CN119119204B

CN119119204B - Temperature-sensitive specific polypeptide and method for non-destructive separation of exosomes based on temperature difference specificity

Info

Publication number: CN119119204B
Application number: CN202411597037.7A
Authority: CN
Inventors: 沈志伟; 胡迈; 辛洪波
Original assignee: Nanchang University
Current assignee: Nanchang University
Priority date: 2024-11-11
Filing date: 2024-11-11
Publication date: 2025-03-18
Anticipated expiration: 2044-11-11
Also published as: CN119119204A

Abstract

The present invention relates to the technical field of separation of biological substances, and in particular to a temperature-sensitive specific polypeptide and a method for non-destructive separation of exosomes based on temperature difference specificity. In the present invention, a temperature-sensitive specific polypeptide having an amino acid sequence as shown in SEQ ID No. 1, SEQ ID No. 2 or SEQ ID No. 3 is provided; nanomagnetic microspheres are modified with the temperature-sensitive specific polypeptide, and then mixed with a liquid containing exosomes, so that the exosomes are specifically bound to the temperature-sensitive specific polypeptide, thereby being able to separate the exosomes by magnetic force.

Description

Temperature-sensitive specific polypeptide and temperature-difference-specificity-based nondestructive exosome separation method

Technical Field

The invention relates to the technical field of biological substance separation, in particular to a temperature-sensitive specific polypeptide and a temperature-difference-specificity-based nondestructive exosome separation method.

Background

Exosomes are double-layer phospholipid membrane vesicles containing complex RNA and protein, play an important role in physiological and pathological processes, and researchers have ascertained that the exosomes have great application potential in the aspects of treatment, detection and the like. High purity exosomes are the basis for exosome research and application, and how to effectively isolate exosomes has been the focus of attention of researchers. Currently, exosome separation techniques mainly include ultracentrifugation, ultrafiltration, polymer precipitation, size exclusion chromatography based on physical separation methods, and affinity separation based on biochemical separation methods.

Separation methods based on physical properties such as density, size, etc., such as ultracentrifugation, are common methods for exosome separation. The method is based on the density gradient difference of various substances in body fluid, and achieves the purpose of separating and enriching exosomes by centrifugal force. The ultracentrifugation method is relatively simple, is not polluted by separation reagents, and has a large number of obtained exosomes, which are still widely used at present. However, the centrifugal instrument required by the method is expensive, the sample amount is large, the time consumption is long, and the purity of the separated exosomes is not high.

Ultrafiltration is a process of using ultra-fine nanofiltration membranes having different interception pore diameters to intercept substances of a specific size on an ultrafiltration membrane by using a specific size of exosomes, and filtering substances of a small particle size to the other side of the membrane. The ultrafiltration method has simple operation, can concentrate exosome samples, and has low cost and high speed. The patent application with the application number of CN202310735921.1 discloses a technical scheme for separating and purifying exosomes, wherein the exosomes can be subjected to mechanical damage force to destroy the active structure of the exosomes in the pressurizing or vibrating process by adopting an ultra-sonar filtering method, and a filtering membrane is easily blocked by macromolecular substances, so that the pressure of the filtering membrane is overlarge and the filtering membrane is broken.

A large number of non-exosome particles having the same physical properties of size, density, etc. can cause a large amount of contamination of exosomes obtained by physical separation methods, such as apoptotic bodies of vesicles, exovesicles, as well as exosomes, superpolymers, lipoproteins, etc.

The affinity separation method is a separation method for capturing another paired molecule by utilizing the specific interaction force between the molecule pairs and covalently modifying the molecule on the solid carrier. The exosome separation by using the principle is to make the exosome surface specific marker binding molecule, such as protein, polypeptide or aptamer, covalently modified on the surface of solid phase carrier (such as magnetic bead or microsphere), after the exosome incubation and combination, the exosome is separated and eluted from the liquid phase, so as to achieve the purpose of separation and enrichment. The method has the advantages of high specificity, simple and convenient operation, and higher purity of the exosomes separated by other separation methods. However, after the exosomes bind to the ligand molecules, extreme or complex dissociation conditions are required, such as immune antibodies binding to exosome marker proteins (CD 63, CD9, CD81, etc.), and the pH is about 3.0, so that the exosomes with complete structure cannot be obtained, the functionality is lost, and there is a risk of damaging activity or aggregation of proteins. In the technical solution of the patent application CN201580066077.1, after the TIM protein specifically binds to phosphatidylserine, high concentration of EDTA-2Na is needed to chelate calcium ions with bridging action between molecules, so that exosomes are eluted, but the free ligand may affect the subsequent use, and further separation will result in lower separation efficiency.

In summary, there is a lack of an efficient and specific exosome nondestructive separation method, so that the development of exosome related research and application is seriously hindered.

Disclosure of Invention

Based on the above, the invention provides a temperature-sensitive specific polypeptide and a temperature-difference-specificity-based method for nondestructively separating exosomes, which at least solve one problem in the prior art.

