CN115678840A - Separation and extraction method of exosome - Google Patents
Separation and extraction method of exosome Download PDFInfo
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Abstract
The invention discloses a separation and extraction method of exosomes, which is characterized in that a liquid sample containing exosomes is filtered by an ultrafiltration membrane with the pore diameter of 0.45nm, the obtained filtrate passes through a polylysine modified cation resin column, the exosomes are adsorbed by polylysine, other foreign proteins, lipids and salts pass through the column, and elution is carried out by eluent to obtain a crude exosome solution; and (3) carrying out fine extraction on the crude exosome solution through a chromatographic column such as CD63-CNBr-Sepharose 4B and the like, and finally carrying out ultrafiltration by using an ultrafiltration membrane with the aperture of 100KD to obtain exosome with the purity of more than 95%. The method can realize the extraction of the high-purity exosome from culture supernatant, dairy products and the like without ultracentrifugation, the whole extraction process is simple, stable and easy to operate, the extracted exosome has a complete structure, high purity and high recovery rate, and the extraction of the exosome can be finished without complex instruments.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a separation and extraction method of exosomes.
Background
Exosomes (Exosomes) are bilayer phospholipid vesicles secreted by cells with a diameter of 30-150 nm and a density of 1.13-1.21 g/mL, usually released into the extracellular environment in exocytic form. The surface of the membrane contains protein and lipid components related to functions, the membrane surface expresses specific proteins such as CD9, CD63, CD81, TSC101, HSP70 and the like, and active substances such as proteins and nucleic acids from donors are wrapped inside the membrane. When the exosome contacts the target cell, the carried biomolecule is received in endocytosed form, released and acted upon in the target cell. In recent years, as research on exosomes has been advanced, it has been found that exosomes derived from different histiocytes exhibit tissue cell specificity and can carry bioactive proteins, lipids, nucleic acids, and the like derived from the cells; in physiological and pathological states of the body, substances contained in exosomes change with the change of cellular environment, and therefore, exosome diagnosis is increasingly emphasized in clinical application.
Exosomes can shuttle among cells, exchange of substances and information among cells is facilitated, functions and states of receptor cells are changed by loading exogenous drugs, and the aim of targeted therapy is achieved. The exosome is used as a loading tool, can carry various types of medicines and reaches each system in a body, in nervous system diseases, the medicines are difficult to enter target cells through a blood brain barrier to play a role, and the exosome can treat the diseases through the realization.
The exosomes are extracted mainly in the following ways: (1) The ultracentrifugation method is a current gold standard method, and the method adopts low-speed centrifugation and high-speed centrifugation to separate exosome alternately, so that the purity is high, but the comprehensive yield is not high; (2) The density gradient centrifugation method enables exosomes to be gathered in a specific density layer, so that the purity is high, the comprehensive yield is good, and the operation is complicated; (3) The ultrafiltration centrifugation method separates samples by using ultrafiltration membranes with different interception relative molecular masses, and is relatively rough and simple to operate; (4) The magnetic bead immunity method uses magnetic beads coated with anti-labeling substances to separate exosomes, and the method has high specificity, and antibodies labeled with the labeling substances are difficult to obtain and have strict requirements. Although different exosome extraction methods exist at present, the problem of large-scale extraction cannot be solved, and the liquid volume obtained by one-time centrifugation through a common ultracentrifugation method is limited; ultrafiltration can be of the above scale but of limited purity; the gel column can achieve the purification effect but has limited sample application amount at one time. The extraction methods have advantages and disadvantages, most methods can only realize small-batch extraction and large-scale extraction, and no effective method exists at present.
Disclosure of Invention
The invention aims to provide a method for extracting exosome with high purity on a large scale, which does not need a centrifuge in the whole operation process, has simple operation process, can realize large-scale extraction only by one ultrafiltration membrane filtration, one gel and one chromatographic column, does not use reagents with strong chemical properties and violent reaction in the whole experiment process, has mild process, does not cause influence on the structural properties of the extracted exosome, has complete structure and stable properties of the extracted exosome, and can realize the extraction of various exosomes.
