Conjugated lipid-binding carrier

Abstract

To provide technology for detecting extracellular vesicles with high sensitivity and technology for isolating high-purity extracellular vesicles in large quantities by a simple operation.SOLUTION: There are provided a method for bringing a complex lipid binding carrier including an insoluble carrier formed by immobilizing protein that includes the amino acid sequence of C2 domain of protein kinase into contact with a sample solution that includes extracellular vesicles and obtaining a complex in which the complex lipid binding carrier and the extracellular vesicles are bound together, and a method for treating the complex with a chelate agent to desorb and recover the extracellular vesicles.SELECTED DRAWING: Figure 11

Description

[Technical Field]

[0001] The present invention relates to a complex lipid-binding carrier in which a protein capable of binding to complex lipids is immobilized on an insoluble carrier, and to a method for detecting and isolating complex lipids and extracellular vesicles using the carrier. [Background technology]

[0002] Complex lipids are major components of the lipid bilayer of cell membranes and extracellular vesicles and are broadly classified into phospholipids with phosphate ester bonds and glycolipids with sugars. Phosphatidylserine, a type of phospholipid, is present on the cytoplasmic side of the cell membrane in normal cells, but in extracellular vesicles, phosphatidylserine is known to be present on the membrane surface (Patent Document 1, Non-Patent Document 1). Extracellular vesicles contain nucleic acids such as microRNA and messenger RNA, as well as proteins, and are thought to be involved in intercellular signaling. Therefore, they are attracting attention as biomarkers for various diseases, including cancer, and are also expected to be used as novel therapeutic agents and drug delivery systems (DDS) (Non-Patent Document 2).

[0003] Known techniques for detecting extracellular vesicles include methods that use heat shock proteins (HSP70, HSP90) and tetraspanins (CD9, CD63, CD81) as markers for extracellular vesicles. However, the expression levels of these markers vary depending on the type of cell releasing the extracellular vesicles, resulting in a lack of sensitivity and quantitation (e.g., Non-Patent Document 1). Furthermore, known techniques for isolating extracellular vesicles include ultracentrifugation, ultrafiltration, density gradient centrifugation, polymer precipitation, and immunoprecipitation. However, there is a problem in that no method satisfies all three of the following requirements: (1) highly purified extracellular vesicles can be obtained; (2) large quantities of extracellular vesicles can be obtained inexpensively; and (3) the operation can be performed quickly and easily. In addition to these methods, methods for detecting and isolating extracellular vesicles using annexin V or Tim4 protein, which bind to phosphatidylserine, are known (Patent Document 2, Non-Patent Document 1). However, the detection and recovery rates of annexin V and Tim4 protein need to be improved. [Prior art documents] [Patent documents]

[0004] [Patent Document 1] Japanese Patent Application Publication No. 2019-164145 [Patent Document 2] WO2016 / 088689 [Non-patent literature]

[0005] [Non-Patent Document 1] Japanese Journal of Pharmacology, Vol. 149, No. 3, pp. 119-122, 2017 [Non-patent document 2] Proteomics vol. 13, pp. 1637-1653, 2013 Summary of the Invention [Problem to be solved by the invention]

[0006] An object of the present invention is to provide a technique for detecting extracellular vesicles with high sensitivity and high accuracy, and a technique for isolating highly pure extracellular vesicles in large quantities using simple procedures, in order to develop the industrial use of extracellular vesicles. [Means for solving the problem]

[0007] As a result of intensive research aimed at solving the above-mentioned problems, the present inventors have prepared a composite lipid-binding carrier in which a protein capable of binding to composite lipids is immobilized on an insoluble carrier, and have found that extracellular vesicles can be detected with high sensitivity and accuracy by contacting the carrier with a biological substance-containing sample solution containing extracellular vesicles to obtain a complex in which the composite lipid-binding carrier and the extracellular vesicles are bound.Furthermore, the present inventors have found that large amounts of highly pure extracellular vesicles can be isolated with a simple procedure by adding a buffer solution containing a chelating agent to the complex to detach the extracellular vesicles from the composite lipid-binding carrier, thereby completing the present invention.

[0008] That is, the present invention provides the inventions described in [1] to

[19] below.

[0009] [1] A complex lipid-binding carrier comprising an insoluble carrier on which a protein containing the amino acid sequence of the C2 domain of a protein kinase is immobilized.

[0010] [2] The complex lipid-binding carrier according to [1], wherein the protein comprising the amino acid sequence of the C2 domain of a protein kinase is any one of the proteins (a) to (d) below: (a) A protein comprising the amino acid sequence shown in SEQ ID NO: 1 and having binding ability to phospholipids and / or extracellular vesicles. (b) A protein having an amino acid sequence in which the amino acid sequence shown in SEQ ID NO: 4 is added to the N-terminal and / or C-terminal sides of the amino acid sequence of the protein (a), and having binding ability to phospholipids and / or extracellular vesicles. (c) A protein having an amino acid sequence in which one or more amino acids are deleted, substituted, inserted, or added in the amino acid sequence of the protein described in (a) or (b), and having binding ability to phospholipids and / or extracellular vesicles. (d) A complex of a protein described in any one of (a) to (c) above with an immobilization assisting protein capable of binding to an insoluble carrier, and a protein having binding ability to phospholipids and / or extracellular vesicles.

[0011] [3] The complex lipid-binding carrier according to [1], wherein the protein comprising the amino acid sequence of the C2 domain of a protein kinase is any one of the proteins (e) to (h) below: (e) A protein comprising the amino acid sequence shown in SEQ ID NO: 2 and having binding ability to phospholipids and / or extracellular vesicles. (f) A protein having an amino acid sequence of the protein (e) in which the amino acid sequence shown in SEQ ID NO: 4 is added to the N-terminal and / or C-terminal sides, and which has binding ability to phospholipids and / or extracellular vesicles. (g) A protein having an amino acid sequence in which one or more amino acids are deleted, substituted, inserted, or added in the amino acid sequence of the protein (e) or (f), and having binding ability to phospholipids and / or extracellular vesicles. (h) A complex of a protein described in any one of (e) to (g) above with an immobilization assisting protein capable of binding to an insoluble carrier, and a protein having binding ability to phospholipids and / or extracellular vesicles.

[0012] [4] The complex lipid-binding carrier according to [1], wherein the protein comprising the amino acid sequence of the C2 domain of a protein kinase is any one of the proteins (i) to (l) below: (i) A protein comprising the amino acid sequence shown in SEQ ID NO: 3 and having binding ability to phospholipids and / or extracellular vesicles. (j) A protein having an amino acid sequence in which the amino acid sequence shown in SEQ ID NO: 4 is added to the N-terminal and / or C-terminal sides of the amino acid sequence of the protein (i), and having binding ability to phospholipids and / or extracellular vesicles. (k) A protein having an amino acid sequence in which one or more amino acids are deleted, substituted, inserted, or added in the amino acid sequence of the protein (i) or (j), and having binding ability to phospholipids and / or extracellular vesicles. (l) A complex of a protein described in any one of (i) to (k) above with an immobilization assisting protein capable of binding to an insoluble carrier, and a protein having binding ability to phospholipids and / or extracellular vesicles.

[0013] [5] A complex lipid-binding carrier according to any one of [1] to [4], wherein the complex lipid is a phospholipid.

[0014] [6] The composite lipid-binding carrier according to [5], characterized in that the phospholipid is a phospholipid containing phosphatidylserine as a component.

[0015] [7] The complex lipid-binding carrier according to any one of [2] to [6], wherein the extracellular vesicles are extracellular vesicles derived from human cells.

[0016] [8] A detection reagent comprising the composite lipid-binding carrier according to any one of [1] to [7] above.

[0017] [9] A kit for detecting extracellular vesicles, comprising the detection reagent described in [8] above.

[0018]

[10] A column packed with the composite lipid-binding carrier according to any one of [1] to [7] above.

[0019]

[11] A kit for isolating extracellular vesicles, the kit comprising the column described in

[10] .

[0020]

[12] A method for producing a composite lipid-binding carrier according to any one of [1] to [7], comprising the following steps (A1) and (A2): (A1) A step of producing a reactive insoluble carrier from an insoluble carrier. (A2) Adding any one of [1] to [4] to the reactive insoluble carrier obtained in the step (A1). A step of immobilizing the protein described in any one of the above.

[0021]

[13] A method for producing a composite lipid-binding carrier according to any one of [1] to [7], characterized in that it includes a step of immobilizing a protein according to any one of (a) to (l) on an insoluble carrier by physical adsorption.

[0022]

[14] A method for detecting phospholipids and / or extracellular vesicles, comprising the following steps (X1) to (X3): (X1) A process of contacting a composite lipid-binding carrier according to any one of [1] to [7] above with a sample solution containing phospholipids and / or extracellular vesicles in the presence of calcium ions to obtain a complex in which the carrier is bound to phospholipids and / or extracellular vesicles. (X2) A step of separating and removing substances that did not bind to the complex lipid-binding carrier from the complex obtained in the step (X1). (X3) A step of detecting the complex obtained in the step (X1) after the step (X2) is completed.

[0023]

[15] The detection method according to

[14] above, characterized in that ELISA or flow cytometry is used as the detection method.

[0024]

[16] A method for isolating phospholipids and / or extracellular vesicles, comprising the following steps (Y1) to (Y3): (Y1) A process of contacting a composite lipid-binding carrier described in any one of [1] to [7] above with a sample solution containing phospholipids and / or extracellular vesicles in the presence of calcium ions to obtain a complex in which the carrier is bound to phospholipids and / or extracellular vesicles. (Y2) A step of separating and removing substances that did not bind to the composite lipid-binding carrier from the complex obtained in the step (Y1). (Y3) After completion of the step (Y2), a step of detaching phospholipids and / or extracellular vesicles from the complex obtained in the step (Y1) and recovering the phospholipids and / or extracellular vesicles.

[0025]

[17] The isolation method according to

[16] above, wherein step (Y3) is carried out using a calcium ion chelating agent.

[0026]

[18] The detection method according to

[14] or

[15] above, characterized in that a detection reagent containing a composite lipid-binding carrier is used.

[0027]

[19] The isolation method according to

[16] or

[17] above, characterized in that a column packed with a composite lipid-binding carrier is used.

[0028] The present invention will be described in further detail below.

[0029] The conjugated lipid-binding carrier of the present invention is characterized in that a protein containing the amino acid sequence of the C2 domain of protein kinase C (hereinafter sometimes abbreviated as PKC) is immobilized on an insoluble carrier.

[0030] The protein used in the complex lipid-binding carrier of the present invention (hereinafter sometimes abbreviated as complex lipid-binding protein) is a protein containing the amino acid sequence of the C2 domain of PKC, more specifically, the protein expressed in transformed Escherichia coli. The C2 domain of PKC is known to bind to complex lipids, particularly phosphatidylserine, a major component of phospholipids (e.g., Biochem Soc Trans. Vol. 42, pp. 1471-1476, 2014), and phosphatidylserine is known to be present on the outside of extracellular vesicles. Furthermore, as shown in the Reference Examples below, recombinant proteins containing the amino acid sequence of the C2 domain of PKC expressed in transformed Escherichia coli have been shown to bind to complex lipids such as phosphatidylserine, phosphatidylinositol 4-phosphate, phosphatidylinositol 4,5-bisphosphate, and sulfatide. Therefore, the complex lipid-binding carrier of the present invention can bind to phospholipids and / or extracellular vesicles by immobilizing the complex lipid-binding protein on an insoluble carrier without impairing its binding ability to complex lipids.

[0031] The term "complex lipid" as used herein refers to a lipid bilayer membrane derived from eukaryotic cells and extracellular vesicles secreted therefrom. Extracellular vesicles are membrane vesicles composed of a lipid bilayer membrane and having a diameter of 20 to 1000 nm, preferably 50 to 500 nm. Specific examples include exosomes, microvesicles, ectosomes, exosome-like vesicles, apoptotic vesicles, and adiposomes. Exosomes are composed of a lipid bilayer membrane derived from late endosomes, have phosphatidylserine on the membrane surface, contain proteins such as tetraspanins (CD63 and CD9), and have a diameter of 50 to 200 nm. Microvesicles are composed of a lipid bilayer membrane derived from cell membranes, have phosphatidylserine on the membrane surface, contain proteins such as integrins, selectins, and CD40 ligand, and have a diameter of 100 to 800 nm. Ectosomes are composed of a lipid bilayer membrane derived from cell membranes, have phosphatidylserine on the membrane surface, contain CR1 and protease enzymes but do not contain the CD63 contained in exosomes, and have a diameter of 50 to 150 nm. Exosome-like vesicles are composed of a lipid bilayer membrane derived from early endosomes, have phosphatidylserine on the membrane surface, contain TNFR1 (Tumor Necrosis Factor Receptor 1), and have a diameter of 20 to 50 nm. Apoptotic vesicles are composed of a lipid bilayer membrane derived from apoptotic cells, have phosphatidylserine on the membrane surface, contain histones, and have a diameter of 50 to 300 nm. Adiposomes are composed of a lipid bilayer membrane derived from adipocytes, have phosphatidylserine on the membrane surface, contain MFG-E8 (Milk Fat Globule-EGF-factor 8), and have a diameter of 100 to 800 nm. As disclosed in a literature (Proteomics, Vol. 13, pp. 1637-1653, 2013), these extracellular vesicles have attracted attention as biomarkers for various diseases, including cancer. Therefore, when the extracellular vesicles are derived from human cells, the composite lipid-binding carrier of the present invention can be suitably used for the diagnosis of various diseases.

[0032] Examples of the PKC C2 domain include the amino acid sequence constituting the C2 domain of PKCα, which has calcium ion-dependent binding to complex lipids (the amino acid sequence of the region from positions 155 to 293 of UniProt Registration No. P17252 shown in SEQ ID NO: 1; hereinafter, sometimes abbreviated as PKCαC2), the amino acid sequence constituting the C2 domain of PKCβ (the amino acid sequence of the region from positions 153 to 293 of UniProt Registration No. P05771 shown in SEQ ID NO: 2; hereinafter, sometimes abbreviated as PKCβC2), and the amino acid sequence constituting the C2 domain of PKCγ (the amino acid sequence of the region from positions 153 to 293 of UniProt Registration No. P05129 shown in SEQ ID NO: 3; hereinafter, sometimes abbreviated as PKCγC2), as well as the C2 domain of PKCε, the C2 domain of PKCη, the C2 domain of PKCδ, and the C2 domain of PKCθ, which have calcium ion-independent binding to complex lipids. Among these PKC C2 domains, PKCαC2, PKCβC2 and PKCγC2 are preferred in that the adsorption and desorption of phospholipids and / or extracellular vesicles to the composite lipid-binding carrier of the present invention can be controlled by adding and capturing calcium ions.

[0033] As long as the complex lipid-binding protein has the ability to bind to phospholipids and / or extracellular vesicles, it may contain two or more amino acid sequences of the C2 domains of PKCα, β, and γ shown in SEQ ID NOs: 1 to 3. When the complex lipid-binding protein contains multiple C2 domains, the C2 domains may be from the same type of PKC or different types of PKC, as long as they have the ability to bind to phospholipids and / or extracellular vesicles. The number of C2 domains is not particularly limited; two or more domains are preferred if the ability to bind to complex lipids and extracellular vesicles is improved, but 10 or fewer domains are preferred so as not to reduce the expression ability in transformed E. coli.

[0034] The binding ability of a multi-lipid-binding protein to phospholipids and / or extracellular vesicles can be improved by adding an amino acid sequence of a protein other than the PKC C2 domain, specifically, the amino acid sequence of glutathione S-transferase (hereinafter sometimes abbreviated as GST) shown in SEQ ID NO: 4 (the amino acid sequence from positions 3 to 218 of UniProt registration number P08515), to the N- and / or C-terminus of each amino acid sequence of the C2 domains of PKCα, β, and γ shown in SEQ ID NOs: 1 to 3. The positional relationship of the PKC C2 domain and GST in the amino acid sequence is not particularly limited as long as they have the ability to bind to phospholipids and / or extracellular vesicles. For example, the PKC C2 domain may be located at the N-terminus and GST at the C-terminus, or the N-terminus may be GST and the PKC C2 domain at the C-terminus. However, a PKC C2 domain located at the N-terminus and GST at the C-terminus is preferred due to its high binding ability to phospholipids and / or extracellular vesicles. Furthermore, the complex lipid-binding protein may contain the GST amino acid sequence shown in SEQ ID NO: 4 at multiple positions; from the viewpoints of binding to complex lipids and extracellular vesicles and expressibility in transformed E. coli, one to three positions are preferred, and one position is more preferred. Furthermore, as long as the complex lipid-binding protein has the ability to bind to phospholipids and / or extracellular vesicles, the amino acid sequence of the PKC C2 domain and the GST amino acid sequence may be directly linked, or one to several linker amino acid sequences may be included. There are no particular restrictions on the number of amino acid residues contained in the linker amino acid sequence; however, a large number of amino acid residues reduces the effect of improving binding to phospholipids and / or extracellular vesicles by the addition of the GST amino acid sequence and also reduces the expressibility of the complex lipid-binding protein in transformed E. coli. Therefore, the number of amino acid residues contained in the linker amino acid sequence is preferably 1 to 50, more preferably 10 to 30.

