Redox-sensitive protein and preparation method and application thereof

Abstract

The present invention relates to a group of redox-sensitive fluorescent protein probes and a preparation method and application thereof. The present invention provides a group of genetically encoded fluorescent protein probes, wherein the fluorescent protein probes are deep red fluorescent protein mKate containing cysteine ​​mutations and a circular rearranged fluorescent protein variant cpmKate thereof. The fluorescent protein probes provided by the present invention can achieve high sensitivity and real-time dynamic detection of redox changes.

Description

Technical Field

[0001] The invention relates to the technical field of fluorescent protein probes, and in particular to redox-sensitive proteins and preparation methods and applications thereof. Background Art

[0002] Redox metabolism plays an extremely important role in the basic characteristics of living organisms. It is involved in many life activities and important events in the body. When redox metabolism is disturbed, it will inevitably interfere with the normal physiological activities of cells and even cause serious damage such as body diseases. Many studies have shown that it is closely related to cancer, inflammatory damage, obesity, diabetes, neurodegenerative diseases, etc. Therefore, it is necessary to detect the redox state of organisms.

[0003] Redox metabolites include macromolecular polypeptides and small molecule substances. Macromolecular polypeptides are mainly substances containing sulfhydryl-disulfide bonds, such as glutathione, glutaredoxin and other proteins with active cysteine ​​residues. Small molecules are mainly active oxygen free radicals, coenzymes and tocopherols. The probes involved in the present invention mainly detect sulfhydryl-disulfide redox metabolites.

[0004] Traditional methods for detecting redox metabolism include: using high performance liquid chromatography, enzymatic methods, capillary enzyme cycle electrophoresis and isotope labeling to detect pyridine nucleotide small molecules including NAD(H) and NADP(H); using thiol chemical dye labeling to determine the content of intracellular glutathione; using non-reducing / reducing diagonal electrophoresis and gel migration methods to detect the metabolic state of thiol-disulfide bonds; using chemical probes to detect H2O2, etc. However, the biochemical methods in traditional methods are extremely invasive and destructive, which will cause sample fragmentation and the introduction of artificial oxidation during the processing process, resulting in inaccurate detection; and chemical probes still have great limitations in terms of quantification, cytotoxicity and subcellular localization.

[0005] Compared with the above methods, genetically encoded probes such as fluorescent protein probes can be combined with different signal peptides through genetic coding and localized to different cell structures. They can achieve in situ, high-temporal, high-resolution, and high-brightness visualization of the activity states of various proteases, changes in the concentration of specific molecules, and various physiological processes at the level of living cells, tissues, and even organisms. Therefore, genetically encoded probes are very suitable for high-temporal and high-resolution monitoring of redox at the level of living cells, and have become an important research tool in the field of redox biology.

[0006] Generally speaking, the shorter the wavelength of the excitation light, the greater the cytotoxicity it brings, and it is more susceptible to the high level of autofluorescence of the organism during imaging, which brings troubles to deep tissue imaging. In addition, in the detection of redox metabolism, the use of probes of different colors for multi-parameter analysis in single cells helps us better understand the compartmentalization characteristics of redox metabolism and the metabolic interactions between organelles. Deep red probes can not only avoid the interference of autofluorescence to the greatest extent, but also can be distinguished spectrally from other color probes. Therefore, this field needs to develop red or deep red redox probes with excellent properties to achieve high spatiotemporal monitoring of dynamic changes in redox in cells. Summary of the invention

[0007] The purpose of the present invention is to provide a redox-sensitive probe. The fluorescent protein probe provided by the present invention can achieve high-sensitivity detection of redox changes.

[0008] The present invention provides a redox-sensitive polypeptide, which has (1) a sequence shown in SEQ ID NO:1 and one or more amino acid residues are mutated to cysteine, (2) a sequence in which the sequence of (1) is connected from beginning to end by a connecting peptide and is disconnected between positions 153 and 154, between positions 169 and 168, or between positions 151 and 150, or (3) a variant having at least 90% sequence identity with (1) or (2) and retaining the redox-sensitive function, wherein the one or more amino acid residues mutated to cysteine ​​in the variant are unchanged relative to the sequence of (1) or (2).

[0009] In one or more embodiments, the connecting peptide is selected from G, GS, GGS, GGSG, GGTG, GGTGG, GGSGG, GGSGGT, GGTGGS.

[0010] In one or more embodiments, the one or more amino acid residues mutated to cysteine ​​are located on the β-fold of the protein formed by the polypeptide. In one or more embodiments, the one or more amino acid residues are amino acid residues on the dimerization interface and / or β-fold of the polypeptide. In one or more embodiments, the one or more amino acid residues are located on the surface of the β-barrel structure of the protein formed by the polypeptide and the amino acid side chains are facing the outside of the β-barrel.

[0011] In one or more embodiments, the one or more amino acid residues are selected from one or more of amino acid residues 138, 139, 141, 142, 144, 146, 148, 157, 159, 162, 163, 192, 194, 196, 198, 200, 202, 212, 214, 216, 218, and 220 of SEQ ID NO: 1.

[0012] In one or more embodiments, the one or more amino acid residues are selected from one or more of amino acid residues 157, 192, 202, and 220 of SEQ ID NO:1.

[0013] In one or more embodiments, the polypeptide has an amino acid sequence as shown in any one of SEQ ID NOs: 3-6, or a variant thereof having at least 90% sequence identity and retaining redox-sensitive function, wherein the cysteine ​​at position 157, 192, 202 or 220 is unchanged. Specifically, the variant is a variant of SEQ ID NO: 3, and the cysteine ​​at position 157 is unchanged; the variant is a variant of SEQ ID NO: 4, and the cysteine ​​at position 192 is unchanged; the variant is a variant of SEQ ID NO: 5, and the cysteine ​​at position 202 is unchanged; the variant is a variant of SEQ ID NO: 6, and the cysteine ​​at position 220 is unchanged.

[0014] In one or more embodiments, the polypeptide has the sequence shown in SEQ ID NO: 2 and wherein the amino acid residues corresponding to the one or more amino acid residues of SEQ ID NO: 1 in (1) are mutated to cysteine.

[0015] In one or more embodiments, the polypeptide has an amino acid sequence as shown in any one of SEQ ID NOs: 7-10, or a variant thereof having at least 90% sequence identity and retaining redox-sensitive function, wherein the cysteine ​​residues at positions 4, 39, 49, and 67 are unchanged. Specifically, the variant is a variant of SEQ ID NO: 7, and the cysteine ​​residue at position 4 is unchanged; the variant is a variant of SEQ ID NO: 8, and the cysteine ​​residue at position 39 is unchanged; the variant is a variant of SEQ ID NO: 9, and the cysteine ​​residue at position 49 is unchanged; the variant is a variant of SEQ ID NO: 10, and the cysteine ​​residue at position 67 is unchanged.

[0016] In one or more embodiments, the polypeptide further comprises other functional polypeptides located at its N-terminus, in the sequence, and / or at its C-terminus. In some embodiments, other functional polypeptides may be localization sequences connected thereto that localize the polypeptide to different organelles (subcellular localization), tags for purification, or tags for immunoblotting, or sequences that can identify targets of interest, such as oxidoreductase Grx, Trx, etc.

[0017] The present invention also provides a nucleic acid molecule comprising (1) a coding sequence of a polypeptide or protein described herein, (2) a sequence having at least 99%, 95%, 90%, 80%, 70% or 50% sequence identity with (1), (3) a complementary sequence or fragment of (1) or (2), preferably the fragment is 5-50 bp in length, more preferably 10-30 bp in length.

[0018] In one embodiment, the nucleic acid molecule of the present invention has a sequence selected from the following: (1) a coding sequence of an amino acid sequence shown in any one of SEQ ID NOs: 1-10 or a complementary sequence thereof, (2) a sequence having at least 99%, 95%, 90%, 80%, 70% or 50% identity with (1), (3) a fragment of (1) or (2).

[0019] The present invention also relates to variants of the above-mentioned nucleic acid molecules, including nucleic acid sequences encoding fragments, analogs, derivatives, soluble fragments and variants of the polypeptide of the present invention or their complementary sequences.

[0020] The present invention also provides a nucleic acid construct comprising the nucleic acid molecules described herein. The nucleic acid molecule encodes the polypeptide of the present invention. In one or more embodiments, the sequence of the nucleic acid molecule is operably connected to an expression control sequence. In one or more embodiments, the nucleic acid construct is a cloning vector, an expression vector or a recombinant vector. In some embodiments, the expression vector is selected from a prokaryotic expression vector, a eukaryotic expression vector and a viral vector.

[0021] The present invention also provides cells expressing the polypeptides described herein, or comprising the nucleic acid molecules or nucleic acid constructs described herein.

[0022] The present invention provides a method for preparing the polypeptide described herein, comprising: providing a cell expressing the polypeptide described herein, culturing the cell under conditions where the cell expresses the polypeptide, and isolating the polypeptide.

[0023] The present invention also provides a method for preparing the polypeptide described herein, comprising the following steps: 1) incorporating a nucleic acid molecule encoding the polypeptide described herein into a nucleic acid construct; 2) transferring the nucleic acid construct into a host cell; 3) culturing the host cell under conditions suitable for expression of the nucleic acid construct; 4) isolating the polypeptide.

[0024] The present invention also provides a detection kit comprising the polypeptide or nucleic acid molecule described herein or the polypeptide prepared by the method described herein, and other reagents required for detecting the redox state.

[0025] The present invention also provides a method for detecting the redox state inside or outside a cell, comprising: causing the cell to express or secrete the polypeptide described herein or incubating the cell described herein; optionally adding a substance of interest; and detecting changes in the polypeptide. Preferably, the change is a change in the optical properties of the polypeptide, and more preferably, the change is a change in the red fluorescence intensity of the polypeptide. In one or more embodiments, the change in the polypeptide indicates that the redox potential is increased or unchanged or decreased. In one or more embodiments, the substance of interest is an oxidizing and / or reducing substance or a substance that changes the redox state of the cell. In one or more embodiments, the substance of interest is a compound and / or a cell. In one or more embodiments, the substance of interest is selected from small molecule compounds, polypeptides, proteins, nucleic acid molecules and cells.

[0026] The present invention also provides a method for detecting a substance of interest in a sample, comprising: contacting the polypeptide or cell described herein with a sample, and detecting changes in the polypeptide. The detection can be performed in vivo, in vitro, intracellular, extracellular, subcellular or in situ. Preferably, the change is a change in the optical properties of the polypeptide, and more preferably, the change is a change in the red fluorescence intensity of the polypeptide. In one or more embodiments, the substance of interest is an oxidizing and / or reducing substance or a substance that changes the redox state of the cell. In one or more embodiments, the substance of interest is a compound and / or a cell. In one or more embodiments, the substance of interest is selected from small molecule compounds, polypeptides, proteins, nucleic acid molecules and cells.

[0027] Also provided herein is a method for quantifying a substance of interest in a sample, comprising: contacting a polypeptide or cell described herein with a sample, and detecting changes in the polypeptide, and quantifying the substance of interest in the sample according to the changes. Preferably, the change is a change in the optical properties of the polypeptide, and more preferably, the change is a change in the red fluorescence intensity of the polypeptide. In one or more embodiments, the substance of interest is an oxidizing and / or reducing substance or a substance that changes the redox potential of a cell. In one or more embodiments, the substance of interest is a compound and / or a cell. In one or more embodiments, the substance of interest is selected from small molecule compounds, polypeptides, proteins, nucleic acid molecules and cells.

