Vesicle and application thereof as standard substance

Abstract

The present disclosure relates to a vesicle, the vesicle comprising: a scaffold protein, the scaffold protein being a nerve growth factor receptor or a fragment thereof; and a tag domain. The vesicles carry unique labels, engineered vesicles are separated through a large-scale purification process, and after quality control, the engineered vesicles can be used as standard substances and / or reference substances to be applied to different scenes.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and in particular relates to a vesicle and an application thereof as a standard product and / or a reference product. Background Art

[0002] Exosomes are a type of vesicle structure secreted by cells, ranging in size from 30 to 200 nm. A cup-shaped structure can be observed under a transmission electron microscope. It has a phospholipid bilayer, which contains a variety of substances, including nucleic acids (DNA and RNA), proteins, lipids, etc., and plays an important role in cell communication and material transport. Due to these characteristics of exosomes, more and more application scenarios have been developed, including in vitro diagnosis, gene therapy, drug delivery, and disease prognosis.

[0003] There are many methods for isolating, purifying and detecting exosomes. Different institutions use different methods. The purity of purified exosomes varies, and the sensitivity of detection methods also varies.

[0004] Therefore, there is an urgent need for exosome standards that can be used as a reference to evaluate different purification or detection methods. Summary of the invention

[0005] In order to solve the above problems existing in the prior art, the present disclosure provides a vesicle, which uses nerve growth factor receptor (NGFR) as a scaffold protein, and the vesicles produced by modifying chassis cells carry unique tags or mRNA. The engineered vesicles are separated by a large-scale purification process and can be used as standard products and / or reference products in different scenarios after quality control.

[0006] According to one aspect of the present disclosure, a vesicle is provided, comprising: a scaffold protein, wherein the scaffold protein is a nerve growth factor receptor or a fragment thereof; and a tag domain.

[0007] In some embodiments, the nerve growth factor receptor has at least the amino acid sequence shown in SEQ ID NO:8 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:8.

[0008] In some embodiments, the nerve growth factor receptor 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.

[0009] In some embodiments, the tag domain includes one or more of a fluorescent protein, a luciferase, a purification tag, a disease marker protein, or a disease marker RNA.

[0010] In some embodiments, the tag domain includes one or more of GST (glutathione sulfhydryltransferase) tag protein, His6 tag protein (His-tag), MBP (maltose binding protein) tag protein, Flag tag protein, SUMO tag protein, HA tag protein, Myc tag protein, eGFP (enhanced green fluorescent protein), eCFP (enhanced cyan fluorescent protein), eYFP (enhanced yellow-green fluorescent protein), mCherry (monomeric red fluorescent protein), T7 tag, V5 tag, cellulose binding domain (CBD) tag, calmodulin tag, AviTag tag protein, Halo Tag tag protein, SNAP-Tag tag protein, firefly luciferase (Fluc), sea cucumber luciferase (Rluc), Guassia luciferase (Gluc), nanoluciferase (Nluc), cancer marker mRNA, and cancer marker protein.

[0011] In some embodiments, the scaffold protein is bound to the tag domain directly or through a linker.

[0012] In some embodiments, the vesicle further comprises an active domain.

[0013] In some embodiments, the active domain comprises a polypeptide, a polynucleotide, a compound, or any combination thereof.

[0014] In some embodiments, the polynucleotide comprises an antisense oligonucleotide (ASO), siRNA, miRNA, shRNA, nucleic acid, or any combination thereof.

[0015] In some embodiments, the polypeptide comprises a peptide, a protein, an antibody or an antigen-binding fragment thereof, a nucleotide-binding protein or a fragment thereof, or any combination thereof.

[0016] In some embodiments, the antigen-binding fragment of the antibody includes scFv, (scFv)2, Fab, Fab', F(ab')2, F(ab1)2, Fv, dAb and Fd fragments, bifunctional antibodies, antibody-related polypeptides or any fragments thereof.

[0017] In some embodiments, the scaffold protein forms a fusion protein with a nucleotide binding protein or a fragment thereof.

[0018] In some embodiments, the nucleotide binding protein or fragment thereof further binds to the polynucleotide sequence.

[0019] In some embodiments, the nucleotide binding protein is selected from Ku protein, Sm7 protein, MS2 coat protein, PP7 coat protein, Com RNA binding protein, aptamer ligand, or any combination, functional variant, fragment or domain thereof.

[0020] In some embodiments, the scaffold protein is bound to the active domain directly or through a linker.

[0021] In some embodiments, the vesicles include one or more of exosomes, microvesicles, apoptotic bodies, tumor vesicles, and nanovesicles.

[0022] In some embodiments, the nerve growth factor receptor or fragment thereof is localized in a vesicle.

[0023] In some embodiments, the tag domain and / or the active domain can be displayed on the outer surface of the vesicle or in the lumen of the vesicle through the scaffold protein nerve growth factor receptor or fragment thereof disclosed in the present invention.

[0024] The present invention produces different types of engineered vesicles by transforming cells, and applies these vesicles as standard products and / or reference products in different scenarios. The vesicles disclosed in the present invention are secreted by engineered cells, have a diameter of 30-200nm, and have a typical cup-shaped structure observed under a transmission electron microscope. The engineered vesicles disclosed in the present invention have similar shapes, structures and sizes to natural vesicles. As standard products and / or reference products, they can well simulate vesicles in actual application scenarios.

[0025] According to another aspect of the present disclosure, an expression system is provided, comprising: a first expression element encoding a scaffold protein, wherein the scaffold protein is a nerve growth factor receptor or a fragment thereof; and a second expression element encoding a tag domain.

[0026] In some embodiments, the nerve growth factor receptor has at least the amino acid sequence shown in SEQ ID NO:8 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:8.

[0027] In some embodiments, the nerve growth factor receptor 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.

[0028] In some embodiments, the tag domain includes one or more of a fluorescent protein, a luciferase, a disease marker protein, or a disease marker RNA.

[0029] In some embodiments, the tag domain includes one or more of Gluc, EGFP, MCP, cancer marker mRNA, and cancer marker protein.

[0030] In some embodiments, the scaffold protein is bound to the tag domain directly or through a linker.

[0031] In some embodiments, the expression system further comprises a third expression element encoding an active domain.

[0032] In some embodiments, the active domain comprises a polypeptide, a polynucleotide, a compound, or any combination thereof.

[0033] In some embodiments, the polynucleotide comprises an antisense oligonucleotide (ASO), siRNA, miRNA, shRNA, nucleic acid, or any combination thereof.

[0034] In some embodiments, the polypeptide comprises a peptide, a protein, an antibody or an antigen-binding fragment thereof, a nucleotide-binding protein or a fragment thereof, or any combination thereof.

