Fusion proteins and their applications in enriching or capturing exosomes

Abstract

The present disclosure provides a fusion protein, which includes a peptide ligand domain that binds to exosomes, a protein purification tag domain, and a water-soluble enhanced tag domain. The present disclosure also provides the use of the fusion protein in enriching, capturing and / or separating exosomes. The fusion protein of the present disclosure can be used to quickly and efficiently specifically capture high-purity, high-activity exosomes from cell supernatants.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and specifically relates to a fusion protein and application of the fusion protein in enriching, capturing and / or separating exosomes. Background Art

[0002] An increasing number of studies have shown that exosomes participate in intercellular communication and are associated with various physiological and pathological behaviors, such as cancer progression and metastasis, immune response, tissue regeneration, etc. Rapid enrichment of highly pure exosomes from cell culture supernatants and body fluids while maintaining their structural integrity and bioactivity remains a very challenging task and a critical step for downstream characterization and clinical studies.

[0003] Exosome separation and enrichment technology has made significant progress in recent years. Ultracentrifugation is the gold standard method and the most widely used method for exosome separation. However, it not only requires expensive equipment and long operation time, but also has a relatively low recovery rate (5-15%), and the shear force generated by ultra-high speed centrifugation can seriously damage the structural integrity of exosomes. Other conventional methods, including polymer-based precipitation, size exclusion chromatography (SEC) and ultrafiltration, also have various disadvantages, such as low purity, time-consuming methods, and possible mechanical damage to exosomes. The physical properties of exosomes allow many microfluidic techniques, such as viscoelasticity, acoustics, dielectrophoresis, and nanoscale deterministic lateral displacement, to be applied to exosome separation. However, the common problem of these separation methods is that they are not suitable for enriching exosomes directly from cell supernatants.

[0004] Recently, several magnetic separation-based methods and commercial immunomagnetic separation kits for exosome enrichment have been reported, with good performance in terms of processing time, purity, and separation efficiency. However, the vast majority of these methods are incompatible with downstream analysis and clinical applications. For example, the capture of exosomes by magnetic nanoparticles is irreversible, making it difficult to release exosomes while maintaining their biological activity.

[0005] It can be seen that the current exosome enrichment or purification methods mostly rely on ultracentrifugation or antibody capture, which have the problems of expensive equipment, low recovery rate, high cost and poor enrichment effect. Summary of the invention

[0006] In view of the deficiencies in the prior art, the present invention combines a peptide ligand that can specifically bind to an exosome membrane surface marker with a protein purification tag system (such as a Twin strep system) to construct a fusion protein, and by expressing the fusion protein in large quantities and utilizing its ability to specifically recognize exosomes and the corresponding protein purification tag (such as a Twin strep tag) to facilitate affinity purification, the exosomes can be quickly and highly purified from the cell supernatant for subsequent clinical research or application.

[0007] According to one aspect of the present disclosure, a fusion protein is provided, comprising: a peptide ligand domain that binds to exosomes, a protein purification tag domain, and a water-solubility enhancing tag domain.

[0008] In some embodiments, the exosome-binding peptide ligand domain includes a peptide ligand that specifically binds to an exosome surface marker, including but not limited to one or more of the peptide ligand CP05 that specifically binds to CD63, the peptide ligand RGD4C that specifically binds to ITGB1, the peptide ligand SP22 that specifically binds to CD59, or other peptide ligands.

[0009] In some embodiments, the protein purification tag domain includes, but is not limited to, one or more of Twin strep tag protein, His tag protein, Strep tag protein, Strep Tag II tag protein, or other protein purification tags.

[0010] In some embodiments, the water-solubility enhancing tag domain includes one or more of a GST tag protein, a His tag protein, a Flag tag protein, an MBP tag protein, or other water-solubility enhancing tags.

[0011] The fusion protein disclosed in the present invention can not only specifically recognize exosome membrane proteins, but also carry a protein purification tag such as a Twinstrep tag to facilitate affinity purification, and also ensure the water solubility of the fusion protein, ensuring that high-purity, high-activity exosomes can be captured efficiently and specifically.

[0012] In some embodiments, the fusion protein includes, from N-terminus to C-terminus, a water-solubility enhancing tag domain, a protein purification tag domain, and an exosome-binding peptide ligand domain.

[0013] In some embodiments, the fusion protein includes, from N-terminus to C-terminus, a peptide ligand domain that binds to exosomes, a protein purification tag domain, and a water-solubility enhancing tag domain.

[0014] In some embodiments, the fusion protein includes, from the N-terminus to the C-terminus, a protein purification tag domain, an exosome-binding peptide ligand domain, and a water-solubility enhancing tag domain.

[0015] In some embodiments, the fusion protein includes, from the N-terminus to the C-terminus, a water-solubility enhancing tag domain, an exosome-binding peptide ligand domain, and a protein purification tag domain.

[0016] In some embodiments, the fusion protein includes, from N-terminus to C-terminus, a protein purification tag domain, a water-solubility enhancing tag domain, and an exosome-binding peptide ligand domain.

[0017] In some embodiments, the fusion protein includes, from N-terminus to C-terminus, a peptide ligand domain that binds to exosomes, a water-solubility enhancing tag domain, and a protein purification tag domain.

[0018] In some embodiments, the water-solubility enhancing tag domain, the protein purification tag domain and / or the exosome-binding peptide ligand domain are directly connected or connected through a linker.

[0019] In some embodiments, the linker is selected from a flexible linker, a rigid linker and / or a cleavable linker.

[0020] In some embodiments, the linker is selected from (G n S) m 、(G) n 、(EAAAK) n or (XP) n , wherein m and n are each independently selected from an integer of 0-5, and X is any amino acid.

[0021] In some embodiments, the linker comprises at least one of G3S, G4S, EA3K, (GS)3, and (G4S)3.

[0022] In some embodiments, other peptide ligands that bind to exosomes may be added to the N-terminus and / or C-terminus of the fusion protein.

