A polypeptide probe and its application

Abstract

The present invention relates to a polypeptide probe and its application. The structure of the polypeptide probe is as follows: peptide chain 1 - L - peptide chain 2; wherein, L is #imgabs0# where m is an integer from 1 to 15. The polypeptide probe provided by the present invention can specifically bind to exosomes, and the operation is simple, saving time and effort.

Description

Technical Field

[0001] The present invention relates to the technical field of molecular detection, and particularly relates to a polypeptide probe and its application. Background Art

[0002] Exosomes are nanoscale vesicles secreted by cells, with a diameter of about 30 - 150 nanometers, having a typical phospholipid bilayer membrane structure, which can protect various biomolecules such as contained proteins, RNAs, and DNAs, and transmit information between cells, participating in various physiological processes such as cell communication, immune response, and cell metabolism. The research on exosomes is of great significance for revealing disease mechanisms, developing new diagnostic markers, and treatment strategies.

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

[0004] In the currently published literature, exosomes are mostly labeled with lipophilic dyes. The principle is that lipophilic fluorescent membrane dyes insert their long aliphatic tails into the lipid bilayer of exosomes and then emit stable and persistent strong fluorescence signals (embedded in a non-covalent manner), which helps to observe the interaction between exosomes and recipient cells / tissues / organs and has many applications both in vivo and in vitro.

[0005] However, the currently commonly used small molecule fluorescent probe PKH has the problem of false positives caused by the aggregation of the fluorescent dye itself and emitting fluorescence; moreover, the exosomes stained with PKH also have the problem of aggregation, and the particle size and distribution of exosomes will change. The unconditional affinity of lipophilic cyanine dyes for lipids, lacking selectivity, may label non-exosomal lipid structures. The method of transfecting fluorescent proteins not only requires a large amount of preparatory genetic engineering work in the early stage, and the expression rate of each cell is also different, resulting in uneven fluorescence intensity of the secreted exosomes, and the movement trajectories of the fusion protein and the original protein are different, affecting cell function analysis. Summary of the Invention

[0006] The present invention provides an exosome enrichment polypeptide probe that has little influence on exosomes, does not change their particle size and distribution, and at the same time does not aggregate itself and has a low false positive rate.

[0007] To achieve the above object, the technical solution adopted by the present invention is:

[0008] On the one hand, the present invention provides a polypeptide probe, and the structure of the polypeptide probe is as follows:

[0009] Peptide chain 1 - L - Peptide chain 2;

[0010] Among them, L is , where m is an integer from 1 to 15;

[0011] The amino acid sequences of the peptide chain 1 and the peptide chain 2 are selected from any one of the following groups:

[0012] (1) The amino acid sequence shown in SEQ ID NO:1 or SEQ ID NO:2;

[0013] (2) An amino acid sequence having at least 80%, 82%, 85%, 87%, 90%, 92%, 95%, 96%, 97%, 98%, 99% identity with the amino acid sequence shown in SEQ ID NO:1 or SEQ ID NO:2, and retaining the biological function of the amino acid sequence shown in SEQ ID NO:1 or SEQ ID NO:2;

[0014] (3) An amino acid sequence with one or more amino acid residues added, substituted, deleted or inserted in the amino acid sequence shown in SEQ ID NO:1 or SEQ ID NO:2, and retaining the biological function of the amino acid sequence shown in SEQ ID NO:1 or SEQ ID NO:2.

[0015] Preferably, the m is an integer from 2 to 10.

[0016] Preferably, the structure of the polypeptide probe is as follows:

[0017] Peptide chain Peptide chain 2;

[0018] Among them, the amino acid sequence of the peptide chain 1 is as shown in SEQ ID NO:1, and the amino acid sequence of the peptide chain 2 is as shown in SEQ ID NO:2.

[0019] Preferably, a fluorescein is connected to the N-terminus of the peptide chain 1.

[0020] Preferably, the fluorescein is selected from one or more of fluorescein isothiocyanate, nitrobenzoxadiazole, 7-amino-4-methylcoumarin, 5-carboxyfluorescein, dansyl chloride, Cy3 and Cy5.

[0021] More preferably, the fluorescein is selected from one or more of fluorescein isothiocyanate, nitrobenzoxadiazole, 5-carboxyfluorescein, Cy3 and Cy5.

[0022] Preferably, the fluorescein is connected to the N-terminus of the peptide chain 1 through 6-aminohexanoic acid.

[0023] On the one hand, the present invention provides the application of the polypeptide probe described in any one of the above in the preparation of a reagent for detecting and / or enriching exosomes.

[0024] On the one hand, the present invention provides a method for detecting exosomes, the method comprising co-incubating the polypeptide probe described in any one of the above with a sample to be tested and detecting the fluorescence signal.

