CN115521895B - Application of water-soluble protein as exosome extraction enhancer and exosome extraction reagent - Google Patents
Application of water-soluble protein as exosome extraction enhancer and exosome extraction reagent Download PDFInfo
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- 108010054147 Hemoglobins Proteins 0.000 claims abstract description 14
- 238000011084 recovery Methods 0.000 claims abstract description 5
- 108091003079 Bovine Serum Albumin Proteins 0.000 claims description 43
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Abstract
The invention discloses application of water-soluble protein as an exosome extraction enhancer and an exosome extraction reagent. The water-soluble protein is selected from one or more of animal-derived protein, plant-derived protein or artificial synthetic protein, and the final concentration of the water-soluble protein in the solution is greater than or equal to 1% when the water-soluble protein is used as an enhancer. The exosome extraction reagent comprises a hydrophilic polymer solution and a water-soluble protein; the mass volume concentration of the water-soluble protein in the extraction reagent is greater than or equal to 1%; the hydrophilic polymer is selected from one or more of polyethylene glycol, dextran or polyvinylpyrrolidone; the water-soluble protein is selected from one or more of albumin, globulin or hemoglobin. When the water-soluble protein and the hydrophilic polymer in the scheme of the invention are matched to extract exosomes by a precipitation method, the recovery rate of the exosomes can be obviously improved, and meanwhile, the method is simple to operate, low in cost and beneficial to practical application and popularization.
Description
Technical Field
The invention belongs to the technical field of biological medicines, and particularly relates to application of water-soluble protein as an exosome extraction enhancer and an exosome extraction reagent.
Background
Exosomes, also known as exosomes, 30-150 nm in diameter, are vesicles coated with a lipid bilayer membrane released into the extracellular environment after fusion of the intracellular multivesicular body (MVB) with the cell membrane, which contain proteins, lipids and nucleic acids, and serve as important mediators of cell-cell communication in various physiological and pathological pathways. Current studies indicate that: the content of exosomes in body fluid such as urine, ascites, amniotic fluid, cerebrospinal fluid and the like is relatively low, so that the content is detected, further clinical application prospect is wide, but the prior art cannot capture the exosomes very efficiently.
The methods for separating exosomes which are commonly used at present mainly comprise the following steps:
1) Ultracentrifugation: the most widely used isolation technique, also known as the gold standard for exosome extraction and isolation, is currently used. The ultracentrifugation method is mainly used for obtaining the required components according to the size and density differences of the components in the original solution, and is suitable for separating large-dose sample components with larger differences in sedimentation coefficients. The method does not need to mark exosomes, avoids cross contamination, but is not suitable for large-scale practical application and popularization due to time consumption, high cost, structural damage and the like.
2) Ultrafiltration method: the method is simple to operate, but the purity of the obtained exosomes is not high.
3) Polymer precipitation process: the method of polymer precipitation generally uses polyethylene glycol (PEG) as a medium, and exosomes are harvested under centrifugation conditions by decreasing their solubility. The polymer precipitation method has simple operation and short analysis time, and is suitable for treating samples with the sample size. But in urine, ascites, amniotic fluid, cerebrospinal fluid and the like and cell culture supernatant, the recovery rate of exosomes is low, which is not beneficial to subsequent experimental analysis.
Disclosure of Invention
The invention aims to provide application of water-soluble protein as an exosome extraction enhancer and an exosome extraction reagent. Aims at solving the technical problem of lower recovery rate of exosomes in the polymer precipitation method in the prior art.
The technical scheme adopted by the invention for solving the technical problems is as follows:
the invention provides application of water-soluble protein as an exosome extraction enhancer.
As a preferred embodiment, the water-soluble protein is used at a mass volume concentration in the extraction solution of greater than or equal to 1%, preferably greater than or equal to 10%.
As a preferred embodiment, the water-soluble protein is completely soluble at a solution pH of 5.0-9.0.
As a preferred embodiment, the solution in which the water-soluble protein is dissolved is an aqueous solution or a salt solution.
As a preferred embodiment, the water-soluble protein is selected from one or more of animal-derived proteins, plant-derived proteins or synthetic proteins.
