CN114891723B - Milk-derived exosome and extraction method - Google Patents
Milk-derived exosome and extraction method Download PDFInfo
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Abstract
The invention relates to an extraction method of milk-derived exosomes, which is sequentially carried out according to the following steps: (1) subpackaging the milk raw materials, centrifuging and taking clear liquid; (2) Centrifuging the clear liquid obtained in the step (1) to obtain a clear liquid in the middle layer; (3) Centrifuging the intermediate layer clear liquid obtained in the step (2), taking the clear liquid, uniformly mixing the clear liquid with a PBS buffer solution and an exosome extraction reagent, and incubating; (4) Centrifuging the product obtained in the step (3), retaining the precipitate, and resuspending the precipitate with PBS buffer; (5) And (4) centrifuging the product obtained in the step (4), and taking the supernatant to obtain the product. Compared with the milk-derived exosomes prepared by other methods, the milk-derived exosomes prepared by the extraction method provided by the invention have more complete forms, less required sample volumes, more protein types of the separated milk-derived exosomes and more contribution to analysis and research operation.
Description
Technical Field
The invention relates to the technical field of dairy products, in particular to a milk-derived exosome and an extraction method thereof.
Background
The exosome is a circular or elliptical vesicle with a double-layer membrane structure and actively secreted by cells, the diameter is 30-200nm, and the density is 1.13-1.19g/mL. In 1983, 50nm vesicles were first found in transferrin receptor studies in reticulocytes. In 1987, the canadian scholare Rose Johnstone created the word "Exosome" and thought that exosomes were the only tool for "excretion" of cells to the external environment. In 2007, valadi et al suggested that exosomes contain mRNA and miRNA and can transmit them to another cell, until the function of exosomes and their mechanism began to be extensively studied. Exosomes are present in various body fluid environments, such as serum, urine, saliva, milk, amniotic fluid, and the like. Exosome in biological fluid shows the functional state of the original cells, and can be used as a biological diagnosis marker for liver cancer, prostate cancer and other diseases medically; since the membrane structure of exosome can also be used as a drug carrier to show the characteristic of high-efficiency drug delivery, for example, the exosome load unstable curcumin can improve the treatment effect.
Milk-derived exosomes are vesicles secreted by mammary epithelial cells, and the main components thereof are proteins, lipids and nucleic acids. The membrane surface of milk-derived exosomes has specific membrane proteins, such as the most abundant tetraspanin proteins (CD 9, CD82, CD81 and CD 63), as well as costimulatory molecules (CD 54) and adhesion molecules (CD 11 b). Lipids (phosphatidylcholine, phosphatidylserine, phosphatidylethanolamine, sphingomyelin, and phosphatidylinositol) are also common in exosomes. The nucleic acid in the milk-derived exosome mainly comprises a non-coding single-stranded RNA (micro RNA, mi RNA), a long-chain non-coding RNA (lnc RNA), a circular RNA (circular RNA), an m RNA, a t RNA and the like. Milk-derived exosomes play important roles in physiological processes, such as mediating cell communication, promoting cell growth, participating in immune reactions, and the like.
The currently used exosome separation technology is centrifugation, but centrifugation can damage the form of exosome.
Therefore, the invention is provided.
Disclosure of Invention
In order to solve the existing technical problems, the invention provides a novel method for extracting milk-derived exosomes, and the extraction method has small influence on the form of the exosomes.
In order to achieve the purpose, the technical scheme adopted by the invention is as follows:
in a first aspect, the invention provides a method for extracting milk-derived exosomes, which is sequentially carried out according to the following steps:
(1) Subpackaging the milk raw materials, centrifuging, and collecting the clear liquid;
(2) Centrifuging the clear liquid obtained in the step (1) and taking the clear liquid in the middle layer;
(3) Centrifuging the intermediate layer clear liquid obtained in the step (2), taking the clear liquid, uniformly mixing the clear liquid with a PBS buffer solution and an exosome extraction reagent, and incubating;
(4) Centrifuging the product obtained in the step (3), retaining the precipitate, and resuspending the precipitate with PBS buffer;
(5) Centrifuging the product of the step (4) and taking
Preferably or alternatively, the centrifugation process in step (1) is 1500-2500 Xg centrifugation at room temperature for 5-15min.
