CN113061571A - Method for separating and identifying Hostan cow milk exosomes - Google Patents
Method for separating and identifying Hostan cow milk exosomes Download PDFInfo
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Abstract
The invention discloses a separation and identification method of a Holstein cow milk exosome, which comprises the steps of collecting a Holstein cow milk sample, centrifugally separating the exosome by density gradient, and respectively carrying out transmission electron microscopy, particle size analysis and specific protein expression identification on the exosome. The method for extracting and identifying the Holstein cow milk exosomes has the advantages of low price, high efficiency and good purity, and can meet various test requirements.
Description
Technical Field
The invention relates to a method for separating and identifying exosomes, in particular to a method for separating and identifying Holstein cow milk exosomes, and belongs to the field of agricultural breeding application.
Background
Exosomes (Exosomes) are lipid bilayer structure membrane vesicles secreted by cells, with the diameter of about 30-150 nm and the density of 1.13-1.19g/mL, are circular or elliptical and have a typical 'cup-and-dish' shape. Exosomes are present in almost all tissues, intercellular spaces, body fluids, including blood, saliva, urine, amniotic fluid, and milk. Exosomes can carry various substances with biological activity such as protein, lipid, DNA and RNA (including mRNA, miRNA, lncRNA and circRNA) and degradation fragments thereof, and the exosomes serving as novel intercellular communication molecules not only participate in the normal information transmission and substance exchange and other biological processes between cells, but also are expected to become early diagnosis markers of various diseases in the aspects of treatment of difficult and complicated diseases such as tumor metastasis, immune regulation and control mechanisms, disease occurrence and development, Alzheimer's disease and the like. It has been found that Exosomes in milk of cows contain many small RNAs (miRNA, lncRNA, circRNA) that can be transported into immune cells, affecting the development of gastrointestinal tract and immune system of the cows; meanwhile, the Exosomes in the cow milk can also carry the medicine to a tumor target, so that the medicine action efficiency and safety are improved. Therefore, the function of exosome in cow's milk is more and more emphasized, and further research is urgently needed. Therefore, the exploration of a low-price, high-efficiency and high-purity cow milk Exosomes extraction and identification method is an important prerequisite for scientific research and disease treatment.
Disclosure of Invention
The invention aims to solve the problems and provide a method for separating and identifying the Holstein cow milk exosomes.
The invention realizes the purpose through the following technical scheme, and a method for separating the exosomes of Holstein cow milk comprises the steps of collecting a Holstein cow milk sample and separating the exosomes through density gradient ultracentrifugation, and specifically comprises the following steps:
a Holstein cow milk sample collection
Selecting healthy Holstein cows in lactation period, collecting 30mL of milk, and storing the collected Holstein cow milk in a low-temperature refrigerator at 4 ℃ for later use;
separation of B Holstein cow's milk exosomes
(1) Centrifuging fresh Holstein cow milk from A at 2,000 Xg and 4 deg.C for 30min, collecting upper layer milk, and removing lower layer precipitate;
(2) transferring the centrifuged upper layer milk into a new sterile 50mL conical centrifuge tube, centrifuging at 4 ℃ and 10,000 Xg for 45min, collecting the upper layer milk, and discarding the lower layer precipitate;
(3) transferring the upper layer milk centrifuged in the step (2) into a new centrifuge tube, selecting an overspeed rotor, performing ultracentrifugation for 120min at the temperature of 4 ℃ and under the condition of 100,000 Xg, discarding the upper layer liquid, and obtaining the lower layer precipitate as a primarily separated holstein cow milk exosome;
(4) adding 20mL of 1 XPBS precooled at 4 ℃ into the precipitate obtained in the step (3), resuspending the precipitate, centrifuging the precipitate again at 4 ℃ and 2,000 Xg for 30min, and discarding the precipitate;
(5) transferring the centrifuged supernatant in the step (4) to a new sharp-bottomed centrifuge tube, centrifuging the centrifuged supernatant at 4 ℃ for 30min at 2,000 Xg, and discarding the precipitate;
(6) repeating the step (5) for 1 time;
(7) transferring the centrifuged supernatant in the step (6) into a new centrifugal tube, selecting an overspeed rotor, and carrying out ultracentrifugation for 120min at the temperature of 4 ℃ and under the condition of 100,000 Xg, removing the supernatant, and keeping the lower-layer precipitate;
(8) resuspending the pellet obtained in (7) in 1mL of precooled PBS, and storing at low temperature of 4 ℃;
(9) respectively preparing 40%, 20%, 10% and 5% iodixanol, sequentially adding 3.6mL of each iodixanol into an ultracentrifuge tube, slowly adding 1mL of heavy suspension obtained in step (8) to the uppermost layer, performing ultracentrifugation at 4 deg.C for 120min to obtain a suspension solution with a concentration of 100,000 Xg;
(10) dividing the centrifuged mixed solution into 12 layers, collecting the liquid in the middle 6-9 layers, performing ultracentrifugation for 120min at 4 ℃ at 100,000 Xg, and removing the supernatant to obtain the purified cow milk exosomes;
(11) the exosomes obtained in step (10) were resuspended in 100. mu.L of precooled PBS and used immediately or frozen in an ultra-low temperature freezer at-80 ℃.