[0035] In some embodiments, the antigen-binding fragment of the antibody includes scFv, (scFv)2, Fab, Fab', F(ab')2, F(ab1)2, Fv, dAb and Fd fragments, bifunctional antibodies, antibody-related polypeptides or any fragments thereof.

[0036] In some embodiments, the scaffold protein forms a fusion protein with a nucleotide binding protein or a fragment thereof.

[0037] In some embodiments, the nucleotide binding protein or fragment thereof further binds to the polynucleotide sequence.

[0038] In some embodiments, the nucleotide binding protein is selected from Ku protein, Sm7 protein, MS2 coat protein, PP7 coat protein, Com RNA binding protein, aptamer ligand, or any combination, functional variant, fragment or domain thereof.

[0039] In some embodiments, the scaffold protein is bound to the active domain directly or through a linker.

[0040] In some embodiments, the expression system can be selected from a prokaryotic expression system, a eukaryotic expression system or a viral expression system. In some embodiments, the expression system can include a prokaryotic vector, a eukaryotic vector or a viral vector, etc.

[0041] According to yet another aspect of the present disclosure, a host cell is provided, wherein the host cell comprises the above expression system.

[0042] In some embodiments, the host cell is a prokaryotic cell or a eukaryotic cell. In some embodiments, the prokaryotic cell can be selected from Escherichia coli or Bacillus subtilis, such as Escherichia coli BL21, T7E, C41, Arctic, etc. In some embodiments, the eukaryotic cell can be selected from yeast cells, insect cells, plant cells, mammalian cells, etc., such as yeast cells, CHO cells, 293 cells, Vero cells, NSO cells, etc.

[0043] According to yet another aspect of the present disclosure, a kit is provided, comprising the vesicles described in the present disclosure.

[0044] In some embodiments, the concentration of the vesicles is 1×10^12 to 3×10^12 particles / mL.

[0045] According to another aspect of the present disclosure, provided is the use of the vesicles described in the present disclosure as a standard and / or reference.

[0046] In some embodiments, the vesicles are used as standards and / or references for vesicle isolation, purification, vesicle particle number detection, RNA detection in in vitro diagnosis, or protein detection in in vitro detection.

[0047] In some embodiments, the protein is a membrane protein.

[0048] According to another aspect of the present disclosure, provided is the use of the vesicles or the kit described in the present disclosure in detecting the purification efficiency of the vesicles, detecting and / or calibrating the number of particles in the vesicles, quantifying the content of the target RNA derived from the vesicles, or quantifying the content of the target protein derived from the vesicles.

[0049] In some embodiments, the target RNA or target protein is an RNA or protein associated with a disease (eg, cancer, etc.).

[0050] In some embodiments, the vesicles are used as vesicle separation and purification standards to detect the purification efficiency of the vesicles.

[0051] In some embodiments, the vesicles are used as vesicle particle number detection standards to detect and / or calibrate the number of vesicle particles. Through this method, human influence can be reduced and detection accuracy can be improved.

[0052] In some embodiments, the vesicles are used as RNA detection standards in in vitro diagnosis to quantify the content of target RNA in the source vesicles.

[0053] In some embodiments, the vesicles are used as protein detection standards in in vitro detection to quantify the content of the target protein in the source vesicles.

[0054] According to another aspect of the present disclosure, a method for detecting exosome purification efficiency is provided, comprising the following steps:

[0055] (1) adding standard vesicles to the culture supernatant of the cells to be tested, wherein the standard vesicles include the vesicles containing the tag domain disclosed in the present invention, and the tag domain includes a fluorescent protein, a luciferase or a purification tag;

[0056] (2) isolating and purifying exosomes to obtain purified exosomes;

[0057] (3) The label signals in the standard vesicles and purified exosomes were detected respectively, and then the exosome purification efficiency was calculated.

[0058] In some embodiments, the calculation is performed by the following formula:

[0059] According to another aspect of the present disclosure, a method for detecting the number of exosome particles is provided, comprising the following steps:

[0060] (1) isolating and purifying exosomes from the culture supernatant of the cells to be tested to obtain purified exosomes;

[0061] (2) Detecting the particle number concentration of purified exosomes;

[0062] (3) mixing the standard vesicles with the purified exosomes (preferably mixing in equal volumes), wherein the standard vesicles include the vesicles containing the tag domain disclosed herein, the tag domain includes a fluorescent protein, a luciferase or a purification tag, and the particle number concentrations of the standard vesicles and the purified exosomes are close (preferably the difference does not exceed 10%);

[0063] (4) Detecting the number concentration of labeled particles and the number concentration of unlabeled particles in the mixed solution of step (3), and then calculating the calibrated exosome particle number concentration.

[0064] In some embodiments, the calculation is performed by the following formula:

[0065]

[0066] According to another aspect of the present disclosure, a method for detecting the content of target RNA derived from a vesicle is provided, comprising the following steps:

[0067] (1) adding a standard vesicle to a test solution suspected of containing the target RNA, wherein the standard vesicle comprises a vesicle containing a tag domain disclosed herein, wherein the tag domain comprises a fusion fragment of the target RNA and a universal sequence;

[0068] (2) separating and purifying exosomes from the test fluid to obtain purified exosomes;

[0069] (3) extracting the total RNA of the purified exosomes and reversely transcribing it to obtain cDNA;

[0070] (4) The copy number of the target RNA and the copy number of the fusion fragment (including the target RNA and part of the universal sequence) are detected respectively, and then the content of the target RNA in the test solution is calculated.

[0071] In some embodiments, the calculation is performed by the following formula:

[0072]

[0073] According to another aspect of the present disclosure, a method for detecting the content of a target protein derived from a vesicle is provided, comprising the following steps:

[0074] (1) adding a standard vesicle to a test solution suspected of containing a target protein, wherein the standard vesicle comprises a vesicle containing a tag domain disclosed herein, and the tag domain comprises the target protein;

[0075] (2) separating and purifying exosomes from the test fluid to obtain purified exosomes;

[0076] (3) The contents of the scaffold protein and the target protein in the purified exosomes and the scaffold protein in an equal amount of standard vesicles are detected respectively, and then the content of the target protein in the test solution is calculated.

[0077] In some embodiments, the calculation is performed by the following formula:

[0078]

[0079] In the above method, the exosomes can be separated and purified by any conventional method in the art, for example, by ultracentrifugation.

[0080] The present invention is based on NGFR, an exosome scaffold protein, and produces different types of engineered vesicles by transforming cells, and makes them carry unique tags. The engineered vesicles are separated through a large-scale purification process and can be used as standards and / or references after quality control in different scenarios. BRIEF DESCRIPTION OF THE DRAWINGS

[0081] Figure 1 TEM results of exosomes overexpressing different scaffold proteins are shown.

[0082] Figure 2 Marker (Western Blot) results of exosomes overexpressing different scaffold proteins are shown.

