Fusion polypeptide and application thereof in capturing extracellular vesicles

Abstract

The invention relates to a fusion polypeptide and application thereof in capturing extracellular vesicles, and particularly provides a fusion polypeptide comprising a binding peptide domain and a precipitating peptide domain, and a method for enriching, capturing and / or separating extracellular vesicles from a fluid sample by using the fusion polypeptide. The method for purifying the extracellular vesicles is simple in operation and short in time consumption, the yield and purity of the extracted extracellular vesicles are high, and the extraction efficiency of the extracellular vesicles is greatly improved.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a fusion polypeptide and its application in capturing extracellular vesicles. Background Technology

[0002] Exosomes are double-membrane vesicles with a diameter of 30-150 nm secreted by cells. Their membranes carry typical transmembrane proteins and receptors, adhesion molecules, lipid raft-related proteins, and immunomodulatory molecules. Exosomes contain various biological components from the host cell, and differences in their contents directly reflect different states of the parent cell. Therefore, exosomes can play an important role as biomarkers in disease diagnosis and monitoring. Furthermore, exosomes are present in all bodily fluids (including blood, urine, saliva, breast milk, cerebrospinal fluid, semen, amniotic fluid, and ascites), and can be easily obtained through biological sampling (liquid biopsy), which is less invasive for patients and is the best option for liquid biopsy. These advantages further highlight the potential of exosomes in the diagnosis and monitoring of various diseases. However, due to the lack of standardized isolation and purification techniques, exosome purification efficiency is low, making it difficult to meet clinical needs. Therefore, finding a rapid and effective exosome isolation method is fundamental for the clinical testing of exosomes.

[0003] Currently, the main methods for exosome extraction include: 1. Ultracentrifugation and suspension-sedimentation density gradient centrifugation. Ultracentrifugation, considered the gold standard for exosome extraction, is widely used for exosome purification. However, due to the large overlap in particle density of extracellular vesicles, the purity of the obtained product is not high. Suspension-sedimentation density gradient centrifugation can improve this problem. Density gradient centrifugation uses a series of continuous density gradients of sucrose or iododiol to promote the separation of exosomes, which increases the purity of exosomes but reduces the yield. Both methods require complex centrifugation steps, are time-consuming, and require large amounts of biological samples, making them unsuitable for clinical analysis. 2. Precipitation methods, such as commercial polymer precipitation kits like ExoQuick (System Biosciences) and Total Exosome Isolation reagent (Thermo Fisher), precipitate exosomes by reducing their solubility. Compared to centrifugation, this method has the advantages of simple operation, high yield, and less time consumption. However, the addition of a precipitant reduces protein solubility, resulting in a high concentration of protein impurities in the separated product, which may adversely affect downstream processing and detection. 3. Size-Exclusion Chromatography (SECCh), such as commercially available pre-packed columns like qEV (Izon Science) and Hilload Superdex (GE Healthcare), utilizes size-exclusion chromatography to separate exosomes by size, offering advantages such as speed and reproducibility. However, compared to precipitation methods, this method results in lower exosome yield and purity, and also leads to a lower concentration of exosomes in the product, typically requiring additional enrichment steps. 4. Immunoaffinity Capture. This method separates exosomes by recognizing unique markers on the exosome membrane, such as transmembrane proteins like CD9 or CD63. Therefore, compared to the methods mentioned above, it yields exosomes with higher specificity, fewer impurities in the product, and is more conducive to subsequent detection. However, this technology has high requirements for markers and corresponding antibodies, resulting in low yields and significant costs. Existing technology discloses a fusion protein that recognizes exosomes, comprising a fusion protein of an exosome-binding peptide and a scaffold protein. However, the use of this fusion peptide for exosome collection requires the assistance of magnetic particles and also involves binding to surface markers on the exosomes, which involves the denaturation and renaturation of the fusion protein, and the collection process is time-consuming. Targeting surface markers has several drawbacks: differences in the specificity and affinity of different batches of antibodies can affect capture efficiency; and the relative abundance of cell surface markers is affected by individual differences, health status, tissue origin, and other factors, leading to significant variations in results. Summary of the Invention

[0004] In order to solve one of the above-mentioned technical problems in the prior art, the present invention provides a fusion polypeptide for enriching, capturing and / or separating extracellular vesicles and its application.

[0005] In a first aspect, the present invention provides a fusion polypeptide comprising a binding peptide domain and a precipitating peptide domain.

[0006] In some embodiments, the binding peptide domain is capable of specifically binding to extracellular vesicles in the sample to form a fusion peptide-extracellular vesicle complex.

[0007] In some embodiments, the binding peptide domain binds to the extracellular vesicle membrane of the extracellular vesicle, preferably the binding peptide domain comprising one or more amphiphilic α-helices.

[0008] Unlike existing technologies that target surface markers of extracellular vesicles, the binding peptide domain of this invention binds to the negative charge on the surface of the extracellular vesicle membrane through electrostatic interactions, thereby specifically recognizing lipid arrangement defects induced by membrane high curvature and thus specifically binding to the extracellular vesicle membrane. This binding is universal and unaffected by the source or environment of the extracellular vesicles.

[0009] In some embodiments, the binding peptide domain binds to the extracellular vesicle membrane.

[0010] In some embodiments, the binding is mediated by extracellular vesicle membrane curvature sensing.

[0011] In some embodiments, the binding peptide domain is derived from a membrane curvature-sensing peptide or a protein domain.

[0012] In some embodiments, the membrane curvature sensing peptide or protein domain is selected from one or more of the following: cardamomylation domain, ESCRT binding motif, palmitoylation domain, isopreneation domain, lysosomal domain, glycosylphosphatidylinositol anchoring protein, and immunoglobulin heavy chain binding protein (BiP) domain.

[0013] In some preferred embodiments, the membrane curvature sensing peptide or protein domain is selected from cardamomylation domains, glycosylphosphatidylinositol anchored proteins, and any combination thereof.

[0014] In some specific embodiments, the binding peptide domain is derived from bradykinin, myristylated alanine-rich C-kinase substrate (MARCKS), MARCKS-like protein 1 (MARCKSL1), synaptic binding protein, Xenopus antimicrobial peptide, BASP1 protein, ADP-ribosylated factor GAP enzyme activator protein 1 (ArfGAP1), α-synuclein, synaptophysin, endophilin, C-reactive protein, or a dual-carrier protein. In some embodiments, the binding peptide domain comprises an amino acid sequence selected from any of the following: (1) The amino acid sequence shown in SEQ ID NOs: 25-34; and (2) An amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequence shown in SEQ ID NOs: 25~34 and maintaining the ability to bind to the extracellular vesicle membrane.

[0015] In some preferred embodiments, the binding peptide domain comprises an amino acid sequence selected from SEQ ID NOs: 25-26; and an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequence shown in SEQ ID NOs: 25-26 and maintaining the ability to bind to the extracellular vesicle membrane. In some more preferred embodiments, the binding peptide domain comprises an amphiphilic α-helix having a positively charged hydrophilic outer surface, such as an outer surface rich in R and / or K residues.

[0016] In some embodiments, the precipitating peptide domain is capable of precipitating the fusion polypeptide-extracellular vesicle complex in response to changes in conditions selected from: pH, temperature, salt concentration, light wavelength, light intensity, charge, or any combination thereof.

[0017] In some embodiments, the binding peptide domain comprises an amino acid sequence selected from RTX toxin peptides, elastin-like peptides, CpA peptides / IpA peptides / MpA peptides, Annexin B1 peptides, CspB peptides, Dronpa peptides, and any combination thereof.

[0018] In some embodiments, the RTX toxin peptide comprises 1-30 tandemly linked repeat sequences, wherein the repeat sequences are selected from SEQ ID NO: 9, SEQ ID NO: 10 and any combination thereof.

[0019] In some embodiments, the RTX toxin peptide comprises 15-20 tandemly linked SEQ ID NO: 9 and / or SEQ ID NO: 10.

[0020] In some embodiments, the elastin-like polypeptide comprises 1-30 tandemly linked repeating pentapeptides, the sequence of which is VPGXG (SEQ ID NO: 38), where X is alanine, glycine, valine, tyrosine, or glutamic acid.

[0021] In some embodiments, the elastin-like polypeptide comprises 15-20 tandemly linked repeating pentapeptides.

[0022] In some implementations, X is valine.

[0023] In some embodiments, the precipitated peptide domain comprises an amino acid sequence selected from any of the following: (1) The amino acids shown in SEQ ID NOs: 11-24; and (2) An amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequence shown in SEQ ID NOs: 11-24 and retaining the ability to form a precipitate in response to changes in conditions selected from: pH, temperature, salt concentration, light wavelength, light intensity, charge, or any combination thereof.

[0024] In some embodiments, the fusion polypeptide includes multiple (e.g., 2, 3, or 4) binding peptide domains.

[0025] In some embodiments, the plurality of binding peptide domains bind to the same extracellular vesicle membrane.

[0026] In some embodiments, the plurality of binding peptide domains bind to different extracellular vesicle membranes.

[0027] In some embodiments, the plurality of binding peptide domains are connected in tandem.

[0028] In some embodiments, the fusion polypeptide includes multiple (e.g., 2, 3, or 4) precipitated peptide domains.

[0029] In some embodiments, the plurality of precipitating peptide domains form precipitates in response to changes in the same conditions.

[0030] In some embodiments, the plurality of precipitating peptide domains precipitate in response to changes in different conditions.

[0031] In some embodiments, the plurality of precipitated peptide domains are connected in series.

[0032] In some embodiments, the fusion peptide is used to enrich, capture, and / or isolate extracellular vesicles from a sample.

[0033] In some embodiments, the precipitated peptide domain and the binding peptide domain are directly connected. In other embodiments, the precipitated peptide domain and the binding peptide domain are connected via linkers.

[0034] In some embodiments, the plurality of precipitated peptide domains are directly connected or connected via linkers.

[0035] In some embodiments, the plurality of binding peptide domains are directly connected or connected via linkers. In some embodiments, the linkers are selected from flexible linkers, rigid linkers, and / or cleavable linkers.

[0036] In some embodiments, the linker comprises the sequence (GmSo)n, (EA3K)n, (PmXo)n1, or (XP)n2, wherein m, n, n1, n2, or o are each independently selected from integers 1-20, and X is any one of common amino acids; specifically, common amino acids are glycine (G), alanine (A), valine (V), leucine (L), isoleucine (I), methionine (M), proline (P), tryptophan (W), serine (S), tyrosine (Y), cysteine ​​(C), phenylalanine (F), asparagine (N), glutamine (Q), threonine (T), aspartic acid (D), glutamic acid (E), lysine (K), arginine (R), and histidine (H).

[0037] In some embodiments, the linker comprises a sequence of (PmXo)n1 or (XP)n2 or a combination thereof, wherein m, n1, n2 or o is at least 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 (or any range thereof), and X is any amino acid.

[0038] In some embodiments, the linker comprises the amino acid sequence shown in SEQ ID NO: 37.

[0039] In some specific embodiments, the connection between the precipitated peptide domain and the binding peptide domain further includes a cleavable linker connection, such as a linker that can be cleaved by a protease.

[0040] In some embodiments, the cleavable linker has an amino acid sequence as shown in SEQ ID NO: 35.

[0041] In some embodiments, the fusion polypeptide may also include, optionally, sequences that promote expression.

[0042] In some embodiments, the fusion polypeptide comprises an amino acid sequence selected from any of the following: (1) The amino acid sequences shown in SEQ ID NOs: 1-8; and (2) An amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequence shown in SEQ ID NOs: 1-8.

[0043] In some embodiments, the fusion peptide can be lyophilized. The lyophilized fusion peptide can then be reformulated.

[0044] In some embodiments, the fusion peptide can be reconstructed using a solution.

[0045] In some embodiments, the solution is selected from acetate buffer, glutamate buffer, citrate buffer, succinate buffer, tartrate buffer, fumarate buffer, maleate buffer, histidine buffer, phosphate buffer (PBS), physiological saline, tris(hydroxymethyl)aminomethane buffer (TBS), or combinations thereof.

[0046] In a second aspect, the present invention provides an isolated nucleic acid molecule encoding the fusion polypeptide described in the first aspect.

[0047] In a third aspect, the present invention provides an expression vector comprising the nucleic acid molecule described in the second aspect.

[0048] In some embodiments, the expression vector is selected from viral expression vectors, bacterial expression vectors, or fungal expression vectors.

[0049] In some embodiments, the expression vector is selected from yeast expression vectors, adenovirus vectors, adeno-associated virus vectors (AAV), retroviral vectors, or lentiviral vectors.

[0050] In some embodiments, the expression vector includes the pGEX plasmid of the Escherichia coli expression system.

[0051] In a fourth aspect, the present invention provides a host cell comprising the nucleic acid molecule described in the second aspect or the expression vector described in the third aspect, or expressing the fusion polypeptide described in the first aspect.

[0052] In some embodiments, the host cell is selected from bacteria, fungi, insect cells, plant cells, or animal cells.

[0053] In some embodiments, the host cell is selected from Escherichia coli, Bacillus subtilis, or yeast.

[0054] In some embodiments, the host cell may be E. coli BL21(DE3) or other Escherichia coli expression systems.

[0055] In a fifth aspect, the present invention provides a method for enriching, capturing, and / or isolating extracellular vesicles from a fluid sample, the method comprising: (a) Providing the fusion polypeptide of the first aspect, incubating the sample with the fusion polypeptide to allow the fusion polypeptide to form a fusion polypeptide-extracellular vesicle complex with extracellular vesicles in the sample. (b) subjecting the sample to the changes in conditions thereby precipitating the fusion polypeptide-extracellular vesicle complex from the sample; and (c) Optionally, the precipitated fusion polypeptide-extracellular vesicle complex is separated.

[0056] In some embodiments, the separation in step (c) is achieved by centrifugation, filtration, decantation, or any combination thereof.

[0057] In some embodiments, the sample includes one or more of cell culture supernatant, samples obtained from animal subjects, or apoplast fluids from plants.

[0058] In some embodiments, the samples obtained from the animal subject include, but are not limited to, whole blood, serum, plasma, urine, saliva, sputum, breast milk, cerebrospinal fluid, semen, amniotic fluid, ascites, lymph, bone marrow, synovial fluid, bronchoalveolar lavage fluid, pleural effusion, or ascites. In some embodiments, the samples obtained from the animal subject can be any sample containing extracellular vesicles, such as lung tissue, peripheral blood mononuclear cells, total white blood cells, lymph node cells, spleen cells, tonsillar cells, cancer cells, tumor cells, bile, pleural effusion, digestive juices, skin, and cultured mammalian cells.

