Extraction and detection method of milk exosome

Abstract

The invention discloses a milk exosome extraction and detection method, and belongs to the technical field of biological detection and extraction. The invention provides a method for extracting milk exosomes, which comprises the following steps of: capturing the milk exosomes: contacting a captured object with a sample of milk exosomes to be separated, so that the captured object captures the milk exosomes in the sample; the capture includes a polynucleotide immobilized to a solid phase carrier. The invention also provides a detection method of the milk exosome, which comprises the following steps: contacting the polynucleotide with a sample to be detected, and combining the polynucleotide with the milk exosome in the sample to be detected. The polynucleotide comprises at least one selected from nucleotide sequences as shown in SEQ ID NO: 5-10 and SEQ ID NO: 13-16. The method provided by the invention can accurately, efficiently and quickly identify the milk exosome, realizes qualitative and quantitative detection of the milk exosome, can also realize extraction of the milk exosome, is simple and quick to operate and low in cost, and does not need to use large instruments such as an ultra-high-speed centrifuge and the like.

Description

Technical Field

[0001] The present invention belongs to the field of biological detection and extraction technology, specifically to the field of exosome detection and extraction; more specifically, it relates to a method for extracting and detecting milk exosomes. Background Art

[0002] Exosomes, also known as extracellular vesicles (EV), are small vesicle structures with a diameter of 30 to 200 nm (reference 1) and a saucer-shaped or elliptical structure. Exosomes can encapsulate proteins, nucleic acids, lipids and a variety of bioactive substances, and play a role in information transmission, immune response and material transport between cells (reference 2, reference 3). They release their contents into receptors through phagocytosis or direct fusion with cell membranes, and play an important role in signal transmission and information exchange in the extracellular environment. In addition, exosomes are also involved in a variety of physiological and pathological processes, including immune regulation, tumor development, and nervous system function. In clinical practice, exosomes are considered to be a potential drug delivery carrier (reference 4). They exist in a variety of tissue fluids, such as cow's milk, blood and urine. Among them, animal milk contains a higher amount of exosomes and has higher biological activity. Exosomes from animal milk are called milk exosomes or milk exosomes. As a relatively stable biologically active carrier, exosomes are very beneficial for promoting infant immune maturation and organ development (reference 5). Cow and goat milk is considered to be one of the most economical and effective sources for obtaining exosomes. The economical and rapid extraction technology of cow and goat milk exosomes is of great value to the development of clinical medicine, infant formula food research and development and other fields. At present, the separation and extraction methods of exosomes cause losses in the quality, yield and purity of exosomes. The yield and purity of different extraction methods are also different.

[0003] At present, the main methods for isolating and extracting milk exosomes include ultracentrifugation, density gradient centrifugation, and size exclusion.

[0004] Reference 6 adopts a method for separating milk exosomes from bovine milk by ultracentrifugation. This method separates milk exosomes by separating particles of different sizes at different centrifugation speeds and times, and can separate cell debris, fat globules and other impurities from exosomes. However, this method is time-consuming and reduces the exosome yield after multiple ultracentrifugation. It also has the problem of low separation efficiency, resulting in a part of the exosomes being unable to be completely separated.

[0005] Reference 7 uses a method for preparing milk exosomes extracted from bovine milk by size exclusion chromatography. Exosomes are separated from other particles according to the different sizes of particles in the bovine milk source, and expensive equipment such as ultracentrifugation is not required for sample preparation. However, in actual applications, samples contain impurities with similar sizes to milk exosomes, such as casein micelles and milk fat globules, which cannot be separated, affecting subsequent analysis and identification.

[0006] Using only a single extraction method may not be able to obtain milk exosomes with high purity. In order to improve the quality and purity of the extracted milk exosomes, most researchers need to purify them based on ultracentrifugation combined with other extraction methods.

[0007] For example, reference 8 uses a density gradient combined with ultracentrifugation, which uses milk-derived exosomes as materials. By adjusting the density gradient, the separation of specific cells or proteins can be improved. For another example, reference 9 uses an ultracentrifugation combined with size exclusion chromatography. Ultracentrifugation can be used to initially separate exosomes, and then further purified using size exclusion chromatography to obtain exosome samples with higher purity. For another example, reference 10 uses an ultracentrifugation combined with a sucrose density gradient method, which uses the density difference of sucrose solutions of different concentrations to improve its separation effect through ultracentrifugation. However, the methods of references 8-10, based on ultracentrifugation, can improve the purity of the product by combining purification with other extraction methods, but the yield of purified exosomes is low, the steps are cumbersome and time-consuming, the machine requirements are relatively high, and additional supplementary methods are required to improve the purification effect. In addition, the separation process under cumbersome steps may increase the risk of loss or contamination of exosomes, thereby affecting the yield, quality and purity of the final product.

[0008] It can be seen that the milk exosome extraction scheme optimized based on ultracentrifugation combined with other methods can obtain milk exosomes with higher purity, but there are still problems to be solved in terms of yield, cost, and cumbersome process. In order to solve these problems, a novel extraction and identification method is needed to reduce the loss cost and obtain high-quality, high-yield and high-purity milk exosomes. It is necessary to improve the rapidity, simplicity, integrity, reliability of the method and reduce cost-effectiveness.

[0009] Citations

[0010] Cited literature 1Milk exosomes:Nature's abundant nanoplatform for theranostic applications. Bioact Mater, 2021,6:2479-2490.

[0011] Cited document 2Exosomes and their roles in immune regulation and cancer[J]..Semin.Cell Dev.Biol,2015,40:72-81.

[0012] Reference 3Unidirectional transfer of microRNA-loaded exosomes from Tcells to antigen-presenting cells.Nat.Commun,2011,2:282.

[0013] References4Recent Advances on the Function and Purification of Milk Exosomes: A Review.Front Nutr,2022,9:871346.

[0014] Reference 5Exosome cargo in milk as a potential marker of cowhealth.JDairy Res,2020,87:79-83.

[0015] Reference 6AMethod for the Isolation of Exosomes from Human and BovineMilk.J Nutr Metab,2019:5764740.

[0016] Cited document 7Pellet-free isolation of human and bovine milk extracellular vesicles by size-exclusion chromatography.J Extracell Vesicles,2017,6:1294340.

