CN117778293A - SEC-based sEV separation and purification reagents, kits and methods - Google Patents

Abstract

The invention discloses a sEV separation and purification reagent, a kit and a method based on SEC, and relates to the technical field of liquid chromatography separation. The sEV separation and purification reagent disclosed by the invention uses Sepharose 4FF and Sepharose 6FF as gel filtration fillers and is used for separating and purifying sEV by a SEC method, and the obtained sEV has the advantages of high purity, high yield, no lipoprotein impurity, complete vesicle structure, stable biological activity and the like, and can be suitable for biological samples from various sources.

Description

SEC-based sEV separation and purification reagents, kits and methods

Technical Field

The invention relates to the technical field of liquid chromatographic separation, in particular to a sEV separation and purification reagent, a kit and a method based on SEC.

Background

Small extracellular vesicles (small Extracellular Vesicles, sEV) are extracellular vesicles with the diameter of 40-150nm, are released into extracellular matrixes by cells, carry abundant RNA, protein, lipid and other contents, participate in biological processes such as cell recognition, immunoregulation, signal transduction and the like, and are novel tools for intercellular communication.

After a Nobel physiological or medical prize was obtained from the disclosure of the cell vesicle transport system in 2013, sEV related studies soon became the focus of attention in subjects such as biology and medicine. sEV has the characteristics of nanoscale volume, high biocompatibility, specific surface markers and the like, making it an important marker for disease diagnosis, treatment and prognosis and an ideal drug carrier. Until now, differential ultracentrifugation, ultrafiltration, density gradient centrifugation, exclusion chromatography, polymer precipitation, immunoaffinity capture, and microfluidic methods have been used to separate and purify sEV.

However, the purification sEV of the prior art still has drawbacks such as impurities (e.g., low density lipoproteins) which have an impact on the proteomic analysis of sEV and its biological function.

In view of this, the present invention has been made.

Disclosure of Invention

The invention aims to provide a SEC-based sEV separation and purification reagent, a kit and a method. The sEV obtained by adopting the reagent provided by the invention has the advantages of high purity, high yield, no lipoprotein impurity, complete vesicle structure, stable biological activity and the like, and can be suitable for biological samples from various sources.

The invention is realized in the following way:

in one aspect, the invention provides a SEC-based sEV isolation and purification reagent comprising: the sEV separation and purification reagent comprises a gel filtration packing, wherein the gel filtration packing comprises agarose gel Sepharose 4FF and Sepharose 6FF.

Size exclusion chromatography (SEC-Exclusion Chromatography) is based on the molecular sieve principle, i.e. a chromatographic column is filled with a porous packing as stationary phase, then the mobile phase (biological sample) is added to the stationary phase, and at this time, different sedimentation paths are generated after molecules of different particle sizes in the sample mobile phase enter the stationary phase. Specifically, particles having a smaller particle size than the stationary phase pore size can pass through the stationary phase pores, the settling path is longer, resulting in delayed elution; large particles which cannot enter the pores of the stationary phase are discharged and directly flow out from the gaps among the porous particles of the stationary phase, the sedimentation path is shorter, and the elution can be carried out from the chromatographic column at the highest speed. Thereby realizing the rapid separation of components with different particle diameters in biological samples.

Commercial kits based on SEC methods such as IZON qEV series, SBI SmartSEC separation systems have been used for the separation and purification of biological samples sEV. However, the impurity such as lipoprotein cannot be removed by sEV separated and purified by the existing kit, so that the method has an influence on the downstream sEV proteomics, metabonomics and other related analyses.

The invention creatively discovers that the combination of agarose gel Sepharose 4FF and Sepharose 6FF is used as gel filtration filler for SEC separation and purification sEV, so that the purification effect can be effectively improved, lipoprotein impurities are reduced, the purity and the yield are effectively improved, the vesicle structure is complete and the bioactivity is stable, and the obtained sEV pure product can meet the analysis requirements of multiple groups of proteomics analysis, transcriptomics analysis and the like.

Alternatively, in some embodiments, the Sepharose 4FF and Sepharose 6FF are present in a ratio of (1-2): 2-1 by volume or are each independently present.