[0059] There is no particular limitation on the volume of the sample. In some embodiments, the volume of the sample is 100 μL to 7 mL, 200 μL to 6.5 mL, 250 μL to 6.5 mL, 200 μL to 6 mL, 250 μL to 6 mL, 200 μL to 5 mL, 200 μL to 4 mL, 200 μL to 3 mL, 200 μL to 2 mL, 100 μL to 1 mL, 150 μL to 1 mL, 200 μL to 1 mL, 250 μL to 1 mL, 200 μL to 500 μL, 200 μL to 500 μL, 200 μL to 400 μL, 250 μL to 500 μL, or 250 μL to 400 μL.

[0060] In some embodiments, the incubation temperature in step (a) is 15~25°C.

[0061] In some implementations, the incubation time in step (a) is 2 to 10 minutes.

[0062] In some preferred embodiments, the incubation time in step (a) is 2 to 5 minutes.

[0063] In some embodiments, the temperature of step (b) is 2~8°C.

[0064] In some embodiments, the temperature of step (b) is 15~25°C.

[0065] In some embodiments, the temperature of step (b) is 35~42°C.

[0066] In some implementations, the time for step (b) is 1 to 70 minutes.

[0067] In some preferred embodiments, the time for step (b) is 1 to 20 minutes.

[0068] In some more preferred embodiments, the time for step (b) is 1 to 10 minutes.

[0069] In some implementations, when the change of the condition is Ca 2+ When the concentration is specified, the method includes step (b): adding Ca to the sample. 2+ This precipitates the fusion peptide-extracellular vesicle complex from the sample; further, the method may include step (d): adding Ca to the precipitated fusion peptide-extracellular vesicle complex. 2+ A chelating solution (e.g., EGTA solution) is used to redissolve the fusion peptide-extracellular vesicle complex to recover the extracellular vesicles.

[0070] In some embodiments, the change in the conditions is the concentration of salt ions (e.g., (NH4)). + Or Na + When the sample is precipitated, the method includes step (b): adding salt ions to the sample to precipitate the fusion polypeptide-extracellular vesicle complex from the sample; further, the method may include step (d): adding a low-salt solution to the precipitated fusion polypeptide-extracellular vesicle complex to redissolve the fusion polypeptide-extracellular vesicle complex to recover the extracellular vesicles.

[0071] In some embodiments, when the condition changes to pH, the method includes step (b): adding an acidic solution to the sample to pH 4.0-6.0 to precipitate the fusion polypeptide-extracellular vesicle complex from the sample; further, the method includes step (d): adding a solution of pH 7.0-8.0 to the precipitated fusion polypeptide-extracellular vesicle complex to redissolve the precipitated fusion polypeptide-extracellular vesicle complex to recover the extracellular vesicles.

[0072] In some embodiments, when the change in conditions is light, the method includes step (b): applying light with a wavelength of 380 nm to 420 nm to the sample to precipitate the fusion peptide-extracellular vesicle complex from the sample; further, the method includes step (d): applying light with a wavelength of 480 nm to 520 nm to the precipitated fusion peptide-extracellular vesicle complex to redissolve the precipitated fusion peptide-extracellular vesicle complex to recover the extracellular vesicles.

[0073] In some preferred embodiments, the wavelength of the light used in step (b) is 400 nm to 405 nm.

[0074] In some preferred embodiments, the wavelength of the light used in step (d) is 500 nm to 505 nm.

[0075] In some embodiments, step (a) includes incubating the sample with the fusion peptide in a solution selected from acetate buffer, glutamate buffer, citrate buffer, succinate buffer, tartrate buffer, fumarate buffer, maleate buffer, histidine buffer, phosphate buffer (PBS), physiological saline, tris(hydroxymethyl)aminomethane buffer (TBS), or combinations thereof.

[0076] In some embodiments, the method increases the yield of extracellular vesicles isolated by at least 27% compared to conventional ultracentrifugation.

[0077] In some embodiments, the purity of the extracellular vesicles isolated by the method is increased by at least 20% compared to conventional ultracentrifugation.

[0078] In a sixth aspect, the present invention provides a kit for enriching, capturing and / or isolating extracellular vesicles from a sample, comprising the fusion peptide described in the first aspect and optional instructions for use.

[0079] In some implementations, the use of the kit includes the following steps: (1) The fusion peptide is incubated with the sample to allow the fusion peptide to form a fusion peptide-extracellular vesicle complex with the extracellular vesicles in the sample. (2) subjecting the sample to the changes in conditions, thereby precipitating the fusion polypeptide-extracellular vesicle complex from the sample; and (3) Separate the precipitated fusion polypeptide-extracellular vesicle complex from the sample.

[0080] In some embodiments, step (3) includes separating the fusion polypeptide-exosome complex by centrifugation, filtration, decantation or any combination thereof.

[0081] In some embodiments, the sample includes one or more of the following: cell culture supernatant (e.g., bacterial cell culture medium, yeast culture medium, or mammalian cell culture medium), fluid sample obtained from an animal object, or apoplast fluid from a plant.

[0082] In some embodiments, the fluid samples obtained from the animal subject include, but are not limited to, whole blood, serum, plasma, urine, saliva, sputum, breast milk, cerebrospinal fluid, semen, amniotic fluid, ascites, lymph, bone marrow, synovial fluid, bronchoalveolar lavage fluid, pleural effusion, or ascites. In some embodiments, the fluid samples obtained from the animal subject may be solutions containing one or more of the following: lung tissue, skin, peripheral blood mononuclear cells, total leukocytes, lymph node cells, spleen cells, tonsillar cells, cancer cells, tumor cells, or any sample containing extracellular vesicles, such as bile, pleural effusion, digestive juices, and cultured mammalian cells.

[0083] In a seventh aspect, the present invention provides a method for preparing the fusion polypeptide described in the first aspect.

[0084] In some embodiments, the method includes: a) Cultivate the host cells described in the fourth aspect to express the fusion polypeptide. b) subjecting the solution containing the expressed fusion peptide to a change in conditions, thereby precipitating the fusion peptide from the solution, said conditions being selected from: pH, temperature, salt concentration, light wavelength, light intensity, charge, or any combination thereof, and c) Separate the precipitated fusion polypeptide from the solution.

[0085] In some embodiments, the solution is selected from: the lysate supernatant of the host cell, the lysate supernatant of the host cell inclusion bodies, the culture medium of the host cell, or a combination thereof.

[0086] In some embodiments, the separation of the fusion peptide in step c) is achieved by centrifugation, filtration, decantation, or any combination thereof.

[0087] In an eighth aspect, the present invention provides a composition comprising a fusion polypeptide-extracellular vesicle complex obtained by the method described in the fifth aspect, preferably, the composition being used for the prevention, diagnosis or treatment of related diseases or conditions.

[0088] In some embodiments, the disease or condition is selected from: cancer, inflammatory diseases, infections, degenerative diseases, diseases caused by pathogens, neurological diseases and conditions, and internal dysfunctions.

[0089] Compared with the prior art, the beneficial effects of the present invention are as follows: 1. The method of the present invention utilizes peptide-specific precipitation to extract exosomes, which is simple to operate, time-saving, and requires no other complex media or instruments, thus greatly increasing the extraction efficiency.

[0090] 2. The binding peptide used in the method of the present invention can specifically bind to exosomes, and the precipitated peptide can specifically respond to Ca. 2+ Precipitation under these conditions greatly reduces contamination from impurities in the product, making it more conducive to subsequent processing and detection of exosomes.

[0091] 3. This polypeptide is expressed and purified by E. coli, resulting in high yield and greatly reducing the cost of exosome purification.

[0092] 4. The method of the present invention utilizes the spontaneous aggregation characteristic of peptide function to actively precipitate and obtain exosomes, without the need for conventional techniques such as magnetic bead enrichment and elution processes, and can achieve rapid extraction of exosomes (as short as 5 minutes), which has great clinical application value. Attached Figure Description

[0093] Figure 1 A schematic diagram of the structure of the fusion polypeptide expression vector is shown.

[0094] Figure 2 A schematic diagram of the structure of the fusion polypeptide is shown.

[0095] Figure 3 A schematic diagram of the structure of the fusion protein-exosome complex is shown.

[0096] Figure 4 The comparison of the time required to extract extracellular vesicles from plasma using different methods is shown.

[0097] Figure 5 The results of different methods for extracting extracellular vesicles from plasma are shown in comparison.

[0098] Figure 6 The purity of extracellular vesicles extracted from plasma by different methods is shown in the comparison.

[0099] Figure 7 The particle distribution of extracellular vesicles extracted from plasma by different methods is shown in comparison.

[0100] Figure 8 The yield of plasma extracellular vesicles extracted using different precipitated peptide domains is shown.

[0101] Figure 9 The relative purity of plasma extracellular vesicles extracted using different precipitated peptide domains is shown. Detailed Implementation

[0102] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. The specific embodiments described herein are for illustrative purposes only and are not intended to limit the invention in any way. Furthermore, descriptions of well-known structures and techniques are omitted in the following description to avoid unnecessarily obscuring the concepts of this disclosure. Such structures and techniques have also been described in many publications.

[0103] definition Unless otherwise defined, all technical and scientific terms used in this invention have the same meaning as commonly used in the field to which this invention pertains. For the purposes of interpreting this specification, the following definitions will apply, and where appropriate, terms used in the singular will also include the plural forms, and vice versa.

[0104] Unless the context clearly indicates otherwise, references to a specific quantity herein include their plural forms. For example, references to "cells" include one or more such cells and equivalents known to those skilled in the art, etc.

[0105] As used herein, the term "about" indicates a range of ±20% of the following value. In some embodiments, the term "about" indicates a range of ±10% of the following value. In some embodiments, the term "about" indicates a range of ±5% of the following value.

[0106] As used herein, "extracellular vesicles (EVs)" refers to cell-derived vesicles, also known as exosomes. Structurally, they are vesicles with a phospholipid bilayer, and their surface includes proteins and carbohydrates. In some embodiments, the diameter of the extracellular vesicles ranges from 1 nm to 1000 nm, such as 10 nm to 1000 nm, 20 nm to 1000 nm, 30 nm to 1000 nm, 1 to 100 nm, 10 to 100 nm, 20 to 100 nm, 30 to 100 nm, 40 to 100 nm, 10 to 200 nm, 20 to 200 nm, 30 to 200 nm, 40 to 200 nm, etc. 200nm, such as 10 to 120nm, such as 20 to 120nm, such as 30 to 120nm, such as 40 to 120nm, such as 10 to 300nm, such as 20 to 300nm, such as 30 to 300nm, such as 40 to 300nm, such as 50 to 1000nm, such as 500 to 2000nm, such as 100 to 500nm, such as 500 to 1000nm, and such as 40nm to 500nm, each range is included. Exosomes can be isolated from any suitable biological sample, including but not limited to whole blood, serum, plasma, urine, saliva, sputum, breast milk, cerebrospinal fluid, amniotic fluid, ascites, bone marrow, and cultured cells (e.g., immature dendritic cells (wild-type or immortalized), induced and uninduced pluripotent stem cells, fibroblasts, platelets, immune cells, reticulocytes, tumor cells, mesenchymal stem cells, satellite cells, hematopoietic stem cells, pancreatic stem cells, white and beige preadipocytes, etc.).

[0107] The binding peptide domain used in this invention is a peptide fragment with affinity for the extracellular vesicle membrane. In some embodiments, the binding peptide domain comprises one or more amphiphilic α-helices. An "amphiphilic α-helix" refers to a secondary structure exhibited by some polypeptides that is associated with a lipid bilayer, wherein amino acids form an α-helical conformation with their opposing polar and nonpolar planes oriented along the long axis of the helix. In some embodiments, the binding peptide domain comprises an amphiphilic α-helix having a positively charged hydrophilic outer surface, such as an outer surface rich in R and / or K residues. A "positively charged hydrophilic outer surface" means, based on α-helix wheel projection, the presence of at least two lysine (K) and / or arginine (R) residues clustered on one side of the amphiphilic α-helix. Such wheel projection can be performed using various programs, such as some online wheel projection tools. In some embodiments, the amphiphilic α-helix may include a positively charged hydrophilic outer surface, based on an α-helix with a rotation angle of 100° between consecutive amino acids and / or an α-helix with 3.6 residues per turn, comprising at least two, three or four adjacent positively charged K and / or R residues when projected onto the helical wheel.

[0108] The term "membrane curvature" as used in this article refers to the degree of bending or shape characteristic of the cell membrane in space, and is a key physical parameter of the cell. Many cellular processes rely heavily on accurate and timely changes in the morphology of biological membranes. These changes can range from dramatic membrane fusion and fission to slight membrane bulges or depressions, i.e., changes in membrane curvature. It is currently widely believed that membrane curvature not only participates in cellular events but has also become an effective regulator of protein-protein interactions and intracellular signal transduction. Extracellular vesicles possess a lipid bilayer membrane structure, with an extremely small radius resulting in a highly curved surface. Furthermore, the presence of a large number of negatively charged unsaturated phospholipids, such as phosphatidylserine, and lipid accumulation defects induce membrane curvature.

[0109] The term "membrane curvature-sensing peptide" used in this article refers to peptide segments that can specifically recognize high-curvature membrane structures. Membrane curvature-sensing peptides have an amphiphilic molecular structure and achieve precise recognition of extracellular vesicles through a dual mechanism of action: first, they anchor to the negative charge on the surface of the extracellular vesicle membrane via electrostatic interactions, and then specifically bind to lipid arrangement defects caused by the high curvature of the extracellular vesicle membrane.

[0110] The binding peptide domain of this invention is derived from a membrane curvature-sensing peptide or protein domain, thereby overcoming the technical bottleneck of lacking universal markers for the separation of extracellular vesicles and making it more suitable for routine clinical testing. In some embodiments, the membrane curvature-sensing peptide or protein domain is selected from myristylation domains, ESCRT binding motifs, palmitoylation domains, isopreneation domains, lysosomal protein domains, glycosylphosphatidylinositol anchoring protein domains, and immunoglobulin heavy chain binding protein (BiP) domains. Exemplarily, the binding peptide domain is derived from bradykinin, myristylated alanine-rich C-kinase substrate (MARCKS), MARCKS-like protein 1 (MARCKSL1), synaptic binding protein, Xenopus antimicrobial peptide (Magainin), ADP-ribosylated factor GAP enzyme activator protein 1 (ArfGAP1), α-synuclein, synaptophysin, endophilin, C-reactive protein, or amphiphysin.

