CN109825472A

CN109825472A - A kind of extracting method and kit of extracellular vesica

Info

Publication number: CN109825472A
Application number: CN201910156434.3A
Authority: CN
Inventors: 陆路
Original assignee: Fang Guowei
Current assignee: Fang Guowei
Priority date: 2019-03-01
Filing date: 2019-03-01
Publication date: 2019-05-31

Abstract

The present invention relates to bio-separation extractive technique fields, and in particular to a kind of extracting method of extracellular vesica.The described method includes: providing biological sample；Under conditions of being enough to make in biological sample impurity to be partially or fully retained on capture surface, make the biological sample by contacting at least twice with the capture surface；Wherein, the impurity is other substances in biological sample some or all of in addition to extracellular vesica, and the capture surface includes inner surface and/or the outer surface of spherical particle porous material；When impurity is partially or fully retained on catches surface, flowing through liquid is to extract the liquid comprising extracellular vesica；Or further the above-mentioned liquid that flows through is concentrated.The present invention being capable of quick isolating and purifying to epicyte vesica, and the lock out operation is simple, and at low cost, separating sample purity is high is intended for single use, sample bioactivity keeps good, apply biomarker discovery, in terms of basic research and industrial production in.

Description

A kind of extracting method and kit of extracellular vesica

Technical field

The present invention relates to bio-separation extractive technique fields, and in particular to a kind of extracting method and reagent of extracellular vesica Box.

Background technique

Extracellular vesica (extracellular vesicles, EVs) is the approach by evolution conservative, by including from original A kind of vesica containing film that core biology is discharged to more advanced eucaryote and plant cell.Past many decades, extracellularly Vesica is due to that with transport protein, lipid, nucleic acid and carbohydrate and then can influence to target the physiology and pathology different from cellulation Function, to be considered as the important carrier exchanged between protokaryon and eukaryotic cells.Based on size, extracellular vesica can divide For the outer vesica>300nm of vesica outside cellule (<300nm, small EVs, sEVs) and middle maxicell (medium/large EVs, m/lEVs)

Excretion body (exosomes) is a subclass of extracellular vesica, and diameter is that 30-150nm belongs to sEVs, excretion body Density is 1.13-1.19g/mL, is formed in intracellular more vesica bodies, is discharged by more vesica body films with cell membrane fusion Extracellularly.Mammal EVs is distributed widely in various body fluid, including cerebrospinal fluid, nasal mucus, saliva, BAL fluid, Knuckle synovia, bile, blood, amniotic fluid, sperm, urine, cervical mucus and excrement.The EVs in body fluid source comes from body fluid Cell and/or body fluid inner cavity surface cell mixture, they pass through lipid film and wrap up a variety of biologies point such as protein nucleic acid Son makes it from the enzymatic hydrolysis of corresponding degrading enzyme in body fluid, to become physiological and pathological information source or realize intercellular signal Remote transmitting.Researcher has found the EVs wide participation biosystem dynamic equilibrium in mammalian cell source, disease The prominent exchange of development and progress, such as nerve and nerve regneration and retrogression, tumour generates and drug resistance.And natural low of sEVs is exempted from Epidemic focus and the biological barrier permeability of height then assign its potentiality for becoming pharmaceutical carrier.

Outer membrane vesicles (outer membrane vesicles, OMVs) are that one kind is derived mainly from protokaryon Gram-negative The extracellular vesica of bacterium, diameter is in 30-300nm.OMVs is sprouted by bacterial outer membrane (outer membrane) generates release To extracellular, it is enriched LPS, nucleic acid, lipid, outer membrane protein, periplasm protein, inner membrane protein and cytoplasmic protein etc..On the one hand, The molecule that OMVs is enriched with prompts it that may play a significant role during curing the disease；On the other hand, multiple studies have shown that, OMVs There is the immunogenicity of height, natural or acquired immunity can be activated simultaneously in vivo, had as the huge of effective vaccine Potentiality.

EVs Molecular Detection or functional analysis either for biological mechanism research, or it is used for biomarker It was found that the biomolecule detections such as nucleic acid-protein, or for small molecule or the tool carrier or treatment of large biological molecule The exploitation of carrier, it is all the demand of the bottom that enough bioactivity, which keep good high-purity EVs,.Therefore, EVs extracting method pair EVs research and application and development field all have considerable influence.

Following several modes are commonly used in the extraction of excretion body at present:

First is that supercentrifugation, mainly includes differential centrifugation and density gradient centrifugation, this is that current EVs extraction is most common One of method.Differential centrifugation is although easy to operate, but (2-3hr) when operating cost, and separation product purity is low, the excretion body rate of recovery Lower, fractional dose amplification is limited, and needs to prepare expensive ultracentrifuge；Density gradient centrifugation then needs to be previously-completed differential Centrifugal concentrating removes outside aforementioned disadvantage although with high purity, cumbersome, more time-consuming (> 16hr).

Second is that the polymer precipitation method, this method is coprecipitated in conjunction with hydrophobic proteins and lipid molecular by polymer (such as PEG) It forms sediment, it is previous to be applied to collect virus from serum equal samples.EVs lock out operation is simple, low in cost, but need to generally grasp overnight Make, take a long time, and maximum problem is still that purity is low, has a large amount of albumen and nucleic acid impurities to be co-precipitated.

Third is that ultrafiltration, this method is easy to operate, but excretion body is big by mechanical shear stress, may influence the biology of excretion body Activity and structure, and purity is more insufficient, is more to play the role of enrichment rather than purification.

Fourth is that affine in immunity method, specific high, easy to operate, without valuableness the instrument and equipment of this kind of method, but due to mesh It is preceding not find that EVs conventional sign object, general used specific antibody identify to the greatest extent EVs subgroup, while experiment reagent yet The cost is relatively high, and is unfavorable for the amplification of separation scale.

Fifth is that the affine method of PS, PS (phosphatidylserine) in conjunction with magnetic bead, is utilized affine principle capture EVs's by this method PS.This method is similar to affine in immunity method, and the excretion volume morphing of acquisition is complete, and purity is higher.Due to not using denaturant, not shadow The bioactivity of excretion body is rung, excretion body can be used for cell co-cultivation and internal injection.2016.9"Scientific Reports " magazine delivered this method latest data, show that PS method can extract the excretion body of suitable high-purity.But this method is still So there are problems that the amplification of separation scale and use cost.

Sixth is that molecular exclusion chromatography, this kind of method utilizes the pore size and sample EVs size according to gel pore Relativeness and solute is separated.Molecule and EVs in sample greater than aperture not can enter gel pore, can only be along porous solidifying It is flowed out first by chromatographic column in gap between micelle；Small-molecular-weight impurity can then enter most holes in gel, More strongly it is detained in column, is flowed out later.The separated excretion body arrived the size under Electronic Speculum of this method is uniform, but separates sample This volume is less, and flow velocity causes disengaging time relatively long slowly, and at least 5-6 times of Sample Dilution, the amplification of separation scale has a fixed limit System effectively uses and needs special equipment, and cannot be distinguished and particulate matter similar in extracellular vesica size.Using phase To limited.