Preferably, the BCD-525WKPZM (E) with the standard of the low-temperature refrigerator in the step (8) for preserving the fresh cow milk is adopted, the model of the low-temperature refrigerator in the step (11) is Eppendorf CryoCube F570, the model of the low-temperature centrifuge in the steps (1) (2) (4) (5) (6) is Eppendorf 5810R, and the model of the low-temperature ultracentrifuge in the steps (3) (7) (9) (10) is Beckman Optima L-100 XP.
A method for identifying Holstein cow milk exosomes comprises transmission electron microscope observation and identification, particle size analysis and identification and Western blot analysis and identification, and specifically comprises the following steps:
transmission Electron Microscope (TEM) observation and identification of A Holstein cow milk exosome
(1) Taking 10 mu L of the separated exosome and PBS mixed solution, dropwise adding the mixed solution on a 100-mesh copper net, standing for 1min, and sucking away the floating liquid by filter paper;
(2) dripping 10 mu L of phosphotungstic acid on the copper net obtained in the step (1), standing for 1min, and sucking away the floating liquid by filter paper;
(3) drying at normal temperature for 10 min;
(4) selecting 100kv of voltage to carry out transmission electron microscope imaging detection and photographing for storage;
identification of particle size analysis (NTA) of B Holstein cow's milk exosomes
(1) Taking 10 mu L of the separated exosome and PBS mixed solution, adding 1.9mL of 1 XPBS, and diluting to a working concentration suspension;
(2) transferring the mixed solution in the step (1) into a flow tube, imaging and observing exosomes and vesicles in the suspension one by one in real time through a nano-flow particle size analyzer, and calculating the particle size distribution and concentration of the exosomes in the suspension through Zeta view 8.04.02 software;
c Holstein cow milk sample marker protein immunoblotting (Western blot) analysis and identification
(1) Preparation of an exosome protein sample: taking out exosome frozen in an ultra-low temperature refrigerator at-80 ℃, unfreezing on ice, adding 100 mu L of RIAP protein lysate, and cracking on ice for 30 min;
(2) centrifuging the cracking mixture in (1) at 12,000 Xg for 15min at 4 deg.C in a low-temperature centrifuge, collecting supernatant, and removing precipitate;
(3) standard curves were made and exosome protein concentrations were detected using the BCA protein concentration kit, the method is outlined below:
adding 20 μ L of protein standard with concentration of 0, 0.025, 0.05, 0.1, 0.2, 0.3, 0.4, 0.5mg/mL and 20 μ L of supernatant in (2) to 96-well plate, each in triplicate; preparing and adding a BCA working solution (solution A: solution B =50: 1) into the holes, oscillating and uniformly mixing, and incubating in an incubator at 37 ℃ for 30 min; measuring the absorbance of A562 by using a full-wavelength microplate reader; drawing a standard curve by using the absorbance measured by protein standard substances with different concentrations and Excel to obtain a regression formula, bringing the absorbance value of the exosome protein into the formula, and calculating the concentration of the exosome protein;
(4) denaturation: mixing 40 μ L of the cracked mixed solution with 10 μ L of 5 ☓ SDS-PAGE protein loading buffer solution, boiling at 100 deg.C for 5min to obtain denatured protein;
(5) loading, electrophoresis and membrane transfer: opening the purchased commercial 12% prefabricated gel, inserting the gel into an electrophoresis tank, filling electrophoresis buffer solution, adding the denatured 20 mu L protein sample into an electrophoresis hole, and carrying out electrophoresis; taking out electrophoresis gel after electrophoresis, soaking the electrophoresis gel in methanol for activation for 10s, then placing the gel in a precooled membrane conversion buffer solution, sequentially filling a sponge, three layers of filter paper and the electrophoresis gel on a pad, covering the membrane on the electrophoresis gel, covering three pieces of filter paper on the membrane, removing bubbles, finally covering another sponge pad, inserting the sponge pad into a membrane conversion groove, turning on a power supply, and carrying out constant-pressure membrane conversion for 90V and 60 min;
