WO2018196831A1 - 一种短肽、荧光探针及其制备方法 - Google Patents

一种短肽、荧光探针及其制备方法 Download PDF

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WO2018196831A1
WO2018196831A1 PCT/CN2018/084718 CN2018084718W WO2018196831A1 WO 2018196831 A1 WO2018196831 A1 WO 2018196831A1 CN 2018084718 W CN2018084718 W CN 2018084718W WO 2018196831 A1 WO2018196831 A1 WO 2018196831A1
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peptide
short peptide
fluorescent
probe
fluorescent probe
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PCT/CN2018/084718
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French (fr)
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张玉慧
韩于冰
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奇点荧光南京生物科技有限公司
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Priority to US16/386,311 priority Critical patent/US11181526B2/en

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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
    • C07K5/10—Tetrapeptides
    • C07K5/1019—Tetrapeptides with the first amino acid being basic
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
    • G01N33/582—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with fluorescent label
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/001—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof by chemical synthesis
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/06—Linear peptides containing only normal peptide links having 5 to 11 amino acids
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/08—Linear peptides containing only normal peptide links having 12 to 20 amino acids
    • C—CHEMISTRY; METALLURGY
    • C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
    • C09K—MATERIALS FOR MISCELLANEOUS APPLICATIONS, NOT PROVIDED FOR ELSEWHERE
    • C09K11/00—Luminescent, e.g. electroluminescent, chemiluminescent materials
    • C09K11/06—Luminescent, e.g. electroluminescent, chemiluminescent materials containing organic luminescent materials
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/6486—Measuring fluorescence of biological material, e.g. DNA, RNA, cells
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/531—Production of immunochemical test materials
    • G01N33/532—Production of labelled immunochemicals
    • G01N33/533—Production of labelled immunochemicals with fluorescent label
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/10—Fusion polypeptide containing a localisation/targetting motif containing a tag for extracellular membrane crossing, e.g. TAT or VP22
    • C—CHEMISTRY; METALLURGY
    • C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
    • C09K—MATERIALS FOR MISCELLANEOUS APPLICATIONS, NOT PROVIDED FOR ELSEWHERE
    • C09K2211/00—Chemical nature of organic luminescent or tenebrescent compounds
    • C09K2211/10—Non-macromolecular compounds
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
    • G01N2021/6439—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes" with indicators, stains, dyes, tags, labels, marks

Definitions

  • the invention belongs to the field of fluorescence imaging, and more particularly to a short peptide, a fluorescent probe and a preparation method thereof.
  • the super-resolution microscopic imaging strategy based on light modulation is more in pursuit of brightness and anti-photobleaching properties in terms of fluorescent labeling.
  • super-resolution microscopy imaging strategy based on single-molecular positioning also requires excellent photoactivation of fluorescent molecules / Light flashing performance for better image quality.
  • the first type uses a fluorescent protein labeling method to express a plasmid containing a specific protein of a fluorescent protein, since the fluorescent protein is lower than the fluorescent dye.
  • the photon yield, large size, and the inability to quantitatively control the transgene expression have certain limitations on imaging resolution and image quality.
  • Another type of fluorescently modified macromolecule is delivered by electroporation or microinjection. Living cells, such as modified quantum dots, nanoparticles, etc., are difficult and expensive to operate, and there are large barriers in the wide application process; the third type is based on a few fluorescent chemical probes.
  • these methods cannot fully meet the requirements for high brightness, high anti-bleaching ability, and excellent light activation/light scintillation capability in super-resolution imaging, so it is difficult to be compatible with multiple strategies of super-resolution microscopic imaging methods. .
  • the “integrated” probe must be designed as a whole to achieve specific recognition, transmembrane and other functions, and to solve steric hindrance, fluorescence quenching, and brightness. And other issues. Therefore, the chemical probe is not suitable for mass production and large-scale production, and can only be customized, resulting in extremely high cost of the chemical probe, and the range of the finished fluorescent probe is extremely narrow.
  • the present invention provides a short peptide, a fluorescent probe and a preparation method thereof, the object of which is to provide a freely coupled most commercial fluorescent dye and exert its excellent fluorescent properties. Therefore, the existing integrated probe is adapted to have a narrow range of fluorescent dyes, has poor effects, and cannot be modularized to realize the technical problems of specific recognition and transmembrane function.
  • a short peptide comprising a peptide having a load-bearing function and a recognition functional peptide; wherein the load-functional peptide is an interval repeat of a lysine residue and a glycine residue
  • the sequence is repeated between 2 and 5 times; the load-functional peptide and the recognition functional peptide are linked by 1 to 2 glycine residues as a linking group.
  • the short peptide has the number of repetitions being three.
  • the short peptide comprises a transmembrane peptide segment linked to a functional peptide or a recognition functional peptide by a linker peptide.
  • the short peptide has a penetrating peptide segment of octameric arginine and the sequence is rRrRrRRR, wherein r is D-type arginine and R is L-type arginine.
  • the short peptide has a glycosylation sequence GG or G.
  • the short peptide which recognizes a functional peptide, is a recognition sequence for a cysteine protease C1 family protease or actin.
  • a fluorescent probe comprising the short peptide provided by the present invention, the lysine residue of the short peptide being bound to a fluorescent dye molecule.
  • the fluorescent probe has a fluorescent dye molecule which is an active fluorescent dye molecule having an NHS reactive group, preferably Alexa Fluor 647 NHS ester, Cy3B NHS ester, Atto 565 NHS ester and/or Atto 488 NHS ester.
  • an NHS reactive group preferably Alexa Fluor 647 NHS ester, Cy3B NHS ester, Atto 565 NHS ester and/or Atto 488 NHS ester.
  • a method of preparing the fluorescent probe comprising the steps of:
  • Fluorescent dye molecular linkage a liquid phase reaction method is used to covalently combine a free amino group on a lysine residue of a short peptide obtained in the step (2) with an NHS reactive group of a fluorescent dye molecule to obtain a right.
  • the fluorescent probe of any one of 1 to 7 is required.
  • the method for preparing the fluorescent probe further comprises the following steps:
  • the fluorescent probe obtained in the step (3) is dissolved and purified by reverse column chromatography to obtain the purified fluorescent probe molecule.
  • the probe is a combination of the main peptide and the fluorescent dye "two-stage", the low-dose linkage reaction solves the high cost of introducing a commercial dye and increases the flexibility of dye selection. ;
  • the short peptide provided by the invention can be applied to fluorescent dye molecules of various sizes, and the fluorescent probe formed by coupling with a commercial fluorescent dye having excellent fluorescence performance can exert its excellent optical performance and has wide application range. Super resolution microscopy with higher resolution, longer time history and better image quality.
  • a preferred embodiment realizes modular specific recognition function and membrane permeation function, and can realize precise positioning of subcellular structure according to specific needs, thereby realizing living cells such as labeled living cell actin fibers or lysosomes. Submicroscopic structure marking.
  • the method for preparing the fluorescent probe provided by the present invention provides the possibility of modular prefabricated semi-finished probes, which is more convenient to use than the conventional integrated probe.
  • Figure 1 is a MS mass spectrometric detection report of the short peptide provided in Example 3.
  • Figure 3 shows the labeling status of cells incubated with different concentrations of lysosomal fluorescent probes based on Alexa Fluor 647 in a final volume of 100 ⁇ L of probe working solution;
  • Figure a is the labeled state containing 14 ⁇ L of probe mother liquor
  • Figure b is The labeled state containing 29 ⁇ L of the probe mother liquor
  • Figure c is the labeled state containing 43 ⁇ L of the probe mother liquor
  • Figure d is the labeled state containing 57 ⁇ L of the probe mother liquor
  • Figure e is the labeled state containing 71 ⁇ L of the probe mother liquor;
  • Figure 4 shows the labeling status of the lysosomal fluorescent probe based on Alexa Fluor 647 and the standard lysosomal marker LysoTracker Red in living cells, wherein panel a shows the labeling state of the lysosomal fluorescent probe based on Alexa Fluor647.
  • Figure b is the marking state of LysoTracker Red, and
  • Figure c is the superposition of the first two channels;
  • Figure 5 is a random optical reconstruction super-resolution microscopic imaging result of a cysteine protease C1 family protease fluorescent probe based on Alexa Fluor 647, wherein the lower left corner of the graph a is an uncalculated total internal reflection imaging overlay, upper right The angle is the calculated random optical reconstruction super-resolution result image, and Figure b is a Gaussian fit image of the full width at half maximum of the lysosome identified by the white square in Figure a;
  • Figure 6 is a structural light illumination super-resolution microscopic imaging result of the Atto 565-based cysteine protease C1 family protease fluorescent probe, wherein Figures a to d show imaging results of 101 lysosomes in four U2OS cells.
  • the first frame, figures e to h, show the trajectory of the corresponding lysosome in the focal plane;
  • Figure 7 is a non-uniform distribution of lysosomal fluorescence of a lysin based on the fluorescent probe of the cysteine protease C1 family of Atto 565, and is a result of super-resolution microscopic imaging of structured light illumination;
  • Figure 8 is a structural light illumination super-resolution microscopic imaging result of a cysteine protease C1 family protease fluorescent probe based on Alexa 647, Atto 565 and Atto 488, respectively, and a standard lysosomal marker LysoTracker Red, wherein Figure a is Imaging results of LysoTracker Red, Figure b is the imaging result of the cysteine protease C1 family protease fluorescent probe based on Alexa Fluor 647, and Figure c is the imaging result of the cysteine protease C1 family protease fluorescent probe based on Atto 565 Figure d is the imaging result of the Atto 488-based cysteine protease C1 family protease fluorescent probe.