[0017] Cited document 8Bovine Milk-Derived Exosomes as a Drug Delivery Vehicle formiRNA-Based Therapy.Int.J.Mol.Sci.2021,22,1105.

[0018] Cited literature 9Omics Analysis of Extracellular Vesicles Recovered fromInfant Formula Products and Milk:Towards Personalized InfantNutrition.Mol.Nutr.Food Res,2023,67,2300404.

[0019] Cited literature 10Isolation and Characterization of Cow-,Buffalo-,Sheep-andGoat-Milk-Derived Extracellular Vesicles.Cells 2023,12,2491 Summary of the invention

[0020] Problem that the invention aims to solve

[0021] In view of the problems of high cost, low product quality and integrity in the current milk exosome extraction methods, the technical problem to be solved by the present invention is to provide a method for practical application of extraction and qualitative and quantitative analysis, which extracts milk exosomes through DNA with a simple process, and further realizes a fast and efficient milk exosome DNA qualitative and quantitative detection and application method.

[0022] Solutions for solving problems

[0023] The present invention provides a method for extracting milk exosomes, wherein the extraction method comprises:

[0024] A step of capturing milk exosomes, wherein a captured object is contacted with a sample from which milk exosomes are to be separated, so that the captured object captures the milk exosomes in the sample;

[0025] The capture object includes a polynucleotide fixed to a solid phase carrier, and the polynucleotide includes at least one nucleotide sequence selected from SEQ ID NO: 5-10, 13-16.

[0026] In some embodiments, the solid phase carrier comprises: magnetic beads or agarose microsphere resin.

[0027] In some embodiments, after the step of capturing milk exosomes, the step of eluting milk exosomes from the capture object is further included, wherein the milk exosomes captured by the capture object are eluted from the capture object using a saline solution.

[0028] In some specific embodiments, the saline solution comprises a sodium chloride solution.

[0029] In some embodiments, the sample to be isolated milk exosomes is milk or a dairy product.

[0030] In some embodiments, before the step of capturing milk exosomes, the method further comprises treating the sample to be separated milk exosomes with rennet to remove casein.

[0031] The present invention also provides a method for detecting milk exosomes, the detection method comprising:

[0032] contacting a polynucleotide with a sample to be detected, wherein the polynucleotide binds to milk exosomes in the sample to be detected;

[0033] Wherein, the polynucleotide comprises at least one nucleotide sequence selected from SEQ ID NO: 5-10, 13-16.

[0034] In some embodiments, the polynucleotide has a fluorescent label.

[0035] In some embodiments, the detection method is a qualitative detection method or a quantitative detection method.

[0036] In some embodiments, after the polynucleotide binds to the milk exosomes in the sample to be detected, electrophoresis is used to determine whether the sample to be detected contains milk exosomes and / or the content thereof.

[0037] In some embodiments, after the electrophoresis, the content of milk exosomes is determined by calculating the gray value of the band of the polynucleotide-milk exosome complex.

[0038] In some specific embodiments, the sample to be tested is milk or a dairy product.

[0039] The present invention also provides the use of polynucleotides in the detection and extraction of milk exosomes, wherein the polynucleotides include at least one nucleotide sequence selected from SEQ ID NOs: 5-10, 13-16.

[0040] The present invention also provides the use of a polynucleotide in the preparation of a reagent for detecting and extracting milk exosomes, wherein the polynucleotide comprises at least one nucleotide sequence selected from SEQ ID NOs: 5-10, 13-16.

[0041] Effects of the Invention

[0042] In some embodiments of the present invention, the present invention provides a rapid qualitative, quantitative and extraction method for milk-derived exosomes, which uses highly specific DNA to achieve accurate, efficient and rapid identification of milk-derived exosomes.

[0043] In some embodiments of the present invention, the qualitative and quantitative detection of milk-derived exosomes is achieved by using the grayscale values ​​of DNA-milk exosome complex bands and composite bands that appear using EMSA technology.

[0044] In some embodiments of the present invention, DNA is fixed to the surface of a solid phase carrier by means of the interaction between milk exosomes and DNA, and biotinylated DNA is coupled to the surface of a streptavidin-modified material (MB or 6FF), and exosomes are captured by the specific binding of DNA and milk exosomes. The NaCl solution is used to destroy the binding of DNA and milk exosomes to achieve the release of milk exosomes, thereby establishing a method for rapid DNA extraction of milk exosomes. The method can achieve simple and rapid extraction of milk exosomes, and promote the improvement of the quality and purity of milk exosome extraction, and has strong practicality, and is also used for the separation and extraction of milk exosomes in actual milk samples. BRIEF DESCRIPTION OF THE DRAWINGS

[0045] Figure 1 :EMSA characterization of the binding of bovine milk exosomes to different DNA sequences;

[0046] In the figure: “-” indicates only a single DNA sequence; “+” indicates a mixture of DNA sequence and bovine milk exosomes.

[0047] Figure 2 :EMSA characterizes the linear range of binding of different DNA sequences to bovine milk exosomes;

[0048] In the figure: "-" indicates only a single DNA sequence; 2, 4, 6, 8, 10, and 12 indicate that the protein concentrations of bovine milk exosomes are 200, 400, 600, 800, 1000, and 1200 ng / μL, respectively; Free-DNA indicates free DNA bands; DNA-EVcomplex indicates bands of DNA-bovine milk exosome complexes.

[0049] Figure 3 :EMSA characterization of DNA5 sequence binding to goat milk exosomes; Figure 3 (a) shows feasibility, Figure 3 (b) shows the linear range and Figure 3 (c) in shows specificity;

[0050] In the figure: "-" indicates only a single DNA5 sequence; "+" indicates a mixture of DNA5 sequence and goat milk exosomes; 2, 4, 6, 8, 10 indicate that the protein concentrations of goat milk exosomes are 200, 400, 600, 800, 1000 ng / μL, respectively; Free-DNA indicates free DNA5 bands; DNA-EV complex indicates the bands of DNA5-goat milk exosome complexes. α-LA indicates α-lactalbumin; β-LG indicates β-lactoglobulin; CM indicates casein micelles; CN indicates casein; IgG indicates immunoglobulin G; EV indicates goat milk exosomes.