[0023] In some embodiments, the water-solubility enhancing tag domain, the protein purification tag domain, the exosome-binding peptide ligand domain and / or the linker can be combined in different numbers and in different orders to construct a fusion protein.

[0024] In some embodiments, the peptide ligand CP05 has the amino acid sequence shown in SEQ ID NO: 1, or an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity with SEQ ID NO: 1.

[0025] In some embodiments, the peptide ligand CP05 is encoded by the nucleotide sequence shown in SEQ ID NO: 2, or by a nucleotide sequence having at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 2.

[0026] In some embodiments, the peptide ligand SP22 has the amino acid sequence shown in SEQ ID NO: 10, or an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity with SEQ ID NO: 10.

[0027] In some embodiments, the peptide ligand SP22 is encoded by the nucleotide sequence shown in SEQ ID NO: 11, or by a nucleotide sequence having at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity with SEQ ID NO: 11.

[0028] In some embodiments, the Twin strep tag protein has the amino acid sequence shown in SEQ ID NO: 3, or an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity with SEQ ID NO: 3.

[0029] In some embodiments, the Twin strep tag protein is encoded by the nucleotide sequence shown in SEQ ID NO: 4, or by a nucleotide sequence having at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 4.

[0030] In some embodiments, the GST tag protein has the amino acid sequence shown in SEQ ID NO: 5, or an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity with SEQ ID NO: 5.

[0031] In some embodiments, the GST tag protein is encoded by the nucleotide sequence shown in SEQ ID NO: 6, or by a nucleotide sequence having at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 6.

[0032] In some embodiments, the fusion protein has the amino acid sequence shown in SEQ ID NO: 7 or 14, or an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity with SEQ ID NO: 7 or 14.

[0033] In some embodiments, the fusion protein is encoded by the nucleotide sequence shown in SEQ ID NO: 8 or 15, or by a nucleotide sequence having at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 8 or 15.

[0034] According to another aspect of the present disclosure, an isolated nucleic acid molecule is provided, wherein the nucleic acid molecule encodes the fusion protein described in the present disclosure.

[0035] In some embodiments, the nucleic acid molecule has the nucleotide sequence shown in SEQ ID NO: 8 or 15, or has a nucleotide sequence with at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 8 or 15.

[0036] According to another aspect of the present disclosure, an expression vector is provided, wherein the expression vector comprises the nucleic acid molecule described in the present disclosure.

[0037] In some embodiments, the expression vector is selected from a viral expression vector, a bacterial expression vector or a fungal expression vector.

[0038] In some embodiments, the expression vector is selected from a yeast expression vector, an adenoviral vector, an adeno-associated viral vector (AAV), a retroviral vector or a lentiviral vector.

[0039] In some embodiments, the expression vector comprises an E. coli expression system pGEX plasmid.

[0040] According to another aspect of the present disclosure, a host cell is provided, characterized in that the host cell comprises the nucleic acid molecule or the expression vector described in the present disclosure, or expresses the fusion protein described in the present disclosure.

[0041] In some embodiments, the host cell is selected from bacteria, fungi, insect cells, plant cells or animal cells.

[0042] In some embodiments, the host cell is selected from Escherichia coli, Bacillus subtilis or yeast.

[0043] In some embodiments, the host cell can be E. coli BL21(DE3) or other E. coli expression systems.

[0044] The fusion protein disclosed in the present invention can be directly expressed in Escherichia coli and quickly obtained by Twin strep tag purification, which is simple to operate, low in cost, and relatively easy to scale up for production.

[0045] In some embodiments, the fusion protein is expressed using pGEX-5X-1 plasmid and E. coli BL21 (DE3), and then IPTG is used to induce the large-scale expression of the fusion protein.

[0046] According to another aspect of the present disclosure, provided is the use of the fusion protein, the nucleic acid molecule, the expression vector or the host cell described in the present disclosure in the enrichment, capture and / or separation of exosomes.

[0047] According to another aspect of the present disclosure, a method for enriching, capturing and / or isolating exosomes is provided, the method comprising the following steps:

[0048] (1) incubating the cell supernatant with the fusion protein described in the present disclosure;

[0049] (2) Purifying the incubated mixed solution by affinity chromatography to obtain an exosome complex coupled with the fusion protein.

[0050] In some embodiments, the cell supernatant is from a culture medium of stem cells, such as a culture medium of mesenchymal stem cells and / or a culture medium of other stem cells.

[0051] In some embodiments, the incubation time is 4-16°C, for example, 4°C, 6°C, 8°C, 10°C, 12°C, 14°C, 15°C, 16°C or any value therebetween.

[0052] In some embodiments, the incubation time is 0.5-5 h, for example, 0.5 h, 1 h, 2 h, 3 h, 5 h or any value therebetween.

[0053] In some embodiments, the method uses stem cells, such as mesenchymal stem cells, and harvests the cell supernatant after culturing in a serum-free medium, and directly incubates with the fusion protein to enrich exosomes by utilizing the specific binding of the peptide ligand to the exosome marker.

[0054] In some embodiments, the method uses human umbilical cord mesenchymal stem cells, harvests the cell supernatant after culturing in serum-free medium for 48 hours, and directly incubates with the fusion protein, and enriches exosomes by utilizing the specific binding of CP05 and CD63, and identifies the enriched exosomes using CD63 and CD81 antibodies.

[0055] The fusion protein disclosed in the present invention can be directly incubated with the cell supernatant, that is, the peptide ligand can specifically bind to the exosome membrane surface marker to achieve rapid, accurate and efficient capture of exosomes, with simple steps, mild conditions, high recovery efficiency and high purity of the obtained exosomes.

[0056] The method for enriching, capturing and / or isolating exosomes disclosed in the present invention does not require expensive precision instruments such as ultracentrifuges, nor expensive reagents such as magnetic beads and antibodies. Exosomes can be easily captured under ordinary experimental conditions and by ordinary technicians.