  • Figure a Imaging results of LysoTracker Red
  • Figure b is the imaging result of the cysteine protease C1 family protease fluorescent probe based on Alexa Fluor 647
  • Figure c is the imaging result of the cysteine
  • Figures a1, b1 and c1 are magnified views of the white solid-line box regions in Figures a, b and c, respectively.
  • Figures b2 are enlarged views of the time-lapse imaging results of the white dotted square area in the graphs a and b, respectively;
  • Figure 9 shows the labeling status of actin fluorescent probes based on Alexa Fluor 647 at different concentrations.
  • Figure a shows the labeling status of 21 ⁇ L of the probe mother liquor
  • Figure b contains The labeled state of 36 ⁇ L of the probe mother liquor
  • Figure c is the labeled state containing 43 ⁇ L of the probe mother liquor
  • Figure d is the labeled state containing 57 ⁇ L of the probe mother liquor;
  • Figure 10 is a marker status of actin fluorescent probe based on Alexa Fluor 647 and standard actin fiber marker GFP-actin in living cells, wherein graph a is the labeling state of GFP-actin, and panel b is based on Alexa Fluor The labeling state of the actin fluorescent probe of 647, and Figure c is the superposition of the first two channels;
  • Figure 11 is a random optical reconstruction super-resolution microscopic imaging result of actin fluorescent probes based on Alexa Fluor 647, Cy3B and Atto 488, in the order of Alexa Fluor 647, Cy3B and Atto 488, Figures a to c One lower left corner is the uncalculated total internal reflection imaging overlay of the corresponding probe, and the upper right corner is the calculated random optical reconstruction super-resolution result image; Figures d to f are respectively identified by the boxes in Figures a to c. a Gaussian fitting image of full width at half maximum of actin fibers;
  • Figure 12 is a structural light illumination super-resolution microscopic imaging result of Atto 488-based actin fluorescent probe and actin fiber standard markers GFP-actin, EGFP-Lifeact, wherein Figure a is an Atto 488-based muscle.
  • the short peptide provided by the present invention comprises a peptide having a load-bearing function and a recognition functional peptide; the load-functional peptide is a spacer repeat of a lysine residue and a glycine residue, and the number of repetitions is from 2 to 5 times.
  • the load functional peptide and the recognition functional peptide are linked by 1 to 2 glycine residues as a linking group.
  • a lysine residue is used to provide a side chain free amino group to bind a fluorescent dye molecule having a NHS reactive group; among several amino acids having a free amino group in a side chain, the side chain structure of lysine K is most Simple, it is possible to combine fluorescent dye molecules with NHS reactive groups without interference from other side chain groups;
  • This ligand pattern repeated at intervals of lysine residues and glycine residues, multiple lysine residues, adaptive single or multiple-loaded fluorescent dye molecules, can accommodate most fluorescent dye molecules,
  • the fluorescent dye molecules are adaptively loaded on the most suitable lysine residue of steric hindrance.
  • the number of repeats therefore determines the load capacity, loading efficiency, and performance of the final probe of the short peptide.
  • the number of repetitions is between 2 and 5 times, preferably 3 times.
  • the larger the value of n the more favorable the loading of the fluorescent dye molecules and the higher the cost; and the comprehensive consideration of the specific luminescent properties of the loaded fluorescent dye, including brightness. , anti-bleaching ability, light scintillation ability, n value is in the above range is suitable.
  • the load-functional peptide which may be (KG) n or (GK) n , binds to the recognition functional peptide in a sequence that facilitates synthesis.
  • G is a glycine residue, as a linking moiety, overcoming steric hindrance. According to the current size of most commercial dye molecules, one glycine is selected as a linking moiety, which can effectively overcome the steric hindrance problem while balancing the probe volume and the membrane. The efficiency of preventing the connection group from being too long leads to a decrease in the efficiency of the membrane.
  • the recognition functional peptide is a recognition sequence for a cysteine protease C1 family protease or actin.
  • the recognition sequence of the cysteine protease C1 family protease is preferably It can specifically recognize the cysteine protease C1 family protease in lysosome, has very good specificity, can be used for purification and mass spectrometric identification of target protease; the recognition sequence of actin, preferably MGCADLIKKFESISKEE amino acid sequence, A marker for living cell actin fibers does not alter the dynamic properties of actin itself.
  • the short peptide preferably further comprises a linking peptide segment and a penetrating peptide segment, and the linking sequence is preferred to facilitate synthesis.
  • the linking peptide is a glycine sequence GG or G;
  • the short peptide When applied to intracellular submicroscopic super-resolution imaging, the short peptide is preferred to have a transmembrane function, that is, a transmembrane peptide segment, and the ligation sequence is preferred to facilitate synthesis.
  • the transmembrane peptide segment preferably octameric arginine, has the sequence rRrRrRRR, wherein r is D-type arginine and R is L-type arginine.
  • the penetrating peptide segment allows the fluorescent probe to penetrate the membrane directly rather than enterocytically, and has a high ability to penetrate the membrane, so that the probe can be effectively positioned above the target within the cell.
  • the linker peptide has only one glycine G; while in the lysosome In the transmembrane organic fluorescent probe of the protease C1 family protease, two glycine Gs are added between the rRrRrRRR and the recognition group.
  • the short peptide can be synthesized by a solid phase peptide synthesis method.
  • the fluorescent probe provided by the present invention comprises the short peptide provided by the present invention, and the lysine residue of the short peptide is bound to a fluorescent dye molecule.
  • the fluorescent dye molecule is a fluorescent dye molecule bearing an NHS reactive group, preferably Alexa Fluor 647 NHS ester, Cy3B NHS ester, Atto 565 NHS ester and/or Atto 488 NHS ester.
  • the short peptide provided by the invention can be widely adapted to various commercial fluorescent dye molecules, and has good adaptability. Since the commercial dye with excellent optical properties enters the design of the probe, after realizing fluorescence imaging of living cells, Get super-resolution microscopy with higher resolution, longer time history and better image quality.
  • the fluorescent probe provided by the present invention preferably uses the short peptide provided by the present invention to react with a fluorescent dye molecule in a liquid phase.
  • the specific fluorescent probe is preferably synthesized by the following method:
  • Fluorescent dye molecular attachment The free amino group on the lysine residue of the short peptide obtained in the step (2) is covalently bonded to the NHS reactive group of the fluorescent dye molecule by a liquid phase reaction method, and the present invention is obtained. Fluorescent probes are provided.
  • the fluorescent probe obtained in the step (3) is dissolved and purified by reverse column chromatography to obtain the purified fluorescent probe molecule.
  • This synthesis method combines the main peptide segment with the fluorescent dye “two-stage”.
  • the low-dose linkage reaction solves the high cost of introducing commercial dyes and increases the flexibility of dye selection.
  • K is a lysine residue
  • G is a glycine residue
  • B is a recognition functional peptide
  • B includes a linker peptide and a recognition functional peptide:
  • the recognition functional peptide is: MGCADLIKKFESISKEE;
  • K is a lysine residue
  • G is a glycine residue
  • B is a recognition functional peptide
  • B includes a linker peptide, a recognition functional peptide, and a penetrating peptide:
  • the recognition functional peptide is:
  • the penetrating peptide segment is: octameric arginine, the sequence is rRrRrRRR, wherein r is D-type arginine and R is L-type arginine.
  • the connecting peptide is: GG
  • the recognition functional peptide is:
  • the transmembrane peptide is: octameric arginine, the sequence is rRrRrRRR, wherein r is D-type arginine and R is L-type arginine.
  • K is a lysine residue
  • G is a glycine residue
  • B is a recognition functional peptide
  • P is a penetrating peptide
  • the recognition functional peptide is: MGCADLIKKFESISKEE
  • the penetrating peptide segment is: octameric arginine, the sequence is rRrRrRRR, wherein r is D-type arginine and R is L-type arginine.
  • the method of solid phase peptide synthesis is obtained, and the MS mass spectrometry detection report is shown in FIG. 2 .
  • a fluorescent probe comprising the short peptide provided in Example 3, the lysine residue of the short peptide being bound to a fluorescent dye molecule.
  • the fluorescent dye molecule is Alexa Fluor 647 NHS ester, Atto 565 NHS ester or Atto 488 NHS ester, respectively named as Alexa Fluor 647-based cysteine protease C1 family protease fluorescent probe, Atto 565 based cysteine Acid protease C1 family protease fluorescent probe, Atto 488-based cysteine protease C1 family protease fluorescent probe.
  • the fluorescent probe was synthesized as follows:
  • the specific steps were as follows: The dye-free probe powder was dissolved in a 0.1 M NaHCO 3 solution at a final concentration of 0.5 mM, and sterilized by filtration. Alexa Fluor 647 NHS ester (1 mg, Thermo Fisher Scientific, Inc.), Atto 565 NHS ester (1 mg, Sigma-Aldrich Co., LLC) or Atto 488 NHS ester (1 mg, Sigma-Aldrich Co., LLC) to be purchased. The dye was dissolved in anhydrous DMSO, and divided into small portions of about 30 nmol/tube, and the solvent in the tube was drained and stored at -20 ° C in a light-shielded manner.