[0051] Figure 4 :Flowchart of DNA enrichment of milk exosomes based on MB and 6FF surface modification ( Figure 4 (a)) and the capture and elution efficiency of milk exosomes ( Figure 4 (b) in the above);

[0052] In the figure: MB represents streptavidin-modified magnetic beads; 6FF represents streptavidin-modified agarose resin microspheres;

[0053] Figure 5 :Flowchart of enrichment of milk exosomes in actual dairy products based on MB and 6FF surface-modified DNA ( Figure 5 (a)) and the transmission electron microscopy characterization results of milk exosomes ( Figure 5 (b) in the above);

[0054] In the figure: MB represents streptavidin-modified magnetic beads; 6FF represents streptavidin-modified agarose resin microspheres; "-Bio DNA" represents MB or 6FF materials without biotin modification; "+Bio DNA" represents MB or 6FF materials with biotin modification.

[0055] Figure 6 : Detection results of milk exosome content extracted from actual dairy products;

[0056] In the figure: MB represents streptavidin-modified magnetic beads; 6FF represents streptavidin-modified agarose resin microspheres. DETAILED DESCRIPTION

[0057] Various exemplary embodiments, features and aspects of the present invention will be described in detail below. The word "exemplary" used here means "used as an example, embodiment or illustrative". Any embodiment described here as "exemplary" is not necessarily interpreted as being superior or better than other embodiments.

[0058] In addition, in order to better illustrate the present invention, numerous specific details are provided in the following specific embodiments. It should be understood by those skilled in the art that the present invention can be implemented without certain specific details. In other examples, methods, means, equipment and steps well known to those skilled in the art are not described in detail in order to highlight the subject matter of the present invention.

[0059] Unless otherwise stated, the units used in this specification are all international standard units, and the numerical values ​​and numerical ranges appearing in the present invention should be understood to include the inevitable systematic errors in industrial production.

[0060] In this specification, the word "may" includes both performing a certain process and not performing a certain process.

[0061] In this specification, the references to "some specific / preferred embodiments", "other specific / preferred embodiments", "embodiments", etc., mean that the specific elements (e.g., features, structures, properties and / or characteristics) described in connection with the embodiments are included in at least one embodiment described herein, and may or may not exist in other embodiments. In addition, it should be understood that the elements may be combined in various embodiments in any suitable manner.

[0062] In this specification, the numerical range expressed using "a numerical value A to a numerical value B" means a range including the endpoints numerical values ​​A and B.

[0063] <Method for extracting milk exosomes>

[0064] In some aspects of the present invention, a method for extracting milk exosomes is provided, the extraction method comprising:

[0065] A step of capturing milk exosomes, wherein a captured object is contacted with a sample from which milk exosomes are to be separated, so that the captured object captures the milk exosomes in the sample;

[0066] The capture object includes a polynucleotide fixed to a solid phase carrier, and the polynucleotide includes at least one nucleotide sequence selected from SEQ ID NO: 5-10, 13-16.

[0067] The method for extracting milk exosomes provided by the present invention is based on the high affinity between polynucleotides (DNA) and milk exosomes to achieve the extraction of milk exosomes. The present invention establishes a method for extracting milk exosomes that is simple to operate, low-cost, fast, high-purity, and does not require large-scale instruments and equipment such as ultracentrifugation. The method is highly practical and is suitable for the separation and extraction of milk exosomes in actual milk and dairy product samples.

[0068] (Samples of milk exosomes to be isolated)

[0069] In the extraction method of the present invention, there is no particular limitation on the sample to be separated milk exosomes, for example, it can be milk, dairy products or any other sample containing milk exosomes, such as food samples.

[0070] In the present invention, "milk" refers to the fluid produced by the mammary glands of mammals, such as bovines (eg, dairy cows), goats, sheep, or camels.

[0071] In the present invention, "dairy product" refers to any food product wherein one of the main ingredients is milk based.

[0072] There is no particular limitation on the source of "dairy products" in the present invention, which may refer to food products produced by animals such as cows, goats, sheep, yaks, horses, camels and other mammals.

[0073] Examples of dairy products are low-fat milk (e.g., 0.1%, 0.5% or 1.5% fat), fat-free milk, milk powder, whole milk, whole milk products, butter, buttermilk, buttermilk products, skim milk, skim milk products, high-fat products, condensed raw milk, fresh cream, cheese, ice cream and dessert products, probiotic beverages or probiotic yogurt-type beverages. Among them, "milk powder" refers to an artificial dairy product made by evaporating milk to dryness.

[0074] In some preferred embodiments of the present invention, the sample of milk exosomes to be separated in the present invention is derived from cow or sheep milk or dairy products.

[0075] (Catch)

[0076] In the present invention, the capture object includes a polynucleotide fixed to a solid phase carrier, and the polynucleotide includes at least one nucleotide sequence selected from SEQ ID NO: 5-10, 13-16.

[0077] According to the present invention, the terms "nucleic acid molecule", "polynucleotide", "polynucleic acid", "nucleic acid" are used interchangeably to refer to a polymeric form of nucleotides of any length, whether deoxyribonucleotides or ribonucleotides, or their analogs. Polynucleotides can have any three-dimensional structure and can perform any known or unknown function. Non-limiting examples of polynucleotides include genes, gene fragments, exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, control regions, isolated RNA of any sequence, nucleic acid probes and primers. Nucleic acid molecules can be linear or circular.

[0078] In some preferred embodiments, the polynucleotide comprises at least one nucleotide sequence selected from SEQ ID NOs: 5-10, 13-15.

[0079] In some more preferred embodiments, the polynucleotide comprises at least one selected from the nucleotide sequences shown in SEQ ID NOs: 5-10.

[0080] In some more preferred embodiments, the polynucleotide comprises the nucleotide sequence shown in SEQ ID NO:5.

[0081] In the present invention, the solid phase carrier may be a microsphere.

[0082] In the present invention, there is no particular limitation on the material of the microspheres. For example, the material of the microspheres may be a polymer (polyethylene, polypropylene, polystyrene, polyamide, polysaccharide, hydrogel), a composite material, silicon or silicon-based material, glass, etc., but is not limited thereto.

[0083] In some specific embodiments, the microspheres can be magnetic beads or agarose microsphere resin.

[0084] In some preferred embodiments, the solid phase carrier is agarose microsphere resin, which has unexpectedly better capture rate and elution rate when extracting milk exosomes compared to other microspheres (such as magnetic beads).