In a first aspect, the invention provides a temperature-sensitive specific polypeptide, the amino acid sequence of which is shown as SEQ ID No.1, SEQ ID No.2 or SEQ ID No. 3.

The temperature-sensitive specific polypeptide is a polypeptide which can specifically and affinitively bind to the surface marker of the exosome, the secondary structure of the polypeptide is regulated and controlled by temperature, so that the polypeptide can specifically bind to the surface marker protein of the exosome at low temperature (2-8 ℃), and the polypeptide can dissociate from the surface marker protein of the exosome at higher temperature (25-37 ℃), and the affinity of the polypeptide has temperature sensitivity. The amino acid sequence of the temperature-sensitive specific polypeptide is shown in table 1, wherein the polypeptide corresponding to SEQ ID No.1 is a CD63 protein specific polypeptide, the polypeptide corresponding to SEQ ID No.2 is a CD9 protein specific polypeptide, and the polypeptide corresponding to SEQ ID No.3 is a CD81 protein specific polypeptide. The polypeptide corresponding to SEQ ID No.1 specifically binds to CD63 protein (one of exosome surface-specific marker proteins) at low temperature (2-8 ℃) and dissociates at higher temperature (25-37 ℃). The polypeptide corresponding to SEQ ID No.2 specifically binds to CD9 protein (one of exosome surface-specific marker proteins) at low temperature (2-8 ℃) and dissociates at higher temperature (25-37 ℃). The polypeptide corresponding to SEQ ID No.3 specifically binds to CD81 protein (one of exosome surface-specific marker proteins) at low temperature (2-8 ℃) and dissociates at higher temperature (25-37 ℃).

TABLE 1 amino acid sequence of temperature sensitive specific polypeptides

In a second aspect, the invention provides the use of the temperature-sensitive specific polypeptide in the isolation of exosomes.

In a third aspect, the present invention provides a method for non-destructive separation of exosomes based on temperature difference specificity, comprising the steps of:

Modifying the nano magnetic microsphere by using the temperature-sensitive specific polypeptide to obtain a polypeptide modified nano magnetic microsphere;

Mixing a liquid containing an exosome with polypeptide-modified nano magnetic microspheres, incubating at 2-8 ℃ to enable the exosome to be combined with the polypeptide-modified nano magnetic microspheres, capturing and separating the exosome from the liquid by the polypeptide-modified nano magnetic microspheres under the action of a magnetic field, and removing non-specific adsorption substances by cleaning;

Wherein the amino acid sequence of the temperature-sensitive specific polypeptide is shown as SEQ ID No.1, SEQ ID No.2 or SEQ ID No. 3.

The liquid containing exosomes refers to a liquid containing exosomes to be separated and purified. For example, the exosome-containing liquid may be a biological fluid such as serum, serum-free cell culture supernatant, urine, or the like. The washing may be performed with physiological saline, and the nonspecific adsorption substance is a substance that is not associated with an exosome protein marker (such as CD63, CD9, and CD 81).

In some alternative embodiments, the incubation time is 1 to 12 hours at 2 to 8 ℃.

In some alternative embodiments, the incubation time is 10 to 15 minutes at 25 to 37 ℃.

In some alternative embodiments, modifying the nanomagnetic microspheres with a temperature-sensitive specific polypeptide comprises:

mixing a polymer monomer, an initiator and oleic acid modified Fe ₃O₄ nano-particles to obtain epoxy polymer magnetic microspheres;

Reacting the temperature-sensitive specific polypeptide with tris (2-carboxyethyl) phosphine (TCEP) to obtain a sulfhydryl reduced temperature-sensitive specific polypeptide;

And reacting the reduced sulfhydryl reduced temperature-sensitive specific polypeptide with the epoxy polymer magnetic microsphere to obtain the polypeptide modified nano magnetic microsphere.

In some alternative embodiments, the polymer monomers include at least one of styrene, divinylbenzene, and at least one of glycidyl acrylate, glycidyl methacrylate. Preferably, the polymer monomers include styrene, divinylbenzene, and glycidyl acrylate.

In some alternative embodiments, the initiator is azobisisobutyronitrile.

In some alternative embodiments, the temperature sensitive specific polypeptide is obtained by:

Screening by using exosome marker proteins CD63, CD9 or CD81 as target proteins through phage display technology to obtain monoclonal phage for displaying polypeptide;

Incubating and combining the monoclonal phage with an exosome marker protein CD63, CD9 or CD81 at 2-8 ℃, then dissociating and eluting at 25-37 ℃, sequencing the eluted monoclonal phage, screening to obtain a positive monoclonal phage which is combined with the exosome marker protein CD63, CD9 or CD81 at 2-8 ℃ and dissociated with the exosome marker protein at 25-37 ℃, and sequencing a nucleic acid substance to obtain the terminal display polypeptide sequence of the positive monoclonal phage;

and synthesizing the temperature-sensitive specific polypeptide according to the terminal display polypeptide sequence of the positive monoclonal phage.