[0111] The "myristoylation" domain used in this paper is a lipid-modified domain in which the myristoyl group, derived from myristic acid, is covalently linked to the α-amino group of the N-terminal glycine residue via an amide bond. Myristoylated proteins begin with the concordant sequence Met-Gly-XXX-Ser / Thr. The initiating Met is removed through co-translation and proteolytic hydrolysis, and myristic acid is added to the exposed N-terminal glycine residue via a stable amide bond.

[0112] The "ESCRT (Endosomal Sorting Complexes Required for Transport)" motif used in this article was initially discovered during the formation of multivecicular bodies (MVBs) through endosome invagination. Its role is to facilitate the degradation of membrane proteins. It comprises five protein complexes, named ESCRT 0, I, II, III, and Vps4 / Vta1. These complexes work synergistically on specialized endosomes to facilitate the movement of specific loads from the restrictive membrane to vesicles within the endosome lumen.

[0113] The term "palmitoylation" as used in this article refers to the covalent attachment of a fatty acid, such as palmitic acid, to cysteine, or, for example, the covalent attachment of palmitic acid to serine and threonine. Palmitoylation involves the covalent attachment of a fatty acid, such as palmitic acid, to residues of a polypeptide.

[0114] The "isoprenelation" domain used in this article refers to isoprenelation modifications that typically occur on cysteine ​​residues. Isoprenelation is commonly found on the C-terminal residues of peptides. In mammals, approximately 2% of proteins are isoprenelated at their C-terminal residues. Isoprenelation of polypeptides significantly affects their hydrophobicity. Furthermore, isoprenelation of peptides increases the strength of interactions with the plasma membrane (e.g., affinity), thereby promoting efficient binding.

[0115] The term "lysosome" as used in this article refers to most structures carrying mannose-6-phosphate (M6P) modification, which mediates the recognition and binding of extracellular vesicles. Exemplary lysosome domains are selected from α-D-mannosidase, N-aspartic-β-glucosidase, lysosomal acid lipase, cystine transporter, lysosome-associated membrane protein-2 (LAMP2), α-galactosidase A, acidic ceramidinase, α-fucosidase, cathepsin A, acidic β-glucosidase, β-galactosidase, β-hexosaminease A, β-hexosaminease B, GlcNAc-1-phosphotransferase, β-galactosylsphingosine, lysosomal acid lipase, arylsulfatase A, α-L-iduronase, idurose-2-sulfatase, pallansulfonamide, acetyl-α-glucosidase, and acetyl-CoA. The domains of α-glucosinolate-N-acetyltransferase, N-acetylglucosamine-6-sulfatase, N-acetylgalactosamine-6-sulfatase, hyaluronidase, acetylgalactosamine-4-sulfatase, β-glucuronidase, α-N-acetylneuraminidase, N-acetylglucosamine-1-phosphotransferase, mucoprotein-1, formylglycine synthase, palmitoyl protein thioesterase-1, tripeptidyl peptidase I, cysteine ​​chain protein, CLN3p, CLN5p, CLN6p, CLN7p, CLN8p, acid sphingomyelinase, NPC1, NPC2, acid α-glucosidase, cathepsin K, sialic acid transporter, α-N-acetylgalactosaminease, GM2 activator, lysosomal acid lipase, and / or any combination thereof.

[0116] The "glycosylphosphatidylinositol" anchoring protein used in this article is the GPI anchoring protein. All GPIs contain a highly conserved core structure: 6-O-phosphoethanolamine-α-D-mannose (1→2)-α-D-mannose (1→6)-α-D-mannose (1→4)-β-D-glucosamine (1→6)-myo-inositol-glycerophospholipid. The amino groups on the conserved phosphoethanolamine bridging chain in the GPI molecule can covalently link to the C-terminus of cell membrane surface proteins or glycoproteins. The fatty chains of the glycerophospholipids on myo-inositol can embed into the lipid bilayer, thus linking the GPI anchoring protein to the plasma membrane.

[0117] The "immunoglobulin heavy chain binding protein (BiP)" used in this article, also known as glucose-regulated protein-78 (GRP78), is an endoplasmic reticulum molecular chaperone protein. BiP belongs to the HsP70 protein family and, in addition to promoting the folding of intrinsic proteins, acting as a mediator in the aggregation process, and triggering the degradation of misfolded proteins by the proteasome, can also bind Ca2+. 2+ It can also serve as a regulator of endoplasmic reticulum stress signals.

[0118] As used herein, "bradykinin (BK)" refers to a 9-peptide substance generated by the action of kallikrein on kininogen, with the amino acid sequence RPPGFSPFR (SEQ ID NO: 29). In some embodiments, variants of the bradykinin, such as PRPPGFSPF (SEQ ID NO: 27), may be used.

[0119] The “myristylated alanine-rich C-kinase substrate (MARCKS)” used in this article refers to a protein of approximately 82 kDa with three evolutionarily conserved regions: an N-terminus, a phosphorylation site domain (PSD), and a multiple homology 2 (MH2) domain. The N-terminus (a 24-amino acid sequence with a myristic acid moiety linked to a terminal glycine residue) is involved in the binding of MARCKS to the membrane and may be involved in the binding of MARCKS to calmodulin. This 24-amino acid sequence is referred to as the MANA peptide (as shown in SEQ ID NO: 30).

[0120] The “synaptotagmin (Syt)” used in this article is a class of transmembrane proteins found on synaptic vesicles of nerve cells and vesicles of endocrine cells. It consists of a short N-terminal sequence, a single-stranded transmembrane region and a tandem C2 domain.

[0121] The "antimicrobial peptides" used in this article are small, bioactive polypeptides produced in vivo through induction, with molecular weights ranging from 2000 to 7000 and composed of 20 to 60 amino acid residues. As a supplementary or additional defense system, this secondary chemoimmune system provides the organism with a complete set of small peptides that are rapidly produced after induction to resist the invasion of opportunistic or obligate pathogens or prevent the uncontrolled proliferation of symbiotic microorganisms. To date, more than 100 different antimicrobial peptides have been isolated and characterized. The largest, and perhaps most studied, family includes those peptides with positive charges and amphiphilic α-helical structures. Extensive research on various natural antimicrobial peptides tends to emphasize the importance of amphiphilic α-helical structures and net positive charges for cytolytic activity. Positive charges promote peptide-negatively charged membrane interactions, which are present in higher concentrations in pathogen cell membranes than in normal eukaryotic cells, while the amphiphilic α-helical structure is crucial for lytic activity. Magainin peptides are a class of natural peptides extracted from the skin of the African clawed frog by Zasloff et al. in 1987. They mainly consist of two structurally very similar peptides, Magainin I and Magainin II. The amino acid sequence of the natural peptide Magainin I is: SKMIEGVFAKGFKGASHLFKGIG (SEQ ID NO: 31); the amino acid sequence of the natural peptide Magainin II is: SNMIEGVFAKGFKKASHLFKGIG (SEQ ID NO: 32).

[0122] The term "ADP-ribosylation factor (ARF)" as used herein refers to a member of the Ras superfamily and a key member of the eukaryotic vesicle transport pathway. ADP-ribosylation factor GAP activator protein 1 (ArfGAP1) is a protein located in the Golgi apparatus that regulates the dynamic changes of the COPI membrane. The ArfGAP gene contains a GAP homologous region with a four-cysteine ​​zinc finger structure at its N-terminus. This domain interacts with the Arf effector region and promotes GTPase activity. The region of ArfGAP1 that binds to the high-curvature lipid membrane is a motif of approximately 40 amino acids (amino acid sequence shown in SEQ ID NO: 33). In some embodiments, the binding peptide domain according to the present invention is derived from the ArfGAP1 domain. In some embodiments, the binding peptide domain according to the present invention has the amino acid sequence shown in SEQ ID NO: 28.

[0123] The BAR (Bin / Amphiphysin / Rvs) domain protein family is an important family in eukaryotic cells, playing a crucial role in various cellular activities such as cell membrane morphogenesis and substance transport. This family is named after its earliest discovered members: Bin1, Amphiphysin, and Rvs167. These proteins structurally possess a conserved BAR domain, which is a dimer structure composed of two to three coiled helical loops. Currently, over 220 BAR family proteins have been identified. Based on subtle differences in the shape and curvature of the BAR domain, these proteins can be divided into three categories: N-BAR proteins, F-BAR proteins, and I-BAR proteins. N-BAR proteins have a typical BAR domain, usually with a short amphiphilic helical structure at the N-terminus. This domain plays a significant role in the dimerization of endocytotoxic proteins and their binding to the membrane, thereby altering membrane curvature. For example, endocytoproteins, as N-bar proteins, have 1-26 residues at their N-terminus in a disordered state in their natural state (their amino acid sequence is shown in SEQ ID NO: 34). They can only fold into amphiphilic helices when inserted into the membrane. As the diameter of liposomes increases, the binding ability of endocytoproteins to liposomes weakens, while the ability of isolated N-terminal amphiphilic helices to sense curvature is comparable to that of full-length endocytoproteins. This indicates that N-terminal amphiphilic helices play a dominant role in endocytoprotein curvature sensing.

[0124] The term "amphiphysin" as used in this article refers to a family of proteins that can bind to various other proteins and participate in clathrin-mediated endocytosis, actin activity, and signal transduction pathways. These proteins are abundant in mammalian nerve endings, serving as connectors between clathrin and actin during synaptic vesicle endocytosis. From primitive yeast and fruit flies to advanced humans, the only conserved structure of amphiphysins and their homologs is the BAR domain at the N-terminus. In addition, numerous homologous proteins exist, such as Arfaptin2, endocytokines, adaptor proteins APPL1 / 2, and SNX.

[0125] The separation method of this invention uses the fusion polypeptide of this invention (comprising a binding peptide domain capable of binding to the extracellular vesicle membrane and a precipitation peptide domain) to precipitate extracellular vesicles bound to its binding peptide domain. In this case, a complex of extracellular vesicles and the fusion polypeptide of this invention can be formed. The desired extracellular vesicles are then separated by precipitation of the precipitation peptide domain. In some embodiments, the binding peptide domain can bind to extracellular vesicles from different cell sources. In this case, the extracellular vesicles can be derived from, for example, but not limited to, epithelial cells, lung tissue cells, breast tissue cells, liver tissue cells, prostate tissue cells, kidney tissue cells, urethral cells, nerve cells, tumor cells, solid tumor cells, lung tumor cells, breast tumor cells, liver tumor cells, prostate tumor cells, kidney tumor cells, urinary system tumor cells, glioblastoma cells, or β-amyloid-expressing cells.

[0126] As used herein, “sample” means any sample in which the presence and / or level of a target object needs to be detected or determined, or any sample containing exosomes or components thereof as described herein. Samples may include liquids, solutions, emulsions, or suspensions. Samples may include any biological fluid or tissue, plant fluids such as apoplasts (the portion of a plant cell contained between the plasma membrane and the cell wall, and including the cell wall and the intercellular spaces of the plant), animal fluids such as blood, whole blood, blood fractions such as plasma and serum, muscle, interstitial fluid, sweat, saliva, urine, tears, synovial fluid, bone marrow, cerebrospinal fluid, nasal secretions, sputum, amniotic fluid, bronchoalveolar lavage fluid, gastric lavage fluid, vomitus, feces, lung tissue, peripheral blood mononuclear cells, total white blood cells, lymph node cells, spleen cells, tonsillar cells, cancer cells, tumor cells, bile, pleural effusion, ascites, digestive juices, skin, or combinations thereof. The sample may be a sample obtained directly from the object or a pre-treated sample, such as through filtration, distillation, extraction, concentration, centrifugation, inactivation of interfering components, addition of reagents, etc. In some embodiments, the sample is blood. In some embodiments, the sample is a serum sample (e.g., the fluid and solute components of blood without clotting factors). In some embodiments, the sample is a plasma sample (e.g., the liquid portion of blood). As used herein, “plasma” refers to the liquid portion of blood and lymph, which constitutes approximately half the volume of blood. Plasma does not contain cells and, unlike serum, does not clot. There is no particular limitation on the volume of the sample. In some embodiments, the volume of the sample is 100 μL to 7 mL, 200 μL to 6.5 mL, 250 μL to 6.5 mL, 200 μL to 6 mL, 250 μL to 6 mL, 200 μL to 5 mL, 200 μL to 4 mL, 200 μL to 3 mL, 200 μL to 2 mL, 100 μL to 1 mL, 150 μL to 1 mL, 200 μL to 1 mL, 250 μL to 1 mL, 200 μL to 500 μL, 200 μL to 500 μL, 200 μL to 400 μL, 250 μL to 500 μL, or 250 μL to 400 μL.

[0127] The terms "aggregation" and "precipitation" used herein are used interchangeably and can refer to any physical interaction or chemical reaction that causes the "aggregation" of the fused polypeptide or precipitated peptide domains. Aggregation processes are well known in the art and are typically affected by a variety of physicochemical stresses, including temperature, pressure, pH, salt concentration, dielectric properties, and light. In some embodiments, the precipitation of the fused polypeptide of the present invention is reversible. In some embodiments, reversible means, for example, responding to Ca... 2+ When a fused polypeptide that has precipitated due to changes in its composition is placed in a solution containing EGTA, the precipitated fused polypeptide will redissolve.

[0128] As used herein, "linker" refers to a peptide or other chemical bond or group that serves to connect other independent functional regions. It is used interchangeably with "connector" herein, referring to the function of connecting other independent functional regions. In some embodiments, the linker is located between multiple binding peptide domains. In some embodiments, the linker is located between a binding peptide domain and a precipitated peptide domain. In some embodiments, the linker is located between multiple precipitated peptide domains. Suitable linkers for coupling two or more linked peptide domains can generally be any linker in the art for connecting peptides, proteins, or other organic molecules. In some embodiments, the linker is a cleavable linker that can be cleaved by a protease selected from the group consisting of: kallikrein, thrombin, chymotrypsin, carboxypeptidase A, cathepsin G, elastase, PR-3, granzyme M, calpains, matrix metalloproteinases (MMPs), plasminogen activator, cathepsin, caspase, trypsin, or tumor cell surface proteases. In this case, the amino acid sequence of the linker is known in the art. In some embodiments, the linker has the amino acid sequence shown in SEQ ID NO: 35, which can be cleaved by a protease.

