CN106632689B - Polypeptide probe, kit containing same and application thereof - Google Patents

Polypeptide probe, kit containing same and application thereof Download PDF

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CN106632689B
CN106632689B CN201611209386.2A CN201611209386A CN106632689B CN 106632689 B CN106632689 B CN 106632689B CN 201611209386 A CN201611209386 A CN 201611209386A CN 106632689 B CN106632689 B CN 106632689B
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probe
polypeptide
polypeptide probe
cells
diagnostic kit
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CN106632689A (en
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杨用
梁岩
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Shenzhen Institute of Advanced Technology of CAS
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    • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/06—Dipeptides
    • C07K5/06008—Dipeptides with the first amino acid being neutral
    • C07K5/06017—Dipeptides with the first amino acid being neutral and aliphatic
    • C07K5/0606—Dipeptides with the first amino acid being neutral and aliphatic the side chain containing heteroatoms not provided for by C07K5/06086 - C07K5/06139, e.g. Ser, Met, Cys, Thr
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    • C07—ORGANIC CHEMISTRY
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    • C07K2319/00—Fusion polypeptide
    • C07K2319/70—Fusion polypeptide containing domain for protein-protein interaction
    • C07K2319/73—Fusion polypeptide containing domain for protein-protein interaction containing coiled-coiled motif (leucine zippers)
    • G—PHYSICS
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Abstract

The invention relates to the field of biological medicine, in particular to a polypeptide probe, a kit containing the probe and application of the polypeptide probe. The polypeptide probe is selected from the following substances and the composition of dimer thereof: x1‑Y1-CC; wherein, X1Is a polypeptide with dimerization capacity; y is1Selected from the sequence of the enzyme cleavage site of the enzyme to be detected. The probe makes full use of the characteristic that the stability and the quantum yield of the double arsenic dye-four cysteine label compound are extremely dependent on the space conformation of the four cysteine label, and further develops a simple and sensitive method for measuring the enzyme activity.

Description

Polypeptide probe, kit containing same and application thereof
Technical Field
The invention relates to the field of biological medicine, in particular to a polypeptide probe, a kit containing the probe and application of the polypeptide probe.
Background
Apoptosis is a cell active death process controlled by genes, has positive and important significance on the development of multicellular organisms, the renewal of normal cells and the maintenance of normal structures and functions of tissues, and abnormal apoptosis can cause diseases such as tumors, neurodegenerative diseases and the like. Apoptosis is performed by a cascade of cysteine proteases (Cold Spring Harb. Perspectrum. biol.,2013,5(4): a008656), among which cysteine protease family, cysteine protease-3 belongs to effector apoptotic enzymes, and activated cysteine protease-3 degrades a series of substrates including apoptosis inhibitors, extracellular matrix and scaffold proteins, and DNA repair-related factors, making biochemical and morphological changes of cells, leading to apoptosis (Oncogene,2008,27(48): 6194-. Cysteine protease-3 is a key protease in the execution of apoptosis and therefore the apoptotic process can be determined by measuring the activity of cysteine protease-3.
The double arsenic dye-four cysteine label system is a fluorescent labeling method for living cell protein with wide application prospect (Science,1998,281(5374): 269-272). In this system, the four cysteine tag is a hexapeptide or dodecapeptide (nat. biotechnol.,2005,23(10): 1308) comprising four cysteine residues, which are bound together in pairs to form cysteine-cysteine pairs separated by proline-glycine pairs. The tetracysteine tag has high specificity and affinity to the diarsenic dye. The bis-arsenic dye does not fluoresce when chelated by 1, 2-ethanedithiol, but fluoresces strongly upon binding to the tetracysteine tag. In addition to being present in a linear fashion, the two cysteine-cysteine doublets in the four-cysteine tag can also be separated, but they must be spatially adjacent (nat. chem. biol.,2007,3(12): 779-. In this labeling system, the spatial conformation of the tetracyste tag has a great influence on the stability and quantum yield of the formed bis-arsenic dye-tetracyste tag complex (J.Am.chem.Soc.,2002,124(21): 6063-.