Due to the adoption of the technical scheme, the embodiment of the invention has at least the following beneficial effects:

(1) The polypeptide modified nano magnetic microsphere can be specifically combined with CD63, CD9 or CD81 proteins on the surface of the exosome, so that most exosome particles in a body fluid sample can be separated, the defect of co-separation of impurities such as lipoprotein and the like in the conventional exosome enrichment technology is effectively overcome, and the purity of the exosome is greatly improved;

(2) The method for non-destructive separation of exosomes based on temperature difference specificity is a specific non-destructive separation method, the separation condition is mild, the integrity of exosomes is not affected, and the defect that exosomes are damaged due to the polarity of the separation condition in the conventional exosome enrichment technology is effectively overcome;

(3) The polypeptide-modified nano magnetic microsphere is combined with the exosome, and the exosome can be quickly and simply separated out by utilizing the low critical temperature regulation affinity effect and an external magnetic field, so that the operation is simple and convenient;

(4) The polypeptide has low price, and effectively overcomes the defects of high instrument price and high reagent cost of the conventional exosome enrichment technology.

Drawings

FIG. 1 shows the binding and dissociation effects of the temperature-sensitive specific polypeptides corresponding to SEQ ID No.1, SEQ ID No.2, SEQ ID No.3 with the exosome marker proteins CD63, CD9 and CD81 at 4℃and 37℃respectively.

Figure 2 shows the binding effect of CD63 antibodies to CD63 protein at 4 ℃ and 37 ℃.

FIG. 3 is a schematic diagram of the operation flow of separation and purification of exosomes in a biological fluid sample based on polypeptide-modified nanomagnetic microspheres.

FIG. 4 shows the identification of the relative content differences of the exosome marker proteins CD63 and CD9 by WB under total amount of homoproteins of urine exosome samples isolated based on different exosome isolation methods.

FIG. 5 is a schematic diagram of the detection of intact exosomes by the antibody-based sandwich method of homoepitope exosome marker protein CD 63.

FIG. 6 shows detection signals of intact exosomes by ELISA immuno-sandwich detection using HRP-labeled anti-CD 63 monoclonal antibody and immuno-magnetic beads covalently coated with the antibody after separation of equal amount of biological fluid samples based on the differential temperature specificity non-destructive separation method and conventional separation method.

FIG. 7 shows total protein concentration in the separation solution measured by BCA method after separation of an equal amount of biological fluid sample based on a temperature difference-specific non-destructive separation method and a conventional separation method.

FIG. 8 shows the ratio of the detected signal value of the intact exosome content to the total protein concentration in the separation solution when exosomes are separated based on the temperature difference specificity non-destructive separation method and the conventional separation method.

FIG. 9 shows the size of the whole exosomes in the separation solution when exosomes are separated based on the temperature difference specificity non-destructive separation method and the conventional separation method.

Detailed Description

The technical solution of the present invention will be described in detail below with reference to specific embodiments of the present invention, but the following examples are only for understanding the present invention and are not intended to limit the present invention, and the examples and features of the examples may be combined with each other, and the present invention may be implemented in various ways defined and covered by the claims.

TABLE 2 amino acid name definition comparison Table

The amino acids in the amino acid sequence are natural L amino acids, or amino acid residue modifications, optical isomers (e.g., D-type amino acids), or chemical molecule or group modifications (e.g., phosphorylation and dephosphorylation, acetylation and deacetylation, methylation and demethylation, adenylation and deadenonylation, -SH and-S-S-tautomerism, lipid group ligation).

Example 1 panning temperature Regulation affinity phage display Polypeptides

1) Directed panning of phage display peptide libraries

① In the first round, exosome marker proteins CD63, CD9 and CD81 were coated overnight with polystyrene dishes, respectively, at a concentration of 100. Mu.g/mL, washed three times with 1 mL Phosphate Buffer (PBST) containing 0.1% Tween 20, added with sodium bicarbonate solution in which 1% Bovine Serum Albumin (BSA) was dissolved, incubated 90 min at room temperature, the samples were thrown off, washed three times with 1 mL PBST, put into phage display peptide library for incubation for 1 hour, washed 10 times with PBST in ice bath, then 150. Mu.L PBS was added and incubated at 37℃for 1h, 120. Mu.L of liquid was removed, and titer measurement was performed and phage amplification was performed;

② In the second round, exosome marker proteins CD63, CD9 and CD81 were coated overnight with polystyrene dishes, respectively, at a concentration of 10. Mu.g/mL, washed three times with 1 mL Phosphate Buffer (PBST) containing 0.1% Tween 20, added with sodium bicarbonate solution in which 1% Bovine Serum Albumin (BSA) was dissolved, incubated 90 min at room temperature, the samples were thrown off, washed three times with 1 mL PBST, put into phage display peptide library for incubation for 1 hour, washed 10 times with PBST in ice bath, then 150. Mu.L PBS was added and incubated at 37℃for 1h, 120. Mu.L liquid was removed, and titer measurement was performed and phage amplification was performed;

③ Third round, exosome marker proteins CD63, CD9 and CD81 were coated overnight with polystyrene dishes, respectively, at a concentration of 10 μg/mL, washed three times with 1 mL Phosphate Buffer (PBST) containing 0.5% tween 20, added with sodium bicarbonate solution dissolved with 1% Bovine Serum Albumin (BSA), incubated 90 min at room temperature, the samples were thrown off, washed three times with 1 mL PBST, placed in phage display peptide library for 1 hour, washed 10 times with PBST under ice bath, then 150 μl PBS was added and incubated 1h at 37 ℃,120 μl of liquid was removed, titer was determined, and monoclonal phage were picked up for identification.