[0085] Methods for fixing / modifying polynucleotides on the surface of solid phase carriers are known in the art. Polynucleotides can be modified on the surface of solid phase carriers by any method known in the art, such as physical adsorption by intermolecular forces such as electrostatic adsorption and hydrophobic interaction; affinity of biotin-streptavidin or protein A / G; cross-linking agent coupling method, etc., but not limited thereto.

[0086] In some preferred embodiments, biotin-streptavidin is used to immobilize / modify the polynucleotide on the surface of a solid support.

[0087] In some specific embodiments, a certain amount of streptavidin-modified magnetic beads or agarose microsphere resin is weighed, washed with PBS (for example, once, twice or three times), and then biotin-modified DNA is added, mixed thoroughly and incubated. After incubation, centrifugation is performed, the solution is discarded, and ultrapure water is added to wash away the DNA not bound to the surface of the material, thereby forming a captured object.

[0088] In some exemplary embodiments, the amount of the streptavidin-modified magnetic beads is preferably 0.5-3 mg, more preferably 1-2.5 mg, such as 1 mg, 1.5 mg, 2 mg, 2.5 mg.

[0089] In some specific embodiments, the concentration of the biotin-modified DNA is preferably 50-300 μM, more preferably 100-250 μM, such as 100 μM, 150 μM, 200 μM.

[0090] In some specific embodiments, the incubation time is preferably 20 to 50 minutes, more preferably 25 to 45 minutes, for example 25 minutes, 30 minutes, 35 minutes. In some embodiments of the present invention, in the step of capturing milk exosomes, the captured object is contacted with a sample of milk exosomes to be separated, so that the captured object captures the milk exosomes in the sample.

[0091] In some specific embodiments, the step of capturing milk exosomes is carried out for 10-60 minutes, that is, the captured object is contacted with the sample of milk exosomes to be separated for 10-60 minutes, preferably 25 to 45 minutes, such as 25 minutes, 30 minutes, 35 minutes, etc.

[0092] (Step of eluting milk exosomes from the captured material)

[0093] In some embodiments, after the step of capturing milk exosomes, the step of eluting milk exosomes from the capture object is further included, wherein the milk exosomes captured by the capture object are eluted from the capture object using a saline solution.

[0094] The present invention has no particular limitation on the type of salt, and the salt may be any salt known in the art that can separate polynucleotides from exosomes, such as sodium chloride.

[0095] In some preferred embodiments, the salt solution comprises a sodium chloride solution.

[0096] In some specific embodiments, the concentration of the sodium chloride solution is 5-20 (w / w)%, more preferably 8-15 (w / w)%, such as 9 (w / w)%, 10 (w / w)%, 11 (w / w)%, etc.

[0097] At this concentration, the best capture and elution rates can be obtained when used with agarose microsphere resin immobilized with polynucleotides.

[0098] Optionally, after the step of eluting the milk exosomes, the method further comprises the step of concentrating the milk exosomes from the eluted solution.

[0099] In some specific embodiments, milk exosomes are collected by concentration using an ultrafiltration tube.

[0100] (Casein removed)

[0101] In some embodiments of the present invention, before the step of capturing milk exosomes, a step of removing casein is further included.

[0102] In some preferred embodiments, the sample (eg, milk or dairy product) from which milk exosomes are to be isolated is treated with rennet to remove casein.

[0103] Specifically, activated rennet is added to the sample of milk exosomes to be separated, and the casein is removed by centrifugation after coagulation to obtain the sample of milk exosomes to be separated from which the casein is removed.

[0104] Before the step of capturing milk exosomes, a step of removing casein is performed to remove the milk protein with the highest content in milk, namely casein, which is more conducive to preventing the presence of casein from interfering with the binding of DNA to exosomes, thereby improving the extraction effect of the milk exosome extraction method of the present invention.

[0105] <Detection method of milk exosomes>

[0106] In some aspects of the present invention, a method for detecting milk exosomes is provided, the detection method comprising:

[0107] contacting a polynucleotide with a sample to be detected, wherein the polynucleotide binds to milk exosomes in the sample to be detected;

[0108] Wherein, the polynucleotide comprises at least one nucleotide sequence selected from SEQ ID NO: 5-10, 13-16.

[0109] The detection method of milk exosomes provided by the present invention is based on the high affinity interaction between polynucleotides (DNA) and milk exosomes, realizing the qualitative and quantitative detection method of milk exosomes. The present invention establishes an accurate, efficient and rapid method for identifying milk exosomes, which is highly practical and realizes the qualitative and quantitative detection of milk exosomes.

[0110] (Samples to be tested)

[0111] In the method for detecting milk exosomes of the present invention, there is no particular limitation on the sample to be detected, for example, it can be milk, dairy products or any other sample containing milk exosomes, such as food samples.

[0112] In the present invention, "milk" refers to the fluid produced by the mammary glands of mammals, such as bovines (eg, dairy cows), goats, sheep, or camels.

[0113] In the present invention, "dairy product" refers to any food product wherein one of the main ingredients is milk based.

[0114] There is no particular limitation on the source of "dairy products" in the present invention, which may refer to food products produced by animals such as cows, goats, sheep, yaks, horses, camels and other mammals.

[0115] Examples of dairy products are low-fat milk (e.g., 0.1%, 0.5% or 1.5% fat), fat-free milk, milk powder, whole milk, whole milk products, butter, buttermilk, buttermilk products, skim milk, skim milk products, high-fat products, condensed raw milk, fresh cream, cheese, ice cream and dessert products, probiotic beverages or probiotic yogurt-type beverages. Among them, "milk powder" refers to an artificial dairy product made by evaporating milk to dryness.

[0116] In some preferred embodiments of the present invention, the sample to be detected is derived from cow or sheep milk or dairy products.

[0117] (Fluorescent marker)

[0118] In the present invention, there is no particular limitation on the binding mode of fluorescent markers to polynucleotides, and those skilled in the art can determine how to bind fluorescent markers to polynucleotides according to the selected fluorescent markers. In some embodiments, fluorescent markers are reversibly bound to polynucleotides. In certain embodiments, fluorescent markers are irreversibly bound to polynucleotides. In some embodiments, the binding between fluorescent markers and polynucleotides can occur by forming one or more covalent bonds. Alternatively, or in addition, fluorescent markers are bound to polynucleotides via one or more non-covalent bonds.