[0028] The present invention also provides a method for screening a substance that can cause a change in redox state, comprising: contacting the polypeptide or cell described herein with a candidate substance, detecting changes in the polypeptide, and screening the substance based on the changes in the polypeptide. Preferably, the change is a change in the optical properties of the polypeptide, and more preferably, the change is a change in the red fluorescence intensity of the polypeptide. In one or more embodiments, the change in the polypeptide indicates that the substance can cause an increase or decrease in redox potential. In one or more embodiments, the substance is a compound and / or a cell. In one or more embodiments, the substance is selected from small molecule compounds, polypeptides, proteins, nucleic acid molecules and cells. The method can screen substances in a high-throughput manner.

[0029] The present invention also provides a method for screening a substance that can stabilize or aggravate a change in a redox state, comprising: contacting the polypeptide or cell described herein with a candidate substance and an optional oxidizing or reducing agent, detecting a change in the polypeptide, and screening a substance based on the change. Preferably, the change is a change in the optical properties of the polypeptide, and more preferably, the change is a change in the red fluorescence intensity of the polypeptide. In one or more embodiments, the change is weakened or enhanced compared to when the candidate substance is not present, indicating that the substance can stabilize or aggravate a change in a redox state.

[0030] The present invention also provides a method for detecting the interaction of substances, comprising: contacting the polypeptide described herein with two or more candidate substances, or contacting the cells described herein with one or more candidate substances, and detecting changes in the polypeptide, wherein the changes in the polypeptide indicate that there is an interaction between the two or more substances, or that there is an interaction between the cell and the one or more candidate substances. Preferably, the change is a change in the optical properties of the polypeptide, and more preferably, the change is a change in the red fluorescence intensity of the polypeptide. Preferably, the interaction causes the redox potential of any or the environment in the cell or the substance to increase or remain unchanged or decrease. In one or more embodiments, the substance is a compound and / or a cell. In one or more embodiments, the substance is selected from small molecule compounds, polypeptides, proteins, nucleic acid molecules and cells.

[0031] The present invention also provides a method for screening compounds for ligands, comprising: contacting the polypeptide or cell described herein with a candidate compound and a ligand, detecting changes in the polypeptide, and screening substances based on the changes in the polypeptide. Preferably, the change is a change in the optical properties of the polypeptide, and more preferably, the change is a change in the red fluorescence intensity of the polypeptide. In one or more embodiments, the change in the polypeptide indicates an interaction between the compound and the ligand, wherein the interaction between the compound and the ligand can cause the redox potential of the compound and / or the ligand and / or the environment to increase or remain unchanged or decrease.

[0032] The present invention also provides the use of the polypeptide described herein in intracellular / extracellular localization. In one or more embodiments, the localization is achieved by detecting redox changes in subcellular structures. In one or more embodiments, the localization is real-time localization.

[0033] Beneficial effects of the present invention: The present invention provides a group of fluorescent protein probes. The present invention replaces part of the nucleotides of the fluorescent protein mKate and its circular rearrangement product cpmKate genes with cysteine, causes changes in the fluorescence intensity of the fluorescent protein through changes in the protein oligomer structure, and finally obtains a fluorescent protein probe that is sensitive to redox. The fluorescent protein probe provided by the present invention has a relatively small molecular weight and is easy to mature, has large dynamic changes, can be expressed in cells through genetic manipulation methods, can achieve spatiotemporal specific detection of redox in cells, and eliminates time-consuming sample processing steps. At the same time, the fluorescent protein probe obtained by the present invention has a longer excitation and emission wavelength, and the emitted light is deep red, which can not only avoid the interference of autofluorescence to the greatest extent, but also can be well distinguished from other color probes in the spectrum, which is very helpful for multi-color combination of probes and multi-parameter analysis in single cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 This is an SDS-PAGE analysis diagram of the exemplary redox fluorescent probe in Example 6.

[0035] Figure 2 The results of the detection of mKate cyclization mutants in Example 2 are shown in Figure 2. A, fluorescence intensity; B, fluorescence change fold of the reduced state compared to the oxidized state.

[0036] Figure 3 The results of screening the fluorescent protein mKate and cpmKate mutants containing thiol mutations in Example 3. A, fluorescence intensity; B, fluorescence change factor of the reduced state compared with the oxidized state.

[0037] Figure 4 The diagram shows the response results of the mKate and cpmKate mutants containing thiol mutations that can respond quickly to oxidants in Example 4. A, mKate; B, cpmKate.

[0038] Figure 5 The results of the effect of the thiol groups of mKate and cpmKate on the redox fluorescent probe in Example 5 are shown in FIG. A, mKate, fluorescence intensity; B, mKate, fluorescence change multiple of the reduced state compared with the oxidized state; C, cpmKate, fluorescence intensity; D, cpmKate, fluorescence change multiple of the reduced state compared with the oxidized state.

[0039] Figure 6The fluorescence spectrum property results of the exemplary redox fluorescent probe in Example 6. A, mKate; B, cpmKate.

[0040] Figure 7 The diagram shows the reversibility detection results of the exemplary redox fluorescent probe in living cells in Example 7. AD, mKate; EH, cpmKate.

[0041] Figure 8 This is a diagram showing the subcellular structure localization analysis of the exemplary redox fluorescent probe in Example 8 in mammalian HEK293 cells.

[0042] Fig. 9 This is a diagram of the analysis of high-throughput compound screening based on an exemplary redox fluorescent probe at the living cell level as described in Example 9.

[0043] Fig.10 The diagram shows the dynamic detection results of the exemplary redox fluorescent probe in mammalian HEK293 cells under different nutritional conditions in Example 10. AD, mKate; EF, cpmKate. Specific implementation plan

[0044] The invention provides a redox-sensitive fluorescent protein probe, wherein the fluorescent protein is an mKate protein or a cpmKate protein containing a cysteine ​​mutation.

[0045] As used herein, the term "about" when giving a value or range means that the value or range is within 20%, within 10%, and within 5% of the given value or range.

[0046] As used herein, the terms "comprising", "including" and equivalent forms thereof include the meanings of "containing" as well as "consisting of", for example, a composition "comprising" X may consist of X alone or may contain other substances, such as X+Y.

[0047] The terms "redox fluorescent probe", "redox sensitive probe", and "redox sensitive polypeptide" used herein refer to a polypeptide that responds to a redox state or a change thereof, and the response includes any response to a chemical, biological, electrical or physiological parameter of the polypeptide. The redox sensitive polypeptide described herein may also include functional variants thereof, including but not limited to variants that can respond to a redox state or a change thereof to undergo the same or similar changes as the parent polypeptide. The redox state may refer to the level of redox potential. The probe of the present invention changes the fluorescence intensity in response to the redox state or a change thereof, and the fluorescence intensity is standardized to obtain a standardized redox state. The change in the fluorescence intensity of the probe of the present invention under different conditions may indicate the redox property / state of the different conditions.

[0048] The redox-sensitive polypeptides of the present invention include, but are not limited to, polypeptide sequences in which one or more amino acid residues of mKate fluorescent protein or its circular rearrangement cpmKate are mutated to cysteine, or variants thereof having at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or at least 99% homology thereto and retaining redox sensitivity. An exemplary mKate fluorescent protein is shown in SEQ ID NO:1. An exemplary cpmKate fluorescent protein is shown in SEQ ID NO:2. Therefore, the redox-sensitive polypeptides described herein have (1) the sequence shown in SEQ ID NO:1 and one or more amino acid residues thereof are mutated to cysteine, or (2) a sequence in which the sequence of (1) is connected at the beginning and the end by a connecting peptide and is disconnected between positions 153 and 154, between positions 169 and 168, or between positions 151 and 150, (3) a variant having at least 90% sequence identity with (1) or (2) and retaining redox-sensitive function, wherein the one or more amino acid residues mutated to cysteine ​​in the variant are unchanged relative to the sequence of (1) or (2).

[0049] In the present invention, the cysteine ​​refers to the cysteine ​​present in the higher structure of the protein and located on the β-fold of the protein. The cysteine ​​introduced on the protein monomer is active in nature and has strong nucleophilicity. It can react with a variety of substances to form disulfides or reversible disulfide bonds. In this process, the spatial conformation of the protein oligomer structure may undergo dynamic changes, which is further manifested as changes in the fluorescence intensity of the fluorescent protein. The cysteine ​​mutation described in the present invention mainly refers to: for the amino acid residues on the dimerization interface and / or β-fold of the mKate or cpmKate fluorescent protein, a cysteine ​​mutation is obtained by directional mutation of a single amino acid residue at a suitable position to cysteine. In the present invention, the cysteine ​​is preferably introduced into the surface of the β-barrel structure of the mKate or cpmKate fluorescent protein and the amino acid branch at the corresponding site faces outward. The mutant produced by the mutation is a variant having the same function as the mKate or cpmKate fluorescent protein but a different sequence. The cysteine ​​mutation can lead to changes in the structure of the protein oligomer, and finally lead to changes in the fluorescence intensity.

[0050] In the polypeptide of the present invention, the one or more amino acid residues mutated to cysteine ​​are selected from one or more of amino acid residues at positions 138, 139, 141, 142, 144, 146, 148, 157, 159, 162, 163, 192, 194, 196, 198, 200, 202, 212, 214, 216, 218, 220. Preferably, the one or more mutated amino acid residues are selected from one or more of amino acid residues at positions 157, 192, 202, 220, for example, preferably amino acid residue 157. The corresponding fluorescent protein probes are mKate157, mKate192, mKate202 and mKate220, respectively. In one or more embodiments, the polypeptide has an amino acid sequence as shown in any one of SEQ ID NOs: 3-6, or a variant thereof having at least 90% sequence identity and retaining redox-sensitive function, wherein cysteine ​​at position 157, 192, 202 or 220 is unchanged.

[0051] In certain embodiments, the redox-sensitive polypeptide of the present invention is a circularly rearranged polypeptide, such as a circularly rearranged product of mKate, cpmKate. The circular rearrangement may be a sequence in which the head and tail of the polypeptide described in the preceding paragraph are connected by a connecting peptide and disconnected at certain positions. Available disconnection positions for obtaining cpmKate from mKate are known in the art, such as sequences disconnected between positions 153 and 154, between positions 169 and 168, or between positions 151 and 150, such as sequences disconnected between positions 153 and 154. In one or more embodiments, the connecting peptide is selected from G, GS, GGS, GGSG, GGTG, GGTGG, GGSGG, GGSGGT, GGTGGS. In certain specific embodiments, the redox-sensitive polypeptide has a sequence shown in SEQ ID NO: 2 and wherein the amino acid residues corresponding to the one or more amino acid residues of SEQ ID NO: 1 are mutated to cysteine. For example, the redox-sensitive polypeptide has a sequence as shown in SEQ ID NO: 2, and one or more of the amino acid residues selected from positions 138, 139, 141, 142, 144, 146, 148, 157, 159, 162, 163, 192, 194, 196, 198, 200, 202, 212, 214, 216, 218, 220 corresponding to SEQ ID NO: 1 are mutated to cysteine. Preferably, the mutated one or more amino acid residues are selected from one or more of amino acid residues 157, 192, 202, 220 corresponding to SEQ ID NO: 1, for example, preferably amino acid residue 157 corresponding to SEQ ID NO: 1. The corresponding fluorescent protein probes are cpmKate157, cpmKate192, cpmKate202, and cpmKate220, respectively. In one or more embodiments, the polypeptide has an amino acid sequence as shown in any one of SEQ ID NOs: 7-10, or a variant thereof having at least 90% sequence identity and retaining redox-sensitive function, wherein cysteine ​​residues at positions 4, 39, 49, and 67 are unchanged.