Alternatively, in some embodiments, sepharose 4FF and Sepharose 6FF are present in a ratio of 2:1 by volume or are each independently present.

It should be noted that, both Sepharose 4FF and Sepharose 6FF in the reagent of the present invention may exist in a mixed form, and it is only necessary to fill it into an air exclusion chromatographic column when in use; or can exist alone, and when in use, the two are mixed according to the required proportion and then refilled into the air exclusion chromatographic column.

The present invention creatively found that when the volume ratio of Sepharose 4FF to Sepharose 6FF is controlled to be (1-2): the ratio of (2-1), preferably the ratio of 2:1, the purity of sEV obtained is the highest and the clearance of lipoproteins is the highest.

Optionally, in some embodiments, the sEV separation and purification reagent further comprises a equilibration solution and/or an elution solution.

Optionally, in some embodiments, the equilibration liquid comprises a PBS solution.

Alternatively, in some embodiments, the equilibration liquid is a1 x PBS solution.

The balancing solution can replace the preservation solution in the sEV separation column, so that the chromatographic column is positioned in the buffer solution of PBS, and the eluting efficiency of sEV is enhanced.

Optionally, in some embodiments, the eluent comprises trehalose, sucrose, and PBS solution.

Alternatively, in some embodiments, the eluate comprises 9-11mM trehalose, 2-3% (w/v) sucrose, and a1 XPBS solution.

Alternatively, in some embodiments, the eluate comprises 10mM trehalose, 2.5% (w/v) sucrose, and a1 XPBS solution.

Trehalose and sucrose may protect the isolated sEV vesicle structure, leaving the resulting sEV structure intact.

In another aspect, the invention provides a SEC-based sEV isolation and purification kit comprising sEV isolation and purification reagents as described above.

In another aspect, the invention provides a method for SEC-based separation sEV, wherein the sample is separated and purified using sEV separation and purification reagents as described above.

Optionally, in some embodiments, the method comprises: and separating and purifying the sample by using an exclusion chromatographic column filled with the gel filtration packing.

Optionally, in some embodiments, the method comprises the steps of:

balancing: balancing the exclusion chromatographic column with a balancing liquid;

separating: adding a sample to be separated to the equilibrium exclusion chromatographic column;

elution: the eluent was added and the fractions were collected to give a purified sEV solution.

Optionally, in some embodiments, the method comprises the steps of:

balancing: equilibrating the exclusion chromatography column with 25-35ml of the equilibration liquid;

separating: adding 0.3-0.7ml of the sample to be separated to the equilibrium exclusion chromatographic column;

elution: adding 3.5-4.5ml of the eluent, discarding the fraction, adding 0.8-1.2ml of the eluent, and collecting the fraction to obtain a purified sEV solution.

Alternatively, in some embodiments, the sample is selected from the group consisting of plasma, serum, cell culture supernatant, saliva, emulsion, and urine.

It should be noted that the reagents used in the present invention may be obtained by commercial methods, such as Sepharose 4FF and Sepharose 6FF, which are available from beijing solebao technologies, inc.

Drawings

In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings that are needed in the embodiments will be briefly described below, it being understood that the following drawings only illustrate some embodiments of the present invention and therefore should not be considered as limiting the scope, and other related drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.

FIG. 1 shows the particle count and protein content of different elution fractions separated sEV using a single packing in example 2.

FIG. 2 shows the particle count and protein content of different elution fractions separated sEV in different volume ratios using the mixed packing material of example 2.

FIG. 3 shows the relative proportions of lipoproteins (HDL, LDL) and total proteins in different elution fractions separated sEV in different volume ratios using a mixed packing material in example 2.

Figure 4 shows statistics of sEV particles isolated by different methodologies in example 3.

FIG. 5 shows the results of sEV Western Blot isolated by different methodologies in example 3.

FIG. 6 shows the sEV TEM results (SEC method, kit method, UC method in order from left to right) isolated according to the different methodologies of example 3.

FIG. 7 shows the results of sEV RNA isolation according to the different methodologies in example 3.

FIG. 8 shows the results of sEV RT-PCR isolated according to the different methodologies of example 3.