2) Identification of monoclonal phage specificity and temperature sensitivity

After the monoclonal phage is amplified, the monoclonal phage is respectively incubated and washed at 4 ℃ with sample holes coated with exosome marker proteins CD63, CD9 and CD81, and is detected by an Anti-M13 antibody coupled with horseradish peroxidase (HRP), and meanwhile, a 37 ℃ washing condition is set as a control group. Screening out positive monoclonal phage with high specificity and obvious response to temperature difference change (binding at 4 ℃ and cleaning dissociation at 37 ℃), and carrying out DNA sequencing on the positive monoclonal phage plasmid to obtain corresponding terminal display polypeptide sequences, namely VATDAHRRELKLGGGSC (SEQ ID No. 1), TQVATAHLHSSHGGGSC (SEQ ID No. 2) and SSAYRTGFFTHSGGGSC (SEQ ID No. 3) respectively.

Example 2 Synthesis of temperature-sensitive specific Polypeptides

1) Weighing and swelling Resin, namely accurately weighing 0.1g king Resin (Wang-Resin, 0.8 mmol/g), placing into a20 mL sand core reactor, adding Dichloromethane (DCM) with the volume of three times that of the Resin, swelling for 30 min, and drying for later use;

2) Detecting ninhydrin by adding ninhydrin solution into a small amount of resin, and heating in an oil bath at 110deg.C for 5min while keeping the color of the solution unchanged;

3) Deprotection, namely, taking three times of resin volume of deprotection liquid (20% of 4-methylpiperidine dissolved by DMF) to react with the resin for 15min, removing the deprotection liquid, repeating the steps once, and then cleaning with 2 times of resin volume of DMF;

4) Detecting ninhydrin by adding ninhydrin solution into a small amount of resin, and heating in 110 deg.C oil bath for 5min until the solution turns into blue-violet;

5) Condensing, namely removing cleaning liquid, adding first protected amino acid (serine), adding condensing reagent, dissolving with a small amount of DMF, shaking, fully mixing and dissolving, and reacting for 30min at room temperature;

6) Detecting ninhydrin by adding ninhydrin solution into a small amount of resin, and keeping the color of the solution unchanged within 5min at 110 ℃;

7) Repeating the operation of the step (3) and the step (6), and carrying out progressive reaction on the protecting amino acids one by one according to the target polypeptide sequence (SEQ ID No.1, SEQ ID No.2 or SEQ ID No. 3);

8) And (3) after all amino acids are connected into a complete peptide chain according to a preset sequence, washing the resin with methanol for 3-4 times, adding a cutting fluid, oscillating for 2.5 hours at room temperature, taking filtrate, dripping ice petroleum ether (-20 ℃ for preservation), magnetically stirring, centrifugally precipitating at 5000rpm, removing supernatant, re-suspending, repeatedly operating for three times, and finally pumping out by using a vacuum reaction kettle to obtain the target temperature-sensitive specific polypeptide.

Example 3 identification of temperature sensitive specific polypeptide specificity and temperature differential affinity

Determining the specific affinity of the temperature-sensitive specific polypeptide to the target protein under different conditions, and verifying whether the polypeptide meets the capability of temperature-sensitive specific binding to the target protein and dissociation, wherein the method comprises the following steps of:

1) Exosome marker proteins CD63, CD9 and CD81 were dissolved in 0.1M sodium bicarbonate solution at 100 μg/mL, 50 μg/mL, 25 μg/mL, 12.5 μg/mL, 6.25 μg/mL, 3.13 μg/mL, 1.56 μg/mL, 0 μg/mL, respectively, coated overnight at 4℃in polystyrene plate wells, then blocked for 2 hours at 37℃with 1% BSA dissolved in 0.1M sodium bicarbonate solution, washed three times with PBST;

2) Respectively reacting 5 mug/mL of temperature-sensitive specific polypeptides corresponding to SEQ ID No.1, SEQ ID No.2 and SEQ ID No.3 with horseradish peroxidase conjugate, putting 100 mug of the temperature-sensitive specific polypeptides into an exosome marker protein coating hole for 4 ℃ to react for 1 hour, setting two groups of the temperature-sensitive specific polypeptides, setting a CD63 antibody horseradish peroxidase conjugate and a CD63 protein reaction control group at the same time, and cleaning three times by using PSBT at 4 ℃ and 37 ℃ after the reaction is finished;

3) After incubation for 10 min in the dark with 150. Mu.L TMB substrate solution, termination was performed with 50. Mu.L 2M HCl solution and absorbance at 450 nm was measured.