[0057] According to another aspect of the present disclosure, an exosome is provided, wherein the exosome is coupled with the fusion protein described in the present disclosure.

[0058] In some embodiments, the exosomes are obtained by the methods described in the present disclosure.

[0059] According to yet another aspect of the present disclosure, a composition is provided, comprising the exosomes described in the present disclosure; and, optionally, a pharmaceutically acceptable carrier. BRIEF DESCRIPTION OF THE DRAWINGS

[0060] Figure 1 A schematic diagram of the GST-Twin strep-CP05 fusion protein of the present disclosure is shown.

[0061] Figure 2 A vector map of the GST-Twin strep-CP05 fusion protein of the present disclosure is shown.

[0062] Figure 3 The expression and purification results of the GST-Twin strep-CP05 fusion protein disclosed in the present invention are shown.

[0063] Figure 4 TEM imaging of the GST-Twin strep-CP05+exosome complex of the present disclosure is shown.

[0064] Figure 5 The HPLC detection results of the GST-Twin strep-CP05+exosome complex disclosed in the present invention are shown.

[0065] Figure 6 The Western blot validation results of the GST-Twin strep-CP05+exosome complex disclosed in the present invention are shown.

[0066] Figure 7 The results of nanoparticle tracking analysis of the GST-Twin strep-CP05+exosome complex concentrate disclosed in the present invention are shown.

[0067] Figure 8 The results of the cellular uptake experiment of the GST-Twin strep-CP05+exosome complex concentrate are shown, where A shows the cell nucleus (blue), B shows the cytoplasm (green), C shows the exosomes (red), and D is a merged image.

[0068] Fig. 9 A schematic diagram of the GST-SP22-Twin strep fusion protein of the present disclosure is shown.

[0069] Fig.10 The vector map of the GST-SP22-Twin strep fusion protein disclosed in the present invention is shown.

[0070] Fig.11 The expression and purification results of the GST-SP22-Twin strep fusion protein disclosed in the present invention are shown.

[0071] Fig.12 TEM imaging of the GST-SP22-Twin strep+exosome complex of the present disclosure is shown.

[0072] Fig.13 The HPLC detection results of the GST-SP22-Twin strep+exosome complex disclosed in the present invention are shown.

[0073] Fig.14 The Western blot validation results of the GST-SP22-Twin strep+exosome complex disclosed in the present invention are shown.

[0074] Fig.15 The results of nanoparticle tracking analysis of the GST-SP22-Twin strep + exosome complex concentrate disclosed herein are shown.

[0075] Fig.16 The results of the cellular uptake experiment of the GST-SP22-Twin strep + exosome complex concentrate are shown, where A shows the cell nucleus (blue), B shows the cytoplasm (green), C shows the exosomes (red), and D is a merged image. DETAILED DESCRIPTION

[0076] Exosomes are typical saucer-shaped structures with diameters ranging from 40 to 160 nm, which are secreted by many types of cells through the fusion of multivesicular bodies (MVBs) with the plasma membrane and are present in various types of body fluids, including urine, plasma, breast milk, and saliva. Exosomes contain cellular components such as DNA, noncoding RNA, mRNAs, and proteins, and can therefore provide important insights into the state of their parent cells. An increasing number of studies have shown that exosomes participate in intercellular communication and are associated with various physiological and pathological behaviors, such as cancer progression and metastasis, immune response, tissue regeneration, etc.

[0077] Exosome biomarkers are also a focus of research. The complexity of exosomes is further amplified by their specific biomarkers, which have characteristic locations of donor cells. The nature of biomarkers indicates that they play an important role in the identification and purification of exosomes and endow exosomes with great potential for clinical applications, such as disease diagnosis and targeted therapy. The exosomal proteome is enriched with the most commonly used biomarkers of exosomes, such as CD63, CD9, and CD81, which show very different levels in exosomes from different cell types. These proteins are often used as markers for exosome identification, quantification, or purification.

[0078] These unique features enable exosomes to be used as diagnostic markers for a variety of clinical diseases and also as delivery vehicles for a variety of contents ranging from anticancer chemotherapeutic drugs to nucleic acids. Current methods for targeting exosomes with therapeutic macromolecules generally involve genetic manipulation of their producing cells or chemical conjugation of peptides to the exosome surface. However, genetic engineering of parental cells is labor-intensive, has variable efficacy depending on transfection efficiency and cell type, and precludes the use of circulating exosomes from patients for therapeutic purposes, while chemical functionalization of natural exosomes, such as click chemistry, can largely alter the surface structure. Transferrin has been used to label patient-derived exosomes with paramagnetic particles, but transferrin binds poorly to exosomes and is disordered because transferrin-binding molecules are present in large quantities in the blood. Electroporation, lyophilization, or transfection of parental cells can load exosomes with macromolecules, but these methods have low loading efficiencies.

[0079] Exosomes can enter recipient cells by adopting different mechanisms such as endocytosis, direct fusion with membranes or binding to receptors on the cell surface, thereby achieving their complex biological effects. The complexity of exosomes is further amplified by their specific biomarkers, which have characteristic locations of donor cells. The nature of biomarkers indicates that they play an important role in the identification and purification of exosomes and endow exosomes with great potential for clinical applications, such as disease diagnosis and targeted therapy.

[0080] The present invention constructs a fusion protein by combining a peptide ligand that can specifically bind to an exosome membrane surface marker with a protein purification system such as a Twin strep system, and expresses the fusion protein in large quantities and utilizes its ability to specifically recognize exosomes and utilizes corresponding protein purification tags such as a Twin strep tag to facilitate affinity purification, thereby achieving rapid and high-purity capture of exosomes from cell supernatant for subsequent clinical research or application.