[0052] The probe described herein can be connected as a basic unit to other proteins or polypeptides that do not affect the properties of the probe. In some embodiments, the redox fluorescent probe described herein also includes other functional polypeptides fused thereto. The other functional polypeptides described herein do not affect the properties of the fluorescent probe. Other functional polypeptides may be located at the N-terminus and / or C-terminus and / or in the sequence of the fluorescent protein probe. In some embodiments, other functional polypeptides include transmembrane signal molecules, polypeptides that locate the fluorescent probe to different organelles, tags for purification, and tags for immunoblotting. The "organelles" or "subcellular structures" described herein include cytoplasm, mitochondria, nuclei, endoplasmic reticulum, cell membranes, etc. Signal peptides and sequences for locating probes to different organelles are known in the art. In some embodiments, tags for purification or tags for immunoblotting include 6-histidine (6*His), glutathione S-transferase (GST), and Flag. A connecting peptide linker may be provided between the fluorescent protein probe and other polypeptides in the fusion polypeptide described herein. A linker may be provided between the fluorescent probe and other proteins or polypeptides, and the linker sequence may be a short peptide chain composed of 0 or more flexible amino acids, such as G, S, and Y.

[0053] When referring to a certain polypeptide or protein, the term "variant" or "mutant" used in the present invention includes variants having the same function of the polypeptide or protein but different sequences. Variants of polypeptides or proteins may include: homologous sequences, conservative variants, allelic variants, natural mutants, induced mutants. These variants include but are not limited to: deletion, insertion and / or substitution of one or more (usually 1-30, preferably 1-20, more preferably 1-10, and most preferably 1-5) amino acids in the sequence of the polypeptide or protein, and sequences obtained by adding one or more (usually within 20, preferably within 10, and more preferably within 5) amino acids to its carboxyl terminal and / or amino terminal. These variants may also include polypeptides or proteins having a sequence identity of at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99% or 100% with the polypeptide or protein. Without wishing to be bound by theory, amino acid residues are changed without changing the overall configuration and function of a polypeptide or protein, i.e., a function conservative mutation. For example, in the art, when amino acids with similar or similar properties are substituted, the function of a polypeptide or protein is usually not changed. In the art, amino acids with similar properties often refer to amino acid families with similar side chains, which are clearly defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), amino acids with acidic side chains (e.g., aspartic acid, glutamic acid), amino acids with uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), amino acids with non-polar side chains (e.g., alanine, valine, leucine, isoleucine, lactic acid, phenylalanine, methionine, tryptophan), amino acids with β-branched side chains (e.g., threonine, valine, isoleucine), and amino acids with aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). For another example, adding one or more amino acids to the amino terminus and / or carboxyl terminus will generally not change the function of a polypeptide or protein. Conservative amino acid substitutions for many common known non-genetically encoded amino acids are known in the art. Conservative substitutions for other non-encoded amino acids can be determined based on a comparison of their physical properties with the properties of the genetically encoded amino acids. In the variants described herein, one or more amino acid residues that have been mutated to cysteine ​​are not changed.

[0054] In the context of two or more polypeptide or nucleic acid molecule sequences, the term "identity" or "percent identity" refers to the fact that two or more sequences or subsequences are identical or that a certain percentage of amino acid residues or nucleotides are identical over a specified region (e.g., at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical) when compared and aligned for maximum correspondence over a comparison window or specified region using methods known in the art, such as sequence comparison algorithms, by manual alignment and visual inspection. For example, preferred algorithms suitable for determining percent sequence identity and percent sequence similarity are the BLAST and BLAST 2.0 algorithms, see Altschul (1997) et al. (Altschul, Madden et al. 1997) and Altschul (1990) et al. (Altschul, Gish et al. 1990), respectively.

[0055] It is well known to those skilled in the art that the introduction of one or more irrelevant residues at the end of the expressed polypeptide or protein due to the need to introduce restriction sites in gene cloning operations does not affect the activity of the target polypeptide or protein. In addition, in order to construct fusion proteins, promote the expression of recombinant proteins, obtain recombinant proteins that are automatically secreted outside the host cell, or facilitate the purification of recombinant proteins, some amino acids can be added to the N-terminus, C-terminus or other suitable regions in the protein of the recombinant protein, for example, including but not limited to, suitable linker peptides, signal peptides, leader peptides, terminal extensions, glutathione S-transferase (GST), maltose E binding protein, protein A, such as 6His or Flag tags, or proteolytic enzyme sites of factor Xa or thrombin or enterokinase.

[0056] The terms "functional fragment", "derivative" and "analog" as used herein refer to proteins that substantially retain the same biological function or activity as the original polypeptide or protein (e.g., the redox-sensitive polypeptide described herein). The functional variant, derivative or analog of the polypeptide or protein of the present invention may be (i) a protein having one or more conservative or non-conservative amino acid residues (preferably conservative amino acid residues) substituted, and such substituted amino acid residues may or may not be encoded by the genetic code, or (ii) a protein having a substitution group in one or more amino acid residues, or (iii) a protein formed by fusion of a mature protein with another compound (e.g., a compound that prolongs the half-life of the protein, such as polyethylene glycol), or (iv) a protein formed by fusion of an additional amino acid sequence to the protein sequence (e.g., a secretory sequence or a sequence used to purify the protein or a proprotein sequence, or a fusion protein formed with an antigen IgG fragment). According to the teachings of this article, these functional variants, derivatives and analogs belong to the well-known scope of those skilled in the art. The analogs may be analogs having residues other than natural L-amino acids (e.g., D-amino acids), as well as analogs having non-naturally occurring or synthetic amino acids (e.g., β, γ-amino acids). It should be understood that the redox-sensitive polypeptides of the present invention are not limited to the representative proteins, variants, derivatives and analogs listed above. Modified forms include: chemical derivatization forms of proteins in vivo or in vitro, such as acetylation or carboxylation. Modifications also include glycosylation, such as those produced by glycosylation modification during protein synthesis and processing or in further processing steps. This modification can be accomplished by exposing the protein to an enzyme that performs glycosylation (such as a mammalian glycosylase or deglycosylation enzyme). Modified forms also include sequences with phosphorylated amino acid residues (such as phosphotyrosine, phosphoserine, phosphothreonine). Also included are proteins that have been modified to improve their resistance to proteolysis or optimize their solubility.

[0057] The "mutation" of the present invention includes the mutation of the above-mentioned amino acid to cysteine ​​and various mutations in the variant. The present invention has no special limitation on the method of the mutation, which can be a molecular biological method. For example, the present invention preferably uses an inverse PCR method to achieve amino acid mutation, that is, a pair of specific primers are matched with both ends of the protein sequence of the mutation site, so that a linearized plasmid containing the nucleotide of the mutation site can be generated by inverse PCR amplification, and the protein encoded by the recombinant plasmid formed thereafter contains the specific mutated amino acid. Alternatively, physical mutagenesis or chemical mutagenesis methods, such as radiation or the addition of mutagens, can be used.

[0058] The present invention includes nucleic acid molecules encoding the polypeptides of the present invention. The terms "nucleic acid molecules" or "nucleotides" or "polynucleotides" or "nucleic acid sequences" used in the present invention can be in the form of DNA or RNA. DNA forms include cDNA, genomic DNA or artificially synthesized DNA. DNA can be single-stranded or double-stranded, coding strands or non-coding strands. When referring to nucleic acids, the term "variant" used herein can be a naturally occurring allelic variant or a non-naturally occurring variant. These nucleotide variants include degenerate variants, substitution variants, deletion variants and insertion variants, but will not substantially change the function of the protein encoded by them. The nucleic acid of the present invention can include a nucleotide sequence having a sequence identity of at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99% or 100% with the nucleic acid sequence. The present invention also relates to nucleic acid fragments hybridized with the above-mentioned sequences. As used herein, a "nucleic acid fragment" is at least 15 nucleotides in length, preferably at least 30 nucleotides, more preferably at least 50 nucleotides, and most preferably at least 100 nucleotides in length. Nucleic acid fragments can be used in nucleic acid amplification techniques (such as PCR), for example, as primers or probes.

[0059] The full-length sequence of the fluorescent probe or fusion protein of the present invention or its fragment can usually be obtained by PCR amplification, artificial synthesis or recombination. The steps and reagents used in conventional PCR, synthesis and recombination are known in the art. In addition, mutations can be introduced into the protein sequence of the present invention by methods such as mutation PCR or chemical synthesis.

[0060] The present invention also relates to nucleic acid constructs comprising the polynucleotides described herein and one or more regulatory sequences operably linked to these sequences. The polynucleotides of the present invention can be manipulated in a variety of ways to ensure expression of the polypeptide or protein. The nucleic acid constructs can be manipulated prior to insertion into a vector depending on the expression vector or requirements. Techniques for altering polynucleotide sequences using recombinant DNA methods are known in the art.

[0061] In certain embodiments, the nucleic acid construct is a vector. The vector may be a cloning vector, an expression vector, or a homologous recombination vector. The polynucleotide of the present invention may be cloned into many types of vectors, for example, plasmids, phagemids, phage derivatives, animal viruses, and cosmids. The cloning vector may be used to provide the coding sequence of the protein or polypeptide of the present invention. The expression vector may be provided to the cell in the form of a bacterial vector or a viral vector. The expression of the polynucleotide of the present invention is usually achieved by operably connecting the polynucleotide of the present invention to a promoter and incorporating the construct into the expression vector. The vector may be suitable for replication and integration of eukaryotic cells. In one or more embodiments, the cloning vector and the expression vector are a kind of vector, i.e., a cloning expression vector. The homologous recombination vector is used to integrate the expression cassette described herein into the host genome.

[0062] Typical expression vectors contain expression control sequences that can be used to regulate the expression of the desired nucleic acid sequence, and are operably connected to the nucleic acid sequence of the present invention or its complementary sequence. The term "expression control sequence" used in the present invention refers to an element that can be operably connected to the target gene to regulate the transcription, translation and expression of the target gene, which can be a replication origin, a promoter, a marker gene or a translation control element, including an enhancer, an operator, a terminator, a ribosome binding site, etc. The selection of the expression control sequence depends on the host cell used. In a recombinant expression vector, "operable connection" means that the nucleotide sequence of the target is connected to the regulatory sequence in a manner that allows the expression of the nucleotide sequence. Those skilled in the art are familiar with methods that can be used to construct expression vectors containing the fusion protein coding sequence of the present invention and suitable transcription / translation control signals. These methods include in vitro recombinant DNA technology, DNA synthesis technology, in vivo recombination technology, etc. The DNA sequence can be effectively connected to an appropriate promoter in the expression vector to guide mRNA synthesis. Representative examples of these promoters include: lac or trp promoters of Escherichia coli; λ phage PL promoter; eukaryotic promoters include CMV immediate early promoter, HSV thymidine kinase promoter, early and late SV40 promoter, LTR of retrovirus and other known promoters that can control gene expression in prokaryotic or eukaryotic cells or their viruses. The expression vector also includes a ribosome binding site for translation initiation and a transcription terminator.

