CN106632689A - Polypeptide probe, kit comprising same and application of polypeptide probe or kit - Google Patents

Polypeptide probe, kit comprising same and application of polypeptide probe or kit Download PDF

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CN106632689A
CN106632689A CN201611209386.2A CN201611209386A CN106632689A CN 106632689 A CN106632689 A CN 106632689A CN 201611209386 A CN201611209386 A CN 201611209386A CN 106632689 A CN106632689 A CN 106632689A
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probe
polypeptide
polypeptide probe
cells
amino acid
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CN106632689B (en
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杨用
梁岩
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Shenzhen Institute of Advanced Technology of CAS
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • 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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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/34—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase
    • C12Q1/37—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase involving peptidase or proteinase
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    • G01—MEASURING; TESTING
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    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
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    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
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    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
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    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5011—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing antineoplastic activity
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5091—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing the pathological state of an organism
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/50—Fusion polypeptide containing protease site
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • 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
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/90—Enzymes; Proenzymes
    • G01N2333/914—Hydrolases (3)
    • G01N2333/948—Hydrolases (3) acting on peptide bonds (3.4)
    • G01N2333/95—Proteinases, i.e. endopeptidases (3.4.21-3.4.99)
    • G01N2333/964—Proteinases, i.e. endopeptidases (3.4.21-3.4.99) derived from animal tissue
    • G01N2333/96402—Proteinases, i.e. endopeptidases (3.4.21-3.4.99) derived from animal tissue from non-mammals
    • G01N2333/96405—Proteinases, i.e. endopeptidases (3.4.21-3.4.99) derived from animal tissue from non-mammals in general
    • G01N2333/96408—Proteinases, i.e. endopeptidases (3.4.21-3.4.99) derived from animal tissue from non-mammals in general with EC number
    • G01N2333/96413—Cysteine endopeptidases (3.4.22)
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    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2500/00—Screening for compounds of potential therapeutic value
    • G01N2500/10—Screening for compounds of potential therapeutic value involving cells
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    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/04—Endocrine or metabolic disorders
    • G01N2800/046—Thyroid disorders
    • G—PHYSICS
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    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/10—Musculoskeletal or connective tissue disorders
    • G01N2800/101—Diffuse connective tissue disease, e.g. Sjögren, Wegener's granulomatosis
    • G01N2800/102—Arthritis; Rheumatoid arthritis, i.e. inflammation of peripheral joints
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/10—Musculoskeletal or connective tissue disorders
    • G01N2800/101—Diffuse connective tissue disease, e.g. Sjögren, Wegener's granulomatosis
    • G01N2800/104—Lupus erythematosus [SLE]
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    • G01—MEASURING; TESTING
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    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/28—Neurological disorders
    • G01N2800/2814—Dementia; Cognitive disorders
    • G01N2800/2821—Alzheimer
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/28—Neurological disorders
    • G01N2800/2835—Movement disorders, e.g. Parkinson, Huntington, Tourette
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/28—Neurological disorders
    • G01N2800/2871—Cerebrovascular disorders, e.g. stroke, cerebral infarct, cerebral haemorrhage, transient ischemic event
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/28—Neurological disorders
    • G01N2800/2878—Muscular dystrophy

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Abstract

The invention relates to the field of biomedical treatment, in particular to a polypeptide probe, a kit comprising the probe and application of the polypeptide probe or the kit. The polypeptide probe is selected from a composition of the following substances and dimers thereof: X<1>-Y<1>-CC, where X<1> is a polypeptide with dimerization capability; Y<1> is selected from a restriction enzyme cutting site sequence of an enzyme to be detected. According to the probe, a simple and sensitive enzyme activity measurement method is further developed by fully utilizing the characteristic of a bi-arsenic dye-tetracysteine tag complex that the stability and the quantum yield greatly depend on the spatial conformation of a tetracysteine tag.

Description

Polypeptide probe, the kit comprising the probe and its application
Technical field
The present invention relates to biologic medical field, in particular to a kind of polypeptide probe, the kit comprising the probe and Its application.