  • a tube of the dye was dispensed, dissolved in 20 ⁇ L of anhydrous DMSO, and added dropwise to 28 ⁇ L of the dye-free probe mother liquor, and mixed well. Under room temperature conditions, shake on a shaker for 2 h or more, overnight.
  • Fluorescent probe purification After the above synthesis step is completed, the liquid in the reaction system is drained, the probe is dissolved in an aqueous solution with 0.5% TFA and 5% acetonitrile, and column chromatography is performed using a C18 reverse column. According to the polarity of the probe, the eluate used 20 ⁇ L of an aqueous solution of acetonitrile in a purity of 50-80% each time. After collecting the product components, the solution was drained to obtain crystals of the product, and 200 ⁇ L of PBS solution was added and placed in a refrigerator at 4 ° C. spare.
  • the Alexa Fluor 647-based cysteine protease C1 family protease fluorescent probe, the Atto 565-based cysteine protease C1 family protease fluorescent probe, and the Atto 488-based cysteine protease C1 family protease were separately prepared. Fluorescent probe.
  • Probe incubation conditions Before the experiment, the medium in the confocal dish was aspirated and the residual serum was washed away with PBS solution. 14 ⁇ L to 71 ⁇ L of the probe mother liquid was taken, diluted to a final volume of 100 ⁇ L with a PBS solution, added to a confocal dish, and incubated at 37 ° C, 5% CO 2 for 30 minutes. The probe solution was removed and 200 ⁇ L of a 1 mg/mL trypan blue solution was added. After standing for one minute, the trypan blue solution was aspirated, and the cells were carefully washed three times with PBS solution, and a colorless DMEM medium containing 10% fetal calf serum was added and taken to an imaging system for observation.
  • Laser scanning confocal microscopy experiments observed by laser scanning confocal microscopy LSM 710 (Zeiss, German).
  • Alexa Fluor 647 has a maximum excitation wavelength of 650 nm and a maximum emission wavelength of 665 nm.
  • LysoTracker Red has a maximum excitation wavelength of 577 nm and a maximum emission wavelength of 590 nm.
  • the results of laser scanning confocal imaging after labeling cells based on the Alexa Fluor 647 cysteine protease C1 family protease fluorescent probe are shown in Figure 3.
  • Figure 3 shows that when the amount of the probe mother liquor is in the range of 14 ⁇ L to 71 ⁇ L, the dot-like structure in the cells can be smoothly labeled; at the same time, the cells are still not stained blue by the trypan blue solution, which proves Cell activity.
  • panel a shows the dot-like structure labeled by the Alexa Fluor 647-based cysteine protease C1 family protease fluorescent probe, which is consistent with the labeling results of LysoTracker Red in Figure b.
  • the two signals can be well co-localized, demonstrating that the Alexa Fluor 647-based cysteine protease C1 family protease fluorescent probe marks the correct living cell lysosomal structure.
  • STORM imaging buffer a) buffer A (pH 8.0) containing 10 mM Tris and 50 mM NaCl; b) catalase Catalase solution: dissolved in buffer A, final concentration 17 mg/mL, Dispense into 10 ⁇ L / tube aliquots, stored in a refrigerator at -20 ° C; c) glucose oxidase Glucose Oxidase solution: dissolved in buffer A, the final concentration of 70mg / mL, divided into 40 ⁇ L / tube of small portions, Store in a refrigerator at -20 °C; d) 1M mercaptoethylamine MEA solution: dissolve in 0.25N hydrochloric acid solution, the final concentration is 70mg/mL, and store in -20 °C refrigerator; e) GLOX solution: c, d two Take one tube of each solution in the step, mix well, a total of 50 ⁇ L, can be stored in the refrigerator at 4 °C for 2 weeks
  • Alexa Fluor 647 has a maximum excitation wavelength of 650 nm and a maximum emission wavelength of 665 nm.
  • the following methods are used in finding the appropriate laser power: the cell sample is placed on the stage and fixed with a clip. Use the bright field field to find the focal plane first, then use the mercury lamp to combine the corresponding filter to find the field of view with clear mark and high signal background ratio, quickly turn off the mercury lamp and reduce the quenching of the sample fluorescence.
  • Figure 5 shows that the random optical reconstruction of the super-resolution image (upper right corner) effectively improves the resolution compared to the uncalculated total internal reflection imaging overlay (lower left corner).
  • the full width at half maximum of the lysosome is 130.38 nm, and its scale is below 200 nm, which is an effective super-resolution microscopic imaging result.
  • This experiment demonstrates that the Alexa Fluor 647-based cysteine protease C1 family protease fluorescent probe has the ability to achieve random optical reconstruction of super-resolution microscopy, enabling high-resolution live cell lysosomal images.
  • Alexa Fluor 647 has a maximum excitation wavelength of 650 nm and a maximum emission wavelength of 665 nm; LysoTracker Red has a maximum excitation wavelength of 577 nm and a maximum emission wavelength of 590 nm; Atto 565 has a maximum excitation wavelength of 563 nm and a maximum emission wavelength of 592 nm; Atto 488 is the largest.
  • the excitation wavelength was 501 nm and the maximum emission wavelength was 523 nm.
  • the sample was taken using the 2D-SIM mode, and a SIM result was calculated from nine original images (three angles, three phases), wherein the exposure time of each original image was set to 30 ms.
  • the monochrome imaging time interval was 1 s and continuously photographed more than 300 frames. After the actual shooting is completed, the time interval between each SIM result map is about 1.15s on average.
  • the imaging time interval was set to 6s in consideration of the module conversion speed of the imaging system itself. .
  • Intra-complex multi-directional movement after which the lysosome may stabilize or re-accelerate and continue to move in a certain direction (two red lines in Figure 6b); (ii) some lysozyme
  • the body performs a motion similar to free diffusion in a certain area.
  • the distance of diffusion is not far from the first type, and the speed is not as fast as the first type, and usually the direction is uncertain, and the motion trajectory is like a scattered wool (Fig. 6c).
  • LysoTracker Red in addition to labeling lysosomes, LysoTracker Red also formed a non-uniform non-specific distribution near the circular dark areas of the nucleus.
  • the emphasis on “unevenness” is due to the fact that uneven backgrounds are difficult to determine the true boundaries of lysosomes due to the high fluorescence intensity at some locations - especially the lysosomal edge.
  • Alexa647-based cysteine protease C1 family protease fluorescent probe in Fig. 8b it can be seen from the labeling of the Alexa647-based cysteine protease C1 family protease fluorescent probe in Fig. 8b that there is a clear boundary as long as the lysosome is on the focal plane.
  • the background difference between the two imaging results is more apparent in the comparison of the series of images a2 and b2 of the time-lapse imaging.
  • the lysosomes labeled with the Alexa Fluor 647-based cysteine protease C1 family of protease fluorescent probes have clear contours and distinct shapes, while the LysoTracker Red-labeled lysosomes remain in a blurred background. It should be noted here that some lysosomes do not exhibit a circular shape during exercise. The main reason is that fast-moving lysosomes may be deformed by force, which is more common in lysosomes in SIM imaging. Traditional microscope imaging is more difficult to find.
  • a fluorescent probe comprising the short peptide provided in Example 4, the lysine residue of which is bound to a fluorescent dye molecule.
  • the fluorescent dye molecule is Alexa Fluor 647 NHS ester, Cy3B NHS ester or Atto 488 NHS ester, respectively named as Alexa Fluor 647-based actin fluorescent probe, Cy3B-based actin fluorescent probe or based on Atto 488 Actin fluorescent probe.
  • the fluorescent probe was synthesized as follows:
  • the specific steps were as follows: The dye-free probe powder was dissolved in a 0.1 M NaHCO 3 solution at a final concentration of 0.5 mM, and sterilized by filtration. Alexa Fluor 647 NHS ester (1 mg, Thermo Fisher Scientific, Inc.), Cy3B NHS ester (1 mg, GE Healthcare shanghai Co., Ltd.) or Atto 488 NHS ester (1 mg, Sigma-Aldrich Co., LLC) dyes to be purchased Dissolve in anhydrous DMSO, dispense into small fractions of about 30 nmol / tube, and drain the solvent in the tube, and store at -20 ° C.
  • a tube of the dye was dispensed, dissolved in 20 ⁇ L of anhydrous DMSO, and added dropwise to 28 ⁇ L of the dye-free probe mother liquor, and mixed well. Under room temperature conditions, shake on a shaker for 2 h or more, overnight.
  • Fluorescent probe purification After the above synthesis step is completed, the liquid in the reaction system is drained, the probe is dissolved in an aqueous solution with 0.5% TFA and 5% acetonitrile, and column chromatography is performed using a C18 reverse column. According to the polarity of the probe, the eluate used 20 ⁇ L of an aqueous solution of acetonitrile in a purity of 50-80% each time. After collecting the product components, the solution was drained to obtain crystals of the product, and 200 ⁇ L of PBS solution was added and placed in a refrigerator at 4 ° C. spare.