Cysteine protease-3 is usually present as an inactive proenzyme in the cell and needs to be cleaved by an upstream activating apoptotic enzyme to be activated. Based on the property that activated caspase-3 specifically cleaves the caspase tetrapeptide sequence, a series of probes based on caspase-Val-Asp have been developed for measuring the activity of caspase-3, and the methods include fluorescence detection, electrochemical method, colorimetric method, surface plasmon resonance, bioluminescence and electrochemiluminescence. With the development of nanotechnology, some nanomaterial-based polypeptide probes such as quantum dot composite polypeptide probes, nanogold composite polypeptide probes, and graphene composite polypeptide probes were also developed to measure the activity of cysteine proteinase-3 (chem. rev.,2015,115(22): 12546-12629). In addition, several protein probes based on fluorescence resonance energy transfer reactions and fluorescence switches (nat. Commun., 2013; 4:2157) were also developed to measure the activity of cystatin-3 in cells. In these methods, the fluorescent label may reduce the cleavage efficiency of cysteine protease-3 to the substrate to some extent, and the preparation of the nanosensor makes the detection process complicated and inefficient. There is therefore an urgent need to develop simple, sensitive and efficient methods for determining cystatin-3 activity in cells to detect apoptotic processes. In addition, the above problems are more or less present in the art when detecting the activities of other enzymes, and a technique that is highly effective for detecting the activities of various enzymes is lacking.
In view of the above, the present invention is particularly proposed.
Disclosure of Invention
In the invention, a novel label-free polypeptide molecular probe is designed by fully utilizing the characteristic that the stability and the quantum yield of the double-arsenic dye-four-cysteine label compound are extremely dependent on the space conformation of the four-cysteine label, and a simple and sensitive method for measuring the enzyme activity is further developed.
Before the present compounds, compositions, proteins, peptides, etc., and methods are described, it is to be understood that these embodiments are not limited to the particular methodology, protocols, and reagents described, as these may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present embodiments or the claims.
In order to achieve the above purpose of the present invention, the following technical solutions are adopted:
a polypeptide probe selected from the group consisting of a combination of:
X1-Y1-CC；
wherein, X1Is a polypeptide with dimerization capacity;
Y1selected from the sequence of the enzyme cleavage site of the enzyme to be detected.
Wherein, X1The dimer formed by the fragmentation can form spontaneously, or can form under other helper proteins or helper conditions (e.g., X)1Some amino acid residues of the peptide segment are modified or the peptide segment is in a hydrophobic environment), preferably the dimer is formed spontaneously.
The probe contains at least three different functional regions, CC, two cysteines, which are binding sites for the diarsenic dye, for reading the activity of the enzyme to be detected. (spontaneous) dimerization between the polypeptide probes when not cleaved by the enzyme to be detected brings the two cysteine-cysteine bigements into close spatial proximity to each other, and thus can bind to the diarsenic dye to emit strong fluorescence; in contrast, enzymatic cleavage of the enzyme to be detected dissociates the cysteine-cysteine bi-mer from the polypeptide, and although the cleaved polypeptide still dimerizes, it lacks a site for anchoring a diarsenic dye, which is therefore unable to emit light. Therefore, the activity of the enzyme to be detected can be quantitatively determined by measuring the change in fluorescence before and after cleavage.
By altering Y1The sequence of the polypeptide fragment, theoretically, the probe provided by the invention can detect the activity of all enzymes with the capability of cutting the peptide fragment.
Preferably, the polypeptide probe as described above, said X1Is selected from D configuration amino acid.
Since amino acids are naturally selected to be in the L-form during the biological evolution process, enzymes in organisms cannot recognize and cleave D-form amino acids. This design prevents X1Non-specific cleavage of the segment by other proteases may also be effective in extending the half-life of the polypeptide probes provided by the present invention.
X1The amino acids in D configuration and L configuration in the segment can be arranged in a crossed way, for example, a D configuration and an L configuration are alternatively connected, and the functions can also be realized; most preferably, X1The amino acid sequences of (a) are all selected from the D configuration amino acids.
Preferably, the polypeptide probe as described above, said X1Is a leucine zipper, or a polypeptide comprising a leucine zipper structure.
A leucine zipper comprises multiple leucine residues, usually occurring once every seven amino acid residues, forming an amphipathic α -helix, with a hydrophobic region on only one side, which provides a dimerizing domain, allowing the motif to pull like a "zipper".