[0081] In order to achieve high-purity capture and gentle recovery of exosomes in a short time, the present disclosure adopts the following technical solutions:

[0082] (1) Construct a fusion protein GST-Twin strep-CP05 with a Twin strep tag that can specifically recognize the peptide ligand CP05 of the exosome membrane surface marker, and obtain a high-purity fusion protein GST-Twin strep-CP05 through protein expression and strep affinity purification technology. The fusion protein disclosed in the present invention can not only be highly expressed in Escherichia coli, but also can be quickly obtained through a simple protein purification method, which saves time and is simple to operate.

[0083] (2) In some embodiments, human umbilical cord mesenchymal stem cells are cultured. When the cell density reaches about 80%, the complete culture medium is removed and replaced with serum-free α-MEM medium for continued culture. The cell supernatant is collected and filtered with a 0.22 μm filter membrane for later use.

[0084] (3) The GST-Twin strep-CP05 fusion protein is directly incubated with the obtained mesenchymal stem cell supernatant at 4°C for 1 hour. The mixed solution is purified and eluted by a strep column to obtain a GST-Twin strep-CP05+exosome complex, and the GST-Twin strep-CP05+exosome complex is ultrafiltrated and concentrated. The fusion protein disclosed in the present invention can be directly incubated with the cell supernatant, and can not only achieve accurate capture and mild enrichment of exosomes by utilizing the characteristics of peptide ligands selectively binding to exosome membrane surface markers; but also achieve high enrichment and rapid elution of exosomes through Twin strep tags. The entire experimental process is mild and basically does not affect the activity of exosomes.

[0085] (4) The obtained GST-Twin strep-CP05+exosome complex was not only subjected to transmission electron microscopy, particle size distribution, Western blot and HPLC purity analysis, but also subjected to cell uptake experiments to verify the activity of the exosomes. The results showed that the exosomes enriched by the method disclosed in the present invention are structurally intact, with obvious membrane protein markers, high purity and high activity, and can be used for downstream clinical research and applications.

[0086] In the present disclosure, the transmembrane protein specific to the surface of exosomes is directly anchored for specific binding, a series of peptide ligands that can specifically bind to the transmembrane protein on the surface of exosomes are synthesized, and then a peptide ligand with relatively high specific binding efficiency is screened through a large number of experiments. The present disclosure maintains physiological conditions throughout the process, uses the GST tag to increase the water solubility of the fusion protein, uses the Twinstrep tag to purify through affinity chromatography to obtain a high-purity fusion protein, and further uses the Twinstrep tag to purify the fusion protein and the exosome complex.

[0087] Unless otherwise defined, all technical and scientific terms used in this disclosure have the same meaning as commonly used in the field to which this disclosure belongs. For the purpose of interpreting this specification, the following definitions will apply, and where appropriate, terms used in the singular will also include the plural form, and vice versa.

[0088] Unless the context clearly dictates otherwise, the expressions "a", "an" and "an" as used herein include plural references. For example, reference to "a cell" includes a plurality of such cells and equivalents thereof known to those skilled in the art, and so forth.

[0089] The term "about" as used herein refers to a range of ±20% of the value that follows. In some embodiments, the term "about" refers to a range of ±10% of the value that follows. In some embodiments, the term "about" refers to a range of ±5% of the value that follows.

[0090] The term "fusion protein" as used herein refers to two or more proteins or fragments thereof co-linearly linked through their respective peptide backbones using genetic expression of polynucleotides encoding the proteins or protein synthesis methods.

[0091] "Percent sequence identity" or "percent identity" between two polynucleotide or polypeptide sequences refers to the number of identical matching positions shared by the sequences over the comparison window, taking into account additions or deletions (i.e., gaps) that must be introduced for optimal alignment of the two sequences. A matching position is any position where the same nucleotide or amino acid is present in both the target sequence and the reference sequence. Gaps present in the target sequence are not counted because gaps are not nucleotides or amino acids. Likewise, gaps present in the reference sequence are not counted because target sequence nucleotides or amino acids are counted and nucleotides or amino acids from the reference sequence are not counted.

[0092] The sequence identity percentage can be calculated by the following process: determine the number of positions where the same amino acid residue or nucleic acid base occurs in both sequences to obtain the number of matching positions, divide the number of matching positions by the total number of positions in the comparison window, and multiply the result by 100 to obtain the sequence identity percentage. The comparison of sequences and the determination of the sequence identity percentage between the two sequences can be completed using software that is easy to use and download online. Suitable software programs are available from various sources for the comparison of protein and nucleotide sequences. A suitable program for determining the sequence identity percentage is bl2seq, which is part of the BLAST program suite available from the BLAST website (blast.ncbi.nlm.nih.gov) of the National Center for Biotechnology Information of the U.S. government. Bl2seq uses BLASTN or BLASTP algorithms to compare between two sequences. BLASTN is used to compare nucleic acid sequences, while BLASTP is used to compare amino acid sequences. Other suitable programs are, for example, Needle, Stretcher, Water or Matcher, part of the EMBOSS suite of bioinformatics programs and also available from the European Bioinformatics Institute (EBI) at www.ebi.ac.uk / Tools / psa.

[0093] As used herein, the term "linker" refers to a peptide or polypeptide sequence (e.g., a synthetic peptide or polypeptide sequence) or a non-polypeptide, such as an alkyl chain. In some embodiments, two or more linkers can be connected in series. When multiple linkers are present, each linker can be the same or different. In general, linkers provide flexibility or prevent / improve steric hindrance.

[0094] In some embodiments, the linker is a peptide linker. In some embodiments, the peptide linker may comprise at least about two amino acids. In some embodiments, the linker comprises one or more amino acids. In some embodiments, the linker comprises a Gly-Ser (GS) linker. In some embodiments, the GS linker comprises (G4S)n, wherein n is an integer between 1 and 10. In some embodiments, the GS linker comprises (G3S)n, wherein n is an integer between 1 and 10.

