CN115354024A - Blood sample exosome extraction kit based on combination of protease treatment, SEC (SEC-activated charcoal) column and membrane filtration technology and application - Google Patents

Abstract

The invention relates to an extraction kit, in particular to a blood sample exosome extraction kit based on combination of protease treatment, SEC (SEC-activated carbon) column and membrane filtration technology and application thereof. The method removes cells, platelets and non-exosome microvesicles by three-step differential centrifugation. Removing impurities above 220nm by pinhole filter, treating with protease to dissolve impurity protein (such as albumin and apolipoprotein) or decompose protein polymer into small peptide fragments, and removing impurities below 10nm by ultrafiltration. And finally, according to the concentration requirement, further performing ultrafiltration, purification and concentration to obtain the high-quality exosome. The combination method is simple and easy to implement, has obvious effect, and successfully solves the problems that the blood sample has complex components, more foreign proteins, high difficulty in separating and extracting exosomes, difficulty in finding clear exosome vesicular structures under an electron microscope and the like.

Description

A blood sample extracorporeal filter based on the combination of protease treatment, SEC column and membrane filtration technology Secretion Extraction Kit and Application

technical field

The invention is an extraction kit, in particular to a blood sample exosome extraction kit and its application based on the combination of protease treatment, SEC column and membrane filtration technology.

Background technique

At present, the most common methods for extracting exosomes from blood samples include ultracentrifugation, PEG precipitation reagent method, SEC column method, ultrafiltration method, and immunomagnetic bead method. Ultracentrifugation and immunomagnetic bead methods are limited by instruments and consumables, and are costly. The extraction kits of several other methods do not perform well in the extraction of blood samples, which is due to the particularity of the blood samples themselves. After removing the cells and platelets from the blood sample, there are still a lot of impurity blood proteins, such as albumin and apolipoprotein, protein aggregates and so on. Some of these impurity proteins in the blood are very similar to the molecular size of exosomes, which has caused great difficulties in the extraction and purification of exosomes. It is difficult to obtain high-quality exosomes by simple sec column method or ultrafiltration method. . Three common detection methods for exosomes are western blot, NTA and electron microscopy. In particular, the electron microscope detection effect in blood samples is easily affected by impurities, and it is difficult to find a clear exosome morphology map.

Exosomes are small membranous vesicles with a diameter of 30-150nm that are released outside the cell after the fusion of multivesicular bodies and cell membranes. They are vesicles with a phospholipid bilayer structure and are widely present in various body fluids. It contains a variety of active biomolecules such as proteins, lipids, and nucleic acids. Exosomes are vehicles for intercellular communication. The patent "Exosome Preparation Kit and Exosome Preparation Method in Serum or Plasma" is applied to PEG precipitation, proteinase K pretreatment and the use of purified microspheres. The principle of the PEG precipitation method is: the polymer hijacks water molecules to reduce the solubility of exosomes, and then settles under low-speed centrifugation conditions. Proteinase K pretreatment: Pretreatment of serum or plasma samples with proteinase K, a serine protease with broad specificity, is useful for reducing albumin and exosome-associated apolipoproteins A-1 and B during purification. Quantity matters. The agarose beads are coupled with organic groups, and the agarose beads coupled with organic groups can adsorb the impurity protein particles that have settled down, making the extracted exosomes more pure. The disadvantage of this method is that PEG will co-sediment with exosomes, resulting in polymers that are difficult to remove, and the agarose beads are coupled with organic groups, which makes the operation complicated and costly. The patent "a kit for extracting plasma exosomes and its application" uses avidin-modified nano magnetic bead solution, biotin-modified specific aptamer solution and magnetic separation device. Disadvantage: High cost of reagents. The patent "rapid and high-purity separation method for human plasma exosomes" firstly uses agarose gel column to pre-separate human plasma to remove most of the small-sized high-abundance proteins; and the collected fractions are sequentially processed Combined, concentrated by ultrafiltration; and then separated by high-performance liquid phase size exclusion chromatography to achieve the separation of exosomes and interfering lipoproteins, and finally obtain high-purity human plasma exosome fractions. High-performance liquid chromatography is required, and the equipment cost is high. The patent "A Method for Efficiently and Quickly Separating Exosomes from Plasma" includes the following steps: Step 1: Prepare a hydrophilic polymer solution; Step 2: Dilute to obtain diluted plasma; Step 3: Centrifuge to remove cellular components in the plasma to obtain Cell-removed plasma; Step 4: Precipitate exosomes by ultra-high-speed centrifugation; Step 5: Suspend exosomes using phosphate buffered saline; Step 6: Use a single-phase solution of phenol and guanidine thiocyanate to obtain Re-dissolved suspended exosomes; Step 7: Add 200 μl chloroform and shake for 15 seconds to obtain the shaken exosome solution; Step 8: Centrifuge the layered exosome solution for 15 minutes; Step 9: Draw the top layer The supernatant liquid was obtained from the colorless liquid; Step 10: Add an equal volume of isopropanol for precipitation to obtain a high-purity exosome precipitate. This method uses organic reagents that are highly toxic and may contaminate and destroy exosomes.