Aiming at the purposes, the method for separating and extracting the exosome comprises the following steps:
1. filtering a liquid sample containing exosomes by using an ultrafiltration membrane with the pore diameter of 0.45nm, and collecting filtrate;
2. passing the filtrate collected in the step 1 through a polylysine modified cation resin column, eluting the resin column with eluent, and collecting a crude exosome solution; wherein the polylysine modified cationic resin is a resin containing-COOH or-NH 2 Obtained by direct attachment of polylysine or will contain-OH or-COOH or-NH 2 The cationic resin is obtained by connecting a Linker and polylysine; the eluent is any one of 3-6 mol/L urea solution, 3-6 mol/L guanidine hydrochloride solution and 3-6 mol/L guanidine isothiocyanate solution;
3. enabling the crude exosome solution to pass through a CD63-CNBr-Sepharose 4B chromatographic column or a CD81-CNBr-Sepharose 4B chromatographic column or a CD9-CNBr-Sepharose 4B chromatographic column, eluting the chromatographic column by using hydrochloric acid-glycine, and carrying out further fine extraction on exosomes;
4. mixing the refined exosome solution with PBS buffer solution in equal volume, then carrying out ultrafiltration by using an ultrafiltration membrane with the aperture of 100KD, and repeating the process for 3-5 times to obtain exosomes with the purity of more than 95%.
In the step 1, the liquid sample containing exosome is any one of cow's milk, goat's milk, human milk, cell culture supernatant, urine and serum.
More preferably, in the step 2, the resin column is firstly equilibrated with 1.5 to 2 times of column volume of 20mmol/L PBS buffer solution with pH =7.35, after the conductance and the ultraviolet absorption value are stabilized, the filtrate collected in the step 1 is loaded, the loading amount is 1.5 to 2 column volumes, the loading speed is 0.5 to 3 mL/min, the mixture is left standing for 5 to 10 min after the loading is finished, and the resin column is equilibrated again with 1 to 2 column volumes of 20mmol/L PBS buffer solution with pH = 7.35; after the base line is stable, eluting the resin column by using eluent with the volume of 1 column, standing for 5-10 minutes, continuously eluting the resin column by using the eluent, and collecting the crude exosome solution.
In the step 2, the-COOH-containing cationic resin is any one of carboxylated silica gel, carboxylated agarose and carboxylated dextran; said group containing-NH 2 The cation resin is any one of amino silica gel, amino agarose and amino dextran; the-OH-containing cationic resin is any one of hydroxylated silica gel, hydroxylated agarose and hydroxylated glucan.
In the step 2, the polylysine modified cationic resin is prepared by reacting-COOH-containing cationic resin with-NH of polylysine under the action of a condensing agent and an additive 2 By condensation reactions or containing-NH 2 The cationic resin of (2) is obtained by condensation reaction with-COOH of polylysine. Or the polylysine-modified cationic resin is: under the action of condensing agent and additive, the product will contain-OH or-NH 2 The cation resin and the resin with both ends of-COOH or one end of-COOH and the other end of-OH or-NH 2 After the condensation reaction of-COOH at one end of the Linker, the-NH of polylysine is added 2 Is condensed with-COOH at the other end of the Linkerthe-COOH of polylysine and-OH or-NH at the other end of the Linker 2 And carrying out condensation reaction to obtain the polylysine modified cationic resin. Or the polylysine-modified cationic resin is: under the action of a condensing agent and an additive, the cationic resin containing-COOH and the cationic resin with both ends being-OH or both ends being NH 2 Or one end is-OH and the other end is-NH 2 Of the Linker on one end of-OH or NH 2 After condensation reaction, the-COOH of polylysine and the-OH or-NH at the other end of the Linker are reacted 2 And carrying out condensation reaction to obtain the polylysine modified cationic resin.