As shown in figure 1, the temperature-sensitive specific polypeptides corresponding to SEQ ID No.1, SEQ ID No.2 and SEQ ID No.3 have obvious specificity with exosome marker proteins CD63, CD9 and CD81, and show obvious affinity temperature-sensitive characteristics, after the temperature-sensitive specific polypeptides are combined with target proteins at 4 ℃, the temperature-sensitive specific polypeptides still have higher detection signals reaching more than 2.0 by using a4 ℃ cleaning solution, and after the temperature-sensitive specific polypeptides are cleaned by using a 37 ℃ PBST cleaning solution, the signal values drop sharply, so that the temperature rises, and the dissociation behavior of the polypeptides and the target proteins occurs. As shown in fig. 2, the CD63 antibody control group showed a better binding effect than the 4 ℃ washing condition after washing at 37 ℃, which accords with the condition that the conventional antigen-antibody needs a certain temperature to have a better binding effect.

Example 4 preparation of polypeptide-modified nanomagnetic microspheres

1) Preparation of epoxy-based Polymer magnetic microspheres

1.5 G styrene, 0.5 g divinylbenzene, 0.5 g glycidyl acrylate, 3.0 g oleic acid modified Fe ₃O₄ superparamagnetic nano particles and 2 g polyvinylpyrrolidone are dissolved in a 90% ethanol water solution of 100mL, 800 rpm is mechanically stirred for 2 hours, then nitrogen is introduced for 30 minutes, an initiator 1g azodiisobutyronitrile is added, the mixture is reacted in an 80 ℃ oil bath for 24 hours, and the mixture is alternately washed for three times by using ethanol and water through magnetic field adsorption to obtain epoxy polymer magnetic microspheres;

2) Preparation of polypeptide-modified magnetic nanospheres

① Incubating 10 mg of the temperature-sensitive specific polypeptide to be modified with 5mM TCEP at room temperature for 10 minutes to reduce disulfide bonds among polypeptide molecules into sulfhydryl groups, and desalting by a desalting column to replace the disulfide bonds with 0.05M Tris-HCl buffer solution with pH of 8.4 for later use;

② Washing the 1 g epoxy polymer magnetic microsphere with 0.05M Tris-HCl buffer solution with pH of 8.4 for three times, adding 10 mg desalted temperature-sensitive polypeptide solution, reversing and uniformly mixing at 37 ℃ for reaction for 24 hours, then sealing with 0.05M Tris-HCl buffer solution containing 50 mM cysteine, reversing and uniformly mixing at 37 ℃ for reaction for 24 hours, then washing with PBST to obtain the polypeptide modified nano magnetic microsphere, and storing in PBS solution for standby.

Example 5 separation and purification of urine exosomes Using polypeptide-modified nanomagnetic microspheres

The affinity of the temperature-sensitive specific polypeptide is regulated and controlled by utilizing the low critical temperature, the polypeptide-modified nano magnetic microsphere is specifically combined with exosomes in urine at 4 ℃, exosomes are separated and captured from a liquid phase, and the microsphere releases the exosomes at 37 ℃, so that the exosomes with high purity and no damage are obtained, and the specific steps are as follows, as shown in figure 3:

1) Sampling and preprocessing urine samples, namely centrifuging the urine samples in the middle of morning at 4 ℃ and 500 xg for 10min, taking the supernatant and discarding the sediment, centrifuging the urine samples at 4 ℃ and 2000 xg for 10min again, taking the supernatant and discarding the sediment, centrifuging the urine samples at 4 ℃ and 10000 xg for 30min again, taking the supernatant and discarding the sediment, and filtering the supernatant and discarding the sediment through a 0.22 mu m filter to obtain preprocessed urine samples;

2) Separating exosomes, namely taking 10 mL pretreatment urine samples, adding 20 mg polypeptide modified nano magnetic microspheres into the pretreatment urine samples, carrying out reverse mixing reaction for 1 hour at the temperature of 4 ℃, carrying out magnetic adsorption on ice, removing supernatant, cleaning three times by using 10 mL 4 ℃ precooled PBS (phosphate buffer solution), finally adding 200 mu L PBS solution, carrying out vortex to disperse the polypeptide modified nano magnetic microspheres, carrying out water bath treatment at the temperature of 37 ℃ for 10 min, carrying out adsorption by using a magnetic frame, and taking supernatant to obtain the exosome solution with temperature difference specificity and nondestructive separation and purification.

