, characterized in that, 所述TtGBP蛋白的变体中，W8突变为R、H、E、A、V、L、F、I、M、C、N、G、K、D或T，W9突变为R、H、E、A、V、L、F、I、M、C、P、D、N、G、K、Y、S或T，A42突变为R、E、W、F、M、C、P、N、G、K、Y或T，H66突变为R、E、A、F、M、C、P、Q或T，K312突变为R、A、Q、G、H或S，V347突变为W，H348突变为T、G或S，Among the variants of the TtGBP protein, W8 is mutated to R, H, E, A, V, L, F, I, M, C, N, G, K, D, or T; W9 is mutated to R, H, E, A, V, L, F, I, M, C, P, D, N, G, K, Y, S, or T; A42 is mutated to R, E, W, F, M, C, P, N, G, K, Y, or T; H66 is mutated to R, E, A, F, M, C, P, Q, or T; K312 is mutated to R, A, Q, G, H, or S; V347 is mutated to W; and H348 is mutated to T, G, or S. 优选地，所述TtGBP蛋白的变体中的突变包含选自以下任一组的突变：(1)V347W和H348T，(2)V347W和H348G，(3)V347W和H348S，(4)V347W、H348T和H66R，(5)V347W、H348T和H66E，(6)V347W、H348T和H66A，(7)V347W、H348T和H66F，(8)V347W、H348T和H66M，(9)V347W、H348T和H66C，(10)V347W、H348T和H66P，(11)V347W、H348T和H66Q，(12)V347W、H348T和H66T，(13)V347W、H348T和W9R，(14)V347W、H348T和W9H，(15)V347W、H348T和W9E，(16)V347W、H348T和W9A，(17)V347W、H348T和W9V，(18)V347W、H348T和W9L，(19)V347W、H348T和W9F，(20)V347W、H348T和W9I，(21)V347W、H348T和W9M，(22)V347W、H348T和W9C，(23)V347W、H348T和W9P，(24)V347W、H348T和W9N，(25)V347W、H348T和W9G，(26)V347W、H348T和W9K，(27)V347W、H348T和W9Y，(28)V347W、H348T和W9S，(29)V347W、H348T和W9T，(30)V347W、H348T和W9D，(31)V347W、H348T和W8R，(32)V347W、H348T和W8H，(33)V347W、H348T和W8E，(34)V347W、H348T和W8A，(35)V347W、H348T和W8V，(36)V347W、H348T和W8L，(37)V347W、H348T和W8F，(38)V347W、H348T和W8I，(39)V347W、H348T和W8M，(40)V347W、H348T和W8C，(41)V347W、H348T和W8N，(42)V347W、H348T和W8G，(43)V347W、H348T和W8K，(44)V347W、H348T和W8T，(45)V347W、H348T和W8D，(46)V347W、H348T和A42R，(47)V347W、H348T和A42E，(48)V347W、H348T和A42W，(49)V347W、H348T和A42F，(50)V347W、H348T和A42M，(51)V347W、H348T和A42C，(52)V347W、H348T和A42P，(53)V347W、H348T和A42N，(54)V347W、H348T和A42G，(55)V347W、H348T和A42K，(56)V347W、H348T和A42Y，(57)V347W、H348T和A42T，(58)V347W、H348T和K312R，(59)V347W、H348T和K312A，(60)V347W、H348T和K312Q，(61)V347W、H348T和K312G，(62)V347W、H348T和K312H，(63)V347W、H348T和K312S。Preferably, the mutations in the variants of the TtGBP protein include mutations selected from any of the following groups: (1) V347W and H348T, (2) V347W and H348G, (3) V347W and H348S, (4) V347W, H348T and H66R, (5) V347W, H348T and H66E, (6) V347W, H348T and H66A, (7) V347W, H348T and H66F, (8) V3 47W, H348T and H66M, (9)V347W, H348T and H66C, (10)V347W, H348T and H66P, (11)V347W, H348T and H66Q, (12)V347W, H348T and H66T, (13)V347W, H348T and W9R, (14)V347W, H348T and W9H, (15)V347W, H348T and W9E, (16)V 347W, H348T and W9A, (17)V347W, H348T and W9V, (18)V347W, H348T and W9L, (19)V347W, H348T and W9F, (20)V347W, H348T and W9I, (21)V347W, H348T and W9M, (22)V347W, H348T and W9C, (23)V347W, H348T and W9P, (24)V347 W, H348T and W9N, (25) V347W, H348T and W9G, (26) V347W, H348T and W9K, (27) V347W, H348T and W9Y, (28) V347W, H348T and W9S, (29) V347W, H348T and W9T, (30) V347W, H348T and W9D, (31) V347W, H348T and W8R, (32) V347W, H 348T and W8H, (33)V347W, H348T and W8E, (34)V347W, H348T and W8A, (35)V347W, H348T and W8V, (36)V347W, H348T and W8L, (37)V347W, H348T and W8F, (38)V347W, H348T and W8I, (39)V347W, H348T and W8M, (40)V347W, H348 T and W8C, (41) V347W, H348T and W8N, (42) V347W, H348T and W8G, (43) V347W, H348T and W8K, (44) V347W, H348T and W8T, (45) V347W, H348T and W8D, (46) V347W, H348T and A42R, (47) V347W, H348T and A42E, (48) V347W, H348T And A42W, (49)V347W, H348T and A42F, (50)V347W, H348T and A42M, (51)V347W, H348T and A42C, (52)V347W, H348T and A42P, (53)V347W, H348T and A42N, (54)V347W, H348T and A42G, (55)V347W, H348T and A42K, (56)V347W, H348T and A42Y, (57) V347W, H348T and A42T, (58) V347W, H348T and K312R, (59) V347W, H348T and K312A, (60) V347W, H348T and K312Q, (61) V347W, H348T and K312G, (62) V347W, H348T and K312H, (63) V347W, H348T and K312S. 