[0035] The complex lipid-binding protein may have one or more amino acid residues deleted, substituted, inserted, or added in an amino acid sequence containing each of the amino acid sequences of the C2 domains of PKCα, β, and γ shown in SEQ ID NOs: 1 to 3, or an amino acid sequence containing each of the amino acid sequences of the C2 domains of PKCα, β, and γ shown in SEQ ID NOs: 1 to 3 and the amino acid sequence of GST shown in SEQ ID NO: 4, so long as it has binding ability to phospholipids and / or extracellular vesicles. The number of residues is not particularly limited, but 20 or fewer are preferred, 10 or fewer are more preferred, and 5 or fewer are even more preferred, so as not to lose binding ability to complex lipids and extracellular vesicles. The deletion, substitution, insertion, or addition of amino acid residues can be performed using genetic engineering methods well known to those skilled in the art.

[0036] The complex lipid-binding protein may be a complex with a non-complex lipid-binding protein, as long as it has the ability to bind to phospholipids and / or extracellular vesicles. There are no restrictions on the number or types of proteins bound to the complex lipid-binding protein, and the complex may be a complex with two or more types of non-complex lipid-binding proteins.

[0037] The non-complex lipid-binding protein constituting the complex is preferably an immobilization assisting protein, which controls the density and orientation of the PKC C2 domain and optimizes the binding of the PKC C2 domain to extracellular vesicles. Immobilization assisting proteins having a trimeric or nonameric structure are even more preferred because they improve affinity due to the multivalent effect of the PKC C2 domain and are easy to prepare. Specific examples of immobilization assisting proteins include proteins consisting of the amino acid sequences set forth in SEQ ID NO: 33 and SEQ ID NO: 37.

[0038] Furthermore, as long as the complex lipid-binding protein has the ability to bind to phospholipids and / or extracellular vesicles, it may contain an additional amino acid sequence other than the amino acid sequence of the GST described above in addition to the amino acid sequence of the C2 domain of PKCα, β, and γ shown in SEQ ID NOs: 1 to 3. Specifically, the N-terminus and / or C-terminus of the amino acid sequence of the C2 domain may be added with an amino acid sequence of a tag for separation and purification useful in producing the complex lipid-binding protein, an amino acid sequence of a tag for carrier immobilization useful in preparing a complex lipid-binding carrier, or a polypeptide useful for carrier immobilization.

[0039] Examples of these tags for separation and purification and tags for carrier immobilization include maltose-binding protein (MBP), cellulose-binding domain (CBD), myc tag, FLAG tag, oligopeptides containing a polyhistidine sequence (polyhistidine tag), oligopeptides containing cysteine ​​residues, oligopeptides containing lysine residues, and oligopeptides containing both a polyhistidine sequence and cysteine ​​residues. Among these additional amino acid sequences, polyhistidine tags and oligopeptides containing both a polyhistidine sequence and cysteine ​​residues are preferred in terms of facilitating separation and purification, while oligopeptides containing cysteine ​​or oligopeptides containing both a polyhistidine sequence and cysteine ​​residues are preferred in terms of being able to immobilize complex lipid-binding proteins to insoluble carriers without impairing their binding to complex lipids, and oligopeptides containing both a polyhistidine sequence and cysteine ​​residues are more preferred. The number of histidine repeats in an oligopeptide containing both a polyhistidine sequence and cysteine ​​residues is preferably a repeat sequence consisting of 5 to 15 histidine residues, and more preferably a repeat sequence consisting of 5 to 10 histidine residues, in order to facilitate purification by nickel chelate affinity chromatography.

[0040] Furthermore, the number of cysteine ​​residues is preferably 1 to 5, and more preferably 1 or 2, cysteine ​​residues, in order to enable highly selective and efficient immobilization to an insoluble carrier. The length of the oligopeptide containing both the polyhistidine sequence and cysteine ​​residues is not particularly limited, as long as it contains the histidine repeat sequence and cysteine ​​residues and does not lose its binding ability to phospholipids and / or extracellular vesicles. Specific examples include oligopeptides consisting of the amino acid sequences set forth in SEQ ID NO: 39 and SEQ ID NO: 40. Furthermore, the position at which the oligopeptide containing both the polyhistidine sequence and cysteine ​​residues is added to the complex lipid-binding protein is not particularly limited, and it may be added to both the N-terminus and the C-terminus, or to either the N-terminus or the C-terminus. However, addition to the C-terminus is preferred in order to enable efficient purification by nickel chelate affinity chromatography and immobilization to an insoluble carrier.

[0041] Examples of polypeptides useful for carrier immobilization include cysteine ​​or lysine residues exposed on the surface of protein molecules, and the protein tags SpyTag and SpyCatcher, which form covalent bonds. Among these, cysteine ​​residues, which generally occur at low frequency in the amino acid sequence of proteins and whose binding site is easily controlled, and SpyTag and SpyCatcher, which have sequences not present in natural proteins, are preferred in terms of being suitable for orientation-controlled immobilization and optimizing binding to extracellular vesicles. SpyTag is more preferred in terms of creating a multimeric structure by binding to the aforementioned immobilization assistant protein, thereby optimizing binding to extracellular vesicles.

[0042] Furthermore, a signal peptide may be added to the N-terminus of the protein used in the adsorbent of the present invention to promote efficient expression in the host. When the host is Escherichia coli, examples of such signal peptides include signal peptides that direct protein secretion into the periplasm, such as PelB, DsbA, MalE, and TorT.

[0043] The binding ability of complex lipid-binding proteins to complex lipids can be evaluated by methods well known to those skilled in the art, such as enzyme-linked immunosorbent assay (hereinafter sometimes abbreviated as ELISA) and flow cytometry. In addition, the binding ability of complex lipid-binding proteins to multiple complex lipids can be evaluated at once by using a membrane lipid strip (P-6002, manufactured by Echelon Bioscience) described in the literature (Immunity, Vol. 27, pp. 927-940, 2007).

[0044] Specific examples of complex lipid-binding proteins include: SEQ ID NO: 5 (the 3rd to 141st positions correspond to the amino acid sequence of SEQ ID NO: 1, and the 155th to 160th positions correspond to a polyhistidine sequence), SEQ ID NO: 7 (positions 3 to 141 correspond to the amino acid sequence of SEQ ID NO: 1, positions 142 to 153 correspond to the sequence derived from the multicloning site of the expression vector pET28a(+), positions 155 to 370 correspond to the amino acid sequence of SEQ ID NO: 4, and positions 373 to 378 correspond to a polyhistidine sequence), SEQ ID NO: 9 (positions 3 to 141 correspond to the amino acid sequence of SEQ ID NO: 1, positions 142 to 179 correspond to the amino acid sequence downstream of the C2 domain of protein kinase α (the amino acid sequence of the region from positions 294 to 331 of Uniprot accession number P17252), positions 193 to 408 correspond to the amino acid sequence of SEQ ID NO: 4, and positions 411 to 416 correspond to a polyhistidine sequence), SEQ ID NO: 11 (positions 3 to 141 correspond to the amino acid sequence of SEQ ID NO: 1, positions 146 to 159 correspond to a GS linker (SEQ ID NO: 41), positions 166 to 381 correspond to the amino acid sequence of SEQ ID NO: 4, and positions 384 to 389 correspond to a polyhistidine sequence), SEQ ID NO: 13 (positions 3 to 141 correspond to the amino acid sequence of SEQ ID NO: 1, positions 152 to 165 correspond to an α-helix linker (SEQ ID NO: 42), positions 171 to 386 correspond to the amino acid sequence of SEQ ID NO: 4, and positions 389 to 394 correspond to a polyhistidine sequence), SEQ ID NO: 15 (positions 4 to 219 correspond to the amino acid sequence of SEQ ID NO: 4, positions 222 to 235 correspond to a GS linker (SEQ ID NO: 41), positions 241 to 379 correspond to the amino acid sequence of SEQ ID NO: 1, and positions 382 to 387 correspond to a polyhistidine sequence), SEQ ID NO: 17 (the 2nd to 142nd positions correspond to the amino acid sequence of SEQ ID NO: 2, and the 156th to 161st positions correspond to a polyhistidine sequence), SEQ ID NO: 19 (the 2nd to 142nd positions correspond to the amino acid sequence of SEQ ID NO: 2, the 147th to 160th positions correspond to a GS linker (SEQ ID NO: 41), the 167th to 382nd positions correspond to the amino acid sequence of SEQ ID NO: 4, and the 385th to 390th positions correspond to a polyhistidine sequence), SEQ ID NO: 21 (the 2nd to 142nd positions correspond to the amino acid sequence of SEQ ID NO: 3, and the 156th to 161st positions correspond to a polyhistidine sequence), SEQ ID NO: 23 (the 2nd to 142nd positions correspond to the amino acid sequence of SEQ ID NO: 3, the 143rd to 154th positions correspond to the sequence derived from the multicloning site of the expression vector pET28a(+), the 156th to 371st positions correspond to the amino acid sequence of SEQ ID NO: 4, and the 374th to 379th positions correspond to a polyhistidine sequence), and SEQ ID NO: 31 (wherein the 3rd to 141st positions correspond to the amino acid sequence of SEQ ID NO: 1, the 146th to 159th positions correspond to a GS linker (SEQ ID NO: 41), the 164th to 279th positions correspond to SpyCatcher (116 amino acid residues of Chain A and B of Protein Data Bank registration number 4MLI), and the 282nd to 287th positions correspond to a polyhistidine sequence).

[0045] Of these complex lipid-binding proteins, the complex lipid-binding proteins consisting of the amino acid sequences shown in SEQ ID NO: 11 and SEQ ID NO: 15 are preferred because of their high extracellular vesicle detection performance, as shown in the Examples below, and the complex lipid-binding protein consisting of the amino acid sequence shown in SEQ ID NO: 31 is preferred because it can concentrate extracellular vesicles contained in cell culture supernatants.

[0046] There are no particular limitations on the method for producing complex lipid-binding proteins, and the method may be carried out by a method commonly used by those skilled in the art, such as that disclosed in JP 2018-000038 A. Specifically, a process of culturing host cells transformed with an expression plasmid containing a polynucleotide encoding a complex lipid-binding protein to obtain a culture (including the cultured transformant itself, secretions, and the medium used for culture), centrifugation of the obtained culture to obtain host cells, suspending the cells in an appropriate buffer, disrupting the cells by physical disruption with ultrasound or disruption with a surfactant or other agent, and then removing the disrupted residue by centrifugation to obtain a soluble protein extract containing the complex lipid-binding protein, and purifying the obtained soluble protein extract by liquid chromatography such as ion exchange chromatography, hydrophobic interaction chromatography, gel filtration chromatography, or affinity chromatography, thereby producing the complex lipid-binding protein of interest. Examples of hosts to be transformed with an expression plasmid containing a polynucleotide encoding the complex lipid-binding protein include animal cells such as COS cells and CHO (Chinese Hamster Ovary) cells, bacteria such as bacteria of the genus Bacillus (including bacteria of the genus Bacillus in a broad sense, such as bacteria of the genus Brevibacillus and Paenibacillus) and Escherichia coli, yeasts such as bacteria of the genus Saccharomyces, Pichia, and Schizosaccharomyces, and filamentous fungi such as Aspergillus oryzae, but Escherichia coli is preferably used as the host because it is easy to handle. Furthermore, when disrupting host cells with a chemical such as a surfactant, the cells may be disrupted using, for example, the method disclosed in JP 2013-252099 A or a commercially available extraction reagent such as BugBuster Protein extraction kit (manufactured by Merck).

[0047] The insoluble carrier used for the composite lipid-binding carrier of the present invention (hereinafter, sometimes abbreviated as the insoluble carrier of the present invention) may be appropriately selected depending on the detection method and isolation method of phospholipids and / or extracellular vesicles of the present invention described below (hereinafter, sometimes abbreviated as the detection method of the present invention and the isolation method of the present invention), and examples thereof include inorganic carriers such as silica gel, porous glass, non-porous glass with a thin gold film deposited thereon, magnetic materials such as alumina, iron, cobalt, nickel, magnetite, and chromite, and metal oxides; polysaccharide carriers such as agarose, cellulose, chitin, chitosan, dextran, pullulan, starch, alginate, and carrageenan; and polysaccharide carriers crosslinked with polysaccharide carriers. Examples include cross-linked polysaccharide carriers cross-linked with a cross-linking agent, polystyrene, poly(meth)acrylate, polyolefins including polyethylene and polypropylene, polyesters including polyethylene terephthalate, polyacrylamide, polyimide, polyvinyl alcohol, polyurethane, polyvinyl chloride, polycarbonate, and other synthetic polymer carriers; cross-linked synthetic polymer carriers obtained by cross-linking synthetic polymer carriers with a cross-linking agent; and hydrophilic surface-modified synthetic polymer carriers obtained by modifying the surface of these with hydrophilic polymers such as dextran, pullulan, and polyvinyl alcohol, and hydrophilic surface-modified cross-linked synthetic polymer carriers obtained by cross-linking these with a cross-linking agent. When immobilizing complex lipid-binding proteins to a carrier by physical adsorption, as described below, synthetic polymer carriers and cross-linked synthetic polymer carriers are preferred because they allow for efficient immobilization of complex lipid-binding proteins. Furthermore, when nonspecific adsorption of complex lipids or extracellular vesicles to the carrier due to hydrophobic interactions adversely affects the detection and isolation of phospholipids and / or extracellular vesicles, polysaccharide carriers, cross-linked polysaccharide carriers, hydrophilic surface-modified synthetic polymer carriers, and hydrophilic surface-modified cross-linked synthetic polymer carriers are preferred.

[0048] The shape of the composite lipid-binding carrier of the present invention may be appropriately selected from among particulate, flat, sponge-like, flat membrane-like, hollow, and fibrous shapes, depending on the detection and isolation methods of phospholipids and / or extracellular vesicles of the present invention described below. When the composite lipid-binding carrier of the present invention is used in the detection method of the present invention, magnetic particulate carriers (hereinafter sometimes referred to as magnetic microparticles) or flat carriers used in conventional immunoassays may be used. The particle size of the magnetic microparticles may be appropriately selected from the range of 0.01 μm to 500 μm, with 0.1 μm to 10 μm being preferred for ease of availability. Furthermore, the number of holes (wells) in the flat carrier may be appropriately selected from the range of 6 to 1536 wells, with 24 to 384 wells being preferred for ease of availability. When the composite lipid-binding carrier of the present invention is used in the isolation method of the present invention, it is preferable to use a particulate carrier that is used in the separation and purification of biological samples such as ordinary lipids and proteins, because isolation can be performed efficiently with simple operations, and the particle size may be appropriately selected from the range of 1 μm to 1000 μm. When used to isolate extracellular vesicles, the particle size is preferably 10 μm to 500 μm, more preferably 20 μm to 200 μm, so that extracellular vesicles can be in sufficient contact with the carrier and extracellular vesicles that are not bound to the carrier can pass through the gaps in the carrier without clogging.

[0049] The method for producing a composite lipid-binding carrier of the present invention is characterized by comprising the following steps (A1) and (A2): (A1) A step of producing a reactive insoluble carrier from an insoluble carrier. (A2) A step of immobilizing the protein according to any one of [1] to [4] above on the reactive insoluble carrier obtained in the step (A1).

[0050] Step (A1) and step (A2) will be described below.

[0051] Step (A1) is a step of introducing a reactive functional group for immobilizing a complex lipid-binding protein onto an insoluble carrier. The reactive functional group is not particularly limited as long as it is a functional group commonly used for protein immobilization, and examples thereof include an epoxy group, a formyl group, a carboxy group, an active ester group, an amino group, a maleimide group, a haloacetyl group, and a mercapto group. Furthermore, the method for introducing the reactive functional group into the carrier is not particularly limited as long as it is a commonly used method for introducing a functional group.

[0052] An example of a method for introducing epoxy groups into a support is a method in which hydroxy groups on the surface of the support are reacted under basic conditions with an epoxy group-containing compound such as diglycidyl ethers (e.g., epichlorohydrin, ethylene glycol diglycidyl ether, 1,4-butanediol diglycidyl ether, 1,6-hexanediol diglycidyl ether, diethylene glycol diglycidyl ether, tetraethylene glycol diglycidyl ether, and resorcinol diglycidyl ether), triglycidyl ethers (e.g., glycerol triglycidyl ether, erythritol triglycidyl ether, and diglycerol triglycidyl ether), or tetraglycidyl ethers (e.g., erythritol tetraglycidyl ether and pentaerythritol tetraglycidyl ether) to introduce epoxy groups into the support, and then the epoxy groups are reacted with hydroxy groups of a hydrophilic polymer under basic conditions.

[0053] Examples of methods for introducing formyl groups into a support include reacting hydroxy groups on the support with bifunctional aldehydes such as glutaraldehyde, or reacting the support with an oxidizing agent such as sodium periodate. Another example is a method in which a support on which an epoxy group has been introduced by the above-mentioned method has been reacted with a compound such as D-glucamine, N-methyl-D-glucamine, or α-thioglycerol to introduce a hydroxy group on the carbon adjacent to the reactive site, and the resulting support is then reacted with an oxidizing agent such as sodium periodate.

[0054] Examples of methods for introducing carboxy groups into a support include reacting hydroxy groups on the support with haloacetic acids such as monochloroacetic acid and monobromoacetic acid under basic conditions, as well as reacting a support into which epoxy groups have been introduced by the above-mentioned method with amino acids such as glycine, alanine, aspartic acid, and glutamic acid, amino group-containing carboxylic acids such as β-alanine, 4-aminobutyric acid, and 6-aminohexanoic acid, and sulfur-containing carboxylic acids such as thioglycolic acid and thiomalic acid under basic conditions. Another example is a method in which the carboxy groups introduced into the support are converted to N-hydroxysuccinimide esters, which are active ester groups, by reacting them with N-hydroxysuccinimide in the presence of a condensing agent such as 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC).

