A vesicle and its use as a standard

Abstract

The present disclosure relates to a vesicle comprising a scaffold protein which is nerve growth factor receptor or a fragment thereof, and a tag domain. The vesicle carries a unique tag, and the engineered vesicle is isolated by a large-scale purification process, and after quality control, it can be applied to different scenarios as a standard and / or reference.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, and in particular relates to a vesicle and its application as a standard and / or reference. Background Technology

[0002] Exosomes are vesicle-like structures secreted by cells, ranging in size from 30 to 200 nm, and can be observed as cup-shaped structures under a transmission electron microscope. They possess a phospholipid bilayer, encapsulating various substances, including nucleic acids (DNA and RNA), proteins, and lipids, playing a crucial role in cell communication and substance transport. Due to these properties, exosomes are finding increasing applications, including in vitro diagnostics, gene therapy, drug delivery, and disease prognosis.

[0003] There are various methods for the isolation, purification, and detection of exosomes, and different institutions use different methods. The purity of the purified exosomes also varies, as do the sensitivity of the detection methods.

[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] To address the aforementioned problems in the prior art, this disclosure provides a vesicle that uses nerve growth factor receptor (NGFR) as a scaffold protein and is generated by modifying chassis cells, carrying a unique tag or mRNA. Engineered vesicles are isolated through a large-scale purification process and, after quality control, can be used as standards and / or references in various applications.

[0006] According to one aspect of this disclosure, a vesicle is provided comprising: a scaffold protein, said scaffold protein being 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, luciferase, purified tag, disease marker protein, or disease marker RNA.

[0010] In some embodiments, the tag domain includes one or more of the following: GST (glutathione thiotransferase) 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 (monomer 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 binds to the tag domain directly or via a linker.

[0012] In some embodiments, the vesicles further include active structural domains.

[0013] In some embodiments, the active domain includes peptides, polynucleotides, compounds, or any combination thereof.

[0014] In some embodiments, the polynucleotide includes antisense oligonucleotides (ASO), siRNA, miRNA, shRNA, nucleic acids, or any combination thereof.

[0015] In some embodiments, the polypeptide includes peptides, proteins, antibodies or antigen-binding fragments thereof, nucleotide-binding proteins or fragments 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-associated peptides or any fragment 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 a fragment thereof further binds to the polynucleotide sequence.

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

[0020] In some embodiments, the scaffold protein binds to the active domain directly or via 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 a fragment thereof is located in a vesicle.

[0023] In some embodiments, the tag domain and / or the active domain can be displayed on the outer surface of a vesicle or within the lumen of an external vesicle using the scaffold protein nerve growth factor receptor or a fragment thereof disclosed herein.

[0024] This disclosure describes the production of different types of engineered vesicles through cell modification, and the application of these vesicles as standards and / or references in various scenarios. The vesicles of this disclosure are secreted by engineered cells, have a diameter between 30-200 nm, and exhibit a typical cup-shaped structure under transmission electron microscopy. The engineered vesicles of this disclosure have similar shape, structure, and size to natural vesicles. As standards and / or references, they can effectively simulate vesicles in real-world application scenarios.

[0025] According to another aspect of this disclosure, an expression system is provided, the 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.

[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 fluorescent proteins, luciferases, disease marker proteins, or disease marker RNAs.

[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 binds to the tag domain directly or via a linker.

[0031] In some embodiments, the expression system further includes a third expression element that encodes an active structural domain.

[0032] In some embodiments, the active domain includes peptides, polynucleotides, compounds, or any combination thereof.

[0033] In some embodiments, the polynucleotide includes antisense oligonucleotides (ASO), siRNA, miRNA, shRNA, nucleic acids, or any combination thereof.

[0034] In some embodiments, the polypeptide includes peptides, proteins, antibodies or antigen-binding fragments thereof, nucleotide-binding proteins or fragments 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-associated peptides or any fragment 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 a fragment thereof further binds to the polynucleotide sequence.

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

[0039] In some embodiments, the scaffold protein binds to the active domain directly or via a linker.

[0040] In some embodiments, the expression system may be selected from prokaryotic expression systems, eukaryotic expression systems, or viral expression systems. In some embodiments, the expression system may include prokaryotic vectors, eukaryotic vectors, or viral vectors, etc.

[0041] According to another aspect of this disclosure, a host cell is provided, the host cell comprising the above-described expression system.

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

[0043] According to another aspect of this disclosure, a kit is provided that includes the vesicles described in this 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 this disclosure, the application of the vesicles described herein as standards and / or references is provided.

[0046] In some embodiments, the vesicles serve as standards and / or references for vesicle isolation, purification, vesicle particle count detection, RNA detection in in vitro diagnostics, or protein detection in in vitro diagnostics.