Background technology
Apoptosis is a kind of cell active death process controlled by gene, to the individual development of multicellular organism, just Often cell turnover, tissue keep normal configuration and function to have positive and important meaning, and aberrant apoptotic then can cause to swell The generation of the diseases such as knurl, nerve degenerative diseases.Apoptotic enforcement is caused by a series of cysteine proteinases Cascade reaction come realize (Cold Spring Harb.Perspect.Biol., 2013,5 (4):A008656), in half Guang ammonia In pepsin family, cysteine proteinase-3 belongs to effect type apoptosis enzyme, and the cysteine proteinase-3 of activation is degradable A series of substrates, including apoptosis inhibitor, extracellular matrix and skelemin and DNA reparation correlation factors, make cellular biochemical And morphology changes, cause Apoptosis (Oncogene, 2008,27 (48):6194-6206).Cysteine protein Enzyme -3 is the key protein enzyme that Apoptosis performs the stage, therefore can be surveyed by determining the activity of cysteine proteinase-3 Determine apoptosis process.
The cysteine label system of double arsenic dyestuffs-four is a kind of mark of the living cells protein fluorescence with broad prospect of application Method (Science, 1998,281 (5374):269-272).Within the system, four cysteine labels are one comprising four Cysteine residues hexapeptide or dodecapeptide (Nat.biotechnol., 2005,23 (10):1308-1314), cysteine is residual Base is combined together two-by-two, forms cysteine-cysteine bigeminy, and the two bigeminy are by proline-glycine bigeminy Son is separated.Four cysteine labels have higher specificity and affinity to double arsenic dyestuffs.Double arsenic dyestuffs are by 1,2- Dithioglycol does not fluoresce in the case of chelating, but will send very strong fluorescence once being combined with four cysteine labels. In addition to existing in a linear fashion, two cysteine-cysteine bigeminy in four cysteine labels can be being divided Leave come, but they spatially must mutually close on (Nat.Chem.Biol., 2007,3 (12):779-784).In the mark In system, the space conformation of four cysteine labels can be stablized to forming double cysteine label compounds of arsenic dyestuff-four Property and quantum yield produce strong influence (J.Am.Chem.Soc., 2002,124 (21):6063-6076).
Cysteine proteinase-3 generally exists in cell with inactive proenzyme, needs by the activated form apoptosis enzyme of upstream Cut and activated.The cysteine proteinase-3 of activation can be with specificity cutting Asp-Glu-valine-day A series of winter propylhomoserin tetrapeptide array, according to this characteristic, probe quilts based on Asp-Glu-valine aspartic acid The activity for determining cysteine proteinase-3 is developed, corresponding method has fluorescence detection, electrochemical process, colorimetric Method, surface plasmon resonance, biloluminescence method, electrogenerated chemiluminescence method.With the development of nanometer technology, some are based on and receive Polypeptide probe such as quantum dot complex polypeptide probe, nm of gold complex polypeptide probe, the Graphene complex polypeptide probe of rice material Be developed for use in determine cysteine proteinase-3 activity (Chem.rev., 2015,115 (22):12546-12629). Additionally, some based on FRET reaction albumen probe and fluorescent switch (Nat.Commun., 2013；, 4: 2157) also it is developed for use in the activity for determining intracellular cysteine proteinase-3.In these methods, fluorescence labeling meeting Cutting efficiency of the cysteine proteinase-3 to substrate is reduced to a certain extent, is prepared nano-sensor and is then caused detection process Become complicated and poorly efficient.Therefore in the urgent need to developing simple, sensitive, efficient method determining intracellular cysteine albumen The activity of enzyme -3 is so as to detecting apoptosis process.Additionally, this area detect other enzymes it is active when also more or less deposit In the problems referred to above, and lack a kind of technology that efficiently can be detected for the activity of various enzymes.
In view of this, it is special to propose the present invention.
The content of the invention
In the present invention, we make full use of the stability and quantum of double cysteine label compounds of arsenic dyestuff-four to produce Rate is very dependent on four cysteine Label space conformation this characteristic, devises a kind of new unmarked peptide molecule probe, A kind of simple, method of sensitive determination enzymatic activity is further developed.
Before compound, composition, protein, peptide in the description present invention etc. and method, it will be appreciated that these enforcements Mode is not limited to described ad hoc approach, scheme and reagent, this is because they can change.It is also understood that herein The term for being used is only used for describing particular implementation purpose, and is not intended to limit the model of present embodiment or claim Enclose.
In order to realize the above-mentioned purpose of the present invention, spy employs the following technical solutions：
A kind of polypeptide probe, the polypeptide probe is selected from following material and its dimeric composition：
X1-Y1-CC；
Wherein, X1It is the polypeptide with dimerization ability；
Y1Selected from the restriction enzyme site sequence of enzyme to be detected.