The Linker with both ends being-COOH is HOOC-CH 2 -CH 2 -CH 2 -COOH, said Linker with-COOH at one end and-OH at the other end is HO-CH 2 -CH 2 -COOH, said one end being-COOH and the other end being-NH 2 Linker of (a) is H 2 N-CH 2 -CH 2 -COOH or HOOC-CH 2 -CH 2 -CH 2 -NH 2 The Linker with both ends being-OH is HO-CH 2 -CH 2 -CH 2 -OH, both said ends being-NH 2 Linker of (a) is H 2 N-CH 2 -CH 2 -CH 2 -NH 2 One end of the catalyst is-OH, and the other end is-NH 2 Linker of (a) is H 2 N-CH 2 -CH 2 -CH 2 -OH。
The condensing agent is any one of N, N' -diisopropylcarbodiimide and dicyclohexylcarbodiimide; the additive is any one or more of 2-oxime ethyl cyanoacetate, 1-hydroxybenzotriazole and N-hydroxysuccinimide.
In the step 2, the urea solution, the guanidine hydrochloride solution and the guanidine isothiocyanate solution are prepared by using a PBS buffer solution or pure water.
The invention has the following beneficial effects:
1. filtering a liquid sample solution containing exosomes by using an ultrafiltration membrane with the pore diameter of 0.45nm, collecting filtrate, enabling the obtained filtrate to pass through a polylysine modified cationic resin column, adsorbing the exosomes by the polylysine, enabling other hetero-proteins, lipids and salts to pass through the column, and eluting by using eluents such as urea solution, guanidine hydrochloride solution, guanidine isothiocyanate solution and the like to obtain a crude exosome solution; the crude exosome solution passes through a CD63/81/9-CNBr-Sepharose 4B chromatographic column, exosomes are combined with the chromatographic column again, elution is carried out by hydrochloric acid-glycine (HCl-Gly) buffer solution, exosomes are collected according to peak shapes, the foreign proteins and apoptotic bodies are separated and removed in one step, and the influence of introduced chemical reagents such as urea, guanidine hydrochloride, guanidine isothiocyanate and the like on exosomes can be removed, so that the fine extraction of exosomes is achieved; and finally, ultrafiltration is carried out by an ultrafiltration membrane with the aperture of 100KD, hydrochloric acid-glycine, partial micromolecule protein and water are removed, and the refined exosome is concentrated to obtain the exosome with the purity of more than 95%.
2. The exosome extracted by the invention comprises the exosome-containing liquid such as milk, goat milk, human milk, cell culture supernatant, urine, serum and the like, the extraction method can realize the extraction of high-purity exosome from the culture supernatant and dairy product without ultracentrifugation, the whole extraction process is simple, stable and easy to operate, the extracted exosome has complete structure, high purity and high recovery rate, and the extraction of exosome can be completed without complex instruments, so that the method is a brand new method for extracting exosome from trace quantity to large scale.
Drawings
FIG. 1 is a graph of particle size distribution (NTA) of the crude exosomes of example 1.
FIG. 2 is an electron microscopy micrograph (TEM) of the crude exosomes of example 1.
FIG. 3 is a graph of particle size distribution (NTA) of exosomes as outlined in example 1.
FIG. 4 shows the protein marker validation (Western blot) of the exosome marker CD63 extracted in example 1.
Detailed Description
The invention will be further described in detail with reference to the following figures and examples, but the scope of the invention is not limited to these examples.
Example 1
Taking the extraction of exosomes from human umbilical cord mesenchymal stem cells as an example
1. Centrifuging a culture medium of the third generation human umbilical cord mesenchymal stem cells to remove impurities, separating culture supernatant, filtering 50mL of the supernatant by using an ultrafiltration membrane with the pore diameter of 0.45nm, removing cells and cell debris, and collecting filtrate.
