Polypeptide probe and application thereof

Abstract

The invention relates to a polypeptide probe and application thereof. The structure of the polypeptide probe is as follows: a peptide chain 1-L-peptide chain 2, wherein L is # imgabs0 #, and m is an integer from 1 to 15. The polypeptide probe provided by the invention can be specifically combined with the exosome, and is simple to operate, trouble-saving and labor-saving.

Description

Technical Field

[0001] The present invention relates to the field of molecular detection technology, and in particular to a polypeptide probe and application thereof. Background Art

[0002] Exosomes are nanoscale vesicles secreted by cells, with a diameter of about 30-150 nanometers and a typical phospholipid bilayer membrane structure. They can protect the proteins, RNA, DNA and other biological molecules contained therein, transmit information between cells, and participate in various physiological processes such as cell communication, immune response, and cell metabolism. The study of exosomes is of great significance for revealing disease mechanisms and developing new diagnostic markers and treatment strategies.

[0003] For the study of exosome function, it is generally necessary to first observe that the exosomes are taken up by the receptor cells or reach the target tissues and organs, and then detect whether the corresponding phenotypic indicators in the receptor cells, tissues or organs have changed. Therefore, exosome labeling and tracing are the basis and key steps for the study of exosome function.

[0004] In the currently published literature, exosomes are mostly labeled with lipophilic dyes. The principle is that the lipophilic fluorescent membrane dye inserts the long tail of the aliphatic group into the lipid bilayer of the exosome and emits a stable and persistent strong fluorescent signal (embedded in a non-covalent manner), which helps to observe the interaction between exosomes and receptor cells / tissues / organs and has many applications both in vivo and in vitro.

[0005] However, the currently more commonly used small molecule fluorescent probe PKH has the problem of self-aggregation of the fluorescent dye to emit fluorescence, resulting in false positives; and the exosomes stained with PKH also have the problem of agglomeration, and the particle size and distribution of the exosomes will change. The lipophilic carbonyl cyanine dye has an unconditional affinity for lipids and lacks selectivity, and may label non-exosomal lipid structures. The transfection of fluorescent proteins not only requires a lot of preliminary genetic engineering preparation work, but also has different expression rates in each cell, resulting in uneven fluorescence intensity of the secreted exosomes, and the movement trajectory of the fusion protein is different from that of the original protein, affecting the analysis of cell function. Summary of the invention

[0006] The present invention provides an exosome enrichment polypeptide probe which has little effect on exosomes, does not change the particle size and distribution thereof, does not aggregate itself, and has low false positive.

[0007] To achieve the above object, the technical solution adopted by the present invention is: In one aspect, the present invention provides a polypeptide probe having the following structure: Peptide chain 1—L—peptide chain 2; Among them, L is , where m is an integer from 1 to 15; The amino acid sequences of the peptide chain 1 and the peptide chain 2 are selected from any one of the following groups: (1) the amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2; (2) an amino acid sequence that is at least 80%, 82%, 85%, 87%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2, and that retains the biological function of the amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2; (3) An amino acid sequence in which one or more amino acid residues are added, substituted, deleted or inserted into the amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2, and which retains the biological function of the amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2.

[0008] Preferably, m is an integer from 2 to 10.

[0009] Preferably, the structure of the polypeptide probe is as follows: Peptide chain Peptide chain 2; Wherein, the amino acid sequence of the peptide chain 1 is shown as SEQ ID NO:1, and the amino acid sequence of the peptide chain 2 is shown as SEQ ID NO:2.

[0010] Preferably, the N-terminus of the peptide chain 1 is connected to fluorescein.

[0011] Preferably, the fluorescein is selected from one or more of fluorescein isothiocyanate, nitrobenzoxadiazole, 7-amino-4-methylcoumarin, 5-carboxyfluorescein, dansyl chloride, Cy3 and Cy5.

[0012] Further preferably, the fluorescein is selected from one or more of fluorescein isothiocyanate, nitrobenzoxadiazole, 5-carboxyfluorescein, Cy3 and Cy5.

[0013] Preferably, the fluorescein is linked to the N-terminus of peptide chain 1 via 6-aminohexanoic acid.

[0014] In one aspect, the present invention provides use of any of the above-mentioned polypeptide probes in the preparation of a reagent for detecting and / or enriching exosomes.