[0119] In some embodiments, fluorescent markers of the present invention include but are not limited to fluorescein, rhodamine, AlexaFluor, Dylight Fluor, ATTO dyes, or any analogs or derivatives thereof.

[0120] In some embodiments, the fluorescent markers of the present invention include but are not limited to fluorescein and chemical derivatives of fluorescein; eosin; carboxyfluorescein; fluorescein isothiocyanate (FITC); fluorescein amidite (FAM); erythrosine; rose bengal; fluorescein secreted from the bacterium Pseudomonas aeruginosa; methylene blue; laser dyes, rhodamine dyes (e.g., rhodamine, rhodamine 6G, rhodamine B, rhodamine 123, auramine O, sulforhodamine 101, sulforhodamine B and Texas Red).

[0121] In some embodiments, the fluorescent marker is attached to the 5' end or the 3' end of the polynucleotide.

[0122] In some preferred embodiments, the fluorescent marker is attached to the 5' end of the polynucleotide.

[0123] In some preferred embodiments, the fluorescent marker is fluoresceinamidite (FAM).

[0124] (Qualitative detection method or quantitative detection method)

[0125] In some embodiments of the present invention, the detection method is a qualitative detection method or a quantitative detection method.

[0126] In some embodiments, after the polynucleotide binds to the milk exosomes in the sample to be detected, the milk exosomes in the sample to be detected are qualitatively or quantitatively detected by fluorescence analysis.

[0127] In the present invention, there is no particular limitation on the fluorescence analysis method, and it may be, for example, fluorescence spectroscopy, atomic fluorescence, chemiluminescence, fluorescence polarization, fluorescence measurement, mercapto fluorescence, laser mercapto fluorescence, grayscale value calculation, etc., but is not limited thereto.

[0128] In some specific embodiments, after the polynucleotide binds to the milk exosomes in the sample to be detected, electrophoresis (eg, agarose gel electrophoresis) is used to determine whether the sample to be detected contains milk exosomes and / or its content.

[0129] In some embodiments, after the electrophoresis, specific identification of milk exosomes is achieved by observing whether there is a DNA-milk exosome complex band.

[0130] In some embodiments, after the electrophoresis, the content of milk exosomes is determined by calculating the gray value of the band of the polynucleotide-milk exosome complex.

[0131] (Casein removed)

[0132] In some embodiments of the present invention, before contacting the polynucleotide with the sample to be detected, a step of removing casein is further included.

[0133] In some preferred embodiments, the sample to be tested (eg, milk or milk products) is treated with rennet to remove casein.

[0134] Specifically, activated rennet is added to the sample to be tested, and after coagulation, the sample is centrifuged to remove casein, thereby obtaining the sample to be tested from which casein has been removed.

[0135] Before the step of contacting the polynucleotide with the sample to be detected, a step of removing casein is performed, which can remove casein, a milk protein with a high content in milk, and is more conducive to preventing the presence of casein from interfering with the binding of DNA and exosomes, thereby improving the detection effect of the method for detecting milk exosomes of the present invention.

[0136] <Purpose>

[0137] In some aspects of the present invention, there is provided the use of polynucleotides in the detection and extraction of milk exosomes, wherein the polynucleotides include at least one nucleotide sequence selected from SEQ ID NOs: 5-10, 13-16.

[0138] In some aspects of the present invention, there is also provided the use of a polynucleotide in the preparation of a reagent for the detection and extraction of milk exosomes, wherein the polynucleotide comprises at least one nucleotide sequence selected from SEQ ID NOs: 5-10, 13-16.

[0139] Example

[0140] The embodiments of the present invention will be described in detail below in conjunction with the examples, but those skilled in the art will appreciate that the following examples are only used to illustrate the present invention and should not be considered to limit the scope of the present invention. If no specific conditions are specified in the examples, they are carried out according to normal conditions or the conditions recommended by the manufacturer. If the manufacturer is not specified for the reagents or instruments used, they are all conventional products that can be obtained commercially.

[0141] Experimental instruments and materials

[0142] Transmission electron microscope: Hitachi, Japan, customized-30;

[0143] Nanoparticle size analyzer: Malvern, NS300;

[0144] Rennet: Doit Company, production batch number A045224A;

[0145] TSG101 antibody: TSG101 protein detection kit (Umibio, UR52301);

[0146] CD9 antibody: GeneTex, GTX76186;

[0147] BCA kit: Shanghai Biotech, P0010;

[0148] The DNA sequence was commissioned to be synthesized by Shanghai Bioengineering Corporation;

[0149] Streptavidin-coated magnetic beads (MB): Thermo Fisher Scientific, 35136, 10 mg / mL;

[0150] Agarose resin microspheres (6FF): Shanghai Bioengineering Company, NO.C600696;

[0151] The sheep milk used in Examples 1 and 2 is goat milk; the cow milk is dairy cow milk-Holstein cow;

[0152] The sheep milk type used in Example 3 is goat milk; the cow milk is dairy cow milk-Holstein cow.

[0153] Example 1 Screening of polynucleotides binding to milk exosomes

[0154] In this example, milk exosomes were extracted, and the extracted samples were identified and characterized by transmission electron microscopy (TEM), nanoparticle size analysis (NTA), and protein immunoblotting (WB), and polynucleotides that specifically bind to milk exosomes (also referred to as DNA that specifically binds to milk exosomes or simply DNA in the present invention) were screened for subsequent detection and extraction of milk exosomes.

[0155] 1. Extraction of milk exosomes

[0156] (1) Mix the milk sample and put it into a centrifuge tube. Centrifuge it twice at 4°C, 3000×g, and 15 min to remove the milk fat.

[0157] (2) Weigh an appropriate amount of rennet by adding 0.035 mg of rennet per ml of milk, dissolve the rennet in 1% (w / w) NaCl solution, and activate it in a 37°C circulating water bath for about 30 min.

[0158] (3) Using 10 (v / v)% acetic acid solution to adjust the pH of skim milk to about 6 (optimal pH for rennet), adding activated rennet to the emulsion, and curdling in a 37°C water bath for 30-35 min;

[0159] (4) After the curdling is completed, the milk is centrifuged at 12,000 × g for 30 min at 4°C to remove casein and collect the whey;

[0160] (5) Filter the supernatant with a 0.45 μm filter membrane, centrifuge the permeate at 120,000 × g and 4 °C for 90 min, discard the supernatant, and resuspend the precipitate in 1× PBS solution;

[0161] (6) The resuspended precipitate was filtered with a 0.22 μm filter membrane, and the permeate was centrifuged again at 150,000 × g and 4°C for 90 min, and the precipitate was collected, resuspended with 1× PBS and stored in a refrigerator at -80°C to obtain the extracted milk exosome-containing sample.