Seven, in interior method, the development phase for being also in early stage, which exists, divides for other methods, including the separation of micro-fluidic field flow etc. It is few from sample, there is higher requirement to detected downstream method sensitivity.

Existing extracellular vesica separation method, is primarily present for sum up, cumbersome time-consuming, separation purity is low, Separation scale can not Linear Amplifer, the high problem of use cost.

Such as, Publication No. 108865983A, the patent of invention that publication date is 2018-11-23, disclose a kind of cell excretion The extracting method of body, method includes the following steps: (1) supernatant separation: by the culture medium after cell culture, centrifugation goes to clean Matter isolates culture supernatant；(2) primary to extract: step (1) culture supernatant to be mixed with excretion body extracting solution laggard Row is incubated for, and the first incubation fluid is obtained after incubation, is carried out centrifugal concentrating to the first incubation fluid, is obtained the concentrate of the body containing excretion；(3) two Secondary extraction: addition excretion body extracting solution is incubated after sterile PBS dissolution is added in the concentrate of the body containing excretion described in step (2) It educates, the second incubation fluid is obtained after incubation, the second incubation fluid is carried out bottom precipitation is collected by centrifugation, obtains cell excretion body.The patent is still It is so to use polymer precipitating centrifugation to extract, inevitably extracts and deposited in the presence of the polymer precipitating centrifugation narrated above The problem of.

In addition other patents disclosed at present have also been all made of above-mentioned one of method, there is a problem of corresponding.

Summary of the invention

The purpose of the present invention is to solve the above problems, provide a kind of extracting method of excretion body.

In order to achieve the above object of the invention, the invention adopts the following technical scheme:

A kind of extracting method of extracellular vesica, which comprises

Biological sample is provided；

Under conditions of being enough to make in biological sample impurity to be partially or fully retained on capture surface, make the biology Sample with the capture surface by contacting at least twice；Wherein, the impurity be biological sample in addition to extracellular vesica Some or all of other substances, it is described capture surface include spherical particle porous material inner surface and/or outer surface；

When impurity is partially or fully retained on catches surface, flowing through liquid is to extract comprising extracellular vesica Liquid；

Or further the above-mentioned liquid that flows through is concentrated.

Preferably, which comprises the biological sample is successively after two concatenated chromatographic columns, the impurity portion The capture surface for dividing or being all retained in filler in chromatographic column, flowing through liquid is the extracellular vesica after purification extracted, institute Stating filler in chromatographic column is spherical particle porous material.

Preferably, in concatenated two chromatographic columns in the first chromatographic column spherical particle porous material inner and outer surfaces For anion exchange groups functionalization, anion exchange groups are quaternary amines or tertiary amine group.Such as-N+ (CH₃)₃Or-O- CH₂COO- or-O-CH₂CHOHCH₂OCH₂CHOHCH₂N+(CH3)₃。

Preferably, the inner and outer surfaces of spherical particle porous material are positively charged in first chromatographic column.

Preferably, spherical particle porous material bore area is in the second chromatographic column in concatenated two chromatographic columns Anion exchange groups functionalization, anion exchange groups are quaternary amines or tertiary amine group.Such as CH₃(CH₂)₇NH-。

Preferably, the spherical particle porous material bore area of second chromatographic column is positively charged.

Preferably, the average grain diameter of the spherical particle porous material is 60-100 μm, and the first chromatographic column spherical particle is more The average pore size of Porous materials is less than or equal to 30nm, and the second chromatographic column spherical particle aperture of porous material maximum exclusion molecular weight is less than 400KD。

Preferably, extracellular vesica is excretion body or outer membrane vesicles.

Preferably, the linear flow rate that the biological sample passes through filler in chromatographic column is 100-480cm/h.

Preferably, the biological sample includes biological fluid, cell injuring model supernatant, the outer culture supernatant of microbial body.

1. preferred, the biological fluid include plasma/serum, urine, cerebrospinal fluid, tears, sperm, amniotic fluid, gastric juice, Saliva, hydrothorax, ascites, nasal mucus, BAL fluid, knuckle synovia, bile, cervical mucus and excrement.

Preferably, the height of filler is 6-35cm in chromatographic column.

Kit used by a kind of extracting method of extracellular vesica, the kit is by the first chromatographic column and the second layer Analysis column is composed in series, wherein the first chromatographic column at least one, the second chromatographic column at least one use standard Shandong between chromatographic column You connect interface.

At least two chromatographic columns are used in the present invention, when using two chromatographic columns, biological sample is with faster linear flow When speed flows through the first chromatographic column, the impurity less than filler aperture enters by partial adsorbates inside filler, miscellaneous greater than filler aperture By adsorption, excretion body is greater than stuffing hole diameter and directly flows through matter；First column flows through liquid and flows through second with same linear flow rate When column, the impurity less than aperture enters inside filler and is adsorbed, and sEVs is directly flowed through.

Carrying out biological substance separation using ion exchange chromatography at present is usually that the substance of needs is carried out absorption capture, And the application is using the form for flowing through liquid.That is, common ion-exchange chromatography separation method is by by purpose object Matter is adsorbed, and impurity flows through, and is then eluted again, and the present processes are then adsorbing contaminants, and target substance is allowed to flow through, because This, flowing through liquid is the mixed liquor containing target substance.Such as, current albumen and viral purification.And the invention land productivity Mode is flowed through with chromatographic column, separation and Extraction is carried out to sEVs.

If extracting using film layer analysis, can not just develop into the formula of flowing through, this is because sEVs and OMVs relative to The flowing of film or be retained in the side of film or be stream across film to the other side.SEVs and OMVs is respectively 30- The non-uniform nanoscaled vesicle of 150nm and 10-300nm, if chromatography membrane aperture be greater than sEVs the or OMVs upper limit, make sEVs or OMVs is flowed across chromatographic film, then sEVs or OMVs then can completely extend across film under the driving of Fluid pressure, in wearing membrane process, SEVs or OMVs all will be largely adsorbed in conjunction with film inner duct inner surface positive charge group, and separation sEVs or OMVs is then It can only be using in conjunction with elution mode；And combine in elution mode, inevitably, elution process is needed with high salt, and with high salt Vesica can be destroyed, bioactivity is influenced.If chromatographing membrane aperture is less than sEVs or OMVs lower limit, either slipstream or dead End flowing, sEVs or OMVs will be unable to directly flow through chromatographic film and become directly collecting, but are trapped in separately by chromatographic film Side.

It is existing slow for excretion body and the separation method of outer membrane vesicles, separating rate: conventional differential centrifugation The sample of one 30ml needs at least to separate 2-3 hours, and the density gradient centrifugation time is longer；And ultracentrifugation equipment price is high Expensive, general value is in 300,000-100 ten thousand RMB；The method that the present invention uses separates, same to measure, most fast only to need not By 5 minutes, and equipment requirement was relatively much lower.Again without FPLC in the case where common plunger pump or peristaltic pump can be used. Usually hypervelocity separates general 6 samples of first separation, and single volume is in 10-40ml, can not be again although reaching as high as 250ml Continue to expand, and needs to be separately configured expensive centrifugal rotor.