[0015] In one aspect, the present invention provides a method for detecting exosomes, comprising incubating any of the above-mentioned polypeptide probes with a sample to be tested, and detecting a fluorescent signal.

[0016] Preferably, the concentration of the polypeptide probe is 400-600 nM.

[0017] Effects of the invention: The exosome detection polypeptide probe provided by the present invention can directly stain the exosome membrane, without the need for complex genetic engineering design, is easy to operate, can complete labeling in a short time, and has high specificity. The polypeptide fragment provided by the present invention is rich in lysine (K) and arginine (R), so it can produce strong electrostatic effects with negatively charged molecules such as PS and PI on the exosome membrane, and then bind to the exosome membrane. At the same time, the phosphatidylserine (PS) on the exosome membrane is usually exposed on the outside (different from the PS distribution on the inside of the normal cell membrane), so the polypeptide probe provided by the present invention can more easily bind to the exosome membrane without producing strong non-specific binding to the ordinary cell membrane. At the same time, the hydrophobic amino acids (such as phenylalanine (F) and leucine (L)) in the polypeptide can be inserted into the hydrophobic core region of the phospholipid bilayer, further enhancing the stability of the binding. Therefore, the present application enables the exosome polypeptide probe to bind to the exosome membrane more stably through electrostatic effects and hydrophobic insertion. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 This is a curve diagram of the fluorescent polypeptide probe concentration in Example 1 of the present invention; Figure 2 This is a curve diagram of the relationship between exosome concentration and fluorescence intensity when the fluorescent probe concentration is 500 nM in Example 1 of the present invention; Figure 3 This is a graph of the nanoflow cytometry results in Example 2 of the present invention, wherein A is a single fluorescent polypeptide probe group, which is used for gating; B is a fluorescent polypeptide probe-labeled exosome group, where the fluorescence is enhanced after the probe binds to the exosome, and the positive rate is improved; Figure 4 The nanoflow particle size distribution diagram in Example 2 of the present invention, wherein A is the particle size distribution diagram of exosomes before fluorescent probe labeling; B is the particle size distribution diagram after the fluorescent probe binds to the exosomes, and there is no significant change in the particle size before and after labeling; Figure 5 These are images taken by confocal microscopy for the tracking of exosomes in Example 3 of the present invention, wherein A is a cy3-modified fluorescent probe labeling exosomes (red), B is a phalloidin-stained cytoskeleton (green), C is a DAPI-stained cell nucleus (blue), and D is a fusion image of the three. DETAILED DESCRIPTION

[0019] In order to make the technical solutions and beneficial effects of the present invention more clearly understandable, the following is a detailed description by listing specific embodiments. The drawings are not necessarily drawn to scale, and local features may be enlarged or reduced to more clearly show the details of the local features; unless otherwise defined, the technical and scientific terms used herein have the same meanings as those in the technical field to which this application belongs.

[0020] After extensive and in-depth research, the inventors have proposed a probe polypeptide that has little effect on exosomes, does not change their particle size and distribution, can specifically and stably bind to exosomes, and can observe living cells through a large number of screening and experimental verifications. When the polypeptide probe is coupled with fluorescein and used to label exosomes, fluorescence enhancement occurs, the half-life is long, and long-term observation is possible.

[0021] As used in the present invention, the term "exosome" refers to small vesicles with a complete membrane structure, with a diameter between 30-150nm, which are secreted by a variety of cells and widely exist and distribute in body fluids including blood, saliva, urine, pleural effusion, etc., and are important media for intercellular information transmission.

[0022] The present invention provides a polypeptide probe, the structure of which is as follows: Peptide chain 1—L—peptide chain 2; Among them, L is , where m is an integer from 1 to 15 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15); The amino acid sequences of the peptide chain 1 and the peptide chain 2 are selected from any one of the following groups: (1) the amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2; (2) an amino acid sequence that is at least 80%, 82%, 85%, 87%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2, and that retains the biological function of the amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2; (3) An amino acid sequence in which one or more amino acid residues are added, substituted, deleted or inserted into the amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2, and which retains the biological function of the amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2.

[0023] In the present invention, the amino acid sequence shown in SEQ ID NO: 1 is: RLRLRLRL.

[0024] In the present invention, the amino acid sequence shown in SEQ ID NO: 2 is: KKFRKKFFKKLFKKR.