Patents mentioned above:

Patent "Exosome Preparation Kit and Exosome Preparation Method in Serum or Plasma" CN 108918228 A

Patent "a kit for extracting plasma exosomes and its application" CN 109266606 A

Patent "Rapid and high-purity separation method of human plasma exosomes" CN 113252808 A

Patent "A method for efficiently and rapidly separating plasma exosomes" CN 111996165 A

Contents of the invention

A blood sample exosome extraction kit and application based on the combination of protease treatment, SEC column and membrane filtration technology. The method uses three steps of differential centrifugation to remove cells, platelets, and non-exosomal microvesicles. Remove impurities above 220nm through a pinhole filter, then dissolve impurity proteins (albumin and apolipoprotein, etc.) by protease treatment or decompose protein aggregates into small peptides, and then remove impurities below 10nm by ultrafiltration . The molecular size of the remaining sample is within the molecular size range of exosomes. Finally, the exosomes are purified in the exosome area through the SEC column. Finally, according to the concentration requirements, further ultrafiltration, purification and concentration are performed to obtain high-quality exosomes. The combination method is simple and easy to implement, and the effect is remarkable. It successfully solves the problems of complex components of blood samples, many impurities, difficult separation and extraction of exosomes, and difficulty in finding clear vesicle-like structures of exosomes under the electron microscope.

Above-mentioned technical problem of the present invention is mainly solved by following technical scheme:

A blood sample exosome extraction kit and application based on the combination of protease treatment, SEC column and membrane filtration technology, according to the following steps:

(1) Human blood is collected in the collection tube: the temperature is 4°C, the weight is 200-500g, the centrifugation time is 5-10min, and the supernatant is transferred to a new centrifuge tube after centrifugation;

(2) Supernatant after transfer: the temperature is 4°C, the weight is 2000-2500g, and the centrifugation time is 10-15min. After the centrifugation, transfer the supernatant to a new centrifuge tube;

Steps (1) and (2) are divided into two steps. The reason is that the first low-speed centrifugation is to centrifuge the complete cells in the blood, and the second centrifugation increases the speed to centrifuge some broken cell contents. Or impurities, etc. If the rotation speed is high for the first time, it is likely that the cells will rupture, and the contents will be released, which cannot be completely precipitated, resulting in contamination of exosome purification in the later stage.

(3) After step (2) is finished, transfer the supernatant to a new centrifuge tube as a human plasma sample;

(4) Dilute the plasma prepared in step (3) with PBS, dilute the plasma to 2 times the volume, and then perform centrifugation: the temperature is 4°C, the weight is 10000-18000g, the centrifugation time is 30min, and the centrifugation is completed. Transfer Transfer the supernatant to a new centrifuge tube;

(5), after step (4), filter the blood of the new centrifuge tube through 0.8um filter membrane and 0.22um filter membrane successively;

(6), carry out protease digestion treatment to the blood filtered in step (5), protease is one or more in pepsin, trypsin or other enzymes with protein digestion function;

Prepare protease solution 50-1000ug/ml, add protease solution 50-1000ul/ml to blood sample, vortex for 30 seconds and mix well: dilute protease with PBS or double distilled water.