The current separation and Extraction mode for excretion body or outer membrane vesicles, including molecular sieve splitter, but which There is also fractional dose is smaller, the lower problem of lock out operation efficiency.

In the field (in basic research and application market) of current entire EVs purifying, existing problem is: purifying it is pure The problem of life cycle costing of degree, purification process period and scale and technique.

The present invention adopts with the aforedescribed process, can be reached higher treating capacity with lower cost, be reached and improve processing Efficiency.

The application uses two chromatography column in series, which has the filler for meeting aperture and surface charge, press The method of more solito is loaded, and meets corresponding pillar height, in use, using corresponding flow velocity.Using two layers of chromatographic column, effect is, First chromatographic column does preliminary purification, and second chromatographic column does middle effect/polishing purification, it is therefore an objective to obtain can be used for the outer of clinic Body is secreted, it is sufficiently pure.

And our method only flows through granular filler since EVs is greater than filler aperture when being that EVs flows through column chromatographic stuffing Between gap reach the big impurity of removal under higher flow velocity only in conjunction with the charged group of the first column granular filler outer surface Outside, EVs loss is reduced.

The filler of column one is that the load capacity of ion-exchange packing impurity is 10 times of the second column packing.And two filler of column is built The pillar height of view is at least 10cm, and the performance of removal supramolecular impurity is not only increased by increasing concatenated column one, while It compensates for and shortens performance degradation brought by column two.

Compared with prior art, the present invention beneficial effect is: the present invention can be quickly to extracellular vesica (such as a variety of body fluid And the outer membrane vesicles in the excretion body or microbial culture medium of cultured cell in vitro) isolated and purified.The lock out operation Simply, single use is at low cost, and separating sample purity is high, sample bioactivity keeps good, can be found in biomarker, It is applied in the basic research such as biological therapy and diagnosis and biotherapeutics production.

Detailed description of the invention

Fig. 1 be the embodiment of the present invention 1 separating mouse blood plasma source excretion body sample marker CD63, CD9 and The WB testing result figure of TSG101；

Fig. 2 be the embodiment of the present invention 1 separating mouse blood plasma source excretion body sample marker CD63, CD9 and The transmission electron microscope aspect graph of TSG101；

Fig. 3 be the embodiment of the present invention 1 separating mouse blood plasma source excretion body sample marker CD63, CD9 and The nano particle trace detection grain size distribution of TSG101；

Fig. 4 embodiment of the present invention 2 separates the WB inspection of human urine source excretion body sample marker CD63, TSG101 and CD9 Result figure is surveyed,

The transmission of the separation of Fig. 5 embodiment of the present invention 2 human urine source excretion body sample marker CD63, TSG101 and CD9 Electron microscopic morphology figure；

The nanometer of the separation of Fig. 6 embodiment of the present invention 2 human urine source excretion body sample marker CD63, TSG101 and CD9 Particle trace detection grain size distribution；

3 separating Escherichia coli culture supernatant source OMV sample transmission electron microscope aspect graph of Fig. 7 embodiment of the present invention；

The nano particle trace detection partial size of 3 separating Escherichia coli culture supernatant source OMV sample of Fig. 8 embodiment of the present invention Distribution map；

The separation of Fig. 9 embodiment of the present invention 4 293T cells and supernatant source excretion body sample marker CD63, CD9, The WB testing result figure of TSG101, ANXA2 and Syntenin and intracellular members protein markers GM130 and Alix,

The separation of Figure 10 embodiment of the present invention 4 293T cells and supernatant source excretion body sample marker CD63, CD9, The transmission electron microscope aspect graph of TSG101, ANXA2 and Syntenin and intracellular members protein markers GM130 and Alix,

The separation of Figure 11 embodiment of the present invention 4 293T cells and supernatant source excretion body sample marker CD63, CD9, The nano particle trace detection partial size of TSG101, ANXA2 and Syntenin and intracellular members protein markers GM130 and Alix Distribution map；

Figure 12 is the result schematic diagram of kit used by method of the invention.

In Figure 12,1 is the first chromatographic column, 2 second chromatographic columns, 3 standard Luer interfaces.

Specific embodiment

Below by specific embodiment the technical scheme of the present invention will be further described explanation.

If raw material employed in the embodiment of the present invention is raw material commonly used in the art without specified otherwise, implement Method employed in example, is the conventional method of this field.

As shown in figure 12, it is extraction element used in the extracting method of the invention i.e. structural schematic diagram of kit.

As shown, the kit is composed in series by the first chromatographic column 1 and the second chromatographic column 2, wherein the first chromatographic column is extremely Few one, the second chromatographic column at least one is connected using standard Luer interface 3 between chromatographic column.

Top connection and lower contact are set on the first chromatographic column 1 and the second chromatographic column 2；

Top connection: one is public female Luer, and one is M6 external screw thread or internal screw thread；

Lower contact: one is female Luer interface, and one is 1/16 external screw thread or internal screw thread.

Application method:

1, by the first chromatographic column and the second chromatographic column, female Luer is sequentially passed through from top to bottom and is connected to become EV purification column, Pay attention to the air at discharge interface；

2、

When A. using the FPLC equipment such as AKTA, by EV purification column upper inlet and lower outlet respectively by accessory (i.e. adapter, As 1/16 inch of external screw thread turns Rule pin end, 3/16 internal screw thread turns Rule female connector) it is connect with FPLC, notice that interface is airtight Property and air discharge；

When B. using peristaltic pump or plunger pump, EV purification column upper inlet is sequentially passed through into accessory and pump discharge pipeline connects It connects, accessory such as pagoda head-Rule revolution connector, internal diameter pagoda head-Rule mother's adapter；Pump intake pipeline is passed through into accessory 1 (two Position threeway hand-operated conversion adapter) it is connect respectively with loading container and column buffer liquid container.

Smooth flow path as formed above empties in flow path after air, can be initially separated EV.

Embodiment 1:

A kind of application method of the excretion body purifying in mouse blood source, steps are as follows (concrete outcome combination Fig. 1-Fig. 3):

(1) purifying of excretion body is prepared with liquid

A. equilibration buffer (PBS, pH7.2) is prepared, 0.025 μm of mixed cellulose ester (MCE) vacuum filtration, balance to 22 ± 2 DEG C spare；

B. washing buffer (2M NaCl, 1M NaOH, 30% isopropanol) is prepared, 0.025 μm of mixed cellulose ester (MCE) Vacuum filtration, balance to 22 ± 2 DEG C it is spare；

d.ddH₂O 0.025 μm of mixed cellulose ester (MCE) vacuum filtration, balance to 22 ± 2 DEG C it is spare；

E. prepare and save 20% ethyl alcohol of liquid, 0.1 μm of mixed cellulose ester (MCE) vacuum filtration, balance to 22 ± 2 DEG C it is standby With；

(2) prepared by mice plasma

A. under aseptic condition, using the anticoagulant vacuum blood collection tube of 3.2% sodium citrate of 3ml specification, from SPF grades of 8 week old male Kunming mice acquires blood, amounts to about 21ml；

B. it after blood collection, softly mixes well at once, it is temporary less than 1 hour that anticoagulation is placed in 22 ± 2 DEG C of environment；

C. in blood collection 1 hour, 4 DEG C of centrifugation 15min of 3000rpm acquire about 12ml supernatant blood plasma.

(3) mice plasma sample excretion body purifies pre-treatment

A. after confirmatory sample is without haemolysis, 0.45 μm of PES membrane micropore filter filtered plasma sample collects about 11ml filtering Blood plasma；

B. the Tri-HCl being pre-chilled using ice bath, pH8.0 buffer 1:2.5 dilute 10ml filtered plasma；

C.0.22 μm PES membrane micropore filter filters diluting plasma sample, collects about 24ml and filters diluting plasma, dilution Blood plasma balances after ten minutes in 4 DEG C of chromatography cabinets, purifies for downstream excretion body.