[0025] In the present application, the peptide chain 1 and the peptide chain 2 may be the same or different.

[0026] As used herein, the terms "polypeptide chain", "peptide" and "protein" are used interchangeably herein to refer to a polymer of amino acid residues. The terms apply to amino acid polymers in which one or more amino acid residues are artificial chemical mimetics of the corresponding naturally occurring amino acids, as well as naturally occurring amino acid polymers and non-naturally occurring amino acid polymers.

[0027] In the present invention, the terms "fluorescent probe" and "fluorescent polypeptide probe" can be used interchangeably, and both refer to polypeptide probes labeled with fluorescein (such as fluorescein isothiocyanate (FITC), nitrobenzoxadiazole, 5-carboxyfluorescein (5-FAM), Cy3 or Cy5).

[0028] As used in this application, the term "identity" is used to refer to the matching of sequences between two polypeptides or between two nucleic acids. When a certain position in the two sequences being compared is occupied by the same base or amino acid monomer subunit (for example, a certain position in each of the two DNA molecules is occupied by adenine, or a certain position in each of the two polypeptides is occupied by lysine), then the molecules are identical at that position. The "percent identity" between two sequences is a function of the number of matching positions shared by the two sequences divided by the number of positions compared × 100. For example, if 6 out of 10 positions of the two sequences match, then the two sequences have 60% identity. For example, the DNA sequences CTGACT and CAGGTT have 50% identity (3 out of a total of 6 positions match). Typically, the comparison is made when the two sequences are aligned to produce maximum identity. Such an alignment can be achieved by using, for example, the method of Needleman et al. (1970) J.Mol.Biol.48:443-453, which can be conveniently performed by a computer program such as the Align program (DNAstar, Inc.). The percent identity between two amino acid sequences can also be determined using the algorithm of E. Meyers and W. Miller (Comput. Appl Biosci., 4: 11-17 (1988)), which has been incorporated into the ALIGN program (version 2.0), using a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4. In addition, the percent identity between two amino acid sequences can be determined using the Needleman and Wunsch (J Mol Biol. 48: 444-453 (1970)) algorithm, which has been incorporated into the GAP program in the GCG software package (available at www.gcg.com), using a Blossum 62 matrix or a PAM250 matrix and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or 6.

[0029] In one embodiment, m is an integer of 2 to 10. In the present invention, the polyethylene glycol segment provides a flexible linker arm, which increases the dynamics of the entire molecular structure, allowing the front segment peptide and the back segment peptide to function independently, avoiding spatial interference between the two functional segments and improving the overall binding efficiency.

[0030] In a specific and preferred embodiment, m is 3, 4, 6 or 8.

[0031] In one embodiment, the structure of the polypeptide probe is as follows: Peptide chain 1 Peptide chain 2; Wherein, the amino acid sequence of the peptide chain 1 is shown as SEQ ID NO:1, and the amino acid sequence of the peptide chain 2 is shown as SEQ ID NO:2.

[0032] In one embodiment, the N-terminus of the peptide chain 1 is connected to fluorescein. In the present invention, when the fluorescein-labeled polypeptide binds to the exosome membrane, the fluorescent group is placed in a more hydrophobic environment, thereby reducing the interaction between the fluorophore and water molecules, thereby reducing the non-radiative attenuation process of the fluorophore, resulting in an increase in fluorescence intensity.

[0033] In the present application, the fluorescein can be selected according to common knowledge, and the present application does not make a sole limitation thereto. In one embodiment, the fluorescein is selected from one or more of fluorescein isothiocyanate (FITC), nitrobenzoxadiazole (NBD), 7-amino-4-methylcoumarin (AMC), 5-carboxyfluorescein (5-FAM), dansyl chloride (Dansyl), Cy3 and Cy5.

[0034] In one embodiment, the fluorescein is selected from one or more of fluorescein isothiocyanate, nitrobenzoxadiazole, 5-carboxyfluorescein, Cy3 and Cy5.

[0035] In one embodiment, the fluorescein is linked to the N-terminus of peptide chain 1 via 6-aminohexanoic acid.

[0036] Specifically, fluorescein is linked to the amino terminus of 6-aminohexanoic acid, and the carboxyl terminus of 6-aminohexanoic acid is linked to the N terminus of peptide chain 1.