2. Firstly, a 2-time column volume of 20mmol/L PBS buffer solution with pH =7.35 is used for balancing an alpha-polylysine modified silica gel column, the length of the column is 10cm, the inner diameter is 16mm, and the balancing speed is 2 mL/min; after the conductance and the ultraviolet absorption value are stable, loading the filtrate collected in the step 1, wherein the loading amount is 50mL, the loading speed is 1 mL/min, standing for 5 min after the loading is finished, and balancing the silica gel column again by using 1 column volume of 20mmol/L PBS buffer solution with the pH = 7.35; after the base line is stable, eluting the silica gel column by using eluent with 1 column volume, standing for 5 minutes, continuously eluting the silica gel column by using the eluent, observing a peak shape chart, and collecting the solution when the peak is started in about 4-7 minutes to obtain a crude exosome solution. Wherein, the alpha-polylysine modified silica gel is the-NH of the alpha-polylysine under the action of N, N' -diisopropylcarbodiimide and 1-hydroxybenzotriazole 2 Carrying out condensation reaction with-COOH of carboxylated silica gel to obtain the product; the eluents are respectively selected from urea solutions with the concentrations of 0.5mol/L, 1mol/L, 3mol/L, 4mol/L, 5mol/L, 6mol/L and 7mol/L, and the selected urea solution is prepared by PBS buffer solution with the concentration of 20mmol/LpH = 7.35. The influence of the urea concentration in the eluent on the exosome recovery rate is examined, and the detection result is shown in table 1. The result of NTA detection of crude exosome is shown in figure 1, and the result of TEM detection of extracted exosome is shown in figure 2. As can be seen from FIGS. 1 and 2, the particle size distribution of the crude exosome particles is between 100-1000 nm, and the later peak is large vesicles.
3. Firstly, washing a CD63-CNBr-Sepharose 4B chromatographic column (the length is 10cm, the inner diameter is 16 mm) by using 2 times of column volume of equilibrium solution (prepared by adding 29g of NaCl into 1000mL of 50mmol/L Tris-HCl buffer solution), wherein the flow rate is 2 mL/min; after the conductance and the ultraviolet absorption value are stable, loading the exosome solution crudely extracted in the step 2, wherein the loading amount is 2 column volumes, the loading speed is 1 mL/min, standing for 15 min after the loading is finished, and flushing the chromatographic column again by using balance liquid with 1 column volume, wherein the flow rate is 0.5 mL/min; after the baseline is stabilized, eluting the chromatographic column by using 1 column volume of hydrochloric acid-glycine (HCl-Gly) buffer solution (16.8mL of 200mol/L HCl aqueous solution and 50mL of 200mol/L glycine aqueous solution which are mixed and then added with 33.2mL of deionized water) with the pH =2.8, wherein the flow rate is 0.5 mL/min, standing for 5 min after the elution is finished, continuously eluting the chromatographic column by using the HCl-Gly buffer solution, observing a peak shape graph, collecting the solution when the peak begins about 4-7 min to obtain a refined exosome solution, and detecting the NTA of the refined exosome as shown in figure 3. As can be seen from FIG. 3, the particle size distribution of the refined exosome particles is between 100-200 nm.
4. Mixing the refined exosome solution with 20mmol/L PBS buffer solution with pH =7.35 in equal volume, then carrying out ultrafiltration by using an ultrafiltration membrane with the aperture of 100KD, removing urea, hydrochloric acid-glycine, partial micromolecule protein and water, and repeating the process for 3 times to obtain the exosome with the purity of more than 95%. Western blot is adopted to verify the qualitative detection result of the obtained exosome, as can be seen from figure 4, the band is clear, and the experimental requirements can be met.
Example 2
In step 2 of this example, the α -polylysine modified silica gel is prepared by reacting an aminated silica gel with-COOH of α -polylysine and-NH of aminated silica gel under the action of N, N' -diisopropylcarbodiimide and 1-hydroxybenzotriazole 2 The product is obtained by condensation reaction, the influence of the concentration of urea in the eluent on the recovery rate of the exosome is shown in table 1, and other steps are the same as the steps in the example 1, so that the exosome with the purity of more than 95% is obtained.