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

[0048] According to another aspect of this disclosure, the vesicles or the kits described herein are provided for use in detecting the purification efficiency of vesicles, detecting and / or calibrating the number of vesicle particles, quantifying the content of target RNA or target protein derived from vesicles.

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

[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 implementations, the vesicles are used as a standard for vesicle particle count detection to detect and / or calibrate the particle count of vesicles. This method can reduce human influence and improve detection accuracy.

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

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

[0055] (1) Add standard vesicles to the cell culture supernatant to be tested, wherein the standard vesicles include vesicles containing a tag domain of the present disclosure, wherein the tag domain includes fluorescent protein, luciferase or purified tag;

[0056] (2) Separate and purify exosomes to obtain purified exosomes;

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

[0058] In some implementations, the calculation is performed using the following formula:

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

[0060] (1) Exosomes were isolated and purified from the cell culture supernatant to obtain purified exosomes;

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

[0062] (3) The standard vesicles are mixed with the purified exosomes (preferably mixed in equal volumes), wherein the standard vesicles include the vesicles containing the tag domain of the present disclosure, the tag domain includes fluorescent protein, luciferase or purified tag, and the particle number concentration of the standard vesicles and the purified exosomes is close (preferably differing by no more than 10%).

[0063] (4) Detect the number concentration of labeled particles and the number concentration of unlabeled particles in the mixture of step (3), and then calculate the calibrated exosome particle number concentration.

[0064] In some implementations, the calculation is performed using the following formula:

[0065]

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

[0067] (1) Add standard vesicles to the test solution suspected of containing target RNA, wherein the standard vesicles include vesicles containing a tag domain of the present disclosure, and the tag domain includes a fusion fragment of target RNA and a universal sequence.

[0068] (2) Separate and purify exosomes from the test solution to obtain purified exosomes;

[0069] (3) Extract total RNA from purified exosomes and reverse transcribe it to obtain cDNA;

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

[0071] In some implementations, the calculation is performed using the following formula:

[0072]

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

[0074] (1) Add standard vesicles to the test solution suspected of containing the target protein, wherein the standard vesicles include the vesicles containing the tag domain of the present disclosure, and the tag domain includes the target protein.

[0075] (2) Separate and purify exosomes from the test solution to obtain purified exosomes;

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

[0077] In some implementations, the calculation is performed using the following formula:

[0078]

[0079] In the above method, exosomes can be isolated and purified by any method conventional in the art, such as ultracentrifugation.

[0080] This disclosure utilizes NGFR, an exosome scaffold protein, to produce different types of engineered vesicles by modifying cells and giving them unique tags. The engineered vesicles are then isolated through a large-scale purification process and, after quality control, can be used as standards and / or references in different scenarios. Attached Figure Description

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

[0082] Figure 2The results of Western Blot show the markers of exosomes overexpressing different scaffold proteins.

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

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

[0085] Figure 5 The results show the characterization of the scaffold distribution ratio of different exosomes.

[0086] Figure 6 The results of scaffold abundance characterization for different exosomes are shown.

[0087] Figure 7 The purity (a), morphology (b), particle size (c), and EGFP-loaded (d) characteristics 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] Figure 9 The results of characterization of exosome purity (a), morphology (b), and particle size (c) of standard 1 are shown.

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

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

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

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

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

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

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

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

[0098] Figure 18 The standard curve of His standard protein, plotted according to Table 11 in Example 9, is shown. Detailed Implementation

[0099] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. The specific embodiments described herein are for illustrative purposes only and are not intended to limit the invention in any way. Furthermore, descriptions of well-known structures and techniques are omitted in the following description to avoid unnecessarily obscuring the concepts of this disclosure. Such structures and techniques have also been described in many publications.

[0100] Unless otherwise defined, all technical and scientific terms used in this invention have the same meaning as commonly used in the field to which this invention pertains. For the purposes of interpreting this specification, the following definitions will apply, and where appropriate, terms used in the singular will also include the plural forms, and vice versa.

[0101] Unless the context clearly indicates otherwise, the terms “a” and “an” as used herein include plural references. For example, reference to “a cell” includes multiple such cells and equivalents known to those skilled in the art, etc.

[0102] As used herein, the term "about" indicates a range of ±20% of the following value. In some embodiments, the term "about" indicates a range of ±10% of the following value. In some embodiments, the term "about" indicates a range of ±5% of the following value.

[0103] The term "nerve growth factor receptor (NGFR)" as used herein belongs to the tumor necrosis factor transmembrane receptor superfamily and is a low-affinity transmembrane receptor of the neurotrophic protein family. NGFR is known to bind to nerve growth factor (NGF) and other neurotrophic factors, stimulating nerve growth and regulating the differentiation of sympathetic neurons and some sensory neurons. This disclosure unexpectedly reveals that NGFR can accumulate on exosomes and can serve as an exosome scaffold protein to transport exogenous proteins or nucleic acids to achieve the desired effects.