(4) excretion body in mice plasma source purifies

A. splitter A and splitter B is directly connected up and down by female Luer；

B. concatenated splitter column is connected in the chromatography cabinet for being placed in 4 DEG C by upper adapter and lower adapter AKTA pure(FPLC)；

C. start FPLC, backpressure valve is kept to close, it is anti-to 500cm/h flow velocity to be stepped up from 40cm/h using ddH2O To chromatographic column is flowed through, flow path ne-leakage is tested and confirmed, until UV curve and stable drain to display at baseline of conductance plots are Air in uniting；

D. splitter is rinsed with 500cm/h linear flow rate forward direction using ddH2O until UV curve and conductance plots are stablized extremely At baseline；

E. Tri-HCl, pH8.0 is used to rinse splitter until UV curve and conductance are bent with 100cm/h linear flow rate forward direction Line is stablized；

F. it is pumped by A and the diluted mice plasma 10.5ml of loading is started with 100cm/h linear flow rate through A1 access, and received Collection flows through liquid, and flowing through liquid is mice plasma source excretion body after purification；

G. take 10ml excretion body sample that 15ml 10KD super filter tube (Millipore) is added, 5000g room temperature is centrifuged 20 minutes；

H. excretion body concentrate about 1ml is collected, can be used to the identification detection of excretion body.

I. divided using regenerated liquid (2M NaCl, 1MNaCl, 30% isopropanol) with 40cm/h flow velocity back flush splitter 5 Clock stands 15min, then reversed 40cm/h flow velocity back flush splitter 10 minutes；

J. ddH is used₂O rinses splitter until UV curve and conductance plots are stablized extremely with 500cm/h linear flow rate forward direction At baseline；

K. e-h step is repeated, excretion body concentrate is collected and amounts to about 2ml, is frozen and direct downstream point for -20 degrees Celsius Analysis and pattern detection.

L. the splitter not used, using regenerated liquid (2M NaCl, 1MNaOH, 30% isopropanol) with 40cm/h flow velocity Back flush splitter 5 minutes, stand 15min, then reversed 40cm/h flow velocity back flush splitter 10 minutes；

M. ddH is used₂O rinses splitter until UV curve and conductance plots are stablized extremely with 500cm/h linear flow rate forward direction At baseline；

N. splitter is rinsed with 100cm/h linear flow rate forward direction using 20% ethyl alcohol until UV curve and conductance plots are stablized To baseline；

O. splitter is removed from FPLC, and splitter upper and lower opening is sealed using former plug, be placed in 4 degree of preservations.

(5) excretion body in mice plasma source is identified

A. transmission electron microscope detects isolated mice plasma excretion volume morphing

A. 255 μ l excretion body samples are drawn into 1.5ml EP pipe, 255 μ l are added dropwise in concussion vortex mixer concussion EP pipe It has balanced to 8% paraformaldehyde solution of room temperature；

B. mixing sample, 12000rpm room temperature brief centrifugation 5s are stored at room temperature and are incubated for 10min；

C. 500 μ l samples are drawn to 0.5ml 10KD super filter tube (Millipore), 14000g, 4 DEG C of centrifugation 25min are discarded Filter liquor；

D. 450 μ l ddH are drawn₂O to 0.5ml 10KD ultrafiltration membrane, 14000g, 4 DEG C of centrifugation 25min discard filter liquor；e. It is primary to repeat step d；

F. filter membrane component in super filter tube is removed, anti-plug enters in new mating EP pipe, 4 DEG C of centrifugation 2min of 2000g, collects dense Contracting excretion body, about 50 μ l；

G. from Electronic Speculum copper mesh box, go out 200 mesh with self-locking elbow tweezer using Electronic Speculum and cover carbon film Electronic Speculum copper mesh, by carbon film one On lateral, tweezers side is turned to be placed horizontally on experimental bench white qualitative filter paper；

H. 10 μ l concentration excretion body sample is drawn, is added dropwise on copper mesh carbon film, is stored at room temperature 1min；

I. straight peen point tweezer takes the quantitative filter paper at a slow speed cut out as fritter, vertical to clamp, and blots copper from tweezers aid position Net liquid；

J. 10 μ l ddH are drawn₂O sucks (such as step i) immediately after being softly added dropwise on copper mesh carbon film；

K. 10 μ l, 1% uranium acetate dye liquor is drawn, 200 mesh Electronic Speculum copper mesh carbon film sides is softly added dropwise to, is stored at room temperature 1min draws rapidly dye liquor；

L. copper mesh is statically placed under infrared lamp, distance about 20cm, copper mesh irradiates dry 5min；

M. it repeats step g-l twice, makes 2, parallel Electronic Speculum sample；

N. next day observes and records the mice plasma source of separation using transmission electron microscope (FEI Tecnai G2Spirit) Excretion body sample.

B. laser nano tracking skill detects isolated mice plasma source excretion body particle diameter distribution

A. 750 μ l excretion body concentrates are drawn into 4ml EP pipe, 750 μ l are added dropwise in concussion vortex mixer concussion EP pipe It has balanced to 8% paraformaldehyde solution of room temperature；

B. mixing sample EP pipe 12000rpm room temperature brief centrifugation 10s is stored at room temperature and is incubated for 10min；

C. (about 2 drops/sec) 25ml is slowly injected to 5ml HiTrap Sephadex desalting column using 50ml syringe DdH2O is to carry out column equilibration；

D. 2ml syringe is used, 1.5ml concentration excretion body sample is injected to desalting column, collects about 2ml efflux；

E. efflux is the fixation excretion body being in ddH2O.

F. sample is respectively 1:2,1:5,1:10,1:20 times, upper machine examination using 2 times of gradient dilution excretion body samples of ddH2O It surveys (Particle Metrix), taking optimum dilution degree value is testing result.