FIG. 9 is a graph showing the ratio of sEV sequencing alignment to mRNA to LncRNA isolated by the different methodologies of example 3.

Figure 10 is a plot of the ratio of sEV sequencing to miRNA isolated by different methodologies in example 3.

FIG. 11 is a graph showing the statistics of the number of particles of the different biological samples sEV isolated according to the present invention in example 4.

FIG. 12 shows Western Blot results of the separation of different biological samples sEV using the present invention in example 4.

Fig. 13 shows TEM results of the separation of different biological samples sEV using the present invention in example 4.

FIG. 14 is a graph showing the comparison of the number of particles before and after membrane rupture of sEV in the supernatant of a cell culture isolated according to the present invention in example 5; a-automatic operation, B-manual operation; EXOSTATION-sEV represents sEV obtained by automated purification; SEC-sEV shows manual purification of sEV.

FIG. 15 shows the results of the biological activity assay of sEV in the supernatant of the cell culture isolated by the invention in example 5.

FIG. 16 shows the particle concentration and protein concentration of each fraction in the comparative example.

FIG. 17 shows the total protein and lipoprotein content of the fractions (F8-F15) in the comparative example.

FIG. 18 shows the results of particle count and purity in the comparative example.

Fig. 19 shows WB detection results of sEV obtained by separating two fillers in the comparative example.

Fig. 20 shows TEM detection results of sEV obtained by separating two fillers in the comparative example.

FIG. 21 shows the RNA content measurement results of sEV obtained by separating two fillers in the comparative example.

FIG. 22 shows the results of detecting the content of the reference gene in sEV obtained by separating two fillers in the comparative example.

Detailed Description

In order to make the objects, technical solutions and advantages of the embodiments of the present invention more clear, the technical solutions of the embodiments of the present invention will be clearly and completely described below. The specific conditions are not noted in the examples and are carried out according to conventional conditions or conditions recommended by the manufacturer. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention.

The features and capabilities of the present invention are described in further detail below in connection with the examples.

Example 1

The sEV separation and purification reagent for exclusion chromatography provided in this example includes gel filtration packing: agarose gel Sepharose 4FF (cat No. S9180) and Sepharose 6FF (cat No. S9190), both purchased from beijing solebone technologies, inc;

balancing solution: 1 x PBS solution;

eluent: 10mM trehalose, 2.5% (w/v) sucrose and 1 XPBS solution.

The method for separating and purifying sEV by adopting the reagent of the embodiment of the invention comprises the following steps:

1. preparation of exclusion chromatography column

Mixing the two fillers uniformly according to the volume ratio of 1:1, 1:2 or 2:1, and then filling the mixture into 12mL empty columns respectively (the filling total amount of the fillers is 9 mL) to prepare the chromatographic column.

In other examples, the packing Sepharose 4FF, sepharose 6FF were packed into 12mL empty columns, respectively.

2. The sample loading, separating and purifying method provided by the embodiment is as follows, and can be suitable for sEV separation and purification of various biological samples, and the steps are as follows:

(1) Adding 30ml of balance liquid to balance the main body;

(2) Adding 0.5ml of biological sample and discarding the fraction;

(3) Adding 4ml of eluent and discarding the fraction;

(4) Adding 1ml of eluent, and collecting to obtain the high-purity sEV solution.

It should be noted that, for different samples, sEV fractions to be collected can be selected autonomously according to different research requirements, and the reference steps are as follows:

(1) Adding 30ml of balance liquid to balance the main body;

(2) Adding 0.5ml of biological sample and discarding the fraction;

(3) Adding 0.5ml of eluent;

(4) Collecting the fraction; repeating the steps (3) - (4) for 10-15 times, and detecting the concentration of fraction particles and the protein content of the collected fraction to confirm the range of the fraction sEV.

Example 2

The clearance of sEV pure lipoprotein obtained as in example 1 was examined

1. Standard preparation

(1) Small extracellular vesicle standard solutions at concentrations E11 (Particles/mL) were prepared according to the instructions of the kit (Creative Biolabs small extracellular vesicle standard kit Purified Exosome (Umbilical Cord Mesenchymal Stem Cells), cat# EXA-0822-CY 1).