(6) and (3) sealing: placing the transferred membrane on a room-temperature decolorizing shaker, soaking in 5% skimmed milk, and sealing for 1 h;
(7) a first antibody: diluting a Calnexin, CD63, CD81 and TSG101 primary anti-concentrated solution and 5% skimmed milk according to a ratio of 1:2000, and incubating a membrane at 4 ℃ for one night;
(8) secondary antibody: membranes were washed 3 times with 1 ☓ TBST on a room temperature decolorizing shaker for 5min each, and secondary antibodies were mixed with 1 ☓ TBS at 1: diluting with 5000, incubating at room temperature for 60min, washing with 1 ☓ TBST for 3 times, each time for 5 min;
(9) exposure: taking out the PVDF membrane, draining, spreading the protein on the preservative film with the protein surface facing upwards, and dropwise adding the ECLA/B mixed liquid with the same volume onto the membrane for reaction for 1min in a dark place; clamping the film and moving the film into a plastic packaging film, wherein the protein surface is always upward in the operation process;
(10) the film was placed in an imager and exposed for 10s, the brightness and contrast were adjusted and the picture was saved.
Preferably, the transmission electron microscope is in a model number of Hitachi 7700, the filter paper is in a model number of Whatman HC0002, the nanoparticle tracking analyzer is in a model number of Particle Metrix ZetaVIEW 17-310, the full-wavelength microplate reader is in a model number of Bio-Tek (Eon), and the incubator is; the imager model is GE LAS 4000.
Preferably, the specification of the protein lysate is Beyotime/P0013, the specification of the BCA protein concentration detection kit is Beyotime/P0010S, the specification of the 5 ☓ SDS-PAGE protein loading buffer is Beyotime/P0015, the specification of the commercialized 12% precast gel is GenScript/M01210C, the specification of the electrophoresis buffer is GenScript/M00138, the specification of the membrane transfer buffer is GenScript/M00139, the specification of the PVDF membrane is MERCK MIIPLLORE IPVH00010, and the specification of the 5% skim milk is Solarbio/D8340.
Preferably, the diameter of exosome in the particle size analysis and identification is 30-150 nm.
Preferably, the voltage of the concentrated gel is set to be 60V and 30min, and the voltage of the separation gel is set to be 120V and 60min in the electrophoresis process.
Preferably, the transmission electron microscope model is FEI Tecnai Spirit T12; the filter paper model is Whatman HC0002, the nanoparticle tracking analyzer model is Particle Metrix ZetaVIEW 17-310, and the imager model is GE LAS4000 mini.
The invention has the beneficial effects that: the invention provides a method for separating and identifying the exosomes of Holstein cow milk, which has the following advantages,
(1) compared with the traditional ultra-high speed centrifugation method and the commercial kit method, the Holstein cow milk exosome separated by the optimized method increases the acquisition amount and concentration of exosome, reduces the content of lipid and vesicles, greatly improves the purity of exosome, and provides a reference basis for extracting high-quality Holstein cow milk exosome;
(2) exosomes are identified by a transmission electron microscope, a nanoparticle tracking analyzer and a protein immunoblotting method respectively, and the shape, the particle size and the specific antigen are identified respectively, so that the reliability of an exosome separation result can be ensured to the maximum extent.
Drawings
FIG. 1 is an electron microscope image of the Holstein cow's milk exosomes of the present invention.