[0104] As used herein, the terms “vesicle,” “extravesicle,” or “EV” 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.) with a diameter smaller than that of the cell from which they originate. In some aspects, vesicles have diameters ranging from 20 nm to 1000 nm and may contain various macromolecular payloads within an internal space (i.e., a lumen), displayed on the outer surface of the vesicle, and / or transmembrane. In some aspects, said payloads may include nucleic acids, proteins, carbohydrates, lipids, small molecules, and / or combinations thereof. In some aspects, the extracellular medium comprises a scaffold portion. By way of example, and not limitation, vesicles include apoptotic bodies, cell debris, cell-derived vesicles obtained through direct or indirect manipulation (e.g., by continuous extrusion or treatment with an alkaline solution), vesicular organelles, and vesicles generated by living cells (e.g., by direct plasma membrane budding or late endosome fusion with the plasma membrane). Vesicles can originate from living or dead organisms, explant tissues or organs, prokaryotic or eukaryotic cells, and / or cultured cells. In some respects, vesicles are produced by cells expressing one or more transgenic products.

[0105] As used herein, the term "exosome" 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, proteins, and lipids, which participates in intercellular molecular transport. Exosomes contain a membrane encapsulating their internal space and, in some respects, can be produced by cells via direct plasma membrane budding or via the fusion of late endosomes or multivesicles with the plasma membrane. In some respects, exosomes contain a scaffold portion. As described below, exosomes can originate from producer cells and be isolated from producer cells based on their size, density, biochemical parameters, or combinations thereof. In some respects, the EVs (e.g., exosomes) of this disclosure are produced by cells expressing one or more transgenic products.

[0106] As used herein, the term "nanovesicle" refers to a vesicle with a diameter between 20 and 250 nm (e.g., between 30 and 150 nm) that is generated by cells (e.g., production cells) through direct or indirect manipulation such that the cells will not generate nanovesicles without manipulation. Suitable manipulation of cells to generate nanovesicles includes, but is not limited to, continuous extrusion, treatment with an alkaline solution, sonication, or combinations thereof. In some aspects, the nanovesicle populations described herein are substantially free of vesicles derived from cells by direct budding from the plasma membrane or fusion of late endosomes with the plasma membrane. In some aspects, the nanovesicles comprise scaffold portions, such as scaffold X and / or scaffold Y. Once derived from production cells, nanovesicles can be isolated from production cells based on their size, density, biochemical parameters, or combinations thereof.

[0107] As used in this article, the term "fusion protein" refers to two or more proteins or fragments thereof that are collinearly linked through their respective peptide backbones using genetic expression or protein synthesis methods that encode protein polynucleotides.

[0108] As used herein, the term "antibody" encompasses immunoglobulins (whether naturally occurring or partially or fully synthetically produced) and fragments thereof. The term also covers any protein having a binding domain homologous to the binding domain of an immunoglobulin. "Antibody" also includes polypeptides containing a framework region derived from an immunoglobulin gene or a fragment thereof that specifically binds to and recognizes an antigen. 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, mouse antibodies, chimeric monoclonal antibodies, mouse-human monoclonal antibodies, mouse-primate monoclonal antibodies, primate-human monoclonal antibodies, anti-idiotype antibodies, antibody fragments, bifunctional antibodies, and antibody-associated polypeptides. Antibodies include both bispecific and multispecific antibodies, provided they exhibit the desired biological activity or function.

[0109] As used in this article, the term "tag domain" refers to a tag molecule that can be linked to a scaffold protein via an anchoring portion, wherein the tag molecule can be used for detection purposes. Therefore, for example, tag domains include detectable fluorescent proteins, luciferases, disease marker proteins, or disease marker RNAs.

[0110] As used herein, the terms "active domain" or "bioactive domain" are used interchangeably to refer to an active molecule that can be linked to a scaffold protein via an anchoring motif, wherein the molecule may have therapeutic or preventative effects in subjects in need, or may be used for diagnostic purposes. Thus, for example, the term bioactive domain includes proteins (e.g., antibodies, proteins, peptides, and their derivatives, fragments, and variants), polynucleotide sequences, or chemical compounds.

[0111] As used herein, the term "polynucleotide" refers to a polymer of nucleotides of any length, including ribonucleotides, deoxyribonucleotides, their analogues, or mixtures thereof. This term refers to the primary structure of the molecule. Therefore, the term includes triple-stranded, double-stranded, and single-stranded deoxyribonucleic acid ("DNA"), and triple-stranded, double-stranded, and single-stranded ribonucleic acid ("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 a positive nucleotide backbone, such as polyamides (e.g., peptide nucleic acid "PNA") and polymorpholino polymers; and other synthetic sequence-specific nucleic acid polymers, provided that the polymer contains nucleosides in a configuration that allows base pairing and base stacking, as found in DNA and RNA.