In addition, according to the teachings of the present invention, X1The fragment polypeptides can be designed based on the protein segments responsible for dimerization among protein dimers commonly found in organisms, such as receptor tyrosine kinases, certain nuclear receptor proteins, 14-3-3 proteins, kinesins, Factor XI, Factor XIII, Toll-like receptors, fibrinogen, β gamma subunit dimers of G protein, and the like, which are easy for those skilled in the art.
Preferably, X1The segment polypeptide can be selected from the following amino acid sequences:
QLEDKVEELLSKNYHLENEVARLKKLVG；
KLEALEGKLEAEGKGKLEAUGICLEALE；
GEIAALKQEIAALKKENAALKWEIAALKQGYY；
ASIARLEEKVKTLKAQNYELASTANMLREQVAQLGAP；
ASAAELEERVKTLKAEIYELQSEANMLREQIAQLGAP；
ASAAELEERVKTLKAEIYELQSEANMLREQGAP；
ASAAELEERVKTLKAEIYELQSEGAP；
ESKVSSLESKVSSLESKVSSLESKVSSLESKVSSLESKVSS。
more preferably, the amino acid sequences are all amino acids in D configuration.
Preferably, the polypeptide probe is an N-terminal acetylated polypeptide probe;
and/or;
the C end of the polypeptide probe is subjected to amidation modification.
Modification of both ends of the polypeptide probe can play a role in increasing the stability of the probe.
Preferably, the polypeptide probe as described above, said X1And said Y1Also comprises a Linker1 and/or the Y1The connector 2 is also arranged between the CC and the shell;
preferably, the number of the amino acids of the Linker1 is 1-30, and more preferably 1-5;
preferably, the number of the amino acids of the Linker2 is 1-30, and more preferably 1-5.
The number of amino acids of Linker1 and Linker2 can be 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30.
Linker1 and Linker2 can also be selected from (GGGGS) n, (GGGS) n, (GGS) n, or Gn, where n can be 1,2, 3, 4, 5, or 6.
Preferably, the polypeptide probe as described above, said Y1Is DEVD.
DEVD is the cleavage site for cysteine protease-3 (Caspase3) and cysteine protease-7 (Caspase 7).
Preferably, the polypeptide probe as described above, said X1The amino acid sequence of (a) is asaaealervkttlkaeiyelrskanmlreqiaqlgap in D configuration.
This sequence is a leucine zipper structure in the D configuration.
Preferably, the amino acid sequence of the Linker2 is G, as in the polypeptide probe described above.
A polypeptide probe for detecting the enzymatic activity of Caspase3 or Caspase7, the sequence of which is asaaelervkttlkaeiyelrkasmlreqiaqlgapDADGCC; wherein the asaaelervkktlkaeiyelrskanmlreqiqlgap is an amino acid with D configuration.
Preferably, the N-terminal and C-terminal of asaaelervktlktlkaeiyelrkaslmrelqiaqlgapDADGCC are acetylated and amidated, respectively.
A diagnostic kit comprises the polypeptide probe, the diarsenic dye, a buffer solution 1 used when a to-be-detected enzyme cuts the polypeptide probe to obtain an enzyme cutting product, and a buffer solution 2 used when the diarsenic dye marks the enzyme cutting product;
preferably, the buffer 1 contains a reducing agent for avoiding disulfide bonds, and more preferably, the reducing agent is selected from dithiothreitol and/or tris (2-carbonylethyl) phosphate;
preferably, the buffer 2 contains an eluent for avoiding the binding of the single CC fragment to the diarsenic dye, and more preferably, the eluent is selected from 2, 3-dimercapto-1-propanol and/or 1, 2-ethanedithiol.
Preferably, the double arsenic dye is selected from the group consisting of ReAsH-EDT2、FlAsH-EDT2One or more of F2FlAsH or F4 FlAsH.
Preferably, the diagnostic kit as described above, wherein when the reducing agent is selected from dithiothreitol, the concentration of the reducing agent is 0.5 to 2 mM.
Preferably, the diagnostic kit as described above, wherein when the eluent is selected from 2, 3-dimercapto-1-propanol, the concentration of the eluent is 0.03 to 0.10 mM.