As a preferred embodiment, the animal-derived proteins include proteins derived from body fluid circulation and tissue-derived proteins; among them, the protein derived from the circulation of body fluid is preferably albumin, globulin or hemoglobin.
As a preferred embodiment, the water-soluble protein is used for exosome extraction in body fluids or cell culture supernatants. The body fluid refers to hydrothorax, urine, cerebrospinal fluid, ascites, amniotic fluid and the like.
As a preferred embodiment, the exosome extraction is by polymer precipitation.
The invention also provides an exosome extraction reagent, which comprises: a hydrophilic polymer solution and a water-soluble protein; the water-soluble protein is present in the extraction reagent at a concentration of greater than or equal to 1% by mass and by volume, preferably greater than or equal to 10%.
As a preferred embodiment, the hydrophilic polymer is selected from one or more of polyethylene glycol, dextran or polyvinylpyrrolidone.
Compared with the prior art, the invention has the beneficial effects that:
1, in the extraction of exosomes by a polymer precipitation method, when the water-soluble protein is matched with a polymer for use, the recovery rate of the exosomes in different body fluids and cell culture supernatants can be obviously improved.
2, the exosome extraction reagent provided by the invention is simple to operate, low in cost and wide in prospect in clinical application.
Drawings
FIG. 1 shows the results of AR-V7 detection using albumin as an enhancer in example 1.
FIG. 2 shows the results of AR-FL detection with albumin as an enhancer in example 1.
FIG. 3 shows the results of AR-V7 detection of immunoglobulins as enhancers in example 2.
FIG. 4 shows the results of AR-FL detection with the immunoglobulin as an enhancer in example 2.
FIG. 5 shows the results of AR-V7 detection of hemoglobin as an enhancer in example 3.
FIG. 6 shows the results of AR-FL detection with hemoglobin as an enhancer in example 3.
FIG. 7 shows the results of AR-V7 detection of different sample types in example 4.
FIG. 8 shows the results of AR-FL detection in example 4 with different sample types.
FIG. 9 shows the results of AR-V7 detection by PEG salt solution extraction in example 5.
FIG. 10 shows the results of AR-FL detection by PEG salt solution extraction in example 5.
FIG. 11 shows the results of AR-V7 detection by aqueous dextran extraction in example 6.
FIG. 12 shows the results of AR-FL detection by aqueous dextran extraction in example 6.
FIG. 13 shows the results of AR-V7 detection by aqueous polyvinylpyrrolidone solution extraction in example 7.
FIG. 14 shows the results of AR-V7 detection by aqueous polyvinylpyrrolidone solution extraction in example 7.
FIG. 15 shows the results of AR-V7 detection in example 8, which is a comparison of the present invention with the patent document.
FIG. 16 shows the results of AR-FL detection in example 8 according to the present invention in comparison with the patent document.
Detailed Description
The following describes the technical scheme of the present invention in detail by referring to examples. The reagents and biological materials used hereinafter are commercial products unless otherwise specified.
Example 1: testing of Albumin as exosome extraction enhancer
1) Configuration of bovine serum albumin with different concentrations
Different amounts of bovine serum albumin (product number: DX201, dening organism, hereinafter referred to as BSA) were dissolved in 20mL of PBS, respectively, to obtain BSA solutions having mass-volume concentrations of 3%,15%,30% and 60%, respectively.
2) Preparation of urine exosomes and extraction of exosome RNA
Urine sample preparation: the required amount is 50mL, the mixture is divided into 5 50mL centrifuge tubes, and AR-V7 (500 cobis) positive 22RV1 exosomes are respectively added, and the serial numbers are S1-S5 in sequence; centrifuging at 4deg.C for 15min at 3000g, and transferring supernatant to a new centrifuge tube;
preparing a urine exosome extraction system: preparing polyethylene glycol aqueous solution, dissolving PEG8000 in water to prepare PEG aqueous solution with mass volume concentration of 30%, and preparing exosome extraction system as shown in table 1.
