CN115558629A - Exosome extraction affinity magnetic bead and extraction kit thereof - Google Patents
Exosome extraction affinity magnetic bead and extraction kit thereof Download PDFInfo
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- CN115558629A CN115558629A CN202210241836.5A CN202210241836A CN115558629A CN 115558629 A CN115558629 A CN 115558629A CN 202210241836 A CN202210241836 A CN 202210241836A CN 115558629 A CN115558629 A CN 115558629A
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- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2509/00—Methods for the dissociation of cells, e.g. specific use of enzymes
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Abstract
The invention relates to an exosome extraction affinity magnetic bead and an extraction kit thereof, wherein the affinity magnetic bead is obtained by solidifying MARCKS-ED polypeptide on the surface layer of the magnetic bead and modifying carboxyl or streptavidin SA. The kit comprises exosome extraction affinity magnetic beads, a washing buffer solution and an elution buffer solution. The affinity magnetic bead has the advantages that the specific surface area of the magnetic bead is large, the steric hindrance of the polypeptide is small, the loading capacity of the magnetic bead is high, and the formed kit extraction scheme is convenient for developing clinical application and assisting the development of a liquid biopsy cause.
Description
Technical Field
The invention belongs to the field of clinical examination, and particularly relates to an exosome extraction affinity magnetic bead and an extraction kit thereof.
Background
Extracellular Vesicles (EVs) are a generic term for microvesicles with membrane structure actively secreted by cells, and are mainly classified into exosomes (exosomes), microvesicles (MVs) and apoptotic bodies (apoptotic bodies) according to their size, biological properties and formation processes. Exosome is EVs with the diameter of 30-150 nm formed by fusing a multivesicular vesicle and a cell membrane, widely exists in body fluids such as blood, saliva, urine, breast milk and the like, carries multiple biomarkers such as nucleic acid, protein, metabolite, phospholipid and the like which represent the properties of maternal cells, has phospholipid bilayer protection and stable inclusion, and is an intercellular basic physiological communication mechanism. The exosome plays an important role in the occurrence and the evolution of various cancers, can promote the formation of the microenvironment of the various cancers, enhance the invasion and metastasis capacities of tumors, participate in tumor immunosuppression and tumor targeted drug resistance, and has application value in the early diagnosis and treatment of the various cancers.
At present, methods commonly used for extracting and purifying exosomes include a differential ultracentrifugation method, a tangential flow ultrafiltration method, a size exclusion chromatography method, a polymer precipitation method, an immunocapture method, a microfluidic chip separation method and the like. The ultracentrifugation method is a recognized gold standard method for exosome extraction, but an ultracentrifuge is required to be equipped, the operation is time-consuming and labor-consuming, the ultracentrifuge is highly dependent on manpower, and the ultracentrifuge is difficult to apply to clinical detection. Furthermore, plasma or serum contains a large amount of high-density lipoproteins whose density distribution overlaps with exosomes, and ultracentrifugation is based on density and particle size separation, and complete separation of exosomes from high-density lipoproteins is difficult to achieve. Although the tangential flow ultrafiltration method can conveniently and rapidly extract exosomes, chylomicron or lipoprotein with the size close to that of the exosomes cannot be distinguished. Exclusion chromatography suffers from the same problems and causes dilution of the sample, often requiring concentration of the resulting sample in combination with other methods. The exosome extracted by the polymer precipitation method has the defects of high impurity protein pollution, uneven particle morphology and influence on downstream analysis. Although the immunocapture method can specifically capture exosomes, and the obtained exosomes have high purity, the method has high cost and low yield, and only can specifically enrich exosomes positive for certain surface proteins. The microfluidic chip separation method has high requirements on materials and technologies, the use cost is generally high, and a sample with a large volume is difficult to process.
The MagCapture exosome extraction kit PS developed by fuji and photonics provides a novel affinity strategy (i.e., PS affinity method), and high-purity exosomes and other extracellular vesicles with PS on the membrane surface can be separated by using Phosphatidylserine (PS) binding protein TIM4 and magnetic beads. The kit captures exosomes in a metal ion dependent manner, and can elute complete exosomes under a neutral condition by using a chelating agent. However, the kit can only process serum and heparin anticoagulated plasma samples, and for the plasma samples anticoagulated by metal chelating agents such as EDTA and sodium citrate, the samples need to be processed by adding heparin, so that the steps are complicated. And the whole operation process from sample pretreatment to affinity reaction to elution to obtain the purified exosome takes about 5 hours, and is basically unacceptable in clinical examination.
Disclosure of Invention
The invention aims to solve the technical problem of providing an exosome extraction affinity magnetic bead and an extraction kit thereof, wherein the affinity magnetic bead has the advantages of large specific surface area of the magnetic bead, small steric hindrance of polypeptide and high magnetic bead load, and the formed kit extraction scheme is convenient for developing clinical application and assisting in the development of liquid biopsy career.
The invention provides an exosome extraction affinity magnetic bead, which is obtained by solidifying MARCKS-ED polypeptide on the surface layer of the magnetic bead and modifying carboxyl, amino or streptavidin SA.