[0129] In some embodiments, the linker may have any amino acid sequence. Various linker lengths and variability can be employed, ranging from flexible linkers in the form of glycine polymers (G)n and glycine-serine polymers (GmSo)n (including, for example, (G4S)n, (G3S)n, (GS)n, and (GSS)n) to more rigid linkers in the form of (EA3K)n and (PmXo)n1 or (XP)n2, to achieve optimal length and rigidity for the fusion protein activity of a specific application, wherein m, n, n1, n2, or o are each independently selected from integers from 1 to 20, and X is any amino acid. Suitable linkers can be readily selected and can have any suitable different lengths. In some embodiments, the linker length is 3-200 amino acids, for example, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 30-35, 35-40, 40-45, 45-50, 50-60, 60-70, 70-80, 80-90, 90-100, 100-150, or 150-200 amino acids. Longer or shorter linkers are also considered. In some embodiments, shorter linkers reduce the overall size or length of the fusion polypeptide or its coding sequence. In some embodiments, longer linkers may be used when it is desirable to ensure that two adjacent peptide fragments do not spatially interfere with each other.

[0130] It should be understood that the length, flexibility, and / or other characteristics of the linker may affect certain properties of the fusion peptide used for binding, such as affinity, specificity, or binding force to extracellular vesicles or other proteins of no interest (i.e., non-target proteins). In some embodiments, two or more linkers are used. In some embodiments, two or more linkers are identical. In some embodiments, two or more linkers are different. In some embodiments, the linkers do not alter (e.g., do not disrupt) the fusion peptide's ability to bind to extracellular vesicles.

[0131] As used herein, the terms "specific binding" or "specific recognition" of a binding peptide refer to the binding peptide domain reacting or associating with extracellular vesicle membranes more frequently, more rapidly, for a longer duration, and with a greater affinity or combination thereof compared to reactions or associations with alternative substances (including unrelated proteins). Due to the similarity of extracellular vesicle membrane structures across different species, specific binding may include recognizing extracellular vesicles in multiple species. In some embodiments, the binding peptide domain preferably specifically binds to extracellular vesicles with greater curvature, while substantially not binding to other larger vesicles.

[0132] The term "RTX toxins (repeated toxins)" used in this article refers to exotoxins (RTXs) produced by a large number of pathogenic Gram-negative bacteria. These toxins belong to the cell pore-forming protein family. They are named RTX based on a series of glycine / aspartic acid-rich non-peptide repeat sequences located at the C-terminus of the toxin protein. The genetic determinant of secretory RTX toxins contains four genes: the rtxA gene is the structural gene encoding the toxin protein; the rtxC gene is responsible for the "activation" process before toxin secretion, and its sequence is highly conserved; the rtxB and rtxD genes encode two secretory proteins. These four RTX genes usually exist in the form of a single transcription unit, "CABD," and are regulated by a common promoter located upstream of the rtxC gene. Different members of the RTX toxin family exhibit significant amino acid homology and common structural features. The N-terminal hydrophobic domain is responsible for pore formation and the formation of transmembrane regions; the C-terminus is a 9-amino acid GD repeat region, rich in glycine (GLy) and aspartic acid (Asp). The glycine-glycine-X-glycine-asparagine / aspartic acid-aspartic acid-X-leucine / isoleucine / phenylalanine-X domain is repeated tandemly 9-10 times in different RTX toxin family members, where X represents any common amino acid. This domain is related to Ca... 2+ Binding is essential for most RTX toxins to exert their activity. Therefore, the action of RTX toxins has Ca... 2+Dependence. Utilizing this characteristic of RTX, RTX fusion peptides can be easily and rapidly purified, or exosomes can be purified using RTX fusion peptides. The purification process eliminates the need for chromatographic separation, avoiding expensive affinity resins and purification equipment. Furthermore, the concentration and buffer replacement of RTX fusion peptides are greatly simplified. By adding Ca... 2+ The aggregation of the RTX fusion peptide is triggered, and the RTX fusion peptide or RTX fusion peptide-exosome complex is separated from the solution by centrifugation, using a Ca-free solution. 2+ The solution redissolves the precipitated RTX fusion peptide or complex. A purer RTX fusion peptide or exosome can be obtained by repeating the precipitation, centrifugation, and reconstitution steps. In some embodiments, the RTX toxin peptide comprises 1-30 (e.g., 1, 5, 10, 15, 20, 25, 30, or any value between) tandemly linked repeat sequences, wherein the repeat sequences are selected from GGAGNDTLY (SEQ ID NO: 9), GGAGIDTLY (SEQ ID NO: 10), and any combination thereof. In some embodiments, the RTX toxin sequence comprises 15-20 (e.g., 17) tandemly linked SEQ ID NO: 9; in other embodiments, the RTX toxin sequence comprises 15-20 (e.g., 17) tandemly linked SEQ ID NO: 10; in still other embodiments, the RTX toxin sequence comprises 15-20 tandemly linked SEQ ID NO: 9 and 1-5 tandemly linked SEQ ID NO: 10. In some specific embodiments, the RTX toxin sequence comprises the amino acid sequence shown in SEQ ID NO: 11.

[0133] The "elastin-like polypeptide (ELP)" and its derivatives used in this article refer to repetitive artificial polypeptides derived from the repeating amino acid sequences in the hydrophobic domains of protoelastin, also known as elastin-like peptides. The most typical characteristics of ELPs are temperature sensitivity and self-assembly. ELPs undergo a reversible phase transition process with changes in ambient temperature. Under certain environmental conditions, if the ambient temperature is below the phase transition temperature, ELPs exhibit high solubility in the liquid phase. However, when the temperature is above the phase transition temperature, the hydrophilic ELPs dehydrate and aggregate. Fusion polypeptides containing ELPs also exhibit this characteristic. As the temperature decreases, they return to a solution state. This phase transition or self-assembly process is mainly temperature-sensitive aggregation; during the phase transition, only the ELPs coagulate, while the fused polypeptides or proteins do not denature or precipitate. Utilizing this characteristic of ELPs, ELP fusion polypeptides can be easily and rapidly purified, or ELP fusion polypeptides can be used to purify exosomes. Similarly, the purification process does not require chromatographic separation, thus avoiding expensive affinity resins and purification equipment. ELP peptide aggregation is triggered by heating or increasing salt ion concentration. ELP peptide fragments, ELP fusion peptides, or ELP fusion peptide-exosome complexes are then separated from the solution by centrifugation. The precipitated ELP peptides are redissolved in a low-salt, low-temperature solution, and centrifuged again to remove insoluble proteins, yielding ELP peptide fragments. Repeating the precipitation, centrifugation, and redissolution steps can yield purer ELP fusion peptides or ELP fusion peptide-exosome complexes. Ambient temperature, protein concentration, salt type, and the length of the ELP can affect the precipitation of the ELP fusion peptide. Various repetitive amino acid sequences are known in the art, such as those described in Chinese patent application CN201680020418.6, which are incorporated herein by reference. In some embodiments, the ELP peptide is primarily composed of tandem pentapeptide repeating units with the sequence VPGXG (SEQ ID NO:38), where X represents any amino acid except proline, and its aggregation characteristics are related to the number of repeating units. In some embodiments, the ELP polypeptide comprises 1-30 (e.g., 1, 5, 10, 15, 20, 25, 30 or any value within this range) tandemly linked repeating pentapeptides. In some embodiments, X is alanine, glycine, valine, tyrosine, or glutamic acid, with a phase transition temperature of 35-45°C. In some embodiments, the ELP polypeptide comprises 15-20 (e.g., 20) tandemly linked repeating pentapeptides. In some embodiments, X is Val. In some specific embodiments, the ELP polypeptide comprises the amino acid sequence shown in SEQ ID NO: 12.

[0134] The "CpA, IpA, MpA" peptides used in this article are different variants of the leucine zipper sequence (MKQLEDKVEELLSK, SEQ ID NO: 36) of the yeast transcription factor GCN4. A leucine zipper is composed of extended amino acids, with leucine as the seventh amino acid in every seven-amino acid sequence. Leucine is a hydrophobic amino acid, arranged on one side of the helix, while all charged amino acid residues are on the other side. When two protein molecules are aligned in parallel, leucine residues interact to form a dimer, commonly referred to in the art as a "leucine zipper." Specifically, the CpA peptide (MKQLEDKVEELLSK...) AA The insertion of two alanine residues (underlined amino acids) into CKQLEDKIEELLSK (SEQ ID NO: 13) causes a phase shift in the C-terminal hydrophobic surface relative to the N-terminal hydrophobic surface. Adding salt enhances the hydrophobic interactions between peptides, thereby initiating the self-assembly of free peptides into aggregation. IpA peptide ( I KQLEDKIEELLSKAA I KQLEDKIEELLSK (SEQ ID NO: 14) is a variant of the CpA peptide that exhibits stronger salt responsiveness due to the substitution of cysteine ​​(the underlined amino acid) with isoleucine at the first position of its two heptapeptide repeat units. MpA peptide ( M KQLEDKIEELLSKAA M KQLEDKIEELLSK (SEQ ID NO: 15) modifies two cysteine ​​residues of the CpA peptide to nonpolar amino acids—methionine—resulting in stronger salt responsiveness. This salt-responsive peptide is then fused with an exosome-binding peptide for exosome purification. Similarly, the purification process eliminates the need for chromatographic separation, avoiding expensive affinity resins and purification equipment. Furthermore, the concentration and buffer replacement of CPA, IPA, or MPA fusion peptides are significantly simplified.

[0135] The "Annexin B1 polypeptide" used in this article refers to an annexin derived from the porcine cysticercosis. The Annexin protein family is a group of structurally similar Ca2+ proteins. 2+ Annexins are phospholipid-dependent binding proteins, abundant in most eukaryotic cells. A typical characteristic of annexins is the presence of four (sometimes eight) repeating sequences of approximately 70 amino acids, each containing a highly conserved "GXGTDE" motif (also known as the intrin fold). All annexins discovered to date contain this conserved domain; the presence of this domain is the core criterion for determining whether a protein belongs to the annexin family. Annexin B1 may have eight amino acids related to Ca2+. 2+The structural regions of the bound amino acids, in addition to GXGT, also exhibit the structural features of TSKIKG LSG ILG amino acids. In some embodiments, the Annexin B1 comprises the amino acid sequence shown in SEQ ID NO: 16. Utilizing the calcium ion concentration dependence of Annexin, Annexin aggregation can be induced simply by adjusting the calcium ion concentration in the chelation solution. Similarly, the purification process eliminates the need for chromatographic separation, thus avoiding expensive affinity resins and purification equipment.

[0136] The “CspB” peptide used in this article refers to a truncated form of cell surface protein B (CspB, also known as PS2, which has the amino acid sequence shown in SEQ ID NO: 17) found in Corynebacterium glutamicum. Fusion peptides containing the CspB sequence exhibit pH-responsive precipitation-redissolution properties, i.e., they precipitate at acidic pH and then completely dissolve at neutral pH. This property enables a simple solid-liquid separation method to separate CspB fusion peptides. In some embodiments, fragments of the N-terminus of the CspB protein at amino acid residues 5, 6, 17, 50, and 250 are selected and named CspB5, CspB6 (SEQ ID NO: 18), CspB17 (SEQ ID NO: 19), CspB50 (SEQ ID NO: 20), and CspB250 (SEQ ID NO: 21), respectively. In some specific embodiments, fusion peptides containing the CspB50 sequence, after binding to exosomes, are in solution at neutral or weakly acidic pH. By adding H₂SO₄ to lower the pH to approximately 4.6, the fusion peptides precipitate, which can be separated by centrifugation. Then, by mixing with 100 mM Tris buffer, all the precipitate immediately redissolves, yielding a clear solution (i.e., precipitate-redissolved sample). This allows for the separation of exosomes contained in the sample.

[0137] The term "photochromic fusion protein" as used herein refers to an assembly formed by the fusion of at least one photochromic peptide with a target peptide via a single amino acid chain that does not exist in nature. The photochromic peptide and other selected peptides can be directly linked by peptide bonds or separated by spacer amino acid sequences or linkers. Such fusions may contain complete proteins or fragments thereof, such as Dronpa, Padron, rsTagRFP, mApple, or their fluorescent variant sequences (e.g., Dronpa-145K, Dronpa-145N, Padron-145N, and mApple-162H-164A). The fusion peptide may also contain exogenous sequences other than the photochromic peptide or other selected peptides, such as targeting sequences, tag sequences, other fluorescent protein sequences (i.e., proteins with fluorescence properties different from Padron, rsTagRFP, or mApple), or other chromophores. Furthermore, the fusion peptide may contain sequences from multiple photochromic proteins or their variants and / or other selected proteins. For example, the fusion protein may be composed of two or more Dronpa, Padron, rsTagRFP, or mApple peptides (these peptides may be the same or different, such as simultaneously containing two or more Dronpa 145K or Dronpa 145N peptides, or simultaneously containing Dronpa 145K and Dronpa 145N peptides in the same fusion). The green fluorescent protein Dronpa (SEQ ID NO: 22) not only exhibits highly efficient reversible switching properties of fluorescence emission, but its immobilized single molecule also achieves a response speed faster than 20 milliseconds. In some embodiments, the reversible photoswitching behavior of Dronpa in solution can be achieved by adjusting the 405 nm irradiation intensity. In some embodiments, the photoresponsive mutant Dronpa protein has the Dronpa 145N domain and has the amino acid sequence shown in SEQ ID NO: 23. In some embodiments, the photoresponsive mutant Dronpa protein domain is embedded in an elastin-like (ELP) backbone, and reversible switching between liquid and gel states is achieved by controlling light at different wavelengths (400nm / 500nm). In this case, the binding peptide domain has the amino acid sequence shown in SEQ ID NO: 24.

[0138] As used herein, “isolated” fusion polypeptides generally refer to polypeptides isolated from other naturally occurring proteins and nucleic acids. They are isolated due to their origin or manipulation: (i) being present as an expression product of a portion of the expression vector in a host cell; or (ii) being linked to a polypeptide or other chemical portion other than its naturally linked portion; or (iii) not existing in nature. Similarly, “isolated” protein refers to a protein that: (i) is chemically synthesized; or (ii) is expressed in a host cell and isolated from the associated protein through purification. Preferably, the fusion polypeptide is isolated from the environment of the host cell from which it was recombinantly generated. In some embodiments, the fusion polypeptide is purified to a purity greater than 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%, and the purity can be determined by, for example, electrophoresis (e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis) or chromatography (e.g., ion exchange or reversed-phase HPLC).