Wherein, X1The dimer that section is formed can be spontaneously formed, and (example also can be formed under other auxilins or subsidiary conditions Such as X1Section some amino acid residues are modified or peptide fragment is in the hydrophobic environment), it is preferred that the dimer is to spontaneously form.
The probe includes at least three different functional areas, and CC is two cysteines, is combined with double arsenic dyestuffs Position, for reading the activity of enzyme to be detected.(spontaneous) when not by cleavage to be detected, between polypeptide probe Dimerization can cause two cysteine-cysteine bigeminy spatially close to each other, so as to be combined with double arsenic dyestuffs Send stronger fluorescence；Conversely, the enzymolysis of enzyme to be detected can dissociate cysteine-cysteine bigeminy from polypeptide Get off, although cut polypeptide still can dimerization, but lacked the position of the double arsenic dyestuff of grappling, double arsenic dyestuffs thus not Can be luminous.Therefore, by the change of fluorescence before and after measure cutting, the activity of enzyme to be detected can be quantitative determined.
By changing Y1The sequence of section polypeptide, the probe that in theory present invention is provided can be to all with cutting peptide fragment ability The activity of enzyme detected.
Preferably, polypeptide probe as above, the X1All or part of amino acid sequence be selected from D configuration amino Acid.
Because during biological evolution, amino acid natural selection is L-type, biological internal enzyme None- identified simultaneously cuts D Amino acids.This design can prevent X1Section also can effectively extend offer of the present invention by other protease Non-specific cleavages Polypeptide probe half-life.
X1In section the amino acid of D configurations and L-configuration can cross arrangement, one L-configuration of such as one D configuration replaces phase Connect, it is also possible to function as described above；Most preferably, X1Amino acid sequence all selected from D amino acids.
Preferably, polypeptide probe as above, the X1For leucine zipper, or comprising many of leucine zipper structure Peptide.
Leucine zipper is a kind of supersecondary structure as Dimerized domain, and the alpha-helix for making to be parallel to each other it Between produce adhesion.One leucine zipper contains multiple leucine residues, generally occurs as soon as one per seven amino acid residue It is secondary, this results in the alpha-helix of a both sexes, hydrophobic region only side wherein.This hydrophobic region provides the region of dimerization, Motif is pulled up as " slide fastener ".
In addition, the enlightenment of the technology contents for being provided according to the present invention, X1Section polypeptide can be according to egg common in biology The albumen section for being responsible for dimerization in white dimer is designed, such as receptor tyrosine kinase, some nuclear receptor proteins, 14- 3-3 albumen, driving albumen, Factor XI, Factor XIII, Toll-like receptor, fiber original albumen, the β γ subunits two of G-protein Aggressiveness etc., this is easy to those skilled in the art.Leucine zipper is only as most preferred technical scheme.
Preferably, X1Section polypeptide may be selected from following amino acid sequences：
QLEDKVEELLSKNYHLENEVARLKKLVG；
KLEALEGKLEAEGKGKLEAUGICLEALE；
GEIAALKQEIAALKKENAALKWEIAALKQGYY；
ASIARLEEKVKTLKAQNYELASTANMLREQVAQLGAP；
ASAAELEERVKTLKAEIYELQSEANMLREQIAQLGAP；
ASAAELEERVKTLKAEIYELQSEANMLREQGAP；
ASAAELEERVKTLKAEIYELQSEGAP；
ESKVSSLESKVSSLESKVSSLESKVSSLESKVSSLESKVSS。
It is furthermore preferred that above-mentioned amino acid sequence is D amino acids.
Preferably, polypeptide probe as above, the N-terminal of the polypeptide probe has carried out acetylation modification；
And/or；
The C-terminal of the polypeptide probe has carried out amidatioon modification.
The modification at polypeptide probe two ends can play a part of the stability for increasing probe.
Preferably, polypeptide probe as above, the X1With the Y1Between also include the Linker1 and/or Y1With Also include Linker2 between the CC；
Preferably, the amino acid number of the Linker1 is 1～30, more preferably 1～5；
Preferably, the amino acid number of the Linker2 is 1～30, more preferably 1～5.
The amino acid quantity of Linker1 and Linker2 can be 1,2,3,4,5,6,7,8,9,10,11,12,13,14, 15th, 16,17,18,19,20,21,22,23,24,25,26,27,28,29 or 30.
The sequence of Linker1 and Linker2 can also be selected from (GGGGS) n, (GGGS) n, (GGS) n or Gn, and wherein n can be with It is 1,2,3,4,5 or 6.
Preferably, polypeptide probe as above, the Y1Sequence be DEVD.