FIG. 2A is a graph of the percent exosome particle size distribution and the mean particle size value of the present invention.
FIG. 2B is a statistical chart of the exosome particle size distribution of the present invention.
FIG. 3A is a graph showing the concentration values of exosomes and the number of detection particles according to the present invention.
FIG. 3B is a two-dimensional scattergram of exosome particles of the present invention.
FIG. 4 is a standard curve of the exosome protein of the present invention.
FIG. 5 is a diagram showing the expression of an exosome-specific protein of the present invention.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
The invention discloses a separation and identification method of Hostan cow milk exosomes
The model of the instrument in the embodiment:
the model of the cryogenic refrigerator is American BCD-525WKPZM (E), the model of the cryogenic refrigerator is Eppendorf CryoCube F570, the model of the cryogenic centrifuge is Eppendorf 5810R, the model of the cryogenic ultracentrifuge is Beckman Optima L-100XP, the model of the transmission electron microscope is Hitachi 7700, the model of the filter paper is Whatman HC0002, the model of the nanoparticle tracking analyzer is Particle Metrix ZetaVIEW 17-310, the model of the full-wavelength microplate reader is Bio-Tek (Eon), and the model of the incubator is ThermFiso her 3111; the imager model is GE LAS 4000.
The specifications of the reagents in the examples are:
the specification of the protein lysate is Beyotime/P0013, the specification of the BCA protein concentration detection kit is Beyotime/P0010S, the specification of the 5 ☓ SDS-PAGE protein loading buffer solution is Beyotime/P0015, the specification of the commercialized 12% precast gel is GenScript/M01210C, the specification of the electrophoresis buffer solution is GenScript/M00138, the specification of the membrane transfer buffer solution is GenScript/M00139, the PVDF membrane is MERCK MILLIPORE IPVH00010, the specification of the 5% skimmed milk is Solarbio/D8340, the specification of the 5% skimmed milk is prepared by TBST, and the primary antibody specifications are CD63 (Proteincech/25682-1-AP), CD81 (Proteich/27855-1-AP), TSG101 (Proteincech/14497-1-AP) and Calteich/1042-AP 27.
Example A method for separation of Holstein cow's milk exosomes
The method comprises the steps of Holstein cow milk sample collection and density gradient ultracentrifugation exosome separation, and specifically comprises the following steps:
a Holstein cow milk sample collection
(1) Milk sample preparation: selecting healthy Holstein cattle in lactation period, collecting 30mL milk during morning milking, and filling into a sterile 50mL pointed-bottom centrifugal tube;
(2) and (3) storage: the collected holstein cow milk was taken back to the laboratory along with an ice bag, stored in a low temperature refrigerator at 4 ℃ and treated for 4 hours.
Separation of B Holstein cow's milk exosomes
(1) Centrifuging the milk sample collected in A at 4 deg.C at 2000 Xg for 10min, collecting the upper layer milk, and removing the lower layer precipitate to remove fat and cell debris;
(2) transferring the upper layer of milk centrifuged in the step (1) into a new sterile conical centrifuge tube, centrifuging at 4 ℃ and 10000 Xg for 45min, collecting the upper layer of milk, and removing the lower layer of sediment to remove larger vesicles and the like;
(3) transferring the upper layer of milk centrifuged in the step (2) into a new pointed-bottom centrifuge tube, selecting an overspeed rotor, performing ultracentrifugation for 120min at the temperature of 4 ℃ and under the condition of 100,000 Xg, discarding the upper layer of liquid, and obtaining a lower layer precipitate which is a primarily separated mixture of the milk exosomes of the Holstein cow;
(4) adding 20mL of 1 XPBS precooled at 4 ℃ into the precipitate obtained in the step (3), resuspending the precipitate, centrifuging the precipitate again at 4 ℃ for 30min at 2,000 Xg, discarding the precipitate, repeating the steps for 3 times, and washing the primarily separated exosome mixture;
(5) transferring the centrifuged supernatant in the step (4) into a new centrifuge tube, selecting an ultracentrifuge rotor, ultracentrifuging for 120min at 4 ℃ under the condition of 100,000 Xg, removing the supernatant, and keeping the lower-layer precipitate;
(6) repeating the step (5) for 1 time;
(7) transferring the centrifuged supernatant in the step (6) into a new centrifuge tube, selecting an ultracentrifuge rotor, ultracentrifuging for 120min at 4 ℃ under the condition of 100,000 Xg, removing the supernatant, and keeping the lower-layer precipitate;
(8) resuspending the pellet obtained in step (7) in 1mL of precooled PBS, and storing at low temperature of 4 ℃;
(9) respectively preparing 40%, 20%, 10% and 5% iodixanol, sequentially adding 3.6mL of the iodixanol into an ultracentrifuge tube, slowly adding 1mL of the heavy suspension obtained in the step (8) to the uppermost layer, performing ultracentrifugation at 4 ℃ for 120min at 100,000 Xg, and separating high-purity exosomes;
(10) separating the mixed solution into 12 layers after centrifugation, collecting the liquid in the middle 6-9 layers, performing ultracentrifugation for 120min at the temperature of 4 ℃ again at 100,000 Xg, and removing the supernatant to obtain the purified cow milk exosome;
(11) and (4) resuspending the exosome obtained in the step (10) in 100 mu L of precooled PBS, and freezing and storing in an ultralow temperature refrigerator at-80 ℃.