[0055] An example of a method for introducing amino groups into a support is to react a support into which epoxy groups have been introduced by the above-mentioned method with a compound having at least two amino groups, such as ethylenediamine, diethylenetriamine, or tris(2-aminoethyl)amine. An example of a method for introducing maleimide groups into a support is to react a support having hydroxy groups and / or amino groups with a carboxylic acid having a maleimide group, such as 3-maleimidopropionic acid, 4-maleimidobutyric acid, 6-maleimidohexanoic acid, or 4-(N-maleimidomethyl)cyclohexanecarboxylic acid, in the presence of a condensing agent such as EDC. Another example is to react an N-hydroxysuccinimide ester or N-hydroxysulfosuccinimide ester of the above-mentioned carboxylic acid having a maleimide group.

[0056] Examples of methods for introducing haloacetyl groups into a support include reacting a support having a hydroxy group or a support into which an amino group has been introduced by the above-mentioned method with an acid halide such as chloroacetic acid chloride, bromoacetic acid chloride, or bromoacetic acid bromide, or reacting a halogenated acetic acid such as chloroacetic acid, bromoacetic acid, or iodoacetic acid in the presence of a condensing agent such as EDC.Further examples include reacting an N-hydroxysuccinimide ester or N-hydroxysulfosuccinimide ester of the halogenated acetic acid.

[0057] A method for introducing a mercapto group into a support can be, for example, by reacting a support into which an amino group has been introduced by the above-mentioned method with cysteine ​​or thioglycolic acid in which the amino group has been protected in the presence of a condensing agent such as EDC.

[0058] The solvent, reaction temperature, and reaction time used in the step of introducing a reactive functional group into the insoluble support described above are not particularly limited as long as the desired reactive functional group can be introduced, and may be appropriately selected from solvents and conditions commonly used by those skilled in the art.

[0059] Step (A2) is a step of immobilizing a complex lipid-binding protein on the reactive insoluble carrier obtained in step (A1) to obtain the complex lipid-binding carrier of the present invention. Specifically, examples include a method of reacting an amino group of a protein with an epoxy group, formyl group, carboxy group, or active ester group such as N-hydroxysuccinimide ester introduced into the carrier, a method of reacting an amino group introduced into the carrier with a carboxy group of a protein, and a method of reacting an epoxy group, maleimide group, haloacetyl group, or haloalkyl group introduced into the carrier with a mercapto group of a protein.

[0060] Among these immobilization methods, the method of reacting a formyl group or active ester group introduced into a carrier with an amino group of a protein, and the method of reacting a maleimide group or haloacetyl group introduced into a carrier with a mercapto group of a protein are preferred, as they allow protein immobilization to a carrier in a short time with high yield. The method of reacting a maleimide group or haloacetyl group introduced into a carrier with a mercapto group of a protein is more preferred, as the immobilization reaction can be carried out at a pH close to neutral and denaturation of the complex lipid-binding protein can be suppressed. The method of reacting a maleimide group introduced into a carrier with a mercapto group of a protein is particularly preferred, as the functional group has high stability.

[0061] Alternatively, in step (A2), the reactive functional group introduced into the complex lipid-binding protein may be reacted with the reactive insoluble carrier obtained in step (A1) to immobilize the complex lipid-binding protein. Specifically, an example of a method is to react the amino group of the protein with the active ester group of a compound having both a maleimide group and an active ester group, such as 4-(N-maleimidomethyl)cyclohexane-1-carboxylic acid or 3-sulfo-N-hydroxysuccinimide ester sodium salt, to introduce a maleimide group or active ester group into the protein, and then react the resulting product with the carrier obtained in step (A1) to which a mercapto group or amino group has been introduced.

[0062] Furthermore, in step (A2), the complex lipid-binding protein may be immobilized on the carrier using affinity binding such as antigen-antibody reaction or avidin-biotin binding. Specifically, a method can be exemplified in which the reactive insoluble carrier containing epoxy groups, formyl groups, carboxy groups, active ester groups, amino groups, etc. obtained in step (A1) is reacted with streptavidin or an antibody such as an anti-MBP antibody, anti-CBD antibody, anti-myc tag antibody, anti-FLAG tag antibody, or anti-His tag antibody to produce an antibody- or streptavidin-immobilized carrier, and then contacted with a complex lipid-binding protein into which MBP, CBD, a myc tag, a FLAG tag, or a polyhistidine tag has been introduced, or a biotinylated complex lipid-binding protein. The method for introducing biotin into a protein may be appropriately selected from methods commonly used by those skilled in the art. Specific examples include a method in which an amino group of a protein is reacted with a biotinylation reagent having an active ester group, such as 9-(biotinamido)-4,7-dioxanonanoate-N-succinimidyl, or a method in which a mercapto group of a protein is reacted with a biotinylation reagent having a maleimide group, such as N-biotinyl-N'-[2-(N-maleimido)ethyl]piperazine hydrochloride.

[0063] In addition, the method for producing a composite lipid-binding carrier of the present invention may also include a step of contacting an insoluble carrier with a composite lipid-binding protein and immobilizing the protein on the insoluble carrier by physical adsorption.The method for immobilizing a protein on an insoluble carrier by physical adsorption may be appropriately selected from methods commonly used by those skilled in the art, and specifically, the method described in Japanese Patent Publication No. 5-41946 can be exemplified.

[0064] The method for producing the complex lipid-binding carrier of the present invention may be selected appropriately depending on the detection method and isolation method of the present invention described below. However, when the carrier is used in the detection method of the present invention, a production method by physical adsorption is preferred because of its simplicity. When the carrier is used in the isolation method of the present invention, a production method in which a reactive insoluble carrier is reacted with a complex lipid-binding protein, or a production method utilizing affinity binding such as the antigen-antibody reaction or avidin-biotin binding described above is preferred.

[0065] The conjugate lipid-binding carrier of the present invention can be produced by reacting or contacting the above-described carrier with a conjugate lipid-binding protein dissolved in a buffer solution. The buffer solution in which the protein is dissolved is not particularly limited, and examples include commercially available buffer solutions such as acetate buffer, phosphate buffer, 2-morpholinoethanesulfonic acid (MES) buffer, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) buffer, tris(hydroxymethyl)aminomethane (Tris) buffer, and D-PBS(-) (manufactured by Fujifilm Wako Pure Chemical Industries). Furthermore, to improve the efficiency of the immobilization reaction, inorganic salts such as sodium chloride or surfactants such as polyoxyethylene sorbitan monolaurate (trade name: Tween 20) may be added to the buffer solution. The reaction or contact temperature and pH in the method for producing a composite lipid-binding carrier of the present invention may be appropriately set within the ranges of 0°C or higher and 50°C or lower, and pH 4 or higher and 10 or lower, taking into consideration the reactivity of the reactive functional group and the stability of the composite lipid-binding protein. In order to prevent protein inactivation, the reaction temperature is preferably set within the ranges of 15°C or higher and 40°C or lower, and pH 5 or higher and 9 or lower.

[0066] The amount of complex lipid-binding protein immobilized on the complex lipid-binding carrier of the present invention may be appropriately selected depending on the detection method of the present invention and the isolation method of the present invention. For example, in the detection method of the present invention, in the case of magnetic particles, the amount is preferably 0.1 μg to 50 μg, more preferably 0.5 μg to 30 μg, and even more preferably 1.0 μg to 20 μg per 1 mg of carrier. In the case of a plate-shaped carrier, the amount is 1 well (2.7 cm 2 ) is preferably 0.05 μg to 5 μg, more preferably 0.1 μg to 2 μg, and even more preferably 0.2 μg to 1 μg. Furthermore, in the isolation method of the present invention, the amount is preferably 0.01 mg to 30 mg, more preferably 0.05 mg to 10 mg, per mL of carrier swollen with an aqueous solution such as water or a buffer solution. The amount of protein immobilized can be adjusted by adjusting the amount of protein used in producing the complex lipid-binding carrier of the present invention or the amount of active functional groups introduced into the insoluble carrier. Furthermore, the amount of protein immobilized on the carrier can be calculated by recovering the protein solution after the immobilization reaction, determining the amount of unreacted protein, and then subtracting the amount of unreacted protein from the amount of protein used in the immobilization reaction.

[0067] The method for detecting phospholipids and / or extracellular vesicles of the present invention is characterized by comprising the following steps (X1) to (X3): (X1) A step of contacting the composite lipid-binding carrier of the present invention with a sample solution containing phospholipids and / or extracellular vesicles in the presence of calcium ions to obtain a complex in which the carrier is bound to phospholipids and / or extracellular vesicles. (X2) A step of separating and removing substances that did not bind to the complex lipid-binding carrier from the complex obtained in the step (X1). (X3) A step of detecting the complex obtained in the step (X1) after the step (X2) is completed.

[0068] Steps (X1) to (X3) will be described below.

[0069] In the detection method of the present invention, it is preferable to use magnetic microparticles and / or a flat-plate carrier, since phospholipids and / or extracellular vesicles can be detected efficiently with a simple operation. Furthermore, in the detection method of the present invention, it is preferable to use a detection reagent containing a composite lipid-binding carrier described below, and an extracellular vesicle detection kit containing the detection reagent, since the operations of steps (X1) to (X3) described below can be easily performed. Using the detection kit, a method used by those skilled in the art in conventional immunoassays can be appropriately selected and performed.

[0070] The sample solution in the detection method of the present invention is not particularly limited as long as it contains complex lipids or extracellular vesicles, and may be derived from a living organism such as biological tissue or cultured cells, or may be a solution in which complex lipids or extracellular vesicles are dissolved or suspended in a medium, buffer solution, or the like.

[0071] Specific examples of sample solutions containing complex lipids include solutions containing extracts or fragments of cells and / or extracellular vesicles, and examples of sample solutions containing extracellular vesicles include body fluids such as blood, saliva, and urine, as well as cell culture supernatants. The solution used to prepare the sample solution containing phospholipids and / or extracellular vesicles is not particularly limited, as long as it does not contain components that bind to calcium to form precipitates, can maintain complex lipids and extracellular vesicles in a stable state, and does not inhibit the binding of the complex lipid-binding carrier to the complex lipids or extracellular vesicles. Specific examples include Tris-buffered saline (TBS) and HEPES-buffered saline (HBS), which have a buffer capacity in the range of pH 7.0 to pH 8.0.

[0072] The buffer concentration in the physiological saline is not particularly limited and may be appropriately selected from the range of 1 mM to 200 mM. The concentration of salts, such as sodium chloride, contained in the physiological saline is also not particularly limited and may be appropriately selected from the range of 100 mM to 200 mM. Furthermore, the physiological saline may contain a surfactant to maintain the stability of complex lipids and extracellular vesicles. Specific examples include nonionic surfactants at 0.00001% (w / v) to 0.5% (w / v), more specifically, Triton X-100 or Tween 20 (both trade names) at 0.00001% (w / v) to 0.5% (w / v).

[0073] Step (X1) in the detection method of the present invention is a step of contacting the composite lipid-binding carrier of the present invention with a sample solution containing phospholipids and / or extracellular vesicles in the presence of calcium ions to obtain a complex in which the carrier and extracellular vesicles are bound. To achieve good detection sensitivity, the calcium ion concentration in step (X1) is preferably 0.1 mM to 100 mM, more preferably 1 mM to 10 mM. Furthermore, when magnetic particles are used as the composite lipid-binding carrier, the amount of sample solution used in step (X1) is preferably 0.1 mL to 1000 mL, more preferably 0.1 mL to 100 mL, per 1 mg of the carrier. When a plate-type carrier is used as the composite lipid-binding carrier, the amount is preferably 25 μL to 300 μL, more preferably 50 μL to 100 μL, per well. In step (X1), the temperature at which the composite lipid-binding carrier of the present invention is brought into contact with a sample solution containing phospholipids and / or extracellular vesicles is preferably 2° C. to 37° C., more preferably 2° C. to 30° C. The contact time is preferably 0.1 to 24 hours, more preferably 1 to 12 hours.

[0074] Specifically, step (X2) in the detection method of the present invention includes a separation step of separating the complex obtained in step (X1) from the sample solution and a washing step of washing the complex. The separation step in step (X2) is not particularly limited as long as it is a B / F (Bound / Free) separation method in a typical immunoassay, and may be appropriately selected from methods commonly used by those skilled in the art. Specifically, when magnetic particles are used as the composite lipid-binding carriers, an example of a method is to perform the complex formation step in step (X1) and then accumulate the complex using magnetic force, followed by removal of the sample solution. Furthermore, when flat-plate carriers are used as the composite lipid-binding carriers, an example of a method is to use a pipette or plate washer to remove the sample solution from the flat-plate carriers that have undergone the complex formation step in step (X1).

[0075] The washing step in step (X2) is a step in which the complex obtained in the separation step is washed with a washing solution containing calcium ions. By performing the washing step, impurities in the sample solution bound to the composite lipid-binding carrier can be removed. The washing step in step (X2) may be appropriately selected from methods commonly used by those skilled in the art, except for using a washing solution containing calcium ions. Specifically, when magnetic microparticles are used as the composite lipid-binding carriers, an example of the method is a method in which the complex is accumulated and washed using magnetic force. Furthermore, when a flat-plate carrier is used as the composite lipid-binding carrier, an example of the method is a method in which the complex is washed using a pipette or plate washer.

[0076] The calcium ion-containing washing solution used in the washing step (X2) is not particularly limited as long as it stabilizes the complex obtained in the separation step. Specific examples include TBS and HBS, which have a buffering capacity in the pH range of 7.0 to 8.0. The calcium ion concentration in the washing solution is preferably 0.1 mM to 100 mM, more preferably 1 mM to 10 mM, in order to stabilize the complex obtained in the separation step. The washing solution may also contain a surfactant to maintain the stability of the complex. Specific examples include a nonionic surfactant at 0.00001% (w / v) to 0.5% (w / v), more specifically, a surfactant such as Triton X-100 or Tween 20 (both trade names) at 0.00001% (w / v) to 0.5% (w / v).

[0077] Step (X3) in the detection method of the present invention is a step of detecting a complex between the composite lipid-binding carrier of the present invention and phospholipids and / or extracellular vesicles in a sample solution. The procedure in step (X3) is not particularly limited as long as it can detect the presence or absence and / or amount of the complex obtained in step (X1), except for using reagents and a measurement solution that contain calcium ions but do not contain components that bind to calcium to form precipitates. A method may be appropriately selected from those commonly used by those skilled in the art. Specific examples include enzyme immunoassay, ELISA, fluorescence / luminescence immunoassay, and flow cytometry. Among these, ELISA and flow cytometry are more preferred in terms of accuracy and sensitivity.

[0078] When step (X3) is performed by ELISA or flow cytometry, it can be performed by a method commonly used by those skilled in the art. Specifically, an antibody having binding affinity to complex lipids or extracellular vesicles can be used to label a primary antibody, a labeled primary antibody, a labeled secondary antibody that binds to the primary antibody, or a biotin-labeled product of the primary antibody and a labeled streptavidin that binds to the biotin-labeled product of the primary antibody. The labeled primary antibody, labeled secondary antibody, and labeled streptavidin can be used as labeled antibodies / streptavidin in ELISA or flow cytometry, as long as they are not particularly limited, and specifically, examples include fluorescently labeled antibodies / streptavidin labeled with fluorescent substances such as Cy3, Cy5, FITC (Fluorescein isothiocyanate), rhodamine, and PE (Phycoerythrin), and enzyme-labeled antibodies / streptavidin labeled with enzymes such as peroxidase and alkaline phosphatase. The fluorescence and the like associated with these labeled antibodies / streptavidin may be measured by a known method corresponding to the labeling method of the labeled antibodies / streptavidin.

[0079] The dilution ratio of the labeled primary antibody, the primary antibody and the labeled secondary antibody, or the biotin-labeled primary antibody and the labeled streptavidin used in the detection step in step (X2) may be appropriately selected depending on the activity and concentration of the antibody, and is preferably 10 times or more and 10 times or less. 6 It is preferable to dilute it to 1000 times or less, and 5 When magnetic microparticles are used as the composite lipid-binding carrier, the volume of the diluted solution of the labeled primary antibody, the primary antibody and labeled secondary antibody, or the biotin-labeled primary antibody and labeled streptavidin solution to be reacted with the complex obtained in the washing step is preferably 0.1 mL to 100 mL, more preferably 0.1 mL to 50 mL, per 1 mg of the carrier.

[0080] When a flat-plate carrier is used as the composite lipid-binding carrier, the amount per well is preferably 10 μL to 300 μL, more preferably 30 μL to 100 μL. The reaction temperature is preferably 2°C to 40°C, more preferably 10°C to 40°C. The reaction time is preferably 0.5 hours to 12 hours, more preferably 1 hour to 4 hours.

[0081] The method for isolating phospholipids and / or extracellular vesicles of the present invention is characterized by comprising the following steps (Y1) to (Y3): (Y1) A step of contacting the composite lipid-binding carrier of the present invention with a sample solution containing phospholipids and / or extracellular vesicles in the presence of calcium ions to obtain a complex in which the carrier is bound to phospholipids and / or extracellular vesicles. (Y2) A step of separating and removing substances that did not bind to the composite lipid-binding carrier from the complex obtained in the step (Y1). (Y3) After completion of the step (Y2), a step of detaching phospholipids and / or extracellular vesicles from the complex obtained in the step (Y1) and recovering the phospholipids and / or extracellular vesicles.

[0082] Steps (Y1) to (Y3) will be described below.