(7), after step (6), add the mixed solution into a water bath with a temperature of 37° C. and incubate for 5 to 60 minutes;

(8), after step (7), transfer to a 60°C water bath and incubate for 3 to 15 minutes;

(9), after step (8), transfer the sample to the pretreated 100KD ultrafiltration tube, the temperature is 4°C, the weight is 3000-4500g, and the centrifugation time is 5-15min; discard the filtrate, and the liquid in the ultrafiltration tube Transfer to a new centrifuge tube, use a pipette gun to take an appropriate amount of PBS to gently flush the filter membrane of the ultrafiltration tube several times, and transfer the solution to the previous centrifuge tube, the final volume of the sample in the centrifuge tube is 0.5-3ml;

(10) Column balance: Rinse the SEC column with PBS solution of 3 times the column volume; after the rinsing is completed, after no liquid flows out from the outlet of the bottom cover, add the sample in the centrifuge tube in step (9), and wait until the sample at the outlet of the bottom cover has flowed out. After the liquid flows out, add the eluent PBS solution. The empty column volume is the first 1.5ml without collection, and the volume of exosomes required for collection is 1-4ml eluent after the empty column volume;

(11) Transfer the collected eluate to a 100KD ultrafiltration tube at a temperature of 4°C, a weight of 3000-4500g, and a centrifugation time of 5-15min; discard the filtrate, and add 0.2-1ml according to the concentration of exosomes Put the PBS solution on the filter membrane of a 100KDa ultrafiltration tube, and let it stand for 1-3 minutes; use a pipette gun to absorb the PBS solution on the filter membrane, gently blow and wash the ultrafiltration membrane several times, and collect the exosomes with the required concentration. Store at -80°C.

As a preference, in step (10), the 3 times column volume is 20ml; the pH of the PBS solution is 7.2-7.4.

The invention discloses a blood sample exosome extraction kit and its application based on the combination of protease treatment, SEC column and membrane filtration technology. Cells, platelets, and non-exosomal microvesicles are removed by three-step differential centrifugation. Remove impurities above 220nm through a pinhole filter, then dissolve impurity proteins (albumin and apolipoprotein, etc.) by protease treatment or decompose protein aggregates into small peptides, and then remove impurities below 10nm by ultrafiltration . The molecular size of the remaining sample is within the molecular size range of exosomes. Finally, the exosomes are purified in the exosome area through the SEC column. Finally, according to the concentration requirements, further ultrafiltration, purification and concentration are performed to obtain high-quality exosomes. The combination method is simple and easy to implement, and the effect is remarkable. In particular, the use of protease before subsequent operations not only dissolves part of the miscellaneous proteins, but also decomposes the protein aggregates into small peptides, and the miscellaneous proteins are then removed one by one by ultrafiltration and SEC columns. Due to the complex composition of blood samples and many miscellaneous proteins, some proteins and protein aggregates are similar in size to exosomes, and they cannot be successfully separated from exosomes simply by SEC column or ultrafiltration. This combination method solves the problems of complex components of blood samples, many impurities, difficult separation and extraction of exosomes, and difficulty in finding clear vesicle-like structures of exosomes under the electron microscope.

Description of drawings

Fig. 1 is the western blot method verification plasma exosome extraction result in the present invention;

In Figure 1, HSP70, TSG101, CD9 and CD63 are human exosome marker proteins;

Fig. 2 is the result of extracting NTA from plasma exosomes in the present invention;

In Figure 2, it was diluted 1000 times on the computer, and the ZetaView nanoparticle tracking analyzer was used for NTA detection; the detection concentration on the computer was 7.7×10 ⁷ particles/ml, and the original exosome concentration was 7.7×10 ¹⁰ particles/ml.

Fig. 3 is the plasma exosome extraction electron microscope result in the present invention;

Fig. 4 is that the western blot method in the present invention verifies the pepsin treatment plasma sample;

Albumin and Apolipoprotein A1 removal effect in Figure 4. 1: before protease treatment 2: after protease treatment.

Fig. 5 is a collection diagram of the SEC column in the present invention to remove foreign protein exosomes.

Detailed ways

The technical solutions of the present invention will be further specifically described below through the embodiments and in conjunction with the accompanying drawings.