[0083] Figure 3 The HPLC purity characterization results after purification of different exosomes are shown, where a is 293Evs, b is NGFR-Evs, and c is CD63-293Evs.

[0084] Figure 4 The HPLC purity characterization results after purification of different exosomes are shown, where a is MFGE8-Evs, b is PTGFRN-Evs, and c is Basp-1-293Evs.

[0085] Figure 5 The characterization results of the scaffold distribution ratios of different exosomes are shown.

[0086] Figure 6 Results of characterization of scaffold abundance of different exosomes are shown.

[0087] Figure 7 The purity (a), morphology (b), particle size (c) and EGFP protein loading (d) characterization results of exosomes overexpressing truncated-NGFR scaffold protein are shown.

[0088] Figure 8 A schematic diagram of the pcDNA3.1(+)-6×his-TEV-NGFR-Flag-Gluc plasmid is shown.

[0089] Fig. 9 The characterization results of exosome purity (a), morphology (b), and particle size (c) of standard 1 are shown.

[0090] Fig.10 A schematic diagram of the pcDNA3.1(+)-6×his-TEV-NGFR-Flag-EGFP plasmid is shown.

[0091] Fig.11 The characterization results of exosome purity (a), morphology (b), and particle size (c) of standard 2 are shown.

[0092] Fig.12 A schematic diagram of the pcDNA3.1(+)-6×his-TEV-NGFR-Flag-MCP plasmid is shown.

[0093] Fig.13 A schematic diagram of the pcDNA3.1(+)-target gene fragment-MS2 plasmid is shown.

[0094] Fig.14 The characterization results of exosome purity (a), morphology (b) and particle size (c) of standard 3 are shown.

[0095] Fig.15A schematic diagram of the pcDNA3.1(+)-6×his-TEV-Flag-NGFR-Flag plasmid is shown.

[0096] Fig.16 The characterization results of exosome purity (a), morphology (b) and particle size (c) of standard 4 are shown.

[0097] Fig.17 The results of Gluc enzyme activity in the exosome standard 1 and purified exosomes in Example 9 are shown.

[0098] Fig.18 The standard curve of the His standard protein prepared according to Table 11 in Example 9 is shown. DETAILED DESCRIPTION

[0099] In order to make the purpose, technical scheme and advantages of the present invention clearer, the present invention is further described in detail below in conjunction with embodiments. The specific embodiments described herein are only used to explain the present invention and are not intended to constitute any limitation of 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.

[0100] Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as commonly used in the field to which the present invention 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.

[0101] 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.

[0102] As used herein, the term "about" 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.

[0103] The term "nerve growth factor receptor (NGFR)" used herein belongs to the tumor necrosis factor transmembrane receptor superfamily and is a transmembrane low-affinity receptor of the neurotrophin family. NGFR is known to bind to nerve growth factor (NGF) and other neurotrophic factors, stimulate nerve growth and regulate the differentiation of sympathetic neurons and some sensory neurons. The present disclosure unexpectedly found that NGFR can be enriched in exosomes and can be used as an exosome scaffold protein to carry exogenous proteins or nucleic acids to achieve the desired effect.

[0104] The terms "vesicle", "exovesicle" or "EV" as used herein refer to cell-derived vesicles comprising a membrane encapsulating an internal space. Vesicles include all membrane-bound vesicles (e.g., exosomes, microvesicles, apoptotic bodies, tumor vesicles, nanovesicles, etc.), whose diameters are smaller than the diameters of the cells from which they are derived. In some aspects, the diameter of the vesicle is in the range of 20nm to 1000nm, and may be contained in the internal space (i.e., cavity), displayed on the outer surface of the vesicle and / or across the membrane. Various macromolecular payloads. In some aspects, the payload may include nucleic acids, proteins, carbohydrates, lipids, small molecules and / or combinations thereof. In some aspects, the extracellular medium comprises a scaffold portion. As an example and not limitation, vesicles include apoptotic bodies, cell fragments, cell-derived vesicles obtained by direct or indirect manipulation (e.g., by continuous extrusion or treatment with an alkaline solution), vesicles of vesicles, and vesicles produced by living cells (e.g., by direct plasma membrane budding or late endosome fusion with the plasma membrane). Vesicles can be derived from living or dead organisms, explanted tissues or organs, prokaryotic or eukaryotic cells, and / or cultured cells. In some aspects, vesicles are produced by cells expressing one or more transgenic products.

[0105] The term "exosome" as used herein refers to a vesicle with a diameter between 20 and 300 nm (e.g., between 40 and 200 nm), rich in RNA (miRNA, lncRNA, circRNA, etc.), DNA, protein and lipid, and is involved in intercellular molecular transmission. Exosomes contain a membrane that encapsulates the internal space, and in some aspects, can be produced by cells by direct plasma membrane budding or by fusion of late endosomes or multivesicular bodies with the plasma membrane. In some aspects, exosomes contain a scaffold portion. As described below, exosomes may be derived from producer cells and separated from producer cells according to their size, density, biochemical parameters, or a combination thereof. In some aspects, EVs (e.g., exosomes) disclosed herein are produced by cells expressing one or more transgenic products.

[0106] The term "nanovesicle" as used herein refers to a vesicle having a diameter between 20-250nm (e.g., between 30-150nm), and is produced by a cell (e.g., a production cell) by direct or indirect manipulation so that the cell will not produce nanovesicles without manipulation. Suitable manipulations of cells for the production of nanovesicles include, but are not limited to, continuous extrusion, treatment with an alkaline solution, ultrasonic treatment, or a combination thereof. In some aspects, the nanovesicle colony described herein is substantially free of vesicles derived from cells by direct budding from the plasma membrane or late endosome fusion with the plasma membrane. In some aspects, the nanovesicle comprises a scaffold portion, such as scaffold X and / or scaffold Y. The nanovesicle, once derived from a production cell, can be separated from the production cell based on its size, density, biochemical parameters, or a combination thereof.

[0107] 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.

[0108] The term "antibody" as used herein encompasses immunoglobulins (whether naturally occurring or partially or completely synthetically produced) and fragments thereof. The term also covers any protein having a binding domain homologous to an immunoglobulin binding domain. "Antibody" also includes polypeptides comprising a framework region that specifically binds and recognizes an antigen from an immunoglobulin gene or a fragment thereof. The use of the term antibody is intended to include complete antibodies, polyclonal antibodies, monoclonal antibodies and recombinant antibodies, fragments thereof, and also includes single-chain antibodies, humanized antibodies, murine antibodies, chimeric monoclonal antibodies, mouse-human monoclonal antibodies, mouse-primate monoclonal antibodies, primate-human monoclonal antibodies, anti-idiotypic antibodies, antibody fragments, bifunctional antibodies, and antibody-related polypeptides. Antibodies include bispecific antibodies and multispecific antibodies as long as they exhibit the desired biological activity or function.