[0083] The sample solution used in the isolation method of the present invention is the same as the sample solution in the detection method of the present invention described above. Furthermore, in the isolation method of the present invention, it is preferable to use a particulate composite lipid-binding carrier, with a particle size of preferably 10 μm to 500 μm, more preferably 20 μm to 200 μm, in order to simplify the operations of steps (Y1) to (Y3) described below. Furthermore, in the isolation method of the present invention, it is preferable to use a column packed with the composite lipid-binding carrier described below, and an extracellular vesicle isolation kit containing the column. Using the isolation kit, a method for isolating biological substances by conventional chromatography can be appropriately selected.

[0084] In step (Y1) of the isolation method of the present invention, the composite lipid-binding carrier of the present invention is contacted with a sample solution containing phospholipids and / or extracellular vesicles in the presence of calcium ions to obtain a complex of the carrier and extracellular vesicles. As in step (X1) of the detection method of the present invention, the calcium ion concentration in step (Y1) is preferably 0.1 mM to 100 mM, more preferably 1 mM to 10 mM.

[0085] The amount of sample solution used in step (Y1) is preferably 1 mL to 1000 mL, more preferably 1 mL to 100 mL, per mL of carrier swollen with water. In step (Y1), the temperature at which the composite lipid-binding carrier of the present invention is contacted with the sample solution containing phospholipids and / or extracellular vesicles is preferably 2°C to 37°C, more preferably 2°C to 30°C. The contact time is preferably 0.5 hours to 24 hours, more preferably 1 hour to 12 hours.

[0086] Step (Y2) in the isolation method of the present invention, like step (X2) in the detection method of the present invention, comprises a separation step of separating the complex obtained in step (Y1) from the sample solution and a washing step of washing the complex. The separation step in step (Y2) is not particularly limited as long as it is a separation method used in ordinary separation and purification procedures for biological substances such as proteins and lipids, and may be appropriately selected from methods commonly used by those skilled in the art.

[0087] The washing step in step (Y2) is a step in which the complex obtained in the separation step is washed with a washing solution containing calcium ions. By performing the washing step, impurities in the sample solution bound to the complex lipid-binding carrier can be removed. The washing step in step (Y2) may be appropriately selected from methods commonly used by those skilled in the art, except for the use of a washing solution containing calcium ions. Specifically, a method in which a washing solution containing calcium ions is added to a column packed with the complex obtained in step (Y1) can be exemplified.

[0088] The calcium ion-containing washing solution used in the washing step (Y2) is not particularly limited as long as it stabilizes the complex obtained in the separation step. Specific examples include TBS and HBS, which have a buffering capacity in the pH range of 7.0 to 8.0. The calcium ion concentration in the washing solution is preferably 0.1 to 100 mM, more preferably 1 to 10 mM, in terms of the stability of the complex obtained in the separation step. The washing solution may also contain a surfactant to maintain the stability of the complex. Specific examples include a nonionic surfactant at 0.00001% (w / v) to 0.5% (w / v), more specifically, Triton X-100 or Tween 20 (both trade names) at 0.00001% (w / v) to 0.5% (w / v).

[0089] Specifically, step (Y3) in the isolation method of the present invention is a step of detaching and recovering phospholipids and / or extracellular vesicles by subjecting the complex obtained in steps (Y1) and (Y2) to a procedure for dissociating the complex. The procedure for dissociating the complex may be any procedure that dissociates the complex from the carrier without destroying the structure of the extracellular vesicles. Specific examples of such a procedure include contacting the complex with a surfactant that can denature proteins, and reducing the calcium ion concentration in the complex.

[0090] In the method of contacting the complex with a detergent, the complex washed in step (Y2) is contacted with a solution of buffered saline such as TBS or HBS to which a detergent has been added, thereby denaturing the complex lipid-binding protein, thereby recovering phospholipids and / or extracellular vesicles. The detergent may be any detergent commonly used by those skilled in the art to denature proteins, and specific examples include anionic detergents such as sodium dodecyl sulfate (SDS), nonionic detergents such as Triton X-100 and Tween 20 (both trade names), and zwitterionic detergents such as CHAPS (3-[(3-Cholamidopropyl)dimethylammonio]propanesulfonate).

[0091] Among these, SDS is preferred because it can denature proteins at low concentrations. The concentration of the surfactant in the buffered saline solution may be appropriately selected from the range of 0.01% (w / v) to 10% (w / v), taking into consideration the stability of the complex lipid-binding protein. There are no particular limitations on the temperature or time for contacting the complex with the buffered saline solution containing the surfactant. The temperature is preferably 2°C to 40°C, more preferably 10°C to 40°C. The time is preferably 1 minute to 60 minutes, more preferably 5 minutes to 30 minutes.

[0092] In the method for reducing the calcium ion concentration in the complex, the complex washed in step (Y2) is contacted with a solution capable of reducing the calcium ion concentration in the complex, and the complex is dissociated, thereby detaching and recovering the phospholipids and / or extracellular vesicles. Examples of solutions capable of reducing the calcium ion concentration in the complex include the aforementioned calcium ion-free buffer solutions such as TBS and HBS, and buffer solutions in which a calcium ion chelating agent has been added to TBS or HBS. Among these, buffer solutions in which a calcium ion chelating agent has been added are preferred because they can reduce the calcium ion concentration in the complex with high efficiency.

[0093] The calcium ion chelating agent is not particularly limited as long as it can form a chelate bond with calcium ions, and specific examples include ethylenediaminetetraacetic acid (EDTA), glycoletherdiaminetetraacetic acid (EGTA), nitrilotriacetic acid (NTA), and glutamic acid diacetic acid (GLDA). Among these, EDTA and EGTA are preferred in terms of their ability to bind to calcium ions.

[0094] The concentration of the calcium ion chelating agent in the buffered saline may be appropriately selected from the range of 0.1 mM to 100 mM, taking into consideration the stability of the complex. The temperature and time for contacting the complex with the buffered saline containing the calcium ion chelating agent are not particularly limited, but the temperature is preferably 2°C to 40°C, more preferably 10°C to 40°C. The time is preferably 1 minute to 60 minutes, more preferably 5 minutes to 30 minutes.

[0095] Whether the phospholipids and / or extracellular vesicles recovered in step (Y3) of the isolation method of the present invention are the target product can be confirmed, as needed, by a method commonly used by those skilled in the art, such as the detection method of the present invention or Western blotting. When the sample liquid is a culture medium of human mesenchymal stem cells (hMSCs), the extracellular vesicles obtained by the isolation method of the present invention are expected to be suitable for use as a source of MSC-exosomes as therapeutic agents, as described in, for example, Drug Delivery System, Vol. 29, pp. 140-151, 2014.

[0096] Next, a detection reagent containing the composite lipid-binding carrier of the present invention (hereinafter referred to as the detection reagent of the present invention) and an extracellular vesicle detection kit containing the detection reagent (hereinafter referred to as the extracellular vesicle detection kit of the present invention) will be described. In addition to the composite lipid-binding carrier of the present invention, the detection reagent of the present invention may contain a labeled primary antibody, a labeled secondary antibody, labeled streptavidin, a preservative such as sodium azide, a stabilizer such as albumin or a surfactant, and the like for detecting the complex between the composite lipid-binding carrier and phospholipids and / or extracellular vesicles in the sample solution.

[0097] In addition to the detection reagents described above, the extracellular vesicle detection kit of the present invention may also contain common reagents typically used in the relevant technical field, such as buffered saline and calcium ion aqueous solutions for preparing sample solutions containing the aforementioned phospholipids and / or extracellular vesicles, calcium ion-containing washing solutions, aqueous solutions such as buffered saline containing surfactants, aqueous solutions such as buffered saline containing calcium ion chelators, substrate solutions and reaction stop solutions for detecting enzyme-labeled antibodies / streptavidin, and standards used to create calibration curves for the substances to be measured. Additionally, the kit may include instructions describing the detection method of the present invention and how to handle the reagents.

[0098] Next, a column packed with the composite lipid-binding carrier of the present invention (hereinafter referred to as the column of the present invention) and an extracellular vesicle isolation kit including the column (hereinafter referred to as the extracellular vesicle isolation kit of the present invention) will be described. The column of the present invention is not particularly limited in its volume or shape, as long as it can carry out the above-mentioned method for isolating extracellular vesicles. The volume can be determined by packing an amount of carrier determined by the amount of sample solution containing extracellular vesicles added and the amount of extracellular vesicles bound to the composite lipid-binding carrier into a column of a size and shape determined taking into account the processing time, etc.

[0099] The column shape can be selected appropriately depending on the amount of sample liquid containing extracellular vesicles added and the processing time, such as an open column with an open top end or a closed column with both ends closed. An open column is preferred when the amount of sample liquid added is small, while a closed column with both ends closed and connectable to an HPLC device is preferred when the amount of sample liquid added is large and processing is required in a short time.

[0100] In addition to the column described above, the extracellular vesicle isolation kit of the present invention may also contain common reagents typically used in the art. Specific examples include buffered saline and aqueous calcium ion solutions for preparing the sample solution containing the phospholipids and / or extracellular vesicles described above, a calcium ion-containing wash solution, an aqueous solution such as buffered saline containing a surfactant, and buffered saline containing a calcium ion chelator. Furthermore, the kit may also contain common tools typically used in the art. Specific examples include a syringe and adapter for injecting the sample solution into the column and a microtube for recovering the extracellular vesicles detached from the column. Additionally, the kit may also include instructions describing the isolation method of the present invention and how to handle the reagents and tools. [Effects of the Invention]

[0101] The composite lipid-binding carrier of the present invention is an insoluble carrier on which a protein capable of binding to a composite lipid such as a phospholipid and / or an extracellular vesicle having a composite lipid such as a phospholipid present on its surface is immobilized, and by using this carrier, it is possible to selectively detect extracellular vesicles from a sample solution containing a biological substance that contains extracellular vesicles with high sensitivity and accuracy.Furthermore, by passing a sample solution containing a biological substance that contains extracellular vesicles through a column packed with the composite lipid-binding carrier of the present invention to adsorb the extracellular vesicles to the carrier, and then treating the sample solution with a chelating agent, it is possible to isolate large quantities of highly pure extracellular vesicles with a simple procedure. [Brief explanation of the drawings]

[0102] [Figure 1]FIG. 10 is a graph showing the calcium ion concentration dependence of the extracellular vesicle detection performance using a microplate on which the recombinant protein rPKCaC2-GST (SEQ ID NO: 7) was immobilized in Reference Example 3. [Figure 2] FIG. 10 is a graph showing the calcium ion concentration dependence of the extracellular vesicle detection performance using a microplate on which the recombinant protein rPKCgC2-GST (SEQ ID NO: 23) was immobilized in Reference Example 3. [Figure 3] FIG. 10 shows a calibration curve for sandwich ELISA for detecting extracellular vesicles using a microplate on which the recombinant protein rPKCaC2-GST (SEQ ID NO: 7) was immobilized in Reference Example 4. [Figure 4] FIG. 10 shows a calibration curve for sandwich ELISA for detecting extracellular vesicles using a microplate on which the recombinant protein rPKCgC2-GST (SEQ ID NO: 23) was immobilized in Reference Example 4. [Figure 5] FIG. 16 shows Western blotting of the solutions recovered from a carrier on which no recombinant protein was immobilized and from a carrier on which the recombinant protein rPKCaC2 (SEQ ID NO: 5) was immobilized in Example 15. [Figure 6] FIG. 13 shows Western blotting of the solutions recovered from a carrier on which no recombinant protein was immobilized and from a carrier on which the recombinant protein rPKCaC2-GST (SEQ ID NO: 7) was immobilized in Example 15. [Figure 7] FIG. 1 shows the results of solid-phase ELISA of recombinant proteins rPKCaC2 (SEQ ID NO: 5), rPKCbC2 (SEQ ID NO: 17), and rPKCgC2 (SEQ ID NO: 21) in Reference Example 5. [Figure 8] FIG. 1 shows the results of solid-phase ELISA of recombinant proteins rPKCaC2-GST (SEQ ID NO: 7), rPKCbC2-gs-GST (SEQ ID NO: 19), and rPKCgC2-GST (SEQ ID NO: 23) in Reference Example 5. [Figure 9] FIG. 10 is a diagram showing the complex lipid binding ability of recombinant protein rPKCaC2 (SEQ ID NO: 5) using membrane lipid strips in Reference Example 6. [Figure 10]FIG. 10 is a diagram showing the complex lipid binding ability of recombinant protein rPKCbC2 (SEQ ID NO: 17) using membrane lipid strips in Reference Example 6. [Figure 11] FIG. 17 shows the results of separation of extracellular vesicles in a culture supernatant using the composite lipid-binding carrier of the present invention in Example 17. [Figure 12] FIG. 10 shows the results of separation of extracellular vesicles in culture supernatant using the composite lipid-binding carrier of the present invention in Example 18. [Example]

[0103] The present invention will be explained in more detail below by giving Preparation Examples, Examples, Comparative Examples and Reference Examples, but the present invention is not limited to these. (1) Construction of protein expression vectors containing the amino acid sequence of the C2 domain of protein kinase α and their transformants in Escherichia coli BL21(DE3) The protein expression vectors containing the amino acid sequence of the C2 domain of protein kinase α are expression vectors for expressing recombinant proteins containing the amino acid sequence of the C2 domain of protein kinase α, and specifically, the six types of expression vectors shown in Table 1. The expression vector used was pET28a(+) (Merck), and each expression vector was prepared using a general method disclosed in JP 2018-000038 A and other publications, and Escherichia coli BL21 strain (DE3) was transformed with the vector.

[0104] [Example 1] Example 1 describes an expression vector for expressing the recombinant protein rPKCaC2, which is the C2 domain of protein kinase α. The amino acid sequence of rPKCaC2 is SEQ ID NO: 5 (the amino acid sequences from positions 3 to 141 correspond to the amino acid sequence of SEQ ID NO: 1, and the amino acid sequences from positions 155 to 160 correspond to a polyhistidine sequence). An expression vector containing the nucleotide sequence encoding this amino acid (SEQ ID NO: 6) was designated pPKCaC2. Furthermore, Escherichia coli BL21(DE3) was transformed with the expression vector pPKCaC2 to produce a transformant EC / pPKCaC2.

[0105] [Example 2] Example 2 describes an expression vector for expressing rPKCaC2-GST, a fusion protein of the C2 domain of protein kinase α and GST (glutathione S-transferase). The amino acid sequence of rPKCaC2-GST is SEQ ID NO:7 (positions 3 to 141 correspond to the amino acid sequence of SEQ ID NO:1, positions 142 to 153 correspond to a sequence derived from the multicloning site of the expression vector pET28a(+), positions 155 to 370 correspond to the amino acid sequence of SEQ ID NO:4, and positions 373 to 378 correspond to a polyhistidine sequence). An expression vector containing the nucleotide sequence encoding this amino acid sequence (SEQ ID NO:8) was designated pPKCaC2-GST. Furthermore, Escherichia coli BL21(DE3) was transformed with the expression vector pPKCaC2-GST to generate the transformant EC / pPKCaC2-GST.

[0106] [Example 3] Example 3 describes an expression vector for expressing rPKCaC2-L-GST, a fusion protein of the C2 domain of protein kinase α and GST. The amino acid sequence of rPKCaC2-L-GST is SEQ ID NO: 9 (positions 3 to 141 correspond to the amino acid sequence of SEQ ID NO: 1, positions 142 to 179 correspond to the amino acid sequence downstream of the C2 domain of protein kinase α (the amino acid sequence of positions 294 to 331 of Uniprot accession number P17252), positions 193 to 408 correspond to the amino acid sequence of SEQ ID NO: 4, and positions 411 to 416 correspond to a polyhistidine sequence). An expression vector containing the nucleotide sequence encoding these amino acids (SEQ ID NO: 10) was designated pPKCaC2-L-GST. Furthermore, Escherichia coli BL21(DE3) was transformed with the expression vector pPKCaC2-L-GST to generate the transformant EC / pPKCaC2-L-GST.

[0107] [Example 4] Example 4 describes an expression vector for expressing rPKCaC2-gs-GST, a fusion protein of the C2 domain of protein kinase α and a GST protein. The amino acid sequence of rPKCaC2-gs-GST is SEQ ID NO: 11 (positions 3 to 141 correspond to the amino acid sequence of SEQ ID NO: 1, positions 146 to 159 correspond to a GS linker (SEQ ID NO: 41), positions 166 to 381 correspond to the amino acid sequence of SEQ ID NO: 4, and positions 384 to 389 correspond to a polyhistidine sequence). An expression vector containing the nucleotide sequence encoding this amino acid sequence (SEQ ID NO: 12) was designated pPKCaC2-gs-GST. Furthermore, Escherichia coli BL21(DE3) was transformed with the expression vector pPKCaC2-gs-GST to prepare a transformant EC / pPKCaC2-gs-GST.

[0108] [Example 5] Example 5 describes an expression vector for expressing rPKCaC2-h-GST, a fusion protein of the C2 domain of protein kinase α and a GST protein. The amino acid sequence of rPKCaC2-h-GST is SEQ ID NO: 13 (positions 3 to 141 correspond to the amino acid sequence of SEQ ID NO: 1, positions 152 to 165 correspond to an α-helical linker (SEQ ID NO: 42), positions 171 to 386 correspond to the amino acid sequence of SEQ ID NO: 4, and positions 389 to 394 correspond to a polyhistidine sequence). An expression vector containing the nucleotide sequence encoding this amino acid sequence (SEQ ID NO: 14) was designated pPKCaC2-h-GST. Furthermore, Escherichia coli BL21(DE3) was transformed with the expression vector pPKCaC2-h-GST to generate the transformant EC / pPKCaC2-h-GST.