DEVD is the digestion position of cysteine proteinase-3 (Caspase3) and Caspase-7 (Caspase7) Point.
Preferably, polypeptide probe as above, the X1Amino acid sequence for D configurations asaaeleervktlkaeiyelrskanmlreqiaqlgap。
This sequence is the leucine zipper structure of D configurations.
Preferably, polypeptide probe as above, the amino acid sequence of the Linker2 is G.
A kind of polypeptide probe, for detecting the enzymatic activity of Caspase3 or Caspase7, its sequence is asaaeleervktlkaeiyelrskanmlreqiaqlgapDEVDGCC；Wherein Asaaeleervktlkaeiyelrskanmlreqiaqlgap is the amino acid of D configurations.
Preferably, the N-terminal and C-terminal of asaaeleervktlkaeiyelrskanmlreqiaqlgapDEVDGCC is carried out respectively Acetylation and amidatioon modification.
A kind of diagnostic kit, it includes polypeptide probe as above, double arsenic dyestuffs, polypeptide described in cleavage to be detected Probe buffer solution 1 used when obtaining digestion products and described pair of arsenic dyestuff are used when being marked to the digestion products Buffer solution 2；
Preferably, the buffer solution 1 is containing the reducing agent for avoiding disulfide bond from occurring, it is furthermore preferred that the reduction Agent is selected from dithiothreitol (DTT) and/or three (2- carbonylethyls) microcosmic salt hydrochlorates；
Preferably, the buffer solution 2 is containing for avoiding the combination of the single CC fragments and described pair of arsenic dyestuff Eluant, eluent, it is furthermore preferred that the eluant, eluent is selected from 2,3- dimercapto -1- propyl alcohol and/or 1,2- dithioglycol.
Preferably, diagnostic kit as above, described pair of arsenic dyestuff is selected from ReAsH-EDT2、FlAsH-EDT2、 One or more in F2FlAsH or F4FlAsH.
Preferably, diagnostic kit as above, when the reducing agent is selected from dithiothreitol (DTT), the reducing agent Concentration is 0.5～2mM.
Preferably, diagnostic kit as above, it is described when the eluant, eluent is selected from 2,3- dimercapto -1- propyl alcohol The concentration of eluant, eluent is 0.03～0.10mM.
Polypeptide probe as above or diagnostic kit as above are in detection Y1In the activity of corresponding enzyme Using；
Preferably, the detection is carried out in cell pyrolysis liquid or in body cell；
It is furthermore preferred that the cell selected from HeLa cells, NIH/3T3 cells, C6 cells, MCF7 cells, people's HCE cells, Any one in Jurkat T cells, Huh-7 cells, DU145 cells, U-937 cells and HepG2 cells.
Polypeptide probe as above or diagnostic kit as described above are in detection individual cells in the activity of certain enzyme Application.
Polypeptide probe as above or diagnostic kit as described above are in detection cysteine proteinase-3 and/or half Guang Application in the activity of serine protease -7.
The application of polypeptide probe as above or diagnostic kit as described above in detection Apoptosis.
Preferably, the Apoptosis can be Apoptosis that abiogenous apoptosis, disease be caused or The cell that the Apoptosis that environment is caused, such as ultraviolet irradiation, hydrogen peroxide, dexamethasone, TNF etc. are induced withers The measure died.
Polypeptide probe as above or diagnostic kit as described above are being prepared and/or evaluated for treating Apoptosis Application in the medicine of relevant disease；
Preferably, application as above, the apoptosis related disease is tumour, autoimmune disease or nerve DD；
It is furthermore preferred that as above apply, the tumour includes：Leukaemia, lymthoma, SCCHN, Lung cancer, cancer of the esophagus, liver cancer, colorectal cancer, breast cancer, oophoroma, cervical carcinoma, clear-cell carcinoma and melanoma；
It is furthermore preferred that as above apply, the autoimmune disease includes：Systemic lupus erythematosis, itself Immunity lymphoproliferative syndrome, rheumatoid arthritis, thyroiditis；
It is furthermore preferred that as above apply, the nerve degenerative diseases include：Alzheimer, Huntington dance Step disease, Parkinson's, apoplexy and amyotrophic lateral sclerosis.
A kind of method of diagnosis cell apoptosis-associated diseases, including：
By the cell pyrolysis liquid or Tissue lysates of main body to be diagnosed and polypeptide probe as above, or examine as mentioned above Composition in disconnected kit is contacted, and the main body is diagnosed according to reaction gained signal value.