[0112] The terms “polypeptide,” “peptide,” and “protein” are used interchangeably herein to refer to an amino acid polymer of any length. The polymer may contain modified amino acids. The term also covers amino acid polymers that have been modified naturally or through intervention; for example, disulfide bond formation, glycosylation, esterification, acetylation, phosphorylation, or any other manipulation or modification, such as conjugation with a labeled component. The definition also includes, for example, polypeptides containing one or more amino acid analogs (including, for example, non-natural amino acids such as homocysteine, ornithine, p-acetylphenylalanine, D-amino acids, and creatine) and other modifications known in the art.

[0113] As used herein, the term "peptide" refers to proteins, polypeptides, and peptides of any size, structure, or function. Peptides include gene products, naturally occurring polypeptides, synthetic polypeptides, homologs, orthogonal homologs, transverse homologs, fragments and other equivalents, variants, and analogs. Polypeptides can be single polypeptides or multi-molecular complexes, such as dimers, trimers, or tetramers. They can also comprise 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, where 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 in length, for example, about 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 amino acids in length.

[0114] As used herein, the term "linker" refers to a peptide or polypeptide sequence (e.g., a synthetic peptide or polypeptide sequence) or a non-peptide, such as an alkyl chain. In some embodiments, two or more linkers may be linked in tandem. When multiple linkers are present, each linker may be the same or different. Generally, linkers provide flexibility or prevent / improve steric hindrance. Linkers are typically not cleaved; however, in some respects, such cleavage may be desired. Thus, in some embodiments, a linker may contain one or more protease-cleavable sites, which may be located within the linker sequence or flanking either end of the linker sequence.

[0115] In some embodiments, the linker is a peptide linker. In some embodiments, the peptide linker may contain at least about two, at least about three, at least about four, at least about five, or at least about ten amino acids. In some embodiments, the linker contains one or more amino acids. In some embodiments, the linker includes a Gly-Ser (GS) linker. In some embodiments, the GS linker includes (G4S)n, where n is an integer between 1 and 10. In some embodiments, the GS linker includes (G3S)n, where n is an integer between 1 and 10.

[0116] As used herein, the term "substitution" or "replacement" of amino acids can refer to the substitution of a conserved amino acid residue, wherein the amino acid residue is replaced by an amino acid residue having a similar side chain. Families of amino acid residues with 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), β-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Therefore, if an amino acid in a polypeptide is replaced by another amino acid from the same side chain family, such substitution is considered conserved. In another respect, a string of amino acids can be conservatively replaced by a structurally similar string that differs in the order and / or composition of its side chain family members.

[0117] The "sequence identity percentage" or "identity percentage" between two polynucleotide or polypeptide sequences refers to the number of identical matching positions shared by sequences within a comparison window, taking into account additions or deletions (i.e., vacancies) that must be introduced for optimal alignment of the two sequences. A matching position is any location where the same nucleotide or amino acid is present in both the target and reference sequences. Vacancies are not nucleotides or amino acids and are not counted in the target sequence. Similarly, vacancies in the reference sequence are not counted because nucleotides or amino acids from the target sequence are included, but those from the reference sequence are excluded.

[0118] The percentage of sequence identity can be calculated as follows: determine the number of positions in both sequences where the same amino acid residue or nucleic acid base appears (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 percentage of sequence identity. Sequence comparison and determination of the percentage of sequence identity between two sequences can be accomplished using software that is readily available online and downloadable. Suitable software programs are available from various sources for protein and nucleotide sequence alignment. A suitable program for determining the percentage of sequence identity is bl2seq, which is part of the BLAST program suite available from the National Center for Biotechnology Information (NCBI) website (blast.ncbi.nlm.nih.gov). Bl2seq uses either the BLASTN or BLASTP algorithm for comparing 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, which are part of the EMBOSS suite of bioinformatics programs and are also available from the European Institute of Bioinformatics (EBI) at www.ebi.ac.uk / Tools / psa.

[0119] The following embodiments and accompanying drawings are provided to aid in understanding the present invention. However, it should be understood that these embodiments and drawings are for illustrative purposes only and do not constitute any limitation. The actual scope of protection of the present invention is set forth in the claims. It should be understood that any modifications and changes can be made without departing from the spirit of the present invention.

[0120] Unless otherwise specified, the technical means used in the embodiments are conventional means and commercially available instruments and reagents well known to those skilled in the art. 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. Construction of expression vector

[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 the expression plasmid pcDNA3.1(+)-scaffold protein-EGFP (SEQ ID NO:6)-Nanoluc (SEQ ID NO:7).

[0123] Example 2. Preparation and Validation of Exosomes

[0124] Exosomes expressing EGFP-Nanoluc fusion proteins with scaffolds NGFR, BASP-1, PTGFRN, CD63, and MFGE8, as well as native Expi 293F exosomes (named NGFR-293Evs, BASP-1-293Evs, PTGFRN-293Evs, CD63-293Evs, MFGE8-293Evs, and 293Evs), were prepared.