Preferably or alternatively, the centrifugation process in step (2) is 8500-10000 Xg centrifugation at room temperature for 25-35min, preferably, the centrifugation time is 30min.
Preferably or alternatively, the centrifugation process in step (3) is 8500-10000 Xg centrifugation at room temperature for 25-35min, and preferably, the centrifugation time is 30min.
Preferably or alternatively, in step (3), the volume ratio of the supernatant, the PBS buffer and the exosome-extracting reagent is 1.
Preferably or alternatively, the exosome-extracting reagent is a total exosome-isolating reagent produced by Thermo Fisher corporation.
Preferably or alternatively, in step (3), the incubation time is 25-35min, preferably, the incubation time is 30min.
Preferably or alternatively, the centrifugation process in the step (4) is 8500-10000 Xg centrifugation at room temperature for 9-11min, and preferably, the centrifugation time is 10min.
Preferably or alternatively, the centrifugation process in the step (5) is 8500-10000 Xg centrifugation at room temperature for 4-6min, and preferably, the centrifugation time is 5min.
In a second aspect, the invention also provides a milk-derived exosome prepared by the extraction method.
Advantageous effects
Compared with the milk-derived exosomes prepared by other methods, the milk-derived exosomes prepared by the extraction method provided by the invention have more complete forms, less required sample volumes, more protein types of the separated milk-derived exosomes and more contribution to analysis and research operation.
Drawings
Fig. 1 is a projection electron microscope image of milk-derived exosomes prepared in example 1;
fig. 2 is a projection electron microscope image of the milk-derived exosomes prepared in example 2;
fig. 3 is a projection electron microscope image of the milk-derived exosomes prepared in comparative example 1;
fig. 4 is a projection electron microscope image of the milk-derived exosomes prepared in comparative example 2;
fig. 5 is a projection electron microscope image of the milk-derived exosomes prepared in comparative example 3;
fig. 6 is a projection electron microscope image of the milk-derived exosomes prepared in comparative example 4;
fig. 7 is a graph of a kind of dairy-derived exosome protein species wien prepared in example 1 and comparative examples 1 and 3.
Detailed Description
In order to facilitate understanding of the present invention, the present invention will be described more fully and in detail below with reference to the accompanying drawings and preferred experimental examples, but the scope of the present invention is not limited to the specific examples below.
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of the present invention.
Unless otherwise specifically stated, various raw materials, reagents, instruments, equipment and the like used in the present invention are commercially available or can be prepared by existing methods.
Example 1
The embodiment provides an extraction method of milk-derived exosomes.
The milk raw material of the milk source exosome is from a fresh milk canned vehicle of Beijing three-component food company Limited.
The milk raw material is respectively loaded in 1mL centrifuge tubes, 1mL sample is added in each tube, centrifugation is carried out for 10min at room temperature and 2000 Xg, clear liquid is taken after centrifugation is finished, centrifugation is carried out again for 30min at room temperature and 10000 Xg, and clear liquid in the middle layer is taken.
Centrifuging the middle layer clear liquid at 10000 Xg for 30min at room temperature, collecting 200 μ L clear liquid, mixing with 200 μ L PBS buffer solution, adding 200 μ L total exosome separating reagent produced by Thermo Fisher company, mixing, incubating at room temperature for 30min, centrifuging at 10000 Xg for 10min at room temperature, and collecting precipitate.
Resuspending the precipitate with 50 μ L PBS buffer solution, centrifuging at 10000 Xg for 5min at room temperature, and collecting supernatant to obtain the extracted exosome.
Example 2
The embodiment provides an extraction method of milk-derived exosomes.
The milk raw material of the milk-derived exosome is Ailiyou milk powder produced by Beijing three-component food Limited.
The extraction process in example 2 was the same as in example 1.
Comparative example 1
This comparative example provides a method for the extraction of milk-derived exosomes.
The milk raw material of the milk source exosome is from a fresh milk canned vehicle of Beijing three-component food company Limited.
The milk raw material is respectively loaded in 1mL centrifuge tubes, 1mL sample is added in each tube, centrifugation is carried out for 10min at room temperature and 2000 Xg, clear liquid is taken after centrifugation is finished, centrifugation is carried out again for 30min at room temperature and 10000 Xg, and clear liquid in the middle layer is taken.