C. excretion body associated protein immune-blotting method

I sample preparation

A. 500 μ l samples are drawn to 0.5ml 10KD super filter tube (Millipore), 14000g, 4 DEG C of centrifugation 25min are discarded Filter liquor；

B. step a is repeated once for the further about 80 μ l of concentration value total volume of whole excretion body concentrates；

C. 20 μ l 5X loading buffer (Thermo) are added into excretion body sample, concussion mixes；

D. sample 95 DEG C of heating 5min in hot lid metal bath, are cooled to room temperature spare；

E. cell scraper scraping routine culture degree of collecting about 60%HeLa cell is pre- with 2ml ice bath as detection positive sample Cell is resuspended in cold PBS and equivalent is transferred in two 1.5ml EP pipes；

F. 4 DEG C of centrifugation 10min of cell sample 200g abandon supernatant to the greatest extent；

G. it is separately added into 300 μ l pre-cooling RIPA (containing protease inhibitors cocktail) into the precipitating of each EP pipe, fills Divide piping and druming cell precipitation；

4 DEG C of blending incubation 30min of h.EP pipe are incorporated in 1 1.5ml EP pipe；

I. broken 2-3 second of sample ice-bath ultrasonic, with cut off release gDNA and further broken inabundant smudge cells and Cell fragment；

J. 4 DEG C of centrifugation 30min of cell sample 12000rpm, honest and upright and thrifty 600 μ l in reservation；

K. 400 μ l cell lysate supernatants are drawn, 100 μ l 5X loading buffer (Thermo) are added, concussion is mixed It is even；

L. sample 95 DEG C of heating 5min in hot lid metal bath, are cooled to room temperature spare；

II immune-blotting method

A.HeLa cell pyrolysis liquid is positive sample (PC) loading and excretion body sample isometric loading 20 μ l, 15%SDS- PAGE (CD63 CD9) 80V electrophoresis 30min, 10%SDS-PAGE (TSG101) 80V electrophoresis 30min；120V electrophoresis is to buffer Dyestuff is away from running gel lower edge about 0.5cm；

B.0.22 μm pvdf membrane (PALL) 90V wet process transferring film；

C.5% skimmed milk power/TBST room temperature shaker is incubated for 1hr；

D.3%BSA/TBST 3 antibody of anti-CD 6 (Abcam, 1:2000), CD9 (Abcam, 1:2000), TSG101 are diluted (Abcam, 1:2000), 4 DEG C of concussions are incubated overnight；

E. 3 antibody of anti-CD 6 uses anti-mouse secondary antibody (Jackson Immunoresearch), and anti-CD9 antibody, anti-TSG101 are anti- Body (Jackson Immunoresearch) is incubated for 1 hour using the 37 DEG C of concussions of anti-rabbit secondary antibody；

F. extemporaneous preparation ECL (Millopore) working solution, pvdf membrane transfer side and are protected from light with ECL working solution contact room temperature It is incubated for 1min altogether, blotting paper exhausts surplus liquid；

G. excretion body marker protein level (Fujifilm) in chemiluminescence detection sample.

Embodiment 2:

A kind of application method of the excretion body purifying in human urine source, steps are as follows (concrete outcome combination Fig. 4-Fig. 6):

(1) purifying of excretion body is prepared with liquid

A. equilibration buffer (PBS, pH6.0) is prepared, 0.025 μm of mixed cellulose ester (MCE) vacuum filtration, balance to 22 ± 2 DEG C spare；

D.ddH2O 0.025 μm of mixed cellulose ester (MCE) vacuum filtration, balance to 22 ± 2 DEG C it is spare；

(2) people middle section urina sanguinis acquires

A. under normal condition, middle section urina sanguinis about 100ml is acquired using urine cup with cover；

B. in the 2-3min after acquiring, urine is poured into 15 minutes superclean benches of ultraviolet irradiation and is previously added In the sterilizing glass beaker of 2% thimerosal of 1ml, while being separately added into 2 protease inhibitors and 2 inhibitors of phosphatases Cocotail (Roche), is quickly stirred as on magnetic stirring apparatus, sufficiently to dissolve；

C. it mixes urine specimen and is immediately available for the separation of downstream excretion body.

(3) human urine sample excretion body is clarified before purification

A. urine specimen is loaded on respectively in 2 50ml sterile centrifugation tubes, and 200g is centrifuged 10min at 4 DEG C, retains supernatant；

B. urine supernatant is transferred to 2 new 50ml sterile centrifugation tubes, and 2000g is centrifuged 15min at 4 DEG C, retains supernatant simultaneously It is transferred to new 50ml sterile centrifugation tube, destruction precipitating is absolutely avoided；

C. urine supernatant, 10000g is centrifuged 15min at 4 DEG C, retains about 85ml supernatant and is simultaneously transferred to the 100ml to sterilize in advance In beaker；

D. it is separately added into 8ml 300mg/ml dithiothreitol (DTT) (DTT) into centrifuge tube, 37 DEG C of incubation 10min, ice bath is cold But；

F. urine supernatant precipitating re-suspension liquid is mixed, is filtered, is collected about using 0.22 μm of PES membrane micropore filter 80ml clarifies urine.

The sample can be used to the purifying of excretion body.

(4) excretion body in human urine source purifies

C. start FPLC, backpressure valve is kept to close, it is anti-to 500cm/h flow velocity to be stepped up from 40cm/h using ddH2O To chromatographic column is flowed through, tests and confirm flow path ne-leakage；

E. phosphate buffer is used, pH6.0 rinses splitter until UV curve and electricity with 500cm/h linear flow rate forward direction Lead curve is stablized；

F. it is pumped by A and loading 80ml clarification urine specimen is started with 500cm/h linear flow rate through A1 access, and collected and flow through Liquid, flowing through liquid is that human urine source excretion body, total collect about collect about 85 milliliters after purification；

G. 450ml PBS, pH7.4 is used to be connected to the 150ml column of plunger pump with 300cm/hr linear flow rate balance in advance Volume sphedex G-25medium desalting column；

H. 50 milliliters of purifying excretion bodies are drawn, are driven by plunger pump, with 300cm/hr linear flow rate through desalting column desalination, are received Collect efflux about 65ml, room temperature is temporary；

I. about 300ml PBS is used, ph7.4 continuation is driven by plunger pump with 300cm/hr linear flow rate cleaning, desalting column, Abandon efflux；

J. sucked away about 35ml purifies excretion body, is driven by plunger pump, de- through desalting column with 300cm/hr linear flow rate Salt collects efflux；

K. after sample loading is complete, using about 340ml PBS, about 65ml, ph7.4 continuation is driven by plunger pump with 300cm/ Hr linear flow rate flows through desalting column, collects about 65ml outflow altogether, efflux abandons it thereafter；

L. merge desalination excretion body sample twice, amount to about 130ml；

M. excretion body sample is concentrated by ultrafiltration extremely with 70ml/min flow velocity by peristaltic pump using 10KD ultrafiltration membrane packet (PALL) About 8ml

N. 15ml 10KD super filter tube (Millipore) is added, 4 DEG C of centrifugation 30min excretion bodies of 5000g are concentrated into about 500ul is identified for excretion body；

O. excretion body concentrate about 1ml is collected, is frozen after packing in -204 DEG C for the identification detection of subsequent excretion body.

P. divided using regenerated liquid (2M NaCl, 1MNaCl, 30% isopropanol) with 40cm/h flow velocity back flush splitter 5 Clock stands 15min, then reversed 40cm/h flow velocity back flush splitter 10 minutes；

Q. splitter is rinsed with 500cm/h linear flow rate forward direction using ddH2O until UV curve and conductance plots are stablized extremely At baseline；

R. the splitter not used, using regenerated liquid (2M NaCl, 1MNaCl, 30% isopropanol) with 40cm/h flow velocity Back flush splitter 5 minutes, stand 15min, then reversed 40cm/h flow velocity back flush splitter 10 minutes；

S. splitter is rinsed with 500cm/h linear flow rate forward direction using ddH2O until UV curve and conductance plots are stablized extremely At baseline；

T. splitter is rinsed with 100cm/h linear flow rate forward direction using 20% ethyl alcohol until UV curve and conductance plots are stablized To baseline；

U. splitter is removed from FPLC, and splitter upper and lower opening is sealed using former plug, be placed in 4 degree of preservations.