[0037] In one aspect, the present invention provides use of any of the above-mentioned polypeptide probes in the preparation of a reagent for detecting and / or enriching exosomes.

[0038] In one aspect, the present invention provides a method for detecting exosomes, comprising incubating any of the above-mentioned polypeptide probes with a sample to be tested, and detecting a fluorescent signal.

[0039] In one embodiment, the concentration of the polypeptide probe is 400-600 nM, for example, 410 nM, 420 nM, 430 nM, 440 nM, 450 nM, 460 nM, 470 nM, 480 nM, 490 nM, 500 nM, 510 nM, 520 nM, 530 nM, 540 nM, 550 nM, 560 nM, 570 nM, 580 nM, 590 nM, 600 nM, etc.

[0040] In one embodiment, the concentration of the polypeptide probe is 450-550 nM.

[0041] In one embodiment, the concentration of the polypeptide probe is 480-520 nM.

[0042] The present invention is further described below by specific examples. Unless otherwise specified herein, "%" represents mass percentage. The materials and reagents in the following examples, unless otherwise specified, are all commonly used materials or reagents in the art, and can be obtained from commercial sources or synthesized by known methods. The experimental methods for which the conditions are not specified in the following implementation cases are usually carried out according to conventional experimental conditions or the conditions recommended by the manufacturers of the relevant reagents (kits).

[0043] Peptide molecules are a class of biologically active molecules. Their amino acid composition, sequence and structure determine their biological effects. Peptide synthesis has become a common technology and commercial service companies can provide synthetic products according to customer needs. We will not repeat the specific details and principles of peptide synthesis and purification here. The polypeptide probe of the present invention is synthesized using conventional technical means or according to known methods.

[0044] The various fluorescent polypeptide probes (fluorescein-Acp-RLRLRLRL KKFRKKFFKKLFKKR) was commissioned to Shenggong Biosynthesis and is in the form of freeze-dried powder. Example 1

[0045] The fluorescent probe freeze-dried powder (5-FAM fluorescent modification) was dissolved in 1╳PBS to prepare a 50μM stock solution. A 10-fold gradient dilution was performed with 1╳PBS to obtain 5μM, 0.5μM, and 0.05μM solutions, which were mixed and incubated with equal amounts of exosomes at 4°C in the dark for 2h. The fluorescence intensity was measured using a fluorescence spectrophotometer (Shanghai Lingguang F98), and the background value of the single fluorescent probe was deducted to calculate the fluorescence gain. The concentration curve of the fluorescent peptide probe is shown in Figure 1 The optimal probe concentration was obtained as 500 nM through the relative fluorescence intensity curve.

[0046] Based on the probe concentration of 500nM, the exosomes were diluted in multiples (initial concentration 6.5μg / mL) to test the corresponding exosome concentration range. The relationship between exosome concentration and fluorescence intensity is shown in Figure 2 , based on the fluorescence enhancement results, the exosome concentration was determined to be 1.6μg / mL-6.5μg / mL. Example 2

[0047] The FITC-modified fluorescent probe was prepared into a 500 nM working solution using 1×PBS.

[0048] The fluorescent probe and mesenchymal stem cell-derived exosomes were mixed in equal amounts of 1:1 and incubated at 4°C in the dark for 1 h. After incubation, the mixture was transferred to a 100 kd ultrafiltration tube (Millipore), 1╳PBS was added to fill the ultrafiltration tube, and centrifuged at 4000 g for 5 min at 4°C. The ultrafiltration tube was removed, and 100 μL of 1╳PBS was added to the inner membrane to resuspend the exosomes.

[0049] The exosomes were diluted appropriately and tested on a nano-flow cytometer (Exoplorer). A single probe group was set as a negative control for gating and the positive rate of the exosome marker group was tested.

[0050] Nanoflow cytometry results Figure 3 As shown, the exosome labeling positive rate is higher than 90%, indicating that the fluorescent probe of the present invention can label exosomes very efficiently.

[0051] The particle size distribution test results are as follows: Figure 4 As shown, according to Figure 4 It can be seen that the median size of exosomes before labeling is 75.9nm, and the average is 85.4nm; the median size of exosomes after labeling is 74.5nm, and the average is 86.1nm. It can be seen that the fluorescent labeling reagent has no significant effect on the exosome particle size distribution, that is, the fluorescently labeled polypeptide probe of the present application has little effect on exosomes and will not change its particle size and distribution. Example 3

[0052] Human dermal fibroblasts were added to a 3.5 cm confocal dish at a density of 200,000 cells / well and cultured for 24 hours.