(2) A high-density lipoprotein standard solution and a low-density lipoprotein (Sigma lyophilized powder, low-density lipoprotein LDL, cat. L8292) standard solution were prepared at a concentration of 1mg/mL according to the instructions of the kit (Sigma lyophilized powder, high-density lipoprotein HDL, cat. L1567), respectively.

(3) Preparing a small extracellular vesicle mixed solution containing 10 mug/mL of lipoprotein (HDL+LDL) as a sample by using the small extracellular vesicle standard substance solution, the HDL standard substance solution and the LDL standard substance solution in the steps (1) and (2) according to a volume ratio of 100:1:1.

2. Sample loading, separating and purifying

(1) The column was equilibrated thoroughly with 30mL of equilibration solution as in example 1, followed by the addition of 0.5mL of 1.3.3 samples of the prepared mixed solution, respectively, followed by elution with eluent as mobile phase, and the 20 fractions were collected in sequence in a volume of 0.5 mL.

(2) The fraction collected at 1.3.4 was subjected to particle concentration detection using nanoparticle tracking analysis (Nanoparticle Tracking Analysis, NTA), the specific steps of: diluting a sample to be tested to a volume of 1mL by using a1 XPBS solution; cleaning the detection module with the filtered sterilized water and removing residual liquid; sucking a small extracellular vesicle sample by using a 1mL syringe and injecting the small extracellular vesicle sample into a detection module; nanoparticle tracking analyzer (Malvern, nanoSight NS 300) was run; recording the detection result. The protein concentration of each fraction was measured according to the kit (BCA protein concentration measurement kit (enhanced), cat No. P0010) instructions. Fractions sEV were identified based on particle concentration and protein concentration distribution of each fraction.

(3) The high-density lipoprotein and low-density lipoprotein contents of the sEV fraction were detected separately according to the instructions of the kit (high-density lipoprotein concentration detection kit-R & D Human Apolipoprotein A-IQuantikine ELISA Kit, cat. DAPA10; low-density lipoprotein concentration detection kit-Human Apolipoprotein B/ApoB Quantikine ELISA Kit, cat. DAPB 00).

Results: particle concentration and protein concentration detection results of each fraction show that, as shown in fig. 1-3 (F1-F15 represent collected sEV elution fractions), a mixed packing is adopted to prepare an exclusion column (fig. 2-3), and the division of each fraction obtained by separation is more obvious than that obtained by adopting a single packing (fig. 1); further, the HDL and LDL contents of each fraction were measured, and the results showed that when the packed Sepharose 4FF and 6FF were mixed in a volume ratio of 2:1 to prepare a column for exclusion, sEV collected fractions were concentrated in fractions F9-F10 (sEV solution volume: 1 mL), at which time the purity of the separated and purified sEV was the highest and the clearance of lipoproteins was the highest.

Example 3

The quality control effect of sEV obtained by the method of example 1 was compared with that obtained by the conventional methods (UC method and ExoEasy method).

1. Description of the purification method

A small extracellular vesicle mixture solution (containing 10. Mu.g/mLHDL+10. Mu.g/mLLDL solution) at a concentration of E11 (Particles/mL) was prepared as in example 2. A sEV separation and purification test was performed using an ultracentrifugation method (UC), a QIAGEN ExoEasy kit method, and a sEV separation and purification method (SEC) of example 1, respectively, from 0.5mL of the mixed solution.

UC method: separation and purification were performed using an ultracentrifuge (Thermo, wX +ultra Series). Adding 0.5mL of the mixed solution into the super-separation tube, and then adding 1 XPBS solution to the position 3-5mm away from the tube orifice; the superlift tube was weighed and trimmed using an analytical balance (Sartorius, BSA 124S-CW); setting parameters of an ultracentrifuge: 110000g at 4 ℃, centrifuging for 90min, re-suspending the precipitate, and centrifuging for 70min at 110000g at 4 ℃ again; the precipitate was resuspended using 1mL of 1 XPBS solution to give a purified sEV solution.

QIAGEN Exoeasy kit method the sEV product isolated was resuspended using 1mL of 1 XPBS solution according to the instructions for use of the kit (QIAGEN exoEasy Maxi Kit, cat. No. 76064).