TABLE 1
| Sample numbering | Urine volume (mL) | PBS(mL) | 30% PEG aqueous solution (mL) | BSA solution (mL) |
| S1 | 10 | 10 | 10 | 0 |
| S2 | 10 | 0 | 10 | 10 |
| S3 | 10 | 0 | 10 | 10 |
| S4 | 10 | 0 | 10 | 10 |
| S5 | 10 | 0 | 10 | 10 |
The mass volume concentrations of the 10mL BSA solutions added with sample numbers of S2, S3, S4 and S5 in Table 1 are 3%,15%,30% and 60%, respectively; the S1 sample was not added with BSA solution, and 10mL of BS solution was added as a blank. The exosome extraction systems of S1-S5 samples are 30mL, and the mass volume concentration of BSA protein in the extraction systems is 0%, 1%, 5%, 10% and 20% respectively. After shaking and mixing the sample system, standing at 4 ℃ overnight, centrifuging for 10min, and extracting RNA of exosomes by using Qiagen miRNeasy Micro Kit from the obtained precipitate.
3) qPCR detection of AR-V7 and AR-FL
The sequences of the primers and probes used for PCR detection are as follows:
AR-V7 forward primer: CAGGGATGACTCTGGGAGAAA (SEQ ID No. 1);
AR-V7 reverse primer: AGTCAGCCTTTCTTCAGGGTC (SEQ ID No. 2);
AR-V7 probe: AGCAGGGATGACTCT (SEQ ID No. 3);
AR forward primer: TGCTCAAGACGCTTCTACCAG (SEQ ID No. 4);
AR reverse primer: AGTGAACTGATGCAGCTCTC (SEQ ID No. 5);
AR probe: TTCTACCAGCTCACCA (SEQ ID No. 6);
the detection results are shown in FIG. 1 and FIG. 2, wherein FIG. 1 is the detection result of AR-V7 and FIG. 2 is the detection result of AR-FL.
The AR-V7 and AR-FL detection results of FIGS. 1 and 2 show that: the S2 group, the S3 group, the S4 group and the S5 group are all better than the S1 group, namely, the addition of BSA has obvious enhancement effect on the extraction of exosomes; the S4 and S5 groups are not much different and both are better than the S3 and S2 groups, indicating that BSA addition to some extent has been optimal for exosome extraction. The detection data result shows that: BSA was used as an exosome enhancer at a concentration of 1% or more, and at an optimal concentration of 10% or more.
Example 2: testing of immunoglobulins as exosome extraction enhancers
1) Bovine immunoglobulin configuration at different concentrations
Bovine immunoglobulin lyophilized powder (product number: SLB66-0100, eimer technology Co., ltd., hereinafter abbreviated as IgG) 3g,6g,12g were dissolved in 20mL PBS, respectively, to obtain IgG solutions having concentrations of 15%,30% and 60% in order.
2) Preparation of urine exosomes and extraction of exosome RNA
Urine sample preparation: the required amount is 40mL, the mixture is divided into 4 50mL centrifuge tubes, and AR-V7 (500 cobis) positive 22RV1 exosomes are respectively added, and the serial numbers are G1-G4 in sequence; centrifuging at 4deg.C for 15min at 3000g, and transferring supernatant to a new centrifuge tube;
preparing a urine exosome extraction system: an aqueous polyethylene glycol solution was prepared, and PEG8000 was dissolved in water to prepare an aqueous PEG solution having a mass volume concentration of 30%, and the system preparation of the exosome extraction system was as shown in Table 2.
TABLE 2
| Sample numbering | Urine volume (mL) | PBS(mL) | 30% PEG aqueous solution (mL) | IgG solution (mL) |
| G1 | 10 | 10 | 10 | 0 |
| G2 | 10 | 0 | 10 | 10 |
| G3 | 10 | 0 | 10 | 10 |
| G4 | 10 | 0 | 10 | 10 |
The mass volume concentrations of the 10mL IgG solutions added with the sample numbers of G2, G3 and G4 in Table 2 are 15%,30% and 60%, respectively; the IgG solution was not added to the G1 sample, and a 10ml pbs solution was added as a blank. The exosome extraction systems of the G1-G4 samples are 30mL, and the mass volume concentration of the IgG protein in the extraction systems is 0%, 5%, 10% and 20% respectively. After shaking and mixing the sample system, standing at 4 ℃ overnight, centrifuging for 10min, and extracting RNA of exosomes by using Qiagen miRNeasy Micro Kit from the obtained precipitate.