The MARCKS-ED polypeptide sequence is as described in any one of the following 1) -6):
1)KKKKKRFSFKKSFKLSGFSFKKNKK(SEQ ID NO:1)；
2)KKSFKLSGFSFKKNKK(SEQ ID NO:2)；
3)KRFSFKK(SEQ ID NO:3)；
4)KKRFSFKK(SEQ ID NO:4)；
5)KKRFSFKKF(SEQ ID NO:5)；
6)KKFSFKKF(SEQ ID NO:6)；
the amino acid in the MARCKS-ED polypeptide sequence is a D-type amino acid.
The magnetic beads are Fe 3 O 4 The surface of the silicon-based magnetic bead with the inner core is modified with carboxyl, amino or streptavidin SA, and the diameter range is 200nm-3000nm.
The invention also provides a preparation method of the exosome extraction affinity magnetic bead, which comprises the following steps:
pretreating carboxyl magnetic beads, coupling with MARCKS-ED polypeptide after EDC activation, and finally sealing and storing to obtain exosome extraction affinity magnetic beads. Wherein, the N end of the MARCKS-ED polypeptide is provided with modified FITC fluorescein or other modifications, such as 5-FAM modification, NBD modification, dansyl modification, HYNIC modification, DTPA modification, MCA modification and the like.
The specific method comprises the following steps:
1) Magnetic bead pretreatment: after mixing the magnetic beads, 100. Mu.L of Mag-COOH magnetic beads were put into a 1.5mL centrifuge tube, the supernatant was removed by magnetic separation, washed 2 times with 200. Mu.L of MEST solution (0.1 MMES, pH 6.0,0.05% Tween 20), and the supernatant was removed;
2) EDC activation: rapidly adding 100 μ L EDC solution (10 mg/mL, using the MEST solution as dispersant) and 100 μ L NHS (10 mg/mL, using the MEST solution as dispersant) into a centrifuge tube containing magnetic beads, vortex-mixing to fully suspend the magnetic beads, activating at 25 deg.C for 60min, and keeping the suspended state of the magnetic beads (reverse-mixing by using a vertical mixer); after the steps, the carboxyl on the surface of the magnetic bead is activated, and covalent coupling can be carried out on the biological ligand with primary amino;
3) Magnetic bead coupling: removing supernatant by magnetic separation, adding 200-800 μ g of MARCKS-ED polypeptide (the appropriate dosage and concentration need to be optimized according to specific experiments, keeping the pH of the solution approximately equal to 8.0, and adding 0.05% Tween 20 to improve the dispersibility of magnetic beads and avoid the existence of reagents containing primary amino groups except biological ligands in a buffer system), gently mixing, coupling at 25 ℃ for 2h, or after coupling at 25 ℃ for 1h, standing at 4 ℃ for overnight, and keeping the suspension state of the magnetic beads during coupling (reverse mixing can be carried out by using a vertical mixer);
4) And (3) sealing: placing the EP tube on a magnetic separation frame, magnetically separating to remove supernatant, adding 200-500 μ L of Tris-HCl (0.1M, pH = 8.0) for resuspending magnetic beads, reacting at 25 ℃ for 3h to block non-specific adsorption sites on the surfaces of the magnetic beads, and after blocking, adding 200-500 μ L of BSA/PBS solution (pH 7.2 containing 1 BSA) for secondary blocking for 1h, wherein the suspension state of the magnetic beads is maintained (reversed and uniform mixing can be carried out by using a vertical mixer);
5) And (3) storage: the centrifuge tube was placed on a magnetic separator and the supernatant was removed by magnetic separation, washed 3 times with 200. Mu.L of PBS (pH 7.2) or a preservation solution each time, resuspended in the preservation solution (the amount of the preservation solution added was determined as necessary to adjust the concentration of the coupled ligand beads), and stored at 4 ℃. 0.02% (w/v) sodium azide (NaN 3) can be added to the preservation solution as a bacteriostatic agent.
The magnetic beads can be used for subsequent operations in combination with the above steps.
The invention also provides a preparation method of the exosome extraction affinity magnetic bead, which comprises the following steps:
and (3) pretreating the amino magnetic beads, coupling with the MARCKS-ED polypeptide activated by EDC, and finally sealing and storing to obtain the exosome extraction affinity magnetic beads. Wherein, the N end of the MARCKS-ED polypeptide is provided with modified FITC fluorescein or other modifications, such as 5-FAM modification, NBD modification, dansyl modification, HYNIC modification, DTPA modification, MCA modification and the like.