Example 1: A blood sample exosome extraction kit and application based on the combination of protease treatment, SEC column and membrane filtration technology, according to the following steps:

(1) Human blood is collected in the collection tube: the temperature is 4°C, the weight is 200g, the centrifugation time is 5min, and the supernatant is transferred to a new centrifuge tube after centrifugation;

(2) Supernatant after transfer: the temperature is 4°C, the weight is 2000g, and the centrifugation time is 10min. After the centrifugation, the supernatant is successively transferred to a new centrifuge tube;

(3) After step (2) is finished, transfer the supernatant to a new centrifuge tube as a human plasma sample;

(4) Dilute the plasma prepared in step (3) with PBS, dilute the plasma to 2 times the volume, and then perform centrifugation: the temperature is 4°C, the weight is 10000-18000g, the centrifugation time is 30min, and the centrifugation is completed. Transfer Transfer the supernatant to a new centrifuge tube;

(5), after step (4), filter the blood of the new centrifuge tube through 0.8um filter membrane and 0.22um filter membrane successively;

(6), carry out protease digestion treatment to the blood filtered in step (5), protease is one or more in pepsin, trypsin or other enzymes with protein digestion function;

Prepare protease solution 50-1000ug/ml, add protease solution 50-1000ul/ml to blood sample, vortex for 30 seconds and mix well: dilute protease with PBS or double distilled water.

(7), after step (6), add the mixed solution into a water bath with a temperature of 37°C and incubate for 5 minutes;

(8), after step (7), transfer to a 60°C water bath and incubate for 3 minutes;

(9), after step (8), transfer the sample to the pretreated 100KD ultrafiltration tube, the temperature is 4°C, the weight is 3000-4500g, and the centrifugation time is 5min; the filtrate is discarded, and the liquid in the ultrafiltration tube is transferred to In a new centrifuge tube, use a pipette gun to take an appropriate amount of PBS to gently rinse the filter membrane of the ultrafiltration tube several times, and transfer the solution to the previous centrifuge tube. The final volume of the sample in the centrifuge tube is 0.5ml;

(10) Column balance: Rinse the SEC column with PBS solution of 3 times the column volume; after the rinsing is completed, after no liquid flows out from the outlet of the bottom cover, add the sample in the centrifuge tube in step (9), and wait until the sample at the outlet of the bottom cover has flowed out. After the liquid flows out, add the eluent PBS solution. The volume of the empty column is the first 1.5ml without collection, and the volume of exosomes required for collection is 1ml of the eluent after the volume of the empty column;

(11) Transfer the collected eluate to a 100KD ultrafiltration tube at a temperature of 4°C, a weight of 3000-4500g, and a centrifugation time of 5-15min; discard the filtrate, and add 0.2ml of Put the PBS solution on the filter membrane of a 100KDa ultrafiltration tube and let it stand for 1min; use a pipette gun to absorb the PBS solution on the filter membrane, gently blow and wash the ultrafiltration membrane several times, collect the exosomes with the required concentration, and store them at -80 Store in Celsius.

In step (10), 3 times the column volume is 20ml; the pH of the PBS solution is 7.2.

Example 2: A blood sample exosome extraction kit based on protease treatment, SEC column and membrane filtration technology and its application, according to the following steps:

(1) Human blood is collected in the collection tube: the temperature is 4°C, the weight is 300g, the centrifugation time is 7min, and the supernatant is transferred to a new centrifuge tube after centrifugation;

(2) Supernatant after transfer: the temperature is 4°C, the weight is 2200g, the centrifugation time is 12min, and the supernatant is successively transferred to a new centrifuge tube after centrifugation;

(3) After step (2) is finished, transfer the supernatant to a new centrifuge tube as a human plasma sample;

(4) Dilute the plasma prepared in step (3) with PBS, dilute the plasma to 2 times the volume, and then perform centrifugation: the temperature is 4°C, the weight is 15000g, the centrifugation time is 30min, and the supernatant is transferred after centrifugation to a new centrifuge tube;

(5), after step (4), filter the blood of the new centrifuge tube through 0.8um filter membrane and 0.22um filter membrane successively;

(6), carry out protease digestion treatment to the blood filtered in step (5), protease is one or more in pepsin, trypsin or other enzymes with protein digestion function;

Prepare 800ug/ml protease solution, add 800ul/ml protease solution to the blood sample, vortex for 30 seconds and mix well: dilute protease with PBS or double distilled water.