(5) excretion body in human urine source is identified

A. transmission electron microscope detects isolated human urine excretion volume morphing

A. 100 μ l excretion body samples are drawn into 1.5ml EP pipe, 100 μ l are added dropwise in concussion vortex mixer concussion EP pipe It has balanced to 8% paraformaldehyde solution of room temperature；

C. 200 μ l samples are drawn to 0.5ml 10KD super filter tube (Millipore), ddH2O supplies 500 μ l, 14000g, 4 DEG C centrifugation 20min, discard filter liquor；

D. filter membrane component in super filter tube is split out, anti-plug enters in new mating EP pipe, and 2000g room temperature is centrifuged 2min, collects dense Contracting excretion body, about 50 μ l；

E. from Electronic Speculum copper mesh box, go out 200 mesh with self-locking elbow tweezer using Electronic Speculum and cover carbon film Electronic Speculum copper mesh, by carbon film one On lateral, tweezers side is turned to be placed horizontally on experimental bench white qualitative filter paper；

F. 10 μ l concentration excretion body sample is drawn, is added dropwise on copper mesh carbon film, is stored at room temperature 1min；

G. straight peen point tweezer takes the quantitative filter paper at a slow speed cut out as fritter, vertical to clamp, and blots copper from tweezers aid position Net liquid；

H. 10 μ l ddH2O are drawn, suck (such as step i) after being softly added dropwise on copper mesh carbon film immediately；

I. 10 μ l, 1% uranium acetate dye liquor is drawn, 200 mesh Electronic Speculum copper mesh carbon film sides is softly added dropwise to, is stored at room temperature 1min draws rapidly dye liquor；

J. copper mesh is statically placed under infrared lamp, distance about 20cm, copper mesh irradiates dry 5min；

K. it repeats step g-l twice, makes 2, parallel Electronic Speculum sample；

L. the same day observes and records the human urine source excretion of separation using transmission electron microscope (FEI Tecnai G2Spirit) Body sample.

B. laser nano tracking skill detects isolated human urine source excretion body particle diameter distribution

A. 150 μ l excretion body concentrates are drawn into 1.5ml EP pipe, 150 μ l are added and have balanced to 8% poly of room temperature Formalin, piping and druming mixes rapidly；

B. the 1.5ml EP pipe equipped with mixing sample is stored at room temperature incubation in 12000rpm room temperature brief centrifugation 10s 10min；

C. sample is respectively 1:10,1:20,1:40 times, upper machine testing using 2 times of gradient dilution excretion body samples of ddH2O (Particle Metrix), taking optimum dilution degree value is testing result.

C. excretion body associated protein immune-blotting method

I sample preparation

A. 250 μ l samples are drawn to 0.5ml 10KD super filter tube (Millipore), ddH2O complements to 500 μ l, 14000g 4 DEG C of centrifugation 25min, discard filter liquor；

B. filter membrane component in super filter tube is split out, anti-plug enters in new mating EP pipe, 4 DEG C of centrifugation 2min of 2000g, collects dense Contracting excretion body, about 50 μ l；

C. 13 μ l 5X loading buffer (Thermo) are added into excretion body sample, concussion mixes；

II immune-blotting method

A.HeLa cell pyrolysis liquid is positive sample (PC) loading 15ul and excretion body sample loading 20 μ l, 15%SDS- PAGE (CD63 CD9) 80V electrophoresis 30min, 10%SDS-PAGE

(TSG101) 80V electrophoresis 30min；120V electrophoresis is to buffer dyestuff away from running gel lower edge about 0.5cm；

B.0.22 μm pvdf membrane (PALL) 90V wet process transferring film；

C.5% skimmed milk power/TBST room temperature shaker is incubated for 1hr；

Embodiment 3:

A kind of application method of Escherichia coli outer membrane vesicles purifying, (concrete outcome combination Fig. 7-Fig. 8) specific as follows:

(1) purifying of Escherichia coli outer membrane vesicles is prepared with liquid

A. equilibration buffer (Tris-HCl, pH8) is prepared, 0.025 μm of mixed cellulose ester (MCE) vacuum filtration, balance It is spare to 22 ± 2 DEG C；

B. washing buffer (2M NaCl, 0.5M NaOH, 30% isopropanol) is prepared, 0.025 μm of mixed cellulose ester (MCE) be filtered by vacuum, balance to 22 ± 2 DEG C it is spare；

(2) E. coli culture supernatant acquires

A. a small amount of Liquid nitrogen storage bacterial strain Escherichia coli TOP10 (the ammonia benzyl of the bamboo toothpick picking of high pressure steam sterilization Penicillin resistance) it is placed in 2ml ammonia benzyl resistance LB culture medium；

B. bacterium solution cultivates about 8hr in 37 DEG C of shaking table 200rpm；

C.1:1000 for inoculation bacterium solution into 220ml ammonia benzyl resistance LB culture medium (1L conical flask), 300rpm cultivates about 15hr；

D. bacterium solution is collected, ice bath is spare to can be used for the clarification of downstream sample.

(3) E. coli culture supernatant sample outer membrane vesicles purify pre-treatment

A. bacterium solution is adjusted to pH8.0；

B. bacterium solution is centrifuged 20min, honest and upright and thrifty 200ml in reservation in 6000g room temperature；

C. 4000U Benzonase (Merk) and final concentration 2mM MgCl2,37 DEG C of incubation 30min are added in supernatant of bacteria solution, Ice bath is cooling afterwards；

D. supernatant of bacteria solution is using vacuum pump successively through 5 μm of single layer, 0.45 μm, 0.22 μm of PES filter membrane filtered off with suction；

The sample can be used to the purifying of Escherichia coli outer membrane vesicles.

(4) purifying of E. coli culture supernatant source outer membrane vesicles and concentration

E. phosphate buffer is used, pH8.0 rinses splitter until UV curve and electricity with 500cm/h linear flow rate forward direction Lead curve is stablized；

F. it is pumped by A and loading 200ml clarification E. coli culture supernatant sample is started with 500cm/h linear flow rate through A1 access This, and collect and flow through liquid, flowing through liquid is E. coli culture supernatant source outer membrane vesicles after purification, collects about 200ml altogether；

G. excretion body sample is concentrated by ultrafiltration extremely with 70ml/min flow velocity by peristaltic pump using 10KD ultrafiltration membrane packet (PALL) About 12ml

H. 15ml 10KD super filter tube (Millipore) is added, 4 DEG C of centrifugation 30min excretion bodies of 5000g are concentrated into about 0.5ml is identified for excretion body；

O. excretion body concentrate 0.5ml is collected, is frozen after packing in -20 DEG C for the identification detection of subsequent excretion body.