Detection of Y with the polypeptide probe or the diagnostic kit1The activity of the corresponding enzyme;
preferably, the detection is performed in a cell lysate or in a somatic cell;
more preferably, the cell is any one selected from the group consisting of HeLa cell, NIH/3T3 cell, C6 cell, MCF7 cell, human HCE cell, Jurkat T cell, Huh-7 cell, DU145 cell, U-937 cell and HepG2 cell.
Use of a polypeptide probe as described above or a diagnostic kit as described above for detecting the activity of a specific enzyme in a single cell.
Use of a polypeptide probe as described above or a diagnostic kit as described above for detecting cystatin-3 and/or cystatin-7 activity.
Use of a polypeptide probe as described above or a diagnostic kit as described above for detecting apoptosis.
Preferably, the apoptosis can be naturally occurring programmed cell death, disease-induced apoptosis or environmental-induced apoptosis, such as measurement of apoptosis induced by ultraviolet radiation, hydrogen peroxide, dexamethasone, tumor necrosis factor, and the like.
Use of a polypeptide probe as described above or a diagnostic kit as described above for the preparation and/or evaluation of a medicament for the treatment of an apoptosis-related disease;
preferably, for use as described above, the apoptosis-related disease is a tumor, an autoimmune disease or a neurodegenerative disease;
more preferably, for use as described above, the tumor comprises: leukemia, lymphoma, head and neck squamous cell carcinoma, lung cancer, esophageal cancer, liver cancer, colorectal cancer, breast cancer, ovarian cancer, cervical cancer, renal cell carcinoma, and melanoma;
more preferably, the autoimmune disease comprises: systemic lupus erythematosus, autoimmune lymphoproliferative syndrome, rheumatoid arthritis, thyroiditis;
more preferably, the neurodegenerative disease includes: alzheimer's disease, Huntington's disease, Parkinson's disease, stroke, and amyotrophic lateral sclerosis.
A method of diagnosing an apoptosis-related disease, comprising:
and contacting the cell lysate or tissue lysate of the main body to be diagnosed with the polypeptide probe or the components in the diagnosis kit, and diagnosing the main body according to the signal value obtained by the reaction.
Preferably, the method for diagnosing apoptosis-related diseases as described above, wherein the signal value is a fluorescence signal value generated by the reaction of the polypeptide probe with a diarsenic dye.
Preferably, the subject is an animal, more preferably a mammal, more preferably a primate, more preferably a human, in the method for diagnosing a disease associated with apoptosis as described above.
Preferably, the method for diagnosing apoptosis-related diseases as described above, wherein the apoptosis-related diseases are tumors, autoimmune diseases or neurodegenerative diseases;
more preferably, the tumor comprises: leukemia, lymphoma, head and neck squamous cell carcinoma, lung cancer, esophageal cancer, liver cancer, colorectal cancer, breast cancer, ovarian cancer, cervical cancer, renal cell carcinoma, and melanoma;
more preferably, the autoimmune disease comprises: systemic lupus erythematosus, autoimmune lymphoproliferative syndrome, rheumatoid arthritis, thyroiditis;
more preferably, the neurodegenerative disease comprises: alzheimer's disease, Huntington's disease, Parkinson's disease, stroke, and amyotrophic lateral sclerosis.
Compared with the prior art, the invention has the beneficial effects that:
1) the polypeptide molecular probe has no any mark, has better biocompatibility with the enzyme to be detected, and can improve the cutting efficiency;
2) in the whole detection reaction process of the kit provided by the invention, separation and cleaning steps are not required, and the operation is simple and convenient;
3) the detection mode has flexibility, and the probe can be marked by double arsenic dyes with different excitation and emission wavelengths, so that the apoptosis level can be measured in various ways;
4) the polypeptide probes specifically provided by the invention for determining Caspase3 and Caspase7 are composed of amino acids, and the apoptosis condition of a single cell can be determined by transferring DNA (deoxyribonucleic acid) encoding the polypeptide probes into the cell.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, and it is obvious that the drawings in the following description are some embodiments of the present invention, and other drawings can be obtained by those skilled in the art without creative efforts.