Example 1 method: 0.5mL of the mixed solution was added to the exclusion chromatography column, and then 4mL of 1 XPBS solution was added to flow out as void volume, yielding 1mL of sEV solution for subsequent detection.

sEV detection method

(1) The resulting particle concentration of sEV was quantified using NS 300; the expression of the positive characterizing proteins CD63, CD9, TSG101, negative proteins Calnexin and lipoproteins Apoli-a and Apoli-B of sEV was detected using protein immunoblotting (WB); film structure observation analysis was performed on sEV using a transmission electron microscope (JEOL, JEM1400 Flash).

(2) The resulting sEV total RNAs were extracted according to the kit (Total RNA extraction purification kit-QIAGEN miRNeasy Micro Kit, cat# 217084) instructions, respectively. The concentration and fragment distribution of total RNA were determined according to the instructions of the kit (Total RNA quality control kit-Agilent RNA 6000Pico kit, cat. No. 5067-1513).

(3) According to the kit (cDNA Synthesis kit-SuperScript) ^TM IV first strand synthesis system, cat No. 18091050) instructions for synthesizing cDNA using total RNA as template. Primers were designed and synthesized, and the abundance of expression of the sEV internal reference genes ACTB and GAPDH was detected using RT-qPCR with the synthesized cDNA as template.

ACTB gene amplification upstream primer (5 '-3'):

TGCGTTACACCCTTTCTT(SEQ ID NO.1)。

ACTB gene amplification downstream primer (5 '-3'):

CTGTCACCTTCACCGTTC(SEQ ID NO.2)。

GAPDH gene amplification downstream primer (5 '-3'):

TCAAGAAGGTGGTGAAGCAGG(SEQ ID NO.3)。

GAPDH gene amplification downstream primer (5 '-3'):

TGGGTGTCGCTGTTGAAGTC(SEQ ID NO.4)。

20. Mu.L RT-qPCR reaction system: 2X TB Green Premix Ex Taq II. Mu.L, 50X ROX Reference Dye II. Mu.L (cat No. RR 820Q), 1. Mu.L each of the upstream and downstream primers at 10mM concentration; 2. Mu.L of cDNA template; nuclease-free water 5.6. Mu.L. The reaction procedure: 95 ℃,30s (1 cycle); signal (40 cycles) c is collected at 95 ℃,5s,60 ℃ and 34 s; 95 ℃,15s,60 ℃,1min,95 ℃,15s (1 cycle).

(4) Preparation of long RNA of sEV according to kit instructionsLibrary (library preparation kit-)Ultra ^TM IIDirectional RNA Library Prep Kit, cat No. E7760L). After library preparation, qubit was used first ^TM The dsDNA quantification kit (cat. No. Q32851) performs preliminary quantification, dilutes the library to 1 ng/. Mu.L, then uses High Sensitivity DNA Kit (cat. No. 5067-4626) to detect the distribution of library fragments, and after the fragment distribution meets the expectations, uses the NGS library quantification kit (Clontech Library Quantification Kit, cat. No. 638324) to accurately quantify the effective concentration of the library.

(5) Small RNA library of sEV total RNA was prepared according to the kit instructions (library preparation kit-Multiplex Small RNA Library Prep Set, cat No. E7300S). Quality control and quantification methods after library preparation were as above.

(6) After the library QC is qualified, the different libraries are sequenced by the Illumina PE150/SE50 according to the effective concentration and the requirement of the target off-machine data volume. Sequencing data were analyzed normalized using bioinformatics means.

The results are shown in FIGS. 4-10: the transmission electron microscopy results showed that typical membrane structures were observed for the sEV solutions obtained by the three methods (fig. 6). Nanoparticle follow-up analysis showed that SEC-sEV particle concentration was significantly higher than UC-sEV and the kit method (fig. 4). The results of protein immunoblotting showed that the three positive proteins of SEC-sEV were significantly higher than the other two methods, and that no calnexin protein contamination was detected by sEV obtained from the three methods (fig. 5). Statistics of the total amount of sEV RNA extracted by the 3 methods show that SEC-sEV is significantly higher than that of the other two methods (FIG. 7). qPCR detection was performed on two reference genes using the RNA reverse transcribed cDNA as a template, and the results showed that the expression levels of ACTB and GAPDH in SEC-sEV were highest (FIG. 8), again demonstrating that sEV isolated and purified by SEC method was significantly higher than the other two methods. A long-chain RNA library was prepared based on sEV RNA extracted by the 3 methods, and the identified transcripts were subjected to classification analysis, so that the mRNA and LncRNA ratios identified by SEC-sEV were found to be optimal (FIG. 9). Similarly, SEC-ev RNA preparation Small RNA libraries identified miRNA ratios that were also optimal (fig. 10).