[0109] The term "tag domain" as used herein refers to a tag molecule that can be connected to a scaffold protein via an anchoring portion, wherein the tag molecule can be used for detection purposes. Thus, for example, the tag domain includes a detectable fluorescent protein, luciferase, disease marker protein or disease marker RNA, etc.

[0110] As used herein, the terms "active domain" or "biologically active domain" are used interchangeably and refer to an active molecule that can be linked to a scaffold protein via an anchoring moiety, wherein the molecule can have a therapeutic or prophylactic effect in a subject in need thereof, or can be used for diagnostic purposes. Thus, for example, the term biologically active domain includes proteins (e.g., antibodies, proteins, polypeptides, and derivatives, fragments, and variants thereof), polynucleotide sequences, or chemical compounds, etc.

[0111] The term "polynucleotide" as used herein refers to a polymer of nucleotides of any length, including ribonucleotides, deoxyribonucleotides, analogs thereof, or mixtures thereof. This term refers to the primary structure of the molecule. Thus, the term includes triple-stranded, double-stranded, and single-stranded deoxyribonucleic acids ("DNA"), and triple-stranded, double-stranded, and single-stranded ribonucleic acids ("RNA"). It also includes modified (e.g., by alkylation and / or by capping) and unmodified forms of polynucleotides. More specifically, the term "polynucleotide" includes polydeoxyribonucleotides (containing 2-deoxy-D-ribose); polyribonucleotides (containing D-ribose), including tRNA, rRNA, hRNA, siRNA, and mRNA, whether spliced ​​or unspliced; any other type of polynucleotide that is an N- or C-glycoside of a purine or pyrimidine base; and other polymers containing an orthonucleotide backbone, such as polyamides (e.g., peptide nucleic acids "PNA") and polymorpholino polymers; and other synthetic sequence-specific nucleic acid polymers, provided that the polymer contains nucleobases in a configuration that permits base pairing and base stacking, such as found in DNA and RNA.

[0112] The terms "polypeptide," "peptide," and "protein" are used interchangeably herein to refer to amino acid polymers of any length. The polymer may comprise modified amino acids. The term also encompasses amino acid polymers that have been modified naturally or by intervention; for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification, such as conjugation with a labeling component. Also included in the definition are, for example, polypeptides containing one or more amino acid analogs (including, for example, unnatural amino acids such as homocysteine, ornithine, p-acetylphenylalanine, D-amino acids, and creatine), as well as other modifications known in the art.

[0113] The term "polypeptide" as used herein refers to proteins, polypeptides and peptides of any size, structure or function. Polypeptides include gene products, naturally occurring polypeptides, synthetic polypeptides, homologues, orthologues, lateral homologues, fragments of the aforementioned and other equivalents, variants and analogs. Polypeptides can be single polypeptides or can be multimolecular complexes, such as dimers, trimers or tetramers. They can also contain single-chain or multi-chain polypeptides. Most commonly, disulfide bonds are present in multi-chain polypeptides. The term polypeptide can also be applied to amino acid polymers in which one or more amino acid residues are artificial chemical analogs of the corresponding naturally occurring amino acids. In some aspects, a "peptide" can be less than or equal to 50 amino acids long, for example, about 5, 10, 15, 20, 25, 30, 35, 40, 45 or 50 amino acids long.

[0114] As used herein, the term "connector" 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 connection sons can be connected in series. When multiple connection sons are present, each connection son can be the same or different. In general, the connection son provides flexibility or prevents / improves steric hindrance. The connection son is usually not cut; however, in some aspects, such cutting may be required. Therefore, in some embodiments, the connection son may include one or more protease cleavable sites, which may be located within the connection son sequence or at the connection son flank at either end of the connection son sequence.

[0115] In some embodiments, the linker is a peptide linker. In some embodiments, the peptide linker may comprise at least about two, at least about three, at least about four, at least about five, at least about 10 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.

[0116] As used herein, the term "replacement" or "substitution" of an amino acid may refer to a conservative amino acid substitution, wherein an amino acid residue is substituted with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art, including basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, if an amino acid in a polypeptide is substituted with another amino acid from the same side chain family, the substitution is considered conservative. In another aspect, a string of amino acids can be conservatively replaced with a structurally similar string that differs in the order and / or composition of side chain family members.

[0117] "Percent sequence identity" or "percent identity" between two polynucleotide or polypeptide sequences refers to the number of identical matching positions shared by the sequences within 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. Since a gap is not a nucleotide or amino acid, gaps present in the target sequence are not counted. Likewise, since target sequence nucleotides or amino acids are counted and nucleotides or amino acids from the reference sequence are not counted, gaps present in the reference sequence are not counted.

[0118] 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 determination of the sequence identity percentage between the comparison of the sequence and the two sequences can be completed using software that is easy to use and download online. Suitable software programs can be obtained from various sources for the comparison of protein and nucleotide sequences. A suitable program for determining the sequence identity percentage is bl2seq, which is a part of the BLAST program suite that can be obtained 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 for comparing nucleic acid sequences, and BLASTP is used for comparing 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.

[0119] Examples and drawings are provided below to help understand the present invention. However, it should be understood that these examples and drawings are only used to illustrate the present invention, but do not constitute any limitation. The actual protection scope 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.

[0120] Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art and commonly used instruments and reagents available on the market, see Welsh JA, et al. Minimal information for studies of extracellular vesicles (MISEV2023): From basic to advanced approaches. J Extracell Vesicles. 2024 Feb; 13(2): e12404.

[0121] Example 1. Expression vector construction

[0122] The NGFR protein (SEQ ID NO: 1) and the control scaffold proteins BASP-1 (SEQ ID NO: 2), PTGFRN (SEQ ID NO: 3), CD63 (SEQ ID NO: 4), and MFGE8 (SEQ ID NO: 5) were used to construct expression plasmids pcDNA3.1 (+) - scaffold protein - EGFP (SEQ ID NO: 6) - Nanoluc (SEQ ID NO: 7), respectively.

[0123] Example 2. Preparation and verification of exosomes

[0124] Exosomes expressing EGFP-Nanoluc fused with scaffold NGFR, BASP-1, PTGFRN, CD63 and MFGE8 expression proteins as well as natural Expi 293F exosomes (named NGFR-293Evs, BASP-1-293Evs, PTGFRN-293Evs, CD63-293Evs, MFGE8-293Evs and 293Evs) were prepared respectively.

[0125] 2.1 293s cell passaging

[0126] Expi 293F cell recovery density 3-10×10 5 cells / ml, and the cell density was controlled at 0.5-5×10 6 cells / ml.