  • Alexa Fluor 647-based actin fluorescent probe Cy3B-based actin fluorescent probe or Atto 488-based actin fluorescent probe were prepared.
  • the cell preparation method was the same as in Example 5.
  • Laser scanning confocal microscopy experiments observed by laser scanning confocal microscopy LSM 710 (Zeiss, German). Alexa Fluor 647 has a maximum excitation wavelength of 650 nm and a maximum emission wavelength of 665 nm; GFP has a maximum excitation wavelength of 488 nm and a maximum emission wavelength of 507 nm. After incubating primary cultured astrocyte cells (Astrocyte) with a 21 ⁇ L probe for 30 min (Fig. 9a), the probes in the cells were mostly diffusely distributed, forming silk only at the cell margin and filopodia.
  • the structure of the labeled label indicates that the concentration of the probe entering the cell is insufficient to reach the threshold of binding of all Lifeact recognition groups to actin; at the same time, the contrast inside and outside the cell is low due to insufficient brightness of the probe inside the cell.
  • the amount of probe used was 36 ⁇ L (Fig. 9b)
  • a distinct filamentous marker structure appeared in the cells, but there was still blurring in the middle of the cells; at the same time, compared with the amount of 21 ⁇ L of the probe, due to intracellular The signal is strong and the extracellular background is relatively small.
  • the amount of the probe was 43 to 57 ⁇ L (Fig.
  • panel a shows the dot-like structure labeled by the actin fluorescent probe based on Alexa Fluor 647, which is consistent with the labeling result of GFP-actin in panel b.
  • the two signals were well colocalized, demonstrating that the Alexa Fluor 647-based actin fiber fluorescent probe marks the correct structure.
  • Alexa Fluor 647 has a maximum excitation wavelength of 650 nm and a maximum emission wavelength of 665 nm; Cy3B has a maximum excitation wavelength of 559 nm and a maximum emission wavelength of 570 nm; Atto 488 has a maximum excitation wavelength of 501 nm and a maximum emission wavelength of 523 nm.
  • the method employed in finding a suitable laser power is in accordance with Embodiment 5.
  • FIG. 11 shows that the random optical reconstruction super-resolution image (upper right corner) effectively improves the resolution compared to the uncalculated total internal reflection imaging overlay (lower left corner), and Alexa 647 can achieve resolutions below 60 nm ( Figure 11a), Cy3B can achieve a spatial resolution of 90nm ( Figure 11b), while Atto 488 can achieve a spatial resolution below 90nm ( Figure 11c).
  • the spatial resolution of several dyes can be different. The main reason is that the number of photons emitted by the dye in each scintillation event is different.
  • This photon number affects the positioning accuracy of the dye molecules and is the most influential spatial resolution.
  • the "bright-dark state” ratio of the light scintillation dye affects the resulting image quality.
  • This experiment demonstrates the excellent properties of commercial dyes in random optical reconstruction of living cells in super-resolution microscopy (excellent scintillation and high brightness, mainly in terms of contribution to image quality and spatial resolution), The importance of introducing commercial dyes into transmembrane organic fluorescent probes.
  • Total internal reflection structure light illumination super-resolution microscopic imaging experiment High-NA TIRF-SIM observation by high-resolution (1.78) total internal reflection structure light illumination super-resolution microscope. Atto 488 has a maximum excitation wavelength of 501 nm and a maximum emission wavelength of 523 nm; EGFP and GFP have a maximum excitation wavelength of 488 nm and a maximum emission wavelength of 507 nm. Samples were taken using the TIRF-SIM mode, and a SIM result was calculated from nine original images (three angles, three phases), wherein the exposure time of each original image was set to 7 ms, and the result is shown in FIG.
  • the result of the Atto 488-based actin fluorescent probe is that the background inside the cell is very low, and the signal itself is bright enough, so the signal background ratio is very high and the image is very clear;