FIG. 1 is a graph showing the performance measurement of a novel unlabeled polypeptide molecular probe; (A) polypeptide probe and double-arsenic dye FlAsH-EDT2Binding the emission spectra of the formed complexes; (B) polypeptide probe and double-arsenic dye FlAsH-EDT2(ii) binding kinetics assay of (a);
FIG. 2 is a diagram showing the quantitative determination of in vitro recombinant cysteine protease-3 using a novel probe; (A) the cleavage of the polypeptide probe by cysteine protease-3 can reduce the fluorescence intensity of the polypeptide probe-double arsenic dye complex; the upper curve represents Jun-CCC, and the lower curve represents Jun-CCC + cystatin-3; (B) quantitative analysis of panel a; (C) specific detection of cysteine proteinase-3 activity by the polypeptide probe; (D) graph of the relationship between the decrease in fluorescence intensity (Δ F) resulting from cystatin-3 cleavage and cystatin-3 concentration;
FIG. 3 is a graph showing the measurement of staurosporine-induced apoptosis of RAW264.7 cells using a novel probe; (A) performing immunoblot analysis on RAW264.7 cell apoptosis induced by staurosporine; (B) detecting the apoptosis of RAW264.7 cells induced by staurosporine by using a polypeptide probe; (C) immunoblot analysis of Ac-DEVD-CHO inhibition of caspase-3 activity in cells; (D) fluorometric assay of Ac-DEVD-CHO inhibition of caspase-3 activity in apoptotic cells.
Detailed Description
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the disclosed embodiments belong. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present embodiments, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting. Other features and advantages of the embodiments will be apparent from the following detailed description and claims.
For the purpose of promoting an understanding of the embodiments described herein, reference will now be made to certain embodiments and specific language will be used to describe the same. The terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of the present disclosure.
Embodiments of the present invention will be described in detail below with reference to examples, but it will be understood by those skilled in the art that the following examples are only illustrative of the present invention and should not be construed as limiting the scope of the present invention. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products available commercially.
In the examples of the present invention, the effect of the probe of the present invention will be described by taking a probe for detecting Caspase3 as an example. The technical scheme of the embodiment comprises four aspects of designing a novel unmarked polypeptide molecular probe, measuring the performance of the novel probe, quantitatively measuring in vitro recombinant cysteine proteinase-3 by the novel probe and measuring the apoptotic cells by the novel probe.
Examples
1. Design of novel label-free polypeptide molecular probe
The designed polypeptide molecular probe is named as Jun-CCC, the sequence of the polypeptide molecular probe is asaaeelervkttlkaeiyelrskanmlreqiqlgapDVDGCC, and the N end and the C end of the probe are respectively acetylated and amidated to improve the stability of the probe. The probe comprises three different functional regions, wherein the asaaelervkktlkaeiyelrskmlreqiaqlgap is a dimerization functional region which is completely composed of D-type amino acids in order to prevent the polypeptide probe from being subjected to non-specific cleavage by other proteases; DEVD is a specific cleavage sequence for cysteine protease-3 and is composed of L-type amino acids; the amino acid G behind the amino acid G is linker, so as to increase the flexibility of the reaction of CC and the double arsenic dye; CC is a site that binds to a bisarsenic dye and is used to read the activity of cystatin-3. When the polypeptide probe is not cut by cysteine protease-3, the self-dimerization between the polypeptide probes can make two cysteine-cysteine bigemins close to each other in space, so that the two cysteine-cysteine bigemins can be combined with a double arsenic dye to emit stronger fluorescence; in contrast, cleavage by cysteine proteinase-3 dissociates the cysteine-cysteine bi-mer from the polypeptide, and although the cleaved polypeptide still dimerizes, it lacks a site for anchoring a bisarsenic dye, which is therefore unable to emit light. Therefore, by measuring the change in fluorescence before and after cleavage, the activity of cystatin-3 can be quantitatively measured, and the apoptosis process can be measured.