[0063] After obtaining the recombinant expression vector, the vector is transformed into a host cell to produce a protein or peptide including the polypeptide described herein. This transfer process can be carried out using conventional techniques known to those skilled in the art such as transformation or transfection. The host cell described in the present invention refers to a cell that can receive and accommodate a recombinant DNA molecule, and is a place for recombinant gene amplification. The ideal recipient cell should meet the two conditions of easy acquisition and proliferation. The "host cell" of the present invention may include prokaryotic cells and eukaryotic cells, specifically including bacterial cells, yeast cells, insect cells and mammalian cells. Specifically, it can be Escherichia coli, Streptomyces, bacterial cells of Salmonella typhimurium, fungal cells such as yeast, plant cells, insect cells of Drosophila S2 or Sf9, CHO, COS, HEK293, HeLa cells, or animal cells of Bowes melanoma cells, etc., including but not limited to those host cells mentioned above. The host cell is preferably a variety of cells that are conducive to the expression or fermentation production of gene products, and such cells are well known and commonly used in the art. The exemplary host cell used in the embodiment of the present invention is the Escherichia coli BL21-DE3 strain. Those skilled in the art are well aware of how to select appropriate vectors, promoters, enhancers and host cells. The host cells of the present invention are preferably Escherichia coli cells.

[0064] The method of transferring to the host cell described in the present invention is a conventional method in the art, including calcium phosphate or calcium chloride coprecipitation, DEAE-mannan-mediated transfection, lipofection, natural competence, chemical-mediated transfer or electroporation. When the host is a prokaryotic organism such as Escherichia coli, the method is preferably treated with CaCl2 or MgCl2, and the steps used are well known in the art. When the host cell is a eukaryotic cell, the following DNA transfection methods can be selected: calcium phosphate coprecipitation, conventional mechanical methods such as microinjection, electroporation, liposome packaging, etc.

[0065] After the expression vector is transferred into the host cell, the host cell into which the expression vector is transferred is cultured for amplification and expression, and the redox fluorescent protein probe is isolated. The host cell amplification and expression culture can be carried out by conventional methods. Depending on the type of host cell used, the culture medium used in the culture can be various conventional culture media. The culture is carried out under conditions suitable for the growth of the host cell.

[0066] In the present invention, the probe is expressed in the cell, on the cell membrane, or secreted outside the cell. If necessary, the recombinant protein can be separated or purified by various separation methods using its physical, chemical and other properties. The present invention does not specifically limit the method for separating the redox fluorescent protein probe, and the conventional protein separation method in the art can be used. These methods are well known to those skilled in the art, including but not limited to: conventional renaturation treatment, salting-out method, centrifugation, osmotic breaking, ultrasonic treatment, ultracentrifugation, molecular sieve chromatography, adsorption chromatography, ion exchange chromatography, high performance liquid chromatography (HPLC) and other various liquid chromatography techniques and combinations of these methods. In one embodiment, the probe is separated using affinity chromatography using a His tag.

[0067] The polypeptide or nucleic acid molecule described herein or the polypeptide prepared by the method described herein can be prepared as a detection kit. The kit also contains other reagents required for detecting the redox state. Other reagents required for detecting the redox state of cells, substances, and the environment are well known in the art.

[0068] The present invention also provides the use of the redox fluorescent protein probe in subcellular localization detection, real-time dynamic detection of redox potential, and high-throughput compound screening. The redox fluorescent protein probe of the present invention can be directly transferred into cells, and in the process of redox detection, time-consuming sample processing is not required, which is more accurate.

[0069] In one aspect, the present invention also provides the use of the polypeptide described herein in localizing intracellular / extracellular substances or redox reactions and in real-time dynamic monitoring of redox states. The localization or monitoring is achieved by detecting redox changes in cells or subcellular structures. For example, the redox fluorescent protein probe is connected to a signal peptide in different parts of a cell, transferred into the cell, and the redox state is detected by detecting the strength of the fluorescent signal in the cell.

[0070] In another aspect, the present invention also provides a method for qualitatively and / or quantitatively detecting a substance of interest in a sample, comprising: contacting the polypeptide or cell described herein with a sample, and detecting changes in the optical properties of the polypeptide, and optionally quantifying the substance of interest in the sample according to the changes. The "substance of interest" described herein mainly refers to a substance with oxidation and / or reduction properties. The substance can be any compound and / or cell with oxidation and / or reduction properties. In one or more embodiments, the substance is selected from small molecule compounds, polypeptides, proteins, nucleic acid molecules and cells. The present invention also provides a method for screening a substance that can cause an increase or decrease in redox potential, comprising: contacting the polypeptide or cell described herein with a candidate substance, detecting changes in the optical properties of the polypeptide, and screening substances according to the changes in the optical properties, wherein the changes in the optical properties indicate that the substance can cause an increase or decrease in redox potential. The present invention also provides a method for screening a substance that can stabilize or aggravate changes in redox potential, comprising: contacting the polypeptide or cell described herein with a candidate substance and an optional oxidizing or reducing agent, detecting changes in the optical properties of the polypeptide, and screening substances according to the changes, wherein the changes are weakened or enhanced compared to when the candidate substance is not present, indicating that the substance can stabilize or aggravate changes in the redox state. The method of the present invention is convenient for high-throughput screening of substances. When the redox fluorescent protein probe is used for high-throughput compound screening, different compounds are added to the cell culture medium to measure the changes in the redox state, thereby screening out compounds that can enter the cell across the membrane and affect the redox state of the cell. The application of the redox fluorescent protein probe described in the present invention in redox detection and high-throughput compound screening is for non-diagnostic and therapeutic purposes and does not involve the diagnosis and treatment of diseases.

[0071] The detection described herein can be performed in vivo, in vitro, intracellular / extracellular, intracellular or in situ. Therefore, the contact described herein includes contact between the polypeptide and the substance of interest inside and outside the cell. For example, the polypeptide can be mixed with a compound of interest; alternatively, the polypeptide can be associated with a transmembrane signal molecule, thereby passing through the cell membrane into the cell and contacting the substance of interest; alternatively, the coding sequence of the polypeptide can be integrated into the cell as described above and expressed by the cell, so that the polypeptide contacts the substance of interest inside the cell; alternatively, the coding sequence of the polypeptide can be associated with a subcellular localization sequence, integrated into the cell as described above and expressed by the cell, and then enters the subcellular structure and contacts the substance of interest. For another example, the polypeptide can be mixed with a cell, and the polypeptide contacts the substance when the cell secretes the substance of interest; alternatively, the coding sequence of the polypeptide can be integrated into the cell as described above and expressed and secreted by the cell, thereby contacting the substance of interest from any source outside the cell; alternatively, the coding sequence of the polypeptide can be associated with a transmembrane signal molecule, integrated into the cell as described above and expressed and secreted by the cell, and then enters other cells with the help of the transmembrane signal molecule and contacts the substance of interest.

[0072] In another aspect, the present invention also provides a method for detecting substance interactions, comprising: contacting the polypeptide described herein with two or more candidate substances or contacting the cells described herein with one or more candidate substances, and detecting changes in the optical properties of the polypeptide, wherein the changes in the polypeptide indicate that there is an interaction between the two or more substances or that there is an interaction between the cell and one or more candidate substances. The "interaction" described herein mainly refers to an interaction that can cause the redox potential of any or the environment in a cell or substance to increase, remain unchanged, or decrease. For example, antigen-antibody reaction, enzymatic or non-enzymatic reaction, binding of ligand to receptor, phosphorylation.

[0073] The present invention also provides a method for detecting the redox state in or outside a cell, comprising: causing the cell to express or secrete the polypeptide described herein or incubating the cell described herein; optionally adding a substance of interest; and detecting changes in the optical properties of the polypeptide. The changes in the polypeptide indicate the redox state, such as an increase, unchanged or decrease in the redox potential.

[0074] In the present invention, concentrations, contents, percentages and other values ​​can be expressed in the form of ranges. It should also be understood that the use of such ranges is only for convenience and brevity, and should be flexibly interpreted as including the values ​​explicitly mentioned at the upper and lower limits of the range, and also including all individual values ​​or sub-ranges included in the range. When giving a value or range, the term "about" used means that the value or range is within 20%, within 10% and within 5% of the given value or range.

[0075] Example

[0076] The probe provided by the present invention is described in detail below in conjunction with the examples, but they should not be construed as limiting the protection scope of the present invention.

[0077] I. Experimental Materials and Reagents

[0078] In the embodiments, conventional genetic engineering molecular biology cloning methods, cell culture and imaging methods are mainly used. These methods are well known to ordinary technicians in the field, for example: "Molecular Biology Laboratory Reference Manual" by Jane Roskams et al., "Molecular Cloning Laboratory Guide" (3rd edition, August 2002, Science Press, Beijing) by J. Sambrook, DW Russell, translated by Huang Peitang et al.; "Animal Cell Culture: Basic Technical Guide" (5th edition) by Frasheny et al., translated by Zhang Jingbo, Xu Cunshuan et al.; "Concise Cell Biology Laboratory Guide" by JS Bonifacion, M. Dassault et al., translated by Zhang Jingbo et al.

[0079] The pRSETB-mKate plasmid used in the examples was constructed by the Protein Laboratory of East China University of Science and Technology, and the pET28a plasmid vector was purchased from Invitrogen. Cloning strains Mach1 and BL21-DE3 were purchased from Invitrogen. The primer sequences are shown in SEQ ID NO: 11-38. For other materials, reagents and instruments, see CN201810215698.7, all of which are incorporated herein by reference.

[0080] II. Molecular Biology Methods and Cell Experimental Methods

[0081] II.1 Fluorescence detection of proteins and live bacteria

[0082] Fluorescence detection of proteins

[0083] 1. After the purified fluorescent protein probe is identified by SDS-PAGE, the probe is diluted with assay buffer (100mM HEPES, 100mM NaCl, pH 7.4) or phosphate buffer PBS to a final concentration of 0.4-1μM protein solution. The reducing agent DTT and the oxidizing agent Diamide are prepared into stock solutions with final concentrations of 20mM and 1mM, respectively, with assay buffer (100mM HEPES, pH 7.4) or phosphate buffer PBS.

[0084] 2. Take 50 μl of 0.4 μM protein solution, incubate at 37°C for 5 min, add reducing agent DTT and oxidizing agent Diamide respectively and mix well to a final concentration of 0.2 μM, and use a multifunctional fluorescence microplate reader to measure the light absorption of the protein at 590 nm.

[0085] 3. Take 50 μl of 0.4 μM protein solution, incubate at 37°C for 5 min, add reducing agent DTT and oxidizing agent Diamide, mix well, and measure the fluorescence intensity of the protein at 645 nm after fluorescence excitation at 590 nm. The fluorescence excitation and emission measurement of the sample is completed using a multifunctional fluorescence microplate reader.

[0086] Fluorescence detection of live bacteria

[0087] 1. After the probe protein is induced to express in E. coli, centrifuge at 4000rpm for 5 minutes to collect the bacteria and discard the supernatant. Use an equal volume of HEPES buffer to resuspend the bacteria for washing. After washing, collect the bacteria again and discard the supernatant. Add an appropriate amount of HEPES buffer to resuspend the bacteria. It is best to use a pipette tip to gently blow or vortex to ensure that the bacteria are evenly mixed.