Preferably, the method for diagnosis cell apoptosis-associated diseases as above, the signal value is the polypeptide probe The fluorescence signal value for producing is reacted with double arsenic dyestuffs.
Preferably, the method for diagnosis cell apoptosis-associated diseases as above, the main body is animal, is more preferably fed Newborn animal, more preferably primate, are more preferably people.
Preferably, the method for diagnosis cell apoptosis-associated diseases as above, the apoptosis related disease is swollen Knurl, autoimmune disease or nerve degenerative diseases；
It is furthermore preferred that the tumour includes：Leukaemia, lymthoma, SCCHN, lung cancer, cancer of the esophagus, liver Cancer, colorectal cancer, breast cancer, oophoroma, cervical carcinoma, clear-cell carcinoma and melanoma；
It is furthermore preferred that the autoimmune disease includes：Systemic lupus erythematosis, LADA lymphocyte increase Raw syndrome, rheumatoid arthritis, thyroiditis；
It is furthermore preferred that the neurodegenerative disorders include：Alzheimer, Huntington's disease, Parkinson's, in Wind and amyotrophic lateral sclerosis.
Compared with prior art, beneficial effects of the present invention are：
1), peptide molecule probe does not have any mark, has preferable biocompatibility between enzyme to be detected, can improve Cutting efficiency；
2), in the whole detection course of reaction of kit provided by the present invention, without separation and cleaning step, operation letter Single and convenience；
3), detection pattern has flexibility, and probe can be marked by exciting with difference with double arsenic dyestuffs of launch wavelength Note, thus there are ways to determine Level of Apoptosis；
4), the polypeptide probe for determining Caspase3 and Caspase7 of specific offer of the invention is by Amino acid profile, The DNA of coded polypeptide probe is proceeded to intracellular, the apoptosis situation of individual cells can be determined.
Description of the drawings
In order to be illustrated more clearly that the specific embodiment of the invention or technical scheme of the prior art, below will be to concrete The accompanying drawing to be used needed for embodiment or description of the prior art is briefly described, it should be apparent that, in describing below Accompanying drawing is some embodiments of the present invention, for those of ordinary skill in the art, before creative work is not paid Put, can be with according to these other accompanying drawings of accompanying drawings acquisition.
Fig. 1 is the performance measurement of new unmarked peptide molecule probe；(A) polypeptide probe and double arsenic dyestuff FlAsH-EDT2 With reference to the emission spectrum of formed compound；(B) polypeptide probe and double arsenic dyestuff FlAsH-EDT2Binding kineticses determine；
Fig. 2 is to quantitative determine vitro recombination cysteine proteinase-3 using novel probe；(A) cysteine proteinase-3 Cutting to polypeptide probe can reduce the fluorescence intensity of polypeptide probe-bis- arsenic dye compositions；What curve above was represented It is Jun-CCC, what underlying curve was represented is Jun-CCC+ cysteine proteinase-3s；(B) to the quantitative analysis of figure A； (C) activity of polypeptide probe specific assay cysteine proteinase-3；(D) cysteine proteinase-3 cutting causes fluorescence strong The reduction (Δ F) of degree and the graph of a relation between cysteine proteinase-3 concentration；
Fig. 3 is to determine the apoptosis that staurosporine induces RAW264.7 cells using novel probe；(A) staurosporine induction The apoptotic immunoblotting assays of RAW264.7；(B) polypeptide probe determines the apoptosis that staurosporine induces RAW264.7 cells； (C) Ac-DEVD-CHO suppresses the immunoblotting assay of intracellular cysteine proteinase-3 active；(D) Ac-DEVD-CHO suppresses The fluorescence analysis of cysteine proteinase-3 active in apoptotic cell.
Specific embodiment
Unless otherwise defined, all technologies for using of the present invention and scientific terminology have with belonging to disclosed embodiment The identical implication that the those of ordinary skill in field is generally understood that.Although similar or equivalent with method of the present invention and material Method and material can be used in the practice or test of present embodiment, but hereafter still describe suitable method and material. All publications, patent application, patent and other bibliography that the present invention is referred to are incorporated into this by quoting full content Wen Zhong.In the case of a conflict, this specification (including definition) will play dominating role.Additionally, material, method and embodiment are only It is merely illustrative, and is not intended to limit.The further feature and advantage of embodiment will be from following detailed description of book and right Become in requirement obvious.