The specific method comprises the following steps:
1. magnetic bead pretreatment: after mixing the magnetic beads evenly, 100. Mu.L of amino magnetic beads were put into a 1mL EP tube, the supernatant was removed by magnetic attraction, washed 3 times with 200. Mu.L of PBS solution (50mM PBS, pH 7.4), and the supernatant was removed by magnetic attraction;
EDC activating polypeptide: adding freshly prepared 10mL of EDC solution (10 mg/mL, MEST (0.1M MES, pH 6.0,0.05% Tween 20) as dispersant) and 10mL of NHS (10 mg/mL, using the above MEST solution as dispersant) into a centrifuge tube containing MARCKS-ED polypeptide, vortexing, and activating at 25 ℃ for 60min;
3. magnetic bead coupling: adding 50-200 mug MARCKS-ED polypeptide (the appropriate dosage and concentration need to be optimized according to specific experiments, keeping the pH of the solution approximately equal to 8.0, and if necessary adding 0.05% -Tween-20 to improve the dispersibility of the magnetic beads) into an EP tube with the magnetic beads for gentle mixing; wrapping with tin foil paper (reaction is away from light), coupling at 25 deg.C for 3 hr, or coupling at 25 deg.C for 1 hr, standing at 4 deg.C for coupling overnight, and maintaining the suspension state of magnetic beads during coupling (vertical mixer can be used for reverse mixing);
4. and (3) sealing after coupling: placing the EP tube on a magnetic separation rack, magnetically separating to remove the supernatant, adding 1000. Mu.L of BSA/PBS solution (pH 7.2, containing 5% BSA) to resuspend the magnetic beads (optionally with sonication), reacting at 25 ℃ for 1h to block non-specific adsorption sites on the surfaces of the beads, while maintaining the magnetic beads in suspension (mixing by inversion using a vertical mixer);
5. and (3) storage: the EP tubes were placed on a magnetic separation rack and the supernatant removed by magnetic separation, washed 3 times with 200. Mu.L PBS (pH 7.2) or preservation solution, resuspended in preservation solution, added with 0.02% (w/v) sodium azide (NaN 3) to inhibit bacterial growth, and finally stored at 4 ℃.
The magnetic beads can be used for subsequent operations in combination with the above steps.
The invention also provides a preparation method of the exosome extraction affinity magnetic bead, which comprises the following steps:
preprocessing streptavidin SA magnetic beads, coupling with biotin-modified MARCKS-ED polypeptide, and storing to obtain exosome extraction affinity magnetic beads.
The specific method comprises the following steps:
1) Magnetic bead pretreatment: the bead bottle was placed on a vortex shaker for 20s and the beads were resuspended by shaking. Remove 100. Mu.L of the beads to a new centrifuge tube using a pipette. Magnetic separation, pipette off the supernatant, and remove the tube from the magnetic separator. Add 1mL of PBST to the centrifuge tube, cover the centrifuge tube lid, and shake the resuspended beads thoroughly. Magnetic separation and removal of supernatant.
The above steps were repeated twice, and washing was repeated three times in total.
2) Magnetic bead coupling: 1mL of MARCKS-ED polypeptide diluted with PBS (to make the concentration of magnetic beads 1 mg/mL) was added, and the magnetic beads were resuspended with sufficient shaking. The centrifuge tube was placed on a rotary mixer and mixed by rotation at room temperature for 60min. Magnetic separation and transfer of the supernatant to a new centrifuge tube. The beads were washed five times as per "pretreatment".
3) And (3) storage: the magnetic beads were resuspended in a magnetic bead storage medium (1 XPBS containing 0.1% (w/v) BSA,0.1% (w/v) proclin-300).
The magnetic beads can be used for subsequent operations in combination with the above steps.
The invention also provides an exosome extraction kit, which comprises exosome extraction affinity magnetic beads, a washing buffer solution and an elution buffer solution.
The washing buffer consists of:
wherein the surfactant may be Tween-20 or TRITONX-100.
Preferably: 10mmol/L Na 2 HPO 4 ，1.75mmol/L KH 2 PO 4 ，137mmol/L NaCl，2.65mmol/L KCl，Tween-20 0.05％w/v，pH 7.2-7.6。
The elution buffer solution is composed of any one solution or a combination solution:
preferably: 20mM Tris-HCl,0.5-1M NaCl, pH 7.2-7.4.
The invention also provides an application of the exosome extraction kit, which comprises the following steps: and respectively preprocessing the sample and the exosome extraction affinity magnetic beads, then extracting exosomes in the sample by the exosome extraction affinity magnetic beads, washing by using a washing buffer solution, and eluting by using an elution buffer solution to obtain exosomes.
The specific method comprises the following steps:
1) Sample processing
The separated serum/plasma samples were centrifuged at 3000g for 10min at 4 ℃ to remove cell debris and the supernatant was transferred to a new tube for use. If high-purity exosome needs to be separated, the sample can be centrifuged again at 10000g for 20 minutes at 4 ℃ to remove impurities, and the supernatant is transferred to a new tube for standby.
If the cell culture solution is a cell culture solution supernatant sample, after the centrifugation, the supernatant is transferred to an ultrafiltration tube (with the molecular weight cutoff of 100 kDa) for ultrafiltration concentration, and the volume is concentrated to about 1mL.
2) Magnetic bead preparation
(1) And (3) after the magnetic bead suspension is fully and uniformly mixed, taking 100 mu L of magnetic beads to a reaction tube of the kit.
(2) Adding 500 mu L of washing liquid, whirling, shaking, mixing uniformly, placing on a magnetic frame, standing for 1 minute, and sucking away the supernatant. And (3) repeating the step (2) twice.
3) Extraction of exosomes
And adding 1mL of the serum/plasma sample treated in the first step into the magnetic beads, mixing uniformly by vortex, and then placing the mixture on a turnover mixing instrument to incubate for 30 minutes in a turnover manner at room temperature (10-25 ℃).
4) Washing magnetic bead
(1) After the incubation, the reaction tube was placed on a magnetic frame and allowed to stand for 1 minute, and the supernatant was aspirated.
(2) Adding 1mL of washing solution, uniformly mixing by vortex, placing on a magnetic frame, standing for 1 minute, and sucking away the supernatant. And (3) repeating the step (2) twice.