[0162] 2. Characterization of milk exosomes

[0163] (1) Characterization of milk exosomes by transmission electron microscopy. The sample extracted in step 1 was diluted to a certain ratio and added to a 300-mesh copper grid. After negative staining with uranyl acetate, the sample was photographed using a transmission electron microscope.

[0164] (2) Characterization of milk exosomes by nanoparticle size analysis The sample extracted in step 1 was diluted with 1×PBS at a ratio of 1:1000 and injected into the analysis tank of a nanoparticle size analyzer (NTA, Malvern, NS300) to detect the size distribution of exosomes.

[0165] (3) Characterization of milk exosomes by western blotting. The sample extracted in step 1 was mixed with the loading buffer and subjected to SDS-PAGE. The target protein was cut and transferred to the membrane, blocked, incubated with the primary antibody (TSG101 and CD9), washed, incubated with the secondary antibody, washed, and imaged with the developer.

[0166] 3. Determination of protein concentration of milk exosomes

[0167] The BCA kit was used to determine the protein concentration of milk exosomes. Cell lysate and phenylmethylsulfonyl fluoride were used to prepare 1 mM lysate, which was mixed with the sample extracted in step 1 at a volume ratio of 1:1 and placed on ice for lysis for 10 to 20 minutes. After sufficient lysis, the supernatant was centrifuged at 4°C and 12,000 × g for 5 minutes, and the protein concentration was subsequently determined using the BCA kit.

[0168] Experimental results: The extracted samples showed a typical saucer-like morphology under an electron microscope, with a particle size ranging from 50 to 200 nm, and the results of protein immunoblotting showed the presence of exosome surface marker proteins CD9 and TSG101. Therefore, it can be confirmed that the extracted samples are milk exosomes. The concentration of exosomes measured by the BCA kit is 6 mg / mL.

[0169] Goat milk can be extracted, characterized and concentration determined using the same methods as steps 1 to 3 above.

[0170] 4. Characterization of the binding effects of different polynucleotides with milk exosomes

[0171] (1) Preparation method of relevant solutions

[0172] (i) Phosphate Buffered Saline (PBS; 20×): Dissolve the contents of one bag in 50 mL of ultrapure water or deionized water to obtain a 20× phosphate buffer solution, which can be diluted with ultrapure water to the working concentration.

[0173] (ii) DNA (100 μM): Take an unopened tube of DNA powder and centrifuge it at 3000-4000 rpm / min and 4°C for 1 min to prevent the DNA powder from being lost when the lid is opened. Add 1×TE buffer according to the label on the tube, shake for 5 min to dissolve it, and centrifuge to collect the solution at the bottom of the tube. The concentration is 100 μM at this time. In order to prevent DNA degradation due to repeated freezing and thawing, store it in aliquots at -20°C and dilute it with 1×PBS as needed when using it.

[0174] (2) Sample preparation

[0175] In order to obtain DNA with the highest affinity and specificity for bovine and goat milk exosomes for detection and extraction of milk exosomes, the present invention uses the candidate DNA shown in Table 1 below as candidate polynucleotides binding to milk exosomes.

[0176] Table 1: Candidate DNA

[0177]

[0178] In Examples 1 and 2, the 5' ends of DNAs 1 to 16 in the above table are modified with a fluorescent marker: fluorescein amide (FAM), thereby forming a DNA probe for the detection of exosomes.

[0179] (3) Sample preparation for the combination of different candidate DNA probes with bovine milk exosomes. Take 1 μM 10 μL of different candidate DNA probes labeled with FAM fluorescence (Table 1) and add 1.0 μL 20×PBS to prepare two tubes. One tube is the control group without adding exosomes, and the other tube is added with 5.0 μL of bovine milk exosomes extracted in step 1. Add ultrapure water to a final volume of 20.0 μL and mix well. Add 6.0 μL of glycerol to each sample, centrifuge and perform agarose electrophoresis.

[0180] (4) Electrophoresis detection of samples of different DNA probes binding to bovine milk exosomes.

[0181] This example characterizes the binding of the aforementioned DNA probe to bovine milk exosomes by using the classic protein-DNA binding characterization technique, electrophoretic mobility shift assay (EMSA). Whether the DNA and bovine exosomes can bind to bovine milk exosomes is determined by whether a DNA-EV complex band appears after electrophoresis.

[0182] Specifically, the electrophoresis parameters were: 0.5% (g / mL) agarose gel, voltage 90V, electrophoresis time 30min, electrophoresis buffer 1×TAE, and electrophoresis was performed after each sample was loaded. The electrophoresis results were obtained by scanning with the TGel Image System (OSE-470P) instrument. DNA probes with high binding affinity and good specificity to bovine milk exosomes were selected as the extraction probes for bovine milk exosomes, such as Figure 1 As shown, after electrophoresis of the DNA probe and bovine exosomes, DNA-EV complex bands appeared, among which DNA5, DNA6, DNA7, DNA8, DNA9, DNA10, DNA13, DNA14, DNA15 and DNA16 were able to bind to bovine milk exosomes.

[0183] Example 2: Application of DNA bound to milk-derived exosomes in qualitative and quantitative detection of milk-derived exosomes.

[0184] 1. Extraction, characterization and quantification of milk exosomes

[0185] Same as steps 1 to 3 in Example 1.

[0186] 2. Combined with DNA from milk exosomes for quantitative detection of milk exosomes

[0187] (1) Prepare the solution.

[0188] (i) Protein solution (10 mg / mL): Weigh a certain amount of powder and dissolve it in 1× PBS to prepare a 10 mg / mL stock solution. Dilute it with 1× PBS as needed when using.

[0189] (ii) The preparation of the remaining solutions is the same as (1) in step 4 of Example 1.