[0125] 2.1 293s cell passage

[0126] Expi 293F cell resuscitation density 3-10×10 5 Cells / ml, with cell density controlled at 0.5-5 × 10⁶ cells / ml during passage. 6 Cells / ml

[0127] 2.2 Cell transfection

[0128] Before transfection, adjust the cell passages from step 2.1 to 1–3 × 10⁶ cells. 6The plasmid concentration was 1–1.5 μg / ml, and the ratio of polyethyleneimine (PEI) to plasmid was controlled at (1:1)–(4:1). The five plasmids obtained in Example 1 were transfected into 293S cells under the above conditions to overexpress the proteins NGFR, BASP-1, PTGFRN, CD63, and MFGE8. Cells were cultured at 37°C with 5% CO2 for 48–72 h, and the cell culture supernatant was collected for downstream purification.

[0129] 2.3 Exosome purification

[0130] Using the cell culture supernatant collected in step 2.2, the exosome sample was obtained by ultrafiltration, density gradient centrifugation, and molecular sieve filtration, and finally, it was used for nanoflow cytometry analysis.

[0131] 2.4 Exosome quality control

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

[0133] In addition, the expression of exosomal markers CD81 and TSG101, as well as Golgi apparatus marker GM130 and endoplasmic reticulum marker Calexin, derived from various cell groups, was detected by Western blotting. The results are as follows: Figure 2 As shown.

[0134] The purity of exosomes was analyzed using a SEC-1000 column (7.8 mm × 150 mm, 7 μm; Thermo Fisher Scientific) combined 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. UV absorbance was measured at 280 nm. Purity (HPLC) characterization results are as follows. 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 no expression of GM130 and Calexin was detected, further indicating that the obtained exosomes were of high purity.

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

[0137] 3.1 Comparison of exosome scaffold distribution characterization

[0138] The proportion of EGFP-positive particles in 20 μL of exosomes from different scaffold proteins and the negative control group was detected using Nano-flow. Figure 5 As shown. Figure 5 The results showed that the NGFR-positive exosome subpopulation accounted for more than 50% of the total exosome distribution, which is higher than that of the currently publicly available universal exosome scaffold CD63.

[0139] 3.2 Comparison of Exosome Scaffold Abundance Characterization

[0140] use Nanolucase activity in exosomes with the same particle number from different scaffold proteins and the negative control group was detected using luciferase reagent, thereby characterizing the abundance of fusion-expressed scaffolds in exosomes with the same particle number. Figure 6 As shown. Figure 6 The results showed that the abundance of NGFR scaffolds was greater than that of CD63 scaffolds.

[0141] Example 4. A novel scaffold protein for localizing and loading exosomes: truncated protein

[0142] To determine the shortest sequence for NGFR to load exogenous substances, this embodiment divides NGFR according to its protein structure, based on the transmembrane domains inside and outside the membrane, and gradually reduces the number of domains outside and inside the membrane. Finally, amino acids 200-338 were named truncated NGFR (SEQ ID NO:8), which is the shortest sequence for exosome localization and loading.

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

[0144] 4.1 Construction and preparation of expression vectors

[0145] (1.1) Carrier Construction

[0146] The C-terminus of truncated-NGFR was fused with EGFP-Nanoluc for expression, and the validation expression vector pcDNA3.1-truncated-NGFR-EGFP-Nanoluc was constructed.

[0147] (1.2) Plasmid preparation

[0148] Plasmid preparation is 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] 4.2 Verification of EGFP loading into exosomes using truncated-NGFR-293Evs

[0152] The high-purity exosomes prepared in (1.3) were characterized. Figure 7 a-7d showed that the exosome purity was close to 100%, with a disc-shaped sinus membrane structure and a particle size distribution within the normal range for exosomes. The exosome EGFP positivity rate was approximately 19%, indicating that truncated-NGFR can load EGFP into exosomes while preserving exosome localization and loading functions.

[0153] Example 5. Engineered exosomes as separation standards

[0154] a.Expi293F cell resuscitation culture.

[0155] b. Two hours before transfection, replace the culture medium (OPM-293CD05 Medium, Shanghai OPM) for Expi293F cells and adjust the density to 3×10^6 cells / mL.

[0156] c. Transfect using PEI (Polysciences, #24765-1) reagent according to the instructions.

[0157] pcDNA3.1(+)-6×his-TEV-NGFR-Flag-Gluc plasmid (e.g.) Figure 8 As shown, the TEV enzyme can cleave the TEV protein (used to remove the his tag) into Expi293F cells.

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

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

[0160] f. The purified exosomes were lyophilized, reconstituted, and then subjected to quality control to obtain standard 1.