In order to promote to understand implementations described herein this purpose, with reference to some embodiments, and will will use Language-specific is describing these embodiments.Term as used herein is only used for describing specific embodiment purpose, and not purport Limiting the scope of the present disclosure.
Embodiment of the present invention is described in detail below in conjunction with embodiment, but those skilled in the art will Understand, the following example is merely to illustrate the present invention, and is not construed as limiting the scope of the present invention.It is unreceipted concrete in embodiment Condition person, the condition advised according to normal condition or manufacturer is carried out.Agents useful for same or the unreceipted production firm person of instrument, are The conventional products that commercially available purchase is obtained can be passed through.
In an embodiment of the present invention, for illustrating the effect of probe of the present invention as a example by the probe for determining Caspase3 Really.It is the design of the technical scheme of the embodiment including new unmarked peptide molecule probe, the performance measurement of novel probe, new Probe quantitative determines four aspects of measure of vitro recombination cysteine proteinase-3, novel probe to apoptotic cell.
Embodiment
1. the design of new unmarked peptide molecule probe
Designed peptide molecule probe is named as Jun-CCC, and its sequence is asaaeleervktlkaeiyelrskanml ReqiaqlgapDEVDGCC, the N-terminal and C-terminal of probe have carried out respectively acetylation and amidatioon modification to increase stablizing for probe Property.The probe includes three different functional areas, and wherein asaaeleervktlkaeiyelrskanmlreqiaqlgap is Dimerization functional areas, to prevent polypeptide probe by other protease Non-specific cleavages, the region is all by D- type amino acid structures Into；DEVD is the specific cutting sequence of cysteine proteinase-3, by L-type Amino acid profile；Thereafter amino acid G is Linker, to increase the flexibility that CC reacts with double arsenic dyestuffs；CC is the position combined with double arsenic dyestuffs, for reading half Guang The activity of serine protease -3.When not cut by cysteine proteinase-3, between polypeptide probe from dimerization Two cysteine-cysteine bigeminy can be caused spatially close to each other, so as to combined with double arsenic dyestuffs send compared with Strong fluorescence；Conversely, the enzymolysis of cysteine proteinase-3 can solve cysteine-cysteine bigeminy from polypeptide From getting off, although cut polypeptide still can dimerization, but lacked the position of the double arsenic dyestuffs of grappling, double arsenic dyestuffs thus Can not light.Therefore, by the change of fluorescence before and after measure cutting, the activity of cysteine proteinase-3 can be quantitative determined, And then measure apoptosis process.
2. the performance measurement of novel probe
To show that the combination of double arsenic dyestuffs and probe depends on the dimerization between probe to act on, we devise two simultaneously Bar does not have the probe Jun of dimerization abilityP- CCC and CCC, wherein JunPThe sequence of-CCC is asaaepeervktpkaeiyeprs The sequence of kanmpreqiaqpgapDEVDGCC, CCC is GCC, JunP- CCC is by the bright ammonia in dimerisation domain in Jun-CCC Sour residue all sports proline residue to lose its dimerization function, and CCC is by the dimerization work(in disappearance Jun-CCC Energy area is allowed to be unable to dimerization.In addition, the N-terminal and C-terminal of two polypeptides have carried out respectively acetylation modification and amidatioon modification.I Determine Jun-CCC, JunPBinding ability between-CCC and CCC and double arsenic dyestuffs.As shown in Figure 1A, Jun-CCC can To be combined with double arsenic dyestuffs, very strong fluorescence signal is produced, conversely, lacking the Jun of dimerization abilityP- CCC and CCC then can only Some background fluorescence signals are produced, this is shown to be the probe of dimerization rather than single probe is in combination with double arsenic dyestuffs.Also, Combination between Jun-CCC probes and double arsenic dyestuffs is that very rapidly the time of about ten minutes or so can just reach maximum Fluorescence intensity (Figure 1B)；Additionally, the combination between Jun-CCC probes and double arsenic dyestuffs is the more stable (equilibrium solution of measure It is 3.17 ± 0.39 per liter of micromoles from constant).