[0139] As used in this article, “isolated” nucleic acid refers to nucleic acid molecules that have been separated from components of their natural environment. Isolated nucleic acids include nucleic acid molecules contained within cells, which typically contain such molecules, but which are located outside chromosomes or at chromosomal locations different from their natural chromosomal locations.

[0140] As used herein, "peptide" refers to a continuous chain of amino acids linked together by peptide bonds. The term "peptide" is used to refer to an amino acid chain of any length, but those skilled in the art will understand that the term is not limited to long chains and can refer to the smallest chain containing two amino acids linked together by peptide bonds. As is known to those skilled in the art, peptides can be processed and / or modified.

[0141] As used herein, “protein” refers to one or more polypeptides that act as discrete units. The terms “polypeptide,” “protein,” or “peptide” are used interchangeably if a single polypeptide is a discrete functional unit and does not require permanent or temporary physical association with other polypeptides to form a discrete functional unit. As used herein, “protein” typically contains naturally occurring or non-naturally occurring amino acids (e.g., alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine).

[0142] When referring to a protein or peptide, a "domain" is a region that possesses the structural and / or functional properties of said protein or peptide. A domain is typically a sequence of three or more, or typically five or seven or more amino acids, such as 10 to 200 amino acid residues, that is structurally and / or functionally distinct from and identifiable from other parts of the molecule. For example, a domain includes portions that can form independently folded structures within a protein or peptide composed of one or more structural motifs and / or are identifiable by functional activity, such as binding activity. A peptide or protein may have one or more distinct domains. For example, a domain can be identified, defined, or distinguished by homology with the primary sequence or structure of a related family member (such as homology with a motif). A domain can be distinguished by its function, such as its ability to interact with biomolecules (such as homologous binding partners). A domain can independently exhibit a biological function or activity, such that the domain can independently perform an activity or fuse with another molecule to perform an activity, such as binding activity. A domain can be a linear sequence of amino acids or a non-linear sequence of amino acids.

[0143] The term "motif" as used in this article refers to a short, conserved region in the sequence of evolution-related proteins. Motifs are often highly conserved portions of a domain, but they can also include only a portion of the domain or be located outside the conserved domain.

[0144] The term "amino acid sequence" as used in this article refers to a list of abbreviations, letters, characters, or words representing amino acid residues. The amino acid abbreviations used in this article are the standard single-letter codes for amino acids and are represented as follows: A, alanine; B, asparagine or aspartic acid; C, cysteine; D, aspartic acid; E, glutamate, glutamic acid; F, phenylalanine; G, glycine; H, histidine; I, isoleucine; K, lysine; L, leucine; M, methionine; N, asparagine; P, proline; Q, glutamine; R, arginine; S, serine; T, threonine; V, valine; W, tryptophan; Y, tyrosine; Z, glutamine or glutamic acid.

[0145] As used herein, “nucleic acid” refers to naturally occurring or synthetic oligonucleotides or polynucleotides, which may be DNA, RNA, or DNA-RNA hybrids, single-stranded or double-stranded, sense or antisense, capable of hybridizing with complementary nucleic acids via Watson-Crick base pairing. Nucleic acids may also contain nucleotide analogs (e.g., BrdU) and non-phosphodiester nucleoside bonds (e.g., peptide nucleic acid (PNA) or thiodiester bonds). In particular, nucleic acids may include, but are not limited to, DNA, RNA, cDNA, gDNA, ssDNA, dsDNA, or any combination thereof. As used herein, “nucleotide” refers to a molecule containing a base moiety, a sugar moiety, and a phosphate moiety. Nucleotides can be bonded together through their phosphate and sugar moieties to form nucleoside bonds.

[0146] The "sequence identity percentage" or "identity percentage" between two polynucleotide or polypeptide sequences refers to the number of identical matching positions shared by sequences within a comparison window, taking into account additions or deletions (i.e., vacancies) that must be introduced for optimal alignment of the two sequences. A matching position is any location in which the same nucleotide or amino acid is present in both the target and reference sequences. Vacancies in the target sequence are not counted because they are not nucleotides or amino acids. Similarly, vacancies in the reference sequence are not counted because nucleotides or amino acids from the target sequence are counted, but those from the reference sequence are not. The sequence identity percentage can be calculated by determining the number of positions in both sequences where the same amino acid residue or nucleic acid base appears, to obtain the number of matching positions; dividing the number of matching positions by the total number of positions in the comparison window; and multiplying the result by 100 to obtain the sequence identity percentage. Sequence comparison and determination of the sequence identity percentage between two sequences can be performed using software that is readily available online and downloadable. Suitable software programs for protein and nucleotide sequence alignment are available from various sources. A suitable procedure for determining the percentage of sequence identity is Bl2 seq, which is part of the BLAST procedure available from the National Center for Biotechnology Information (NCBI) website (blast.ncbi.nlm.nih.gov). Bl2 seq uses either the BLASTN or BLASTP algorithm to compare two sequences. BLASTN is used to compare nucleic acid sequences, while BLASTP is used to compare amino acid sequences. Other suitable procedures are, for example, Needle, Stretcher, Water, or Matcher, which are part of the bioinformatics procedure EMBOSS and are also available from the European Institute of Bioinformatics (EBI) at www.ebi.ac.uk / Tools / psa.

[0147] Fusion Peptides This invention provides fusion peptides for enriching, capturing, or separating extracellular vesicles.

[0148] In some embodiments, the fusion peptide includes (a) a precipitated peptide domain and (b) an extracellular vesicle-binding peptide domain from the N-terminus to the C-terminus.

[0149] In some embodiments, the extracellular vesicle-binding peptide domain is directly fused to the precipitated peptide domain. In other embodiments, the extracellular vesicle-binding peptide domain is fused to the precipitated peptide domain via a linker.

[0150] In some embodiments, the fusion polypeptide may include one or more additional domains or regions. Such additional regions may be sequences that assist in the expression of the fusion polypeptide. For example, a leader sequence (also referred to as a "signal peptide") at the N-terminus for secreting the expressed fusion polypeptide. Exemplary leader peptides of the present invention include natural leader sequences, such as the PelB signal peptide fragment known in the art.

[0151] In some embodiments, the fusion peptide can be lyophilized. The lyophilized fusion peptide can then be reformulated.

[0152] In some embodiments, the fusion peptide can be reconstructed in solution. In some embodiments, the fusion peptide of the present invention can be provided in solution. In some embodiments, the solution may optionally be acetate buffer, glutamate buffer, citrate buffer, succinate buffer, tartrate buffer, fumarate buffer, maleate buffer, histidine buffer, phosphate buffer (PBS), physiological saline, tris(hydroxymethyl)aminomethane buffer (TBS), or a combination thereof. Optionally, the solution is TBS. In some aspects, the pH of the solution is about 6 to about 8, or about 6 to about 7, or about 7 to about 8. Suitable solution concentrations cover about 200 mM or lower. In some embodiments, the concentration of the solution is (i) at least about 1 mM, at least about 2 mM, at least about 3 mM, at least about 4 mM, at least about 5 mM, at least about 6 mM, at least about 7 mM, at least about 8 mM, at least about 9 mM, at least about 10 mM, at least about 11 mM, at least about 12 mM, at least about 13 mM, at least about 14 mM, or at least about 15 mM, and (ii) not more than about 20 mM, not more than about 30 mM, not more than about 40 mM, not more than about 50 mM, not more than about 60 mM, not more than about 70 mM, not more than about 80 mM, not more than about 90 mM, not more than about 100 mM, or not more than about 200 mM. In some embodiments, the solution is present at a concentration of about 190 mM, about 180 mM, about 170 mM, about 160 mM, about 150 mM, about 140 mM, about 130 mM, about 120 mM, about 110 mM, about 100 mM, about 80 mM, about 70 mM, about 60 mM, about 50 mM, about 40 mM, about 30 mM, about 20 mM, about 19 mM, about 18 mM, about 17 mM, about 16 mM, about 15 mM, about 14 mM, about 13 mM, about 12 mM, about 11 mM, about 10 mM, or about 5 mM.

[0153] Recombinant expression of fusion peptides In some embodiments, the fusion polypeptide is "recombinant-generated" (i.e., generated using recombinant DNA technology). Exemplary recombination methods that can be used to synthesize the fusion polypeptide include, but are not limited to, polymerase chain reaction (PCR) based synthesis, tandemization, seamless cloning, and recursive directed ligation.

[0154] The present invention also provides a polynucleotide sequence encoding the fusion polypeptide. Those skilled in the art can readily deduce the polynucleotide sequence encoding the amino acid sequence. Furthermore, commercially available products can be used to codon-optimize the polynucleotide sequence for expression in host cells.

[0155] The polynucleotides of the present invention optionally further comprise one or more expression control elements. For example, the polynucleotide may comprise one or more promoters or transcriptional enhancers, ribosome binding sites, transcription termination signals, and polyadenylation signals as expression control elements. The polynucleotides can be inserted into any suitable vector, which can be contained in any suitable host cell for expression. Expression of the nucleic acid encoding the fusion polypeptide is typically achieved by operably linking the nucleic acid encoding the fusion polypeptide to a promoter in an expression vector. Typical expression vectors contain transcription and translation terminators, initiation sequences, and promoters that can be used to regulate the expression of the desired nucleic acid sequence. Exemplary promoters that can be used for expression in *E. coli* include, for example, the T7 promoter.

[0156] Methods known in the art can be used to construct expression vectors containing a nucleic acid sequence encoding the fusion polypeptide and appropriate transcription / translation control signals. These methods include, but are not limited to, in vitro recombinant DNA techniques, synthetic techniques, and in vivo recombination / gene recombination. Expression of the polynucleotide can be performed in any suitable expression host known in the art, including but not limited to bacterial cells, yeast cells, insect cells, plant cells, or mammalian cells. In some embodiments, the nucleic acid sequence encoding the fusion polypeptide is operatively linked to a suitable promoter sequence such that the nucleic acid sequence is transcribed and / or translated into the fusion polypeptide in the host.

[0157] A variety of host expression vector systems can be used to express nucleic acids encoding the fusion polypeptide. Vectors containing nucleic acids encoding the fusion polypeptide or a portion or fragment thereof include plasmid vectors, single-stranded and double-stranded phage vectors, and single-stranded and double-stranded RNA or DNA viral vectors. Phage and viral vectors can also be introduced into host cells in the form of packaged or encapsulated viruses using known infection and transduction techniques. Furthermore, viral vectors can be replication-capable or replication-deficient. Optionally, cell-free translation systems can also be used to produce proteins using RNA derived from DNA expression constructs.

[0158] Generally, any type of cell or cultured cell line can be used to express the fusion peptide described herein. Suitable host cells include, but are not limited to, microorganisms, such as bacteria transformed with recombinant phage DNA, plasmid DNA, or coliform DNA expression vectors containing the coding sequence of the fusion peptide (e.g., *Escherichia coli*, *Bacillus subtilis*); yeast transformed with recombinant yeast expression vectors containing the coding sequence of the fusion peptide (e.g., *Saccharomyces*, *Pichia*); insect cell systems infected with recombinant viral expression vectors containing the coding sequence of the fusion peptide (e.g., baculovirus); and plant cell systems infected with recombinant viral expression vectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or transformed with recombinant plasmid expression vectors containing the coding sequence of the fusion peptide (e.g., Ti plasmid).

[0159] Prokaryotes that can serve as host cells in the production of the fusion polypeptide include Gram-negative or Gram-positive organisms such as *Escherichia coli* and *Bacillus subtilis*. Expression vectors used for prokaryotic host cells typically contain one or more phenotypic selection marker genes (e.g., genes encoding proteins that confer antibiotic resistance or provide autotrophic requirements). Examples of useful prokaryotic host expression vectors include the pKK223-3, pGEM1, pET, and pRSET family of vectors. Exemplary promoter sequences commonly used in prokaryotic host cell expression vectors include T7, β-lactamase (penicillinase), lactose promoter systems, tryptophan (trp) promoter systems, and tac promoters. A gene construct or expression cassette obtained according to the methods described herein is inserted into an expression system optimized for high-level expression of the heterologous protein in *E. coli* cells. This expression system is based on *E. coli* cell growth to significant densities and subsequent intracellular induction of T7 polymerase, which induces high-level transcription of the gene construct. The fusion polypeptide is then expressed and accumulated in inclusion bodies within the *E. coli* cells.

[0160] In some embodiments, a eukaryotic host cell system is used, including yeast cells transformed with a recombinant yeast expression vector containing or encoding a nucleic acid sequence of the fusion polypeptide. Exemplary yeasts that can be used to generate the fusion polypeptide of the present invention include yeasts from the genera *Saccharomyces*, *Pichia pastoris*, *Actinomyces*, and *Kluyveromyces*. Yeast vectors typically contain a replication origin sequence, an autonomous replication sequence (ARS), a promoter region, a polyadenylated sequence, a transcription termination sequence, and a selection marker gene from a 2 μm yeast plasmid. Examples of promoter sequences in yeast expression constructs include promoters derived from metallothioneins, glycerol-3-phosphate kinase, and other glycolytic enzymes such as enolase, glyceraldehyde-3-phosphate dehydrogenase, hexokinase, pyruvate decarboxylase, phosphofructokinase, glucose-6-phosphate isomerase, glycerol-3-phosphate mutase, pyruvate kinase, triose phosphate isomerase, phosphoglucose isomerase, and glucoskinase. Other suitable vectors and promoters for yeast expression, as well as yeast transformation protocols, are known in the art.

[0161] Insect and plant host cell culture systems can also be used to generate the fusion peptides described herein. Such host cell systems include, for example, insect cell systems infected with recombinant viral expression vectors (e.g., baculoviruses) containing or encoding nucleic acid sequences of the fusion peptides; and plant cell systems infected with recombinant viral expression vectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or transformed with recombinant plasmid expression vectors (e.g., Ti plasmids) containing or encoding nucleic acid sequences of the fusion peptides.