  • the microfilament fiber, or the thicker fiber at the periphery of the cell is clear and clear, especially in the TIRF overlay.
  • the thicker fiber that is unclear in the TIRF overlay shows that the fine fibers are intertwined into a bundle. As a result, it was proved that SIM imaging did improve the resolution effectively.
  • the GFP-actin-labeled microfilaments have a poor signal-to-background ratio, and the cells have a turbid background in addition to the coarser fibers; the filamentous pseudopods outside the cells can be seen radially. Fiber; overall, the SIM reconstruction map has no significant resolution improvement over the TIRF overlay.
  • the EGFP-Lifeact used in Figure 12c has a strong signal-to-background ratio, and despite its high intracellular background, it still achieves higher quality images.
  • this experiment demonstrates that Atto 488-based actin fluorescent probes in structured light illumination super-resolution microscopy, due to their excellent brightness, very low background (controllable concentration of probe-incubated cells) and The best performance with clear resolution.

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Abstract

本发明公开了一种短肽、荧光探针及其制备方法。所述短肽包括负载功能肽段和识别功能肽段；所述负载功能肽段为赖氨酸残基和甘氨酸残基的间隔重复序列，其重复次数在2次至5次之间；所述负载功能肽段和所述识别功能肽段以1至2个甘氨酸残基为连接基团连接。所述荧光探针为所述短肽的赖氨酸残基结合荧光染料分子，其制备方法包括以下步骤：(1)短肽合成；(2)短肽纯化；(3)荧光染料分子连接。

Description

一种短肽、荧光探针及其制备方法 技术领域
本发明属于荧光成像领域，更具体地，涉及一种短肽、荧光探针及其制备方法。
背景技术
在超分辨率显微成像技术中，基于光调制的超分辨率显微成像策略(结构光照明超分辨率显微成像技术等)在荧光标记方面，更追求亮度和抗光漂白能力等性质，以得到更高的分辨率和更久的成像时长；而基于单分子定位的超分辨率显微成像策略(随机光学重构超分辨率显微成像技术等)还要求荧光分子具有优秀的光激活/光闪烁性能，以获得更好的成像质量。
目前有多种方法可以实现对活细胞的荧光标记，其主要分为三类：第一类采用荧光蛋白标记方法，表达连接了荧光蛋白的特定蛋白质的质粒，由于荧光蛋白与荧光染料相比较低的光子产量、较大的尺寸、转基因表达无法定量控制等问题，对成像分辨率和图像质量产生了一定限制；另一类将荧光修饰后的大分子通过电穿孔或显微注射的方法送入活细胞，例如修饰后的量子点、纳米颗粒等，这类标记方法操作难度较大且成本昂贵，在广泛应用的过程中存在着较大的壁垒；第三类为化学荧光探针基于少数自身可以透膜的荧光染料，利用探针中特异性的识别基团或染料自身对某些亚细胞结构的亲和力实现对目标的标记，这类探针可能存在一些非特异性标记的情况。但是截至目前，这些方法都无法完全满足超分辨成像中对于高亮度、高抗漂白能力、优异的光激活/光闪烁能力的要求，因此很难兼容于多种策略的超分辨率显微成像方法。
由于许多商业化的荧光染料的同时具有高亮度和高抗漂白能力，且在 加入STORM成像缓冲液时可以表现出优秀的闪烁性能，因此与抗体偶联后可以应用于不同策略的超分辨率显微成像中。然而，尽管具备了优秀的超分辨性能，大多数商业染料受限于其不透膜的性质，无法特异性地应用于活细胞内的标记中；同时由于现有化学荧光探针“一体式”的结构设计和商业染料昂贵的价格，使商业染料极大地增加了化学荧光探针的构建成本。针对不同的成像要求，需要选择不同的荧光染料，在此基础之上“一体式”探针必须整体设计，来实现特异性识别、穿膜等功能，并解决空间位阻、荧光淬灭、亮度等问题。因此化学探针不适合批量化、规模化制作，只能个性化定制，造成化学探针成本极高，同时成品荧光探针适应范围极窄。
发明内容
针对现有技术的以上缺陷或改进需求，本发明提供了一种短肽、荧光探针及其制备方法，其目的在于提供一种可自由偶联大部分商业化荧光染料并发挥其优秀荧光特性的，由此解决现有一体式探针适应的荧光染料范围极窄、效果不佳、不能模块化的实现特异性识别、穿膜功能的技术问题。
为实现上述目的，按照本发明的一个方面，提供了一种短肽，包括具有负载功能肽段和识别功能肽段；所述负载功能肽段为赖氨酸残基和甘氨酸残基的间隔重复序列，其重复次数在2次至5次之间；所述负载功能肽段和所述识别功能肽段以1至2个甘氨酸残基为连接基团连接。
优选地，所述短肽，其所述重复次数为3。
优选地，所述短肽，所述短肽包括穿膜肽段，通过连接肽段与负载功能肽段或识别功能肽段相连。
优选地，所述短肽，其穿膜肽段为八聚精氨酸，序列为rRrRrRRR，其中r为D型精氨酸，R为L型精氨酸。
优选地，所述短肽，其连接肽段为甘氨酸序列GG或G。
优选地，所述短肽，其识别功能肽段为半胱氨酸蛋白酶C1家族蛋白酶 或肌动蛋白的识别序列。
按照本发明的另一个方面，提供了一种荧光探针，包括本发明提供的短肽，所述短肽的赖氨酸残基结合有荧光染料分子。
优选地，所述荧光探针，其荧光染料分子为具有NHS活性基团的活性荧光染料分子，优选Alexa Fluor 647 NHS ester、Cy3B NHS ester、Atto 565 NHS ester和/或Atto 488 NHS ester。
按照本发明的另一个方面提供了所述的荧光探针的制备方法，其特征在于，包括以下步骤：
(1)短肽合成：采用固相合成法，合成如权利要求1至6任意一项所述的短肽；
(2)短肽纯化：将步骤(1)中获得的短肽从用于固相合成法的树脂上剪切，去掉所述氨基酸侧链的保护基团，并纯化浓缩得到经纯化后的所述短肽；
(3)荧光染料分子连接：采用液相反应法，将步骤(2)中获得的短肽的赖氨酸残基上的游离氨基与荧光染料分子的NHS活性基团共价结合，获得如权利要求1至7任意一项所述的荧光探针。
优选地，所述荧光探针的制备方法，其还包括以下步骤：
(4)荧光探针纯化：将步骤(3)中获得的荧光探针溶解，采用反向柱层析纯化，得到纯化后的所述荧光探针分子。
总体而言，通过本发明所构思的以上技术方案与现有技术相比，能够取得下列优异效果：
(1)由于探针为主体肽段和荧光染料“两段式”相结合的方式，小剂量的连接反应解决了引入商业化染料时带来的高成本问题，同时增加了染料选择的灵活性；
(2)本发明提供的短肽，可适用各种大小的荧光染料分子，与荧光性能优越的商业化荧光染料偶联后形成的荧光探针，能发挥其优异的光学性 能，适用范围广泛，能得到具有更高分辨率，更长时程和更好图像质量的超分辨率显微成像。
(3)优选方案，实现了模块化的特异性识别功能和透膜功能，可按照具体需要实现亚细胞结构的精确定位，从而实现诸如标记活细胞肌动蛋白纤维或溶酶体这样的活细胞亚显微结构标记。
(4)本发明提供的所述荧光探针的制备方法，提供了模块化预制半成品探针的可能，突破以往的一体式探针，使用更加方便。
附图说明
图1是实施例3提供的短肽的MS质谱检测报告；
图2是实施例4提供的短肽的MS质谱检测报告；
图3是基于Alexa Fluor 647的溶酶体荧光探针以不同浓度孵育细胞的标记状况，在终体积100μL的探针工作液中；其中图a为含有14μL探针母液的标记状态，图b为含有29μL探针母液的标记状态，图c为含有43μL探针母液的标记状态，图d为含有57μL探针母液的标记状态，图e为含有71μL探针母液的标记状态；
图4是基于Alexa Fluor 647的溶酶体荧光探针与标准溶酶体标记物LysoTracker Red在活细胞内的标记状况，其中图a显示的是基于Alexa Fluor647的溶酶体荧光探针的标记状态，图b为LysoTracker Red的标记状态，图c为前两个通道的叠加情况；
图5是基于Alexa Fluor 647的半胱氨酸蛋白酶C1家族蛋白酶荧光探针的随机光学重构超分辨率显微成像结果，其中图a左下角为未计算前的全内反射成像叠加图，右上角为计算后的随机光学重构超分辨结果图像，图b为图a中白色方框所标识的溶酶体的半高全宽的高斯拟合图像；
图6是基于Atto 565的半胱氨酸蛋白酶C1家族蛋白酶荧光探针的结构光照明超分辨率显微成像结果，其中图a至d展示了四个U2OS细胞中101个溶酶体的成像结果的第一帧，图e至h展示了对应的溶酶体在焦平面的 轨迹图；
图7是基于Atto 565的半胱氨酸蛋白酶C1家族蛋白酶荧光探针表征的溶酶体内荧光的不均匀分布情况，为结构光照明超分辨率显微成像结果图；