[0088] 2. Take 100 μL into a 96-well transparent flat-bottom plate and measure the OD value of the bacterial solution at 600 nm using an ELISA reader. Unify the different samples to OD 600 The concentration was 0.2.

[0089] 3. Spread the diluted samples with the same concentration into a 96-well opaque black plate, incubate at 37°C for 5 minutes, and add the reducing agent DTT and the oxidizing agent Diamide for detection.

[0090] II.2 Transfection of mammalian cells and fluorescence detection

[0091] 1. The PAAV-based fluorescent protein probe plasmid was transfected into HEK293 cells using the transfection reagent Lipofectamine 2000 (Invitrogen), and cultured in a cell culture incubator at 37°C and 5% CO2. Fluorescence detection was performed after the exogenous gene was fully expressed for 24 to 36 hours.

[0092] 2. After the induction of expression was completed, the attached HEK293 cells were rinsed three times with PBS and placed in HBSS solution for fluorescence microscopy detection.

[0093] II.3 Other experiments

[0094] For other experimental processes involved in the embodiments, please refer to CN201810215698.7, all the contents of which are incorporated herein by reference.

[0095] Example 1: Construction of pET28a-mKate plasmid

[0096] The mKate gene was amplified using pRSETB-mKate as a template. The PCR product was recovered by gel electrophoresis and then double-digested with BamHI and NotI. The pET28a vector was also double-digested. After ligation with T4 DNA ligase, the ligation product was transformed into competent Trans 5α. The transformed Trans 5α was spread on an LB plate (kanamycin 100 μg / mL) and cultured at 37°C overnight. The grown Trans 5α transformants were subjected to plasmid extraction and PCR identification. The positive plasmid was sequenced correctly for subsequent plasmid construction.

[0097] Primers for constructing pET28a-mKate plasmid: SEQ ID NOs: 11-14.

[0098] Example 2: Plasmid construction and selection of mKate cyclization mutants

[0099] In this embodiment, we selected three groups of sites, 169 / 168, 151 / 150, and 154 / 153, to construct circularly rearranged cpmKate mutants. Using pET28a-mKate as a template, short fragments 1-168 and 169-231, 1-150 and 151-231, 1-153 and 154-231 were amplified respectively, and they were connected through flexible connecting peptides using overlapping PCR methods to further amplify the rearranged long gene fragments. After the PCR product was recovered by gel electrophoresis, it was double-digested with BamHI and NotI, and the pET28a vector was subjected to the same double digestion. After connection with T4 DNA ligase, the ligation product was transformed into the competent Trans 5α, and the transformed Trans 5α was coated on an LB plate (kanamycin 100 μg / mL) and cultured at 37°C overnight. After the growing Trans 5α transformant was subjected to plasmid extraction, PCR identification was performed. Primers for constructing pET28a-cpmKate169 / 168, pET28a-cpmKate151 / 150, and pET28a-cpmKate154 / 153 plasmids: SEQ ID NOs: 15-22.

[0100] After the positive plasmid sequencing is correct, the recombinant plasmid is transformed into competent BL21 (DE3) to induce expression and compare their fluorescence intensity and their response to oxidants and reductants. The results are normalized to mKate. The specific test results are as follows Figure 2 shown.

[0101] The test results showed that the fluorescence brightness of the mutants at sites 169 / 168 and 151 / 150 of the three recombinant circularized mutants was weaker than that of mKate, and the fluorescence brightness of the mutant at site 154 / 153 was basically consistent with that of mKate. mKate itself has a response of about 1.5 times to redox, the mutant at site 169 / 168 has a slightly higher response to redox than mKate, and the mutants at sites 151 / 150 and 154 / 153 have a basically consistent response to redox with that of mKate. On the whole, mKate and the circularized mutants of mKate at sites 154 / 153 were selected for subsequent plasmid construction.

[0102] Example 3: Construction and detection of plasmids with cysteine ​​introduced at different sites in pET28a-mKate and pET28a-cpmKate154 / 153

[0103] In this example, we used pET28a-mKate and pET28a-cpmKate154 / 153 as the basic plasmids and selected 22 cysteine ​​introduction sites, including 138, 139, 141, 142, 144, 146, 148, 157, 159, 162, 163, 192, 194, 196, 198, 200, 202, 212, 214, 216, 218, and 220, according to the crystal structure of mKate. Among them, the sites with relatively large redox response are 157, 192, 202, and 220, and the primers used for point mutation are: SEQ ID NO: 23-30.

[0104] By designing appropriate primers and then using PCR to generate mKate and cpmKate154 / 153 nucleic acid fragments containing cysteine ​​mutations, the 5' end of the linearized fragment was phosphorylated with PNK enzyme, and then connected under the action of PEG 4000 and T4 ligase to form a recombinant plasmid. After the plasmid sequencing is correct, the recombinant plasmid is transformed into competent BL21 (DE3) to induce expression, and their fluorescence intensity is compared at the viable bacterial level and their changes in fluorescence intensity after incubation with the oxidant Diamide and the reducing agent DTT. The specific test results are as follows Figure 3 shown.

[0105] The results showed that the samples with relatively large redox responses among these mutants include mKate157, mKate192, mKate202, mKate220, cpmKate157, cpmKate192, cpmKate202 and cpmKate220 (sequences are shown in SEQ ID NO: 3-10). In terms of fluorescence brightness and change multiple, cpmKate157 has the best properties, followed by mKate157. Among them, the fluorescence intensity of cpmKate157 changes by up to 5 times at the viable bacteria level compared with other samples in both oxidation and reduction states, and the fluorescence change multiple of mKate157 reaches up to 4 times. Since cpmKate154 / 153 and mKate proteins themselves have a response of about 1.5 times, the fluorescence change multiples of cpmKate157 and mKate157 after normalization with mKate are about 3 times and 2.5 times, respectively.

[0106] Example 4: Oxidation kinetics detection of fluorescent probes at four sites: 157, 192, 202, and 220

[0107] In this example, we selected fluorescent probes at four sites: 157, 192, 202, and 220: mKate157, mKate192, mKate202, mKate220, cpmKate157, cpmKate192, cpmKate202, and cpmKate220 (SEQ ID NOs: 3-10) to further study the kinetic characteristics of the oxidative response.

[0108] The probe plasmid was transformed into competent BL21 (DE3) to induce expression. The bacterial solution was uniformly diluted to OD600 0.2. After reading the initial fluorescence value, the oxidant Diamide was quickly added to a final concentration of 300 μM and the change process of the fluorescence value was recorded. The specific test results are as follows: Figure 4 shown.

[0109] The results showed that the fluorescence values ​​of these mutants decreased immediately after the addition of oxidants. The cpmKate-based mutants responded faster than the mKate-based mutants. Among them, cpmKate157 had the fastest response and the largest change. It could react completely in about 5 minutes, and the maximum fluorescence change could reach 5 times. At the same time, the results showed that these mutant proteins of mKate were mainly in a reduced state in E. coli, so when the redox potential of the microenvironment increased, it could be indicated by the change of fluorescence intensity.

[0110] Example 5: Effect of cysteine ​​in mKate and cpmKate154 / 153 on the probe

[0111] In this example, we analyzed the effect of the cysteine ​​residues in mKate and cpmKate154 / 153 on the probes.

[0112] mKate and cpmKate154 / 153 have four cysteine ​​residues, corresponding to positions 26, 114, 172, and 222 of the mKate protein. In redox detection, the cysteine ​​residues of the fluorescent protein itself are likely to interfere or participate in the response of the probe. In order to explore the effect of these four cysteines on the properties of the probe, we made combined mutations of C26A-C114M-C172V-C222S, C26A-C114M-C172V, and C26A-C114M based on mKate157, mKate192, mKate202, mKate220, cpmKate157, cpmKate192, cpmKate202, and cpmKate220, respectively. Primers used for point mutations: SEQ ID NO: 31-38.

[0113] After the combined mutant plasmid sequencing is correct, the recombinant plasmid is transformed into the competent BL21 (DE3) to induce expression, and their fluorescence intensity and the change multiple of their fluorescence intensity after incubation with the oxidant Diamide and the reducing agent DTT are compared at the viable bacteria level. Figure 5 shown.

[0114] The results showed that: (1) The change trends of the mKate and cpmKate154 / 153 combination mutants were consistent, but the latter had a larger overall change. (2) The change folds of the C26A / C114M / C172V / C222S and C26A / C114M / C172V combination mutants of the 192 site mutation probe were significantly affected; the change fold of the C26A / C114M combination mutant of the 192 site mutation probe was basically unaffected. (3) The combined mutation results of the 220 site mutation probe and the 202 site mutation probe were similar to those of the 192 site. (4) The change folds of the C26A / C114M / C172V / C222S and C26A / C114M / C172V combination mutants of the 157 site mutation probe were greatly affected; the change fold of the C26A / C114M combination mutant of the 157 site mutation probe was basically unaffected. (5) Based on the fluorescence brightness and dynamic changes, the best one is cpmKate157 which retains four cysteines, followed by mKate157 which retains four cysteines.

[0115] In summary, we believe that the response of mutants at the four sites 157, 192, 202 and 220 to redox stimulation is related to protein dimerization or oligomerization, and the cysteines at positions 172 and 222 of mKate itself are also involved in and enhance this process because they are located at the dimerization interface.

[0116] Example 6: Properties of mKate157 and cpmKate157 probes

[0117] In this example, we selected the mutants mKate157 and cpmKate157 with the greatest redox response for further study of their properties.

[0118] First, the purified mKate157 and cpmKate157 as well as mKate and cpmKate154 / 153 were identified by SDS-PAGE electrophoresis to be around 35 kDa. The size of cpmKate157 was slightly larger than that of mKate157 due to the addition of a connecting peptide in the middle. Their sizes were consistent with the size of the proteins containing the His-tag purification tag expressed by pET28a-mKate and pET28a-cpmKate. The SDS-PAGE results of the proteins are shown in the figure. Figure 1 shown.

[0119] Subsequently, we incubated the purified mKate157 and cpmKate157 probe proteins with a final concentration of 1 mM oxidant Diamide and a final concentration of 10 mM reducing agent DTT at 37°C for 30 minutes, and then measured the fluorescence spectra using a fluorescence spectrophotometer. When measuring the excitation spectrum, the emission was fixed at 660 nm, and the excitation spectrum was recorded in the excitation range of 400 nm to 630 nm; when measuring the emission spectrum, the excitation was fixed at 560 nm, and the emission spectrum was recorded in the emission range of 590 nm to 730 nm. The fluorescence spectral properties of the purified mKate157 and cpmKate157 probes are shown in the following figure. Figure 6 As shown. The spectra of mKate157 and cpmKate157 are similar to the wild-type mKate, and the spectra in the oxidized and reduced states do not shift, only the fluorescence intensity changes. It is worth noting that the change multiples of mKate157 and cpmKate157 after adding oxidants and reductants to the protein are slightly smaller than the change multiples in living bacteria, indicating that the redox probe involved in the present invention is more suitable for use in living cell environments.