5) Elution of exosomes
(1) Add 50. Mu.L of the eluent to the beads, vortex and mix well, incubate for 10 minutes at room temperature (10-25 ℃) with several vortexes being interrupted.
(2) The reaction tube was placed on a magnetic stand and allowed to stand for 1 minute, and the supernatant was aspirated into a new centrifuge tube, which was the purified exosome.
Repeating the steps (1) and (2) once, and combining the exosomes obtained twice.
The exosome extraction affinity magnetic beads can also be directly used for extracting exosomes.
Advantageous effects
The affinity magnetic bead has the advantages of large specific surface area, small steric hindrance of polypeptide and high magnetic bead load. The rich lysine side chain and the polypeptide N-terminal of MARCKS-ED can be subjected to condensation reaction with magnetic beads, so that the coupling efficiency is improved. The affinity magnetic beads have strong capturing specificity to exosomes, the extraction purity can meet the requirement of clinical diagnosis, and the method has the advantages of simple operation, short time consumption and low cost. The formed kit extraction scheme can realize automatic extraction by adapting to corresponding equipment, is convenient for developing clinical application and assists in the development of liquid biopsy career.
Drawings
FIG. 1 is a schematic diagram of the use of the extraction kit of the present invention.
FIG. 2a shows the percent positivity of FITC-labeled magnetic beads in method 1 of example 1.
FIG. 2b is a graph comparing the coupling effect of method 3 in example 1.
Figure 3 is exosome nanoparticle tracking analysis.
Figure 4 is an exosome transmission electron microscope.
FIG. 5 is an exosome protein marker assay; wherein, exosome is an exosome sample, cell is a cell sample, and plasma is a plasma sample.
FIG. 6 shows the effect of different exosome extraction methods.
FIG. 7 shows electron microscopy results for different exosome extraction methods.
Fig. 8 shows WB results for different exosome extraction methods.
FIG. 9 is a graph showing NTA results of exosomes in cell culture supernatant.
FIG. 10 is a TEM image of exosomes in cell culture supernatant.
FIGS. 11a and 11b are comparisons of exosome nucleomics extracted by different methods.
Detailed Description
The invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. Further, it should be understood that various changes or modifications of the present invention may be made by those skilled in the art after reading the teaching of the present invention, and such equivalents may fall within the scope of the present invention as defined in the appended claims.
Example 1
Preparation of MARCKS-ED polypeptide coupled magnetic beads
Method 1
Polypeptide probe synthesis: the MARCKS-ED polypeptide is biosynthesized by the committee, and FITC fluorescein is modified at the N terminal. The sequence of the decorated MARCKS-ED polypeptide is
FITC-acp-KKKKKRFSFKKSFKLSGFSFKKNKK。
Reagents such as carboxyl magnetic beads, MES, NHS, EDC and the like are purchased from solitary organisms, tween-20, PBS and the like are purchased from biological organisms.
1) Magnetic bead pretreatment: after mixing the magnetic beads, 10mL of carboxyl magnetic beads were placed in a 50mL centrifuge tube, the supernatant was removed by magnetic separation, washed 2 times with 20mL of MEST solution (0.1M MES, pH 6.0,0.05% Tween 20) by magnetic separation, and the supernatant was removed;
2) EDC activation: rapidly adding 10mL of freshly prepared EDC solution (10 mg/mL, the MEST solution is used as a dispersing agent) and 10mL of NHS (10 mg/mL, the MEST solution is used as a dispersing agent) solution into a centrifuge tube filled with magnetic beads, carrying out vortex mixing to fully suspend the magnetic beads, activating at 25 ℃ for 60min, and using a turnover mixing machine to keep the suspended state of the magnetic beads during the period;
3) Magnetic bead coupling: magnetically separating to remove supernatant, adding 40mg MARCKS-ED polypeptide, gently mixing, coupling at 25 deg.C for 2 hr, or coupling at 25 deg.C for 1 hr, standing at 4 deg.C overnight, and maintaining the suspension state of magnetic beads with a turnover mixing instrument during coupling;
4) And (3) sealing: placing the centrifuge tube on a magnetic separation rack, magnetically separating to remove supernatant, adding 20-50mL Tris-HCl (0.1M, pH = 8.0) to resuspend the magnetic beads, reacting at 25 ℃ for 3h to block non-specific adsorption sites on the surfaces of the magnetic beads, after blocking, adding 20-50mL BSA/PBS solution (pH 7.2, containing 1% BSA) to block for two times for 1h, and using a tumbling homogenizer to maintain the suspension state of the magnetic beads during the blocking;
5) And (3) storage: placing the centrifuge tube on a magnetic separator, magnetically separating to remove supernatant, washing with 20mL of preservation solution 3 times, resuspending in 10mL of preservation solution (PBS, pH7.2, containing 0.1% BSA and 0.02% (w/v) NaN 3 ) And stored at 4 ℃.
Measuring the polypeptide coupling efficiency by a flow cytometer:
the method 1 is adopted to mix the magnetic beads evenly, 20uL is taken to be put into a centrifuge tube, supernatant is removed by magnetic separation, washed 3 times by PBS and resuspended in 500-1000 uL PBS. The carboxyl magnetic beads which are not coupled with the polypeptide are used as negative control, and a flow cytometer is loaded and provided with a gate. And then loading the polypeptide to couple with the magnetic beads, and detecting the positive proportion of FITC fluorescence labeled magnetic beads. As can be seen from FIG. 2a, the coupling efficiency was good when the proportion of the positive magnetic beads was 99%.