图8是分别基于Alexa 647、Atto 565和Atto 488的半胱氨酸蛋白酶C1家族蛋白酶荧光探针与标准溶酶体标记物LysoTracker Red的结构光照明超分辨率显微成像结果，其中图a为LysoTracker Red的成像结果，图b为基于Alexa Fluor 647的半胱氨酸蛋白酶C1家族蛋白酶荧光探针的成像结果，图c为基于Atto 565的半胱氨酸蛋白酶C1家族蛋白酶荧光探针的成像结果，图d为基于Atto 488的半胱氨酸蛋白酶C1家族蛋白酶荧光探针的成像结果，图a1、b1和c1分别为图a、b、c中白色实线方框区域的放大图，图a2、b2分别为图a、b中白色虚线方框区域的延时成像结果放大图；
图9是基于Alexa Fluor 647的肌动蛋白荧光探针以不同浓度孵育细胞的标记状况，在终体积100μL的探针工作液中，图a为含有21μL探针母液的标记状态，图b为含有36μL探针母液的标记状态，图c为含有43μL探针母液的标记状态，图d为含有57μL探针母液的标记状态；
图10是基于Alexa Fluor 647的肌动蛋白荧光探针与标准肌动蛋白纤维标记物GFP-actin在活细胞内的标记状况，其中图a为GFP-actin的标记状态，图b是基于Alexa Fluor 647的肌动蛋白荧光探针的标记状态，图c为前两个通道的叠加情况；
图11是基于Alexa Fluor 647、Cy3B和Atto 488的肌动蛋白荧光探针的随机光学重构超分辨率显微成像结果，其中按照Alexa Fluor 647、Cy3B和Atto 488的顺序，图a至c每一幅左下角为对应探针的未计算前的全内反射成像叠加图，右上角为计算后的随机光学重构超分辨结果图像；图d至f分别为图a至c中方框所标识的肌动蛋白纤维的半高全宽的高斯拟合图像；
图12是基于Atto 488的肌动蛋白荧光探针与肌动蛋白纤维的标准标记物GFP-actin、EGFP-Lifeact的结构光照明超分辨率显微成像结果，其中图 a为基于Atto 488的肌动蛋白纤维荧光探针的成像结果，图b为GFP-actin的成像结果，图c为EGFP-Lifeact的成像结果。
在所有的附图中，图3、图9和图10的标尺为20μm，图4的标尺为10μm，图5、图6、图11和图12的标尺为5μm，图7和图8自带标尺。
具体实施方式
为了使本发明的目的、技术方案及优点更加清楚明白，以下结合附图及实施例，对本发明进行进一步详细说明。应当理解，此处所描述的具体实施例仅仅用以解释本发明，并不用于限定本发明。此外，下面所描述的本发明各个实施方式中所涉及到的技术特征只要彼此之间未构成冲突就可以相互组合。
本发明提供的短肽，包括具有负载功能肽段和识别功能肽段；所述负载功能肽段为赖氨酸残基和甘氨酸残基的间隔重复序列，其重复次数在2次至5次之间；所述负载功能肽段和所述识别功能肽段以1至2个甘氨酸残基为连接基团连接。
其中：赖氨酸残基用于提供侧链游离氨基，以结合带有NHS活性基团的荧光染料分子；在几种侧链带有游离氨基的氨基酸中，赖氨酸K的侧链结构最为简单，可以结合有NHS活性基团的荧光染料分子而不受其他侧链基团的干扰；
以赖氨酸残基和甘氨酸残基的间隔重复的这种配基模式，多个赖氨酸残基，自适应的单一或者多重负载荧光染料分子，可以适应绝大部分的荧光染料分子，使得荧光染料分子自适应地负载在空间位阻最合适的赖氨酸残基上。因此重复数量决定了所述短肽的负载能力、负载效率以及最终探针的性能。重复次数在2次至5次之间，优选3次，n取值越大，对于荧光染料分子的负载越有利，成本也越高；同时综合考虑负载的荧光染料特异性的发光性能，包括亮度、抗漂白能力、光闪烁能力，n取值在以上范围为宜。所述负载功能肽段，可为(KG) n或(GK) n，按照便于合成的顺序与识别 功能肽段结合。G为甘氨酸残基，作为连接部分，克服空间位阻，根据目前大多数商业染料分子大小确定，选取1个甘氨酸作为连接部分，能有效克服空间位阻问题，同时平衡了探针体积与透膜效率，防止连接基团过长导致透膜效率的降低。
所述识别功能肽段为半胱氨酸蛋白酶C1家族蛋白酶或肌动蛋白的识别序列。所述半胱氨酸蛋白酶C1家族蛋白酶的识别序列，优选为
Figure PCTCN2018084718-appb-000001
可以特异性识别溶酶体中半胱氨酸蛋白酶C1家族蛋白酶，具有非常好的特异性，可以用于目标蛋白酶的纯化和质谱鉴定；所述肌动蛋白的识别序列，优选MGVADLIKKFESISKEE氨基酸序列，其为活细胞肌动蛋白纤维的标记物，不会改变肌动蛋白本身的动力学性质。
所述短肽，优选还包括连接肽段和穿膜肽段，连接顺序以方便合成为优。
所述连接肽段为甘氨酸序列GG或G；
当应用于胞内亚显微结构超分辨率成像时，优选所述短肽，还具有穿膜功能，即包括穿膜肽段，连接顺序以方便合成为优。所述穿膜肽段，优选为八聚精氨酸，序列为rRrRrRRR，其中r为D型精氨酸，R为L型精氨酸。所述穿膜肽段，使得荧光探针直接穿膜而不是以胞吞的方式进入细胞，且具有高效的穿膜能力，可以使探针有效地定位到细胞内的目标之上。
考虑到探针的长度、合成成本和空间位阻之间的平衡，在针对肌动蛋白纤维的透膜有机荧光探针中，连接肽段只有一个甘氨酸G；而在针对溶酶体中半胱氨酸蛋白酶C1家族蛋白酶的透膜有机荧光探针中，rRrRrRRR与识别基团之间添加了两个甘氨酸G。
所述短肽，可采用固相肽合成方法合成。
本发明提供的荧光探针，包括本发明提供的短肽，所述短肽的赖氨酸残基结合有荧光染料分子。所述荧光染料分子，为带有NHS活性基团的荧 光染料分子，优选为Alexa Fluor 647 NHS ester、Cy3B NHS ester、Atto 565 NHS ester和/或Atto 488 NHS ester。
本发明提供的短肽能广泛地适应各种商业化的荧光染料分子，具有良好的适应性，由于将光学性质优异的商业化染料进入探针的设计中，能够在实现活细胞荧光成像之后，得到具有更高分辨率、更长时程和更好图像质量的超分辨率显微成像。
本发明提供的荧光探针，优选采用本发明提供的短肽与荧光染料分子液相反应。具体的所述荧光探针优选按照以下方法合成：
(1)短肽合成：采用固相合成法，合成本发明提供的短肽；
(2)短肽纯化：将步骤(1)中获得的短肽从用于固相合成法的树脂上剪切，去掉所述氨基酸侧链的保护基团，并纯化浓缩得到经纯化后的所述短肽；
(3)荧光染料分子连接：采用液相反应法，将步骤(2)中获得的短肽的赖氨酸残基上的游离氨基与荧光染料分子的NHS活性基团共价结合，获得本发明提供的荧光探针。
(4)荧光探针纯化：将步骤(3)中获得的荧光探针溶解，采用反向柱层析纯化，得到纯化后的所述荧光探针分子。
这种合成方法由于探针为主体肽段和荧光染料“两段式”相结合的方式，小剂量的连接反应解决了引入商业化染料时带来的高成本问题，同时增加了染料选择的灵活性；
以下为实施例：
实施例1
一种短肽，具有式I的结构：
(KG) 2-GG-B
式I
其中：K为赖氨酸残基，G为甘氨酸残基，B为识别功能肽段。
B包括连接肽段和识别功能肽段：
所述识别功能肽段为：MGVADLIKKFESISKEE；
采用固相合成法合成。
实施例2
一种短肽，具有式I的结构：
(KG) 5-G-B
式I
其中：K为赖氨酸残基，G为甘氨酸残基，B为识别功能肽段。
B包括连接肽段、识别功能肽段、以及穿膜肽段：
所述识别功能肽段为：
Figure PCTCN2018084718-appb-000002
所述穿膜肽段为：八聚精氨酸，序列为rRrRrRRR，其中r为D型精氨酸，R为L型精氨酸。
采用固相合成法合成。
实施例3
一种短肽，具有式I的结构：
Figure PCTCN2018084718-appb-000003
其中：
所述连接肽段为：GG
所述识别功能肽段为：
Figure PCTCN2018084718-appb-000004
所述穿膜肽段为：八聚精氨酸，序列为rRrRrRRR，其中r为D型精氨 酸，R为L型精氨酸。
按照以下方法合成：
Figure PCTCN2018084718-appb-000005
通过固相肽合成的方法得到，其MS质谱检测报告如图1所示。
实施例4
一种短肽，具有式I的结构：
B-G-(KG) 3-P
式I
其中：K为赖氨酸残基，G为甘氨酸残基，B为识别功能肽段，P为穿膜肽段：
所述识别功能肽段为：MGVADLIKKFESISKEE
所述穿膜肽段为：八聚精氨酸，序列为rRrRrRRR，其中r为D型精氨酸，R为L型精氨酸。
其具体结构如下式
MGVADLIKKFESISKEEGKGKGKGrRrRrRRR
按照以下方法合成：
Figure PCTCN2018084718-appb-000006
通过固相肽合成的方法得到，其MS质谱检测报告如图2所示。
实施例5
一种荧光探针，包括实施例3提供的短肽，所述短肽的赖氨酸残基结合有荧光染料分子。所述荧光染料分子，为Alexa Fluor 647 NHS ester、Atto 565 NHS ester或Atto 488 NHS ester，分别命名为基于Alexa Fluor 647的半胱氨酸蛋白酶C1家族蛋白酶荧光探针、基于Atto 565的半胱氨酸蛋白酶C1家族蛋白酶荧光探针、基于Atto 488的半胱氨酸蛋白酶C1家族蛋白酶荧光探针。
所述荧光探针按照如下方法合成：
(1)短肽合成：运用固相合成法，按照顺序分别连接侧链保护的氨基酸或小分子；
Figure PCTCN2018084718-appb-000007
(2)短肽纯化：去掉氨基酸侧链的保护，并将无染料肽段从树脂上切下来，经过HPLC纯化至95％纯度以上，收集产物组分后冷冻干燥，浓缩得到所需的无染料肽段的晶体；
(3)荧光染料分子连接，合成路线如下：
Figure PCTCN2018084718-appb-000008
具体步骤如下：使用0.1M浓度的NaHCO 3溶液溶解无染料的探针粉 末，溶液终浓度为0.5mM，过滤除菌。将购买的Alexa Fluor 647 NHS ester(1mg,Thermo Fisher Scientific,Inc.)、Atto 565 NHS ester(1mg,Sigma-Aldrich Co.,LLC)或Atto 488 NHS ester(1mg,Sigma-Aldrich Co.,LLC)染料经无水DMSO溶解，分装成约30nmol/管的小份，并将管中的溶剂抽干，置于-20℃遮光保存。取一管分装后的染料，加入20μL无水DMSO溶解，并逐滴加入到28μL无染料的探针母液中，混合均匀。室温条件下，在摇床上遮光摇动2h以上，可过夜。