Example 3
In step 2 of this example, the column of silica gel modified with α -polylysine of example 1 was replaced with a column of silica gel modified with ε -polylysine, which was prepared by reacting carboxylated silica gel with N, N' -diisopropylcarbodiimide, 1-hydroxybenzotriazole, and the like to yield-NH of ε -polylysine 2 The obtained product is subjected to condensation reaction with-COOH of carboxylated silica gel, the influence of the concentration of urea in eluent on the recovery rate of exosomes is shown in table 1, and other steps are the same as those in example 1, so that exosomes with the purity of more than 95% are obtained.
Example 4
In step 2 of this example,. Epsilon. -polyPolylysine-modified silica gel column in place of the α -polylysine-modified silica gel column of example 2, the ∈ -polylysine-modified silica gel was prepared by reacting the-COOH group of ∈ -polylysine with the-NH group of the aminated silica gel under the action of N, N' -diisopropylcarbodiimide, 1-hydroxybenzotriazole 2 The exosome was obtained by condensation reaction, the influence of the urea concentration in the eluate on the exosome recovery rate is shown in table 1, and the exosome with a purity of 95% or more was obtained by the same procedure as in example 1.
Example 5
In step 2 of this embodiment, HOOC-CH is used 2 -CH 2 -CH 2 the-COOH is Linker, under the action of N, N' -diisopropylcarbodiimide and 1-hydroxybenzotriazole, the-COOH at one end of Linker and-OH of hydroxylated silica gel are subjected to condensation reaction, and then-NH of alpha-polylysine 2 And carrying out condensation reaction with-COOH at the other end of the Linker to obtain alpha-polylysine modified silica gel, wherein the influence of the concentration of urea in eluent on the recovery rate of exosome is shown in Table 1, and other steps are the same as those in example 1 to obtain exosome with purity of more than 95%.
Example 6
In step 2 of this example, H is used 2 N-CH 2 -CH 2 the-COOH is Linker, under the action of N, N' -diisopropylcarbodiimide and 1-hydroxybenzotriazole, the-COOH at one end of the Linker and the-OH of hydroxylated silica gel are subjected to condensation reaction, and then the-COOH of alpha-polylysine and the-NH at the other end of the Linker are subjected to condensation reaction 2 The condensation reaction is carried out to obtain the alpha-polylysine modified silica gel, the influence of the concentration of urea in the eluent on the recovery rate of the exosome is shown in the table 1, and other steps are the same as the example 1 to obtain the exosome with the purity of more than 95%.
Example 7
In step 2 of this embodiment, HOOC-CH is used 2 -CH 2 -CH 2 the-COOH is Linker, under the action of N, N' -diisopropylcarbodiimide and 1-hydroxybenzotriazole, the-COOH at one end of Linker and-OH of hydroxylated silica gel are subjected to condensation reaction, and then-NH of epsilon-polylysine 2 Performing condensation reaction with-COOH at the other end of Linker to obtain epsilon-polylysine modified silica gel, elutingThe influence of the medium urea concentration on the exosome recovery rate is shown in table 1, and the other steps are the same as those in example 1, so that exosomes with the purity of more than 95% are obtained.
Example 8
In step 2 of this example, H is used 2 N-CH 2 -CH 2 the-COOH is Linker, under the action of N, N' -diisopropylcarbodiimide and 1-hydroxybenzotriazole, the-COOH at one end of the Linker and-OH of hydroxylated silica gel are subjected to condensation reaction, and then the-COOH of epsilon-polylysine and-NH at the other end of the Linker are subjected to condensation reaction 2 The condensation reaction is carried out to obtain epsilon-polylysine modified silica gel, the influence of the concentration of urea in eluent on the recovery rate of exosome is shown in table 1, other steps are the same as the example 1, and exosome with the purity of more than 95% is obtained.