Polypeptide probe synthesis: the MARCKS-ED polypeptide is biosynthesized by the committee, and FITC fluorescein is modified at the N terminal. The sequence of the decorated MARCKS-ED polypeptide is FITC-acp-KKKKKRFSFKKSFKLSGFSFKKNKK.
Reagents such as magnetic beads, MES, NHS, EDC, etc. were purchased from Solierge, tween-20, PBS, etc. were purchased from biologies.
The specific method comprises the following steps:
1. magnetic bead pretreatment: after mixing the magnetic beads evenly, 100. Mu.L of amino magnetic beads were put into a 1mL EP tube, the supernatant was removed by magnetic attraction, washed 3 times with 200. Mu.L of PBS solution (50mM PBS, pH 7.4), and the supernatant was removed by magnetic attraction;
EDC activating polypeptide: adding freshly prepared 10mL of EDC solution (10 mg/mL, MEST (0.1M MES, pH 6.0,0.05% Tween 20) as dispersant) and 10mL of NHS (10 mg/mL, using the above MEST solution as dispersant) into a centrifuge tube containing MARCKS-ED polypeptide, vortexing, and activating at 25 ℃ for 60min;
3. magnetic bead coupling: adding 400. Mu.g of activated MARCKS-ED polypeptide (0.05% Tween-20 was added to improve bead dispersibility) to an EP tube containing beads, gently mixing; wrapping with tin foil paper (reaction is away from light), coupling at 25 deg.C for 3 hr, or coupling at 25 deg.C for 1 hr, standing at 4 deg.C for coupling overnight, and maintaining the suspension state of magnetic beads during coupling (vertical mixer can be used for reverse mixing);
4. and (3) sealing after coupling: placing the EP tube on a magnetic separation rack, magnetically separating to remove the supernatant, adding 1000. Mu.L of BSA/PBS solution (pH 7.2, containing 5% BSA) for resuspending magnetic beads (optionally with ultrasound), reacting at 25 deg.C for 1h to block the non-specific adsorption sites on the surface of the magnetic beads, and keeping the magnetic beads in suspension (reversed and mixed by using a vertical mixer);
5. and (3) storage: the EP tube was placed on a magnetic separation rack and the supernatant removed by magnetic separation, washed 3 times with 200. Mu.L PBS solution (pH 7.2) or preservation solution, resuspended in preservation solution, added 0.02% (w/v) sodium azide (NaN 3) to inhibit bacterial growth, and finally stored at 4 ℃.
The magnetic beads can be used for subsequent operations in combination with the above steps.
The magnetic bead coupling efficiency is detected in the same way as in the method 1, and the result shows that the coupling efficiency is good.
Polypeptide probe synthesis: venetian biosynthesizes MARCKS-ED polypeptide, and N terminal has biotin modification. The sequence of the decorated MARCKS-ED polypeptide is biotin-KKKKKRFSFKKSFKLSGFSFKKNKK.
Streptavidin SA magnetic beads were purchased from solitary organisms, tween-20, PBS, etc.
The specific method comprises the following steps:
1) Magnetic bead pretreatment: the bead bottle was placed on a vortex shaker for 20s and the beads were resuspended by shaking. Remove 100. Mu.L of the beads to a new centrifuge tube using a pipette. Magnetic separation, pipette off the supernatant, and remove the tube from the magnetic separator. Add 1mL of PBST to the centrifuge tube, cover the centrifuge tube, and shake the resuspension beads well. Magnetic separation and removal of supernatant.
The above steps were repeated twice, and washing was repeated three times in total.
2) Magnetic bead coupling: 1mL of MARCKS-ED polypeptide diluted with PBS (to make the concentration of magnetic beads 1 mg/mL) was added, and the magnetic beads were resuspended with sufficient shaking. The centrifuge tube was placed on a rotary mixer and mixed by rotation at room temperature for 60min. Magnetic separation and transfer of the supernatant to a new centrifuge tube. The beads were washed five times as per "pretreatment".
3) And (3) storage: the magnetic beads were resuspended in a magnetic bead storage medium (1 XPBS containing 0.1% (w/v) BSA,0.1% (w/v) proclin-300).
The magnetic beads can be used for subsequent operations after the steps are combined.
ELISA for measuring magnetic bead coupling efficiency
SA magnetic beads not coupled with polypeptide were used as blank control, SA magnetic beads coupled with MARCKS-ED polypeptide were used as test groups, 5ul of each magnetic bead was taken for each group, washed 3 times according to the above pretreatment method, and 1mL of PBST solution containing 5% BSA was added for blocking for 1h, during which time the suspension was maintained with a tumble mixer. The supernatant was removed by magnetic separation, 1mL of HRP-labeled rabbit anti-biotin antibody was added, the mixture was inverted and mixed well and incubated at room temperature for 1h. Removing supernatant through magnetic separation, washing the PBST solution for 5 times, adding 100ul ELISA developer, uniformly mixing, incubating for 5-30 min at room temperature in a dark place, and adding 50ul stop solution. And (4) after magnetic separation, absorbing the supernatant into an enzyme label plate, and detecting at the double wavelengths of 450nm and 620 nm.