(5) E. coli culture supernatant source outer membrane vesicles are identified

A. transmission electron microscope detects isolated E. coli culture supernatant outer membrane vesicles form

A. 100 μ l outer membrane vesicles samples are drawn into 1.5ml EP pipe, 110 μ are added dropwise in concussion vortex mixer concussion EP pipe L has been balanced to 8% paraformaldehyde solution of room temperature；

D. filter membrane component in super filter tube is split out, anti-plug enters in new mating EP pipe, and 2000g room temperature is centrifuged 2min, collects dense Contracting outer membrane vesicles, about 65 μ l；

F. 10 μ l concentration outer membrane vesicles sample is drawn, is added dropwise on copper mesh carbon film, is stored at room temperature 1min；

K. it repeats step g-l twice, makes 2, parallel Electronic Speculum sample；

L. the same day is observed and recorded in the Escherichia coli culture of separation using transmission electron microscope (FEI Tecnai G2Spirit) Clear source outer membrane vesicles sample.

B. laser nano tracking skill detects isolated E. coli culture supernatant source outer membrane vesicles particle diameter distribution

A. 350 μ l outer membrane vesicles concentrates are drawn into 1.5ml EP pipe, concussion EP pipe is added dropwise 350 μ l and has balanced To 8% paraformaldehyde solution of room temperature；

C. sample is respectively 1:5,1:10,1:20,1:40 times using 2 times of gradient dilution outer membrane vesicles samples of ddH2O, on Machine testing (Particle Metrix), taking optimum dilution degree value is testing result.

Embodiment 4:

A kind of application method of 293T cell excretion body purifying, steps are as follows (concrete outcome combination Fig. 9-Figure 11):

(1) 293T cell excretion body purifying is prepared with liquid

(2) 293T cells and supernatant acquires

I excretion body removes FBS concentration screening

A.DMEM culture medium be separately added into FBS (GIBCO) and excretion body removal FBS (GIBCO) to final concentration of 10%, 5%, 2.5%, 1.25% and 0.63%, it is spare；

B. 2000 every hole 293T cells to 96 orifice plates are inoculated with, amounting to 60 parallel holes, (0.1ml is only added in the outermost collar aperture of culture plate Sterile PBS)；

C. 24 hours after cell inoculation, the various concentration blood serum medium prepared in a, every group of 6 parallel holes are changed to；

D. r and 48hr uses CellTiter-Glo (Promega) detection cell living respectively for 24 hours after cell replacement culture medium Power, more each concentration level and culture duration FBS and go excretion body FBS culture cell viability and conventional culture conditions under cell live The difference of power；

E. 48 hours culture durations are selected according to cell viability result, minimum nontoxicity goes excretion body FBS concentration 2.5% to make Culture medium, which is prepared, for excretion body goes excretion body FBS condition.

The acquisition of II cells and supernatant

A. recovery freezes 293T cell (2,000,000 /), Trypan Blue, and cell counting board counts, and shows cell survival rate 90%；

B. 10ml DEME culture medium, 37 DEG C, 5%CO2 routine culture is added in 10cm culture dish in inoculation 293T cell；

C. every 2-3 days, when cell degree of collecting is up to 80%, cell 1:4-6 was passed on, every ware 10ml culture medium；

D. preparing total 12 ware 10cm Tissue Culture Dish, 293T cell degree of collecting about 30%, PBS is softly rinsed cell 2 times, Every ware is changed to 10ml 2.5% and removes excretion body FBS-DMEM, 37 DEG C, 5%CO2 routine culture；

E. after cell culture 48 hours, all about 110ml 293T cells and supernatants are collected, it is spare.

(3) 293T cells and supernatant sample excretion body is clarified before purification

A.293T cells and supernatant is centrifuged 10min, honest and upright and thrifty 105ml in reservation in room temperature 200g；

B. cells and supernatant is centrifuged 15min, honest and upright and thrifty 100ml in reservation in room temperature 2000g；

C. the MgCl2 of 2000U Benzonase (Merk) and final concentration 2mM, 37 DEG C of incubations are added in cells and supernatant 20min ice bath is cooling；

D. cells and supernatant uses vacuum pump filtered off with suction, and filter membrane diameter is that 50mm is round, aperture is 5 μm, 0.45 μm, Successively single overlays are fixed in detachable filter 0.22 μm of PES filter membrane from top to bottom；

E. it adjusts sample pH value and collects about 100ml cells and supernatant sample altogether to pH8.0.

Sample can be used to the purifying of excretion body.

(4) 293T cells and supernatant source excretion body purifies

E. use TrisHCl buffer, pH8.0 with 500cm/h linear flow rate forward direction rinse splitter until UV curve and Conductance plots are stablized；

F. it is pumped by A and loading 100ml clarified cell culture supernatant sample is started with 350cm/h linear flow rate through A1 access, And collect and flow through liquid, flowing through liquid is 293T cells and supernatant source excretion body after purification, collects about 95 milliliters altogether；

J. sucked away about 45ml purifies excretion body, and PBS supplies 50ml, driven by plunger pump, with 300cm/hr linear flow Speed collects efflux through desalting column desalination；

K. merge desalination excretion body sample twice, amount to about 130ml；

L. excretion body sample is concentrated by ultrafiltration extremely with 70ml/min flow velocity by peristaltic pump using 10KD ultrafiltration membrane packet (PALL) About 10ml；

M. 15ml 10KD super filter tube (Millipore) is added, PBS supplies 12ml, 4 DEG C of centrifugation 30min of 5000g, excretion Body is concentrated into about 500ul, identifies for excretion body；

N. excretion body concentrate about 0.5ml is collected, is frozen after packing in -204 DEG C for the identification detection of subsequent excretion body.

O. divided using regenerated liquid (2M NaCl, 1MNaOH, 30% isopropanol) with 40cm/h flow velocity back flush splitter 5 Clock stands 15min, then reversed 40cm/h flow velocity back flush splitter 10 minutes；

P. splitter is rinsed with 500cm/h linear flow rate forward direction using ddH2O until UV curve and conductance plots are stablized extremely At baseline；

Q. the splitter not used, using regenerated liquid (2M NaCl, 1MNaOH, 30% isopropanol) with 40cm/h flow velocity Back flush splitter 5 minutes, stand 15min, then reversed 40cm/h flow velocity back flush splitter 10 minutes；

R. splitter is rinsed with 500cm/h linear flow rate forward direction using ddH2O until UV curve and conductance plots are stablized extremely At baseline；

S. splitter is rinsed with 100cm/h linear flow rate forward direction using 20% ethyl alcohol until UV curve and conductance plots are stablized To baseline；

T. splitter is removed from FPLC, and splitter upper and lower opening is sealed using former plug, be placed in 4 degree of preservations.