(7), after step (6), add the mixed solution into a water bath with a temperature of 37°C and incubate for 25 minutes;

(8), after step (7), transfer to a 60°C water bath and incubate for 8 minutes;

(9), after step (8), transfer the sample to the pretreated 100KD ultrafiltration tube, the temperature is 4°C, the weight is 3500g, and the centrifugation time is 10min; the filtrate is discarded, and the liquid in the ultrafiltration tube is transferred to a new In the centrifuge tube, use a pipette gun to take an appropriate amount of PBS to gently flush the filter membrane of the ultrafiltration tube several times, and transfer the solution to the centrifuge tube just now. The final volume of the sample in the centrifuge tube is 2ml;

(10) Column balance: Rinse the SEC column with PBS solution of 3 times the column volume; after the rinsing is completed, after no liquid flows out from the outlet of the bottom cover, add the sample in the centrifuge tube in step (9), and wait until the sample at the outlet of the bottom cover has flowed out. The liquid flows out, then add the eluent PBS solution, the volume of the empty column is the first 1.5ml without collecting, and the volume of exosomes required for collection is the 2ml eluent after the empty column volume;

(11) Transfer the collected eluate to a 100KD ultrafiltration tube at a temperature of 4°C, a weight of 3500g, and a centrifugation time of 10 minutes; discard the filtrate, and add 0.8ml of PBS solution to 100KDa according to the concentration of exosomes On the filter membrane of the ultrafiltration tube, let it stand for 2 minutes; use a pipette gun to absorb the PBS solution on the filter membrane, gently blow and wash the ultrafiltration membrane several times, collect the exosomes of the required concentration, and store them at -80 degrees Celsius.

In step (10), 3 times the column volume is 20ml; the pH of the PBS solution is 7.3.

Example 3: A blood sample exosome extraction kit based on protease treatment, SEC column and membrane filtration technology and its application, according to the following steps:

(1) Human blood is collected in the collection tube: the temperature is 4°C, the weight is 500g, the centrifugation time is 10min, and the supernatant is transferred to a new centrifuge tube after centrifugation;

(2) Supernatant after transfer: the temperature is 4°C, the weight is 2500g, and the centrifugation time is 15min. After the centrifugation, the supernatant is successively transferred to a new centrifuge tube;

(3) After step (2) is finished, transfer the supernatant to a new centrifuge tube as a human plasma sample;

(4) Dilute the plasma prepared in step (3) with PBS, dilute the plasma to 2 times the volume, and then perform centrifugation: the temperature is 4°C, the weight is 18000g, the centrifugation time is 30min, and the supernatant is transferred after centrifugation to a new centrifuge tube;

(5), after step (4), filter the blood of the new centrifuge tube through 0.8um filter membrane and 0.22um filter membrane successively;

(6), carry out protease digestion treatment to the blood filtered in step (5), protease is one or more in pepsin, trypsin or other enzymes with protein digestion function;

Prepare protease solution 1000ug/ml, add protease solution 1000ul/ml to the blood sample, vortex for 30 seconds and mix well: dilute protease with PBS or double distilled water.

(7), after step (6), add the mixed solution into a water bath with a temperature of 37°C and incubate for 60 minutes;

(8), after step (7), transfer to a 60°C water bath and incubate for 15 minutes;

(9), after step (8), transfer the sample to the pretreated 100KD ultrafiltration tube, the temperature is 4°C, the weight is 4500g, and the centrifugation time is 15min; the filtrate is discarded, and the liquid in the ultrafiltration tube is transferred to a new In the centrifuge tube, use a pipette gun to take an appropriate amount of PBS to gently wash the filter membrane of the ultrafiltration tube several times, and transfer the solution to the centrifuge tube just now. The final volume of the sample in the centrifuge tube is 3ml;