3) qPCR detection of AR-V7 and AR-FL
The specific detection method and the detection primer and probe sequences used are the same as in example 1.
The detection results are shown in FIG. 3 and FIG. 4, wherein FIG. 3 is the detection result of AR-V7 and FIG. 4 is the detection result of AR-FL.
The AR-V7 and AR-FL detection results in the figures 3 and 4 show that the G2 group, the G3 group and the G4 group are all superior to the G1 group, namely, the addition of IgG has obvious enhancement effect on the extraction of exosomes; the group G3 and the group G4 are not much different and both are better than the group G2, indicating that IgG addition to some extent is optimal for exosome extraction. The detection data result shows that: igG is used in exosome enhancer at an optimal concentration of 10% or more.
Example 3: testing of hemoglobin as exosome extraction enhancer
1) Bovine hemoglobin configuration of different concentrations
Different amounts of hemoglobin (product number: SS8024-25g, van der Waals, hereinafter abbreviated as Hem) 3g,6g,12g were dissolved in 20mL PBS, respectively, to obtain Hem solutions with mass volume concentrations of 15%,30% and 60% in order.
2) Preparation of urine exosomes and extraction of exosome RNA
Urine sample preparation: the required amount is 40mL, the mixture is divided into 4 50mL centrifuge tubes, and AR-V7 (500 cobis) positive 22RV1 exosomes are respectively added, and the serial numbers are H1-H4 in sequence; centrifuging at 4deg.C for 15min at 3000g, and transferring supernatant to a new centrifuge tube;
preparing a urine exosome extraction system: an aqueous polyethylene glycol solution was prepared, and PEG8000 was dissolved in water to prepare an aqueous PEG solution having a mass volume concentration of 30%, and the preparation of the exosome extraction system was as shown in Table 3.
TABLE 3 Table 3
The mass volume concentrations of 10mL of Hem solution added with the sample numbers of H2, H3 and H4 in the table 3 are 15%,30% and 60% respectively; no Hem solution was added to the H1 sample, and a 10mL BS solution was added as a blank. The exosome extraction systems of the H1-H4 samples are 30mL, and the mass volume concentration of the Hem protein in the extraction systems is 0%, 5%, 10% and 20% respectively. After shaking and mixing the sample system, standing at 4 ℃ overnight, centrifuging for 10min, and extracting RNA of exosomes by using Qiagen miRNeasy Micro Kit from the obtained precipitate.
3) qPCR detection of AR-V7 and AR-FL
The specific detection method and the detection primer and probe sequences used are the same as in example 1.
The detection results are shown in FIG. 5 and FIG. 6, wherein FIG. 5 is the detection result of AR-V7 and FIG. 6 is the detection result of AR-FL.
The AR-V7 and AR-FL detection results in the figures 5 and 6 show that the H2 group, the H3 group and the H4 group are all superior to the H1 group, namely, the addition of hemoglobin has obvious enhancement effect on the extraction of exosomes; the group H3 is not much different from the group H4 and both are better than the group H2, which indicates that the exosome extraction is optimized to a certain extent by adding hemoglobin. The detection data result shows that: i.e. the optimal use concentration of hemoglobin for exosome enhancers is 10% and above.
Example 4: albumin as an enhancer for testing of exosomes extracted from different samples
1) 30% bovine serum albumin BSA configuration
Bovine serum albumin (cat# DX201, dening organism) 6g was dissolved in 20mL PBS to prepare a 30% BSA solution by mass volume.
2) Sample preparation from different sources
2 cases of hydrothorax samples (No. P1 and P2), 2 cases of ascites samples (No. A1 and A2), 2 cases of cerebrospinal fluid samples (No. C1 and C2), and 20mL of serum-free cell culture supernatants (No. L1 and L2) of cell lines H1975 and A549 were respectively taken, centrifuged at 4℃for 10min, and the supernatants were collected and transferred to new centrifuge tubes.
3) Exosome preparation and exosome RNA extraction
Respectively adding AR-V7 (500 caps) positive 22RV1 exosomes into different types of samples, and vibrating and uniformly mixing;
exosome extraction system preparation: the preparation of the aqueous solution of PEG8000 (30% by mass/volume concentration) and the preparation of the exosome extraction system with albumin is shown in table 4.