[0109] [Example 6] Example 6 describes an expression vector for expressing rGST-gs-PKCaC2, a fusion protein of the C2 domain of protein kinase α and a GST protein. The amino acid sequence of rGST-gs-PKCaC2 is SEQ ID NO: 15 (positions 4 to 219 correspond to the amino acid sequence of SEQ ID NO: 4, positions 222 to 235 correspond to a GS linker (SEQ ID NO: 41), positions 241 to 379 correspond to the amino acid sequence of SEQ ID NO: 1, and positions 382 to 387 correspond to a polyhistidine sequence). An expression vector containing the nucleotide sequence encoding this amino acid sequence (SEQ ID NO: 16) was designated pGST-gs-PKCaC2. Furthermore, Escherichia coli BL21(DE3) was transformed with the expression vector pGST-gs-PKCaC2 to prepare a transformant EC / pGST-gs-PKCaC2.

[0110] [Table 1]

[0111] (2) Construction of protein expression vectors containing the amino acid sequence of the C2 domain of protein kinase β and their transformants in Escherichia coli BL21(DE3). The protein expression vectors containing the amino acid sequence of the C2 domain of protein kinase β are expression vectors for expressing recombinant proteins containing the amino acid sequence of the C2 domain of protein kinase β, and specifically, the two types of expression vectors shown in Table 2. The expression vector used was pET28a(+) (Merck), and each expression vector was prepared in the same manner as in (1), and Escherichia coli BL21 strain (DE3) was transformed with them.

[0112] [Example 7] Example 7 describes an expression vector for expressing the recombinant protein rPKCbC2, which is the C2 domain of protein kinase β. The amino acid sequence of rPKCbC2 is SEQ ID NO: 17 (the amino acid sequences from the second to the 142nd correspond to the amino acid sequence of SEQ ID NO: 2, and the sequences from the 156th to the 161st correspond to a polyhistidine sequence). An expression vector containing the nucleotide sequence encoding this amino acid (SEQ ID NO: 18) was designated pPKCbC2. Furthermore, Escherichia coli BL21(DE3) was transformed with the expression vector pPKCbC2 to produce a transformant EC / pPKCbC2.

[0113] [Example 8] Example 8 describes an expression vector for expressing rPKCbC2-gs-GST, a fusion protein of the C2 domain of protein kinase β and a GST protein. The amino acid sequence of rPKCbC2-gs-GST is SEQ ID NO: 19 (the amino acid sequence from positions 2 to 142 corresponds to the amino acid sequence of SEQ ID NO: 2, the 147th to 160th corresponds to a GS linker (SEQ ID NO: 41), the 167th to 382nd corresponds to the amino acid sequence of SEQ ID NO: 4, and the 385th to 390th corresponds to a polyhistidine sequence). An expression vector containing the nucleotide sequence encoding this amino acid sequence (SEQ ID NO: 20) was designated pPKCbC2-gs-GST. Furthermore, Escherichia coli BL21(DE3) was transformed with the expression vector pPKCbC2-gs-GST to prepare a transformant EC / pPKCbC2-gs-GST.

[0114] [Table 2]

[0115] (3) Construction of protein expression vectors containing the amino acid sequence of the C2 domain of protein kinase γ and their transformants in Escherichia coli BL21(DE3) The protein expression vectors containing the amino acid sequence of the C2 domain of protein kinase γ are expression vectors for expressing recombinant proteins containing the amino acid sequence of the C2 domain of protein kinase γ, and specifically, the two types of expression vectors shown in Table 3. The expression vector used was pET28a(+) (Merck), and each expression vector was prepared in the same manner as in (1), and Escherichia coli BL21 strain (DE3) was transformed with them.

[0116] [Example 9] Example 9 describes an expression vector for expressing the recombinant protein rPKCgC2 of the C2 domain of protein kinase γ. The amino acid sequence of rPKCgC2 is SEQ ID NO: 21 (the amino acid sequences from the second to the 142nd correspond to the amino acid sequence of SEQ ID NO: 3, and the sequences from the 156th to the 161st correspond to a polyhistidine sequence). An expression vector containing the nucleotide sequence encoding this amino acid (SEQ ID NO: 22) was designated pPKCgC2. Furthermore, Escherichia coli BL21(DE3) was transformed with the expression vector pPKCgC2 to produce a transformant EC / pPKCgC2.

[0117] [Example 10] Example 10 describes an expression vector for expressing rPKCgC2-GST, a fusion protein of the C2 domain of protein kinase γ and a GST protein. The amino acid sequence of rPKCgC2-GST is SEQ ID NO: 23 (positions 2 to 142 correspond to the amino acid sequence of SEQ ID NO: 3, positions 143 to 154 correspond to a sequence derived from the multicloning site of the expression vector pET28a(+), positions 156 to 371 correspond to the amino acid sequence of SEQ ID NO: 4, and positions 374 to 379 correspond to a polyhistidine sequence). An expression vector containing the nucleotide sequence encoding this amino acid sequence (SEQ ID NO: 24) was designated pPKCgC2-GST. Furthermore, Escherichia coli BL21(DE3) was transformed with the expression vector pPKCgC2-GST to generate the transformant EC / pPKCgC2-GST.

[0118] [Table 3]

[0119] [Example 11] Production of recombinant protein The Escherichia coli transformants (Tables 1 to 3) prepared in (1) to (3) above were inoculated into LB medium (10 g / L tryptone, 5 g / L yeast extract, and 5 g / L NaCl) supplemented with 30 μg / mL kanamycin, and pre-cultured by shaking at 37°C overnight. The pre-culture solutions were inoculated into LB medium supplemented with 30 μg / mL kanamycin, and cultured with shaking at 37°C. The turbidity (OD 600 When the chromatogram (ΔΨ) reached approximately 0.6, the culture temperature was changed to 30°C, 0.5 mM IPTG (Isopropyl β-D-1-thiogalactopyranoside) was added, and the culture was continued for 5 hours to express each recombinant protein (10 types of recombinant proteins listed in Tables 1 to 3). Soluble protein extracts were collected from each strain using ultrasonic disruption or a BugBuster Protein extraction kit (Merck). The recombinant proteins were purified from the soluble protein extracts by nickel chelate affinity chromatography.

[0120] (4) Construction of various protein expression vectors and their transformants in Escherichia coli BL21(DE3) An expression vector for a protein containing the amino acid sequence of the C2 domain of protein kinase α (Comparative Example 2) and an expression vector for a protein not containing the C2 domain of protein kinase α (Comparative Examples 1 and 3) were constructed. Specifically, these are three types of expression vectors, designated Comparative Examples 1 to 3, as shown in Table 4. The expression vector used was pET28a(+) (Merck), and each expression vector was constructed in the same manner as in (1), and Escherichia coli BL21 strain (DE3) was transformed with them.

[0121] [Comparative Example 1] Comparative Example 1 is an expression vector for expressing the GST recombinant protein rGST, whose amino acid sequence is SEQ ID NO: 25 (positions 3 to 218 correspond to the amino acid sequence of SEQ ID NO: 4, and positions 221 to 226 correspond to a polyhistidine sequence), and an expression vector containing the nucleotide sequence encoding these amino acids (SEQ ID NO: 26) was designated pGST. Furthermore, Escherichia coli BL21(DE3) was transformed with the expression vector pGST to prepare a transformant EC / pGST.

[0122] Comparative Example 2 Comparative Example 2 is an expression vector for expressing rPKCaC2-gs-MBP, a fusion protein of the C2 domain of protein kinase α and the amino acid sequence constituting maltose binding protein (MBP) (the sequence from positions 27 to 392 of GenPept registration number EER8245063). The amino acid sequence of rPKCaC2-gs-MBP is SEQ ID NO: 27 (positions 3 to 141 correspond to the amino acid sequence of SEQ ID NO: 1, positions 146 to 159 correspond to a GS linker (SEQ ID NO: 41), positions 165 to 530 correspond to the amino acid sequence of MBP protein, and positions 533 to 538 correspond to a polyhistidine sequence). An expression vector containing the nucleotide sequence encoding this amino acid (SEQ ID NO: 28) was designated pPKCaC2-gs-MBP. Furthermore, Escherichia coli BL21(DE3) was transformed with the expression vector pPKCaC2-gs-MBP to generate the transformant EC / pPKCaC2-gs-MBP.

[0123] Comparative Example 3 Comparative Example 3 is an expression vector for expressing the recombinant MBP protein rMBP, whose amino acid sequence is SEQ ID NO: 29 (positions 3 to 368 correspond to the amino acid sequence of the MBP protein, and positions 371 to 376 correspond to a polyhistidine sequence). An expression vector containing the nucleotide sequence encoding these amino acids (SEQ ID NO: 30) was designated pMBP. Furthermore, Escherichia coli BL21(DE3) was transformed with the expression vector pMBP to produce the transformant EC / pMBP.

[0124] [Table 4]

[0125] Comparative Example 4: Production of recombinant protein The Escherichia coli transformants (Table 4) prepared in (4) above were cultured for expression by the method described in Example 11 above, and recombinant proteins were prepared.

[0126] [Reference Example 1] Preparation of extracellular vesicles derived from various cell culture supernatants The cell lines listed under conditions a to g in Table 5 were cultured for 48 hours at 37°C and 5% CO2 in the medium listed in Table 5 supplemented with 5% (v / v) fetal bovine serum (FBS) ultrafiltered through AMICON ULTRA-15, 100 KDa (Merck). Extracellular vesicles (large EVs and small EVs) were then collected using the following method.

[0127] [1] 60 mL of the culture medium was centrifuged at 2000 × g for 30 minutes at 4°C, and the supernatant was collected.

[0128] [2] The supernatant was centrifuged at 16,000 xg for 30 minutes at 4°C to separate the supernatant from the precipitate.

[0129] [3] 60 mL of PBS was added to the precipitate obtained in [2] above, and the mixture was centrifuged at 16,000 × g at 4°C for 30 minutes. The supernatant was removed and the precipitate was collected.

[0130] [4] 200 μL of PBS was added to the precipitate obtained in [3] above, and the mixture was suspended by pipetting. The 16,000×g fraction of extracellular vesicles (large EVs) was collected.

[0131] [5] The supernatant obtained in [2] above was ultracentrifuged at 100,000×g for 16 hours at 4°C, and the supernatant was removed and the precipitate was collected.

[0132] [6] 60 mL of PBS was added to the ultracentrifugal precipitate obtained in [5] above, and ultracentrifugation was carried out at 4°C and 100,000 xg for 3 hours. The supernatant was removed and the precipitate was collected.

[0133] [7] 200 μL of PBS was added to the ultracentrifugal precipitate obtained in [6] above, and the mixture was suspended by pipetting. The 100,000×g fraction of extracellular vesicles (small EVs) was collected.

[0134] [Table 5]

[0135] [Reference Example 2] Quantification of protein concentration in extracellular vesicles The protein concentration of the extracellular vesicles prepared in Reference Example 1 was measured using the Pierce BCA Protein Assay Kit (Thermo Fisher Scientific) by the following method.

[0136] [1] Working Reagent (WR) was prepared by mixing BCA Reagent A and BCA Reagent B at a ratio of 50:1.

[0137] [2] A standard solution of bovine serum albumin (BSA) of known concentration was diluted with PBS to prepare a dilution series with final concentrations of 1000, 500, 250, 125, 62.5, 31.25, 15.625, and 0 μg / mL.

[0138] [3] The dilution series of the BSA standard solution prepared in [2] above and the extracellular vesicle solution were dispensed into two wells of a 96-well microplate, 25 μL per well, for each sample.

[0139] [4] 200 μL / well of the WR prepared in [1] above was added, and the plate was immediately shaken for 30 seconds, after which the plate was covered and incubated at 37°C for 30 minutes.

[0140] [5] After the plate was returned to room temperature, the absorbance at 562 nm was measured using an absorbance plate reader.

[0141] [6] A calibration curve was created from the dilution series of the BSA standard solution using the corrected absorbance value obtained by subtracting the blank (absorbance of 0 μg / mL BSA standard solution) from the absorbance. This calibration curve was used to calculate the protein concentration in the extracellular vesicle solution.

[0142] [Example 12] Evaluation of extracellular vesicle detection performance using an ELISA plate immobilized with a recombinant protein containing the amino acid sequence of the C2 domain of a protein kinase Using a microplate onto which the recombinant protein produced in Example 11 was immobilized and a commercially available biotin-labeled anti-tetraspanin antibody (CD9, manufactured by Frontier Laboratories), a sandwich ELISA was performed to detect extracellular vesicles (small EVs) prepared from the culture supernatant of 293T cells in Reference Example 1. The specific procedure for the sandwich ELISA is described below.

[0143] [1] Protein diluted with TBS (Tris-buffered saline) (pH 7.4) was dispensed into a Maxisorp 96-well microplate (Thermo Fisher Scientific) at 50 μL per well and left to stand overnight at 4°C for immobilization.

[0144] [2] After washing three times with TBS, 300 μL / well of blocking solution (SuperBlock (PBS), Thermo Fisher Scientific) was added and allowed to stand at room temperature for 1 hour.

[0145] [3] After washing three times with TBS, 50 μL of the measurement sample diluted with diluent (TBS containing 1% (w / v) BSA and 2 mM CaCl2, pH 7.4) was added to each well containing the immobilized ligand protein, and the plate was left to stand at room temperature for 2 hours.

[0146] [4] The plate was washed three times with washing solution (TBS containing 2 mM CaCl2, pH 7.4), and 50 μL / well of biotin-labeled detection antibody diluted with diluent was added and left to stand at room temperature for 1 hour.

[0147] [5] After washing three times with washing solution, 50 μL / well of Streptavidin-PolyHRP40 (manufactured by Stereospecific Detection Technologies) diluted 50,000 times with diluent was added and the plate was left to stand at room temperature for 1 hour.

[0148] [6] The plate was washed three times with washing solution, and 50 μL / well of substrate solution (SureBlue Reserve TMB, manufactured by SeraCare Life Sciences) was added, followed by leaving the plate to stand at room temperature for 10 minutes.

[0149] [7] The reaction was stopped by adding 50 μL / well of 1 M phosphoric acid solution.

[0150] [8] The absorbance at 450 nm was measured using an absorbance plate reader.

[0151] [Comparative Example 5] Evaluation of extracellular vesicle detection performance using ELISA plates immobilized with various recombinant proteins produced in Comparative Example 4 Sandwich ELISA was performed to detect extracellular vesicles (small EVs) prepared from the culture supernatant of 293T cells in Reference Example 1 using a microplate on which the recombinant protein produced in Comparative Example 4 was immobilized or a microplate on which no protein was immobilized, and a commercially available biotin-labeled anti-tetraspanin antibody (CD9, manufactured by Frontier Laboratories). The procedure for sandwich ELISA was the same as in Example 12.

[0152] The evaluation conditions and results of Example 12 and Comparative Example 5 are shown in Table 6.

[0153] [Table 6]

[0154] Among conditions a to f, in which the recombinant protein immobilized on the microplate contained the amino acid sequence of the C2 domain of protein kinase α, conditions b to f, in which recombinant proteins fused to the amino acid sequence of GST protein on the N- or C-terminus of the C2 domain, increased absorbance in the presence of extracellular vesicles, demonstrating that sandwich ELISA using these recombinant proteins is possible. Among these, the recombinant proteins used in conditions b to e differed in the amino acid sequence sandwiched between the N-terminal C2 domain of protein kinase α (corresponding to the amino acid sequence of SEQ ID NO: 1) and the C-terminal GST protein (corresponding to the amino acid sequence of SEQ ID NO: 4). Comparing these conditions, the highest extracellular vesicle detection performance was obtained when the C2 domain of protein kinase α and the GST protein were linked with a GS linker (SEQ ID NO: 41) (condition d, SEQ ID NO: 11). Conditions d and f compared a recombinant protein in which the GST protein was fused to the C-terminus of the C2 domain of protein kinase α (condition d, sequence number 11) with a recombinant protein in which the GST protein was fused to the N-terminus (condition f, sequence number 15), and both were able to detect extracellular vesicles with similar sensitivity.

[0155] Of conditions g and h, in which the recombinant protein contained the amino acid sequence of the C2 domain of protein kinase β, condition h (sequence number 19) used a recombinant protein in which the amino acid sequence of GST protein was fused to the C-terminus of the C2 domain. In this condition, the absorbance increased when extracellular vesicles were present, demonstrating that extracellular vesicles could be detected by sandwich ELISA.

[0156] Of conditions i and j, in which the recombinant protein contained the amino acid sequence of the C2 domain of protein kinase γ, condition j (sequence number 23) used a recombinant protein in which the amino acid sequence of GST protein was fused to the C-terminus of the C2 domain. In this condition, the absorbance increased when extracellular vesicles were present, demonstrating that extracellular vesicles could be detected by sandwich ELISA.

[0157] On the other hand, when the recombinant protein immobilized on the microplate did not contain the C2 domain of protein kinase (conditions k (sequence number 25) and m (sequence number 29)), when the amino acid sequence of MBP protein was fused to the C-terminus of the C2 domain of protein kinase α (condition l, sequence number 27), and when the protein was not immobilized on the microplate (condition n), the absorbance did not increase even in the presence of extracellular vesicles.

[0158] [Example 13] Evaluation of detection performance of extracellular vesicles (small EVs) prepared from various cell lines A sandwich ELISA was performed to detect extracellular vesicles (small EVs) prepared from the culture supernatant of various cells in Reference Example 1 using a microplate onto which the recombinant protein rPKCaC2-gs-GST (SEQ ID NO: 11) produced in Example 11 was immobilized and three types of biotin-labeled commercially available anti-tetraspanin antibodies (CD9, CD63, and CD81, all manufactured by Frontier Laboratories). The sandwich ELISA procedure was the same as in Example 12. The evaluation conditions and results are shown in Table 7.