[0190] (2) Sample preparation

[0191] Take 10 μL of different DNAs (1 μM) labeled with FAM fluorescence and add 1.0 μL 20× PBS, and add proteins at final concentrations of 200 ng / μL, 400 ng / μL, 600 ng / μL, 800 ng / μL, 1000 ng / μL, 1200 ng / μL ( Figure 3 and Figure 4 2, 4, 6, 8, 10, 12) were added to the exosomes, ultrapure water was added to the final volume of 20.0 μL, and mixed; 10 μL of different DNAs (1 μM) labeled with FAM fluorescence were added to 1.0 μL 20×PBS, and 5.0 μL of milk exosomes, β-lactoglobulin (β-LG), α-lactalbumin (α-LA), bovine lactoferrin (BLF), bovine serum albumin (BSA), casein (CN), casein micelles (CM), and immunoglobulin G (IgG) were added, and ultrapure water was added to make up the final volume of 20.0 μL. Except for exosomes, the concentrations of other proteins were all 1 mg / mL, and 6.0 μL of glycerol was added to each sample, and electrophoresis was performed after centrifugation.

[0192] (3) Electrophoresis

[0193] Same as (4) in step 4 of Example 1.

[0194] (4) Qualitative and quantitative detection results of the binding of DNA probes to bovine milk exosomes.

[0195] The gray value of the band was obtained using Image J software. Different concentrations of milk exosomes were added to the DNA probe labeled with the same concentration. As the concentration of milk exosomes increased, the gray value of the free DNA band decreased, such as Figure 2 As shown, the increase in the gray value of the DNA-EV complex bands indicates that DNA5, DNA6, DNA7, DNA8, DNA9, DNA10, DNA13, DNA14 and DNA15 can all be used for the quantitative detection of bovine milk exosomes.

[0196] (5) Qualitative and quantitative detection results of the binding of DNA5 probe to goat milk exosomes.

[0197] After mixing the DNA5 probe with goat milk exosomes, Figure 3 In (a), the DNA5 probe can bind to goat milk exosomes. Different concentrations of goat milk exosomes were added to the same concentration of labeled DNA probes, such as Figure 3 In (b), as the number of goat milk exosomes increases, the grayscale value of the band of the DNA-goat milk exosome complex increases, and the grayscale value of the band of the free DNA decreases. After the DNA5 probe was mixed with β-lactoglobulin, α-lactalbumin, bovine lactoferrin, bovine serum albumin, casein, casein micelles, immunoglobulin G and goat milk exosomes, as shown in Figure 3 As shown in (c), the DNA probe only binds to goat milk exosomes and the DNA5 probe can specifically recognize goat milk exosomes, with good specificity.

[0198] The DNA probes screened in this way are suitable for the rapid qualitative and quantitative detection of bovine and goat milk exosomes.

[0199] Example 3: Application of DNA combined with milk exosomes in rapid separation and extraction of milk exosomes

[0200] In this example, DNA5 was selected for feasibility verification of milk exosome extraction.

[0201] 1. Rapid DNA extraction of milk exosomes

[0202] (1) Prepare the solution.

[0203] Same as (1) in step 4 in Example 1.

[0204] (2) DNA-modified magnetic beads for milk exosome extraction

[0205] See also Figure 4 (a) in the figure: (i) Coupling of DNA with streptavidin-modified magnetic beads (MB). The DNA was fixed to the MB surface using the biotin-streptavidin interaction. Weigh 200 μL of streptavidin-modified magnetic beads, wash them three times with 200-300 μL 1×PBS, add 1 μL (100 μM) of biotin-modified DNA, mix thoroughly, and place them on a decolorizing shaker for 30 min;

[0206] (ii) Capture of bovine milk exosomes. Incubate and centrifuge the product obtained in step (i) and place it on a magnetic rack, and discard the solution. Wash away the DNA that is not bound to the MB with 200-300 μL of ultrapure water. Add 1 mL of milk exosomes with an exosome protein concentration of 0.1 mg / mL (extracted and quantified according to the method of Example 1), mix thoroughly, and place it on a decolorizing shaker for incubation for 30 minutes. Use the BCA kit to determine the protein concentration of the captured milk exosomes;

[0207] (iii) Release of milk exosomes. The binding interaction between milk exosomes and DNA can be destroyed by using NaCl solution. After incubation and centrifugation of the product obtained in step (ii), place it on a magnetic rack and discard the solution. Wash away the exosomes that are not bound to the MB surface with 200-300 μL of ultrapure water. Add NaCl solution with a final concentration of 10 (w / w)%, mix well, and place it on a decolorizing shaker for 30 minutes to release milk exosomes;

[0208] (vi) Collection of milk exosomes. The product obtained in step (iii) was incubated and centrifuged, and then placed on a magnetic rack to collect the solution. The milk exosomes were concentrated using an ultrafiltration tube, and the protein concentration of the collected milk exosomes was determined using a BCA kit.

[0209] (3) DNA-modified agarose resin microspheres (6FF) for the extraction of exosomes from bovine and goat milk

[0210] (i) Coupling of DNA with streptavidin-modified agarose resin microspheres. Weigh 200 μL of streptavidin-modified 6FF, wash three times with 200-300 μL 1× PBS, add 1 μL (100 μM) of biotin-modified DNA, mix thoroughly, and incubate on a decolorizing shaker for 30 min;

[0211] (ii) Capture of milk exosomes. Incubate the product obtained in step (i), centrifuge and discard the solution. Wash away the DNA that is not bound to 6FF with 200-300 μL ultrapure water. Add 1 mL of milk exosomes with an exosome protein concentration of 0.1 mg / mL (extracted and quantified according to the method of Example 1), mix thoroughly, and place it on a decolorizing shaker for incubation for 30 minutes. Use the BCA kit to measure the protein concentration of the captured milk exosomes;

[0212] (iii) Release of milk exosomes. The binding interaction between milk exosomes and DNA can be destroyed by using NaCl solution. Incubate and centrifuge the mixture obtained in step (ii) and discard the solution. Wash away the exosomes that are not bound to the 6FF surface with 200-300 μL of ultrapure water. Add NaCl solution with a final concentration of 10% (w / w), mix thoroughly, and place it on a decolorizing shaker for incubation for 30 minutes to release milk exosomes;

[0213] (vi) Collection of milk exosomes. The mixture obtained in step (iii) was incubated and centrifuged to collect the solution. The milk exosomes were concentrated using an ultrafiltration tube and the protein concentration of the collected milk exosomes was determined using a BCA kit.