(10) Column balance: Rinse the SEC column with PBS solution of 3 times the column volume; after the rinsing is completed, after no liquid flows out from the outlet of the bottom cover, add the sample in the centrifuge tube in step (9), and wait until the sample at the outlet of the bottom cover has flowed out. After the liquid flows out, add the eluent PBS solution, the volume of the empty column is the first 1.5ml without collecting, and the volume of exosomes required for collection is the 4ml eluent after the volume of the empty column;

(11) Transfer the collected eluate to a 100KD ultrafiltration tube at a temperature of 4°C, a weight of 4500g, and a centrifugation time of 15 minutes; discard the filtrate, and add 1ml of PBS solution to a 100KDa ultrafiltration tube according to the concentration of exosomes. On the filter membrane of the filter tube, let it stand for 3 minutes; use a pipette gun to absorb the PBS solution on the filter membrane, gently blow and wash the ultrafiltration membrane several times, collect the exosomes with the required concentration, and store them at -80 degrees Celsius.

In step (10), 3 times the column volume is 20ml; the pH of the PBS solution is 7.4.

Claims (2)

1. A blood sample exosome extraction kit and application based on the combination of protease treatment, SEC column and membrane filtration technology, characterized in that it is carried out in the following steps: (1) Human blood is collected in the collection tube: the temperature is 4°C, the weight is 200-500g, the centrifugation time is 5-10min, and the supernatant is transferred to a new centrifuge tube after centrifugation; (2) Supernatant after transfer: the temperature is 4°C, the weight is 2000-2500g, and the centrifugation time is 10-15min. After the centrifugation, transfer the supernatant to a new centrifuge tube; (3) After step (2) is finished, transfer the supernatant to a new centrifuge tube as a human plasma sample; (4) Dilute the plasma prepared in step (3) with PBS, dilute the plasma to 2 times the volume, and then perform centrifugation: the temperature is 4°C, the weight is 10000-18000g, the centrifugation time is 30min, and the centrifugation is completed. Transfer Transfer the supernatant to a new centrifuge tube; (5), after step (4), filter the blood of the new centrifuge tube through 0.8um filter membrane and 0.22um filter membrane successively; (6), carry out protease digestion treatment to the blood filtered in step (5), protease is one or more in pepsin, trypsin or other enzymes with protein digestion function; Prepare protease solution 50-1000ug/ml, add protease solution 50-1000ul/ml to blood sample, vortex for 30 seconds and mix well: dilute protease with PBS or double distilled water. (7), after step (6), add the mixed solution into a water bath with a temperature of 37° C. and incubate for 5 to 60 minutes; (8), after step (7), transfer to a 60°C water bath and incubate for 3 to 15 minutes; (9), after step (8), transfer the sample to the pretreated 100KD ultrafiltration tube, the temperature is 4°C, the weight is 3000-4500g, and the centrifugation time is 5-15min; discard the filtrate, and the liquid in the ultrafiltration tube Transfer to a new centrifuge tube, use a pipette gun to take an appropriate amount of PBS to gently flush the filter membrane of the ultrafiltration tube several times, and transfer the solution to the previous centrifuge tube, the final volume of the sample in the centrifuge tube is 0.5-3ml; (10) Column balance: Rinse the SEC column with PBS solution of 3 times the column volume; after the rinsing is completed, after no liquid flows out from the outlet of the bottom cover, add the sample in the centrifuge tube in step (9), and wait until the sample at the outlet of the bottom cover has flowed out. After the liquid flows out, add the eluent PBS solution. The empty column volume is the first 1.5ml without collection, and the volume of exosomes required for collection is 1-4ml eluent after the empty column volume; (11) Transfer the collected eluate to a 100KD ultrafiltration tube at a temperature of 4°C, a weight of 3000-4500g, and a centrifugation time of 5-15min; discard the filtrate, and add 0.2-1ml according to the concentration of exosomes Put the PBS solution on the filter membrane of a 100KDa ultrafiltration tube, and let it stand for 1-3 minutes; use a pipette gun to absorb the PBS solution on the filter membrane, gently blow and wash the ultrafiltration membrane several times, and collect the exosomes with the required concentration. Store at -80°C. 2. A blood sample exosome extraction kit and application based on protease treatment, SEC column and membrane filtration technology combined according to claim 1, characterized in that: in step (10), 3 times the column volume is 20ml; the pH of the PBS solution is 7.2-7.4.

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