Example two Holstein cow milk exosome identification method Transmission Electron Microscopy (TEM) observation identification
(1) Taking out the exosome and PBS mixed solution frozen in the ultra-low temperature refrigerator at minus 80 ℃ in the first embodiment, thawing at room temperature until the exosome and PBS mixed solution are completely melted, taking 10 mu L of the exosome and PBS mixed solution, dripping the exosome and PBS mixed solution on a copper net of 100 meshes, standing for 1min, and sucking away the floating liquid by filter paper;
(2) dripping 10 mu L of phosphotungstic acid on the copper net in the step (1), standing for 1min, and sucking away the floating liquid by filter paper;
(3) drying at normal temperature for 10 min;
(4) the voltage is selected to be 100kv for transmission electron microscope imaging detection and photographing and storing, and the result is shown in fig. 1: the separated exosome is circular, such as a cup and tray structure, and the diameter of the exosome is concentrated to about 80 nm.
Example particle size analysis (NTA) identification of the method of Sanhostan cow milk exosome identification
(1) Taking out the exosome and PBS mixed solution frozen in the ultra-low temperature refrigerator at minus 80 ℃ in the first embodiment, thawing the mixture on ice until the exosome and PBS mixed solution are completely thawed, taking 10 mu L of the exosome and PBS mixed solution, adding 1.9mL of 1 XPBS, and diluting the mixture to a working concentration suspension;
(2) transferring the mixed solution in the step (1) into a flow tube, imaging and observing exosomes and vesicles with the diameters of 30-150 nm in the suspension one by one in real time through a nano-flow particle size analyzer, and calculating the particle size distribution and concentration of the exosomes in the suspension through Zeta view 8.04.02 software, wherein the result is shown in figure 2: the average grain diameter of the cow milk exosome is 73.82 nm; the exosome concentration was 8.76E +9 pellets/mL as shown in figure 3.