(4)荧光探针纯化：在上述合成步骤完成之后，将反应体系中的液体抽干，使用带有0.5％TFA和5％乙腈的水溶液溶解探针，用C18反向柱进行柱层析，根据探针的极性，洗脱液使用每次20μL的纯度为50～80％的乙腈水溶液体系，收集产物组分后将溶液抽干，得到产物晶体，加入200μL PBS溶液，置于4℃冰箱备用。
至此，分别制得基于Alexa Fluor 647的半胱氨酸蛋白酶C1家族蛋白酶荧光探针、基于Atto 565的半胱氨酸蛋白酶C1家族蛋白酶荧光探针、基于Atto 488的半胱氨酸蛋白酶C1家族蛋白酶荧光探针。
本实施例提供的荧光探针，成像实验如下：
细胞准备：将生长状态良好的U2OS细胞(2x 10 4cells/well)接种于无菌的共聚焦小皿(glass bottomΦ15mm,NEST Biotechnology Co.,LTD.,China)中。在含有10％胎牛血清的Mcboy’s 5A培养基中，以37℃、5％CO 2的条件培养过夜。
探针孵育条件：实验前，吸出共聚焦小皿中的培养基，用PBS溶液洗去残余血清。取14μL～71μL探针母液，用PBS溶液稀释到终体积100μL，加入到共聚焦小皿中，在37℃、5％CO 2的条件孵育30分钟。去掉探针溶液，加入200μL的1mg/mL的台盼蓝溶液。静置一分钟后吸去台盼蓝溶液，用PBS溶液小心清洗细胞三次，加入含有10％胎牛血清的无色DMEM培养基，带到成像系统观察。
激光扫描共聚焦显微成像实验：通过激光扫描共聚焦显微镜LSM 710(Zeiss,German)观察。Alexa Fluor 647的最大激发波长为650nm，最大发射波长为665nm；LysoTracker Red的最大激发波长为577nm，最大发射波长为590nm。基于Alexa Fluor 647的半胱氨酸蛋白酶C1家族蛋白酶荧光探针标记细胞后，激光扫描共聚焦成像的结果见图3。图3显示，探针母液的使用量在14μL到71μL范围内时，都能顺利标记到细胞内的点状结构；同时，经过台盼蓝溶液检测，细胞仍然没有被染成蓝色，证明了细胞的活性。图4中，图a显示的是基于Alexa Fluor 647的半胱氨酸蛋白酶C1家族蛋白酶荧光探针所标记到的点状结构，可以与图b中LysoTracker Red的标记结果一致。在两个通道的叠加图c中，两种信号可以良好地共定位，证明基于Alexa Fluor 647的半胱氨酸蛋白酶C1家族蛋白酶荧光探针标记到的是正确的活细胞溶酶体结构。
活细胞STORM成像缓冲液的配置：a)缓冲液A(pH 8.0)，含有10mM的Tris和50mM的NaCl；b)过氧化氢酶Catalase溶液：使用缓冲液A溶解，终浓度为17mg/mL，分装成10μL/管的小份，置于-20℃冰箱保存；c)葡萄糖氧化酶Glucose Oxidase溶液：使用缓冲液A溶解，终浓度为70mg/mL，分装成40μL/管的小份，置于-20℃冰箱保存；d)1M的巯基乙胺MEA溶液：使用0.25N的盐酸溶液溶解，终浓度为70mg/mL，置于-20℃冰箱保存；e)GLOX溶液：c、d两步中的溶液各取一管，混合均匀，共50μL，在4℃冰箱可保存2星期，现用现取；f)活细胞成像缓冲液：取700μL DMEM培养基，分别加入0.0125g的HEPES(终浓度为75mM)和0.014g的Glucose(终浓度为2％)溶解；g)活细胞STORM成像缓冲液：分别将1.2μL的巯基乙胺MEA溶液以及2μL的GLOX溶液加入到200μL的活细胞成像缓冲液中，配制完成后可用60min。
随机光学重构超分辨率显微成像实验：通过超分辨率显微镜N-STORM(Nikon,Japan)观察。Alexa Fluor 647的最大激发波长为650nm，最大发 射波长为665nm。在寻找合适的激光功率时均采用以下方法：将细胞样品置于载物台上，用夹子固定好。使用明场视野先找到焦面，再用汞灯结合相应的滤光片，寻找标记清晰、信号背景比高的视野，快速关掉汞灯，减少对样本荧光的淬灭。选用合适的激发光波长，以极小的功率(能看清视野里的荧光样品即可)观察，调节合适的TIRF角度以得到最佳信号背景比的图像。将激发光的功率缓慢提高，当超过一定阈值后，视野中的荧光分子将开始闪烁，记录此时使用的激光功率大小，即为当前探针的默认激发光功率。依据样本的闪烁程度，选用低密度定位重建算法MaLiang来处理图像，得到随机光学重构超分辨显微成像的结果(图5)。图5显示，与未计算前的全内反射成像叠加图(左下角)相比，随机光学重构超分辨图像(右上角)有效地提高了分辨率，我们测量了图中白色方框所标识的溶酶体的半高全宽为130.38nm，其尺度在200nm以下，是有效的超分辨率显微成像结果。本实验证明，基于Alexa Fluor 647的半胱氨酸蛋白酶C1家族蛋白酶荧光探针具有实现随机光学重构超分辨率显微成像的能力，能够获得高分辨率的活细胞溶酶体图像。
结构光照明超分辨率显微成像实验：通过超分辨率显微镜N-SIM(Nikon,Japan)观察。Alexa Fluor 647的最大激发波长为650nm，最大发射波长为665nm；LysoTracker Red的最大激发波长为577nm，最大发射波长为590nm；Atto 565的最大激发波长为563nm，最大发射波长为592nm；Atto 488的最大激发波长为501nm，最大发射波长为523nm。使用2D-SIM模式拍摄样品，由九张原始图像(三个角度，三个相位)计算出一张SIM结果，其中每张原始图像的曝光时间设定为30ms。考虑到溶酶体在活细胞中的快速运动能力，我们将单色成像时间间隔设置为1s，对细胞连续进行了300帧以上的拍摄。实际拍摄完成后，每张SIM结果图之间的时间间隔平均在1.15s左右。在基于Alexa Fluor 647的半胱氨酸蛋白酶C1家族蛋白酶荧光探针与标准溶酶体标记物LysoTracker Red的双色成像实验中，考虑 到成像系统本身的模块转换速度，我们将成像时间间隔设置为6s。
基于拍摄到的SIM延时成像结果，我们以溶酶体的圆心为基准，使用ImageJ软件记录溶酶体坐标，导入MATLAB后，绘制了四个U2OS细胞中101个溶酶体在焦平面的轨迹图(图6)。从图6中可以看出，溶酶体的运动也有很多种类：(i)有些溶酶体的运动路线长而复杂，它们可能会经历一个快速的、有方向性的运动，之后，在一定范围内进行复杂的多方向运动，在这之后，这个溶酶体可能会稳定下来，也可能重新加速，继续朝某个方向快速运动(如图6b中的两条红线)；(ii)有些溶酶体在一定的区域内进行着类似于自由扩散的运动，扩散的距离没有第一类远，速度也没有第一类快，而且通常方向不确定，运动轨迹犹如一团散乱的毛线(如图6c中的橙线)；(iii)还有一类溶酶体几乎没有位移，只在原地进行非常微小的运动，考虑到手动追踪的误差，我们认为这类溶酶体是几乎不动的(图6a-d中均有典型的几乎没有位移的轨迹，例如c图右上角的橙色、蓝色线)。尽管从以上分析可以看出，绝大多数溶酶体都处于相对稳定的状态，但仍有小部分溶酶体存在着较快的运动速度和较大的位移，如果成像的速度过慢、总时间过短都可能丢失很多运动细节。因此，这对探针的亮度、抗漂白能力都提出了要求，实践检验，我们所开发的基于Atto 565的半胱氨酸蛋白酶C1家族蛋白酶荧光探针可以满足以上要求，意味着其在对溶酶体的快速、长时程超分辨成像中有着极强的潜力。
此外，我们所开发的基于Atto 565的半胱氨酸蛋白酶C1家族蛋白酶荧光探针对溶酶体的透膜有机荧光探针除了可以应用在溶酶体的轨迹追踪中之外，我们还发现由于其特异性地标记溶酶体内的半胱氨酸蛋白酶C1家族蛋白酶，因此可以依据探针荧光强度在溶酶体中的不均匀分布，来推测其在溶酶体中的分布。除了图6中的大多数溶酶体的均匀的荧光分布图案之外，我们还在图7中举例显示了几种不均匀荧光分布的种类：一个溶酶体中有两个亮点、一个不均匀的亮点、一个圆形的亮点或者半月形的亮点。 这些亮点在整个溶酶体圆形或者椭圆的轮廓里非常明显，并且可以随着溶酶体的运动或晃动在溶酶体内移动位置。这些溶酶体内荧光的不均匀分布很可能暗示着不同的溶酶体中半胱氨酸蛋白酶C1家族蛋白酶的含量和分布不同，鉴于半胱氨酸蛋白酶C1家族蛋白酶在溶酶体酶中的重要作用，很可能意味着它们消化、水解底物的能力有所不同。本实验证明了我们所开发的基于Atto 565的半胱氨酸蛋白酶C1家族蛋白酶荧光探针在结构光照明超分辨成像中具有优异的图像质量，可以描述出半胱氨酸蛋白酶C1家族蛋白酶在溶酶体内的不均匀分布。
如图8a所示，LysoTracker Red除了标记溶酶体外，还在实际上为细胞核的圆形黑暗区域附近形成了一片不均匀的非特异性分布。在此强调“不均匀”是因为，不均匀的背景由于有些位置的荧光强度很高——特别是溶酶体边缘的部分，很难确定溶酶体真实的边界。然而从图8b中基于Alexa647的半胱氨酸蛋白酶C1家族蛋白酶荧光探针的标记可以看出，只要是在焦面上的溶酶体都存在着清晰的边界。两者成像结果的背景差别，在延时成像的系列图a2和图b2的对比中更为明显。在运动中，基于Alexa Fluor 647的半胱氨酸蛋白酶C1家族蛋白酶荧光探针标记的溶酶体有着清晰的轮廓和明显的形状，而LysoTracker Red标记的溶酶体则一直处于模糊的背景中。在此需要注意的是，有些溶酶体在运动中不呈现圆形，主要原因是快速运动的溶酶体可能由于力的作用变形，这在SIM成像中的溶酶体中比较常见，而在传统显微镜成像时较难发现。为了证明我们所设计的溶酶体探针的优秀特异性并非偶然，我们又分别使用了基于Atto 565的半胱氨酸蛋白酶C1家族蛋白酶荧光探针和基于Atto 488的半胱氨酸蛋白酶C1家族蛋白酶荧光探针标记了U2OS细胞，由图8c-d可以看到，两者标记到的溶酶体同样都有着清晰的边界轮廓。