[0120] Example 7: Reversible detection of fluorescent probes in living cells

[0121] In this example, we selected fluorescent probes at four sites, 157, 192, 202, and 220 (mKate157, mKate192, mKate202, mKate220, cpmKate157, cpmKate192, cpmKate202, and cpmKate220) to study their reversibility in living cells.

[0122] The eight probe plasmids with the best changes were transformed into competent BL21 (DE3) to induce expression, and the bacterial solution was uniformly diluted to OD 600 After the fluorescence value is 0.2, the plate is plated on a 96-well full black plate. The oxidant Diamide is first added to read and record the change in fluorescence value, and then the reducing agent DTT is added to read and record the change in fluorescence value. The specific test results are as follows Figure 7 As shown. According to the test results, the fluorescence of the eight probes decreased to varying degrees after the addition of the oxidant, reaching the maximum change at about 5 minutes, among which cpmKate157 and mKate157 changed the most. When the fluorescence of the eight probes decreased to the maximum due to oxidation, the reducing agent was added, and their fluorescence recovered immediately, indicating that the response of the redox probe involved in the present invention is reversible.

[0123] Example 8: Subcellular localization of fluorescent probes

[0124] In this example, we used different localization signal peptides to fuse to the C-terminus or N-terminus of the exemplary redox fluorescent probe cpmKate157.

[0125] HEK293 cells were transfected with probe plasmids fused with different localization signal peptides for 36 hours, then washed with PBS and placed in HBSS solution for fluorescence detection using a 561nm excitation of an inverted fluorescence microscope. We found that the redox fluorescent probe cpmKate157 can be localized to subcellular structures such as cytoplasm, mitochondria, endoplasmic reticulum, cell membrane, and cell nucleus by fusing with different specific localization signal peptides. The results are shown in Figure 8 As shown, fluorescence is displayed in different subcellular structures, and the distribution and intensity of the fluorescence are different.

[0126] Example 9: High-throughput compound screening based on probes at the living cell level

[0127] In this example, we expressed the exemplary redox fluorescent probe cpmKate157 in the cytoplasm of HeLa cells for high-throughput compound screening.

[0128] After the HeLa cells were transfected with the cpmKate157 gene, they were washed with PBS, placed in HBSS solution for 1 hour, and then incubated with 10 μM of different compounds for 1 hour. The fluorescence intensity changes at 590 nm excitation and 645 nm emission were recorded using an ELISA reader. The sample not treated with any compound was used as the standard. The results are shown in Figure 2. Fig. 9 As shown in the results, we found that most of the compounds treated with 1,200 compounds had little effect on the redox potential of the cells. Among them, 24 compounds could cause obvious oxidative stress in cells, resulting in increased redox potential; and 18 compounds could cause obvious reductive stress in cells, resulting in decreased redox potential.

[0129] Example 10: Dynamic monitoring of fluorescent probes in mammalian cells under different nutritional conditions

[0130] In this example, we used exemplary redox fluorescent probes mKate157 and cpmKate157 to monitor the redox status in mammalian HEK293 cells in real time under different nutrient conditions.

[0131] 25mM glucose was added or not added to the detection buffer to change the nutritional conditions of the cells (sugar or sugar-free), and exogenous oxidants (200μM Diamide) or reductants (10mM DTT) were added to regulate the redox state in the cells. The fluorescence value of the sample was detected using a multifunctional microplate reader. The test results are shown in Figure 2. Fig.10 As shown, firstly, the experimental results show that the probe of the present invention can detect the dynamic changes of redox in mammalian cells in real time; in addition, the results show that under nutrient-rich (sugar-containing) conditions, HEK293 cells have a stronger ability to maintain redox homeostasis, and the decrease in probe fluorescence caused by oxidative stimulation can be recovered in about 15-20 minutes, while under nutrient-deficient (sugar-free) conditions, the decrease in probe fluorescence caused by oxidative stress in HEK293 cells is difficult to recover.

[0132] As can be seen from the above examples, the redox fluorescent protein probe provided by the present invention has a small protein molecular weight and is easy to mature, has a large dynamic change in fluorescence, and can be expressed in cells by genetic manipulation methods, can achieve spatiotemporal specific detection of redox in cells, and eliminates the time-consuming sample processing steps. In addition, the fluorescent protein probe provided by the present invention can be used for high-throughput compound screening.