[0127] 2.2 Cell transfection

[0128] Before transfection, adjust the number of cells in step 2.1 to 1-3×10 6cells / ml, the plasmid concentration was 1-1.5 μg / ml, and the concentration ratio of polyethyleneimine (PEI) to plasmid was controlled to be (1:1)-(4:1). The five plasmids obtained in Example 1 were transfected into 293s cells according to the above conditions to overexpress proteins NGFR, BASP-1, PTGFRN, CD63 and MFGE8 in the cells. The cells were cultured at 37°C and 5% CO2 for 48-72 hours, and the cell culture supernatant was collected for downstream purification.

[0129] 2.3 Exosome purification

[0130] The cell culture supernatant collected in step 2.2 was used for ultrafiltration, density gradient centrifugation, and molecular sieve filtration to obtain exosome samples that can be used for nanoflow cytometry analysis.

[0131] 2.4 Exosome quality inspection

[0132] The purified exosomes were characterized, and the morphological (TEM) characterization results were as follows Figure 1 As shown, the purified product obtained has a typical exosome disc-shaped sinus membrane layer structure.

[0133] In addition, the expression of exosome markers CD81 and TSG101, Golgi marker GM130 and endoplasmic reticulum marker Calexin derived from each group of cells was detected by Western Blot. Figure 2 shown.

[0134] The purity of exosomes was analyzed using a SEC-1000 column (7.8 mm × 150 mm, 7 μm; Thermo Fisher Scientific) coupled with a UPLC H-Class system (Waters). The column was eluted with 150 mM NaCl and 20 mM phosphate buffer (pH 7.2) at a flow rate of 0.3 mL / min. The UV absorbance was measured at a wavelength of 280 nm. Purity (HPLC) characterization results are shown in Figure 3 a-3c and Figure 4 As shown in a-4c.

[0135] from Figures 2 to 4 It can be seen that the exosomes obtained from each group of cells highly expressed CD81 and TSG101, while the expression of GM130 and Calexin was not detected, further indicating that the exosomes obtained were of high purity.

[0136] Example 3. Distribution and abundance characterization of exosomes engineered with new scaffold proteins on exosomes

[0137] 3.1 Comparison of exosome scaffold distribution characterization

[0138] Nano-flow was used to detect the proportion of EGFP-positive particles in 20 μL exosomes of different scaffold proteins and the negative control group. Figure 5 shown. Figure 5 It was shown that the NGFR-positive exosome subpopulation accounted for more than 50% of the entire exosome distribution, which was higher than the currently disclosed exosome universal scaffold CD63.

[0139] 3.2 Comparison of exosome scaffold abundance characterization

[0140] use The luciferase reagent was used to detect the Nanoluc enzyme activity in exosomes with the same number of particles of different scaffold proteins and the negative control group, and then the abundance of the fusion expression scaffold in the exosomes with the same number of particles was characterized. Figure 6 shown. Figure 6 It was shown that the abundance of NGFR scaffolds was greater than that of CD63 scaffolds.

[0141] Example 4. A new scaffold protein truncated protein that achieves exosome localization and loading of foreign substances

[0142] In order to determine the shortest sequence of NGFR to achieve exogenous substance loading, this example is divided according to the protein structure of NGFR, based on the internal and external domains of the transmembrane segment, and gradually reduces the domains outside and inside the membrane. Finally, the amino acids 200-338 are determined and named as truncated-NGFR (SEQ ID NO: 8), which is the shortest sequence to achieve exosome localization loading.

[0143] The following is the verification of its distribution and abundance in exosomes.

[0144] 4.1 Expression vector construction and preparation

[0145] (1.1) Vector construction

[0146] The C-terminus of truncated-NGFR was fused with EGFP-Nanoluc to construct and verify the expression vector pcDNA3.1-truncated-NGFR-EGFP-Nanoluc.

[0147] (1.2) Plasmid preparation

[0148] Plasmid preparation was the same as before.

[0149] (1.3) Exosome preparation

[0150] The plasmid prepared in (1.2) was transfected into Expi 293 cells, and the preparation, purification and quality control process were the same as in Example 2. It was named truncated-NGFR-293Evs.

[0151] 4.2Verification of truncated-NGFR-293Evs loading EGFP into exosomes

[0152] The high-purity exosomes prepared in (1.3) were characterized. Figure 7 a-7d shows that the purity of exosomes is close to 100%, the morphology is the disc-shaped membrane structure of exosomes, and the particle size distribution range is within the normal range of exosomes. The exosome EGFP positive rate is about 19%, indicating that truncated-NGFR can load EGFP into exosomes and retain the localization and loading function of exosomes.

[0153] Example 5. Engineered exosomes as isolation standards

[0154] a. Expi293F cell recovery culture.

[0155] b. 2 h before transfection, fresh culture medium (OPM-293CD05 Medium, Shanghai Aopumai) was replaced for Expi293F cells and the density was adjusted to 3×10^6 cells / mL.

[0156] c. Use PEI (polysciences, #24765-1) reagent to transfect according to the instructions

[0157] pcDNA3.1(+)-6×his-TEV-NGFR-Flag-Gluc plasmid (such as Figure 8 As shown, TEV enzyme is able to cleave TEV protein for removal of his tag) into Expi293F cells.

[0158] d. 24 hours after transfection, wash the cells with PBS and replace the medium. After replacing the medium, adjust the cell density to 1.5×10^6 cells / mL.

[0159] e. Continue to culture for 48 h. Centrifuge at 3000 rpm for 20 min, collect the cell culture supernatant, and filter using a 0.45 μm PVDF membrane. The filtrate is centrifuged at 100,000 g for 105 min using an ultracentrifuge (Beckman Coulter, USA), resuspend the precipitate with DPBS, and then purify the resuspended exosomes using affinity chromatography (nickel column) (Ni SEPHAROSE 6FF, Cytiva).

[0160] f. The purified exosomes are freeze-dried, and after the exosomes are re-dissolved, quality control is performed to obtain standard product 1.

[0161] The protein concentration (BCA method, #A65453, Thermo Fisher Scientific) and particle number concentration (NanoFCM Inc., Xiamen, China) are shown in Table 1 below.

[0162] Exosome morphology (transmission electron microscopy, Hitachi High-Technologies Corporation, Tokyo, Japan), exosome purity (high-performance liquid chromatography, SEC-1000 column (Thermo Fisher Scientific), combined with UPLC H-Class system (Waters)), and particle size distribution (NanoFCM Inc., Xiamen, China) were examined, e.g. Fig. 9 The results of a-9c showed that the purity of exosomes was close to 100%, the morphology was an exosome disc-shaped sinus membrane layer structure, and the particle size distribution range was within the normal range of exosomes. The results of exosome marker detection (proteome analysis, Easy NLC 1200-QExactive Orbitrap mass spectrometer (Thermo Fisher Scientific)) are shown in Table 2 below.

[0163] Table 1

[0164] sample Exosome protein concentration (μg / mL) Particle number concentration (particles / mL) Standard 1 298 1.35×10^12

[0165] Table 2

[0166]

[0167] Example 6. Engineered exosomes as particle number detection standards

[0168] a. Expi293F cell recovery culture.