一种融合多肽，包含权利要求1-3中任一项所述的光学探针和其它多肽，所述其他多肽包括定位序列、便于纯化的标签或者用于免疫反应的标签。A fusion polypeptide comprising the optical probe of any one of claims 1-3 and other polypeptides, said other polypeptides including a localization sequence, a tag for easy purification, or a tag for an immune response. 一种核酸分子，包含选自以下的序列：A nucleic acid molecule comprising sequences selected from the following: (1)编码权利要求1-3中任一项所述的光学探针或权利要求4所述的融合多肽的多核苷酸序列，和(1) A multinucleotide sequence encoding the optical probe of any one of claims 1-3 or the fusion polypeptide of claim 4, and (2)(1)的互补序列。(2)(1) complementary sequences. 一种核酸构建物，其包含权利要求5所述的核酸分子，A nucleic acid construct comprising the nucleic acid molecule of claim 5. 优选地，所述核酸构建物是表达载体。Preferably, the nucleic acid construct is an expression vector. 一种宿主细胞，所述宿主细胞：A host cell, said host cell: (1)包含、表达或分泌权利要求1-3中任一项所述的光学探针或权利要求4所述的融合多肽；(1) Containing, expressing or secreting the optical probe of any one of claims 1-3 or the fusion polypeptide of claim 4; (2)包含权利要求5所述的核酸分子；或(2) Contains the nucleic acid molecule as described in claim 5; or (3)包含权利要求6所述的核酸构建物。(3) It includes the nucleic acid construct of claim 6. 一种制备权利要求1-3中任一项所述的光学探针或权利要求4所述的融合多肽的方法，包括培养权利要求7所述的宿主细胞，和由培养物分离所述光学探针。A method for preparing the optical probe of any one of claims 1-3 or the fusion polypeptide of claim 4, comprising culturing the host cell of claim 7 and isolating the optical probe from the culture. 权利要求1-3中任一项所述的光学探针、权利要求4所述的融合多肽、权利要求5所述的核酸分子或权利要求6所述的核酸构建物在制备检测样品中的D-葡萄糖、筛选化合物或D-葡萄糖的细胞内和/或细胞外定位的试剂盒中的应用。The use of the optical probe of any one of claims 1-3, the fusion peptide of claim 4, the nucleic acid molecule of claim 5, or the nucleic acid construct of claim 6 in a kit for preparing a sample to detect D-glucose, screening compounds, or intracellular and/or extracellular localization of D-glucose. 一种检测试剂盒，其包含A test kit comprising (1)权利要求1-3中任一项所述的光学探针或权利要求4所述的融合多肽；(1) The optical probe according to any one of claims 1-3 or the fusion polypeptide according to claim 4; (2)权利要求5所述的核酸分子；(2) The nucleic acid molecule according to claim 5; (3)权利要求6所述的核酸构建物；或(3) The nucleic acid construct according to claim 6; or (4)权利要求7所述的宿主细胞；(4) The host cell as described in claim 7; 所述检测试剂盒任选还包含利用光学探针检测D-葡萄糖所需的其他试剂，The detection kit may optionally also include other reagents required for the detection of D-glucose using an optical probe. 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、D-葡萄糖标准品。Preferably, the test kit further comprises one or more reagents selected from the following: buffer solution, culture medium, and D-glucose standard.
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Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20020004217A1 (en) * 1997-12-31 2002-01-10 Duke University Biosensor
US20030232383A1 (en) * 2001-11-02 2003-12-18 Sylvia Daunert Novel reagentless sensing system for measuring carbohydrates based on the galactose/glucose binding protein
US20110117661A1 (en) * 2007-08-06 2011-05-19 Sylvia Daunert Polypeptides, systems, and methods useful for detecting glucose
US20120232251A1 (en) * 2009-11-13 2012-09-13 King's College London Glucose sensor

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20020004217A1 (en) * 1997-12-31 2002-01-10 Duke University Biosensor
US20030232383A1 (en) * 2001-11-02 2003-12-18 Sylvia Daunert Novel reagentless sensing system for measuring carbohydrates based on the galactose/glucose binding protein
US20110117661A1 (en) * 2007-08-06 2011-05-19 Sylvia Daunert Polypeptides, systems, and methods useful for detecting glucose
US20120232251A1 (en) * 2009-11-13 2012-09-13 King's College London Glucose sensor

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