Example Western blot assay identification of the method for the identification of four Holstein cow's milk exosomes
(1) Preparation of an exosome protein sample: taking out exosome frozen in an ultra-low temperature refrigerator at-80 ℃, unfreezing on ice, adding 100 mu L of RIAP protein lysate, and cracking on ice for 30 min;
(2) centrifuging: centrifuging the cracking mixture in (1) at 12,000 Xg for 15min at 4 deg.C in a low-temperature centrifuge, collecting supernatant, and removing precipitate;
(3) protein standard yeast preparation and protein concentration detection: adding 20 mul of protein standard substance with the concentration of 0, 0.025, 0.05, 0.1, 0.2, 0.3, 0.4 and 0.5mg/mL and 20 mul of cracked mixed liquid into a 96-well plate, and repeating for three times in each group; preparing and adding a BCA working solution (solution A: solution B =50: 1) into the holes, oscillating and uniformly mixing, and incubating in an incubator at 37 ℃ for 30 min; measuring the absorbance of A562 by using a full-wavelength microplate reader; the absorbance measured by using protein standards with different concentrations and Excel are used for drawing a standard curve, and the result is shown in figure 4:
obtaining a regression formula: y =0.4287x +0.0683, R2=0.9982；
Substituting the exosome protein absorbance y =0.711 to the above formula, the calculation yields: exosome protein concentration
；
(4) Denaturation: mixing 40 μ L of the cracked mixed solution with 10 μ L of 5 ☓ SDS-PAGE protein loading buffer solution, boiling at 100 deg.C for 5min to obtain denatured protein;
(5) loading, electrophoresis and membrane transfer: opening the purchased commercial 12% prefabricated gel, inserting the gel into an electrophoresis tank, filling electrophoresis buffer solution, adding the denatured 20 mu L protein sample into an electrophoresis hole, and carrying out electrophoresis; taking out electrophoresis gel after electrophoresis, soaking the electrophoresis gel in methanol for activation for 10s, then placing the gel in a precooled membrane conversion buffer solution, sequentially filling a sponge, three layers of filter paper and the electrophoresis gel on a pad, covering the membrane on the electrophoresis gel, covering three pieces of filter paper on the membrane, removing bubbles, finally covering another sponge pad, inserting the sponge pad into a membrane conversion groove, turning on a power supply, and carrying out constant-pressure membrane conversion for 90V and 60 min;
(6) and (3) sealing: placing the transferred membrane on a room-temperature decolorizing shaker, soaking in 5% skimmed milk, and sealing for 1 h;
(7) a first antibody: diluting a Calnexin, CD63, CD81 and TSG101 primary anti-concentrated solution and 5% skimmed milk according to a ratio of 1:2000, and incubating a membrane at 4 ℃ for one night;
(8) secondary antibody: membranes were washed 3 times with 1 ☓ TBST on a room temperature decolorizing shaker for 5min each, and secondary antibodies were mixed with 1 ☓ TBS at 1: diluting with 5000, incubating at room temperature for 60min, washing with 1 ☓ TBST for 3 times, each time for 5 min;
(9) exposure: taking out the PVDF membrane, draining, spreading the protein on the preservative film with the protein surface facing upwards, and dropwise adding the ECLA/B liquid mixture mixed in the same volume onto the membrane for reaction for 1min in a dark place; clamping the film and moving the film into a plastic packaging film, wherein the protein surface is always upward in the operation process;
(10) the film was placed in the imager and exposed for 10s, the brightness and contrast were adjusted and the picture was saved with the results shown in fig. 5: the exosome samples 1,2 and 3 can detect specific proteins CD63, CD81 and TSG101, and can not detect Calnexin protein, while ordinary Cell Samples (BMECs) can only detect Calnexin protein, which indicates that the exosome extracted by the method has good purity.
It will be evident to those skilled in the art that the invention is not limited to the details of the foregoing illustrative embodiments, and that the present invention may be embodied in other specific forms without departing from the spirit or essential attributes thereof. The present embodiments are therefore to be considered in all respects as illustrative and not restrictive, the scope of the invention being indicated by the appended claims rather than by the foregoing description, and all changes which come within the meaning and range of equivalency of the claims are therefore intended to be embraced therein.
Furthermore, it should be understood that although the present description refers to embodiments, not every embodiment may contain only a single embodiment, and such description is for clarity only, and those skilled in the art should integrate the description, and the embodiments may be combined as appropriate to form other embodiments understood by those skilled in the art.