TABLE 1
As can be seen from the table 1, when the eluent is selected from urea solution with the concentration of 3-7 mol/L, higher exosome recovery rate can be obtained, wherein the exosome recovery rate reaches the peak value when the urea concentration is 5-6 mol/L. And, as can be seen from the results of comparative examples 1 to 8, the α -polylysine-modified silica gel column used in example 5 has the best exosome separation and recovery effect, and the recovery rate reached 80% at a urea concentration of 6mol/L, as compared with the other 7 examples.
Example 9
In step 2 of this example, 0.5mol/L, 1mol/L, 3mol/L, 4mol/L, 5mol/L, 6mol/L, 7mol/L guanidine hydrochloride solution and guanidine isothiocyanate solution were selected as eluents, and the other steps are the same as example 5, and the influence of the urea concentration in the eluents on the exosome recovery rate is shown in Table 2.
TABLE 2
As can be seen from Table 2, the high recovery rate of exosomes can be obtained when the concentration of guanidine hydrochloride and guanidine thiocyanate is 3-7 mol/L, wherein the recovery rate of exosomes reaches the peak value when the concentration of guanidine hydrochloride is 5-6 mol/L.
Example 10
Taking the extraction of exosomes from fresh milk as an example
1. Filtering 50mL of fresh cow milk by using an ultrafiltration membrane with the pore diameter of 0.45nm, and collecting filtrate.
2. Firstly, a 2-time column volume of 20mmol/L PBS buffer solution with pH =7.35 is used for balancing an alpha-polylysine modified silica gel column, the inner diameter of the column is 16mm, the length of the column is 10cm, and the balancing speed is 2 mL/min; after the conductance and the ultraviolet absorption value are stable, loading the filtrate collected in the step 1 at the loading speed of 0.5 mL/min, standing for 10 min after the loading is finished, and balancing the silica gel column again by using 1 column volume of 20mmol/L PBS (phosphate buffer solution) with the pH = 7.35; after the base line is stable, eluting the silica gel column by using eluent with 1 column volume, standing for 5 minutes, continuously eluting the silica gel column by using the eluent, observing a peak shape chart, and collecting the solution when the peak is started in about 4-7 minutes to obtain a crude exosome solution. Wherein, the alpha-polylysine modified silica gel is the same as in example 5; the eluent is urea solution with the concentration of 5mol/L, and the selected urea solution is prepared by PBS buffer solution with the pH of 20mmol/L = 7.35.
3. This step was the same as in step 3 of example 1.
4. The steps are the same as the step 4 of the embodiment 1, and the milk exosome with the purity of more than 95 percent is obtained and is frozen.
Claims (9)
1. A separation and extraction method of exosome is characterized by comprising the following steps:
(1) Filtering a liquid sample containing exosomes by using an ultrafiltration membrane with the pore diameter of 0.45nm, and collecting filtrate;
(2) Passing the filtrate collected in the step (1) through a polylysine modified cationic resin column, eluting the resin column with eluent, and collecting a crude exosome solution;
the polylysine modified cationic resin is a resin containing-COOH or-NH 2 Obtained by direct attachment of polylysine or will contain-OH or-COOH or-NH 2 The cationic resin is obtained by connecting a Linker and polylysine;
the eluent is any one of 3-6 mol/L urea solution, 3-6 mol/L guanidine hydrochloride solution and 3-6 mol/L guanidine isothiocyanate solution;
(3) Enabling the crude exosome solution to pass through a CD63-CNBr-Sepharose 4B chromatographic column or a CD81-CNBr-Sepharose 4B chromatographic column or a CD9-CNBr-Sepharose 4B chromatographic column, eluting the chromatographic column by using hydrochloric acid-glycine, and carrying out further fine extraction on exosomes;
(4) Mixing the refined exosome solution and a PBS buffer solution in equal volume, then carrying out ultrafiltration by using an ultrafiltration membrane with the aperture of 100KD, repeating the process for 3-5 times, and obtaining the exosome with the purity of more than 95%.
2. The method for separating and extracting exosomes according to claim 1, wherein in the step (1), the exosome-containing liquid sample is any one of cow's milk, goat's milk, human milk, cell culture supernatant, urine and serum.