3. quantitative determination of the novel probe to vitro recombination cysteine proteinase-3
Can be cut by cysteine proteinase-3 to determine probe Jun-CCC, we add 0.1 microlitre of 1 mmoles Your per liter of Jun-CCC and 0.2 microlitre of cysteine proteinase-3 to 10 microlitres cutting buffer (25mM HEPES, 100mM NaCl, 1mM EDTA, 10%sucrose, 0.1%CHAPS, 1mM DTT, pH=7.4) in, and it is incubated one at 37 degree Hour.The mixture is subsequently added to 40 microlitres and includes every liter of FlAsH-EDT of 1 micromole2Double arsenic dye marker buffer solutions In (100mM TrisCl, 75mM NaCl, 1mM EDTA, 1mM DTT, 0.05mM BAL, pH=7.4), and in room temperature lucifuge Incubation 30 minutes.We determine the fluorescence level (Fig. 2A) before and after enzymolysis.The cutting of cysteine proteinase-3 can cause The fluorescence intensity of the double arsenic dye compositions of Jun-CCC- declines 30 times (Fig. 2 B).Further, we determine the probe can be special Property determine cysteine proteinase-3 activity, it is general that we determine trypsase, fibrin ferment, class in identical reaction system Dissection of the fibroin enzyme 1 to Jun-CCC, it will be seen that trypsase, fibrin ferment, ubiquitin-like proteins from Fig. 2 C Enzyme 1 can not cut Jun-CCC, therefore, probe Jun-CCC can be with the activity of specific assay cysteine proteinase-3.Enter one Step, we are mutually incubated Jun-CCC probes with variable concentrations cysteine proteinase-3, and determine every kind of concentration cysteine egg White enzyme -3 causes the decline Δ F of fluorescence, the relation between Δ F and cysteine proteinase-3 concentration as shown in Figure 2 D, to calculate The lowest detection for going out is limited to 0.128 nanograms per milliliter, better than existing most of detection method.
4. measure of the novel probe to apoptotic cell
Further, we determine the Apoptosis of staurosporine induction using probe Jun-CCC.We adopt RAW264.7 cells induce the apoptosis of RAW264.7 cells using the staurosporine of 1 per liter of micromole as research model, Simultaneously by not through any process cell as a control group.The apoptosis of cell is simultaneously using Western blot and based on spy The fluorescence method of pin Jun-CCC is determining.Staurosporine is added to be clearly visible the appearance (figure of cysteine proteinase-3 large subunit 3A), show that intracellular cysteine proteinase-3 is successfully activated.It is consistent with immunoblot results, at staurosporine Its fluorescence signal will be weaker than control group (Fig. 3 B) in the cell managed.Also, staurosporine induces the apoptosis of RAW264.7 cells It is time dependent, with the increase of induction time, it may appear that more cysteine proteinase-3 large subunits and weaker Fluorescence signal.For further verify fluorescence it is less be due to activating cutting institute of the cysteine proteinase-3 to probe Jun-CCC Caused, we add cysteine proteinase-3 specific inhibitor Ac-DEVD-CHO to suppress intracellular cysteine The activity of proteinase-3.As illustrated in figures 3 c and 3d, Ac-DEVD-CHO is added to change the albumen of cysteine proteinase-3 Amount, but can but cause fluorescence to recover, and the reduction for showing fluorescence is because cysteine proteinase-3 is cut to probe Jun-CCC Cut what is caused.Therefore, probe Jun-CCC can be effectively to determine apoptotic.
Finally it should be noted that：Various embodiments above only to illustrate technical scheme, rather than a limitation；To the greatest extent Pipe has been described in detail with reference to foregoing embodiments to the present invention, but it will be understood by those within the art that：Its Still the technical scheme described in foregoing embodiments can be modified, either to which part or all technical characteristic Carry out equivalent；And these modifications or replacement, do not make the essence disengaging various embodiments of the present invention skill of appropriate technical solution The scope of art scheme.

Claims (10)

1. a kind of polypeptide probe, it is characterised in that the polypeptide probe is selected from following material and its dimeric composition：
X1-Y1-CC；
Wherein, X1It is the polypeptide with dimerization ability；
Preferably, the dimer is to spontaneously form；
Preferably, the X1All or part of amino acid sequence be selected from D amino acids；
Preferably, the X1For leucine zipper, or the polypeptide comprising leucine zipper structure；
Y1Selected from the restriction enzyme site sequence of enzyme to be detected；
Preferably, the N-terminal of the polypeptide probe has carried out acetylation modification；
And/or；
The C-terminal of the polypeptide probe has carried out amidatioon modification.
2. polypeptide probe according to claim 1, it is characterised in that the X1With the Y1Between also include Linker1 And/or the Y1Also include Linker2 between the CC；
Preferably, the amino acid number of the Linker1 is 1～5；
Preferably, the amino acid number of the Linker2 is 1～5.
3. polypeptide probe according to claim 1 and 2, it is characterised in that the Y1Sequence be DEVD.