Claims (7)
1. A method for separating exosomes from Holstein cow milk is characterized by comprising the steps of collecting Holstein cow milk samples and separating exosomes through density gradient ultracentrifugation, and specifically comprises the following steps:
a Holstein cow milk sample collection
Selecting healthy Holstein cows in lactation period, collecting 30mL of milk, and storing the collected Holstein cow milk in a low-temperature refrigerator at 4 ℃ for later use;
separation of B Holstein cow's milk exosomes
(1) Centrifuging fresh Holstein cow milk from A at 2,000 Xg and 4 deg.C for 30min, collecting upper layer milk, and removing lower layer precipitate;
(2) transferring the centrifuged upper layer milk into a new sterile 50mL conical centrifuge tube, centrifuging at 4 ℃ and 10,000 Xg for 45min, collecting the upper layer milk, and discarding the lower layer precipitate;
(3) transferring the upper layer milk centrifuged in the step (2) into a new centrifuge tube, selecting an overspeed rotor, performing ultracentrifugation for 120min at the temperature of 4 ℃ and under the condition of 100,000 Xg, discarding the upper layer liquid, and keeping the precipitate;
(4) adding 20mL of 1 XPBS precooled at 4 ℃ into the precipitate obtained in the step (3), resuspending the precipitate, centrifuging the precipitate again at 4 ℃ and 2,000 Xg for 30min, and discarding the precipitate;
(5) transferring the centrifuged supernatant in the step (4) into a new centrifuge tube, centrifuging the centrifuged supernatant at 4 ℃ for 30min at 2,000 Xg, and discarding the precipitate;
(6) repeating the step (5) for 1 time;
(7) transferring the centrifuged supernatant in the step (6) into a new centrifugal tube, selecting an overspeed rotor, and carrying out ultracentrifugation for 120min at the temperature of 4 ℃ and under the condition of 100,000 Xg, removing the supernatant, and keeping the lower-layer precipitate;
(8) resuspending the pellet obtained in (7) in 1mL of precooled PBS, and storing at low temperature of 4 ℃;
(9) respectively preparing 40%, 20%, 10% and 5% iodixanol, sequentially adding 3.6mL of each iodixanol into an ultracentrifuge tube, slowly adding 1mL of heavy suspension obtained in step (8) to the uppermost layer, performing ultracentrifugation at 4 deg.C for 120min to obtain a suspension solution with a concentration of 100,000 Xg;
(10) separating the centrifuged solution into 12 layers, collecting the liquid in the middle 6-9 layers, performing ultracentrifugation at 4 ℃ for 120min at 100,000 Xg, and removing the supernatant to obtain the purified cow milk exosomes;
(11) the exosomes obtained in step (10) were resuspended in 100. mu.L of precooled PBS and used immediately or frozen in an ultra-low temperature freezer at-80 ℃.
2. The method of claim 1, wherein the fresh milk preservation and the step (8) of low temperature freezer model number BCD-525wkpzm (e), the step (11) of low temperature freezer model number Eppendorf CryoCube F570, the step (1) (2) (4) (5) (6) of low temperature centrifuge model number Eppendorf 5810R, the step (3) (7) (9) (10) of low temperature ultracentrifuge model number Beckman Optima L-100 XP.
3. A method for identifying Holstein cow milk exosomes is characterized by comprising transmission electron microscope observation identification, particle size analysis identification and Western blot analysis identification, and specifically comprises the following steps:
transmission Electron Microscope (TEM) observation and identification of A Holstein cow milk exosome
(1) Taking 10 mu L of the exosome + PBS mixed solution separated in the claim 1, dripping the mixed solution on a copper net with 100 meshes, standing for 1min, and sucking away the floating liquid by filter paper;
(2) dripping 10 mu L of phosphotungstic acid on the copper net obtained in the step (1), standing for 1min, and sucking away the floating liquid by filter paper;
(3) drying at normal temperature for 10 min;
(4) selecting 100kv of voltage to carry out transmission electron microscope imaging detection and photographing for storage;
identification of particle size analysis (NTA) of B Holstein cow's milk exosomes
(1) Taking 10 mu L of the separated exosome and PBS mixed solution, adding 1.9mL of 1 XPBS, and diluting to a working concentration suspension;
(2) transferring the step (1) into a flow tube, imaging and observing exosomes and vesicles in the suspension one by one in real time through a nano-flow particle size analyzer, and calculating the particle size distribution and concentration of the exosomes in the suspension through Zeta view 8.04.02 software;
c Holstein cow milk sample marker protein immunoblotting (Western blot) analysis and identification