(5) 293T cells and supernatant source excretion body is identified

A. transmission electron microscope detects isolated 293T cells and supernatant excretion volume morphing

A. 100 μ l excretion body samples are drawn into 1.5ml EP pipe, 200 μ l are added dropwise in concussion vortex mixer concussion EP pipe It has balanced to 8% paraformaldehyde solution of room temperature；

C. 300 μ l samples are drawn to 0.5ml 10KD super filter tube (Millipore), ddH2O supplies 500 μ l, 14000g, 4 DEG C centrifugation 25min, discard filter liquor；

H. 10 μ l, 1% uranium acetate dye liquor is drawn, 200 mesh Electronic Speculum copper mesh carbon film sides is softly added dropwise to, is stored at room temperature 1min draws rapidly dye liquor；

I. copper mesh is placed in situ in copper mesh box, is waited to be detected；

J. it repeats step e-i twice, makes 2, parallel Electronic Speculum sample；

K. the same day is observed and recorded in the 293T cell culture of separation using transmission electron microscope (FEI Tecnai G2Spirit) Clear source excretion body sample.

B. laser nano tracking skill detects isolated 293T cells and supernatant source excretion body particle diameter distribution

A. 100 μ l excretion body concentrates are drawn into 1.5ml EP pipe, 100 μ l are added and have balanced to 8% poly of room temperature Formalin, piping and druming mixes rapidly；

C. excretion body associated protein immune-blotting method

I sample preparation

A. 300 μ l samples are drawn to 0.5ml 10KD super filter tube (Millipore), ddH2O complements to 500 μ l, 14000g 4 DEG C of centrifugation 15min, discard filter liquor；

B. filter membrane component in super filter tube is split out, anti-plug enters in new mating EP pipe, 4 DEG C of centrifugation 2min of 2000g, collects dense Contracting excretion body, about 100 μ l；

C. 25 μ l 5X loading buffer (Thermo) are added into excretion body sample, concussion mixes；

II immune-blotting method

A.HeLa cell pyrolysis liquid is positive sample (PC) loading 15ul and excretion body sample loading 20 μ l, 15%SDS- PAGE (CD63 CD9) 80V electrophoresis 30min, 10%SDS-PAGE (TSG101) 80V electrophoresis 30min；120V electrophoresis is to buffer Dyestuff is away from running gel lower edge about 0.5cm；

B.0.1 μm pvdf membrane (PALL) 90V wet process transferring film；

C.5% skimmed milk power/TBST room temperature shaker is incubated for 1hr；

D.3%BSA/TBST 3 antibody of anti-CD 6 (Abcam, 1:2000), CD9 (Abcam, 1:2000), ANNEXIN are diluted (Abcam, 1:3000), 4 DEG C of Syntenin (Abcam, 1:4000), GM130 (Abcam, 1:2500), Alix (1:5000) shaking tables It is incubated overnight；

E. 3 antibody of anti-CD 6 and anti-Alix antibody use anti-mouse secondary antibody (Jackson Immuno-research), and anti-CD9 is anti- Body, anti-Syntenin antibody, uses anti-rabbit secondary antibody (Jackson Immunoresearch) room temperature shaker at anti-ANNEXIN antibody Concussion is incubated for 2 hours；

F. Fresh ECL (Millopore) working solution, pvdf membrane transfer side and are protected from light with ECL working solution contact room temperature It is incubated for 1-5min altogether, blotting paper exhausts surplus liquid；

Excretion body product purity parameter of the invention, specifically see the table below:

Sample	Mice plasma	Human urine	293T culture supernatant	E.Coli culture medium
					Albumen removal rate	95.4%	98.6?	97.7%	98.1%

The above is only a better embodiment of the present invention and the applied technical principle.It will be appreciated by those skilled in the art that this hair It is bright to be not limited to specific embodiment described here, be able to carry out for a person skilled in the art it is various it is apparent variation, again Adjustment and substitution are without departing from protection scope of the present invention.Therefore, although by above embodiments to the present invention carried out compared with For detailed description, but the present invention is not limited to the above embodiments only, without departing from the inventive concept, can be with Including more other equivalent embodiments, and the scope of the invention is determined by the scope of the appended claims.

Claims

1. a kind of extracting method of extracellular vesica, which is characterized in that the described method includes:

Biological sample is provided；

Under conditions of being enough to make in biological sample impurity to be partially or fully retained on capture surface, make the biological sample By being contacted at least twice with the capture surface；Wherein, the impurity is the portion in biological sample in addition to extracellular vesica Divide or other whole substances, the capture surface include inner surface and/or the outer surface of spherical particle porous material；

When impurity is partially or fully retained on catches surface, flowing through liquid is to extract the liquid comprising extracellular vesica Body；

Or further the above-mentioned liquid that flows through is concentrated.

2. a kind of extracting method of extracellular vesica according to claim 1, which is characterized in that the described method includes: institute Biological sample is stated successively after two concatenated chromatographic columns, the impurity is partially or fully retained in catching for filler in chromatographic column Surface is obtained, flowing through liquid is the extracellular vesica after purification extracted, and filler is the porous material of spherical particle in the chromatographic column Material.

3. a kind of extracting method of extracellular vesica according to claim 2, which is characterized in that concatenated two layers The inner and outer surfaces of spherical particle porous material in the first chromatographic column in column are analysed as anion exchange groups functionalization, anion is handed over Changing group is quaternary amines or tertiary amine group.

4. a kind of extracting method of extracellular vesica according to claim 3, which is characterized in that in first chromatographic column The inner and outer surfaces of spherical particle porous material are positively charged.

5. a kind of extracting method of extracellular vesica according to claim 2, which is characterized in that concatenated two layers Analysing spherical particle porous material bore area in the second chromatographic column in column is anion exchange groups functionalization, anion exchange Group is quaternary amines or tertiary amine group.

6. a kind of extracting method of the outer vesica of cellule according to claim 5, which is characterized in that second chromatographic column Spherical particle porous material bore area it is positively charged.

7. a kind of extracting method of extracellular vesica according to claim 1 or 2, which is characterized in that the spherical particle The average grain diameter of porous material is 60-100 μm, and the average pore size of the first chromatographic column spherical particle porous material is less than or equal to 30nm, the second chromatographic column spherical particle aperture of porous material maximum exclusion molecular weight are less than 400KD.

8. a kind of extracting method of extracellular vesica according to claim 1, which is characterized in that extracellular vesica is excretion Body or outer membrane vesicles.

9. a kind of extracting method of extracellular vesica according to claim 2, which is characterized in that the biological sample passes through The linear flow rate of filler is 100-480cm/h in chromatographic column.

10. a kind of extracting method of extracellular vesica according to claim 1, which is characterized in that the biological sample packet Include the outer culture supernatant of biological fluid, cell injuring model supernatant, microbial body.

11. a kind of extracting method of extracellular vesica according to claim 10, which is characterized in that the biological fluid Including plasma/serum, urine, cerebrospinal fluid, tears, sperm, amniotic fluid, gastric juice, saliva, hydrothorax, ascites, nasal mucus, bronchovesicular Irrigating solution, knuckle synovia, bile, cervical mucus and excrement.

12. a kind of extracting method of extracellular vesica according to claim 2, which is characterized in that filler in chromatographic column Height is 6-35cm.

13. kit used by a kind of extracting method of extracellular vesica according to any of the above-described claim, It is characterized in that, the kit is composed in series by the first chromatographic column and the second chromatographic column, wherein the first chromatographic column at least one, Second chromatographic column at least one is connected using standard Luer interface between chromatographic column.