[0095] An expression vector may contain a nucleic acid as described herein in a form suitable for expressing the nucleic acid in a host cell, which means that the expression vector includes one or more regulatory elements, which may be selected based on the host cell used for expression, and which are operably linked to the nucleic acid sequence to be expressed. Within an expression vector, "operably linked" is intended to link the nucleotide sequence of interest to the regulatory elements in a manner that allows the nucleotide sequence to be expressed (e.g., in an in vitro transcription / translation system or when the vector is introduced into a host cell, in a host cell).

[0096] The term "pharmaceutically acceptable carrier" as used herein refers to a component of a pharmaceutical preparation other than an active ingredient that is non-toxic to a subject. Pharmaceutically acceptable carriers include, but are not limited to, buffers, excipients, stabilizers or preservatives.

[0097] In order to make the purpose, technical scheme and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the accompanying drawings and embodiments. The specific embodiments described herein are only used to explain the present invention and are not intended to constitute any limitation to the present invention. In addition, in the following description, the description of known structures and technologies is omitted to avoid unnecessary confusion of the concepts of the present disclosure. Such structures and technologies are also described in many publications.

[0098] The reagents and / or kits used in the following examples are all commercially available or can be synthesized by known methods. Example 1

[0099] Follow the steps below to capture exosomes:

[0100] 1. Obtain the amino acid sequence of the peptide ligand CP05 that can specifically bind to the exosome surface marker CD63.

[0101] The main part of the fusion protein Twin strep-CP05 is constructed using the Twin strep tag and the peptide ligand CP05, which contains the Twin strep tag protein and the specific binding peptide ligand CP05 of the exosome surface marker CD63. The above fusion protein Twin strep-CP05 is placed at the C-terminus, and the N-terminus is the GST tag carried by the vector. The GST tag can enhance the water solubility of the fusion protein to construct the GST-Twin strep-CP05 fusion protein, such as Figure 1 and Figure 2 Among them, the Twinstrep tag can be used for strep column purification, and CP05 is a peptide ligand that can specifically bind to the exosome surface marker CD63.

[0102] The sequence information is shown in Table 1 below.

[0103]

[0104] 2. The amino acid sequence of the above fusion protein GST-Twin strep-CP05 is combined as follows:

[0105] MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKKFELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRYGVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLC HKTYLNGDHVTHPDFMLYDALDVVLYMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPPKSDLIEGRGIPSAWSHPQFEKGGGSGGGSGGSAWSHPQFEKEFCRHSQMTVTSRL (SEQ ID NO: 7).

[0106] The gene sequence of the above fusion protein GST-Twin strep-CP05 after optimization based on E. coli BL21 (DE3) codons is:

[0107] atgtcccctatactaggttattggaaaattaagggccttgtgcaacccactcgacttcttttggaatatcttgaagaaaaatatgaagagcatttgtatgagcgcgatgaaggtgataaatggcgaaacaaaaagtttgaattgggtttggagtttcccaatcttccttattatattgatggtgatgttaaattaacacagtctatggccatcatacgttatatagctgacaagcacaacatgttgggtggttgtccaaaagagcgtgcagagatttcaatgcttgaaggagcggttttggatattagatacggtgtttcgagaattgcatatagtaaagactttgaaactctcaaagttgattttcttagcaagctacctgaaatgctgaaaatgttcgaagatcgtttatgtcataaaacatatttaaatggtgatcatgtaacccatcctgacttcatgttgtatgacgctcttgatgttgttttatacatggacccaatgtgcctggatgcgttcccaaaattagtttgttttaaaaaacgtattgaagctatcccacaaattgataagtacttgaaatccagcaagtatatagcatggcctttgcagggctggcaagccacgtttggtggtggcgaccatcctccaaaatcggatctgatcgaaggtcgtgggatccccagtgcttggtcacacccccaatttgaaaaaggcggtggctccggtggtggctctggtggcagcgcgtggtcgcatccgcaatttgaaaaggaattctgccgccacagccagatgaccgtgacgagccgtctg(SEQ ID NO:8)。

[0108] 3. According to the gene fragment of the synthetic fusion protein Twin strep-CP05 (SEQ ID NO: 9), the Twin strep-CP05 gene fragment was inserted into the 3' end of the GST of the vector pGEX-5X-1 by PCR technology to obtain the recombinant plasmid connected with the vector pGEX-5X-1 and the fusion protein. Use E. coli DH5α (DE3) competent state for transformation, select single clones for PCR verification and send for sequencing. Shake 3mL of the colony with correct sequencing for expansion and perform plasmid mini-extraction. Use E. coli BL21 (DE3) competent state for protein expression, select single clones for sequencing, and the strain with correct sequencing is the GST-Twin strep-CP05 expression strain.

[0109] Among them, the gene sequence of Twin strep-CP05 after codon optimization of E. coli BL21 (DE3) is:

[0110] agtgcttggtcacacccccaatttgaaaaaggcggtggctccggtggtggctctggtggcagcgcgtggtcgcatccgcaatttgaaaaggaattctgccgccacagccagatgaccgtgacgagccgtctg (SEQ ID NO: 9).

[0111] 4. Inoculate the GST-Twin strep-CP05 expression bacteria into 3 mL of 2YT medium with ampicillin resistance, culture at 37℃220rpm overnight, transfer it to 200 mL of 2YT medium with ampicillin resistance at a ratio of 1:100, culture at 37℃220rpm for about 2 hours, take 100μL of bacterial solution to measure its OD value, wait until its OD value reaches between 0.8-1.0, first place the bacterial solution on a 25℃220rpm shaker for 10 minutes, then add IPTG with a final concentration of 0.5mM to induce fusion protein expression, and continue to culture at 25℃220rpm overnight. Collect the bacteria by centrifugation at 4℃10000rpm, add 15mL of Buffer W, 150μL of lysozyme (1:100) and 1.5μL of DNA degrading enzyme (1:10000), fully resuspend and lyse the bacteria by low temperature ultra-high pressure crusher to release the peptide ligand fusion protein.