[0159] [Table 7]

[0160] It was demonstrated that the plate onto which the recombinant protein rPKCaC2-gs-GST was immobilized could detect small EVs from all of the different cell lines used in this example by using anti-tetraspanin antibodies CD9, CD63, or CD81 as biotin-labeled detection antibodies.

[0161] [Example 14] Evaluation of extracellular vesicle (large EV) detection performance A sandwich ELISA was performed to detect extracellular vesicles (large EVs) prepared from the culture supernatant of 293T cells (condition a in Table 5) and AGS cells (condition d in Table 5) in Reference Example 1 using a microplate immobilized with the recombinant protein rPKCaC2-gs-GST (SEQ ID NO: 11) produced in Example 11 and three commercially available biotin-labeled anti-tetraspanin antibodies (CD9, CD63, and CD81, all manufactured by Frontier Laboratories). The sandwich ELISA procedure was the same as in Example 12. The evaluation conditions and results are shown in Table 8.

[0162] [Table 8]

[0163] Plates immobilized with the recombinant protein rPKCaC2-gs-GST were shown to be capable of detecting large EVs purified from the culture supernatants of 293T and AGS cells by using anti-tetraspanin antibodies against CD9, CD63, or CD81 as biotin-labeled detection antibodies.

[0164] [Reference Example 3] Calcium ion concentration dependence of extracellular vesicle detection performance using recombinant protein-immobilized microplates A sandwich ELISA was performed to detect extracellular vesicles (small EVs) prepared from the culture supernatant of AGS cells (condition d in Table 5) in Reference Example 1 using a microplate onto which the recombinant protein rPKCaC2-GST (SEQ ID NO: 7) or rPKCgC2-GST (SEQ ID NO: 23) produced in Example 11 was immobilized and a commercially available biotin-labeled anti-tetraspanin antibody (CD9, Frontier Laboratories). The sandwich ELISA procedure was the same as in Example 12, except that the concentrations of CaCl2 contained in the diluent and washing solution were as shown in Table 9. The evaluation conditions and results are shown in Table 9.

[0165] [Table 9]

[0166] The absorbance values ​​of the recombinant proteins rPKCaC2-GST and rPKCgC2-GST were normalized so that the minimum value was 0 and the maximum value was 1, and plotted against the CaCl2 concentration. These values ​​are shown in Figures 1 and 2, respectively.

[0167] Under conditions a to h, the plate on which rPKCaC2-GST (SEQ ID NO: 7) was immobilized contained CaCl2 in the dilution solution and the washing solution at a concentration of 1 × 10 -5 M, i.e., 0.01 mM or higher, extracellular vesicles can be detected, and the number of extracellular vesicles is 2 × 10 -3 It was shown that the detection performance was maximized when M, i.e., 2 mM or more.

[0168] In addition, under conditions i to p, the plate on which rPKCgC2-GST (SEQ ID NO: 23) was immobilized had a CaCl concentration of 1 × 10 in the diluent and washing solution, as shown in FIG. -5 M, i.e., 0.01 mM or more, extracellular vesicles can be detected, and 1 × 10 -4 It was shown that the detection performance was maximized when M, i.e., 0.1 mM or more.

[0169] [Reference Example 4] Calibration curve of sandwich ELISA for detecting extracellular vesicles using a recombinant protein-immobilized microplate A sandwich ELISA was performed to detect extracellular vesicles (small EVs) prepared from the culture supernatant of 293T cells (condition a in Table 5) in Reference Example 1 using a microplate onto which the recombinant protein rPKCaC2-GST (SEQ ID NO: 7) or rPKCgC2-GST (SEQ ID NO: 23) produced in Example 11 was immobilized and a commercially available biotin-labeled anti-tetraspanin antibody (CD9, Frontier Laboratories). The sandwich ELISA procedure was the same as in Example 12. The evaluation conditions and results are shown in Table 10.

[0170] [Table 10]

[0171] The calibration curves prepared for the recombinant proteins rPKCaC2-GST and rPKCgC2-GST are shown in Figures 3 and 4, respectively. Under the sandwich ELISA conditions in this Reference Example, it was demonstrated that extracellular vesicles could be quantitatively detected in the range of 1 ng to 1000 ng.

[0172] [Example 15] Isolation of extracellular vesicles using a composite lipid-binding carrier with a polyethylene glycol-immobilized carrier (1) Production of recombinant proteins A recombinant protein rPKCaC2-6HC was produced by adding a polyhistidine sequence consisting of six histidine residues and an amino acid sequence (SEQ ID NO: 39) consisting of one cysteine ​​residue to the C-terminus of the recombinant protein rPKCaC2 using methods similar to those in Examples 1 and 11. Similarly, a recombinant protein rPKCaC2-GST-6HC was produced by adding a polyhistidine sequence consisting of six histidine residues and an amino acid sequence (SEQ ID NO: 39) consisting of one cysteine ​​residue to the C-terminus of the recombinant protein rPKCaC2 using methods similar to those in Examples 2 and 11. The two proteins produced were subjected to ultrafiltration using an AMICON ULTRA-0.5, 10 KDa (Merck) to dissolve the residues in D-PBS(-) (Fujifilm Wako Pure Chemical Industries, Ltd.).

[0173] (2) Preparation of polyethylene glycol-immobilized reactive insoluble carrier Sephadex G-25 Fine (Cytiva, particle size 20 μm to 80 μm) was used after swelling with water and filtering through a glass filter. 2.5 g (wet weight after filtration) of Sephadex G-25 Fine (Cytiva, 5.0 mL of water), 0.5 mL of tetraethylene glycol diglycidyl ether (Denacol EX-821, Nagase ChemteX), and 52 μL of 48% (w / v) aqueous NaOH solution were added to a 100 mL polytetrafluoroethylene (PTFE) container (final concentration: 0.5% (w / v)). Epoxidation was then carried out by shaking in a shaker at 50 °C and 120 rpm for 6 hours.

[0174] After the reaction was complete, the carrier was washed with water on a glass filter until the filtrate became neutral, and then the entire amount of epoxidized Sephadex G-25 Fine was added to a 100 mL PTFE container. Next, 5.0 mL of water and 166.5 μL of ethylenediamine (concentration: 0.5 M, prepared from anhydrous ethylenediamine manufactured by Tokyo Chemical Industry Co., Ltd.) were added to the reaction container, and the mixture was shaken in a shaker at 40 °C and 120 rpm for 15 hours to carry out amination. After the reaction was complete, the carrier was washed with water on a glass filter until the filtrate became neutral, and then the entire amount of aminated Sephadex G-25 Fine was added to a 100 mL PTFE container.

[0175] Next, 5.0 mL of a DMSO solution (concentration: 10 mg / mL) of N-succinimidyl 3-maleimidopropionate (Fujifilm Wako Pure Chemical Industries, Ltd.) was added to the reaction vessel, and the mixture was shaken in a shaker at 35°C and 120 rpm for 4 hours to perform maleimidation. After the reaction was complete, the carrier was washed three times with 20 mL of DMSO and five times with 30 mL of water on a glass filter to produce the desired polyethylene glycol-immobilized reactive insoluble carrier. Maleimidated Sephadex G-25 Fine was then prepared.

[0176] (3) Preparation of complex lipid-bound carriers A solution of recombinant protein rPKCaC2-6HC and recombinant protein rPKCaC2-GST-6HC used in the production of composite lipid-bound carriers was diluted in D-PBS(-) and tris(2-carboxyethyl)phosphine (TCEP) solution (concentration: 0.1 M) was added to a final concentration of 0.5 mM, and the mixture was left at room temperature for 1 hour for reduction to prepare a recombinant protein solution for the production of composite lipid-bound carriers.

[0177] A 50% (w / v) slurry (100 μL) of maleimide-modified Sephadex G-25 Fine was added to a reaction vessel (Mini Biospin chromatography column, Bio-Rad) (gel volume: 50 μL). 150 μL of immobilization buffer (0.2 M Na3PO4, 0.5 M NaCl, 20 mM EDTA, pH 7.4) was added, and the reactive insoluble carrier was replaced with the buffer by centrifugation (this procedure was repeated four times).

[0178] Next, 50 μL of the previously prepared recombinant protein solution for producing the composite lipid-bound carrier (concentration: 0.5 mg / mL-carrier) was added to the reaction vessel containing the maleimide-modified Sephadex G-25 Fine, and the mixture was stirred and shaken overnight at 4°C to produce Sephadex G-25 Fine on which the recombinant protein rPKCaC2-6HC was immobilized (hereinafter referred to as rPKCaC2-Sephadex) and Sephadex G-25 Fine on which the recombinant protein rPKCaC2-GST-6HC was immobilized (hereinafter referred to as rPKCaC2-GST-Sephadex).

[0179] After the immobilization reaction, the reaction vessel was spun down to recover the reaction solution. Subsequently, 150 μL of D-PBS(-) was added to the reaction vessel, and the vessel was stirred and shaken for 5 minutes. This process was repeated three times to recover a total of 0.5 mL of reaction solution and wash solution. The concentration of the recombinant protein in the recovered wash solution was measured using a Micro BCA Protein Assay Kit (Thermo Fisher Scientific). The amount of recombinant protein immobilized per mL of carrier was calculated by subtracting the amount of recombinant protein recovered from the amount of recombinant protein charged during carrier production. As shown in Table 11, the amount of rPKCaC2-6HC immobilized on rPKCaC2-Sephadex was 0.34 mg / mL, and the amount of rPKCaC2-GST-6HC immobilized on rPKCaC2-GST-Sephadex was 0.24 mg / mL, demonstrating that the desired complex lipid-bound carriers were produced.

[0180] [Table 11]

[0181] (4) Isolation of extracellular vesicles using conjugated lipid-binding carriers [4-1] Washing of the conjugated lipid-binding carrier A 50% (w / v) slurry (40 μL) of rPKCaC2-Sephadex and rPKCaC2-GST-Sephadex prepared in (3) above was added to a 1.5 mL tube (carrier volume: 20 μL). 150 μL of TBS solution containing 2 mM CaCl2 (TBS solution composition: 137 mM NaCl, 2.68 mM KCl, 25 mM Tris-HCl, pH 8.0) was added, and the carrier was washed by tapping, spinning down, and discarding the supernatant four times. As a control, the same procedure was performed on Sephadex G-25 Fine without immobilized recombinant protein.

[0182] [4-2] Adsorption of extracellular vesicles onto composite lipid-binding carriers Next, 20 μL of a suspension containing purified human extracellular vesicles (lyophilized exosomes from K562 cell culture supernatant, manufactured by HansaBioMed) (a mixture of 2 μL of the exosomes and 18 μL of TBS solution containing 2 mM calcium chloride) was added to the washed rPKCaC2-Sephadex and rPKCaC2-GST-Sephadex, and the mixture was incubated for 3 hours using a tube rotator (manufactured by AS ONE, model number TR-350) placed in a refrigerator at 4 ° C. As a control, the same procedure was also performed on Sephadex G-25 Fine without immobilized recombinant protein.

[0183] After the reaction, the carrier (rPKCaC2-Sephadex, rPKC aC2-GST-Sephadex and Sephadex without recombinant protein immobilization Adex G-25 Fine) was allowed to settle, and the supernatant was recovered (recovered solution 1).

[0184] [4-3] Recovery of extracellular vesicles adsorbed on conjugated lipid-binding carriers by chelating agent treatment Next, 150 μL of TBS-T solution containing 2 mM calcium chloride (composition of TBS-T solution: 137 mM NaCl, 2.68 mM KCl, 25 mM Tris-HCl, 0.05% Tween 20 (trade name), pH 8.0) was added, and the carrier was washed by repeating the process of tapping, separation by spinning down, and discarding the supernatant four times.

[0185] Next, 20 μL of TBS solution containing 5 mM EDTA was added to the washed carrier as an eluent, and the mixture was suspended in a vortex mixer, spun down in a tabletop centrifuge, and left to stand at room temperature for 15 minutes. After 15 minutes, the mixture was suspended again in a vortex mixer, the carrier was precipitated by spinning down, and the supernatant was collected (recovery solution 2). After collecting recovery solution 2, the following series of steps were repeated: adding 20 μL of TBS solution containing 5 mM EDTA to the carrier, suspending in a vortex mixer, spinning down in a tabletop centrifuge, leaving the mixture at room temperature for 15 minutes, resuspending in a vortex mixer, and precipitating the carrier by spinning down. The supernatant was collected (recovery solution 3).

[0186] Next, 20 μL of 2% (w / v) sodium dodecyl sulfate (SDS) aqueous solution was added to the carrier as an eluent, and the mixture was suspended in a vortex mixer, spun down, and allowed to stand at room temperature for 15 minutes. After 15 minutes, the mixture was suspended again in a vortex mixer and spun down to precipitate the carrier, and the supernatant was collected (recovery solution 4).

[0187] [4-4] Western blotting of recovered liquid Next, Recovery Solutions 1 to 4 were denatured by treatment at 95°C for 5 minutes, and then electrophoresed at 30 mA for 55 minutes using a commercially available polyacrylamide gel for electrophoresis (e-PAGEL E-R520L, manufactured by ATTO). EzProtein Ladder (WSE-7020, manufactured by ATTO) was used as a molecular weight marker for electrophoresis.

[0188] After electrophoresis, the gel was washed with transfer buffer (EzFastBlot, ATTO). A filter paper soaked in transfer buffer was placed on a blotting apparatus (PoweredBlot Ace, WSE-4115, ATTO). A PVDF membrane (Clear Blot P Plus membrane, WSE-4051, ATTO), gel, and filter paper soaked in transfer buffer were then placed on top of the filter paper. The gel was transferred to the PVDF membrane in Fast mode for 10 minutes. After transfer, the PVDF membrane was washed with TBS-T and blocked overnight at 4°C with TBS-T containing 3% (w / v) skim milk.

[0189] The blocked PVDF membrane was then placed in a polypropylene container with an anti-CD81 antibody (Novus Biologicals, product code: NB100-65805H, HRP (horseradish peroxidase)-conjugated) solution prepared using TBS-T solution containing 3% (w / v) skim milk. The membrane was then shaken at room temperature for 1 hour to bind to the antibody. After the antibody reaction, the PVDF membrane was immersed in TBS-T solution to wash away unreacted antibody, and the membrane was shaken at room temperature for 5 minutes three times.

[0190] After washing, bands representing extracellular vesicles were detected by the luminescence of an HRP detection reagent (EzWestLumiOne, WSE-7110, ATTO) using a high-resolution chemiluminescence analyzer, GeneGnome (GGNOME-XRQ-NPC, SYNGENE) (Figures 5 and 6).

[0191] As is clear from Figures 5 and 6, when Sephadex G-25 Fine, on which no recombinant protein was immobilized, was used, a band derived from extracellular vesicles was detected in recovery solution 1 (lane number 2 in Figures 5 and 6), which was recovered immediately after contact with extracellular vesicles, demonstrating that extracellular vesicles do not nonspecifically adsorb to the carrier.

[0192] On the other hand, in the case of rPKCaC2-Sephadex and rPKCaC2-GST-Sephadex, almost no bands derived from extracellular vesicles were detected in recovery solution 1 (lane 7 in Figures 5 and 6), which was collected immediately after contact with extracellular vesicles. However, bands derived from extracellular vesicles were confirmed in recovery solution 2 (lane 8 in Figures 5 and 6), which was collected after adding a buffer containing the chelating agent EDTA. This demonstrates that extracellular vesicles adsorbed to the composite lipid-binding carrier can be recovered by treatment with a chelating agent.

[0193] [Reference Example 5] Evaluation of phosphatidylserine binding ability of recombinant proteins According to the method described in the literature (Immunity, Vol. 27, pp. 927-940, 2007), the binding ability of the six recombinant proteins produced in Example 11 (rPKCaC2 (SEQ ID NO: 5), rPKCbC2 (SEQ ID NO: 17), rPKCgC2 (SEQ ID NO: 21), rPKCaC2-GST (SEQ ID NO: 7), rPKCbC2-gs-GST (SEQ ID NO: 19), and recombinant protein rPKCgC2-GST (SEQ ID NO: 23)) to phosphatidylserine (hereinafter referred to as PS) was evaluated by solid-phase ELISA.

[0194] A 25 μg / mL PS solution (Sigma-Aldrich 1,2-Diacyl-sn-glycero-3-phospho-L-serine dissolved in chloroform to a concentration of 5 mg / mL, then diluted 200-fold with methanol) was added at 100 μL / well to an ELISA plate (MaxiSorp 96-well plate, Thermo Fisher Scientific), and the plate was air-dried to immobilize PS onto the plate.

[0195] Next, 200 μL / well of 20 mM Tris-HCl (pH 7.5) containing 0.5% (w / v) BSA was added and the plate was left at 30°C for 2 hours to block the plate. After that, 350 μL / well of Buffer W (0.05% (w / v) Tween 20 (trade name), 150 mM NaCl, 20 mM Tris-HCl, pH 7.5) was added and the plate was washed a total of three times.

[0196] Next, the six recombinant proteins diluted with buffer A (20 mM Tris-HCl, pH 7.5, containing 2 mM CaCl2) were added and heated at 30°C for 90 minutes. After that, 350 μL / well of buffer WC (20 mM Tris-HCl, pH 7.5, containing 0.05% (w / v) Tween 20 (trade name), 150 mM NaCl, and 2 mM CaCl2) was added and washed three times.