[0161] Protein concentration (BCA method, #A65453, Thermo Fisher Scientific) and particle number concentration (NanoFCMInc., 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 determined. Figure 9 The results of a-9c showed that the exosome purity 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 for exosomes. The results of exosome marker detection (proteomic analysis, Easy NLC 1200-Q Exactive Orbitrap mass spectrometer (Thermo Fisher Scientific)) are shown in Table 2 below.

[0163] Table 1

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

[0165] Table 2

[0166]

[0167] Example 6. Engineered exosomes as particle count standards

[0168] a.Expi293F cell resuscitation culture.

[0169] b. Two hours before transfection, replace the culture medium of Expi293F cells with fresh medium and adjust the density to 3 × 10^6 cells / mL.

[0170] c. Transfect the pcDNA3.1(+)-6×his-TEV-NGFR-Flag-EGFP plasmid (e.g., using PEI (Polysciences, #24765-1) reagent according to the manufacturer's instructions) Figure 10 (As shown) into Expi293F cells.

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

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

[0173] f. The purified exosomes were lyophilized, and after being reconstituted with DPBS solution, they were subjected to quality control to obtain standard 2.

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

[0175] Table 3

[0176] sample Exosomal protein concentration (μg / mL) Particle number concentration (particles / mL) Standard Product 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 resuscitation culture.

[0181] b. Two hours before transfection, replace the culture medium of Expi293F cells with fresh medium and adjust the density to 3 × 10^6 cells / mL.

[0182] c. Transfect the pcDNA3.1(+)-6×his-TEV-NGFR-Flag-MCP plasmid (e.g., using PEI (Polysciences, #24765-1) reagent according to the manufacturer's instructions) Figure 12 As shown, the MCP sequence is shown in SEQ ID NO:10) and the pcDNA3.1(+)-universal sequence-target gene fragment-MS2 plasmid (as shown in SEQ ID NO:10) are also present. Figure 13As shown, MS2 (SEQ ID NO:11) is not translated into protein; during RNA transcription, it forms a neck loop structure for binding MCP; the universal sequence (SEQ ID NO:12) is a sequence located to the left of the target gene fragment and is transcribed into Expi293F cells. The target gene is a disease-related gene fragment in IVD detection; in this embodiment, the target gene is the PCA3 mRNA fragment (SEQ ID NO:13).

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

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

[0185] f. The purified exosomes were lyophilized, reconstituted, and then subjected to quality control to obtain standard 3.

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

[0187] Table 5

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

[0189] Table 6

[0190]

[0191] g. Take 200 μL of exosome standard 3 and extract RNA from the exosomes using the Qiagen miRNeasy Serum / Plasma Kit (catalog number 217184). Finally, elute with 14 μL of nuclease-free water.

[0192] h. Using TB Premix Ex Taq TMcDNA was obtained by reverse transcription using II (Tli RNase H Plus) (catalog number RR820B) (reverse transcription primer: 5'-TGGGTGATCCTCATGTTTCGATG-3', SEQ ID NO:14).

[0193] After diluting the cDNA 10-fold, 2 μL was used for qPCR using PrimeScript RT Master Mix (Perfect RealTime) (catalog number RR036A). The qPCR primers were: Forward: 5'-TAACAACTCCGCTCCAAGGC-3' (SEQ ID NO: 15), Reverse: 5'-TGGGTGATCCTCATGTTTCGATG-3' (SEQ ID NO: 14), and the qPCR reference plasmid was: pcDNA3.1(+)-target gene sequence-MS2. The copy number of the marker RNA was determined by repeating the test three times. 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 resuscitation and culture (NGFR protein knockout strain). The NGFR protein knockout strain was obtained as follows: NGFR gene knockout lentivirus (L29036, Beyotime) was used to infect Expi293F cells according to the manufacturer's instructions. Positive cells were selected using puromycin and seeded in 96-well plates for monoclonal selection. Two weeks later, the cells were digested and seeded into 24-well plates. After confluence, qPCR was used for identification, and finally, the NGFR knockout cell line was selected.

[0198] b. Two hours before transfection, replace the culture medium of Expi293F cells with fresh medium and adjust the density to 3 × 10^6 cells / mL.

[0199] c. Transfect the pcDNA3.1(+)-6×his-TEV-marker protein-NGFR-Flag plasmid (e.g., using PEI (polysciences, #24765-1) reagent according to the manufacturer's instructions) Figure 15 (As shown) into Expi293F cells. The marker protein is a specific protein on the surface of natural exosomes or exosomes under pathological conditions in the IVD detection sample. In this example, the marker protein is GPC1 protein (SEQ ID NO:16).

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

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

[0202] f. The purified exosomes were lyophilized, reconstituted, and then subjected to quality control to obtain standard 4.