Centrifuging the intermediate layer clear liquid at 10000 Xg for 30min at room temperature, collecting 200 μ L clear liquid, mixing with 200 μ L PBS buffer solution, adding 200 μ L high-efficiency exosome precipitation reagent produced by English special biotechnology (Beijing) Co., ltd, mixing, incubating at room temperature for 30min, centrifuging at 10000 Xg for 10min at room temperature, and collecting precipitate.
Resuspending the precipitate with 50 μ L PBS buffer solution, centrifuging at 10000 Xg for 5min at room temperature, and collecting supernatant to obtain the extracted exosome.
Comparative example 2
The comparative example provides an extraction method of milk-derived exosomes.
The milk raw material of the milk-derived exosome is Ailiyou milk powder produced by Beijing three-component food company Limited.
The extraction process in comparative example 2 was the same as in comparative example 1.
Comparative example 3
This comparative example provides a method for the extraction of milk-derived exosomes.
The milk raw material of the milk source exosome is from a fresh milk canned vehicle of Beijing three-component food company Limited.
Placing 50mL milk raw material in an ultracentrifuge tube, centrifuging at 4 deg.C and 3000rpm for 60min, collecting supernatant, centrifuging at 4 deg.C and 16000rpm for 20min, removing superficial fat, collecting supernatant, centrifuging at 4 deg.C and 30000rpm for 60min, and collecting loose granules.
Re-suspending the collected loose particles by PBS, dissolving by 0.3mol/L sucrose solution, centrifuging at 20000rpm for 120min at 4 ℃, taking the precipitate, re-suspending by PBS, filtering by a 0.22 mu membrane, and collecting the filtered clear liquid, namely the exosome.
Comparative example 4
This comparative example provides a method for the extraction of milk-derived exosomes.
The milk raw material of the milk-derived exosome is from a fresh milk canned vehicle of Beijing three-component food company Limited.
Placing 50mL milk raw material in an ultracentrifuge tube, centrifuging at 4 deg.C and 3000rpm for 60min, collecting supernatant, centrifuging at 4 deg.C and 16000rpm for 20min, removing superficial fat, and collecting supernatant.
Mixing 200 μ L of the clear solution with 200 μ L of PBS buffer solution, adding 200 μ L of high efficiency exosome precipitation reagent produced by Tokyo Biotechnology (Beijing) Limited, mixing, incubating at room temperature for 30min, centrifuging at 10000 Xg for 10min at room temperature, and collecting the precipitate.
The precipitate was resuspended in 50. Mu.L PBS buffer, centrifuged at 10000 Xg for 5min at room temperature, and the supernatant was taken as the exosome extracted.
Effect example 1
The morphology of the milk-derived exosomes obtained in examples 1 to 2 and comparative examples 1 to 4 was observed by a projection electron microscope (TEM), and the results are shown in fig. 1 to 3.
As can be seen from FIGS. 1 to 6, the milk-derived exosomes obtained in examples 1 to 2 and comparative examples 1 to 3 each had a diameter range between 30 to 150nm and each had a cup-packed bilayer membrane structure. The milk-derived exosomes obtained by the methods provided in comparative examples 2-3 have incomplete morphology and disordered background, while the milk-derived exosomes obtained by the methods in comparative example 1 and example 1 have complete morphology and present complete "cup-and-tray" structures. The milk-derived exosome morphology obtained in the method of comparative example 4 was completely disrupted.
Effect example 2
The milk-derived exosomes obtained in example 1 and comparative examples 1 and 3 are respectively treated, and the specific treatment method comprises the following steps: adding 0.1% Rapidest SF reagent produced by Waters into milk exosome 100 μ L, mixing well, ultrasonic dissolving for 30min, loading DTT reagent 100 μ L, and reacting in water bath at 56 deg.C for 1h. After the reaction is finished, naturally cooling to room temperature, adding 100 mu L of 0.1mol/L IAA, reacting for 40min in a dark place, adding 50 mu L of 1mg/mL trypsin, performing enzyme digestion overnight, and adding once more; after the enzymolysis is finished, adding a proper amount of 0.5mol/L HCl to terminate the reaction for 50min, and precipitating RapidGest SF reagent. Desalting with HLB column, removing precipitate generated by RapidGest SF reagent under acidic condition, collecting sample, lyophilizing, re-dissolving with 0.1% formic acid water solution, centrifuging at 10000 × g for 10min, and collecting supernatant.