[0112] Protein purification tags were performed using strep affinity columns and eluted with low concentrations of biotin as follows:

[0113] (1) Centrifuging the bacterial solution after lysing the cells;

[0114] (2) After centrifugation, the supernatant of the bacterial solution is filtered through a 0.45um filter membrane and transferred to a new 50mL centrifuge tube to wait for the IBA strep column. (At this time, 80μL of the supernatant needs to be taken as a sample and marked as GST-Twin strep-CP05 supernatant). Take the bacterial precipitate in the centrifuge tube and dissolve the precipitate with 80μL of 8M urea, and mark it as GST-Twin strep-CP05 precipitate.

[0115] (3) Column loading: Fill the column with 2 mL of strep resin (Strep-Tactin® XT 4Flow resin; brand: IBA; catalog number: 2-5010-025). After draining off the ethanol, add 2 times the volume of 1X Buffer W (Strep-Tactin® XT 4Flow resin; brand: IBA; catalog number: 2-5010-025) to the column. ® XT Buffer Set; Brand: IBA; Product Number: 2-1043-000) was used to elute three times to balance the column.

[0116] (4) Transfer the filtered bacterial solution in step (2) to the equilibrated strep column in batches, and collect the bacterial solution that flows down after the column. At this time, most of the fusion protein with Twin Strep tag has bound to the resin in the strep column. Take 80 μL of the bacterial solution after the column and mark it as GST-Twin Strep-CP05-flow-through.

[0117] (5) After filtering the bacterial solution, add 5 volumes (5 mL) of 1X Buffer W to the strep gel column to rinse the column. When washing with 1X Buffer W for the fifth time, collect about 80 μL of the effluent bacterial solution and mark it as GST-Twin strep-CP05-wash solution 1. Continue to rinse with 1X Buffer W, and then take 100 μL of Quick Start Brandford 1x DyeReagent (commercial Strep-Tactin ® XT Buffer Set; Brand: IBA; Product No.: 2-1043-000)) on the spotting plate, and then take one or two drops of the effluent from the strep column for detection. When the effluent spotting shows no obvious change, take about 80 μL of the effluent and mark it as GST-Twin strep-CP05-equilibrium.

[0118] (6) Use 6×0.5 times 1X Buffer BXT (50mM biotin elution buffer, commercial Strep-Tactin ®XTBuffer Set; Brand: IBA; Product No.: 2-1043-000) was used to elute the strep column. Each time, an eluent twice the volume of the strep resin was added. The eluent obtained by elution three times was collected. 80 μL of the eluent was retained and marked as GST-Twin strep-CP05-eluent 1 and stored in a -20°C refrigerator.

[0119] (7) Repeat step (6), take 80 μL of sample and label it as GST-Twin strep-CP05-elution buffer 2, and store the eluate in a -20°C refrigerator.

[0120] (8) Rinse the column with 2 mL of 1× Buffer W. After resuspending, take about 80 μL of the resin suspension, mark it as GST-Twin strep-CP05-strep column, and save it for running gel.

[0121] (9) Ultrafiltration: Filter GST-Twin strep-CP05-elution 1 and GST-Twin strep-CP05-elution 2 with a 0.22 μm filter membrane, transfer the filtered liquid to the inner tube of a 10KD ultrafiltration tube (brand: Millipore; catalog number: UFC901096), centrifuge at 4°C and 3500 rpm for 15 min, collect the ultrafiltrate, replace the buffer in the eluate with 1× PBS, collect the liquid at the bottom of the ultrafiltration tube, mix evenly, and sample 80 μL, labeled as GST-Twin strep-CP05-elution ultrafiltration. The ultrafiltration concentrated protein was stored in a -20°C refrigerator.

[0122] (10) Add 20 μL of 5X loading buffer to each of the 9 samples taken in the above steps and cook the protein at 95°C for 10 min. Figure 3 .

[0123] Figure 3 The results showed that a high concentration of fusion protein GST-Twin strep-CP05 could be obtained after protein purification tag was used through strep affinity column and eluted with low concentration of biotin.

[0124] 5. Culture human umbilical cord mesenchymal stem cells (UMSC) in complete medium (DMEM / F-12 (brand: GIBCO, catalog number: C11330500BT) + 10% serum (brand: VivaCell, catalog number: 04-400-1A) + 100X P / S double antibody (brand: GIBCO, catalog number: 15140-122)) at 37°C and 5.0% CO2. When the cell density reaches about 80%, remove the complete medium and replace it with serum-free α-MEM medium for 48 hours. Collect the cell supernatant and centrifuge it at 4000 rpm at 4°C for 20 minutes to remove cell debris and dead cells. Collect the supernatant and filter it with a 0.22μm filter membrane and store it at -80°C or perform subsequent operations.

[0125] 6. Take 200 mL of the above human umbilical cord mesenchymal stem cell supernatant and 1.5 mg of the GST-Twinstrep-CP05 fusion protein obtained in step 4 above and incubate them on a 4°C rotary mixer for 1 h. After the mixture is purified and eluted by a strep column, the GST-Twin strep-CP05+exosome complex can be obtained. The GST-Twin strep-CP05+exosome complex is concentrated to 1 mL by ultrafiltration.

[0126] 7. Take 10 μl of GST-Twin strep-CP05+exosome complex concentrate for transmission electron microscopy analysis. The particle size and morphology determine whether this peptide ligand fusion protein can successfully capture exosomes, such as Figure 4 shown.

[0127] 8. Take 20 μL of GST-Twin strep-CP05+exosome complex concentrate for HPLC detection. The mobile phase is 10 mM HEPES, the flow rate is 0.4 mL / min, and the detection time is 20 min. Figure 5 The peak appeared at 6-8.5min, indicating that the peptide ligand fusion protein can accurately capture exosomes, and the obtained exosomes have a single component and high purity.