Example 4

The method of example 1 was used for different types of biological samples.

The procedure of example 1 was used to isolate and purify sEV from different samples, namely plasma, serum, cell culture supernatant, saliva, emulsion, urine, and the resulting purification sEV was tested. The results are shown in figures 12-13, and the quality control identification of SEC-sEV of plasma, serum, cell culture supernatant, saliva, emulsion and urine shows that sEV for separating different types of biological samples by the method is better.

Example 5

The procedure of example 1 was used for manual operation consistent with the automated separation and purification sEV effect.

sEV uptake assay method: lysis was completed by incubating 0.1% Triton-X100 with sEV for 10min at room temperature using BD FACSymphony ^TM The A1 flow cytometer detects sEV particle numbers before and after lysis. Labeling the obtained sEV with DiO dye at 1.5.1, and incubating at room temperature in dark place for 20min; ultracentrifuge 120000×g for 90min, discard supernatant, collect DIO-labeled sEV, re-suspend with FBS complete medium without sEV; co-culturing DiO-labeled sEV and 293T cells for 12 hours, and fixing the cells after co-incubation by using 4% paraformaldehyde solution; the nuclei were then labelled with DAPI and uptake of sEV by the cells was observed under a fluorescent microscope.

The results of the manual and automated separation and purification as described in example 1 showed that sEV separated and purified by the two methods were substantially identical in particle count and purity (membrane particle fraction) for a general biological sample (cell culture supernatant) sEV membrane disruption (FIG. 14). sEV uptake experiments show (FIG. 15) that sEV obtained by both methods has good biological activity and can be used for studying the function and regulatory mechanism of sEV in biological processes.

Comparative example

And comparing the separation and purification effects of different fillers.

Preparation of a 1-row chromatography column:

5mL of Sepharose 2B packing (product No. S8701, available from Beijing Soy Bao technology Co., ltd.) was mixed thoroughly and packed into a12 mL-exclusion chromatography column, sepharose 4FF and Sepharose 6FF packing were mixed thoroughly in a 2:1 ratio and packed into a12 mL-exclusion chromatography column, and then the column was equilibrated thoroughly with 30mL of 1 XPBS solution.

2, testing the separation and purification effect of a column with a resistance-removing chromatographic column:

2.1 Small extracellular vesicle Standard solution, HDL Standard solution and LDL Standard solution were prepared in a volume ratio of 100:1:1 to prepare a small extracellular vesicle mixture solution containing 10. Mu.g/mL of lipoprotein (HDL+LDL) as a sample.

2.2 sample separation and purification

The chromatographic column was fully equilibrated with 30mL of equilibration solution according to the method of example 1, then 0.5mL of the prepared mixed solution sample was added to the exclusion column prepared by mixing the Sepharose 2B as the filler and Sepharose 4FF and Sepharose 6FF as the fillers in a ratio of 2:1, and then elution was carried out using the eluent as the mobile phase, and 15 fractions were collected in sequence in a volume of 0.5mL and subjected to detection (the related detection method was the same as the example), with the following results.

3 results

3.1 the particle concentration and protein concentration detection results of each fraction show that the separation of each fraction is more remarkable than the separation of each fraction by using a Sepharose 2B filler (see FIG. 16) by using a mixed filler to prepare an exclusion column; the HDL and LDL contents of each fraction were tested, and the results showed that the lipoprotein clearance of the mixed filler was higher (see FIG. 17).