[0133] Other Implementations

[0134] The above is only a preferred embodiment of the present invention. However, it should be understood that for those skilled in the art, several improvements and modifications can be made without departing from the concept and principle of the present invention, and these improvements and modifications should also be regarded as the protection scope of the present invention. Sequence Listing <110> East China University of Science and Technology <120> Redox-sensitive protein and preparation method and application thereof <130> 204708 <160> 38 <170> SIPOSequenceListing 1.0 <210> 1 <211> 466 <212> PRT <213> Artificial Sequence <400> 1 Val Ser Glu Leu Ile Thr Glu Asn Met His Met Lys Leu Tyr Met Glu 1 5 10 15 Gly Thr Val Asn Asn His His Phe Lys Cys Thr Ser Glu Gly Glu Gly 20 25 30 Lys Pro Tyr Glu Gly Thr Gln Thr Met Arg Ile Lys Val Val Glu Gly 35 40 45 Gly Pro Leu Pro Phe Ala Phe Asp Ile Leu Ala Thr Ser Phe Met Tyr 50 55 60 Gly Ser Lys Thr Phe Ile Asn His Thr Gln Gly Ile Pro Asp Phe Phe 65 70 75 80 Lys Gln Ser Phe Pro Glu Gly Phe Thr Trp Glu Arg Val Thr Thr Tyr 85 90 95 Glu Asp Gly Gly Val Leu Thr Ala Thr Gln Asp Thr Ser Leu Gln Asp 100 105 110 Gly Cys Leu Ile Tyr Asn Val Lys Ile Arg Gly Val Asn Phe Pro Ser 115 120 125 Asn Gly Pro Val Met Gln Lys Lys Thr Leu Gly Trp Glu Ala Ser Thr 130 135 140 Glu Met Leu Tyr Pro Ala Asp Gly Gly Leu Glu Gly Arg Ser Asp Met 145 150 155 160 Ala Leu Lys Leu Val Gly Gly Gly His Leu Ile Cys Asn Leu Lys Thr 165 170 175 Thr Tyr Arg Ser Lys Lys Pro Ala Lys Asn Leu Lys Met Pro Gly Val 180 185 190 Tyr Tyr Val Asp Arg Arg Leu Glu Arg Ile Lys Glu Ala Asp Lys Glu 195 200 205 Thr Tyr Val Glu Gln His Glu Val Ala Val Ala Arg Tyr Cys Asp Leu 210 215 220 Pro Ser Lys Leu Gly His Lys Leu Asn Val Ser Glu Leu Ile Thr Glu 225 230 235 240 Asn Met His Met Lys Leu Tyr Met Glu Gly Thr Val Asn Asn His His 245 250 255 Phe Lys Cys Thr Ser Glu Gly Glu Gly Lys Pro Tyr Glu Gly Thr Gln 260 265 270 Thr Met Arg Ile Lys Val Val Glu Gly Gly Pro Leu Pro Phe Ala Phe 275 280 285 Asp Ile Leu Ala Thr Ser Phe Met Tyr Gly Ser Lys Thr Phe Ile Asn 290 295 300 His Thr Gln Gly Ile Pro Asp Phe Phe Lys Gln Ser Phe Pro Glu Gly 305 310 315 320 Phe Thr Trp Glu Arg Val Thr Thr Tyr Glu Asp Gly Gly Val Leu Thr 325 330 335 Ala Thr Gln Asp Thr Ser Leu Gln Asp Gly Cys Leu Ile Tyr Asn Val 340 345 350 Lys Ile Arg Gly Val Asn Phe Pro Ser Asn Gly Pro Val Met Gln Lys 355 360 365 Lys Thr Leu Gly Trp Glu Ala Ser Thr Glu Met Leu Tyr Pro Ala Asp 370 375 380 Gly Gly Leu Glu Gly Arg Ser Asp Met Ala Leu Lys Leu Val Gly Gly 385 390 395 400 Gly His Leu Ile Cys Asn Leu Lys Thr Thr Tyr Arg Ser Lys Lys Pro 405 410 415 Ala Lys Asn Leu Lys Met Pro Gly Val Tyr Tyr Val Asp Arg Arg Leu 420 425 430 Glu Arg Ile Lys Glu Ala Asp Lys Glu Thr Tyr Val Glu Gln His Glu 435 440 445 Val Ala Val Ala Arg Tyr Cys Asp Leu Pro Ser Lys Leu Gly His Lys 450 455 460 Leu Asn 465 <210> 2 <211> 239 <212> PRT <213> Artificial Sequence <400> 2 Leu Glu Gly Arg Ser Asp Met Ala Leu Lys Leu Val Gly Gly Gly His 1 5 10 15 Leu Ile Cys Asn Leu Lys Thr Thr Tyr Arg Ser Lys Lys Pro Ala Lys 20 25 30 Asn Leu Lys Met Pro Gly Val Tyr Tyr Val Asp Arg Arg Leu Glu Arg 35 40 45 Ile Lys Glu Ala Asp Lys Glu Thr Tyr Val Glu Gln His Glu Val Ala 50 55 60 Val Ala Arg Tyr Cys Asp Leu Pro Ser Lys Leu Gly His Lys Leu Asn 65 70 75 80 Gly Gly Thr Gly Gly Ser Val Ser Glu Leu Ile Thr Glu Asn Met His 85 90 95 Met Lys Leu Tyr Met Glu Gly Thr Val Asn Asn His His Phe Lys Cys 100 105 110 Thr Ser Glu Gly Glu Gly Lys Pro Tyr Glu Gly Thr Gln Thr Met Arg 115 120 125 Ile Lys Val Val Glu Gly Gly Pro Leu Pro Phe Ala Phe Asp Ile Leu 130 135 140 Ala Thr Ser Phe Met Tyr Gly Ser Lys Thr Phe Ile Asn His Thr Gln 145 150 155 160 Gly Ile Pro Asp Phe Phe Lys Gln Ser Phe Pro Glu Gly Phe Thr Trp 165 170 175 Glu Arg Val Thr Thr Tyr Glu Asp Gly Gly Val Leu Thr Ala Thr Gln 180 185 190 Asp Thr Ser Leu Gln Asp Gly Cys Leu Ile Tyr Asn Val Lys Ile Arg 195 200 205 Gly Val Asn Phe Pro Ser Asn Gly Pro Val Met Gln Lys Lys Thr Leu 210 215 220 Gly Trp Glu Ala Ser Thr Glu Met Leu Tyr Pro Ala Asp Gly Gly 225 230 235 <210> 3 <211> 233 <212> PRT <213> Artificial Sequence <400> 3 Val Ser Glu Leu Ile Thr Glu Asn Met His Met Lys Leu Tyr Met Glu 1 5 10 15 Gly Thr Val Asn Asn His His Phe Lys Cys Thr Ser Glu Gly Glu Gly 20 25 30 Lys Pro Tyr Glu Gly Thr Gln Thr Met Arg Ile Lys Val Val Glu Gly 35 40 45 Gly Pro Leu Pro Phe Ala Phe Asp Ile Leu Ala Thr Ser Phe Met Tyr 50 55 60 Gly Ser Lys Thr Phe Ile Asn His Thr Gln Gly Ile Pro Asp Phe Phe 65 70 75 80 Lys Gln Ser Phe Pro Glu Gly Phe Thr Trp Glu Arg Val Thr Thr Tyr 85 90 95 Glu Asp Gly Gly Val Leu Thr Ala Thr Gln Asp Thr Ser Leu Gln Asp 100 105 110 Gly Cys Leu Ile Tyr Asn Val Lys Ile Arg Gly Val Asn Phe Pro Ser 115 120 125 Asn Gly Pro Val Met Gln Lys Lys Thr Leu Gly Trp Glu Ala Ser Thr 130 135 140 Glu Met Leu Tyr Pro Ala Asp Gly Gly Leu Glu Gly Cys Ser Asp Met 145 150 155 160 Ala Leu Lys Leu Val Gly Gly Gly His Leu Ile Cys Asn Leu Lys Thr 165 170 175 Thr Tyr Arg Ser Lys Lys Pro Ala Lys Asn Leu Lys Met Pro Gly Val 180 185 190 Tyr Tyr Val Asp Arg Arg Leu Glu Arg Ile Lys Glu Ala Asp Lys Glu 195 200 205 Thr Tyr Val Glu Gln His Glu Val Ala Val Ala Arg Tyr Cys Asp Leu 210 215 220 Pro Ser Lys Leu Gly His Lys Leu Asn 225 230 <210> 4 <211> 233 <212> PRT <213> Artificial Sequence <400> 4 Val Ser Glu Leu Ile Thr Glu Asn Met His Met Lys Leu Tyr Met Glu 1 5 10 15 Gly Thr Val Asn Asn His His Phe Lys Cys Thr Ser Glu Gly Glu Gly 20 25 30 Lys Pro Tyr Glu Gly Thr Gln Thr Met Arg Ile Lys Val Val Glu Gly 35 40 45 Gly Pro Leu Pro Phe Ala Phe Asp Ile Leu Ala Thr Ser Phe Met Tyr 50 55 60 Gly Ser Lys Thr Phe Ile Asn His Thr Gln Gly Ile Pro Asp Phe Phe 65 70 75 80 Lys Gln Ser Phe Pro Glu Gly Phe Thr Trp Glu Arg Val Thr Thr Tyr 85 90 95 Glu Asp Gly Gly Val Leu Thr Ala Thr Gln Asp Thr Ser Leu Gln Asp 100 105 110 Gly Cys Leu Ile Tyr Asn Val Lys Ile Arg Gly Val Asn Phe Pro Ser 115 120 125 Asn Gly Pro Val Met Gln Lys Lys Thr Leu Gly Trp Glu Ala Ser Thr 130 135 140 Glu Met Leu Tyr Pro Ala Asp Gly Gly Leu Glu Gly Arg Ser Asp Met 145 150 155 160 Ala Leu Lys Leu Val Gly Gly Gly His Leu Ile Cys Asn Leu Lys Thr 165 170 175 Thr Tyr Arg Ser Lys Lys Pro Ala Lys Asn Leu Lys Met Pro Gly Cys 180 185 190 Tyr Tyr Val Asp Arg Arg Leu Glu Arg Ile Lys Glu Ala Asp Lys Glu 195 200 205 Thr Tyr Val Glu Gln His Glu Val Ala Val Ala Arg Tyr Cys Asp Leu 210 215 220 Pro Ser Lys Leu Gly His Lys Leu Asn 225 230 <210> 5 <211> 233 <212> PRT <213> Artificial Sequence <400> 5 Val Ser Glu Leu Ile Thr Glu Asn Met His Met Lys Leu Tyr Met Glu 1 5 10 15 Gly Thr Val Asn Asn His His Phe Lys Cys Thr Ser Glu Gly Glu Gly 20 25 30 Lys Pro Tyr Glu Gly Thr Gln Thr Met Arg Ile Lys Val Val Glu Gly 35 40 45 Gly Pro Leu Pro Phe Ala Phe Asp Ile Leu Ala Thr Ser Phe Met Tyr 50 55 60 Gly Ser Lys Thr Phe Ile Asn His Thr Gln Gly Ile Pro Asp Phe Phe 65 70 75 80 Lys Gln Ser Phe Pro Glu Gly Phe Thr Trp Glu Arg Val Thr Thr Tyr 85 90 95 Glu Asp Gly Gly Val Leu Thr Ala Thr Gln Asp Thr Ser Leu Gln Asp 100 105 110 Gly Cys Leu Ile Tyr Asn Val Lys Ile Arg Gly Val Asn Phe Pro Ser 115 120 125 Asn Gly Pro Val Met Gln Lys Lys Thr Leu Gly Trp Glu Ala Ser Thr 130 135 140 Glu Met Leu Tyr Pro Ala Asp Gly Gly Leu Glu Gly Arg Ser Asp Met 145 150 155 160 Ala Leu Lys Leu Val Gly Gly Gly His Leu Ile Cys Asn Leu Lys Thr 165 170 175 Thr Tyr Arg Ser Lys Lys Pro Ala Lys Asn Leu Lys Met Pro Gly Val 180 185 190 Tyr Tyr Val Asp Arg Arg Leu Glu Arg Cys Lys Glu Ala Asp Lys Glu 195 200 205 Thr Tyr Val Glu Gln His Glu Val Ala Val Ala Arg Tyr Cys Asp Leu 210 215 220 Pro Ser Lys Leu Gly His Lys Leu Asn 225 230 <210> 6 <211> 233 <212> PRT <213> Artificial Sequence <400> 6 Val Ser Glu Leu Ile Thr Glu Asn Met His Met Lys Leu Tyr Met Glu 1 5 10 15 Gly Thr Val Asn Asn His His Phe Lys Cys Thr Ser Glu Gly Glu Gly 20 25 30 Lys Pro Tyr Glu Gly Thr Gln Thr Met Arg Ile Lys Val Val Glu Gly 35 40 45 Gly Pro Leu Pro Phe Ala Phe Asp Ile Leu Ala Thr Ser Phe Met Tyr 50 55 60 Gly Ser Lys Thr Phe Ile Asn His Thr Gln Gly Ile Pro Asp Phe Phe 65 70 75 80 Lys Gln Ser Phe Pro Glu Gly Phe Thr Trp Glu Arg Val Thr Thr Tyr 85 90 95 Glu Asp Gly Gly Val Leu Thr Ala Thr Gln Asp Thr Ser Leu Gln Asp 100 105 110 Gly Cys Leu Ile Tyr Asn Val Lys Ile Arg Gly Val Asn Phe Pro Ser 115 120 125 Asn Gly Pro Val Met Gln Lys Lys Thr Leu Gly Trp Glu Ala Ser Thr 130 135 140 Glu Met Leu Tyr Pro Ala Asp Gly Gly Leu Glu Gly Arg Ser Asp Met 145 150 155 160 Ala Leu Lys Leu Val Gly Gly Gly His Leu Ile Cys Asn Leu Lys Thr 165 170 175 Thr Tyr Arg Ser Lys Lys Pro Ala Lys Asn Leu Lys Met Pro Gly Val 180 185 190 Tyr Tyr Val Asp Arg Arg Leu Glu Arg Ile Lys Glu Ala Asp Lys Glu 195 200 205 Thr Tyr Val Glu Gln His Glu Val Ala Val Ala Cys Tyr Cys Asp Leu 210 215 220 Pro Ser Lys Leu Gly His Lys Leu Asn 225 230 <210> 7 <211> 239 <212> PRT <213> Artificial Sequence <400> 7 Leu Glu Gly Cys Ser Asp Met Ala Leu Lys Leu Val Gly Gly Gly His 1 5 10 15 Leu Ile Cys Asn Leu Lys Thr Thr Tyr Arg Ser Lys Lys Pro Ala Lys 20 25 30 Asn Leu Lys Met Pro Gly Val Tyr Tyr Val Asp Arg Arg Leu Glu Arg 35 40 45 Ile Lys Glu Ala Asp Lys Glu Thr Tyr Val Glu Gln His Glu Val Ala 50 55 60 Val Ala Arg Tyr Cys Asp Leu Pro Ser Lys Leu Gly His Lys Leu Asn 65 70 75 80 Gly Gly Thr Gly Gly Ser Val Ser Glu Leu Ile Thr Glu Asn Met His 85 90 95 Met Lys Leu Tyr Met Glu Gly Thr Val Asn Asn His His Phe Lys Cys 100 105 110 Thr Ser Glu Gly Glu Gly Lys Pro Tyr Glu Gly Thr Gln Thr Met Arg 115 120 125 Ile Lys Val Val Glu Gly Gly Pro Leu Pro Phe Ala Phe Asp Ile Leu 130 135 140 Ala Thr Ser Phe Met Tyr Gly Ser Lys Thr Phe Ile Asn His Thr Gln 145 150 155 160 Gly Ile Pro Asp Phe Phe Lys Gln Ser Phe Pro Glu Gly Phe Thr Trp 165 170 175 Glu Arg Val Thr Thr Tyr Glu Asp Gly Gly Val Leu Thr Ala Thr Gln 180 185 190 Asp Thr Ser Leu Gln Asp Gly Cys Leu Ile Tyr Asn Val Lys Ile Arg 195 200 205 Gly Val Asn Phe Pro Ser Asn Gly Pro Val Met Gln Lys Lys Thr Leu 210 215 220 Gly Trp Glu Ala Ser Thr Glu Met Leu Tyr Pro Ala Asp Gly Gly 225 230 235 <210> 8 <211> 239 <212> PRT <213> Artificial Sequence <400> 8 Leu Glu Gly Arg Ser Asp Met Ala Leu Lys Leu Val Gly Gly Gly His 1 5 10 15 Leu Ile Cys Asn Leu Lys Thr Thr Tyr Arg Ser Lys Lys Pro Ala Lys 20 25 30 Asn Leu Lys Met Pro Gly Cys Tyr Tyr Val Asp Arg Arg Leu Glu Arg 35 40 45 Ile Lys Glu Ala Asp Lys Glu Thr Tyr Val Glu Gln His Glu Val Ala 50 55 60 Val Ala Arg Tyr Cys Asp Leu Pro Ser Lys Leu Gly His Lys Leu Asn 65 70 75 80 Gly Gly Thr Gly Gly Ser Val Ser Glu Leu Ile Thr Glu Asn Met His 85 90 95 Met Lys Leu Tyr Met Glu Gly Thr Val Asn Asn His His Phe Lys Cys 100 105 110 Thr Ser Glu Gly Glu Gly Lys Pro Tyr Glu Gly Thr Gln Thr Met Arg 115 120 125 Ile Lys Val Val Glu Gly Gly Pro Leu Pro Phe Ala Phe Asp Ile Leu 130 135 140 Ala Thr Ser Phe Met Tyr Gly Ser Lys Thr Phe Ile Asn His Thr Gln 145 150 155 160 Gly Ile Pro Asp Phe Phe Lys Gln Ser Phe Pro Glu Gly Phe Thr Trp 165 170 175 Glu Arg Val Thr Thr Tyr Glu Asp Gly Gly Val Leu Thr Ala Thr Gln 180 185 190 Asp Thr Ser Leu Gln Asp Gly Cys Leu Ile Tyr Asn Val Lys Ile Arg 195 200 205 Gly Val Asn Phe Pro Ser Asn Gly Pro Val Met Gln Lys Lys Thr Leu 210 215 220 Gly Trp Glu Ala Ser Thr Glu Met Leu Tyr Pro Ala Asp Gly Gly 225 230 235 <210> 9 <211> 239 <212> PRT <213> Artificial Sequence <400> 9 Leu Glu Gly Arg Ser Asp Met Ala Leu Lys Leu Val Gly Gly Gly His 1 5 10 15 Leu Ile Cys Asn Leu Lys Thr Thr Tyr Arg Ser Lys Lys Pro Ala Lys 20 25 30 Asn Leu Lys Met Pro Gly Val Tyr Tyr Val Asp Arg Arg Leu Glu Arg 35 40 45 Cys Lys Glu Ala Asp Lys Glu Thr Tyr Val Glu Gln His Glu Val Ala 50 55 60 Val Ala Arg Tyr Cys Asp Leu Pro Ser Lys Leu Gly His Lys Leu Asn 65 70 75 80 Gly Gly Thr Gly Gly Ser Val Ser Glu Leu Ile Thr Glu Asn Met His 85 90 95 Met Lys Leu Tyr Met Glu Gly Thr Val Asn Asn His His Phe Lys Cys 100 105 110 Thr Ser Glu Gly Glu Gly Lys Pro Tyr Glu Gly Thr Gln Thr Met Arg 115 120 125 Ile Lys Val Val Glu Gly Gly Pro Leu Pro Phe Ala Phe Asp Ile Leu 130 135 140 Ala Thr Ser Phe Met Tyr Gly Ser Lys Thr Phe Ile Asn His Thr Gln 145 150 155 160 Gly Ile Pro Asp Phe Phe Lys Gln Ser Phe Pro Glu Gly Phe Thr Trp 165 170 175 Glu Arg Val Thr Thr Tyr Glu Asp Gly Gly Val Leu Thr Ala Thr Gln 180 185 190 Asp Thr Ser Leu Gln Asp Gly Cys Leu Ile Tyr Asn Val Lys Ile Arg 195 200 205 Gly Val Asn Phe Pro Ser Asn Gly Pro Val Met Gln Lys Lys Thr Leu 210 215 220 Gly Trp Glu Ala Ser Thr Glu Met Leu Tyr Pro Ala Asp Gly Gly 225 230 235 <210> 10 <211> 239 <212> PRT <213> Artificial Sequence <400> 10 Leu Glu Gly Arg Ser Asp Met Ala Leu Lys Leu Val Gly Gly Gly His 1 5 10 15 Leu Ile Cys Asn Leu Lys Thr Thr Tyr Arg Ser Lys Lys Pro Ala Lys 20 25 30 Asn Leu Lys Met Pro Gly Val Tyr Tyr Val Asp Arg Arg Leu Glu Arg 35 40 45 Ile Lys Glu Ala Asp Lys Glu Thr Tyr Val Glu Gln His Glu Val Ala 50 55 60 Val Ala Cys Tyr Cys Asp Leu Pro Ser Lys Leu Gly His Lys Leu Asn 65 70 75 80 Gly Gly Thr Gly Gly Ser Val Ser Glu Leu Ile Thr Glu Asn Met His 85 90 95 Met Lys Leu Tyr Met Glu Gly Thr Val Asn Asn His His Phe Lys Cys 100 105 110 Thr Ser Glu Gly Glu Gly Lys Pro Tyr Glu Gly Thr Gln Thr Met Arg 115 120 125 Ile Lys Val Val Glu Gly Gly Pro Leu Pro Phe Ala Phe Asp Ile Leu 130 135 140 Ala Thr Ser Phe Met Tyr Gly Ser Lys Thr Phe Ile Asn His Thr Gln 145 150 155 160 Gly Ile Pro Asp Phe Phe Lys Gln Ser Phe Pro Glu Gly Phe Thr Trp 165 170 175 Glu Arg Val Thr Thr Tyr Glu Asp Gly Gly Val Leu Thr Ala Thr Gln 180 185 190 Asp Thr Ser Leu Gln Asp Gly Cys Leu Ile Tyr Asn Val Lys Ile Arg 195 200 205 Gly Val Asn Phe Pro Ser Asn Gly Pro Val Met Gln Lys Lys Thr Leu 210 215 220 Gly Trp Glu Ala Ser Thr Glu Met Leu Tyr Pro Ala Asp Gly Gly 225 230 235 <210> 11 <211> 36 <212> DNA <213> Artificial Sequence <400> 11 ccgggatcca tggtgagcga gctgatcacc gagaac 36 <210> 12 <211> 37 <212> DNA <213> Artificial Sequence <400> 12 gccgcggccg cttaattaag cttgtgcccc agtttgc 37 <210> 13 <211> 24 <212> DNA <213> Artificial Sequence <400> 13 ccggcggccg cactcgagca ccac 24 <210> 14 <211> 22 <212> DNA <213> Artificial Sequence <400> 14 catggatccg cgacccattt gc 22 <210> 15 <211> 41 <212> DNA <213> Artificial Sequence <400> 15 ggagccacca gtgccaccat taagcttgtg ccccagtttg c 41 <210> 16 <211> 37 <212> DNA <213> Artificial Sequence <400> 16 ggtggcactg gtggctccgt gagcgagctg atcaccg 37 <210> 17 <211> 33 <212> DNA <213> Artificial Sequence <400> 17 ccgggatcca tgcacctgat ctgcaacttg aag 33 <210> 18 <211> 33 <212> DNA <213> Artificial Sequence <400> 18 cgggcggccg cttagccccc gcccacgagc ttc 33 <210> 19 <211> 32 <212> DNA <213> Artificial Sequence <400> 19 ccgggatcca tggacggcgg cctggaaggc ag 32 <210> 20 <211> 33 <212> DNA <213> Artificial Sequence <400> 20 gccgcggccg cttaagcggg gtacagcatc tcg 33 <210> 21 <211> 30 <212> DNA <213> Artificial Sequence <400> 21 ccgggatcca tgctggaagg cagaagcgac 30 <210> 22 <211> 33 <212> DNA <213> Artificial Sequence <400> 22 cgggcggccg cttagccgcc gtcagcgggg tac 33 <210> 23 <211> 22 <212> DNA <213> Artificial Sequence <400> 23 cagcgacatg gccctgaagc tc 22 <210> 24 <211> 20 <212> DNA <213> Artificial Sequence <400> 24 cagccttcca ggccgccgtc 20 <210> 25 <211> 27 <212> DNA <213> Artificial Sequence <400> 25 ctactatgtg gacagaagac tggaaag 27 <210> 26 <211> 21 <212> DNA <213> Artificial Sequence <400> 26 cagccgggca tcttgaggtt c 21 <210> 27 <211> 22 <212> DNA <213> Artificial Sequence <400> 27 caaggaggcc gacaaagaga cc 22 <210> 28 <211> 30 <212> DNA <213> Artificial Sequence <400> 28 catctttcca gtcttctgtc cacatagtag 30 <210> 29 <211> 26 <212> DNA <213> Artificial Sequence <400> 29 ctactgcgac ctccctagca aactgg 26 <210> 30 <211> 20 <212> DNA <213> Artificial Sequence <400> 30 caggccacag ccacctcgtg 20 <210> 31 <211> 19 <212> DNA <213> Artificial Sequence <400> 31 cacatccgag ggcgaaggc 19 <210> 32 <211> 25 <212> DNA <213> Artificial Sequence <400> 32 gccttgaagt ggtggttgtt cacgg 25 <210> 33 <211> 27 <212> DNA <213> Artificial Sequence <400> 33 ctcatctaca acgtcaagat cagaggg 27 <210> 34 <211> 20 <212> DNA <213> Artificial Sequence <400> 34 catgccgtcc tggaggctgg 20 <210> 35 <211> 30 <212> DNA <213> Artificial Sequence <400> 35 gtgaacttga agaccacata cagatccaag 30 <210> 36 <211> 18 <212> DNA <213> Artificial Sequence <400> 36 gatcaggtgg cccccgcc 18 <210> 37 <211> 22 <212> DNA <213> Artificial Sequence <400> 37 gacctcccta gcaaactggg gc 22 <210> 38 <211> 24 <212> DNA <213> Artificial Sequence <400> 38 gctgtatctg gccacagcca cctc 24