将这四种探针放在一起比较，我们可以看到LysoTracker Red的不均匀背景非常明显，而基于Alexa Fluor 647的半胱氨酸蛋白酶C1家族蛋白酶荧 光探针、基于Atto 565的半胱氨酸蛋白酶C1家族蛋白酶荧光探针和基于Atto 488的半胱氨酸蛋白酶C1家族蛋白酶荧光探针几乎没有可察觉的背景，证明这三种针对溶酶体的透膜有机荧光探针在结构光照明超分辨率显微成像当中比LysoTracker Red更适合作为溶酶体的标记物。
实施例6
一种荧光探针，包括实施例4提供的短肽，所述短肽的赖氨酸残基结合有荧光染料分子。所述荧光染料分子，为Alexa Fluor 647 NHS ester、Cy3B NHS ester或Atto 488 NHS ester，分别命名为基于Alexa Fluor 647的肌动蛋白荧光探针、基于Cy3B的肌动蛋白荧光探针或基于Atto 488的肌动蛋白荧光探针。
所述荧光探针按照如下方法合成：
(1)短肽合成：运用固相合成法，按照顺序分别连接侧链保护的氨基酸或小分子；合成路线如下：
Figure PCTCN2018084718-appb-000009
(2)短肽纯化：去掉氨基酸侧链的保护，并将无染料肽段从树脂上切下来，经过HPLC纯化至95％纯度以上，收集产物组分后冷冻干燥，浓缩得到所需的无染料肽段的晶体；
(3)荧光染料分子连接，合成路线如下：
具体步骤如下：使用0.1M浓度的NaHCO 3溶液溶解无染料的探针粉末，溶液终浓度为0.5mM，过滤除菌。将购买的Alexa Fluor 647 NHS ester(1mg,Thermo Fisher Scientific,Inc.)、Cy3B NHS ester(1mg,GE Healthcare  shanghai Co.,Ltd)或Atto 488 NHS ester(1mg,Sigma-Aldrich Co.,LLC)染料经无水DMSO溶解，分装成约30nmol/管的小份，并将管中的溶剂抽干，置于-20℃遮光保存。取一管分装后的染料，加入20μL无水DMSO溶解，并逐滴加入到28μL无染料的探针母液中，混合均匀。室温条件下，在摇床上遮光摇动2h以上，可过夜。
(4)荧光探针纯化：在上述合成步骤完成之后，将反应体系中的液体抽干，使用带有0.5％TFA和5％乙腈的水溶液溶解探针，用C18反向柱进行柱层析，根据探针的极性，洗脱液使用每次20μL的纯度为50～80％的乙腈水溶液体系，收集产物组分后将溶液抽干，得到产物晶体，加入200μL PBS溶液，置于4℃冰箱备用。
至此，分别制得基于Alexa Fluor 647的肌动蛋白荧光探针、基于Cy3B的肌动蛋白荧光探针或基于Atto 488的肌动蛋白荧光探针。
本实施例提供的荧光探针，成像实验如下：
细胞准备方法与实施例5同理。
激光扫描共聚焦显微成像实验：通过激光扫描共聚焦显微镜LSM 710(Zeiss,German)观察。Alexa Fluor 647的最大激发波长为650nm，最大发射波长为665nm；GFP的最大激发波长为488nm，最大发射波长为507nm。使用21μL的探针孵育原代培养的星形神经胶质细胞(Astrocyte)30min后(图9a)，细胞内的探针多数为弥散状分布，仅在细胞边缘和丝状伪足处形成了丝状标记的结构，这说明进入细胞的探针浓度不够，不能达到全部Lifeact识别基团结合肌动蛋白的阈值；同时，由于细胞内的探针亮度不足，导致细胞内外的对比度很低。当探针的使用量为36μL时(图9b)，细胞内出现了明显的丝状标记结构，但在细胞中间的部分仍有模糊；同时，与21μL的探针用量时相比，由于胞内信号比较强，因而胞外背景也相对较小。当探针的用量为43～57μL时(图9c-d)，细胞内的丝状结构的亮度进一步提高，而胞外背景几乎可以忽略；同时，使用较高的浓度(57μL)孵育细胞 后，经过台盼蓝溶液处理，明场下的细胞没有被台盼蓝染色，说明细胞在探针孵育后仍然保持活性。鉴于探针的用量为43～57μL时，不论是胞内还是胞外，探针的标记都有着较高的对比度，因此我们推荐将探针的用量确定在这个范围之内。同时，本实验也证明了我们设计合成的新型微丝探针可以应用于难转染的原代星形胶质细胞的标记当中。图10中，图a显示的是基于Alexa Fluor 647的肌动蛋白荧光探针所标记到的点状结构，可以与图b中GFP-actin的标记结果一致。在两个通道的叠加图中，两种信号可以良好地共定位，证明基于Alexa Fluor 647的肌动蛋白纤维荧光探针标记到的是正确的结构。
活细胞STORM成像缓冲液的配置与实施例9一致。
成像条件：通过超分辨率显微镜N-STORM(Nikon,Japan)或ELYRA P.1(Zeiss,German)观察。Alexa Fluor 647的最大激发波长为650nm，最大发射波长为665nm；Cy3B的最大激发波长为559nm，最大发射波长为570nm；Atto 488的最大激发波长为501nm，最大发射波长为523nm。在寻找合适的激光功率时采用的方法与实施例5一致。依据样本的闪烁程度，选用低密度定位重建算法MaLiang来处理图像，得到随机光学重构超分辨显微成像的结果(图11)。图11显示，与未计算前的全内反射成像叠加图(左下角)相比，随机光学重构超分辨图像(右上角)有效地提高了分辨率，Alexa 647可以达到60nm以下的分辨率(图11a)，Cy3B可以达到90nm的空间分辨率(图11b)，而Atto 488可以达到90nm以下的空间分辨率(图11c)。几种染料能够达到的空间分辨率不同，其原因主要在于染料在每个闪烁事件当中发出的光子数不同，此光子数影响到的是算法对染料分子的定位精度，是影响空间分辨率的最主要的直接因素。而光闪烁染料的“亮-暗态”比率影响着结果的图像质量。本实验证明了商业染料在活细胞随机光学重构超分辨率显微成像中优秀的性质(优秀的闪烁能力和高亮度，主要体现在对图像质量和空间分辨率的贡献两方面)，说明在透膜有机荧光 探针中引入商业染料的重要性。
全内反射结构光照明超分辨率显微成像实验：通过高数值孔径(1.78)的全内反射结构光照明超分辨率显微镜High-NA TIRF-SIM观察。Atto 488的最大激发波长为501nm，最大发射波长为523nm；EGFP和GFP的最大激发波长为488nm，最大发射波长为507nm。使用TIRF-SIM模式拍摄样品，由九张原始图像(三个角度，三个相位)计算出一张SIM结果，其中每张原始图像的曝光时间设定为7ms，结果如图12所示。图12a中，是基于Atto 488的肌动蛋白荧光探针的标记结果，细胞内的背景非常低，而信号本身又足够亮，因此信号背景比很高，图像很清晰；不论是细胞内部较细的微丝纤维，还是细胞外围较粗的纤维都很明显和清晰，尤其是在TIRF叠加图内分辨不清的较粗的纤维，在SIM重建图中会显示出多根细纤维交缠成一束的结果，证明SIM成像的确有效地提高了分辨率。图12b中，GFP-actin标记的微丝有着较差的信号背景比，细胞内部除了可以分辨出较粗的纤维之外，均是浑浊的背景；细胞外部的丝状伪足可以看到放射状的纤维；整体上讲，SIM重建图比TIRF叠加图没有明显的分辨率提升。图12c所使用的EGFP-Lifeact则有着较强的信号背景比，尽管其胞内的背景很高，但仍然能获得较高质量的图像。总结来说，本实验证明基于Atto 488的肌动蛋白荧光探针在结构光照明超分辨率显微成像中，由于其出色的亮度、很低的背景(探针孵育细胞的浓度可控)以及清晰的分辨能力而表现最佳。
本领域的技术人员容易理解，以上所述仅为本发明的较佳实施例而已，并不用以限制本发明，凡在本发明的精神和原则之内所作的任何修改、等同替换和改进等，均应包含在本发明的保护范围之内。

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  1. 一种短肽，其特征在于，包括具有负载功能肽段和识别功能肽段；所述负载功能肽段为赖氨酸残基和甘氨酸残基的间隔重复序列，其重复次数在2次至5次之间；所述负载功能肽段和所述识别功能肽段以1至2个甘氨酸残基为连接基团连接。
  2. 如权利要求1所述的短肽，其特征在于，所述重复次数为3。
  3. 如权利要求1所述的短肽，其特征在于，所述短肽包括穿膜肽段，通过连接肽段与负载功能肽段或识别功能肽段相连。
  4. 如权利要求3所述的短肽，其特征在于，所述穿膜肽段为八聚精氨酸，序列为rRrRrRRR，其中r为D型精氨酸，R为L型精氨酸。
  5. 如权利要求3所述的短肽，其特征在于，所述连接肽段为甘氨酸序列GG或G。
  6. 如权利要求3所述的短肽，其特征在于，所述识别功能肽段为半胱氨酸蛋白酶C1家族蛋白酶或肌动蛋白的识别序列。
  7. 一种荧光探针，其特征在于，包括如权利要求1至6任意一项所述短肽，所述短肽的赖氨酸残基结合有荧光染料分子。
  8. 如权利要求7所述的荧光探针，其特征在于，所述荧光染料分子为具有NHS活性基团的活性荧光染料分子，优选Alexa Fluor 647 NHS ester、Cy3B NHS ester、Atto 565 NHS ester和/或Atto 488 NHS ester。
  9. 如权利要求7或8所述的荧光探针的制备方法，其特征在于，包括以下步骤：
    (1)短肽合成：采用固相合成法，合成如权利要求1至6任意一项所述的短肽；
    (2)短肽纯化：将步骤(1)中获得的短肽从用于固相合成法的树脂上剪切，去掉所述氨基酸侧链的保护基团，并纯化浓缩得到经纯化后的所 述短肽；
    (3)荧光染料分子连接：采用液相反应法，将步骤(2)中获得的短肽的赖氨酸残基上的游离氨基与荧光染料分子的NHS活性基团共价结合，获得如权利要求1至7任意一项所述的荧光探针。
  10. 如权利要求9所述的荧光探针的制备方法，其特征在于，还包括以下步骤：
    (4)荧光探针纯化：将步骤(3)中获得的荧光探针溶解，采用反向柱层析纯化，得到纯化后的所述荧光探针分子。
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