[0162] In some embodiments, host cell systems may be used, including animal cell systems infected with recombinant viral expression vectors (e.g., adenovirus, retrovirus, adeno-associated virus, herpesvirus, lentivirus), including cell lines (e.g., mouse cell lines) engineered to contain multiple copies of DNA encoding the fusion polypeptide, either stably amplified (CHO / dhfr) or unstablely amplified in two microchromosomes. In some embodiments, the vector containing the polynucleotide encoding the fusion polypeptide is polycistronic. Exemplary mammalian cells that can be used to produce these compositions include 293 cells (e.g., 293T and 293F), CHO cells, BHK cells, NSO cells, SP2 / 0 cells, YO myeloma cells, P3X63 mouse myeloma cells, PER cells, PER.C6 cells, HeLa cells, COS cells, MDCK cells, 3T3 cells, W138 cells, BT483 cells, Hs578T cells, HTB2 cells, BT20 cells, T47D cells, CRL7O30 cells, HsS78Bst cells, hybridoma cells, and other mammalian cells. Other exemplary mammalian host cells that can be used to implement the invention include, but are not limited to, T cells. Exemplary expression systems and selection methods are well known in the art. The transcriptional and translational control sequences of mammalian host cell expression vectors are typically derived from viral genomes. Commonly used promoter and enhancer sequences in mammalian expression vectors include sequences derived from polyomaviruses, adenovirus 2, simian virus 40 (SV40), and human cytomegalovirus (CMV). Exemplary commercially available expression vectors for use in mammalian host cells include pCEP4 and pcDNA3.

[0163] Physical methods for introducing nucleic acids into host cells (e.g., mammalian host cells) include calcium phosphate precipitation, lipid transfection, particle bombardment, microinjection, electroporation, etc. Methods for generating cells containing vectors and / or exogenous nucleic acids are well known in the art.

[0164] Biological methods for introducing polynucleotides of interest into host cells include the use of DNA and RNA vectors. Viral vectors, and especially retroviral vectors, have become the most widely used method for inserting genes into mammalian (e.g., human) cells. Other viral vectors may be derived from lentiviruses, poxviruses, herpes simplex virus I, adenoviruses, and adeno-associated viruses, among others.

[0165] Methods for introducing DNA and RNA polynucleotides of interest into host cells include cell electroporation, in which an electric field is applied to the cell to increase cell membrane permeability, thereby allowing the introduction of chemicals, drugs, or polynucleotides into the cell. Electroporation can be used to introduce said fusion peptides containing DNA or RNA constructs into mammalian or prokaryotic cells.

[0166] In some embodiments, electroporation of the cells results in the expression of the fusion peptide on the surface of T cells, NK cells, and / or NKT cells. This expression can be transient or stable throughout the cell's lifespan. Electroporation can be performed using methods known in the art, including the MaxCyte transfection system.

[0167] Regardless of the method used to introduce exogenous nucleic acids into host cells, the presence of recombinant nucleic acid sequences in host cells can be routinely confirmed by a variety of assays known in the art. Such assays include, for example, “molecular biology” assays known in the art, such as DNA and RNA blotting, RT-PCR, and PCR; and “biochemical” assays, such as detecting the presence or absence of specific peptides, for example by immunological means (ELISA and Western blotting) or by assays described herein.

[0168] Purification of fusion peptides The fusion polypeptide, generated through DNA recombination technology, comprises one or more precipitating peptide domains and one or more binding peptide domains. The fusion polypeptide of the present invention can be directly isolated and purified from, for example, crude cell extracts or culture media, i.e., simply by subjecting the extract containing the fusion polypeptide to conditions that allow the precipitating peptide domains to precipitate, such as a certain concentration of Ca. 2+ The precipitated fusion peptides can be easily separated from the liquid in a highly purified form using various methods such as centrifugation, decantation, filtration, or any combination thereof. Specifically, after cell harvesting, the fusion peptides are purified to remove bacterial proteins and prepared as a dissolved fusion peptide extract. The purified fusion peptides obtained thereby can be used to form extracts specifically for the purification of extracellular vesicles or exosomes. In some embodiments, a method for recovering recombinant peptides from inclusion bodies within prokaryotic host cells is provided, the method comprising cross-flow filtration of a suspension containing the inclusion bodies with unwanted substances (such as fragments of broken cells), and subjecting the solution to a set of conditions that allow the fusion peptides to precipitate from the solution. In some embodiments, the precipitated fusion peptides are recovered from the solution.

[0169] Fusion peptides can be used in a fused form. In some embodiments, the precipitated peptide domain can also be hydrolyzed or cleaved from the fusion peptide. This can be achieved through various methods. For example, a DNA fragment encoding the fusion peptide (e.g., a linker sequence) can be used to link DNA fragments encoding the binding peptide domain and the precipitated peptide domain. The linker sequence, in addition to providing a convenient proteolytic cleavage site, can also serve as a multiple linker, i.e., providing multiple DNA restriction sites to facilitate the fusion of the DNA fragments encoding the binding peptide domain and the precipitated peptide domain, and / or as a spacer separating the binding peptide domain and the precipitated peptide domain, allowing the proteolytic agent to approach and cleave the fusion peptide. In some embodiments, the precipitated fusion peptide can be separated by centrifugation, decantation, filtration, or any combination thereof.

[0170] In some embodiments, the fusion polypeptide is optionally fused with a heterologous polypeptide sequence specifically disclosed herein or known in the art to promote expression.

[0171] In addition to recombination methods, various liquid-phase and solid-phase chemical methods known in the art can be used to generate the fusion peptides through organic chemical synthesis of the desired peptides. Various automated synthesizers are commercially available and can be used according to known protocols. They allow the incorporation of non-natural amino acid residues into the amino acid sequence of the fusion peptides.

[0172] As used herein, the terms “capture,” “separation,” and “enrichment” are used interchangeably to refer to the state (e.g., multiple known or unknown quantities and / or concentrations) of a desired population of extracellular vesicles after one or more separation processes (e.g., selection or enrichment of desired extracellular vesicles). As used herein, “separation” refers to a method for separating extracellular vesicles from other materials in a sample. Conventional separation of extracellular vesicles may include differential centrifugation via ultracentrifugation, sucrose gradient purification, and combined filtration / concentration, etc. Separation of exosomes may also utilize commercially available kits and the use of lectin affinity or affinity chromatography. In this invention, the separation of extracellular vesicles from a sample in a short time, with few steps, and with high purity can be achieved using fusion peptides that specifically recognize and bind to extracellular vesicles or exosomes. In some embodiments, the separated extracellular vesicles do not have detectable undesirable activity, or alternatively, the level or amount of undesirable activity is at or below an acceptable level or amount. In other embodiments, the isolated extracellular vesicle composition has a desired amount and / or concentration of extracellular vesicles at or above an acceptable amount and / or concentration. In some embodiments, the isolated extracellular vesicle composition is enriched compared to the starting material (e.g., plasma) from which the composition was obtained. This enrichment compared to the starting material can be 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, 99.9%, 99.99%, 99.999%, 99.9999%, or greater than 99.9999%.

[0173] The term "analysis" for extracellular vesicles as used in this article includes any method that allows for direct or indirect visualization of extracellular vesicles and can be in vivo or ex vivo. For example, analysis may include, but is not limited to, in vitro microscopy or cell counting to detect and visualize isolated extracellular vesicles, as well as flow cytometry, fluorescence imaging, etc. Extracellular vesicles can be analyzed by: flow cytometry expression of surface markers of extracellular vesicles: CD63, CD47, CD9, CD81, TSG101, HSP70, and ALIX, and / or transmission electron microscopy (TEM). Additionally, nanoparticle tracking analysis and microBCA assays can be used to determine the number of extracellular vesicles.

[0174] Compared to other extracellular vesicle separation methods known in the art (particularly ultracentrifugation and polyethylene glycol precipitation), the fusion peptides and methods described herein offer several advantages. In fact, the method for separating extracellular vesicles using the fusion peptides described herein provides high-throughput separation, ease of handling, and reduced costs. Furthermore, the method of the present invention allows for rapid separation of extracellular vesicles, as the binding and precipitation of the fusion peptide with the extracellular vesicles can be completed in 30 minutes or less.

[0175] The following embodiments and accompanying drawings are provided to aid in understanding the present invention. However, it should be understood that these embodiments and drawings are for illustrative purposes only and do not constitute any limitation. The actual scope of protection of the present invention is set forth in the claims. It should be understood that any modifications and changes can be made without departing from the spirit of the present invention.

[0176] Example Unless otherwise stated, the present invention will be carried out using conventional techniques of cell biology, cell culture, molecular biology, microbiology, recombinant DNA, protein chemistry, and immunology, techniques described in the literature or performed in accordance with product instructions. See, for example, J. Sambrook's *Molecular Cloning: A Laboratory Manual* (4th edition, Science Press). Unless otherwise specified, the materials, reagents, or instruments used in the examples are all commercially available conventional products.

[0177] This invention provides a method for extracting exosomes from a condition-responsive fusion peptide. The C-terminus of the peptide binds to the exosome bilayer membrane structure, and its N-terminus can precipitate in response to conditions, thereby rapidly and efficiently separating exosomes from solution.

[0178] Basic principle: The fusion peptide contains a binding peptide domain at its C-terminus that can bind to the exosome bilayer membrane structure, and a precipitation peptide domain at its N-terminus that responds to conditional precipitation. After the fusion peptide binds to the exosome at its C-terminus, precipitation is carried out using the N-terminus, thereby separating the exosome from the body fluid by low-speed centrifugation.

[0179] The main steps of this method are: 1. Design and construct fusion peptides.

[0180] 2. High-purity peptides were obtained through in vitro recombination and purification.

[0181] 3. Incubate the peptide and the solution from which the exosomes are to be extracted, and use the binding peptide to bind the exosomes.

[0182] 4. Add Ca 2+ Other conditions may promote the aggregation of precipitated peptides and precipitate polypeptide-exosome complexes, which can be obtained by centrifugation.

[0183] 5. Elute exosomes.

[0184] 6. Analyze the eluted exosomes.

[0185] The method of separating and purifying extracellular vesicles from plasma using fusion peptides includes the acquisition of the fusion peptides and the extraction of extracellular vesicles from plasma. The main steps are as follows: 1) Construct a fusion peptide expression vector, the main components of which include: a T7 promoter, a precipitating peptide sequence, a linker sequence, a protease cleavage site sequence, a binding peptide sequence, and a T7 terminator. A schematic diagram of the expression vector is shown below. Figure 1 As shown.

[0186] 2) The fusion peptide was expressed using *E. coli* to obtain a high-purity fusion peptide. The structural diagram of the fusion peptide is shown below. Figure 2 As shown.

[0187] 3) Add the peptide to the test solution and incubate at room temperature for 5 minutes.

[0188] 4) Apply the responsive precipitation conditions to the solution and continue incubation at room temperature for 2 minutes.

[0189] 5) Centrifuge at low speed to obtain the fusion peptide-exosome complex. A schematic diagram of the structure of the fusion peptide-exosome complex is shown below. Figure 3 As shown.

[0190] 6) Use elution buffer or protease to elute the exosomes to obtain high-purity exosomes.

[0191] Example 1 Ca 2+ Response precipitation RTX fusion peptide 1.1 Ca 2+ Expression of precipitated fusion peptides The specific process of this embodiment is as follows: (1) Construct an RTX fusion peptide sequence (SEQ ID NO: 1), including a precipitated peptide RTX repeat sequence, a linker sequence, a protease cleavage site linker sequence (SEQ ID NO: 35), and an exosome binding peptide sequence (SEQ ID NO: 25).

[0192] In this embodiment, the precipitated peptide selected is the one that responds to Ca. 2+ The precipitated RTX sequence. In other embodiments, the selected precipitating peptide may also be a sequence that precipitates in response to temperature, pH, salt concentration, and light.

[0193] (2) The gene was synthesized according to the above sequence, and a large number of the synthesized gene was amplified by PCR. The gene fragment was ligated to the linearized pET-28a plasmid using T4 DNA ligase. The product was transformed into DH5α competent cells, cultured on agarose plates, and single clones were selected for PCR verification and sequencing.

[0194] (3) Transform the correctly sequenced plasmid into the E. coli BL21(DE3) expression system. Select suitable single clones and inoculate them into 5 mL of LB medium. Incubate at 37°C and 220 rpm for 3 h, then transfer to 1 L of LB medium and wait for OD. 600 Once the value reached 0.6, IPTG was added and the bacteria were expressed at 16℃ and 220 rpm for 16 h. The bacteria were then collected by centrifugation.

[0195] (4) Add Tris buffer to the collected bacteria and sonicate to lyse the bacteria. Centrifuge at 8000 rpm to remove cell debris. Centrifuge at 150000g to remove residual E. coli-derived exosomes from the lysate. Add 50 mM CaCl2 to the supernatant after centrifugation, mix well, let stand for 5 min, and centrifuge at 8000 rpm to collect the protein precipitate. Wash the precipitate twice with Tris buffer, add 1 mL of Tris buffer containing 50 mM EDTA to dissolve the precipitate, and after the precipitate is dissolved, add 10 times the volume of Tris buffer to dilute the protein. Identify the target protein using SDS-PAGE. After successful identification, store at -80℃ for later use.

[0196] 1.2 Ca 2+ Response precipitation fusion peptide extraction exosomes Experimental apparatus: mixer, low-temperature centrifuge Experimental reagents: Tris buffer, CaCl2, EGTA, fusion peptide (1) Take 200 μL of the plasma to be tested, centrifuge at 3000g for 5 min to remove cell debris. Add 800 μL of Tris buffer to the supernatant as the test sample.

[0197] (2) Add 800 μL of Tris buffer to the supernatant, mix well, then add 80 μL of the purified fusion peptide and incubate at room temperature for 5 min. Add 25 mM CaCl2 to the mixture, mix well, and let stand at room temperature for 2 min. Centrifuge at 10000 rpm for 3 min. Collect the protein precipitate.

[0198] (3) Add 100 μL of 25mM EGTA buffer to the above precipitate, mix well with a pipette, and store at -80℃ or perform subsequent detection.

[0199] Example 2: Temperature- and salt ion-responsive precipitated peptide (ELP fusion peptide) 2.1 Expression and purification of fusion peptides (1) Construct an ELP fusion peptide sequence (SEQ ID NO: 2), including the precipitated peptide ELP sequence, the linker sequence, the protease cleavage site linker sequence, and the exosome binding peptide sequence.