3. The exosome separation and extraction method according to claim 1, characterized in that, in the step (2), the resin column is equilibrated with 1.5-2 times of column volume of 20mmol/L PBS buffer solution with pH =7.35, after the conductance and the ultraviolet absorption value are stabilized, the filtrate collected in the step (1) is loaded, the loading amount is 1.5-2 column volumes, the loading speed is 0.5-3 mL/min, after the loading is finished, the solution is left for 5-10 min, and the resin column is equilibrated again with 1-2 column volumes of 20mmol/L PBS buffer solution with pH = 7.35; after the base line is stable, eluting the resin column by using eluent with the volume of 1 column, standing for 5-10 minutes, continuously eluting the resin column by using the eluent, and collecting the crude exosome solution.
4. Method for the isolation and extraction of exosomes according to claim 1, characterized in that said-COOH-containingThe cation resin is any one of carboxylated silica gel, carboxylated agarose and carboxylated dextran; said group containing-NH 2 The cation resin is any one of aminated silica gel, aminated agarose and aminated dextran; the-OH-containing cationic resin is any one of hydroxylated silica gel, hydroxylated agarose and hydroxylated glucan.
5. The method for separating and extracting exosomes according to claim 4, wherein the polylysine modified cationic resin is prepared by reacting polylysine modified cationic resin with-COOH-containing cationic resin and-NH of polylysine under the action of a condensing agent and an additive 2 By condensation reactions or containing-NH 2 The cationic resin of (2) is obtained by condensation reaction with-COOH of polylysine.
6. A method for separating and extracting exosomes according to claim 5, wherein the polylysine-modified cationic resin is: under the action of condensing agent and additive, the product will contain-OH or-NH 2 The cation resin and the resin with both ends of-COOH or one end of-COOH and the other end of-OH or-NH 2 After the condensation reaction of-COOH at one end of the Linker, the-NH of polylysine is added 2 Condensation reaction with-COOH at the other end of the Linker or-COOH of polylysine and-OH or-NH at the other end of the Linker 2 Performing condensation reaction to obtain polylysine modified cationic resin;
or the polylysine-modified cationic resin is: under the action of a condensing agent and an additive, the cationic resin containing-COOH and the cationic resin with both ends being-OH or both ends being NH 2 Or one end is-OH and the other end is-NH 2 Of the Linker on one end of-OH or NH 2 After condensation reaction, the-COOH of polylysine and the-OH or-NH at the other end of the Linker are reacted 2 And carrying out condensation reaction to obtain the polylysine modified cationic resin.
7. The method for separating and extracting exosomes according to claim 6, wherein both ends are-COThe Linker of OH is HOOC-CH 2 -CH 2 -CH 2 -COOH, said Linker with-COOH at one end and-OH at the other end is HO-CH 2 -CH 2 -COOH, said one end being-COOH and the other end being-NH 2 Linker of (a) is H 2 N-CH 2 -CH 2 -COOH or HOOC-CH 2 -CH 2 -CH 2 -NH 2 The Linker with both ends being-OH is HO-CH 2 -CH 2 -CH 2 -OH, both said ends being-NH 2 Linker of (a) is H 2 N-CH 2 -CH 2 -CH 2 -NH 2 One end of the catalyst is-OH, and the other end is-NH 2 Linker of (a) is H 2 N-CH 2 -CH 2 -CH 2 -OH。
8. The method for separating and extracting exosome according to claim 5 or 6, wherein the condensing agent is any one of N, N' -diisopropylcarbodiimide and dicyclohexylcarbodiimide; the additive is any one or more of 2-oxime ethyl cyanoacetate, 1-hydroxybenzotriazole and N-hydroxysuccinimide.
9. The method for separating and extracting exosomes according to claim 1, wherein in the step (2), the urea solution, the guanidine hydrochloride solution, the guanidine isothiocyanate solution are prepared by PBS buffer solution or pure water.
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