In order to compare the effect of the separation method in this example with that of the separation method in the prior art, the following control group was set:

① Ultracentrifugation by taking pretreated urine sample 10mL, centrifuging at 4deg.C for 1 hr at 100000 xg, removing supernatant, re-suspending with 10mL PBS to obtain precipitate, centrifuging again at 4deg.C for 1 hr at 100000 xg, re-suspending with 200 μl PBS solution to obtain exosome solution;

② PEG precipitation method comprises preparing 50% PEG 8000 solution with PBS containing 0.1M NaCl, adding 2.5mL into 10mL pretreated urine sample, vortex mixing, standing at 4deg.C overnight, centrifuging at 4deg.C and 10000 xg for 30 min, removing supernatant, re-suspending the precipitate with 1 mL 10% PEG 8000 PBS solution, standing on ice for 1 hr, centrifuging at 4deg.C and 10000 xg for 30 min, re-suspending the precipitate with 200 μl PBS solution to obtain exosome solution;

③ The immune magnetic bead affinity separation method comprises the steps of adding 100 mu L of Dynabeads' CD63 ⁺ exosome separation magnetic beads into a10 mL pretreatment urine sample, mixing and incubating the mixture at 4 ℃ overnight, removing supernatant through magnetic adsorption, washing the magnetic beads three times through PBST, washing the magnetic beads once through PBS, adding 100 mu L of 0.1M Gly-HCl buffer solution with the pH of 2.2, re-suspending the magnetic beads, incubating the magnetic beads at room temperature for 10min, and neutralizing the magnetic beads by using 0.1M Tris-HCl buffer solution with the pH of 8.6 to obtain the purified exosome.

The urine exosome samples separated by the different methods are subjected to a Western Blotting (WB) experiment with the total protein amount of 4 mug, and the concentration of the CD63 protein is identified, and the result is shown in figure 4, and the method has the highest specificity with the immunomagnetic bead affinity separation method. Meanwhile, the exosomes contained CD9, indicating that exosomes were bound instead of the CD63 protein alone.

Example 6 isolation and purification of cell culture supernatant exosomes Using polypeptide-modified nanomagnetic microspheres

The affinity of the temperature-sensitive specific polypeptide is regulated and controlled by utilizing the low critical temperature, the polypeptide-modified nano magnetic microsphere is specifically combined with exosomes in cell culture supernatant at 4 ℃, the exosomes are separated and captured from a liquid phase, and the microsphere releases the exosomes at 37 ℃, so that the exosomes with high purity and no damage are obtained, and the specific steps are as follows:

1) Pretreatment of cell culture supernatant, namely taking a serum-free cultured cell culture supernatant. Centrifuging at 4 deg.C and 300 xg for 10min, collecting supernatant, removing free cells, centrifuging again at 4 deg.C and 2000 xg for 10min, collecting supernatant, removing large cell debris, centrifuging again at 4 deg.C and 10000 xg for 30min, collecting supernatant, removing large vesicle and cell debris, and filtering with 0.22 μm filter to obtain pretreated cell culture supernatant;

2) Separating exosomes, namely taking a 10mL pretreated cell culture supernatant sample, adding 20 mg polypeptide modified nano magnetic microspheres into the pretreated cell culture supernatant sample, carrying out reverse mixing reaction for 1 hour at the temperature of 4 ℃, carrying out magnetic adsorption on ice, removing the supernatant, washing three times by using 10mL 4 ℃ precooled PBS, finally adding 200 mu L PBS solution, carrying out vortex to disperse the polypeptide modified nano magnetic microspheres, carrying out water bath treatment at the temperature of 37 ℃ for 10 min, carrying out adsorption by using a magnetic frame, and taking the supernatant to obtain the exosomes solution with temperature difference specificity and nondestructive separation and purification.

① Ultracentrifugation by taking 10 mL of pretreated cell culture supernatant, centrifuging at 4deg.C for 1 hr at 100000 xg, removing supernatant, re-suspending with 10 mL PBS to obtain precipitate, centrifuging again at 4deg.C for 1 hr at 100000 xg, re-suspending the precipitate with 200 μl PBS solution to obtain exosome solution;

② PEG precipitation method comprises preparing 50% PEG 8000 solution with PBS containing 0.1M NaCl, adding 2.5 mL into 10mL pretreated cell culture supernatant, mixing under vortex, standing overnight at 4deg.C, centrifuging at 4deg.C and 10000 xg for 30min, removing supernatant, suspending the precipitate with 1mL 10% PEG 8000 PBS solution, standing on ice for 1 hr, centrifuging at 4deg.C and 10000 xg for 30min, and suspending the precipitate with 200 μl PBS solution to obtain exosome solution;

③ The immunomagnetic bead affinity separation method comprises adding 100 μl Dynabeads' CD63 ⁺ exosome separation magnetic beads into 10 mL pretreated cell culture supernatant sample, mixing and incubating at 4deg.C overnight, removing supernatant by magnetic adsorption, washing the magnetic beads with PBST three times, washing with PBS once, adding 100 μl of 0.1M Gly-HCl buffer solution with pH of 2.2 to resuspend the magnetic beads, incubating at room temperature for 10 min, and neutralizing with 0.1M Tris-HCl buffer solution with pH of 8.6 to obtain purified exosome.