Claims

1. A redox-sensitive polypeptide having an amino acid sequence (1) a sequence as shown in SEQ ID NO: 1 and any one of the following amino acid residues is mutated to cysteine: amino acid residues 138, 139, 141, 142, 148, 157, 159, 162, 192, 194, 196, 198, 202, 216, 220, or (2) A sequence as shown in SEQ ID NO: 1 and any one of the following amino acid residues is mutated to cysteine: amino acid residues 139, 141, 142, 148, 157, 159, 162, 192, 196, 198, 202, 216, 220, and the sequence is connected from beginning to end by a connecting peptide and disconnected between positions 153 and 154.

2. The polypeptide according to claim 1, characterized in that The connecting peptide is selected from G, GS, GGS, GGSG, GGTG, GGTGG, GGSGG, GGSGGT, and GGTGGS.

3. The polypeptide according to claim 1, characterized in that The polypeptide has a sequence as shown in SEQ ID NO: 2, wherein the amino acid residue corresponding to the one amino acid residue in SEQ ID NO: 1 in (2) is mutated to cysteine.

4. The polypeptide according to claim 1 or 2, characterized in that The amino acid sequence of the polypeptide is shown in any one of SEQ ID NOs: 3-6, or The amino acid sequence of the polypeptide is shown in any one of SEQ ID NOs: 7-10.

5. A nucleic acid molecule consisting of any of the following: (1) a coding sequence of the polypeptide according to any one of claims 1 to 4, (2) The complementary sequence of (1). A nucleic acid construct comprising the nucleic acid molecule according to claim 5 .

7. The nucleic acid construct according to claim 6, wherein The nucleic acid construct is a cloning vector, an expression vector or a recombinant vector.

8. A host cell, wherein the host cell (1) expressing the polypeptide according to any one of claims 1 to 4; (2) comprising the nucleic acid molecule of claim 5; or (3) Comprising the nucleic acid construct according to claim 6 or 7.

9. A detection kit comprising one or more selected from the following: (1) The polypeptide according to any one of claims 1 to 4; (2) The nucleic acid molecule according to claim 5; (3) The nucleic acid construct according to claim 6 or 7; (4) the host cell according to claim 8; and Other reagents required for detection of redox state.

10. Use of the polypeptide according to any one of claims 1 to 4, the nucleic acid molecule according to claim 5, the nucleic acid construct according to claim 6 or 7, or the host cell according to claim 8 in detecting intracellular or extracellular redox states, detecting substances of interest, detecting substance interactions, screening substances that can cause redox state changes, screening substances that can stabilize or aggravate redox state changes, and screening substances that interact with ligands.

11. A method for detecting intracellular or extracellular redox state, comprising: Allowing the cell to express or secrete the polypeptide according to any one of claims 1 to 4 or incubating the host cell according to claim 8; optionally adding a substance of interest; and detecting a change in an optical property of the polypeptide, the change in the polypeptide indicating an increase, unchanged or decrease in the redox state.

12. A method for screening a substance that can cause a change in redox state, comprising: The polypeptide according to any one of claims 1 to 4 or the host cell according to claim 8 is contacted with a candidate substance, a change in the optical property of the polypeptide is detected, and a substance is screened according to the change in the optical property, wherein the change in the optical property indicates that the substance can cause an increase or decrease in the redox state.

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