[0197] Next, 100 μL / well of HRP-labeled anti-6-His antibody (manufactured by Bethyl Laboratories, diluted 5000-fold with 0.5% (w / v) BSA-containing buffer WC) was added, and the plate was heated at 30°C for 1.5 hours. After that, 350 μL / well of buffer WC was added and the plate was washed a total of three times.

[0198] TMB Microwell Peroxidase Substrate (KPL) was added at 50 μL / well to develop color, and then 1 M phosphoric acid was added at 50 μL / well to stop the reaction, and the absorbance at 450 nm was measured.

[0199] Figure 7 shows the results of solid-phase ELISA for the recombinant proteins rPKCaC2 (sequence number 5), rPKCbC2 (sequence number 17), and rPKCgC2 (sequence number 21), and Figure 8 shows the results for the recombinant proteins PKCaC2-GST (sequence number 7), rPKCbC2-gs-GST (sequence number 19), and rPKCgC2-GST (sequence number 23). The absorbance of each recombinant protein increased in a concentration-dependent manner, demonstrating that all six recombinant proteins evaluated bound to PS.

[0200] [Reference Example 6] Evaluation of lipid binding properties of recombinant proteins The binding of the recombinant proteins rPKCaC2 (SEQ ID NO: 5) and rPKCbC2 (SEQ ID NO: 17) produced in Example 11 to complex lipids was evaluated using commercially available membrane lipid strips (P-6002, Echelon Biosciences) according to the method described in the literature (Immunity, Vol. 27, pp. 927-940, 2007).

[0201] The membrane lipid strip and 10 mL of Blocking Buffer (20 mM Tris-HCl (pH 7.5), 150 mM NaCl, 3% (w / v) BSA) were placed in a container and shaken at room temperature for 1 hour to block the membrane lipid strip.

[0202] Next, after discarding the blocking buffer, 9 mL of recombinant protein solution (blocking buffer containing 0.5 μg / mL recombinant protein and 2 mM CaCl 2 ) was added and the mixture was shaken at room temperature for 1 hour to bind the recombinant protein to the membrane lipid strip.

[0203] Next, after discarding the recombinant protein solution, 10 mL of washing solution (20 mM Tris-HCl (pH 7.5), 150 mM NaCl, 0.05% (w / v) Tween 20 (trade name), 2 mM CaCl) was added and washed by shaking at room temperature for 10 minutes. This washing procedure was repeated three times.

[0204] After three washes, the washing solution was discarded, and then 10 mL of an anti-polyhistidine tag antibody solution (Rabbit anti-6-His Antibody HRP conjugated (hereinafter also referred to as "anti-6-His antibody"), catalog number A190-114P, Bethyl Laboratories) and a blocking buffer containing 2 mM CaCl2 were added and shaken at room temperature for 1 hour to allow the anti-6-His antibody to bind to the recombinant protein bound to the membrane lipid strip.

[0205] After shaking for 1 hour, the excess anti-6-His antibody was discarded, and 10 mL of the washing solution was added. The membrane lipid strip was washed by shaking at room temperature for 10 minutes, which was repeated three times.

[0206] After washing, the membrane lipid strip was subjected to high-resolution chemiluminescence detection using a dedicated instrument (GeneGnome, manufactured by SYNGENE) to detect the recombinant proteins bound to the membrane lipid strip by luminescence from an HRP detection reagent (EzWestLumiOne, model number WSE-7110, manufactured by ATTO).

[0207] The results of the lipid-binding assay for the recombinant protein rPKCaC2 (SEQ ID NO: 5) are shown in Figure 9, and the results of the lipid-binding assay for rPKCbC2 (SEQ ID NO: 17) are shown in Figure 10. In Figures 9 and 10, the darkly colored spots indicate the binding of the recombinant protein. As is clear from Figure 9, the recombinant protein rPKCaC2 also binds to phosphatidylserine (PS), phosphatidylinositol 4-phosphate, phosphatidylinositol 4,5-bisphosphate, sulfatide, and cardiolipin. As is also clear from Figure 10, the recombinant protein rPKCbC2 strongly binds to phosphatidylserine (PS) and also to phosphatidylinositol 4-phosphate and cardiolipin.

[0208] Example 16: Expression vector for fusion protein of protein containing the amino acid sequence of the C2 domain of protein kinase α and SpyCatcher, Escherichia coli BL21(DE3) transformant thereof, and production of the fusion protein [1] Construction of expression vector pPKCaC2-SpyC and its transformant EC / pPKCaC2-SpyC First, we constructed an expression vector for producing rPKCaC2-SpyC, a fusion protein of the C2 domain of PKCα and SpyCatcher (SpyC). The amino acid sequence of rPKCaC2-SpyC is SEQ ID NO: 31 (positions 3 to 141 correspond to the amino acid sequence of SEQ ID NO: 1, positions 146 to 159 correspond to a GS linker (SEQ ID NO: 41), positions 164 to 279 correspond to SpyCatcher (116 amino acid residues of Chains A and B of Protein Data Bank registration number 4MLI), and positions 282 to 287 correspond to a polyhistidine sequence). The nucleotide sequence encoding this amino acid (SEQ ID NO: 32) was inserted into the multicloning site of pET28a(+) (Merck) to construct the vector pPKCaC2-SpyC, which can express rPKCaC2-SpyC in E. coli. Furthermore, Escherichia coli BL21(DE3) was transformed with the expression vector pPKCaC2-SpyC to generate a transformant EC / pPKCaC2-SpyC capable of producing the recombinant protein rPKCaC2-SpyC.

[0209] [2] Production of recombinant protein rPKCaC2-SpyC The transformant EC / pPKCaC2-SpyC prepared in [1] above was cultured for expression and a recombinant protein was prepared by the method described in Example 11. The prepared protein was ultrafiltered using an AMICON ULTRA-0.5, 10 KDa (Merck) and replaced with TBS buffer (25 mM Tris-HCl, 137 mM NaCl, 26.8 mM KCl, pH 8.0).

[0210] [Preparation Example 1] Preparation of immobilization assisting protein [1] Preparation of Cys-(SpA-Z)-3SpyT [1-1] Construction of expression vector pGEX_GST-Cys-(SpA-Z)-3SpyT and its transformant EC / pGEX_GST-Cys-(SpA-Z)-3SpyT The expression vector pGEX_GST-Cys-(SpA-Z)-1SpyT contains the Z domain of Protein A from Staphylococcus aureus (SpA-Z, a polypeptide consisting of amino acid residues 4 to 61 of GenBank Accession No. AL052730) with a cysteine ​​residue (Cys) for biotin labeling added to the N-terminus, and SpyTag (Chain 1 of Protein Data Bank Accession No. 4MLI). This is an expression vector for producing the fusion protein Cys-(SpA-Z)-3SpyT with a polypeptide containing three copies of SpA-B [13 residues], SpyT) arranged in tandem. The amino acid sequence is SEQ ID NO: 33, and the base sequence encoding this amino acid (SEQ ID NO: 34) was inserted into the multicloning site of the pBR322-based plasmid pGEX (Cytiva) to produce the vector pGEX_GST-Cys-(SpA-Z)-3SpyT, which can express Cys-(SpA-Z)-3SpyT in E. coli.

[0211] pGEX contains a polynucleotide encoding GST (Glutathione S-transferase, a polypeptide consisting of amino acid residues 1 to 218 of GenBank Accession Number QLV95778) and a protease recognition site upstream of the multiple cloning site. The expression vector pGEX_GST-Cys-(SpA-Z)-3SpyT constructed by this insertion is a vector capable of expressing the fusion protein GST-Cys-(SpA-Z)-3SpyT (SEQ ID NO: 35) of GST and Cys-(SpA-Z)-3SpyT in E. coli. The nucleotide sequence encoding GST-Cys-(SpA-Z)-3SpyT is shown in SEQ ID NO: 36.

[0212] Escherichia coli BL21 was transformed with the expression vector pGEX_GST-Cys-(SpA-Z)-3SpyT to generate the transformant EC / pGEX_GST-Cys-(SpA-Z)-3SpyT, which is capable of producing the recombinant protein GST-Cys-(SpA-Z)-3SpyT.

[0213] [1-2] Production of recombinant proteins The Escherichia coli transformant EC / pGEX_GST-Cys-(SpA-Z)-3SpyT prepared in [1-1] was inoculated into LB medium supplemented with 60 μg / mL of carbenicillin and pre-cultured by shaking at 37°C overnight. The pre-culture solutions were inoculated into LB medium supplemented with 60 μg / mL of carbenicillin and cultured with shaking at 37°C. The turbidity (OD 600 When the chromatogram (σ) reached approximately 0.6, the culture temperature was changed to 30°C, 0.5 mM IPTG was added, and the culture was continued for 5 hours to express the recombinant protein GST-Cys-(SpA-Z)-3SpyT. Soluble protein extracts were collected from each strain by ultrasonic disruption or using a BugBuster Protein extraction kit (Merck).

[0214] The recombinant protein was purified from the soluble protein extract by affinity chromatography using a glutathione-immobilized support. The bound GST-Cys-(SpA-Z)-3SpyT was digested with GST-fused HRV 3C protease (Merck) according to the manufacturer's protocol, and the supernatant was collected to obtain a Cys-(SpA-Z)-3SpyT (SEQ ID NO: 33) solution. The resulting protein was transferred to D-PBS(-) (Fujifilm Wako Pure Chemical Industries) by ultrafiltration using an AMICON ULTRA-0.5, 10 KDa (Merck).

[0215] Preparation of [2](BC2LCNm2)-3SpyT [2-1] Construction of expression vector p(BC2LCNm2)-3SpyT and its transformant EC / p(BC2LCNm2)-3SpyT The expression vector p(BC2LCNm2)-3SpyT is used to produce a fusion protein (BC2LCNm2)-3SpyT consisting of the mutant BC2LCN lectin BC2LCNm2 (GenBank Accession No. WP_006490828), which forms a homotrimer of a polypeptide consisting of amino acid residues 2 through 156, except that the glutamine at position 40 is replaced by leucine and the glutamic acid at position 82 is replaced by cysteine ​​(for biotin labeling)). The amino acid sequence of this protein is SEQ ID NO: 37. The nucleotide sequence (SEQ ID NO: 38) encoding this amino acid sequence was inserted into the multicloning site of pET28a(+) (Merck) to produce the vector p(BC2LCNm2)-3SpyT, which can express (BC2LCNm2)-3SpyT (SEQ ID NO: 37) in E. coli. Escherichia coli BL21(DE3) was transformed with the expression vector p(BC2LCNm2)-3SpyT to generate a transformant EC / p(BC2LCNm2)-3SpyT capable of producing the recombinant protein (BC2LCNm2)-3SpyT.

[0216] [2-2] Production of recombinant proteins The Escherichia coli transformant EC / p(BC2LCN-m2)-3SpyT prepared in [2-1] was inoculated into LB medium (10 g / L tryptone, 5 g / L yeast extract, and 5 g / L NaCl) supplemented with 30 μg / mL carbenicillin and precultured overnight at 37°C with shaking. The preculture was then inoculated into Terrific broth (TB) medium (24 g / L yeast extract, 12 g / L tryptone, 9.4 g / L K2HPO4, 2.2 g / L KH2PO4, and 4 mL / L glycerol) supplemented with 30 μg / mL kanamycin and cultured at 37°C with shaking.

[0217] The turbidity of the culture medium (OD 600When the pH reached approximately 0.6, the culture temperature was changed to 20°C, 0.1 mM IPTG was added, and the mixture was cultured overnight to express the recombinant protein (BC2LCN-m2)-3SpyT. The recombinant protein (BC2LCN-m2)-3SpyT was prepared from the collected cells in the same manner as in Example 11, and the solution was replaced with D-PBS(-) to obtain a (BC2LCN-m2)-3SpyT (SEQ ID NO: 37) solution.

[0218] [Example 17] Isolation of extracellular vesicles from a solution containing extracellular vesicles using a conjugated lipid-binding carrier with an immobilized auxiliary protein [1] Preparation of a recombinant protein construct containing the amino acid sequence of the C2 domain of PKCα and an immobilization assisting protein. The immobilization assisting proteins Cys-(SpA-Z)-3SpyT (sequence number 33) and (BC2LCN-m2)-3SpyT (sequence number 37) prepared in Preparation Example 1 were each replaced with D-PBS(+) buffer using an ultrafiltration filter, and then the sulfhydryl groups of the cysteine ​​residues present in the immobilization assisting proteins were labeled with biotin using EZ-Link Maleimide-PEG2-Biotin (manufactured by Thermo Fisher Scientific) according to the manufacturer's protocol.

[0219] The prepared biotin-labeled immobilization assisting protein was bound to the recombinant protein rPKCaC2-SpyC (SEQ ID NO: 31) containing the amino acid sequence of the C2 domain of PKCα prepared in Example 16 by the method described below to prepare a construct in which the immobilization assisting protein and rPKCaC2-SpyC were bound (hereinafter referred to as the "PKCα C2 domain construct") and immobilized on an insoluble support. The combinations of rPKCaC2-SpyC and immobilization assisting proteins used to prepare the PKCα C2 domain construct and the resulting PKCα C2 domain constructs are summarized in Table 12.

[0220] [Table 12]

[0221] Based on the combinations shown in Table 12, the biotin-labeled immobilized assisting proteins were bound to the recombinant protein rPKCaC2-SpyC prepared in Example 16. Specifically, the biotin-labeled immobilized auxiliary proteins Cys-(SpA-Z)-3SpyT (SEQ ID NO: 33) and (BC2LCNm2)-3SpyT (SEQ ID NO: 37) and the recombinant protein rPKCaC2-SpyC (SEQ ID NO: 31) were suspended in D-PBS(+) or TBS-T (25 mM Tris-HCl (pH 7.2) containing 0.15 M NaCl and 0.05% (w / v) Tween 20 (trade name)) at a concentration of 0.3 mg / mL or higher, respectively. The biotin-labeled immobilized auxiliary proteins and rPKCaC2-SpyC solutions were mixed in the combination described in Condition a or b of Example 17 in Table 12, and then allowed to stand at 4°C for at least one day. This produced a PKCαC2 domain structure through a binding reaction between the protein tags SpyT and SpyC.

[0222] The molar ratio of the mixture was 1 part biotin-labeled immobilized support protein to 9 parts rPKCaC2-SpyC. The recombinant protein rPKCaC2-SpyC, which contains the SpyC sequence, was added in excess to ensure sufficient binding between the SpyT protein tag and SpyC. Because the immobilized support proteins Cys-(SpA-Z)-3SpyT and (BC2LCNm2)-3SpyT contain three copies of the SpyTag sequence per molecule, a molar ratio of 3 parts SpyC recombinant protein rPKCaC2-SpyC containing the SpyC sequence was required to bind to all SpyTag sequences within the molecule. To ensure smooth binding, 9 parts rPKCaC2-SpyC was added, which is three times the required molar ratio.

[0223] The binding reaction between SpyT and SpyC results in a PKCα C2 domain structure formed by the binding of the biotin-labeled immobilized auxiliary protein Cys-(SpA-Z)-3SpyT and the recombinant protein rPKCaC2-SpyC (condition a, Example 17, Table 12, rPKCaC2 / 3SpyT-Biotin), in which three molecules of the C2 domain of PKCα are bound per molecule of immobilized auxiliary protein. Furthermore, the PKCα C2 domain structure formed by the binding of biotin-labeled immobilized auxiliary protein (BC2LCNm2)-3SpyT and the recombinant protein rPKCaC2-SpyC (condition b of Example 17 in Table 12, rPKCaC2 / 3SpyT_BC2LCNm2-Biotin) has three molecules of the C2 domain of PKCα bound to one molecule of immobilized auxiliary protein, and the amino acid sequence of the BC2LCN lectin contained in the immobilized auxiliary protein forms a homotrimeric structure, resulting in a nonameric PKCα C2 domain structure.

[0224] [2] Preparation of complex lipid-binding carriers Magnoshere MS300 / Streptavidin (manufactured by JSR Life Sciences), which are streptavidin-immobilized magnetic microparticles, was used as the insoluble carrier, and 100 μL of a slurry solution of the magnetic microparticles (microparticle content 1% (w / v)) was collected, and the supernatant was removed.

[0225] The supernatant was removed from the magnetic particles, which were then washed with a fixing buffer (10 mM Tris-HCl (pH 7.4) containing 0.5 mM EDTA, 1 M NaCl, and 0.05% (w / v) Tween 20 (trade name)).

[0226] The PKCαC2 domain structure rPKCaC2 / 3SpyT-Biotin (condition a of Example 17 in Table 12) or rPKCaC2 / 3SpyT_BC2LCNm2-Biotin (condition b of Example 17 in Table 12) prepared in [1], which had been previously replaced with an immobilization buffer using an ultrafiltration membrane or the like, was adjusted to a concentration of 0.1 mg / mL or more in terms of the concentration of immobilization assisting protein, and then 4.5 μg in terms of the amount of immobilization assisting protein was added to the washed magnetic microparticles and mixed at room temperature for 10 minutes.

[0227] The supernatant was removed, and the mixture was washed three times with immobilization buffer, followed by one wash with exosome binding buffer (10 mM HEPES [4-(2-HydroxyEthyl)-1-PiperazineEthaneSulfonic acid], pH 7.3) to produce a carrier with the PKCαC2 domain structure immobilized.