Example 7 polypeptide-modified nanomagnetic microspheres separation and purification of exosomes in peripheral blood

The affinity of the temperature-sensitive specific polypeptide is regulated and controlled by utilizing the low critical temperature, the polypeptide-modified nano magnetic microsphere is specifically combined with exosomes in peripheral blood at 4 ℃, exosomes are separated and captured from a liquid phase, and the microsphere releases the exosomes at 37 ℃, so that the exosomes with high purity and no damage are obtained, and the specific steps are as follows:

1) Extracting and pre-treating peripheral blood sample by using sampling tube of anticoagulant (such as sodium citrate or EDTA-2 Na) to receive peripheral blood, centrifuging at 20deg.C and 2500 xg for 15min, collecting supernatant above blood cell layer, removing red blood cells and blood platelets, centrifuging at 20deg.C and 2500 xg for 15min again, collecting supernatant, removing large cell fragments, and obtaining sample with platelets and red blood cells removed;

2) Separating exosomes, namely taking a 10 mL pretreated plasma sample, putting 20 mg polypeptide modified nano magnetic microspheres into the pretreated plasma sample, carrying out reverse mixing reaction for 1 hour at the temperature of 4 ℃, carrying out magnetic adsorption on ice, removing supernatant, washing three times by using 10 mL 4 ℃ precooled PBS, finally adding 200 mu L PBS solution, carrying out vortex to disperse the polypeptide modified nano magnetic microspheres, carrying out water bath treatment at the temperature of 37 ℃ for 10 min, carrying out adsorption by using a magnetic frame, and taking supernatant to obtain the exosome solution with temperature difference specificity and nondestructive separation and purification.

① Ultracentrifugation by taking pretreated plasma sample 10 mL, centrifuging at 4deg.C for 1 hr at 100000 xg, removing supernatant, re-suspending the precipitate with 10 mL PBS, centrifuging again at 4deg.C for 1 hr at 100000 xg, re-suspending the precipitate with 200 μl PBS solution to obtain purified peripheral blood exosomes;

② PEG precipitation method comprises preparing 50% PEG 8000 solution with PBS containing 0.1M NaCl, adding 2.5 mL into 10mL pretreated plasma sample, vortex mixing, standing at 4deg.C overnight, centrifuging at 4deg.C and 10000 xg for 30min, removing supernatant, re-suspending the precipitate with 1mL 10% PEG 8000 PBS solution, standing on ice for 1 hr, centrifuging at 4deg.C and 10000 xg for 30min, re-suspending the precipitate with 200 μl PBS solution to obtain purified peripheral blood exosome;

③ The immunomagnetic bead affinity separation method comprises the steps of adding 100 mu L of Dynabeads' CD63 ⁺ exosome separation magnetic beads into a 10 mL pretreated plasma sample, reversing and mixing the mixture overnight at 4 ℃, incubating the mixture, removing supernatant through magnetic adsorption, washing the magnetic beads by PBST three times, washing the magnetic beads once by PBS, adding 100 mu L of 0.1M Gly-HCl buffer solution with the pH of 2.2, re-suspending the magnetic beads, incubating the magnetic beads at room temperature for 10 min, and neutralizing the magnetic beads by using 0.1M Tris-HCl buffer solution with the pH of 8.6 to obtain purified peripheral blood exosome.

Example 8 identification of the content and purity of exosomes of intact Structure

The same CD63 monoclonal antibody is used as both solid phase binding antibody and detecting antibody to form sandwich immune complex with exosome. Only when the exosome structure is complete and more than two exosome marker proteins CD63 exist, the exosome cells will be revealed, and the schematic diagram is shown in fig. 5, and the complete exosome content is estimated by signal intensity. Purity was assessed by comparing the ratio of total exosome protein concentration extracted by different methods to exosome CD63 detection signal. The specific steps for obtaining the CD63 detection signal ratio are as follows:

1) CD63 antibody was taken and dissolved at 10. Mu.g/mL in 0.1M NaHCO ₃ solution at pH 8.6, coated overnight in 96 well plates, washed three times with PBST, and fully blocked by addition of 1% BAS dissolved in 0.1M NaHCO ₃ solution at pH 8.6;

2) Urine, cell culture supernatant and peripheral blood exosomes obtained by separation of different separation methods are taken, diluted by PBS (phosphate buffer solution) in half, filtered by a 0.22 mu m filter, 50 mu L of exosome sample to be detected is added into each hole, after incubation for 30 minutes at 37 ℃, 50 mu L of CD63-HRP complex is added for continuous reaction for 1 hour, PBST is used for cleaning three times, 150 mu L of TMB substrate solution is added for color development, incubation is carried out for 10 minutes at 25 ℃ in a dark place, 50 mu L of 2M H ₂SO₄ solution is added for termination, and signal values are read at a position of 450: 450 nm by an enzyme-labeling instrument, so that the content information of the whole exosomes obtained by separation of different separation methods is obtained.