As can be seen from FIG. 2b, the absorbance of the test group is significantly higher than that of the blank group, and the coupling efficiency is higher.
Example 2
Extracting and purifying plasma exosome
1) Sample pretreatment: the plasma from Creams was centrifuged at 3000g and 4 deg.C for 10min, the supernatant was transferred to a new centrifuge tube, and then centrifuged at 10000g and 4 deg.C for 20min, and the supernatant was collected for further use.
2) Exosome extraction and separation: washing MARCKS-ED coupled magnetic beads for three times by washing buffer, adding 100ul of magnetic beads into 1ml of treated plasma supernatant, mixing uniformly, and performing overturning incubation for 30min at room temperature. And (4) placing the magnetic beads on a magnetic frame, standing for 1min, and removing a supernatant. The beads were washed three times with 1ml washing buffer and the supernatant was discarded by magnetic separation. Adding 50ul of eluent, carrying out vortex oscillation, eluting for 10min at room temperature, placing on a magnetic frame for 1min, and transferring supernatant to a new tube after magnetic separation, thus obtaining the purified exosome. The elution step was repeated once and the exosomes obtained in two times were pooled.
3) Exosome identification-Nanoparticle Tracking Analysis (NTA)
The extracted exosomes were analyzed for particle size and concentration using a NanoSight NS300 (Malvern) instrument.
1. Washing the sample cell with deionized water;
2. the instrument was calibrated with polystyrene microspheres (110 nm);
3. the cuvette was washed with 1 XPBS buffer (Biological Industries, israel);
samples were diluted with 1 × PBS buffer (BI, israel) and assayed by injection. The results are shown in FIG. 3, sample XC0965-10 dilution factor 20 test, and the original concentration was found to be 3.37e +09+/-1.83e +08particles/ml. The average particle size is found to be 82.2+/-1.5nm, and the particle concentration in the distribution range of 30-200nm is found to be 3.35E +9particles/ml, accounting for 99.3 percent.
4) Exosome identification-Transmission Electron Microscope (TEM)
1. Taking the exosome sample out of a refrigerator at minus 80 ℃, placing the exosome sample in an ice box, slightly centrifuging after dissolving, and sucking 15ul of exosome sample by using a pipette gun to stand on a copper mesh for 1min.
2. The exosome samples on the copper mesh were blotted dry using filter paper, then 15ul of 2% uranyl acetate staining solution was blotted using a pipette gun for 1min at room temperature.
3. And (4) sucking the exosome sample on the copper mesh by using filter paper, placing the dyed sample under a lamp for baking for 10min, observing and taking a picture, and storing the picture.
As shown in FIG. 4, the structure of the saucer-shaped extracellular vesicle can be seen under the microscope, and the particle size of the vesicle conforms to the range of exosomes.
5) Exosome protein concentration determination
And detecting the protein concentration of the extracted exosome by using a BCA method protein concentration determination kit. Adding the exosome into the lysis solution, then cracking the exosome on ice for 10min-30min, and then measuring the protein concentration of the exosome according to the operation of the BCA kit instruction. Combining the NTA test results, the particle concentration is divided by the protein concentration to obtain the number of particles contained in each mg of protein, and the exosome purity is indirectly reflected, wherein the purity is higher when the ratio is larger. The results are shown in the following table.
| Protein concentration (mg/ml) | 0.127 |
| Particle concentration (particles/ml) | 7.55E+9 |
| Exosome purity (particles/mg) | 5.9E+10 |
From the above results, it was found that the purity of the extracted product was high.
6) Exosome protein marker western blot
Adding a proper amount of loading buffer into the collected exosome, and heating in a boiling water bath for 10min to fully denature the protein. And (3) preparing SDS-PAGE gel, loading the exosome protein denaturation sample into a loading hole of the SDS-PAGE gel, and electrophoretically separating a protein sample and transferring the protein sample to a membrane. And after the film is completely transferred, washing the film transfer liquid. And then the protein membrane is placed in 5% of sealing liquid and sealed for 1 hour at room temperature. After blocking was complete, primary antibodies (CD 9, CD63, TSG101, etc.) were formulated in the appropriate proportions and added to the blocked protein membrane and incubated overnight at 4 ℃. Then preparing an enzyme-labeled secondary antibody according to a proper proportion, putting the membrane in a diluent of the enzyme-labeled secondary antibody, and incubating for 1h at room temperature. And finally, placing the protein membrane in 1ml of mixed ECL luminescent liquid, reacting for 2min, and then placing the protein membrane into a chemiluminescence imaging system for color development imaging, wherein the image is shown in figure 5. As can be seen from fig. 5, the extracted product contains an exosome positive marker and does not contain negative and contaminating markers, and the extracted product meets the exosome characteristics according to the industry standard MISEV2018 issued by the international association of extracellular vesicles ISEV.
Example 3
Comparison of different exosome extraction methods
Compared with a commercial kit (A, B, C, D) with different methodology principles on the market and a gold standard ultracentrifugation method (UC)
The exosomes in 1ml of serum/plasma were extracted according to the kit instructions, and the exosomes were identified according to the method in example 2, and the extraction effects of different products were compared. The results are shown in FIG. 6, where MARCKS-ED extract particle concentration is comparable to the gold standard ultracentrifugation, but particle size is smaller.