[0214] Figure 4 As shown in (a), the DNA that can bind to milk exosomes in Example 1 is selected, and the DNA is fixed on the MB or 6FF surface using biotin-streptavidin interaction, which can effectively capture milk exosomes; Figure 4 As shown in (b), the capture rates of MB and 6FF modified DNA were 30.6% and 54.35%, respectively. The use of NaCl solution can destroy the binding effect between milk exosomes and DNA, and successfully release milk exosomes, with elution rates of 5.89% and 74%, respectively.

[0215] Capture efficiency calculation method: (original exosome concentration - unbound exosome concentration) / original exosome concentration

[0216] Elution efficiency calculation method: exosome concentration in the eluate / (original exosome concentration - unbound exosome concentration)

[0217] The present invention is a technology for extracting bovine and goat milk exosomes based on DNA-milk exosome interaction. The method is simple and fast to operate and does not require the use of large-scale instruments and equipment such as ultra-high-speed centrifuges.

[0218] 2. Rapid DNA extraction of bovine milk exosomes in actual samples.

[0219] (1) Extraction of milk exosomes using traditional methods.

[0220] Same as step 1 in Example 1.

[0221] (2) The method of the present invention is used to extract milk exosomes from whey milk.

[0222] First, rennet is used to remove casein from milk. The rennet treatment process is the same as step 1 (1) to (4) in Example 1.

[0223] 10 mL of bovine whey was collected and added to MB and 6FF modified with DNA (the modification steps were the same as those in Example 3, step 1 (2) (i) and step 1 (3) (i)), and the mixture was placed on a decolorization shaker for incubation for 30 min. After the incubation, the mixture was centrifuged and placed on a magnetic rack, the solution was discarded, and after washing with ultrapure water, 10% (w / w) NaCl was added and mixed thoroughly. The mixture was incubated on a decolorization shaker for 30 min to collect the solution. The mixture was concentrated using an ultrafiltration tube and the concentrate was collected.

[0224] (3) Characterization of milk exosomes.

[0225] Same as (1) in step 2 of Example 1.

[0226] (4) Determination of the content of milk exosomes.

[0227] Same as step 3 in Example 1.

[0228] The method of the present invention is applied to the rapid extraction of milk exosomes in fresh milk. The specific process is as follows: Figure 5 As shown in (a) in Figure 5 As shown in (b), MB and 6FF materials (-Bio DNA) that were not coupled to milk exosome-bound DNA were used to extract milk exosomes from samples that had been induced to remove casein by rennet. Scanning electron microscopy (TEM) identification did not reveal the typical saucer-shaped vesicle structure of exosomes; Figure 5 As shown in (b), the sample extracted from the complex of modified DNA and MB, 6FF (+Bio DNA) contained a typical complete saucer-shaped vesicle structure and a clean background color through TEM. The number of bovine milk exosomes extracted from 6FF-DNA material was more than that from MB-DNA material. The comparison showed that the content of milk exosomes extracted from 6FF material was higher and the purity was higher. Figure 6 The milk exosomes extracted from fresh milk were quantitatively detected in the experiment. The contents of milk exosomes extracted by 6FF and MB were 398.37 μg / mL and 148.36 μg / mL, respectively.

[0229] It should be noted that, although the technical solution of the present invention is introduced with specific examples, those skilled in the art will appreciate that the present invention should not be limited thereto.

[0230] The embodiments of the present invention have been described above, and the above description is exemplary, not exhaustive, and is not limited to the disclosed embodiments. Many modifications and variations will be apparent to those of ordinary skill in the art without departing from the scope and spirit of the described embodiments. The selection of terms used herein is intended to best explain the principles of the embodiments, practical applications, or technical improvements in the market, or to enable other persons of ordinary skill in the art to understand the embodiments disclosed herein.

Claims

1. A method for extracting milk exosomes, characterized in that: The extraction method comprises: A step of capturing milk exosomes, wherein a captured object is contacted with a sample from which milk exosomes are to be separated, so that the captured object captures the milk exosomes in the sample; The capture object includes a polynucleotide fixed to a solid phase carrier, and the polynucleotide includes at least one nucleotide sequence selected from SEQ ID NO: 5-10, 13-16.

2. The extraction method according to claim 1, characterized in that The solid phase carrier includes magnetic beads or agarose microsphere resin.

3. The extraction method according to claim 1 or 2, characterized in that After the step of capturing milk exosomes, the method further comprises a step of eluting the milk exosomes from the captured object, wherein the milk exosomes captured by the captured object are eluted from the captured object using a salt solution.

4. The extraction method according to claim 3, characterized in that The salt solution includes a sodium chloride solution.

5. The extraction method according to any one of claims 1 to 4, characterized in that The sample to be separated milk exosomes is milk or dairy products.

6. The extraction method according to any one of claims 1 to 5, characterized in that Before the step of capturing milk exosomes, the method further includes treating the sample to be separated milk exosomes with rennet to remove casein.

7. A method for detecting milk exosomes, characterized in that: The detection method comprises: contacting a polynucleotide with a sample to be detected, wherein the polynucleotide binds to milk exosomes in the sample to be detected; Wherein, the polynucleotide comprises at least one nucleotide sequence selected from SEQ ID NO: 5-10, 13-16.

8. The detection method according to claim 7, characterized in that: The polynucleotide has a fluorescent label.

9. The detection method according to claim 7 or 8, characterized in that: The detection method is a qualitative detection method or a quantitative detection method.

10. The detection method according to any one of claims 7 to 9, characterized in that: After the polynucleotide is combined with the milk exosomes in the sample to be detected, electrophoresis is used to determine whether the sample to be detected contains milk exosomes and / or its content.

11. The detection method according to claim 10, characterized in that: After the electrophoresis, the content of milk exosomes is determined by calculating the gray value of the band of the polynucleotide-milk exosome complex.

12. The detection method according to any one of claims 7 to 11, characterized in that: The sample to be detected is milk or dairy products.

13. Application of polynucleotides in the detection and extraction of milk exosomes, including: The polynucleotide comprises at least one nucleotide sequence selected from the group consisting of nucleotide sequences shown in SEQ ID NOs: 5-10 and 13-16.

14. Use of polynucleotides in the preparation of reagents for the detection and extraction of milk exosomes, wherein: The polynucleotide comprises at least one nucleotide sequence selected from the group consisting of nucleotide sequences shown in SEQ ID NOs: 5-10 and 13-16.

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