And detecting the treated milk-derived exosomes by adopting a nanoliter liquid chromatography-tandem Q active Orbitrap mass spectrometer, and drawing a protein species wien graph according to a detection result, wherein the graph is shown in figure 7.
As can be seen from fig. 7, 315 bovine milk exosome protein can be isolated from the milk-derived exosome prepared in example 1, 267 bovine milk exosome protein can be isolated from the milk-derived exosome prepared in comparative example 1, and 307 bovine milk exosome protein can be isolated from the milk-derived exosome prepared in comparative example 3.
In summary, according to the extraction method of the milk-derived exosomes provided by the invention, through the optimization of the process and the selection of the reagents, compared with the traditional high-speed centrifugation method, the prepared milk-derived exosomes are more complete in form, the required sample volume is less, and compared with the similar reagents, the prepared milk-derived exosomes are more in protein types after being separated, so that the method is more suitable for the analysis operation of the subsequent exosomeproteomics.
Finally, it should be noted that: the above examples are only intended to illustrate the technical solution of the present invention, and not to limit it; although the present invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some technical features may be equivalently replaced; and such modifications or substitutions do not depart from the spirit and scope of the corresponding technical solutions of the embodiments of the present invention.
Claims (5)
1. The method for extracting the milk-derived exosome is characterized by comprising the following steps of:
(1) Subpackaging the milk raw materials, centrifuging at room temperature of 2000 Xg for 10min, and collecting the clear liquid;
(2) Centrifuging the clear liquid obtained in the step (1) at room temperature at 10000 Xg for 30min, and taking the clear liquid of the middle layer;
(3) Centrifuging the intermediate layer clear liquid obtained in the step (2) at room temperature of 10000 Xg for 30min, taking the clear liquid, uniformly mixing the clear liquid with a PBS buffer solution and an exosome extraction reagent, and incubating, wherein the volume ratio of the clear liquid to the PBS buffer solution to the exosome extraction reagent is 1;
(4) Centrifuging the product of step (3) at 8500-10000 Xg for 9-11min at room temperature, retaining the precipitate, and resuspending with PBS buffer;
(5) Centrifuging the product of the step (4) at 8500-10000 Xg for 4-6min at room temperature, and taking the supernatant to obtain the product.
2. The method for extracting milk-derived exosomes according to claim 1, wherein in step (3), the incubation time is 25-35min.
3. The method for extracting milk-derived exosomes according to claim 2, wherein in step (3), the incubation time is 30min.
4. The method for extracting milk-derived exosomes according to claim 1, wherein the centrifugation time in step (4) is 10min.
5. The method for extracting milk-derived exosomes according to claim 1, wherein the centrifugation time in step (5) is 5min.
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| CN202210755949.7A CN114891723B (en) | 2022-06-30 | 2022-06-30 | Milk-derived exosome and extraction method |
| PCT/CN2023/089769 WO2024001457A1 (en) | 2022-06-30 | 2023-04-21 | Milk-derived exosome and extraction method |
| CA3205241A CA3205241A1 (en) | 2022-06-30 | 2023-04-21 | Milk-derived exosome and extraction method therefor |
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| CN109468265A (en) * | 2018-11-06 | 2019-03-15 | 广州市创唯曦旺生物科技有限公司 | A method of extracting newborn excretion body |
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| CA2453198A1 (en) * | 2004-01-07 | 2005-07-07 | Wei-Ping Min | Quantification and generation of immune suppressive exosomes |
| CA2960161A1 (en) * | 2014-09-05 | 2016-03-10 | Exerkine Corporation | Exosome isolation |
| US10166259B1 (en) * | 2016-03-15 | 2019-01-01 | 3P Biotechnologies, Inc. | Isolation of exosomes from colostrum powder and exosomal drug formulations using the same |
| CN114717181A (en) * | 2022-04-26 | 2022-07-08 | 陕西师范大学 | Separation method of goat milk exosomes and oral liquid |
| CN114891723B (en) * | 2022-06-30 | 2023-04-14 | 北京三元食品股份有限公司 | Milk-derived exosome and extraction method |
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| CN109468265A (en) * | 2018-11-06 | 2019-03-15 | 广州市创唯曦旺生物科技有限公司 | A method of extracting newborn excretion body |
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