The results of protein concentration measurement by the BCA method are as follows:
the electron microscope results are shown in fig. 7, except for C, which is an atypical image and may be aggregated protein, the tea-saucer-like extracellular vesicles can be observed under the microscope. And D, lipoprotein particles like white round balls can be seen.
WB results are shown in FIG. 8, in which calnexin is an exosome-negative marker, apoB is a lipoprotein contamination marker, and CD63, TSG101, CD81 are exosome-positive markers. P is serum/plasma lysate control and 293T is cell control. Except for A, other commercial kit products have lipoprotein pollution, and A has no lipoprotein pollution but contains a small amount of calnexin pollution, and the extraction concentration is not high. The exosome extracted from MARCKS-ED has high purity and can replace the gold standard ultracentrifugation method. The comprehensive evaluation results are summarized as follows:
example 4
Extraction of exosomes from cell culture supernatant
Materials: MARCKS-ED exosome extraction kit (hundred million microorganisms), hela cells (purchased from ATCC)
1. Collecting cell culture supernatant: after the cells are subcultured until the confluency is about 80%, the cells are continuously cultured for 48h by replacing the cells with a serum-free culture medium, and cell culture solution supernatants are collected.
2. Placing the culture supernatant into a centrifuge tube, centrifuging at 4 deg.C for 10min at 3000g to remove cell debris, transferring the supernatant into a new tube, centrifuging at 4 deg.C for 20min at 10000g, and filtering the supernatant with 0.22um filter membrane for use.
500ul MARCKS-ED affinity magnetic beads are added into 3.10ml of culture supernatant, and after mixing evenly, the mixture is turned over and incubated for 30min. And magnetically separating and discarding the supernatant, washing the supernatant for three times by using a washing buffer, eluting the supernatant for 10min by using 100ul of elution buffer, repeating the elution for one time, and combining the eluates obtained in two times to obtain the exosome of the cell culture supernatant.
4. Exosomes were identified as in example 2.
5. And (3) identification result:
the NTA results are shown in figure 9 and the table below.
| 1 | 2 | |
| BCA(mg/mL) | 0.021 | 0.012 |
| NTA(particles/mL) | 2.2E+8 | 6.2E+8 |
| SIZE(nm) | 129.4 | 125.6 |
| Ratio(particles/mg) | 1.05E+10 | 5.17E+10 |
The TEM results are shown in FIG. 10.
From the above results, it was found that exosomes were successfully extracted from the kit in the cell culture supernatant.
Example 5
Exosome nucleic acid omics comparison extracted by different methods
Materials: MARCKS-ED exosome extraction Kit, ultracentrifugation exosome extraction Kit, serum/plasma dissociation and exosome RNA extraction Kit (NORGEN biotech), scientific plasma (Xinfan organism), miRNeasy mini Kit (Qiagen), QIAseq miRNA Library Kit (Qiagen), miSeq sequencing Kit v3 (Illumina)
In order to research the difference of exosomes extracted by a MARCKS-ED exosome extraction kit and other methods in nucleic acid omics, the MARCKS-ED exosome extraction kit, an ultracentrifugation method and an NORGEN kit with higher industrial utilization rate are respectively used for extracting exosome RNA of the same plasma, miRNA is subjected to library construction and sequencing, and miRNA expression difference is compared.
The pretreatment process of the sample is consistent, namely, the sample is centrifuged at 3000g and 4 ℃ for 10min, the supernatant is taken and transferred into a new centrifugal tube, and then the sample is centrifuged at 10000g and 4 ℃ for 20min, and the supernatant is taken for standby application.
The extraction process of the MARCKS-ED exosome extraction kit comprises the following steps: washing 500ul MARCKS-ED affinity magnetic beads for three times, adding the washed beads into 3ml of plasma supernatant, uniformly mixing, and performing turnover incubation for 30min. Removing supernatant by magnetic separation, washing magnetic beads for three times by washing buffer without elution, adding 700ul QIAzol for cracking for 5min, sucking supernatant by magnetic separation to a new tube, and extracting total RNA according to the instruction of a qiagen kit.
Ultracentrifugation extraction process: mixing 2ml of plasma sample and 16ml of PBS uniformly, filtering the mixture through a 0.22um filter membrane, filling the mixture into a large centrifuge tube, and centrifuging the mixture for 2 hours at 100000g and 4 ℃ after balancing. The supernatant was aspirated off, 500ul PBS was added, the pellet resuspended by gentle pipetting, and transferred to a fresh centrifuge tube. The reaction is repeated once, and the two heavy suspensions are combined to obtain 1ml. Trim, centrifuge at 100000g 4 ℃ for 2h. The supernatant was aspirated, 100ul PBS was added, and the pellet was resuspended by gentle pipetting. 700ul QIAzol was added for lysis for 5min, followed by total RNA extraction according to the instructions of the qiagen kit.
The NORGEN kit was used as described to directly extract free and exosome total RNA from 2ml plasma.
Results
And (5) constructing a library and sequencing in sequence according to the instruction of the corresponding kit.