2. Performance measurement of novel probes
To show that the binding of the diarsenic dye to the probe depends on dimerization between the probes, we designed two probes Jun without dimerization ability simultaneouslyPCCC and CCC, where JunPThe sequence of CCC is asaaepeervktpkkaeiyeprskanmpreqiaqpgapdevdgcc, and the sequence of CCC is GCC, JunPCCC is a method in which all leucine residues in the dimerization domain of Jun-CCC are mutated to proline residues to lose the dimerization function, and CCC is a method in which the dimerization domain of Jun-CCC is deleted to make it impossible to dimerize. In addition, the N end and the C end of the two polypeptides are respectively subjected to acetylation modification and amidation modification. We determined Jun-CCC, JunPCCC and the binding capacity between CCC and a diarsenic dye. As shown in FIG. 1A, Jun-CCC can bind to a diarsenic dye, producing a strong fluorescent signal, in contrast to Jun which lacks dimerization capacityPCCC and CCC then only produce some background fluorescence signal, indicating that it is a dimerized probe and not a single probe that binds to the bis-arsenic dye. Furthermore, the binding between the Jun-CCC probe and the diarsenic dye was very rapid, reaching maximum fluorescence intensity in about ten minutes (FIG. 1B); furthermore, the binding between the Jun-CCC probe and the bisarsenic dye was relatively stable (equilibrium dissociation constant determined to be 3.17. + -. 0.39 micromoles per liter).
3. Quantitative determination of in vitro recombinant cysteine proteinase-3 by novel probe
To determine whether the probe Jun-CCC was cleaved by caspase-3, we added 0.1. mu.l of 1mM Jun-CCC per liter and 0.2. mu.l of caspase-3 to 10. mu.l of cleavage buffer (25mM HEPES,100mM NaCl,1mM EDTA, 10% sucrose, 0.1% CHAPS,1mM DTT, pH 7.4) and incubated at 37 ℃ for one hour. The mixture was then added to 40. mu.l of a solution containing 1. mu. mol per liter of FlAsH-EDT2In the bisarsenic dye-labeled buffer (100mM Tris · Cl,75mM NaCl,1mM EDTA,1mM DTT,0.05mM BAL, pH 7.4) and incubated at room temperature for 30 minutes in the absence of light. We measured the fluorescence levels before and after enzymatic digestion (fig. 2A). Cleavage of caspase-3 resulted in a 30-fold decrease in the fluorescence intensity of the Jun-CCC-bis-arsenic dye complex (FIG. 2B). Further, we determinedWhether the probe can specifically measure the activity of the cysteine protease-3 or not, the cleavage effect of trypsin, thrombin and ubiquitin-like protease 1 on Jun-CCC is measured in the same reaction system, and as can be seen from FIG. 2C, the trypsin, thrombin and ubiquitin-like protease 1 can not cleave Jun-CCC, so that the activity of the cysteine protease-3 can be specifically measured by the probe Jun-CCC. Further, we incubated the Jun-CCC probe with different concentrations of cystatin-3 and measured the decrease of fluorescence Δ F caused by each concentration of cystatin-3, the relationship between Δ F and cystatin-3 concentration is shown in fig. 2D, and the calculated minimum detection limit is 0.128 ng/ml, which is superior to most existing detection methods.
4. Novel probe for determination of apoptotic cells
Further, we used the probe Jun-CCC to measure staurosporine-induced apoptosis. We used RAW264.7 cells as a study model and induced apoptosis of RAW264.7 cells with 1 micromole per liter of staurosporine, while cells without any treatment were used as a control group. Apoptosis of cells was determined by immunoblotting and fluorescence based on the probe Jun-CCC. The appearance of the large subunit of cystatin-3 was clearly seen with the addition of staurosporine (FIG. 3A), indicating that cystatin-3 was successfully activated in the cell. Consistent with the immunoblotting results, the fluorescence signal was weaker in the staurosporine-treated cells than in the control group (FIG. 3B). Furthermore, the induction of apoptosis of RAW264.7 cells by staurosporine is time-dependent, and more cystatin-3 large subunit and weaker fluorescence signal appear with the increase of induction time. To further verify that the reduced fluorescence was due to cleavage of the probe Jun-CCC by activated caspase-3, we added the caspase-3 specific inhibitor Ac-DEVD-CHO to inhibit intracellular caspase-3 activity. As shown in FIGS. 3C and 3D, the addition of Ac-DEVD-CHO did not change the protein content of caspase-3, but resulted in a recovery of fluorescence, indicating that the decrease in fluorescence is due to cleavage of the probe Jun-CCC by caspase-3. Therefore, the probe Jun-CCC is effective for determining apoptosis.