[0169] b. 2 h before transfection, replace the culture medium with fresh one and adjust the density to 3×10^6 cells / mL.

[0170] c. Use PEI (polysciences, #24765-1) reagent to transfect pcDNA3.1(+)-6×his-TEV-NGFR-Flag-EGFP plasmid (such as Fig.10 as indicated) into Expi293F cells.

[0171] d. 24 hours after transfection, wash the cells with PBS and replace the medium. After replacing the medium, adjust the cell density to 1.5×10^6 cells / mL.

[0172] e. Continue to culture for 48 hours. Centrifuge at 3000 rpm for 20 minutes, collect the cell culture supernatant, and filter it using a 0.45 μm PVDF membrane. Centrifuge the filtrate at 100,000 g for 105 minutes using an ultracentrifuge, resuspend the precipitate with DPBS, and then purify the resuspended exosomes using affinity chromatography (nickel column).

[0173] f. The purified exosomes were freeze-dried, re-dissolved with DPBS solution, and then quality controlled to obtain standard product 2.

[0174] The characterization was performed in the same manner as in Example 5. The protein concentration and particle number concentration are shown in Table 3 below, and the exosome morphology, exosome purity and particle size distribution are shown in Fig.11 As shown in a-11c, the results show that the purity of exosomes is close to 100%, the morphology is an exosome disc-shaped sinus membrane layer disc-shaped sinus membrane layer structure, and the particle size distribution range is within the normal range of exosomes. The results of exosome marker detection are shown in Table 4 below.

[0175] Table 3

[0176] sample Exosome protein concentration (μg / mL) Particle number concentration (particles / mL) Standard 2 259 1.03×10^12

[0177] Table 4

[0178]

[0179] Example 7. Engineered exosomes as RNA detection standards in in vitro diagnostic products (IVD)

[0180] a. Expi293F cell recovery culture.

[0181] b. 2 h before transfection, replace the culture medium with fresh one and adjust the density to 3×10^6 cells / mL.

[0182] c. Use PEI (polysciences, #24765-1) reagent to transfect pcDNA3.1(+)-6×his-TEV-NGFR-Flag-MCP plasmid according to the instructions (such as Fig.12 The sequence of MCP is shown in SEQ ID NO: 10) and pcDNA3.1(+)-universal sequence-target gene fragment-MS2 plasmid (as shown in Fig.13As shown, MS2 (SEQ ID NO: 11) will not be translated into protein. When RNA is transcribed, it will form a neck ring structure for binding to MCP; the universal sequence (SEQ ID NO: 12) is a sequence located on the left side of the target gene fragment) into Expi293F cells. The target gene is a gene fragment related to the disease in IVD detection. In this embodiment, the target gene is a PCA3 mRNA fragment (SEQ ID NO: 13).

[0183] d. 24 hours after transfection, wash the cells with PBS and replace the medium. After replacing the medium, adjust the cell density to 1.5×10^6 cells / mL.

[0184] e. Continue to culture for 48 hours. Centrifuge at 3000 rpm for 20 minutes, collect the cell culture supernatant, and filter it using a 0.45 μm PVDF membrane. Centrifuge the filtrate at 100,000 g for 105 minutes using an ultracentrifuge, resuspend the precipitate with DPBS, and then purify the resuspended exosomes using affinity chromatography (nickel column).

[0185] f. The purified exosomes were freeze-dried, and after the exosomes were re-dissolved, quality control was performed to obtain standard product 3.

[0186] The characterization was performed in the same manner as in Example 5. The protein concentration and particle number concentration are shown in Table 5 below, and the exosome morphology, exosome purity and particle size distribution are shown in Fig.14 As shown in a-14c, the results show that the purity of exosomes is close to 100%, the morphology is an exosome disc-shaped sinus membrane layer disc-shaped sinus membrane layer structure, and the particle size distribution range is within the normal range of exosomes. The results of exosome marker detection are shown in Table 6 below.

[0187] Table 5

[0188] sample Exosome protein concentration (μg / mL) Particle number concentration (particles / mL) Standard 3 336 1.52×10^12

[0189] Table 6

[0190]

[0191] g. Take 200ul of exosome standard 3, use Qiagen miRNeasy Serum / Plasma Kit (Cat. No. 217184) to extract RNA in exosomes, and finally elute with 14μL nuclease-free water.

[0192] h. Use TB Premix Ex Taq TMII (Tli RNase H Plus) (Cat. No. RR820B) was used for reverse transcription to obtain cDNA (reverse transcription primer: 5'-TGGGTGATCCTCATGTTTCGATG-3', SEQ ID NO: 14).

[0193] i. After the cDNA was diluted 10 times, 2 μL was taken and PrimeScript RT Master Mix (Perfect RealTime) (Cat. No. RR036A) was used for qPCR (qPCR primers: Forward: 5'-TAACAACTCCGCTCCAAGGC-3' (SEQ ID NO: 15), Reverse: 5'-TGGGTGATCCTCATGTTTCGATG-3' (SEQ ID NO: 14), qPCR reference plasmid: pcDNA3.1 (+) - target gene sequence - MS2) to detect the copy number of the marker RNA. The test was repeated 3 times to determine the copy number of the marker RNA in the exosome standard. The results are shown in Table 7 below.

[0194] Table 7

[0195]

[0196] Example 8. Engineered exosomes as protein detection standards in IVD

[0197] a. Expi293F cell recovery culture (NGFR protein knockout strain). The NGFR protein knockout strain was obtained by the following method: using NGFR gene knockout lentivirus (L29036, Bio-Tech) according to the instructions, after infecting Expi293F cells, using puromycin to screen positive cells, inoculating them in 96-well plates for monoclonal screening, digesting the cells and inoculating them in 24-well plates two weeks later, and identifying them by qPCR after the cells were fully grown, and finally screening out the NGFR knockout strain cells.

[0198] b. 2 h before transfection, replace the culture medium with fresh one and adjust the density to 3×10^6 cells / mL.

[0199] c. Use PEI (polysciences, #24765-1) reagent to transfect pcDNA3.1(+)-6×his-TEV-flag protein-NGFR-Flag plasmid according to the instructions (such as Fig.15 The marker protein is a specific protein on the surface of natural exosomes or exosomes under pathological conditions in the IVD test project sample. In this embodiment, the marker protein is GPC1 protein (SEQ ID NO: 16).

[0200] d. 24 hours after transfection, wash the cells with PBS and replace the medium. After replacing the medium, adjust the cell density to 1.5×10^6 cells / mL.

[0201] e. Continue to culture for 48 hours. Centrifuge at 3000 rpm for 20 minutes, collect the cell culture supernatant, and filter it using a 0.45 μm PVDF membrane. Centrifuge the filtrate at 100,000 g for 105 minutes using an ultracentrifuge, resuspend the precipitate with DPBS, and then purify the resuspended exosomes using affinity chromatography (nickel column).