[0203] Characterization was performed using the same method as in Example 5. Protein concentration and particle number concentration are shown in Table 8 below, and exosome morphology, exosome purity, and particle size distribution are shown in Table 8 below. Figure 16 As shown in a-16c, the results indicate that the exosome purity is close to 100%, the morphology is an exosome discoid sinus membrane structure, and the particle size distribution range is within the normal range for exosomes. The results of exosome marker detection are shown in Table 9 below.

[0204] Table 8

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

[0206] Table 9

[0207]

[0208] g. Take 100 μL of exosomes and use the ELISA method (ab155436, Abcam) to detect NGFR protein on the surface of 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 1.62 ng / mL 1.65 ng / mL 1.63 ng / mL 1.63 ng / mL

[0211] Example 9. Used to detect exosome purification efficiency

[0212] Engineered exosomes were used as standards for exosome isolation and purification to assess purification efficiency. This example uses exosome isolation from commonly used 293T cells.

[0213] a. Culture 293T cells in T225 flasks at a seeding density of 60,000 cells / cm². 2 45 mL of culture volume (using exosome-free FBS) was placed in a CO2 incubator for incubation.

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

[0215] c. Use common ultracentrifugation methods to separate and purify exosomes: centrifuge at 300g for 10 min, take the supernatant and centrifuge at 2000g for 10 min, take the supernatant and centrifuge at 10000g for 30 min, take the supernatant and centrifuge at 100000g for 70 min, resuspend the precipitate in PBS and centrifuge at 100000g for 70 min, resuspend the precipitate in 500μL PBS to obtain purified exosomes.

[0216] d. The activity of Glucase in 5 μL of exosome standard (with 45 μL of PBS added to bring the total to 50 μL) and 50 μL of purified exosomes was detected using the Gauss luciferase reporter gene assay kit (Beyotime, #RG021S). Finally, the exosome purification efficiency was calculated.

[0217]

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

[0219] e. Optionally, the His tag content in 5 μL of exosome standard (with 45 μL of PBS added to bring the volume to 50 μL) and 50 μL of purified exosome sample was detected using the His Tag ELISA Detection Kit (GenScript, #L00436), and the exosome purification efficiency was calculated.

[0220]

[0221] The OD450 detection results (3 replicates) of the his protein serially diluted in the ELISA kit are shown in Table 11 below.

[0222] Table 11

[0223]

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

[0225] Table 12

[0226]

[0227] Example 10. Used for detecting exosome particle count

[0228] The application of engineered exosomes as particle count standards for calibrating exosome particle count detection reduces human intervention and improves accuracy. This example uses exosome isolation from commonly used 293T cells.

[0229] a. Culture 293T cells in T225 flasks at a seeding density of 60,000 cells / cm². 2 45 ml culture volume (using FBS with exosomes removed), 3 groups were run in parallel and incubated in a CO2 incubator.

[0230] b. Collect the cell culture supernatant after 72 hours.

[0231] c. Use common ultracentrifugation methods to separate and purify exosomes: centrifuge at 300g for 10 min, take the supernatant and centrifuge at 2000g for 10 min, take the supernatant and centrifuge at 10000g for 30 min, take the supernatant and centrifuge at 100000g for 70 min, resuspend the precipitate in PBS and centrifuge at 100000g for 70 min, resuspend the precipitate in 500μL PBS to obtain purified exosomes.

[0232] d. The particle number concentration of the three exosome samples was determined using a nanoflow cytometer (NanoFCM).

[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 approximately 3.00E+11 particles / mL.

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

[0235] g. The number concentrations of exosomes exhibiting green fluorescence and those without color were detected using nanofluid cytometry (NanoFCM). The number concentrations of the separated exosomes were then calculated using the following formula. The results are shown in Table 13.

[0236]

[0237] Table 13

[0238]

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

[0240] Example 11. Detection of RNA in IVD

[0241] Engineered exosomes are used as RNA detection standards in IVD to quantify the content of target RNA in exosomes from specific sources. For example, based on the mRNA copy numbers of PCA3, SPEDF, and ERG in exosomes from a subject's urine, combined with a specific algorithm, it is possible to assess whether a subject has prostate cancer, which can greatly reduce the risks associated with unnecessary prostate biopsies. This embodiment uses the detection of PCA3 mRNA copy number in exosomes derived from a subject's urine as an example.

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

[0243] b. Use common ultracentrifugation methods to separate and purify exosomes: centrifuge at 300g for 10 min, take the supernatant and centrifuge at 2000g for 10 min, take the supernatant and centrifuge at 10000g for 30 min, take the supernatant and centrifuge at 100000g for 70 min, resuspend the precipitate in PBS and centrifuge at 100000g for 70 min, resuspend the precipitate in 500μL PBS to obtain purified urine exosome samples.

[0244] c. Total RNA was extracted from 200 μL of urinary exosomes using the Qiagen miRNeasy Serum / Plasma Kit (catalog number 217184) and finally eluted with 14 μL of nuclease-free water.