[0025] Preferably, the concentration of the polypeptide probe is 400-600 nM.

[0026] Effects of the invention:

[0027] The exosome detection polypeptide probe provided by the present invention can directly stain the exosome membrane, without complex genetic engineering design, is easy to operate, can be labeled in a short time, and has high specificity. The polypeptide fragment provided by the present invention is rich in lysine (K) and arginine (R). Therefore, it can produce strong electrostatic interactions with negatively charged molecules such as PS and PI on the exosome membrane, and then bind to the exosome membrane. At the same time, phosphatidylserine (PS) on the exosome membrane is usually exposed on the outside (different from the distribution of PS on the inner side of the normal cell membrane). Therefore, the polypeptide probe provided by the present invention can more easily bind to the exosome membrane without producing strong non-specific binding to ordinary cell membranes. At the same time, hydrophobic amino acids in the polypeptide (such as phenylalanine (F), leucine (L)) can be inserted into the hydrophobic core region of the phospholipid bilayer, further enhancing the stability of the binding. Therefore, through the electrostatic interaction and hydrophobic insertion, the exosome polypeptide probe of the present application can bind to the exosome membrane more stably. Description of the drawings

[0028] Figure 1 It is the fluorescence polypeptide probe concentration curve graph in Example 1 of the present invention;

[0029] Figure 2 In Example 1 of the present invention, it is the relationship curve graph between the exosome concentration and the fluorescence intensity under the condition that the fluorescence probe concentration is 500 nM;

[0030] Figure 3 It is the nano-flow cytometry result graph in Example 2 of the present invention. Among them, A is the group of the single fluorescence polypeptide probe, used for gating; B is the group of the exosomes labeled with the fluorescence polypeptide probe. After the probe binds to the exosomes, the fluorescence increases and the positive rate improves;

[0031] Figure 4 It is the nano-flow cytometry particle size distribution graph in Example 2 of the present invention. Among them, A is the particle size distribution graph of the exosomes before being labeled with the fluorescence probe; B is the particle size distribution graph after the fluorescence probe binds to the exosomes. There is no significant change in the particle size before and after labeling;

[0032] Figure 5This is the confocal microscopy image of exosome tracing in Example 3 of the present invention. Among them, A is the exosome labeled with a cy3-modified fluorescent probe (red), B is the phalloidin-stained cytoskeleton (green), C is the DAPI-stained cell nucleus (blue), and D is the merged image of the three. Detailed implementation mode

[0033] To make the technical solutions and beneficial effects of the present invention more obvious and understandable, the following will be described in detail by listing specific examples. Among them, the drawings are not necessarily drawn to scale, and local features can be enlarged or reduced to more clearly show the details of local features; unless otherwise defined, the technical and scientific terms used herein have the same meanings as those in the technical and scientific fields to which this application belongs.

[0034] Through extensive and in-depth research, a large number of screenings and experimental verifications, the present inventors have proposed a probe polypeptide that has little influence on exosomes, does not change their particle size and distribution, can specifically and stably bind to exosomes, and can be used for live cell observation. When the polypeptide probe is conjugated with a fluorophore and used to label exosomes, a fluorescence enhancement phenomenon occurs, and the half-life is long, enabling long-term observation.

[0035] As used in the present invention, the term "exosome" refers to small vesicles with a complete membrane structure, with a diameter between 30 and 150 nm, secreted by various cells and widely present and distributed in body fluids including blood, saliva, urine, and pleural and peritoneal effusions, etc., and is an important medium for intercellular information transmission.

[0036] The present invention provides a polypeptide probe, and the structure of the polypeptide probe is as follows:

[0037] Peptide chain 1 - L - peptide chain 2;

[0038] Among them, L is , where m is an integer from 1 to 15 (for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15);

[0039] The amino acid sequences of the peptide chain 1 and the peptide chain 2 are selected from any one of the following groups:

[0040] (1) The amino acid sequences shown in SEQ ID NO:1 or SEQ ID NO:2;

[0041] (2) An amino acid sequence having at least 80%, 82%, 85%, 87%, 90%, 92%, 95%, 96%, 97%, 98%, 99% identity to the amino acid sequence shown in SEQ ID NO:1 or SEQ ID NO:2, and retaining the biological function of the amino acid sequence shown in SEQ ID NO:1 or SEQ ID NO:2;

[0042] (3) An amino acid sequence in which one or more amino acid residues are added, substituted, deleted or inserted in the amino acid sequence shown in SEQ ID NO:1 or SEQ ID NO:2, and retaining the biological function of the amino acid sequence shown in SEQ ID NO:1 or SEQ ID NO:2.

[0043] In the present invention, the amino acid sequence shown in SEQ ID NO:1 is: RLRLRLRL.