[0200] (2) The gene was synthesized according to the above sequence, and a large number of the synthesized gene was amplified by PCR. The gene fragment was ligated to the linearized pET-28a plasmid using T4 DNA ligase. The product was transformed into DH5α competent cells, cultured on agarose plates, and single clones were selected for PCR verification and sequencing.

[0201] (3) The ELP fusion peptide was expressed by *E. coli* (BL21). After expression, the bacteria were collected by centrifugation. PBS buffer was added to the collected bacteria, and the bacteria were lysed by sonication. Cell debris was removed by centrifugation at 8000 rpm. *E. coli*-derived exosomes were removed from the lysate by centrifugation at 150,000 g. 1M NaCl was added to the protein supernatant, and the mixture was heated at 37°C for 15 min. The protein precipitate was collected by centrifugation at 8000 rpm. The protein precipitate was dissolved in PBS buffer, and the target protein was identified by SDS-PAGE. After successful identification, the protein was stored at -80°C.

[0202] 2.2 Extraction of exosomes from fusion peptides Experimental apparatus: mixer, low-temperature centrifuge, constant-temperature metal bath Experimental reagents: Tris buffer, NaCl, fusion peptide (1) Take 200 μL of the plasma to be tested, centrifuge at 3000 g for 5 min to remove cell debris. Add 800 μL of Tris buffer to the supernatant as the test sample.

[0203] (2) Add 80 μL of the purified fusion peptide to the supernatant and incubate at room temperature for 5 min. Add 1M NaCl to the mixture and heat at 37℃ for 15 min. Centrifuge at 10000 rpm for 3 min. Collect the protein precipitate.

[0204] (3) Add 100 μL of Tris buffer to the above precipitate, mix well with a pipette, and store at -80℃ or perform subsequent detection.

[0205] Example 3: Salt ion-responsive precipitated peptide (MPA fusion peptide) 3.1 Expression and purification of fusion peptides (1) Construct an MPA fusion peptide sequence (SEQ ID NO: 3), including the precipitated peptide MPA sequence, the linker sequence, the protease cleavage site linker sequence, and the exosome binding peptide sequence.

[0206] (2) The gene was synthesized according to the above sequence, and a large number of the synthesized gene was amplified by PCR. The gene fragment was ligated to the linearized pET-28a plasmid using T4 DNA ligase. The product was transformed into DH5α competent cells, cultured on agarose plates, and single clones were selected for PCR verification and sequencing.

[0207] (3) The MPA fusion peptide was expressed by *E. coli* (BL21). After expression, the bacteria were collected by centrifugation. The collected bacteria were resuspended in low-salt buffer (20 mM Tris-HCl, 1 mM EDTA, pH 8.0), sonicated, and centrifuged at 8000 rpm to remove cell debris. The lysate was then centrifuged at 150,000 g to remove residual *E. coli*-derived exosomes. 0.7 M Na2SO4 / (NH4)2SO4 or 3 M NaCl was added to the supernatant after centrifugation, and the mixture was incubated at 4°C for 20 min. The protein precipitate was collected by centrifugation at 8000 rpm. The protein precipitate was dissolved in low-salt buffer (20 mM Tris-HCl, 1 mM EDTA, pH 8.0), and the target protein was identified by SDS-PAGE. After successful identification, the protein was stored at -80°C.

[0208] 3.2 Extraction of exosomes from fusion peptides: Experimental apparatus: mixer, low-temperature centrifuge Experimental reagents: low-salt Tris buffer, NaCl / Na2SO4 / (NH4)2SO4, fusion peptide (1) Take 200 μL of the plasma to be tested, centrifuge at 3000 g for 5 min to remove cell debris. Add 800 μL of Tris buffer to the supernatant as the test sample.

[0209] (2) Add 80 μL of the purified fusion peptide to the supernatant and incubate at room temperature for 5 min. Add 0.7M Na2SO4 / (NH4)2SO4 or 3M NaCl to the mixture, incubate at 4℃ for 20 min, and then centrifuge at 10000 rmp for 3 min. Collect the protein precipitate.

[0210] (3) Add 100 μL of low-salt Tris buffer to the above precipitate, mix well with a pipette, and store at -80℃ or perform subsequent detection.

[0211] Example 4 Ca 2+ Ion-responsive precipitated peptide (Annexin B1 fusion peptide) 4.1 Expression and purification of fusion peptides (1) Construct the Annexin B1 fusion peptide sequence (SEQ ID NO: 4), including the Annexin B1 precipitated peptide sequence, the linker sequence, the protease cleavage site linker sequence, and the exosome binding peptide sequence.

[0212] (2) The gene was synthesized according to the above sequence, and a large number of the synthesized gene was amplified by PCR. The gene fragment was ligated to the linearized pET-28a plasmid using T4 DNA ligase. The product was transformed into DH5α competent cells, cultured on agarose plates, and single clones were selected for PCR verification and sequencing.

[0213] (3) The Annexin B1 fusion peptide was expressed by E. coli (BL21). After expression, the bacteria were collected by centrifugation. PBS buffer was added to the collected bacteria, and the bacteria were lysed by sonication. Cell debris was removed by centrifugation at 8000 rpm. Exosomes of E. coli remaining in the lysate were removed by centrifugation at 150000g.

[0214] Add 20 mM CaCl2 to the supernatant after centrifugation, mix well, and incubate for 1 h. Collect the protein precipitate by centrifugation at 8000 rpm. Wash the precipitate twice with Tris buffer, and dissolve the precipitate in 1 mL of Tris buffer containing 20 mM EGTA. Identify the target protein using SDS-PAGE. After successful identification, store at -80℃.

[0215] 4.2 Extraction of exosomes from fusion peptides: Experimental apparatus: mixer, low-temperature centrifuge Experimental reagents: Tris buffer, CaCl2, EGTA, fusion peptide (1) Take 200 μL of the plasma to be tested, centrifuge at 3000 g for 5 min to remove cell debris. Add 800 μL of Tris buffer to the supernatant as the test sample.

[0216] (2) Add 80 μL of the purified fusion peptide to the supernatant and incubate at room temperature for 5 min. Add 50 mM CaCl2 to the mixture and incubate at 4 °C for 10 min. Centrifuge at 10,000 rpm for 3 min. Collect the protein precipitate.

[0217] (3) Add 100 μL of 20 mM EGTA buffer to the above precipitate, mix well with a pipette, and store at -80℃ or perform subsequent detection.

[0218] Example 5: pH-responsive precipitating peptide (CspB50 fusion peptide) 5.1 Expression and purification of fusion peptides (1) Construct a CspB50 fusion peptide sequence (SEQ ID NO: 5), including the precipitated peptide CspB50 sequence, the linker sequence, the protease cleavage site linker sequence, and the exosome binding peptide sequence.

[0219] (2) The gene was synthesized according to the above sequence, and a large number of the synthesized gene was amplified by PCR. The gene fragment was ligated to the linearized pET-28a plasmid using T4 DNA ligase. The product was transformed into DH5α competent cells, cultured on agarose plates, and single clones were selected for PCR verification and sequencing.

[0220] (3) The CspB50 fusion peptide was expressed and secreted into the culture medium by *C. glutamicum*. After expression, the culture medium was collected by centrifugation. 0.5 M H2SO4 was added to the supernatant to adjust the pH of the medium to 5.0, and then the medium was incubated at 4°C for 20 min. The protein precipitate was collected by centrifugation at 10,000 g for 10 min. The supernatant was discarded, and the protein precipitate was dissolved in Tris buffer at pH 8.0. The protein solution was then centrifuged at 150,000 g to remove any remaining exosomes. The protein solution was collected, and the target protein was identified by SDS-PAGE. After successful identification, the protein was stored at -80°C.

[0221] 5.2 Extraction of exosomes from fusion peptides: Experimental apparatus: mixer, low-temperature centrifuge Experimental reagents: Tris buffer, 0.5M H2SO4, fusion peptide (1) Take 200 μL of the plasma to be tested, centrifuge at 3000 g for 5 min to remove cell debris. Add 800 μL of Tris buffer to the supernatant as the test sample.

[0222] (2) Add 80 μL of the purified fusion peptide to the supernatant, incubate at room temperature for 5 min, then add 0.5 M H2SO4 to the mixture until the pH of the solution is 5.0, incubate at 4℃ for 20 min, and centrifuge at 10000 rmp for 3 min. Collect the protein precipitate.

[0223] (3) Add 100 μL of Tris buffer (pH 8.0) to the above precipitate, mix well with a pipette, and store at -80℃ or perform subsequent detection.

[0224] Example 6: Light-responsive precipitated peptide (ELP-Dronpa 145N-ELP fusion peptide) 6.1 Expression and purification of fusion peptides (1) Construct the ELP-Dronpa 145N-ELP fusion peptide sequence (SEQ ID NO: 6), including the precipitated peptide ELP-Dronpa 145N-ELP sequence, the linker sequence, the protease cleavage site linker sequence, and the exosome binding peptide sequence.

[0225] (2) The gene was synthesized according to the above sequence, and a large number of the synthesized gene was amplified by PCR. The gene fragment was ligated to the linearized pET-28a plasmid using T4 DNA ligase. The product was transformed into DH5α competent cells, cultured on agarose plates, and single clones were selected for PCR verification and sequencing.

[0226] (3) The fusion peptide was expressed by *E. coli* (BL21). After expression, the bacteria were collected by centrifugation. PBS buffer was added to the collected bacteria, and the bacteria were lysed by sonication. Cell debris was removed by centrifugation at 8000 rpm. *E. coli*-derived exosomes in the lysate were removed by centrifugation at 150,000 g. The protein was purified using the ITC method. The protein supernatant was heated at 60°C for 1 h, followed by centrifugation at 40°C and 10,000 g for 10 min. The protein precipitate was collected and dissolved in 1×PBS. To improve protein purity, the above purification steps were repeated twice. The final protein solution was identified by SDS-PAGE. After verification, the solution was stored at -80°C.

[0227] 6.2 Extraction of exosomes from fusion peptides: Experimental apparatus: Low-temperature centrifuge, 400 nm / 500 nm custom light source Experimental reagents: Tris buffer, fusion peptide (1) Take 200 μL of the plasma to be tested, centrifuge at 3000 g for 5 min to remove cell debris. Add 800 μL of Tris buffer to the supernatant as the test sample.

[0228] (2) Add 100 μg of the purified fusion peptide to the supernatant, incubate at room temperature for 5 min, then irradiate the protein solution with a custom light source of 500 mW (400 nm) for 20 min, and then let it stand at room temperature for 30 min to stabilize the reaction. Wash the gel three times with Tris buffer.

[0229] (3) Irradiate the protein gel with a custom light source of 250 mW (500 nm) for 20 min to dissolve the gel and obtain the exosome solution.

[0230] Example 7 Other salt ion-responsive precipitated peptides (CPA fusion peptide and IPA fusion peptide) 7.1 Expression and purification of fusion peptides (1) Construct a CPA fusion peptide sequence (SEQ ID NO: 7) or an IPA fusion peptide sequence (SEQ ID NO: 8), including a precipitated peptide CPA sequence or IPA sequence, a linker sequence, a protease cleavage site linker sequence, and an exosome binding peptide sequence.

[0231] (2) The gene was synthesized according to the above sequence, and a large number of the synthesized gene was amplified by PCR. The gene fragment was ligated to the linearized pET-28a plasmid using T4 DNA ligase. The product was transformed into DH5α competent cells, cultured on agarose plates, and single clones were selected for PCR verification and sequencing.

[0232] (3) The CPA fusion peptide or IPA fusion peptide was expressed by *E. coli* (BL21). After expression, the bacteria were collected by centrifugation. The collected bacteria were resuspended in low-salt buffer (20 mM Tris-HCl, 1 mM EDTA, pH 8.0), lysed by sonication, and centrifuged at 8000 rpm to remove cell debris. The exosomes derived from *E. coli* were removed by centrifugation at 150,000 g. 1.4 M Na2SO4 / (NH4)2SO4 or 3 M NaCl were added to the supernatant after centrifugation, and the mixture was incubated at 4°C for 1 h. The protein precipitate was collected by centrifugation at 8000 rpm. The protein precipitate was dissolved in low-salt buffer (20 mM Tris-HCl, 1 mM EDTA, pH 8.0), and the target protein was identified by SDS-PAGE. After successful identification, the protein was stored at -80°C.

[0233] 7.2 Extraction of exosomes from fusion peptides: Experimental apparatus: mixer, low-temperature centrifuge Experimental reagents: low-salt Tris buffer, NaCl / Na2SO4 / (NH4)2SO4, fusion peptide (1) Take 200 μL of the plasma to be tested, centrifuge at 3000 g for 5 min to remove cell debris. Add 800 μL of Tris buffer to the supernatant as the test sample.

[0234] (2) Add 80 μL of the purified fusion peptide to the supernatant and incubate at room temperature for 5 min. Add 1.4M Na2SO4 / (NH4)2SO4 or 3M NaCl to the mixture, incubate at 4℃ for 1 h, and then centrifuge at 10000 rmp for 3 min. Collect the protein precipitate.

[0235] (3) Add 100 μL of low-salt Tris buffer to the above precipitate, mix well with a pipette, and store at -80℃ or perform subsequent detection.

[0236] Comparative Example 1: Extraction of exosomes by ultracentrifugation (UC) Ultracentrifugation is the recognized gold standard for EV extraction.

[0237] Experimental apparatus: Low-temperature centrifuge, ultracentrifuge Experimental reagent: Tris buffer Experimental methods: (1) Take 200 μL of the plasma to be tested, centrifuge at 3000g for 5 min to remove cell debris.

[0238] (2) Add 800 μL of Tris buffer to the supernatant above, mix well, and transfer to a 1 mL ultracentrifuge tube. Centrifuge at 150000 g for 2 h.

[0239] (3) Discard the supernatant, add 100 μL of Tris buffer to resuspend the exosomes, and store at -80℃ or perform subsequent detection.

[0240] Comparative Example 2: Extraction of exosomes using polyethylene glycol (PEG) precipitation method The polyethylene glycol precipitation method is the most successful commercially available reagent kit solution.

[0241] Experimental apparatus: mixer, low-temperature centrifuge Experimental reagents: Tris buffer, commercial precipitation kit (Thermo Fisher, catalog number: 4484450) Experimental methods: (1) Take 200 μL of the plasma to be tested, centrifuge at 3000g for 5 min to remove cell debris.

[0242] (2) Add 200 μL of Tris buffer to the supernatant above, mix well, and then add 200 μL of precipitation reagent (commercial kit). Shake at 4℃ for 30 min and then centrifuge at 10000 rpm for 5 min.