TABLE 4 Table 4
| Sample numbering | Sample size (mL) | PBS(mL) | 30% PEG aqueous solution (mL) | 30% BSA solution (mL) |
| P1 | 10 | 0 | 10 | 10 |
| P2 | 10 | 0 | 10 | 10 |
| A1 | 10 | 0 | 10 | 10 |
| A2 | 10 | 0 | 10 | 10 |
| C1 | 10 | 0 | 10 | 10 |
| C2 | 10 | 0 | 10 | 10 |
| L1 | 10 | 0 | 10 | 10 |
| L2 | 10 | 0 | 10 | 10 |
The preparation of the exosome extraction system without albumin is shown in table 5.
TABLE 5
After shaking and mixing the sample system uniformly, standing at 4 ℃ overnight, centrifuging for 10min, and extracting RNA of exosomes by using Qiagen miRNeasy Micro Kit on the obtained precipitate;
4) qPCR detection of AR-V7 and AR-FL
The specific detection method and the detection primer and probe sequences used are the same as in example 1.
The detection results are shown in FIG. 7 and FIG. 8, wherein FIG. 7 is the detection result of AR-V7 and FIG. 8 is the detection result of AR-FL.
The results of the AR-V7 and AR-FL assays in fig. 7 and 8 show that adding BSA protein as an exosome enhancer to different sample types provides better exosome extraction than the non-added group.
Example 5: albumin as enhancer for testing extraction of exosomes from PEG and inorganic salts
1) Configuration of bovine serum albumin with different concentrations
Different amounts of bovine serum albumin (cat# DX201, dening organism) 3g,6g,12g were dissolved in 20mL PBS, respectively, to give Bovine Serum Albumin (BSA) solutions with concentrations of 15%,30% and 60% in order.
2) Preparation of urine exosomes and extraction of exosome RNA
Urine sample preparation: the required amount is 40mL, the mixture is divided into 4 50mL centrifuge tubes, and AR-V7 (500 cobis) positive 22RV1 exosomes are respectively added, and the serial numbers are Y1-Y4 in sequence; centrifuging at 4deg.C for 15min at 3000g, and transferring supernatant to a new centrifuge tube;
urine exosome preparation: the preparation of the exosome extraction system was formulated as shown in Table 6, with a 0.5M NaCl solution of PEG8000 (30% by mass and volume concentration of PEG).
TABLE 6
| Sample numbering | Urine volume (mL) | PBS(mL) | 30% PEG salt solution (mL) | BSA solution (mL) |
| Y1 | 10 | 10 | 10 | 0 |
| Y2 | 10 | 0 | 10 | 10 |
| Y3 | 10 | 0 | 10 | 10 |
| Y4 | 10 | 0 | 10 | 10 |
The mass volume concentrations of 10mL BSA solutions added for sample numbers Y2, Y3, and Y4 in Table 6 were 15%,30%, and 60%, respectively; the Y1 sample was not added with BSA solution, and 10mL of BS solution was added as a blank. The exosome extraction systems of the Y1-Y4 samples are 30mL, and the mass volume concentration of BSA protein in the extraction systems is 0%, 5%, 10% and 20% respectively. After shaking and mixing the sample system, standing at 4 ℃ overnight, centrifuging for 10min, and extracting RNA of exosomes by using Qiagen miRNeasy Micro Kit from the obtained precipitate.
Shaking and mixing the extraction system, standing at 4 ℃ overnight, centrifuging for 10min, and extracting RNA of exosomes by using Qiagen miRNeasy Micro Kit;
3) qPCR detection of AR-V7 and AR-FL
The specific detection method and the detection primer and probe sequences used are the same as in example 1.
The detection results are shown in FIG. 9 and FIG. 10, wherein FIG. 9 is the detection result of AR-V7 and FIG. 10 is the detection result of AR-FL.
The AR-V7 and AR-FL detection results in the figures 9 and 10 show that the Y2 group, the Y3 group and the Y4 group are all superior to the Y1 group, namely, the addition of bovine serum albumin has obvious enhancement effect on the extraction of exosomes by PEG salt solution; the group Y3 is not much different from the group Y4 and both are better than the group Y2, which indicates that the extraction of exosomes is optimized to a certain extent by adding bovine serum albumin. The detection data result shows that: bovine serum albumin is used as an exosome enhancer at a concentration of 5% or more, preferably at a concentration of 10% or more.