[0245] d. Take 1.4 μL of RNA from each sample and use TB. Premix Ex Taq TM cDNA was obtained by reverse transcription using II (Tli RNase H Plus) (catalog number RR820B) (primer: 5'-AGAGAGGATTGGTAAGCGATGTG-3', SEQ ID NO:17).

[0246] After diluting the e.cDNA 10-fold, 2 μL was used for qPCR with PrimeScript RT Master Mix (Perfect RealTime) (catalog number RR036A) (primer sequences are shown in Table 14 below). The copy number of PCA3 and the fusion fragment (containing the PCA3 fragment and a universal sequence, the universal sequence being located on the pcDNA3.1(+)-target gene fragment-MS2 plasmid) were detected. Figure 13 The copy number of the universal sequence to the left of the target gene fragment.

[0247] Table 14

[0248]

[0249] f. Calculate the PCA3 copy number in 50 mL of urine exosomes according to the formula. The results are shown in Table 15.

[0250]

[0251] Table 15

[0252]

[0253]

[0254] Example 11. Detection of proteins in IVD

[0255] Engineered exosomes are used as exogenous references for protein detection in in vitro diagnostics (IVD) to quantify the content of target proteins in exosomes from specific sources. For example, studies have shown that the proportion of glypican-1 (GPC1) positive exosomes is significantly higher in pancreatic cancer patients compared to healthy individuals, and the serum GPC1 level in exosomes of early-stage pancreatic cancer patients is significantly higher than that in healthy individuals. GPC1-positive exosomes have potential clinical application value as a diagnostic marker for early pancreatic cancer. This example uses 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 a patient at the Fifth Central Hospital of Tianjin 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 from Example 8 (the surface of this standard exosome contains GPC1 and NGFR proteins).

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

[0259] d. The GPC1 protein content in 100 μL of exosomes extracted from c was detected using a GPC1 protein ELISA kit (ab270217, Abcam). The NGFR protein content in 100 μL of exosomes extracted from c and 2 μL of standard exosomes (with 98 μL of PBS added to make up to 100 μL) was detected using an NGFR protein ELISA kit (ab155436, Abcam).

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

[0261]

[0262] Table 16

[0263]

[0264] The technical solutions of the present invention are not limited to the specific embodiments described above. Any technical modifications made in accordance with the technical solutions of the present invention fall within the protection scope of the present invention.

Claims

1. Use of a vesicle as a standard and / or reference, characterized in that, The vesicle comprises: a scaffold protein, which is a nerve growth factor receptor fragment; and a tag domain; The nerve growth factor receptor or fragment thereof is located in a vesicle, which is used as a standard and / or reference for vesicle isolation, purification, vesicle particle number detection, RNA detection in in vitro diagnosis, or protein detection in in vitro detection, The amino acid sequence of the nerve growth factor receptor fragment is shown in SEQ ID NO: 8, The scaffold protein is directly or through a linker combined with the tag domain.

2. Use according to claim 1, characterized in that, The tag domain comprises one or more of a fluorescent protein, a luciferase, a purification tag, a disease marker protein, or a disease marker RNA.

3. Use according to claim 2, characterized in that, The tag domain comprises one or more of a GST (glutathione S-transferase) tag protein, a His6 tag protein (His-tag), a MBP (maltose binding protein) tag protein, a Flag tag protein, a SUMO tag protein, a HA tag protein, a Myc tag protein, an eGFP (enhanced green fluorescent protein), an eCFP (enhanced cyan fluorescent protein), an eYFP (enhanced yellow green fluorescent protein), an mCherry (monomeric red fluorescent protein), a T7 tag, a V5 tag, a cellulose binding domain (CBD) tag, a calmodulin tag, an AviTag tag protein, a Halo Tag tag protein, a SNAP-Tag tag protein, a firefly luciferase (Fluc), a sea pansy luciferase (Rluc), a Guassia luciferase (Gluc), a Nano luciferase (Nluc), a cancer marker protein.

4. Use according to claim 1, characterized in that, The vesicle further comprises an active domain, which is directly or through a linker combined with the scaffold protein.

5. Use according to claim 4, characterized in that, The active domain comprises a polypeptide.

6. Use according to claim 5, characterized in that, 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.

7. Use according to claim 1, characterized in that, The vesicle comprises one or more of an exosome, a microvesicle, an apoptotic body, a tumor vesicle, and a nanovesicle.

8. Use according to claim 1, characterized in that, The concentration of the vesicle is 1x10^12 ~ 3x10^12 particles / mL.

9. The use according to claim 1, characterized in that, The vesicle is used for detecting the purification efficiency of the vesicle, detecting and / or calibrating the particle number of the vesicle, quantifying the content of a target RNA derived from the vesicle, or quantifying the content of a target protein derived from the vesicle.

10. Use according to claim 9, characterized in that, The target RNA or target protein is a disease-related RNA or protein.

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