As shown in FIG. 6, the PEG precipitation method has relatively high exosome content, the method for specifically separating exosome based on temperature difference (the method of the invention) has the next content, the ultracentrifugation method has relatively low content, the complete exosome signal obtained by the immunomagnetic bead affinity separation method is also relatively weak, and the correlation with the WB result in FIG. 4 is poor, which indicates that the exosome structure is damaged by acid washing treatment.

The step of obtaining exosome purity information is as follows:

1) 180 mu L of BCA working solution reagent is added into a 96-well plate, then 20 mu L of exosome samples (urine, cell culture supernatant and peripheral blood) to be detected and standard concentration work pieces are added, and after shaking and mixing, incubation is carried out for 30 minutes at 37 ℃ to enable the reaction to be fully carried out;

2) Reading a light absorption value at 562 nm by using an enzyme-labeled instrument, establishing a standard curve by using the concentration of the standard substance and the light value, and further calculating the total protein concentration information of the exosomes according to the standard curve;

3) And dividing the exosome content signal value by the total protein concentration value to obtain exosome purity information.

As shown in FIG. 7, the PEG precipitation method is used for separating exosomes of various samples, the total protein concentration is relatively high, the ultracentrifugation method is used for obtaining the total protein concentration, and the temperature difference specificity-based nondestructive exosomes separation method (the method of the invention) is similar to the total protein concentration obtained by the immunomagnetic bead affinity separation method.

As shown in fig. 8, the results show that the purity of the whole exosome obtained by the method for non-destructive separation of exosome based on temperature difference specificity (the method of the invention) is highest, which indicates that the separation method has unique advantages compared with the ultracentrifugation method, the PEG precipitation method and the immunomagnetic bead affinity separation method, and combines the advantages of the exosome such as the integrity, the purity, the content and the like.

As shown in FIG. 9, the method for non-destructive separation of exosomes based on temperature difference specificity (the method of the present invention) gave a standard exosomes size of about 115 nm, which was relatively close to the exosomes particle size by ultracentrifugation, but the exosomes particle size by PEG precipitation was significantly increased, which could be aggregation caused by polymer-induced precipitation.

The present invention is not limited to the above embodiments, but is merely preferred embodiments of the present invention, and the present invention should be considered as being within the scope of the present invention as long as the technical effects of the present invention are achieved by the same or equivalent means. Various modifications and variations are possible in the technical solution and/or in the embodiments within the scope of the invention.

Claims

1. A temperature-sensitive specific polypeptide, characterized in that the amino acid sequence of the temperature-sensitive specific polypeptide is shown in SEQ ID No.1, SEQ ID No.2 or SEQ ID No.3.

2. Use of the temperature-sensitive specific polypeptide according to claim 1 in isolating exosomes.

3. A method for non-destructive separation of exosomes based on temperature difference specificity, characterized in that it comprises the following steps:

Modifying nanomagnetic microspheres with temperature-sensitive specific polypeptides to obtain polypeptide-modified nanomagnetic microspheres;

The liquid containing exosomes is mixed with the polypeptide-modified nanomagnetic microspheres, and incubated at 2-8°C to allow the exosomes to bind to the polypeptide-modified nanomagnetic microspheres. Under the action of a magnetic field, the polypeptide-modified nanomagnetic microspheres capture and separate the exosomes from the liquid, and non-specific adsorbed substances are removed by washing. Thereafter, the liquid is incubated at 25-37°C to allow the exosomes to dissociate from the polypeptide-modified nanomagnetic microspheres, and the polypeptide-modified nanomagnetic microspheres are removed by magnetic separation to obtain separated and purified exosomes.

Wherein, the amino acid sequence of the temperature-sensitive specific polypeptide is shown as SEQ ID No.1, SEQ ID No.2 or SEQ ID No.3.

4. The method according to claim 3, characterized in that the incubation time at 2-8°C is 1-12 hours.

5. The method according to claim 3, characterized in that the incubation time at 25-37°C is 10-15 minutes.

6. The method according to claim 3, characterized in that modifying the nanomagnetic microspheres with the temperature-sensitive specific polypeptide comprises:

The polymer monomer, initiator and oleic acid-modified Fe ₃ O ₄ nanoparticles are mixed to obtain epoxy polymer magnetic microspheres;

reacting the temperature-sensitive specific polypeptide with tris(2-carboxyethyl)phosphine to obtain a temperature-sensitive specific polypeptide in a thiol-reduced state;

The thermosensitive specific polypeptide in the thiol-reduced state is reacted with the epoxy polymer magnetic microspheres to obtain polypeptide-modified nano magnetic microspheres.

7. The method according to claim 6, characterized in that the polymer monomer comprises at least one of styrene and divinylbenzene, and also comprises at least one of glycidyl acrylate and glycidyl methacrylate.

8. The method according to claim 7, wherein the polymer monomers include styrene, divinylbenzene and glycidyl acrylate.

9. The method according to claim 6, characterized in that the initiator is azobisisobutyronitrile.