[0128] 9. Take 20 μg of GST-Twin strep-CP05+exosome complex concentrate for Western blotting identification, and use CD63 and CD81, common surface markers of stem cell exosomes, for detection. The results are as follows: Figure 6 shown.

[0129] 10. Take 10 μL of GST-Twin strep-CP05+exosome complex concentrate, dilute to 1 mL with PBS, and determine the particle size distribution and particle concentration of the complex by Nano sight 300. The results are as follows: Figure 7 shown.

[0130] 11. Take 200 μg of GST-Twin strep-CP05+exosome complex concentrate to conduct cell uptake experiment: (1) Use PKH26 (PKH26 is a red fluorescent dye that can stably bind to the exosome lipid bilayer and emit red fluorescence) to label the exosomes in the GST-Twin strep-CP05+exosome complex; (2) Co-incubate the red fluorescent labeled exosomes with human umbilical cord mesenchymal stem cells grown to 80% in a 6-well plate for 3 hours. If the exosomes are active and can be normally internalized by the cells, the red fluorescence will enter the cells; (3) Wash the cells with PBS and fix the cells with 4% formaldehyde; (4) Use Actin-Tracker Green (green) to stain the microfilament skeleton in the cytoplasm; (5) DAPI to stain the cell nucleus. Then perform confocal imaging, in which PKH26 (red) labels the exosomes, Actin (green) labels the cytoplasm, and DAPI (blue) labels the cell nucleus.

[0131] Figure 8 The results in AD showed that the exosomes captured by the peptide-ligand fusion protein could be normally internalized by UMSC cells, thus verifying that the exosomes enriched by this method could maintain high activity. Example 2

[0132] 1. Obtain the amino acid sequence of the peptide ligand SP22 that can specifically bind to the exosome surface marker CD59.

[0133] The main part of the fusion protein Twin strep-SP22 is constructed using the Twin strep tag and the peptide ligand SP22, which contains the Twin strep tag protein and the specific binding peptide ligand SP22 of the exosome surface marker CD59. In the present invention, the peptide ligand SP22 is placed between the GST tag and the Twin strep tag, and the N-terminus is the GST tag carried by the vector, the GST tag can enhance the water solubility of the fusion protein, and the C-terminus is the Twin strep tag carried by the vector, which improves the purification effect of the fusion protein, and constructs the GST-SP22-Twin strep fusion protein, such as Fig. 9 and Fig.10 Among them, Twin strep tag can be used for strep column purification, and SP22 is a peptide ligand that can specifically bind to the exosome surface marker CD59.

[0134] The sequence information is shown in Table 2 below.

[0135]

[0136] 2. The amino acid sequence of the above fusion protein GST-SP22-Twin strep is combined as follows:

[0137] MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKKFELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRYGVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDAL DVVLYMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPKSDLEVLFQGPGSGGGGSACHWPWCHGWHSACDLPMHPMCGGGGSGGGGSGGGGSKLGSGSGSGSSASAWSHPQFEKGGGSGGGSGGSAWSHPQFEK (SEQ ID NO: 14).

[0138] The gene sequence of the above fusion protein GST-SP22-Twin strep after optimization based on E. coli BL21 (DE3) codons is:

[0139] atgtcccctatactaggttattggaaaattaagggccttgtgcaacccactcgacttcttttggaatatcttgaagaaaaatatgaagagcatttgtatgagcgcgatgaaggtgataaatggcgaaacaaaaagtttgaattgggtttggagtttcccaatcttccttattatattgatggtgatgttaaattaacacagtctatggccatcatacgttatatagctgacaagcacaacatgttgggtggttgtccaaaagagcgtgcagagatttcaatgcttgaaggagcggttttggatattagatacggtgtttcgagaattgcatatagtaaagactttgaaactctcaaagttgattttcttagcaagctacctgaaatgctgaaaatgttcgaagatcgtttatgtcataaaacatatttaaatggtgatcatgtaacccatcctgacttcatgttgtatgacgctcttgatgttgttttatacatggacccaatgtgcctggatgcgttcccaaaattagtttgttttaaaaaacgtattgaagctatcccacaaattgataagtacttgaaatccagcaagtatatagcatggcctttgcagggctggcaagccacgtttggtggtggcgaccatcctccaaaatcggatctggaagttctgttccaggggcccggatccggagggggaggttcagcttgtcactggccctggtgtcatggttggcacagcgcgtgcgacctgccgatgcacccgatgtgcggtggcggcggctctggtggcggcggtagcggtggtggtggctccaagcttggaagcggatctggatctggaagcagcgccagcgcttggagtcatccccaatttgagaaaggtggaggttccggaggtggatcgggaggttcggcgtggagccacccgcagttcgaaaaa(SEQ ID NO: 15)。

[0140] 3. According to the gene fragment of the synthetic peptide ligand SP22, the SP22 gene fragment was inserted into the 3' end of the GST of the vector 21pCOLI_N-GST_C-STREP by PCR technology to obtain the recombinant plasmid connected with the vector and the peptide ligand. Use E. coli DH5α (DE3) competent state for transformation, select single clones for PCR verification and send for sequencing. Shake 3mL of the colony with correct sequencing for expansion culture and perform plasmid mini-extraction. Use E. coli BL21 (DE3) competent state for protein expression, select single clones for sequencing, and the strain with correct sequencing is the GST-SP22-Twin strep expression strain.

[0141] 4. Inoculate the GST-SP22-Twin strep expression bacteria into 3 mL of 2YT medium with ampicillin resistance, culture at 37℃220rpm overnight, transfer it to 200 mL of 2YT medium with ampicillin resistance at a ratio of 1:100, culture at 37℃220rpm for about 2 hours, take 100μL of bacterial solution to measure its OD value, and when its OD value reaches between 0.8-1.0, add IPTG with a final concentration of 0.5mM to induce fusion protein expression, and continue to culture at 37℃220rpm for 4-6 hours. Collect the bacteria by centrifugation at 4℃10000rpm, add 15mL of Buffer W, 150μL of lysozyme (1:100) and 1.5μL of DNA degrading enzyme (1:10000), fully resuspend and lyse the bacteria by low temperature ultra-high pressure crusher to release the peptide ligand fusion protein.