[0202] f. The purified exosomes were freeze-dried, and after the exosomes were re-dissolved, quality control was performed to obtain standard product 4.

[0203] The characterization was performed in the same manner as in Example 5. The protein concentration and particle number concentration are shown in Table 8 below, and the exosome morphology, exosome purity and particle size distribution are shown in Fig.16 As shown in a-16c, the results show that the purity of exosomes is close to 100%, the morphology is an exosome disc-shaped sinus membrane layer disc-shaped sinus membrane layer structure, and the particle size distribution range is within the normal range of exosomes. The results of the exosome marker detection are shown in Table 9 below.

[0204] Table 8

[0205] sample Exosome protein concentration (μg / mL) Particle number concentration (particles / mL) Standard 4 285 1.30×10^12

[0206] Table 9

[0207]

[0208] g. Take 100uL of exosomes and use the ELISA method (ab155436, Abcam) to detect the NGFR protein on the surface of the exosomes. Repeat 3 times to determine the content of NGFR protein in the exosome standard. The results are shown in Table 10 below.

[0209] Table 10

[0210] Repeat 1 Repeat 2 Repeat 3 average Exosomal NGFR protein concentration 1.62ng / mL 1.65 ng / mL 1.63ng / mL 1.63ng / mL

[0211] Example 9. Detection of exosome purification efficiency

[0212] The engineered exosomes were used as the standard for exosome separation and purification to detect the exosome purification efficiency. This example takes the commonly used 293T cell exosome separation as an example.

[0213] a. 293T cells were cultured in T225 flasks at a seeding density of 60,000 cells / cm 2 , 45 mL culture volume (using exosome-depleted FBS) and cultured in a CO2 incubator.

[0214] b. After 72 hours, the cell culture supernatant was collected and 50 μL of the exosomes of the standard sample 1 prepared in Example 5 was added thereto.

[0215] c. Use the commonly used ultracentrifugation method to separate and purify exosomes: centrifuge at 300g for 10 minutes, take the supernatant and centrifuge at 2000g for 10 minutes, take the supernatant and centrifuge at 10000g for 30 minutes, take the supernatant and centrifuge at 100000g for 70 minutes, resuspend the precipitate with PBS and centrifuge at 100000g for 70 minutes, resuspend the precipitate with 500μL PBS to obtain purified exosomes.

[0216] d. Gaussia luciferase reporter gene detection kit (Beyotime, #RG021S) was used to detect the Gluc enzyme activity in 5 μL exosome standard (plus 45 μL PBS, make up to 50 μL) and 50 μL purified exosomes, and finally the exosome purification efficiency was calculated.

[0217]

[0218] like Fig.17 As shown, exosome purification efficiency = 206244.3333÷737191.6667×100% = 29.97%

[0219] e. Optionally, His Tag ELISA Detection Kit (GenScript, #L00436) was used to detect the His tag content in 5 μL of exosome standard (plus 45 μL PBS to make up to 50 μL) and 50 μL of purified exosome sample to calculate the exosome purification efficiency.

[0220]

[0221] After the his protein in the ELISA kit was graded diluted, the OD450 test results (3 replicate wells) of each concentration were shown in Table 11 below.

[0222] Table 11

[0223]

[0224] A standard curve for his protein detection was prepared based on the data in Table 11 ( Fig.18 ) and the average OD450 values ​​of the samples in Table 12, and the concentration of his protein in the samples was calculated according to the standard curve equation. The test results are shown in Table 12.

[0225] Table 12

[0226]

[0227] Example 10. Detection of exosome particle count

[0228] The application of engineered exosomes as particle number detection standards is used to calibrate the detection of exosome particle number. Through this method, human influence can be reduced and detection accuracy can be improved. This example takes the commonly used 293T cell exosome separation as an example.

[0229] a. 293T cells were cultured in T225 flasks at a seeding density of 60,000 cells / cm 2 , 45 ml culture volume (using exosome-depleted FBS), three groups were made in parallel and cultured in a CO2 incubator.

[0230] b. Collect cell culture supernatant after 72 h.

[0231] c. Use the commonly used ultracentrifugation method to separate and purify exosomes: centrifuge at 300g for 10 minutes, take the supernatant and centrifuge at 2000g for 10 minutes, take the supernatant and centrifuge at 10000g for 30 minutes, take the supernatant and centrifuge at 100000g for 70 minutes, resuspend the precipitate with PBS and centrifuge at 100000g for 70 minutes, resuspend the precipitate with 500μL PBS to obtain purified exosomes.

[0232] d. Nano flow cytometry (NanoFCM) was used to detect the particle number concentration of three exosome samples.

[0233] e. In this example, the average particle number concentration of exosomes directly measured was 3.17E+11 particles / mL, so the concentration of the standard in Example 6 was adjusted to about 3.00E+11 particles / mL.

[0234] f. Take an equal volume of the adjusted concentration standard exosomes and mix them with the three isolated exosome samples respectively.

[0235] g. The particle number concentration of green fluorescent exosomes and the particle number concentration of colorless exosomes in the mixed solution were detected using a nano flow cytometer (NanoFCM), and the particle number concentration of the separated exosomes was calculated according to the following formula. The results are shown in Table 13.

[0236]

[0237] Table 13

[0238]

[0239] From the results in Table 13, it can be seen that the CV value of the particle number concentration detection dropped from 7.89% to 1.91%.

[0240] Example 11. Detection of RNA in IVD

[0241] The application of engineered exosomes as RNA detection standards in IVD is used to quantify the content of target RNA in exosomes from a specific source. For example, based on the mRNA copy number of the three genes PCA3, SPEDF, and ERG in the subject's urine exosomes, combined with a specific algorithm, it can be assessed whether the subject has prostate cancer, which can greatly reduce the risk of unnecessary prostate biopsy of the subject. This example takes the detection of the copy number of PCA3 mRNA in exosomes from the subject's urine as an example.

[0242] a. Collect 50 mL of urine from 3 male patients from Tianjin Fifth Central Hospital after rectal examination, and add 15.24 μL of the exosome standard in Example 7 (this standard exosome contains PCA3 mRNA fragment) to each patient.

[0243] b. Use the commonly used ultracentrifugation method to separate and purify exosomes: centrifuge at 300g for 10 minutes, take the supernatant and centrifuge at 2000g for 10 minutes, take the supernatant and centrifuge at 10000g for 30 minutes, take the supernatant and centrifuge at 100000g for 70 minutes, resuspend the precipitate with PBS and centrifuge at 100000g for 70 minutes, resuspend the precipitate with 500μL PBS to obtain the purified urine exosome sample.