[0228] As controls, biotin-labeled immobilized auxiliary proteins Cys-(SpA-Z)-3SpyT and (BC2LCNm2)-3SpyT, which were not bound to the PKCαC2 domain structure, were immobilized to magnetic microparticles in a similar manner to prepare immobilized carriers of biotin-labeled immobilized auxiliary proteins 3SpyT-Biotin (condition c of Comparative Example 6 in Table 12) and 3SpyT_BC2LCNm2-Biotin (condition d of Comparative Example 6 in Table 12).

[0229] [3] Preparation of extracellular vesicle solution Prostate cancer cell line (PC3 cells) was cultured at 37°C in Ham's F-12K medium (Fujifilm Wako Pure Chemical Industries, Ltd.) supplemented with 15% fetal bovine serum (FBS). 2.5 × 10 5 The cells were suspended in Ham's F-12K medium to a concentration of 100 cells / mL, then seeded and suspended in a 6-well plate at 2 mL / well, and further cultured for 3 days.

[0230] After incubation, the entire culture supernatant (approximately 2 mL) was collected and centrifuged at 300 × G for 10 minutes at room temperature to remove floating cells, and 1.5 mL of the supernatant was collected. The collected supernatant was further centrifuged at 3,000 × G for 10 minutes at 4°C to remove cell debris, and 1.2 mL of the supernatant was collected and further centrifuged at 16,000 × G for 60 minutes at 4°C. 1 mL of the supernatant was transferred to another tube.

[0231] The supernatant (1 mL) transferred to another tube was mixed with 1 mL of phosphate buffered saline (PBS) and ultracentrifuged at 259,000 × G for 70 minutes at 4°C. 1.8 mL of the supernatant was removed. The remaining precipitate was suspended in 1.8 mL of PBS and washed by ultracentrifugation at 259,000 × G for 70 minutes at 4°C. 1.8 mL of the supernatant was removed, and 0.2 mL of the remaining precipitate was used as the extracellular vesicle solution.

[0232] [4] Isolation of extracellular vesicles To a container containing the PKCαC2 domain structure immobilization carrier prepared in [2] above (conditions a and b of Example 17 in Table 12) and the immobilization assistant protein immobilization carrier of Comparative Example 6 (conditions c and d of Comparative Example 6 in Table 12), 20 μL of the extracellular vesicle solution prepared in [3] above was added, and the container was then shaken at 10° C. for 3 hours. After shaking, 7.5 μL of the supernatant was collected and designated as the "supernatant."

[0233] After removing the remaining supernatant, the mixture was washed twice with TBS-T buffer. 7.5 μL of 5% (w / v) EDTA aqueous solution was added, and the mixture was vigorously stirred using a vortex mixer. 7.5 μL of the supernatant was collected and used as the "EDTA eluate."

[0234] After removing the remaining supernatant, 7.5 μL of 2% (w / v) SDS aqueous solution was added, and the mixture was again stirred using a vortex mixer. 7.5 μL of 2x sample buffer (DTT-free) (ATTO) was then added, and the entire volume was recovered. This was designated as the "SDS eluate."

[0235] The supernatant, EDTA eluate, and SDS eluate were heat-treated and then electrophoresed using polyacrylamide gel (e-PAGEL E-R520L, ATTO). Western blotting was performed as in (4)[4-4]. However, the detection antibodies for extracellular vesicles were anti-CD9 mouse antibody as the primary antibody and HRP-conjugated anti-mouse antibody as the secondary antibody.

[0236] The results of extracellular vesicle separation using the immobilization carriers of Example 17 and Comparative Example 6 are shown in Figure 11.

[0237] When extracellular vesicles were eluted from the composite lipid-binding carrier immobilized with the biotin-labeled trimer PKCαC2 domain construct rPKCaC2 / 3SpyT-Biotin (Example 17, Condition a, Table 12) using an EDTA eluate containing the chelating agent EDTA (lane 5), a clear band corresponding to the extracellular vesicles contained in the extracellular vesicle solution (lane 4) was confirmed, indicating that the composite lipid-binding carrier of Example 17 adsorbed the extracellular vesicles contained in the extracellular vesicle solution. Furthermore, when the eluate containing the extracellular vesicles was eluted from the composite lipid-binding carrier using EDTA and then the adsorbed proteins on the carrier surface were eluted using an SDS eluate containing the denaturant SDS (lane 6), almost no band corresponding to the extracellular vesicles was confirmed, indicating that most of the extracellular vesicles adsorbed to the composite lipid-binding carrier could be recovered using EDTA.

[0238] On the other hand, when cell separation was performed using a carrier prepared as a control, on which only the biotin-labeled immobilized auxiliary protein 3SpyT-Biotin was immobilized (condition c of Comparative Example 6 in Table 12), almost no band corresponding to the extracellular vesicles (lane number 1) contained in the extracellular vesicle solution was observed in the EDTA eluate (lane number 2) and SDS eluate (lane number 3), indicating that the extracellular vesicles do not nonspecifically adsorb to the insoluble carrier and 3SpyT-Biotin used.

[0239] The composite lipid-binding carrier on which the biotin-labeled nonamer PKCαC2 domain construct rPKCaC2 / 3SpyT_BC2LCNm2-Biotin of Example 17 was immobilized (condition b of Example 17 in Table 12) and its control carrier on which only the immobilization auxiliary protein was immobilized (condition d of Comparative Example 6 in Table 12) also showed results similar to those of the triamer PKCαC2 domain construct. The signal intensity of the band in the EDTA eluate (lane 11) of the nonamer PKCαC2 domain construct-immobilized carrier was significantly stronger than that of the supernatant (lane 10) and the SDS eluate (lane 12), indicating that extracellular vesicles were adsorbed to the nonamer PKCαC2 domain construct-immobilized carrier and that most of them were recovered by EDTA.

[0240] On the other hand, when cell separation was performed using a control carrier immobilized with only the biotin-labeled immobilized support protein 3SpyT_BC2LCNm2-Biotin (condition d, Comparative Example 6, Table 12), a band was observed in the EDTA eluate (lane 8), and this band had a stronger signal intensity than the carrier immobilized with the control for the trimeric PKCα C2 domain construct (condition c, lane 2, Comparative Example 6), suggesting some nonspecific adsorption on the carrier immobilized with 3SpyT_BC2LCNm2-Biotin. However, the amount of adsorbed extracellular vesicles was greater on the carrier containing the PKC C2 domain (condition b, Example 17, Table 12), demonstrating that extracellular vesicles can also be adsorbed and recovered on the composite lipid-binding carrier immobilized with the nonameric PKCα C2 domain construct.

[0241] [Example 18] Isolation of extracellular vesicles from cell culture supernatant using a composite lipid-binding carrier containing an immobilized auxiliary protein [1] A recombinant protein structure containing an immobilization auxiliary protein and the amino acid sequence of the C2 domain of a protein kinase, and its immobilization carrier In the same manner as in Examples 17[1] and [2], the kinase C2 domain construct PKCaC2 / 3SpyT (condition a of Example 17 in Table 12) and the immobilization assistant protein Cys-(SpA-Z)-3SpyT (condition c of Comparative Example 6 in Table 12) were prepared.

[0242] [2] Immobilization of the PKCαC2 domain structure PKCaC2 / 3SpyT-Biotin on an insoluble carrier Magnoshere MS300 / Streptavidin (JSR Life Sciences), a streptavidin-immobilized magnetic particle, was used as the insoluble carrier. 100 μL of the magnetic particle slurry (particle content 1% (w / v)) was collected, and the supernatant was removed. The magnetic particles from which the supernatant was removed were washed with immobilization buffer (10 mM Tris-HCl (pH 7.4) containing 0.5 mM EDTA, 1 M NaCl, and 0.05% (w / v) Tween 20 (trade name)) containing 2 mM CaCl2.

[0243] The solution of the PKCαC2 domain structure rPKCaC2 / 3SpyT-Biotin (condition a of Example 17 in Table 12) prepared in [1], which had been previously replaced with a fixation buffer containing CaCl2 using an ultrafiltration membrane or the like, was adjusted to a concentration of 0.1 mg / mL or more in terms of the concentration of the fixation assisting protein, and then 4.5 μg in terms of the amount of fixation assisting protein was added to the washed magnetic microparticles and mixed at room temperature for 10 minutes.

[0244] The supernatant was removed, and the mixture was washed three times with immobilization buffer containing 2 mM CaCl2, and then once with TBS-T buffer containing 2 mM CaCl2 to prepare a PKCαC2 domain structure PKCaC2 / 3SpyT-Biotin immobilized carrier.

[0245] As a control, a biotin-labeled immobilized auxiliary protein, Cys-(SpA-Z)-3SpyT, which was not bound to a recombinant protein containing the amino acid sequence of the C2 domain of PKCα, was immobilized to magnetic microparticles in a similar manner to prepare a biotin-labeled immobilized auxiliary protein, 3SpyT-Biotin immobilized carrier (condition c of Comparative Example 6 in Table 12).

[0246] [3] Preparation of cell culture supernatant Prostate cancer cell line (PC3 cells) was cultured at 37°C in Ham's F-12K medium (Fujifilm Wako Pure Chemical Industries, Ltd.) supplemented with 15% fetal bovine serum (FBS). After culturing the PC3 cells for 2 days in Ham's F-12K medium containing 15% (v / v) FBS (exosomes had been removed in advance by ultrafiltration), the culture supernatant was collected.

[0247] The collected culture supernatant was centrifuged at 300 × G for 10 minutes at room temperature to remove floating cells, and the supernatant was collected. It was further centrifuged at 1200 × G for 20 minutes at 4°C to remove cell debris, and the supernatant was collected. The collected supernatant was further centrifuged at 10,000 × G for 30 minutes at 4°C to remove large extracellular vesicles, and 1.2 mL of the supernatant was collected and centrifuged at 16,000 × G for 60 minutes at 4°C. The supernatant was collected and used as the cell culture supernatant.

[0248] [4] Isolation of extracellular vesicles 1 mL of the cell culture supernatant prepared in [3] above, supplemented with 2 mM CaCl2, was placed in a container containing the PKCαC2 domain construct PKCaC2 / 3SpyT-immobilized carrier prepared in [2] above and the carrier on which the immobilization assisting protein Cys-(SpA-Z)-3SpyT was immobilized, and the container was shaken for 3 hours at 10°C. After shaking, 7.5 μL of the supernatant was collected and designated as the "supernatant."

[0249] After removing the remaining supernatant, the mixture was washed twice with TBS-T buffer containing 2 mM CaCl. 7.5 μL of 5% (w / v) EDTA (sodium dodecyl sulfate) aqueous solution was added, and the mixture was vigorously stirred using a vortex mixer. 7.5 μL of the supernatant was collected and used as the "EDTA eluate."

[0250] After removing the remaining supernatant, 7.5 μL of 2% (w / v) SDS (Sodium Dodecyl Sulfate) aqueous solution was added, and the mixture was again stirred using a vortex mixer. 7.5 μL of 2x sample buffer (DTT-free) (ATTO) was then added, and the entire volume was recovered. This was designated as the "SDS eluate."

[0251] The supernatant, EDTA eluate, SDS eluate, and the extracellular vesicle solution prepared in Example 17[3] and the cell culture supernatant prepared in [3] as extracellular vesicle preparations were heat-treated, and then electrophoresis, Western blotting, and antibody detection of extracellular vesicles were performed in the same manner as in Example 17[4].

[0252] The results of extracellular vesicle separation using a composite lipid-binding carrier immobilized with the PKCαC2 domain structure PKCaC2 / 3SpyT-Biotin (condition a, Example 17 in Table 12) and a carrier immobilized with the immobilized auxiliary protein Cys-(SpA-Z)-3SpyT of Comparative Example 6 (condition c, Comparative Example 6 in Table 12) are shown in Figure 12.

[0253] When extracellular vesicles were eluted from the composite lipid-binding carrier immobilized with rPKCaC2 / 3SpyT-Biotin (Table 12, Example 17, Condition a) using an EDTA eluate (lane 3) containing the chelating agent EDTA, a clear band corresponding to extracellular vesicles (lane 0 in Figure 8) was observed. This demonstrated that treatment with a chelating agent allows the recovery of extracellular vesicles adsorbed to the composite lipid-binding carrier containing the PKCαC2 domain. Furthermore, the significantly stronger signal intensity than the band of extracellular vesicles contained in the cell culture supernatant (lane 1) suggests that extracellular vesicles contained in the cell culture supernatant can be selectively concentrated on the composite lipid-binding carrier. Furthermore, when an eluate containing extracellular vesicles was obtained from the above-mentioned composite lipid-binding carrier using EDTA, and then the proteins adsorbed on the carrier surface were eluted with an SDS eluate (lane number 5) containing the denaturing agent SDS, the band corresponding to extracellular vesicles was fainter than in the EDTA eluate, indicating that most of the extracellular vesicles adsorbed to the composite lipid-binding carrier could be recovered using EDTA.

[0254] On the other hand, when cell separation was performed using a carrier on which only 3SpyT-Biotin was immobilized (condition c of Comparative Example 6 in Table 12), bands corresponding to extracellular vesicles could be confirmed in the EDTA eluate (lane number 2) and SDS eluate (lane number 4), but they were fainter than when the above-mentioned composite lipid-binding carrier was used (EDTA eluate: lane number 3, SDS eluate: lane number 4).This indicates that although extracellular vesicles adsorb to a certain extent to the insoluble carrier used and the immobilization auxiliary protein immobilized on it, the amount was very small compared to the carrier on which a recombinant protein containing the amino acid sequence of the C2 domain of PKCα was immobilized.

Claims

1. A composite lipid-binding carrier comprising an insoluble carrier on which a protein comprising the C2 domain of a protein kinase and the amino acid sequence of glutathione S-transferase is immobilized, A conjugated lipid-binding carrier, wherein the protein comprising the C2 domain of a protein kinase and an amino acid sequence of glutathione S-transferase is any one of the following proteins (a) to (c): (a) A protein comprising an amino acid sequence in which the amino acid sequence shown in SEQ ID NO: 4 is added to the N-terminal and / or C-terminal sides of the amino acid sequence shown in SEQ ID NO: 1, and having binding properties to phospholipids and / or extracellular vesicles. (b) A protein having an amino acid sequence in which 1 to 20 amino acids are deleted, substituted, inserted, or added in the amino acid sequence of the protein described in (a), and having binding ability to phospholipids and / or extracellular vesicles. (c) A complex of the protein described in (a) or (b) with an immobilization assisting protein capable of binding to an insoluble carrier, and a protein having binding ability to phospholipids and / or extracellular vesicles.

2. The protein according to (a), The complex lipid-binding carrier according to claim 1, which is a protein that contains the amino acid sequence shown in SEQ ID NO: 41 between the amino acid sequence shown in SEQ ID NO: 1 and the amino acid sequence shown in SEQ ID NO: 4, and has binding ability to phospholipids and / or extracellular vesicles.

3. The complex lipid-binding carrier according to claim 1 or 2, wherein the complex lipid is a phospholipid.

4. The complex lipid-binding carrier according to claim 3, wherein the phospholipid is a phospholipid containing phosphatidylserine as a component.

5. The complex lipid-binding carrier according to any one of claims 1 to 4, wherein the extracellular vesicles are extracellular vesicles derived from human cells.

6. A detection reagent comprising the composite lipid-binding carrier according to any one of claims 1 to 5.

7. A kit for detecting extracellular vesicles, the kit being used for detecting extracellular vesicles, comprising the detection reagent according to claim 6.

8. A column packed with the composite lipid-binding carrier according to any one of claims 1 to 5.

9. A kit for isolating extracellular vesicles, comprising the column according to claim 8.

10. The method for producing a composite lipid-binding carrier according to any one of claims 1 to 5, comprising the following steps (A1) and (A2): (A1) A step of producing a reactive insoluble carrier from an insoluble carrier. (A2) A step of immobilizing the protein according to claim 1 or 2 on the reactive insoluble carrier obtained in the step (A1).

11. A method for producing a composite lipid-binding carrier according to any one of claims 1 to 5, characterized in that it comprises a step of immobilizing a protein according to any one of (a) to (c) on an insoluble carrier by physical adsorption.

12. A method for detecting phospholipids and / or extracellular vesicles, comprising the following steps (X1) to (X3): (X1) A step of contacting a composite lipid-binding carrier according to any one of claims 1 to 5 with a sample solution containing phospholipids and / or extracellular vesicles in the presence of calcium ions to obtain a complex in which the carrier is bound to phospholipids and / or extracellular vesicles. (X2) A step of separating and removing substances that did not bind to the complex lipid-binding carrier from the complex obtained in the step (X1). (X3) A step of detecting the complex obtained in the step (X1) after the step (X2) is completed.

13. 13. The detection method according to claim 12, wherein the detection method is an ELISA method or a flow cytometry method.

14. A method for isolating phospholipids and / or extracellular vesicles, comprising the following steps (Y1) to (Y3): (Y1) A step of contacting a composite lipid-binding carrier according to any one of claims 1 to 5 with a sample solution containing phospholipids and / or extracellular vesicles in the presence of calcium ions to obtain a complex in which the carrier is bound to phospholipids and / or extracellular vesicles. (Y2) A step of separating and removing substances that did not bind to the composite lipid-binding carrier from the complex obtained in the step (Y1). (Y3) After completion of the step (Y2), a step of detaching phospholipids and / or extracellular vesicles from the complex obtained in the step (Y1) and recovering the phospholipids and / or extracellular vesicles.

15. 15. The isolation method according to claim 14, wherein step (Y3) is carried out using a calcium ion chelating agent.

16. The detection method according to claim 12 or 13, characterized in that a detection reagent containing a conjugated lipid-binding carrier is used.

17. 16. The isolation method according to claim 14 or 15, characterized in that a column packed with a composite lipid-binding carrier is used.

Images