[0243] (3) Discard the supernatant, add 100 μL of Tris buffer to resuspend the exosomes, and store at -80℃ or perform subsequent detection.

[0244] Comparison of results from Example 1, Comparative Example 1, and Comparative Example 2 Ca from Example 1 2+ The extraction process and the extracellular vesicles obtained from the precipitated RTX fusion peptide and Comparative Examples 1 and 2 were analyzed and compared. The yield and purity of the obtained extracellular vesicles were detected by Western blotting and transmission electron microscopy (TEM); the particle size distribution of the extracellular vesicles was confirmed by nanoparticle tracking analysis (NTA).

[0245] 1. Extraction process Figure 4 The Ca of Example 1 is shown. 2+A comparison of the extraction times required for extracellular vesicles from plasma using the fusion peptide precipitation method, ultracentrifugation, and polyethylene glycol precipitation. Ultracentrifugation typically requires 3 hours; polyethylene glycol precipitation typically takes 50 minutes; and the fusion peptide method of Example 1 only requires 15 minutes to complete the extraction process. The extraction time required by the fusion peptide method of Example 1 is significantly shorter than that of ultracentrifugation and polyethylene glycol precipitation.

[0246] 2. Production of extracellular vesicles Figure 5 The Ca of Example 1 is shown. 2+ The yields of extracellular vesicles extracted from plasma were compared using the RTX fusion peptide precipitation method, ultracentrifugation, and polyethylene glycol precipitation. Alix was used as a marker for extracellular vesicles, and the extraction yields were compared via Western blotting. Figure 5 The middle left figure shows Ca of Example 1. 2+ A comparison of the yields of extracellular vesicles in plasma extracted by the RTX fusion peptide precipitation method, ultracentrifugation, and polyethylene glycol precipitation method. Figure 5 As can be seen in the middle right figure, Ca 2+ The yield of extracellular vesicles extracted by the RTX fusion peptide precipitation method was recorded as 100%. Compared with the yield of extracellular vesicle products obtained by ultracentrifugation and polyethylene glycol precipitation, the yield of extracellular vesicles extracted by the fusion peptide method in Example 1 was significantly higher than that obtained by ultracentrifugation and polyethylene glycol precipitation.

[0247] 3. Purity of extracellular vesicles Figure 6 This paper compares the purity results of extracellular vesicles extracted from plasma using the fusion peptide method, ultracentrifugation, and polyethylene glycol precipitation, respectively, as shown in Example 1. The purity of the obtained extracellular vesicle products was determined using transmission electron microscopy. Figure 6 As shown in the left-middle figure, the extracellular vesicles in the plasma extracted using the fusion peptide method of Example 1 are almost free of lipoprotein contamination, indicating good purity; Figure 6 As can be seen in the middle right figure, Ca 2+ The purity of extracellular vesicles extracted by the RTX fusion peptide precipitation method was recorded as 100%. Compared with the purity of extracellular vesicle products obtained by ultracentrifugation and polyethylene glycol precipitation, the purity of extracellular vesicles extracted by the fusion peptide method in Example 1 was significantly higher than that obtained by ultracentrifugation and polyethylene glycol precipitation.

[0248] 4. Granule distribution of extracellular vesicles Figure 7 The Ca of Example 1 is shown. 2+The particle distribution of extracellular vesicles in plasma extracted by RTX fusion peptide precipitation, ultracentrifugation, and polyethylene glycol precipitation were compared. Nanoparticle tracking analysis (NTA) was used to detect the particle distribution of extracellular vesicles obtained by different extraction methods. Figure 7 It can be seen that, using Ca from Example 1 2+ The extracellular vesicles extracted from plasma by the RTX fusion peptide precipitation method exhibited concentrated and uniform particle distribution peaks and good surface condition, which were similar to those of extracellular vesicles extracted by the polyethylene glycol precipitation method, and were superior to the ultracentrifugation method.

[0249] Comparison of results from Examples 1 to 7 The extraction process of extracellular vesicles from the fusion peptides in Examples 1 to 7 and the extracellular vesicles obtained were analyzed and compared.

[0250] 1. Extraction process Figure 8 The results show a comparison of the yields of the fusion peptides from Examples 1 to 7 when used to extract extracellular vesicles from plasma. Figure 8 As can be seen in the right figure of Example 1, Ca 2+ The yield of extracellular vesicles extracted in response to the fusion peptide was recorded as 100%, relative to Ca in Example 1. 2+ In response to the fusion peptides, the yield of extracellular vesicles extracted from the fusion peptides of Examples 2 to 7 of this invention is all higher than 60%.

[0251] Table 1 shows a comparison of the extraction times required by the fusion peptides of Examples 1-7 for extracellular vesicles from plasma. The extraction process using the fusion peptides of Examples 1-5 can be completed in approximately 30 minutes. The extraction time required by the fusion peptide method of the present invention for extracellular vesicles is significantly shorter than that of ultracentrifugation and polyethylene glycol precipitation.

[0252] Table 1

[0253] 2. Purity of extracellular vesicles Figure 9 The purity comparison results of extracellular vesicles obtained from the fusion peptides of Examples 1 to 7 are shown. Figure 9 It can be seen that Ca in Example 1 2+ The purity of the extracellular vesicles extracted from the fusion peptide was recorded as 100%, relative to Ca in Example 1. 2+ In response to the fusion peptides, the purity of the extracellular vesicles extracted from the fusion peptides of Examples 2 to 7 of this invention is mostly close to or greater than 50%.

[0254] In summary, compared to traditional extraction methods, peptide extraction methods can achieve large-scale, economical, and rapid extraction (excluding light-induced extraction), with improved yield and purity (CPA peptide purification also yields higher purity than UC and PEG). While light-induced precipitation takes longer than other peptide extraction methods, the induction process is gentler and has minimal impact on the extracted exosomes.

[0255] This invention provides a technical approach and scheme for in vitro purification of exosomes based on peptides, using the membrane structure of exosomes as binding targets and utilizing Ca... 2+ Exosomes are separated by precipitation under specific conditions. The method of this invention is unaffected by the heterogeneity of exosomes, allowing for specific purification and maximizing the preservation of exosome information. It also enables low-cost and rapid large-scale purification of exosomes. Currently, exosomes, as valuable clinical testing materials, are limited by complex extraction processes or the ability to extract only a single form of exosome, hindering their effective clinical use. This invention aims to provide a rapid, high-purity, and user-friendly method for efficient extraction of total exosomes from plasma / serum, particularly suitable for clinical applications, and facilitating the promotion of exosomes as truly usable specimens for physiological and disease diagnosis.

[0256] The technical solutions of the present invention are not limited to the specific embodiments described above. Any technical modifications made in accordance with the technical solutions of the present invention fall within the protection scope of the present invention.

Claims

1. A fusion polypeptide comprising a binding peptide domain and a precipitation peptide domain, wherein the binding peptide domain is capable of specifically binding extracellular vesicles in a sample to form a fusion polypeptide-extracellular vesicle complex, and the precipitation peptide domain is capable of precipitating the fusion polypeptide-extracellular vesicle complex in response to a change in conditions selected from: pH, temperature, salt concentration, light wavelength, light intensity, charge, or any combination thereof.

2. The fusion polypeptide according to claim 1, wherein, The precipitated peptide domain contains an amino acid sequence selected from RTX toxin peptide, elastin-like peptide, CpA peptide / IpA peptide / MpA peptide, Annexin B1 peptide, CspB peptide, Dronpa peptide, and any combination thereof. Preferably, the RTX toxin peptide comprises 1-30 tandemly linked repeating sequences, wherein the repeating sequences are selected from SEQ ID NO: 9, SEQ ID NO: 10 and any combination thereof, and / or the elastin-like polypeptide comprises 1-30 tandemly linked repeating pentapeptides, wherein the repeating pentapeptide sequence is SEQ ID NO: 38; Preferably, the precipitated peptide domain comprises an amino acid sequence selected from any of the following: (1) The amino acid sequences shown in SEQ ID NOs: 11-24; and (2) An amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequence shown in SEQ ID NOs: 11-24 and retaining the ability to form a precipitate in response to changes in conditions selected from: pH, temperature, salt concentration, light wavelength, light intensity, charge, or any combination thereof, and / or Wherein, the binding peptide domain binds to the extracellular vesicle membrane of the extracellular vesicle, and preferably the binding peptide domain comprises one or more amphiphilic α-helices.

3. The fusion polypeptide according to claim 1 or 2, wherein the binding peptide domain is derived from a membrane curvature-sensing peptide or protein domain, the membrane curvature-sensing peptide or protein domain being selected from one or more of a cardiomylated domain, an ESCRT binding motif, a palmitoylated domain, an isoprenelated domain, a lysosomal domain, a glycosylphosphatidylinositol anchoring protein domain, and an immunoglobulin heavy chain binding protein domain, preferably a cardiomylated domain, a glycosylphosphatidylinositol anchoring protein domain, or any combination thereof, more preferably, the binding peptide domain comprises an amino acid sequence selected from any of the following: (1) The amino acid sequence shown in SEQ ID NOs: 25-34; and (2) An amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequence shown in SEQ ID NOs: 25~34 and maintaining the ability to bind to the extracellular vesicle membrane.

4. The fusion polypeptide according to any one of claims 1 to 3, wherein the fusion polypeptide comprises a plurality of binding peptide domains, preferably, the plurality of binding peptide domains bind to the same extracellular vesicle membrane, or bind to different extracellular vesicle membranes, and even more preferably, the plurality of binding peptide domains are connected in series; and / or The fusion polypeptide comprises multiple precipitating peptide domains. Preferably, the multiple precipitating peptide domains form a precipitate in response to changes in the same conditions or in response to changes in different conditions. More preferably, the multiple precipitating peptide domains are connected in series.

5. The fusion polypeptide according to claim 4, wherein the plurality of precipitated peptide domains are directly connected or connected via linkers to each other, the plurality of binding peptide domains are directly connected to each other, and / or the precipitated peptide domains are directly connected to the binding peptide domains. Preferably, the connector is selected from flexible connectors, rigid connectors, and / or cuttable connectors. Preferably, the linker comprises the sequence (GmSo)n, (EA3K)n, (PmXo)n1, or (XP)n2, wherein m, n, n1, n2, or o are each independently selected from integers 1-20, and X is any amino acid. Preferably, the linker comprises a sequence of (PmXo)n1 or (XP)n2 or a combination thereof, wherein m, n1, n2 or o is at least 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, and X is any amino acid.

6. The fusion polypeptide according to any one of claims 1-5, wherein the fusion polypeptide comprises an amino acid sequence selected from any one of the following: (1) The amino acid sequences shown in SEQ ID NOs: 1-8; and (2) An amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequence shown in SEQ ID NOs: 1-8.

7. An isolated nucleic acid molecule encoding a fusion polypeptide according to any one of claims 1-6.

8. An expression vector comprising the nucleic acid molecule of claim 7. Preferably, the expression vector is selected from viral expression vectors, bacterial expression vectors, or fungal expression vectors. Preferably, the expression vector comprises the pET 28a plasmid of the Escherichia coli expression system.

9. A host cell comprising the nucleic acid molecule of claim 7 or the expression vector of claim 8.

10. A method for enriching, capturing, and / or isolating extracellular vesicles from a fluid sample, the method comprising: (a) Providing a fusion polypeptide according to any one of claims 1-6, wherein the sample is incubated with the fusion polypeptide to allow the fusion polypeptide to form a fusion polypeptide-extracellular vesicle complex with extracellular vesicles in the sample. (b) subjecting the sample to the changes in conditions thereby precipitating the fusion polypeptide-extracellular vesicle complex from the sample; and (c) Optionally, the precipitated fusion peptide-extracellular vesicle complex is separated. Preferably, the separation is achieved by centrifugation, filtration, decantation, or any combination thereof.

11. The method of claim 10, wherein the sample comprises one or more of cell culture supernatant, a sample obtained from an animal subject, or an apoplast fluid from a plant, preferably, the sample obtained from the animal subject is selected from one or more of whole blood, serum, plasma, urine, saliva, sputum, breast milk, cerebrospinal fluid, amniotic fluid, ascites, lymph, bone marrow, synovial fluid, bronchoalveolar lavage fluid, pleural effusion, or ascites.

12. The method according to claim 10 or 11, wherein the incubation temperature in step (a) is 15~25°C, and / or The incubation time in step (a) is 2-10 min, preferably 2-5 min, and / or The time for step (b) is 1 to 70 minutes, preferably 1 to 20 minutes, and more preferably 1 to 10 minutes.

13. The method according to any one of claims 10-12, wherein the method further comprises step (d): subjecting the precipitated fusion polypeptide-extracellular vesicle complex to a further change in conditions, the further change in conditions causing the precipitated fusion polypeptide-extracellular vesicle complex to dissolve.

14. A kit for enriching, capturing and / or isolating extracellular vesicles from a sample, comprising the fusion peptide of any one of claims 1-6, and optionally instructions for use.

15. The kit according to claim 14, wherein, The sample includes one or more of cell culture supernatant, samples obtained from animal subjects, or apoplast fluids from plants. Preferably, the sample obtained from animal subjects is selected from one or more of whole blood, serum, plasma, urine, saliva, sputum, breast milk, cerebrospinal fluid, semen, amniotic fluid, ascites, lymph, bone marrow, synovial fluid, bronchoalveolar lavage fluid, pleural effusion, or ascites.

16. A method for preparing the fusion polypeptide according to any one of claims 1-6, wherein the method comprises: a) Cultivate the host cells according to claim 9 to express the fusion polypeptide. b) subjecting a solution containing the expressed fusion polypeptide to a change in conditions, thereby precipitating the fusion polypeptide from the solution, said conditions being selected from: pH, temperature, salt concentration, light wavelength, light intensity, charge, or any combination thereof, and c) Separate the precipitated fusion polypeptide from the solution. The solution is selected from: the lysate supernatant of the host cells, the lysate supernatant of the host cell inclusion bodies, the culture medium of the host cells, or a combination thereof. Preferably, the separation of the fusion peptide in step c) is achieved by centrifugation, filtration, decantation, or any combination thereof.

17. A composition comprising a fusion polypeptide-extracellular vesicle complex obtained by the method of any one of claims 10-13, preferably, the composition being used for the prevention, diagnosis or treatment of related diseases or conditions.

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