[0244] c. Use Qiagen miRNeasy Serum / Plasma Kit (Cat. No. 217184) to extract 200 μL of total RNA from urine exosomes, and finally elute with 14 μL of nuclease-free water.

[0245] d. Take 1.4 μL RNA from each tube and use TB Premix Ex Taq TM II (Tli RNase H Plus) (Cat. No. RR820B) was used for reverse transcription to obtain cDNA (primer: 5'-AGAGAGGATTGGTAAGCGATGTG-3', SEQ ID NO: 17).

[0246] e. After the cDNA was diluted 10 times, 2 μL was taken and qPCR was performed using PrimeScript RT Master Mix (Perfect RealTime) (Cat. No. RR036A) (primer sequences are shown in Table 14 below) to detect the copy number of PCA3 and the fusion fragment (including the PCA3 fragment and the universal sequence, which is located on the pcDNA3.1(+)-target gene fragment-MS2 plasmid ( Fig.13 ), the copy number of the universal sequence on the left side of the target gene fragment).

[0247] Table 14

[0248]

[0249] f. According to the formula, the copy number of PCA3 in 50 mL of urine exosomes was calculated. The results are shown in Table 15.

[0250]

[0251] Table 15

[0252]

[0253]

[0254] Example 11. Detection of proteins in IVD

[0255] The application of engineered exosomes as an external reference for protein detection in IVD is used to quantify the content of target proteins in exosomes of specific sources. For example, studies have shown that the proportion of glypican-1 (GPC1) positive exosomes in pancreatic cancer patients is significantly increased compared with healthy individuals, and the level of exosome GPC1 in the serum of early pancreatic cancer patients is significantly higher than that of healthy people. GPC1 positive exosomes have potential clinical application value as a marker for early pancreatic cancer diagnosis. This example takes the detection of GPC1 protein content in exosomes derived from pancreatic cancer serum as an example.

[0256] a. Collect 10 mL of whole blood from patients in Tianjin Fifth Central Hospital and centrifuge at 2500 g for 10 min at 4°C to obtain approximately 5 mL of serum.

[0257] b. Add 10 μL of the exosome standard in Example 8 (the surface of the exosomes in this standard contains GPC1 and NGFR proteins).

[0258] c. Use the commonly used ultracentrifugation method to separate and purify exosomes: centrifuge at 300g for 10 minutes, take the supernatant and centrifuge at 2000g for 10 minutes, take the supernatant and centrifuge at 10000g for 30 minutes, take the supernatant and make up the volume with PBS, centrifuge at 100000g for 70 minutes, resuspend the precipitate with PBS and centrifuge at 100000g for 70 minutes, resuspend the precipitate with 500μL PBS to obtain purified exosomes.

[0259] d. The GPC1 protein ELISA kit (ab270217, Abcam) was used to detect the GPC1 protein content in 100 μL of exosomes extracted from c, and the NGFR protein ELISA kit (ab155436, Abcam) was used to detect the NGFR protein content in 100 μL of exosomes extracted from c and 2 μL of standard exosomes (plus 98 μL PBS to make up 100 μL).

[0260] e. According to the formula, the content of GPC1 protein on exosomes in 2 mL of blood was calculated. The results are shown in Table 16.

[0261]

[0262] Table 16

[0263]

[0264] 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. A vesicle, characterized in that The vesicles include: A scaffold protein, wherein the scaffold protein is a nerve growth factor receptor or a fragment thereof; and Tag domain.

2. The vesicle according to claim 1, characterized in that The nerve growth factor receptor has at least the amino acid sequence shown in SEQ ID NO: 8 or an amino acid sequence having at least 85% sequence identity with SEQ ID NO: 8; Preferably, the nerve growth factor receptor has the amino acid sequence shown in SEQ ID NO:1 or an amino acid sequence having at least 85% sequence identity with SEQ ID NO:

1.

3. The vesicle according to claim 1 or 2, characterized in that The tag domain includes one or more of a fluorescent protein, a luciferase, a purification tag, a disease marker protein, or a disease marker RNA; Preferably, the tag domain includes one or more of GST (glutathione sulfhydryl transferase) tag protein, His6 tag protein (His-tag), MBP (maltose binding protein) tag protein, Flag tag protein, SUMO tag protein, HA tag protein, Myc tag protein, eGFP (enhanced green fluorescent protein), eCFP (enhanced cyan fluorescent protein), eYFP (enhanced yellow-green fluorescent protein), mCherry (monomeric red fluorescent protein), T7 tag, V5 tag, cellulose binding domain (CBD) tag, calmodulin tag, AviTag tag protein, Halo Tag tag protein, SNAP-Tag tag protein, firefly luciferase (Fluc), sea cucumber luciferase (Rluc), Guassia luciferase (Gluc), nanoluciferase (Nluc), cancer marker mRNA, and cancer marker protein; Preferably, the scaffold protein is bound to the tag domain directly or through a linker.

4. The vesicle according to any one of claims 1 to 3, characterized in that The vesicle also includes an active domain; Preferably, the active domain comprises a polypeptide, a polynucleotide, a compound or any combination thereof; More preferably, the polynucleotide comprises an antisense oligonucleotide (ASO), siRNA, miRNA, shRNA, nucleic acid or any combination thereof, More preferably, the polypeptide comprises a peptide, a protein, an antibody or an antigen-binding fragment thereof, a nucleotide-binding protein or a fragment thereof, or any combination thereof; More preferably, the scaffold protein is bound to the active domain directly or through a linker.

5. The vesicle according to any one of claims 1 to 4, characterized in that The vesicles include one or more of exosomes, microvesicles, apoptotic bodies, tumor vesicles and nanovesicles; and / or The nerve growth factor receptor or fragment thereof is localized in the vesicle.

6. An expression system, comprising: A first expression element encoding a scaffold protein, wherein the scaffold protein is a nerve growth factor receptor or a fragment thereof; and a second expression element encoding a tag domain; Optionally, the expression system further comprises a third expression element encoding an active domain; Preferably, the scaffold protein, the tag domain and the active domain are the same as defined in claims 2-4.

7. A host cell comprising the expression system according to claim 6.

8. A kit comprising the vesicle according to any one of claims 1 to 5; Preferably, the concentration of the vesicles is 1×10^12 to 3×10^12 particles / mL.

9. Use of the vesicle according to any one of claims 1 to 5 as a standard and / or reference substance, Preferably, the vesicles are used as standards and / or references for vesicle isolation, purification, vesicle particle number detection, RNA detection in in vitro diagnosis, or protein detection in in vitro detection.

10. Use of the vesicle according to any one of claims 1 to 5 or the kit according to claim 8 in detecting the purification efficiency of vesicles, detecting and / or calibrating the number of particles in vesicles, quantifying the content of target RNA derived from vesicles, or quantifying the content of target protein derived from vesicles; Preferably, the target RNA or target protein is an RNA or protein associated with a disease.

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