3.2 nanoparticle tracking analysis showed that the mixed filler separation resulted in sEV particle concentrations significantly higher than Sepharose 2B filler (fig. 18) with 92% recovery. The Western Blot (WB) results showed that the three positive protein contents of sEV obtained by mixed-filler separation were significantly higher than the other two methods, none of which detected calnexin contamination, but the filler Sepharose 2B was able to detect weak lipoprotein contamination (fig. 19). The sEV membrane structure resulting from separation of the two fillers shows that the mixed filler is able to capture the typical sEV membrane structure, whereas the filler Sepharose 2B is able to detect the sEV membrane structure but atypical (fig. 20).

Statistics of the total amount of sEV RNA obtained by separating the two fillers show that the mixed filler is significantly higher than the filler Sepharose 2B (FIG. 21). qPCR detection is carried out on two reference genes by taking cDNA of reverse transcription of the RNA as a template, and the result shows that the mixed filler is obviously higher than the filler Sepharose 2B (figure 22), and sEV separated and purified by the mixed filler is better than the Sepharose 2B filler.

In summary, the embodiment of the invention provides a reusable SEC separation kit suitable for various body fluids for the first time. The kit comprises SEC columns and related reagents, not only supports manual operation, but also supports automatic extraction by a matched extractor, can be widely used for separating various body fluids sEV, and can be used for analysis of upstream proteomics and sEV transcriptomics on the premise of ensuring high recovery rate and high purity.

The above description is only of the preferred embodiments of the present invention and is not intended to limit the present invention, but various modifications and variations can be made to the present invention by those skilled in the art. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims (15)

1. A SEC-based sEV isolation and purification reagent, comprising: the sEV separation and purification reagent comprises a gel filtration packing, wherein the gel filtration packing comprises agarose gel Sepharose 4FF and Sepharose 6FF.

2. The sEV isolation and purification reagent according to claim 1, wherein the Sepharose 4FF and the Sepharose 6FF are present in a mixed form in a volume ratio of (1-2): 2-1 or are present independently of each other.

3. The sEV isolation and purification reagent according to claim 1 or 2, wherein the Sepharose 4FF and Sepharose 6FF are present in a mixed form in a volume ratio of 2:1 or are present independently of each other.

4. A sEV separation and purification reagent according to claim 3, wherein the sEV separation and purification reagent further comprises a equilibration solution and/or an elution solution.

5. The sEV isolation and purification reagent of claim 4, wherein the equilibration solution comprises a PBS solution.

6. The sEV isolation and purification reagent of claim 5, wherein the equilibration solution is a1 x PBS solution.

7. The sEV isolation and purification reagent of claim 4, wherein the eluent comprises trehalose, sucrose and PBS solution.

8. The sEV isolation and purification reagent of claim 7, wherein the eluent comprises 9-11mM trehalose, 2-3% (w/v) sucrose and 1 x PBS solution.

9. The sEV isolation and purification reagent of claim 8, wherein the eluent comprises 10mM trehalose, 2.5% (w/v) sucrose and 1 x PBS solution.

10. A SEC-based sEV isolation and purification kit comprising the sEV isolation and purification reagent of any one of claims 1-9.

11. A method for SEC-based separation sEV, characterized in that the sample is isolated and purified using the sEV isolation and purification reagent of any one of claims 1-9.

12. The method according to claim 11, characterized in that the method comprises: and separating and purifying the sample by using an exclusion chromatographic column filled with the gel filtration packing.

13. The method according to claim 12, characterized in that it comprises the steps of:

balancing: balancing the exclusion chromatographic column with a balancing liquid;

separating: adding a sample to be separated to the equilibrium exclusion chromatographic column;

elution: the eluent was added and the fractions were collected to give a purified sEV solution.

14. The method according to claim 13, characterized in that it comprises the steps of:

balancing: equilibrating the exclusion chromatography column with 25-35ml of the equilibration liquid;

separating: adding 0.3-0.7ml of the sample to be separated to the equilibrium exclusion chromatographic column;

elution: adding 3.5-4.5ml of the eluent, discarding the fraction, adding 0.8-1.2ml of the eluent, and collecting the fraction to obtain a purified sEV solution.

15. The method of any one of claims 11-14, wherein the sample is selected from the group consisting of plasma, serum, cell culture supernatant, saliva, emulsion, and urine.