Example 6: albumin as an enhancer for testing dextran extraction exosomes
1) 30% bovine serum albumin BSA configuration
Bovine serum albumin (cat# DX201, dening organism) 6g was dissolved in 20mL PBS, 30% BSA solution.
2) Sample preparation from different sources
Respectively taking 2 urine samples, dividing into two tubes (with serial numbers of U1, U1-1, U2 and U2-1 in sequence), centrifuging at 4 ℃ for 10min at 20mL each tube and 3000g each tube, taking the supernatant, and transferring to a new centrifuge tube.
3) Exosome preparation and exosome RNA extraction
Adding AR-V7 (500 caps) positive 22RV1 exosomes into different types of samples respectively, and shaking and mixing uniformly.
Preparation of exocrine: an aqueous solution of 100mg/mL dextran (D1662-100G, SIGMA) was prepared, and the albumin addition groups were U1-1 and U2-1, and the no albumin addition groups were U1 and U2. The formulation of the extraction system is shown in table 7.
TABLE 7
| Sample numbering | Sample size (mL) | PBS(mL) | Dextran solution (mL) | BSA solution (mL) |
| U1 | 10 | 10 | 10 | 0 |
| U2 | 10 | 10 | 10 | 0 |
| U1-1 | 10 | 0 | 10 | 10 |
| U2-2 | 10 | 0 | 10 | 10 |
Shaking and mixing the extraction system, standing at 4 ℃ overnight, centrifuging for 10min, and extracting RNA of exosomes by using Qiagen miRNeasy Micro Kit;
4) qPCR detection of AR-V7 and AR-FL As described in example 1
The detection results are shown in FIG. 11 and FIG. 12, wherein FIG. 11 is the detection result of AR-V7, and FIG. 12 is the detection result of AR-FL.
The AR-V7 and AR-FL detection results in FIG. 11 and FIG. 12 show that the extraction efficiency of the exosomes in the U1-1 group and the U2-1 group is significantly better than that of the exosomes in the U1 group and the U2 group, namely, when glucan is used as a precipitator for exosome extraction, the addition of albumin as an enhancer has a significant enhancement effect on exosome extraction.
Example 7: albumin as an enhancer for testing of polyvinylpyrrolidone extracted exosomes
1) 30% bovine serum albumin BSA configuration
Bovine serum albumin (cat# DX201, dening organism) 6g was dissolved in 20mL PBS, 30% BSA solution.
2) Sample preparation from different sources
Respectively taking 2 urine samples, dividing into two tubes (with serial numbers of V1, V1-1, V2 and V2-1 in sequence), centrifuging at 4 ℃ for 10min at 20mL each tube and 3000g each tube, taking the supernatant, and transferring to a new centrifuge tube.
3) Exosome preparation and exosome RNA extraction
Respectively adding AR-V7 (500 caps) positive 22RV1 exosomes into different types of samples, and vibrating and uniformly mixing;
preparation of exocrine: an aqueous solution (LUVITEC@K90, shanghai Chengcheng fine chemical Co., ltd.) with mass and volume concentration of polyvinylpyrrolidone of 20% was prepared, and the albumin addition groups were V1-1 and V2-1, and the albumin no-addition groups were V1 and V2. The formulation of the extraction system is shown in table 8.
TABLE 8
| Sample numbering | Sample size (mL) | PBS(mL) | Polyvinylpyrrolidone solution (mL) | BSA solution (mL) |
| V1 | 10 | 10 | 10 | 0 |
| V2 | 10 | 10 | 10 | 0 |
| V1-1 | 10 | 0 | 10 | 10 |
| V2-2 | 10 | 0 | 10 | 10 |
Shaking and mixing the extraction system, standing at 4 ℃ overnight, centrifuging for 10min, and extracting RNA of exosomes by using Qiagen miRNeasy Micro Kit;
4) qPCR detection of AR-V7 and AR-FL As described in example 1
The detection results are shown in FIG. 13 and FIG. 14, wherein FIG. 13 shows the detection result of AR-V7, and FIG. 14 shows the detection result of AR-FL.