[0142] Fig.11 The results showed that after protein purification tag was used through strep affinity column and eluted with 50mM biotin (same as Example 1), a high concentration of fusion protein GST-SP22-Twin strep could be obtained.

[0143] 5. Cultivate human umbilical cord mesenchymal stem cells in complete medium (DMEM / F-12 (brand: GIBCO, catalog number: C11330500BT) + 10% serum (brand: VivaCell, catalog number: 04-400-1A) + 100X P / S double antibody (brand: GIBCO, catalog number: 15140-122)) at 37°C and 5.0% CO2. When the cell density reaches about 80%, remove the complete medium and replace it with serum-free α-MEM medium for 48 hours. Collect the cell supernatant and centrifuge at 4000rpm for 20 minutes at 4°C to remove cell debris and dead cells. Collect the supernatant and filter it with a 0.22μm filter membrane and store it at -80°C or perform subsequent operations.

[0144] 6. Take 200 mL of the above human umbilical cord mesenchymal stem cell supernatant and 1.5 mg of the GST-Twin-SP22strep fusion protein obtained in step 4 above and incubate them on a 4°C rotary mixer for 1 h. After the mixture is purified and eluted by a strep column, the GST-SP22-Twin strep + exosome complex can be obtained. The GST-SP22-Twin strep + exosome complex is concentrated to 1 mL by ultrafiltration.

[0145] 7. Take 10 μl of GST-SP22-Twin strep+exosome complex concentrate for transmission electron microscopy analysis. The particle size and morphology determine whether this peptide ligand fusion protein can successfully capture exosomes, such as Fig.12 shown.

[0146] 8. Take 20 μL of GST-SP22-Twin strep+exosome complex concentrate for HPLC detection. The mobile phase is 10 mM HEPES, the flow rate is 0.4 mL / min, and the detection time is 20 min. Fig.13 The peak appeared at 6-8.5min, indicating that the peptide ligand fusion protein can accurately capture exosomes, and the obtained exosomes have a single component and high purity.

[0147] 9. Take 20 μg of GST-SP22-Twin strep+exosome complex concentrate for Western blotting identification, and use common surface markers CD63 and CD81 of stem cell exosomes for detection. The results are as follows: Fig.14 shown.

[0148] 10. Take 10 μL of GST-SP22-Twin strep+exosome complex concentrate, dilute to 1 mL with PBS, and determine the particle size distribution and particle concentration of the complex by Nano sight 300. The results are as follows: Fig.15 shown.

[0149] 11. Take 200 μg of GST-SP22-Twin strep+exosome complex concentrate to conduct cell uptake experiment: first label the exosomes in the GST-SP22-Twin strep+exosome complex with PKH26 dye; then co-incubate the red fluorescent labeled exosomes with human umbilical cord mesenchymal stem cells grown to 80% in a 6-well plate for 3 hours. If the exosomes are active and can be normally internalized by the cells, the red fluorescence will enter the cells; then wash the cells with PBS and fix the cells with 4% formaldehyde, then stain the microfilament skeleton in the cytoplasm with Actin-Tracker Green (green); finally, stain the cell nucleus with DAPI. Then perform confocal imaging, in which PKH26 (red) labels the exosomes, Actin (green) labels the cytoplasm, and DAPI (blue) labels the cell nucleus.

[0150] Fig.16 The results in AD showed that the exosomes captured by the peptide-ligand fusion protein could be normally internalized by UMSC cells, thus verifying that the exosomes enriched by the peptide-ligand fusion protein could maintain high activity.

[0151] The technical solution of the present invention is not limited to the above-mentioned specific embodiments. All technical variations made according to the technical solution of the present invention fall within the protection scope of the present invention.

Claims

1. Fusion protein, comprising: Peptide ligand domains that bind to exosomes, a protein purification tag domain, and a water-solubility enhancing tag domain; The peptide ligand domain binding to exosomes is selected from the peptide ligand CP05 that specifically binds to CD63 or the peptide ligand SP22 that specifically binds to CD59; the protein purification tag domain is selected from the Twin strep tag protein; the water-soluble enhanced tag domain is selected from the GST tag protein; The amino acid sequence of the fusion protein is shown in SEQ ID NO: 7 or 14.

2. The fusion protein according to claim 1, characterized in that The amino acid sequence of the peptide ligand CP05 is shown in SEQ ID NO: 1; The amino acid sequence of the peptide ligand SP22 is shown in SEQ ID NO: 10; The amino acid sequence of the Twin strep tag protein is shown in SEQ ID NO: 3; The amino acid sequence of the GST-tagged protein is shown in SEQ ID NO:

5.

3. An isolated nucleic acid molecule, characterized in that The nucleic acid molecule encodes the fusion protein according to claim 1 or 2.

4. An expression vector, characterized in that The expression vector comprises the nucleic acid molecule of claim 3.

5. A host cell, characterized in that The host cell comprises the nucleic acid molecule of claim 3 or the expression vector of claim 4, or expresses the fusion protein of claim 1 or 2.

6. Use of the fusion protein according to claim 1 or 2, the nucleic acid molecule according to claim 3, the expression vector according to claim 4 or the host cell according to claim 5 in enriching, capturing and / or separating exosomes.

7. A method for enriching, capturing and / or isolating exosomes, characterized in that: The method comprises the following steps: (1) incubating the cell supernatant with the fusion protein according to claim 1 or 2; (2) Purifying the incubated mixed solution by affinity chromatography to obtain an exosome complex coupled with the fusion protein.

8. The method according to claim 7, characterized in that The cell supernatant is from the culture medium of stem cells; and / or The incubation temperature is 4-16°C; The incubation time is 0.5-5h.

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