4. polypeptide probe according to claim 3, it is characterised in that the X1Amino acid sequence for D configurations asaaeleervktlkaeiyelrskanmlreqiaqlgap。
5. polypeptide probe according to claim 3, it is characterised in that the amino acid sequence of the Linker2 is G.
6. a kind of diagnostic kit, it includes polypeptide probe, double arsenic dyestuffs, the enzyme to be detected described in any one of Claims 1 to 5 Cutting the polypeptide probe and obtaining buffer solution 1 used during digestion products and described pair of arsenic dyestuff is carried out to the digestion products Buffer solution 2 used during mark；
Preferably, the buffer solution 1 is containing the reducing agent for avoiding disulfide bond from occurring, it is furthermore preferred that reducing agent choosing From dithiothreitol (DTT) and/or three (2- carbonylethyls) microcosmic salt hydrochlorates；Most preferably, the reducing agent is selected from dithiothreitol (DTT), and The concentration of the reducing agent is 0.5～2mM；
Preferably, the buffer solution 2 be containing for avoiding the eluant, eluent that the single CC fragments are combined with described pair of arsenic dyestuff, It is furthermore preferred that the eluant, eluent is selected from 2,3- dimercapto -1- propyl alcohol and/or 1,2- dithioglycol；Most preferably, the eluant, eluent Selected from 2,3- dimercapto -1- propyl alcohol, and the concentration of the eluant, eluent is 0.03～0.10mM.
7. diagnostic kit according to claim 6, it is characterised in that described pair of arsenic dyestuff is selected from ReAsH-EDT2、 FlAsH-EDT2, one or more in F2FlAsH or F4FlAsH.
8. the diagnostic kit described in polypeptide probe described in any one of Claims 1 to 5 or any one of claim 6～7 exists Detection Y1Application in the activity of corresponding enzyme；
Preferably, the detection is carried out in cell pyrolysis liquid or in body cell；
It is furthermore preferred that the cell selected from HeLa cells, NIH/3T3 cells, C6 cells, MCF7 cells, people's HCE cells, Any one in Jurkat T cells, Huh-7 cells, DU145 cells, U-937 cells and HepG2 cells.
9. polypeptide probe or diagnostic kit described in any one of claim 6～7 described in any one of claim 3～5 is being examined Application in surveying cysteine proteinase-3 and/or Caspase-7 activity.
10. polypeptide probe or diagnostic kit described in any one of claim 6～7 described in any one of claim 3～5 is being made It is standby and/or evaluate for the application in the medicine for treating apoptosis related disease；
Preferably, the apoptosis related disease is tumour, autoimmune disease or nerve degenerative diseases；
It is furthermore preferred that the tumour includes：Leukaemia, lymthoma, SCCHN, lung cancer, cancer of the esophagus, liver cancer, knot The carcinoma of the rectum, breast cancer, oophoroma, cervical carcinoma, clear-cell carcinoma and melanoma；
It is furthermore preferred that the autoimmune disease includes：Systemic lupus erythematosis, LADA lymphocytosis are comprehensive Simulator sickness, rheumatoid arthritis, thyroiditis；
It is furthermore preferred that the nerve degenerative diseases include：Alzheimer, Huntington's disease, Parkinson's, apoplexy and Amyotrophic lateral sclerosis.
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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2023178704A1 (en) * 2022-03-25 2023-09-28 京东方科技集团股份有限公司 Polypeptide probe, and preparation method therefor and use thereof

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101768625A (en) * 2010-02-23 2010-07-07 厦门大学 Pathogen detection method
CN103667194A (en) * 2013-12-06 2014-03-26 华中科技大学同济医学院附属同济医院 Human liver cancer cell line for observing life period of hepatitis B virus in cells and applications thereof

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101768625A (en) * 2010-02-23 2010-07-07 厦门大学 Pathogen detection method
CN103667194A (en) * 2013-12-06 2014-03-26 华中科技大学同济医学院附属同济医院 Human liver cancer cell line for observing life period of hepatitis B virus in cells and applications thereof

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
JODY M.MASON等: "Improved Stability of the Jun-Fos Activator Protein-1 Coiled Coil Motif", 《THE JOURNAL OF BIOLOGY CHEMISTRY》 *
陈磊，姚祝军: "特异性的蛋白小分子荧光探针及其标记技术", 《生命科学》 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2023178704A1 (en) * 2022-03-25 2023-09-28 京东方科技集团股份有限公司 Polypeptide probe, and preparation method therefor and use thereof

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