The detection results of AR-V7 and AR-FL in FIG. 13 and FIG. 14 show that the extraction efficiency of the exosomes in the V1-1 group and the V2-1 group is significantly better than that of the exosomes in the V1 group and the V2 group, namely, when polyvinylpyrrolidone is used as a precipitant for exosome extraction, albumin is added as an enhancer, and the exosomes extraction has a remarkable enhancement effect.
Example 8: comparison test of the Water-soluble proteins of the present invention as enhancers with patent 201810865076.9
1) Urine sample preparation: the required amount is 40mL, the mixture is divided into 4 50mL centrifuge tubes, and AR-V7 (500 cobis) positive 22RV1 exosomes are respectively added, and the serial numbers are Z1-Z4 in sequence; centrifuge at 4 ℃,3000g, for 15min, transfer supernatant into a new centrifuge tube.
2) Patent 201810865076.9 related reagent configuration
Extraction reagent P1 configuration: glucose sulfate 20000 (Sigma-Aldrich) 10g, basic protein (Sigma-Aldrich) 0.5 in 100mL PBS solution;
purified microsphere B1 configuration: protein A (Tiandi Renzhen, inc.).
3) Preparation of patent 201810865076.9 exosomes and extraction of exosome RNA
Taking urine samples Z1 and Z2, respectively adding 5mL of P1, reversing and uniformly mixing, standing at 4 ℃ for 30 minutes, centrifuging at 10,000g for 10 minutes, discarding the supernatant, collecting the precipitate, centrifuging again for 10,000g for 5 minutes, discarding the residual supernatant, adding 20mL of PBS solution, repeatedly blowing and uniformly mixing, adding 1/2 purified microsphere Protein A, centrifuging at 4,000g for 3 minutes, discarding the supernatant, retaining microspheres, adding PBS solution into the microspheres to obtain exosome solution, and extracting the RNA of the exosome by using Qiagen miRNeasy Micro Kit.
4) Preparation of urine exosome and extraction of exosome RNA
Taking a urine sample Z3 and Z4, respectively adding 10mL of an aqueous solution (30% concentration) of PEG8000 and 10mL of a 30% BSA solution, shaking and mixing uniformly, standing at 4 ℃ overnight, centrifuging for 10min with 3000g, and extracting RNA of an exosome by using Qiagen miRNeasy Micro Kit;
5) qPCR detection of AR-V7 and AR-FL As described in example 1
The detection results are shown in FIG. 15 and FIG. 16, wherein FIG. 15 shows the detection result of AR-V7, and FIG. 16 shows the detection result of AR-FL.
The AR-V7 and AR-FL detection results of fig. 15 and 16 show that the extraction efficiency of the exosomes in the Z3 group and the Z4 group is significantly better than that of the exosomes in the Z1 group and the Z2 group, i.e., the present patent is more advantageous in the exosome enrichment effect compared with the patent 201810865076.9.
The foregoing is only a part of the preferred embodiments of the present invention, and the present invention is not limited to the contents of the embodiments. It will be apparent to those skilled in the art that various changes and modifications can be made within the scope of the technical solution of the present invention, and any changes and modifications are within the scope of the present invention.
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Claims (5)
1. The application of bovine serum albumin, bovine immunoglobulin or bovine hemoglobin in extracting exosomes by polymer precipitation method to improve exosome recovery rate is provided.
2. The use according to claim 1, wherein: the mass volume concentration of the bovine serum albumin, bovine immunoglobulin or bovine hemoglobin in the extraction solution is greater than or equal to 1% when the bovine serum albumin, bovine immunoglobulin or bovine hemoglobin is used.
3. The use according to claim 2, wherein: the mass volume concentration of the bovine serum albumin, bovine immunoglobulin or bovine hemoglobin in the extraction solution is more than or equal to 10 percent when the bovine serum albumin, bovine immunoglobulin or bovine hemoglobin is used.
4. The use according to claim 1, wherein: the bovine serum albumin, bovine immunoglobulin or bovine hemoglobin is completely soluble at a solution pH of 5.0-9.0.
5. The use according to claim 1, wherein: the bovine serum albumin, bovine immunoglobulin or bovine hemoglobin is used for exosome extraction in body fluids or cell culture supernatants.
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