[0053] Take 5 μg of mesenchymal stem cell-derived exosomes and dilute them to 0.5 mL with 1×PBS solution.

[0054] The cy3-modified fluorescent probe was prepared into a 500 nM working solution with 1×PBS. 500 μL of the fluorescent probe solution was mixed with 500 μL of exosomes and incubated at 4°C for 1 h in the dark.

[0055] After completion, the mixture was added to a 100 kd ultrafiltration tube, supplemented with 1╳PBS to 4 mL, and centrifuged at 4000 g for 5 min.

[0056] Resuspend the exosomes in the ultrafiltration tube with 1╳PBS, gently blow with a pipette, transfer to a centrifuge tube containing serum-free medium and mix well for later use. Fluorescently labeled exosomes were added to human dermal fibroblasts at 50ng / mL and incubated for 24h. After incubation, the cells were fixed with 4% paraformaldehyde for 10min at room temperature, washed with 1╳PBS, and the cytoskeleton was stained with phalloidin for 30min. After adding DAPI to stain the nucleus, the cells were observed and photographed with a confocal microscope.

[0057] The results are as follows Figure 5 As shown, it can be observed that mesenchymal stem cell exosomes can be taken up into human dermal fibroblasts. Therefore, the fluorescently labeled polypeptide probe provided by the present invention can trace and observe exosomes without affecting cell activity and the uptake of exosomes.

[0058] It should be understood that the above embodiments are exemplary and are not intended to include all possible implementations included in the claims. Various modifications and changes may be made on the basis of the above embodiments without departing from the scope of the present disclosure. Similarly, the various technical features of the above embodiments may be arbitrarily combined to form other embodiments of the present invention that may not be explicitly described. Therefore, the above embodiments only express several implementations of the present invention and do not limit the scope of protection of the patent of the present invention.

Claims

1. A polypeptide probe, characterized in that: The structure of the polypeptide probe is as follows: Peptide chain 1—L—peptide chain 2; Among them, L is , where m is an integer from 1 to 15; The amino acid sequences of the peptide chain 1 and the peptide chain 2 are selected from any one of the following groups: (1) the amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2; (2) an amino acid sequence that is at least 80%, 82%, 85%, 87%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2, and that retains the biological function of the amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2; (3) An amino acid sequence in which one or more amino acid residues are added, substituted, deleted or inserted into the amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2, and which retains the biological function of the amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO:

2.

2. The polypeptide probe according to claim 1, characterized in that The m is an integer from 2 to 10.

3. The polypeptide probe according to claim 1, characterized in that The structure of the polypeptide probe is as follows: Peptide chain 1 Peptide chain 2; Wherein, the amino acid sequence of the peptide chain 1 is shown as SEQ ID NO:1, and the amino acid sequence of the peptide chain 2 is shown as SEQ ID NO:

2.

4. The polypeptide probe according to claim 1, characterized in that The N-terminus of the peptide chain 1 is connected to fluorescein.

5. The polypeptide probe according to claim 4, characterized in that The fluorescein is selected from one or more of fluorescein isothiocyanate, nitrobenzoxadiazole, 7-amino-4-methylcoumarin, 5-carboxyfluorescein, dansyl chloride, Cy3 and Cy5.

6. The polypeptide probe according to claim 5, characterized in that The fluorescein is selected from one or more of fluorescein isothiocyanate, nitrobenzoxadiazole, 5-carboxyfluorescein, Cy3 and Cy5.

7. The polypeptide probe according to claim 4, characterized in that The fluorescein is linked to the N-terminus of peptide chain 1 via 6-aminohexanoic acid.

8. Use of the polypeptide probe according to any one of claims 1 to 7 in the preparation of a reagent for detecting and / or enriching exosomes.

9. A method for detecting exosomes, characterized in that: The method comprises co-incubating the polypeptide probe according to any one of claims 1 to 7 with a sample to be tested, and detecting a fluorescent signal.

10. The method according to claim 9, characterized in that The concentration of the polypeptide probe is 400-600 nM.

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