(1) Preparation of an exosome protein sample: taking out exosome frozen in an ultra-low temperature refrigerator at-80 ℃, unfreezing on ice, adding 100 mu L of RIAP protein lysate, and cracking on ice for 30 min;
(2) centrifuging the cracking mixture in (1) at 12,000 Xg for 15min at 4 deg.C in a low-temperature centrifuge, collecting supernatant, and removing precipitate;
(3) standard curves were made and exosome protein concentrations were detected using the BCA protein concentration kit, the method is outlined below:
adding 20 μ L of protein standard with concentration of 0, 0.025, 0.05, 0.1, 0.2, 0.3, 0.4, 0.5mg/mL and 20 μ L of supernatant from step (2) to 96-well plate, each in triplicate; preparing and adding a BCA working solution (solution A: solution B =50: 1) into the holes, oscillating and uniformly mixing, and incubating in an incubator at 37 ℃ for 30 min; measuring the absorbance of A562 by using a full-wavelength microplate reader; drawing a standard curve by using the absorbance measured by protein standard substances with different concentrations and Excel to obtain a regression formula and the concentration of the exosome protein;
(4) denaturation: mixing 40 μ L of the cracked mixed solution with 10 μ L of 5 ☓ SDS-PAGE protein loading buffer solution, boiling at 100 deg.C for 5min to obtain denatured protein;
(5) loading, electrophoresis and membrane transfer: opening the purchased commercial 12% prefabricated gel, inserting the gel into an electrophoresis tank, filling electrophoresis buffer solution, adding the denatured 20 mu L protein sample into an electrophoresis hole, and carrying out electrophoresis and membrane conversion; taking out electrophoresis gel after electrophoresis, soaking the electrophoresis gel in methanol for activation for 10s, then placing the gel in a precooled membrane conversion buffer solution, sequentially filling a sponge, three layers of filter paper and the electrophoresis gel on a pad, covering the membrane on the electrophoresis gel, covering three pieces of filter paper on the membrane, removing bubbles, finally covering another sponge pad, inserting the sponge pad into a membrane conversion groove, turning on a power supply, and carrying out constant-pressure membrane conversion for 90V and 60 min;
(6) and (3) sealing: placing the transferred membrane on a room-temperature decolorizing shaker, soaking in 5% skimmed milk, and sealing for 1 h;
(7) a first antibody: diluting a Calnexin, CD63, CD81 and TSG101 primary anti-concentrated solution and 5% skimmed milk according to a ratio of 1:2000, and incubating a membrane at 4 ℃ for one night;
(8) secondary antibody: membranes were washed 3 times with 1 ☓ TBST on a room temperature decolorizing shaker for 5min each, and secondary antibodies were mixed with 1 ☓ TBS at 1: diluting with 5000, incubating at room temperature for 60min, washing with 1 ☓ TBST for 3 times, each time for 5 min;
(9) exposure: taking out the PVDF membrane, draining, spreading the protein on the preservative film with the protein surface facing upwards, and dropwise adding the ECLA/B liquid mixture mixed in the same volume onto the membrane for reaction for 1min in a dark place; clamping the film and moving the film into a plastic packaging film, wherein the protein surface is always upward in the operation process;
(10) the film was placed in an imager and exposed for 10s, the brightness and contrast were adjusted and the picture was saved.
4. The method of claim 3, wherein the TEM model is Hitachi 7700; the filter paper is in a Whatman HC0002 model, and the nanoparticle tracking analyzer is in a Particle Metrix ZetaVIEW 17-310 model; the model of the full-wavelength microplate reader is Bio-Tek (Eon), and the incubator is Thermo Fisher 3111; the imager model is GE LAS 4000.
5. The method for identification of Hestein cow's milk exosomes according to claim 3, wherein said protein lysate specification is Beyotime/P0013, said BCA protein concentration detection kit specification is Beyotime/P0010S, said 5 ☓ SDS-PAGE protein loading buffer specification is Beyotime/P0015, said commercial 12% pre-gel specification is GenScript/M01210C, said electrophoresis buffer specification is GenScript/M00138, said transmembrane buffer specification is Script/M00139, said PVDF membrane specification is MERCK MILLIPORE IP00010, said 5% skim milk specification is Solarbio/D8340.
6. The method for identifying the Holstein cow's milk exosomes according to claim 3, wherein the diameter of the exosomes in the particle size analysis and identification is 30-150 nm.
7. The method for identification of Hostan cow's milk exosomes according to claim 3, wherein the voltage of the concentrated gel is set at 60V for 30min and the voltage of the separated gel is set at 120V for 60min during electrophoresis.
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