[0044] In the present invention, the amino acid sequence shown in SEQ ID NO:2 is: KKFRKKFFKKLFKKR.

[0045] In the present application, the peptide chain 1 and the peptide chain 2 may be the same or different.

[0046] As used herein, the terms "polypeptide chain", "peptide" and "protein" are used interchangeably herein to denote a polymer of amino acid residues. The term applies to amino acid polymers in which one or more amino acid residues are artificial chemical mimics of the corresponding naturally occurring amino acids, as well as to naturally occurring amino acid polymers and non-naturally occurring amino acid polymers.

[0047] In the present invention, the terms "fluorescent probe" and "fluorescent polypeptide probe" are used interchangeably, and both refer to a polypeptide probe labeled with a fluorophore (such as fluorescein isothiocyanate (FITC), nitrobenzoxadiazole, 5-carboxyfluorescein (5-FAM), Cy3 or Cy5).

[0048] As used in this application, the term "identity" is used to refer to the sequence matching between two polypeptides or two nucleic acids. When a position in two sequences being compared is occupied by the same base or amino acid monomer subunit (e.g., a position in each of two DNA molecules is occupied by adenine, or a position in each of 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 being compared × 100. For example, if 6 of 10 positions in two sequences match, then the two sequences have 60% identity. For example, the DNA sequences CTGACT and CAGGTT have 50% identity (3 of 6 positions in total match). Generally, the comparison is made by aligning the two sequences to yield maximum identity. Such 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 algorithm of E. Meyers and W. Miller (Comput. Appl. Biosci., 4:11 - 17 (1988)), incorporated into the ALIGN program (version 2.0), can also be used, with a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4 to determine the percent identity between two amino acid sequences. In addition, the algorithm of Needleman and Wunsch (J. Mol. Biol. 48:444 - 453 (1970)) incorporated into the GAP program in the GCG software package (available at www.gcg.com) can be used, with a Blossum 62 matrix or a PAM250 matrix and gap weights of 16, 14, 12, 10, 8, 6, or 4 and length weights of 1, 2, 3, 4, 5, or 6 to determine the percent identity between two amino acid sequences.

[0049] In one embodiment, the m is an integer from 2 to 10. In the present invention, the polyethylene glycol chain segment provides a flexible linker arm, increasing the dynamics of the entire molecular structure, enabling the front - segment peptide and the rear - segment peptide to function independently, avoiding spatial interference between the two functional segments, and enhancing the overall binding efficiency.

[0050] In a specific and preferred embodiment, the m is 3, 4, 6, or 8.

[0051] In one embodiment, the structure of the polypeptide probe is as follows:

[0052] Peptide chain 1 Peptide chain 2;

[0053] Among them, the amino acid sequence of the peptide chain 1 is as shown in SEQ ID NO:1, and the amino acid sequence of the peptide chain 2 is as shown in SEQ ID NO:2.

[0054] In one embodiment, a fluorescein is connected to the N-terminus of the peptide chain 1. In the present invention, when the fluorescein-labeled polypeptide binds to the exosome membrane, the fluorophore is placed in a more hydrophobic environment, thereby reducing the interaction between the fluorophore and water molecules, thus reducing the non-radiative decay process of the fluorophore and resulting in an increase in fluorescence intensity.

[0055] In the present application, the fluorescein can be selected according to common general knowledge, and the present application does not make a unique 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.

[0056] In one embodiment, the fluorescein is selected from one or more of fluorescein isothiocyanate, nitrobenzoxadiazole, 5-carboxyfluorescein, Cy3 and Cy5.

[0057] In one embodiment, the fluorescein is connected to the N-terminus of the peptide chain 1 through 6-aminohexanoic acid.

[0058] Specifically, the fluorescein is connected to the amino terminus of 6-aminohexanoic acid, and the carboxyl terminus of 6-aminohexanoic acid is connected to the N-terminus of the peptide chain 1.

[0059] On the one hand, the present invention provides the application of the polypeptide probe as described in any one of the above in the preparation of a reagent for detecting and / or enriching exosomes.

[0060] On the one hand, the present invention provides a method for detecting exosomes, the method comprising co-incubating the polypeptide probe as described in any one of the above with a sample to be tested and detecting a fluorescence signal.

[0061] In one embodiment, the concentration of the polypeptide probe is 400-600 nM, such as 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.

[0062] In one embodiment, the concentration of the polypeptide probe is 450-550 nM.

[0063] In one embodiment, the concentration of the polypeptide probe is 480-520 nM.

[0064] 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).

[0065] 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.

[0066] 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

[0067] 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.

[0068] 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

[0069] The FITC-modified fluorescent probe was prepared into a 500 nM working solution using 1×PBS.