Finally, it should be noted that: the above embodiments are only used to illustrate the technical solution of the present invention, and not to limit the same; while the invention has been described in detail and with reference to the foregoing embodiments, it will be understood by those skilled in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some or all of the technical features may be equivalently replaced; and the modifications or the substitutions do not make the essence of the corresponding technical solutions depart from the scope of the technical solutions of the embodiments of the present invention.

Claims (22)

1. A polypeptide probe selected from the group consisting of:
X1-Y1-CC；
said X1The amino acid sequence of (A) is asaaelervktlkleiyeilrskanmlreqiqlgap; said X1All or part of the amino acids in (A) are D-configuration amino acids;
said Y is1Is DEVD.
2. The polypeptide probe of claim 1, wherein the N-terminus of the polypeptide probe is modified by acetylation;
and/or;
the C end of the polypeptide probe is subjected to amidation modification.
3. The polypeptide probe of claim 1, wherein X is1And said Y1Through the first linker and/or the Y1And the CC is connected with the second connector through a second connector.
4. The polypeptide probe of claim 3, wherein the number of amino acids in the first linker is 1 to 5.
5. The polypeptide probe of claim 3, wherein the number of amino acids in the second linker is 1 to 5.
6. A polypeptide probe as claimed in any one of claims 3 to 5 characterised in that the amino acid sequence of the second linker is G.
7. A diagnostic kit, which comprises the polypeptide probe of any one of claims 1-6, a diarsenic dye, a buffer solution 1 used when the enzyme to be detected cuts the polypeptide probe to obtain an enzyme cutting product, and a buffer solution 2 used when the diarsenic dye marks the enzyme cutting product.
8. The diagnostic kit according to claim 7, wherein the buffer 1 contains a reducing agent for avoiding the occurrence of disulfide bonds.
9. The diagnostic kit of claim 8, wherein the reducing agent is selected from dithiothreitol and/or tris (2-carbonylethyl) phosphate.
10. The diagnostic kit according to claim 8, wherein the reducing agent is selected from dithiothreitol and the concentration of the reducing agent is 0.5 to 2 mM.
11. The diagnostic kit of claim 7, wherein said buffer 2 contains an eluent to avoid binding of a single said CC fragment to said bis-arsenic dye.
12. The diagnostic kit of claim 11, wherein the eluent is selected from 2, 3-dimercapto-1-propanol and/or 1, 2-ethanedithiol.
13. The diagnostic kit as claimed in claim 11, wherein the eluent is selected from 2, 3-dimercapto-1-propanol and the concentration of the eluent is 0.03-0.10 mM.
14. The diagnostic kit of claim 11, wherein the diarsenic dye is selected from the group consisting of ReAsH-EDT2、FlAsH-EDT2One or more of F2FlAsH or F4 FlAsH.
15. Use of a polypeptide probe according to any one of claims 1 to 6 or a diagnostic kit according to any one of claims 7 to 14 for detecting cystatin-3 and/or cystatin-7 activity.
16. The use according to claim 15, wherein the detection is carried out in a cell lysate or in a somatic cell.
17. The use according to claim 16, wherein the cell is selected from any one of HeLa cells, NIH/3T3 cells, C6 cells, MCF7 cells, human HCE cells, Jurkat T cells, Huh-7 cells, DU145 cells, U-937 cells and HepG2 cells.
18. Use of a polypeptide probe according to any one of claims 1 to 6 or a diagnostic kit according to any one of claims 7 to 14 for the manufacture of a medicament for the evaluation and/or treatment of a disease associated with apoptosis.
19. The use of claim 18, wherein the apoptosis-related disorder is a tumor, an autoimmune disease, or a neurodegenerative disease.
20. The use of claim 19, wherein the tumor comprises: leukemia, lymphoma, head and neck squamous cell carcinoma, lung cancer, esophageal cancer, liver cancer, colorectal cancer, breast cancer, ovarian cancer, cervical cancer, renal cell carcinoma, and melanoma.
21. The use according to claim 19, wherein the autoimmune disease comprises: systemic lupus erythematosus, autoimmune lymphoproliferative syndrome, rheumatoid arthritis, and thyroiditis.
22. The use of claim 19, wherein the neurodegenerative disease comprises: alzheimer's disease, Huntington's disease, Parkinson's disease, stroke, and amyotrophic lateral sclerosis.
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