Using | log2 (FC) | > =0.584962500721156; FDR < =0.05 is used as a screening standard, a MARCKS-ED and super-separation method miRNA difference volcanic map is shown in figure 11a, a MARCKS-ED and NORGEN miRNA difference volcanic map is shown in figure 11b, and exosome miRNA extracted by a MARCKS-ED kit is only partially different from other two mainstream research methods, and can be comparable to a mainstream classical method.
Sequence listing
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Claims (10)
1. An exosome extraction affinity magnetic bead, which is characterized in that: the polypeptide is obtained by solidifying MARCKS-ED polypeptide on the surface layer of magnetic beads and modifying carboxyl, amino or streptavidin SA.
2. Exosome-extracting affinity magnetic beads according to claim 1, characterized in that: the MARCKS-ED polypeptide sequence is as described in any one of the following 1) -6):
1)KKKKKRFSFKKSFKLSGFSFKKNKK(SEQ ID NO:1)；
2)KKSFKLSGFSFKKNKK(SEQ ID NO:2)；
3)KRFSFKK(SEQ ID NO:3)；
4)KKRFSFKK(SEQ ID NO:4)；
5)KKRFSFKKF(SEQ ID NO:5)；
6)KKFSFKKF(SEQ ID NO:6)；
the amino acid in the MARCKS-ED polypeptide sequence is a D-type amino acid.
3. Exosome-extracting affinity magnetic beads according to claim 1, characterized in that: the magnetic beads are Fe 3 O 4 Silica-based magnetic beads of an inner core.
4. A preparation method of affinity magnetic beads for exosome extraction is characterized by comprising the following steps:
pretreating carboxyl magnetic beads, coupling with MARCKS-ED polypeptide after EDC activation, and finally sealing and storing to obtain exosome extraction affinity magnetic beads; or
Pretreating amino magnetic beads, coupling with MARCKS-ED polypeptide activated by EDC, and finally sealing and storing to obtain exosome extraction affinity magnetic beads; or
Preprocessing streptavidin SA magnetic beads, coupling with MARCKS-ED polypeptide, and storing to obtain exosome extraction affinity magnetic beads.
5. The method for preparing affinity magnetic beads for exosome extraction according to claim 4,
when carboxyl magnetic beads are used, the N end of the MARCKS-ED polypeptide is provided with modified FITC fluorescein;
when amino magnetic beads are used, the N end of the MARCKS-ED polypeptide is provided with modified FITC fluorescein;
when streptavidin SA magnetic beads are used, the N-terminus of the MARCKS-ED polypeptide is modified by biotin.
6. An exosome extraction kit, characterized in that: comprising exosome-extracting affinity magnetic beads according to any one of claims 1-3, and a wash buffer and an elution buffer.
7. Exosome extraction kit according to claim 6, characterized in that: the washing buffer consists of:
8. exosome extraction kit according to claim 6, characterized in that: the elution buffer is composed of any one or a combination of the following solutions:
9. use of an exosome extraction kit according to claims 6-8.
10. Use according to claim 9, characterized in that: the method comprises the following steps: and respectively preprocessing the sample and the exosome extraction affinity magnetic beads, then extracting exosomes in the sample by the exosome extraction affinity magnetic beads, washing by using a washing buffer solution, and eluting by using an elution buffer solution to obtain exosomes.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN116555160A (en) * | 2023-06-29 | 2023-08-08 | 亿航(苏州)生物医药有限公司 | Extraction method of radix paeoniae alba exosome and decoction |
| CN117420298A (en) * | 2023-10-19 | 2024-01-19 | 江苏格诺生物科技有限公司 | Storage solution of magnetic microspheres and preparation method thereof |
| CN118253292A (en) * | 2024-05-28 | 2024-06-28 | 亿航(苏州)生物医药有限公司 | Affinity chromatographic column packing and extraction method of extracellular vesicles |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105934670A (en) * | 2013-12-03 | 2016-09-07 | 拜奥默里克斯公司 | Method for isolating exosomes |
| CN112394169A (en) * | 2020-11-05 | 2021-02-23 | 清华大学 | Exosome probe applied to cancer detection, diagnosis and treatment |
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2022
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105934670A (en) * | 2013-12-03 | 2016-09-07 | 拜奥默里克斯公司 | Method for isolating exosomes |
| CN112394169A (en) * | 2020-11-05 | 2021-02-23 | 清华大学 | Exosome probe applied to cancer detection, diagnosis and treatment |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN116555160A (en) * | 2023-06-29 | 2023-08-08 | 亿航(苏州)生物医药有限公司 | Extraction method of radix paeoniae alba exosome and decoction |
| CN116555160B (en) * | 2023-06-29 | 2023-10-13 | 亿航(苏州)生物医药有限公司 | Extraction method of radix paeoniae alba exosome and decoction |
| CN117420298A (en) * | 2023-10-19 | 2024-01-19 | 江苏格诺生物科技有限公司 | Storage solution of magnetic microspheres and preparation method thereof |
| CN118253292A (en) * | 2024-05-28 | 2024-06-28 | 亿航(苏州)生物医药有限公司 | Affinity chromatographic column packing and extraction method of extracellular vesicles |
| CN118253292B (en) * | 2024-05-28 | 2024-09-20 | 亿航(苏州)生物医药有限公司 | Affinity chromatographic column packing and extraction method of extracellular vesicles |
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