[0070] The fluorescent probe was mixed with mesenchymal stem cell-derived exosomes in an equal volume ratio of 1:1 and incubated at 4°C in the dark for 1 h. After incubation, the mixture was transferred into a 100 kD ultrafiltration tube (Millipore), filled with 1×PBS to the brim of the ultrafiltration tube, and centrifuged at 4000 g for 5 min at 4°C. The ultrafiltration tube was taken out, 100 μL of 1×PBS was added to the inner membrane to resuspend the exosomes.

[0071] The exosomes were diluted appropriately and loaded onto a nano flow cytometer (Exoplorer by Weidu Biosciences) to detect the fluorescence intensity in the B525 channel. A separate probe group was set as a negative control for gating, and the positive rate of the exosome-labeled group was detected.

[0072] The results of the nano flow cytometry were as Figure 3 shown. The positive rate of exosome labeling was higher than 90%, indicating that the fluorescent probe of the present invention could label exosomes very efficiently.

[0073] The results of the particle size distribution detection were as Figure 4 shown. According to Figure 4 it was known that the median particle size of exosomes before labeling was 75.9 nm and the average value was 85.4 nm; the median particle size of exosomes after labeling was 74.5 nm and the average value was 86.1 nm. It can be seen that the fluorescent labeling reagent had no significant effect on the particle size distribution of exosomes, that is, the fluorescently labeled polypeptide probe of the present application had little effect on exosomes and did not change their particle size and distribution. Example 3

[0074] Human dermal fibroblasts were added to a 3.5 cm confocal dish at 200,000 cells / well and cultured for 24 h for standby.

[0075] 5 μg of mesenchymal stem cell-derived exosomes were taken and diluted to 0.5 mL with 1×PBS solution.

[0076] The cy3-modified fluorescent probe was formulated 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 in the dark for 1 h.

[0077] After completion, the mixture was added to a 100 kD ultrafiltration tube, filled with 1×PBS to 4 mL, and centrifuged at 4000 g for 5 min.

[0078] The exosomes in the ultrafiltration tube were resuspended with 1×PBS, gently pipetted, transferred to a centrifuge tube containing serum-free medium and mixed well for standby. The fluorescently labeled exosomes were added to human dermal fibroblasts at 50 ng / mL and incubated for 24 h. After incubation, the cells were fixed with 4% paraformaldehyde at room temperature for 10 min. After washing with 1×PBS, the cell skeleton was stained with phalloidin for 30 min, and then DAPI was added to stain the cell nucleus. Then, the cells were observed and photographed under a confocal microscope.

[0079] The results are as Figure 5 shown, and it can be observed that mesenchymal stem cell exosomes can be taken up by human dermal fibroblasts into the cells. Therefore, the fluorescently labeled polypeptide probe provided by the present invention can be used to trace and observe exosomes without affecting cell viability and the uptake of exosomes.

[0080] It should be understood that the above embodiments are all exemplary and do not cover all possible implementation manners included in the claims. Without departing from the scope of the present disclosure, various deformations and changes can be made on the basis of the above embodiments. Similarly, the various technical features of the above embodiments can also be arbitrarily combined to form additional embodiments of the present invention that may not be explicitly described. Therefore, the above embodiments only represent several implementation manners of the present invention and do not limit the protection scope of the present invention patent.

Claims

1. A polypeptide probe, characterized in that, The structure of the polypeptide probe is as follows: Peptide chain 1—L—peptide chain 2; where L is , where m is 3, 4, 6 or 8; The amino acid sequence of the peptide chain 1 is as shown in SEQ ID NO:1, and the amino acid sequence of the peptide chain 2 is as shown in SEQ ID NO:

2.

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

2.

3. The polypeptide probe according to claim 1, wherein The N-terminus of the peptide chain 1 is connected with a fluorescein.

4. The polypeptide probe according to claim 3, wherein The fluorescein is selected from one or more of fluorescein isothiocyanate, nitrobenzoxadiazole, 7-amino-4-methylcoumarin, 5-carboxyfluorescein, dansyl chloride, Cy3 and Cy5.

5. The polypeptide probe according to claim 4, wherein The fluorescein is selected from one or more of fluorescein isothiocyanate, nitrobenzoxadiazole, 5-carboxyfluorescein, Cy3 and Cy5.

6. The polypeptide probe according to claim 3, wherein The fluorescein is connected to the N-terminus of the peptide chain 1 through 6-aminohexanoic acid.

7. Use of the polypeptide probe according to any one of claims 1 to 6 in the preparation of a reagent for detecting and / or enriching exosomes.

8. A method for detecting exosomes for non-diagnostic and non-therapeutic purposes, characterized in that, The method includes co-incubating the polypeptide probe according to any one of claims 1 to 6 with a sample to be tested and detecting the fluorescence signal.

9. The method according to claim 8, wherein The concentration of the polypeptide probe is 400 to 600 nM.

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