CN110343664A - The method for extracting excretion body and excretion body protein - Google Patents
The method for extracting excretion body and excretion body protein Download PDFInfo
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- CN110343664A CN110343664A CN201810287204.6A CN201810287204A CN110343664A CN 110343664 A CN110343664 A CN 110343664A CN 201810287204 A CN201810287204 A CN 201810287204A CN 110343664 A CN110343664 A CN 110343664A
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Abstract
The invention discloses the methods that excretion body and excretion body protein are extracted from the culture solution of human body fluid or cell.The excretion body and the method for excretion body protein provided by the invention of extracting includes: that extraction material extraction is added into the liquid to be extracted containing excretion body to obtain excretion body;Extracting material is metal oxide or the functional material for being modified with the metal oxide;Double coordinations can occur with phosphate anion for metal oxide;The liquid to be extracted is the body fluid, the dilution of the body fluid or the cultured products of cell from human body.Experiments have shown that, the high efficiency extraction of excretion body in the culture solution to human body fluid or cell may be implemented in excretion body extracting method of the invention, and extraction time is short, the excretion body purity is high of acquisition, complete excretion body, and the recyclable recycling of extraction material used can be obtained by elution;In addition extracting gained excretion body protein can find that gained excretion body protein type is more and closer with excretion body function.
Description
Technical field
The present invention relates in field of biotechnology, from extracting excretion body and excretion in the culture solution of human body fluid or cell
The method of body protein.
Background technique
Excretion body, which is mammalian cell, is secreted into extracellular a kind of capsule balloon-shaped structure by " endocytosis-outlet " process,
Diameter is about 30-150nm.Excretion body is not only present in cell culture fluid, exists in blood, urine, amniotic fluid, breast milk, essence
In the body fluid such as liquid, saliva, pleural effusion, composition include lipid bilayer and the relevant protein of cell origin, RNA,
The substances such as DNA.Almost all of cell can generate excretion body, both include and cell shape in the excretion body of different cell origins
It also include special molecular relevant to the biological function of derived cell at, structure and the relevant shared ingredient of substance transportation.Excretion
Body is paid more and more attention as the important way of a kind of intercellular communication, a large amount of biologically functional molecule of transmitting.Studies have shown that outer
Secrete body play the role of in a series of physiology and pathologic process it is vital.Tumour correlation excretion body being capable of induced cancer hair
Raw, participation cancer target transfer, inhibits immunological surveillance and mediation of the immune system to tumour at the microenvironment for improving tumour growth
Drug resistant tumor cells, early diagnosis, prognostic analysis and the curative effect that analysis and detection to tumour excretion body are conducive to tumour are commented
Valence.
Currently used excretion body extracting method includes supercentrifugation, density-gradient centrifugation method, the polymer precipitation method, surpasses
Filter method, size exclusion chromatograph, affine in immunity method.Preceding four kinds of methods are based on the physics parameters such as the unique density of excretion body, size
It realizes and extracts, lack specificity, be not able to satisfy the requirement of tumour cell excretion body specific extraction and analysis in actual sample.Especially
In its human body fluid sample, contain the particles such as a large amount of low-density lipoprotein bletilla chylomicron, the partial size and density of these particles
Close with excretion body, most methods can not separate it with excretion body, to cause to do to the subsequent analysis of excretion body
It disturbs.
Summary of the invention
The technical problem to be solved by the present invention is to how extract excretion body.
In order to solve the above technical problems, present invention firstly provides the extracting methods of excretion body, which comprises Xiang Han
There is addition extraction material extraction in the liquid to be extracted of excretion body to obtain excretion body;The extraction material is metal oxide or modification
There is the functional material of the metal oxide;
Double coordinations can occur with phosphate anion for the metal oxide.
In the above method, the metal oxide can be TiO2、ZrO2、Al(OH)3、Ga2O3、Fe3O4、Fe2O3、Nb2O3、
SnO2、HfO2And/or Ta2O5;
The functional material for being modified with the metal oxide is concretely modified with the oxidation of the metal oxide
Graphene, magnetic nanoparticle or silicon ball.
The partial size of the material can be 1~50 μm.
In the above method, the liquid to be extracted can be for from the body fluid of human body, the dilution of the body fluid or cell
Cultured products.
The dilution of the body fluid can be by the way that the body fluid to be diluted to obtain using buffer.The buffer can be
With pH buffer capacity and it is able to maintain that the solution of excretion body osmotic pressure.The buffer concretely DMEM buffer
(solvent is water, and solute and its concentration are respectively 50mM 4- hydroxyethyl piperazine by (Gibico company, article No. 31053-028), HEPS
Ethanesulfonic acid and 150Mm NaCl, pH 8) or PBS (Corning company, article No.: 21-040-CVR).The body fluid is utilized into buffering
The volume ratio of the body fluid and the buffer can be 1:1~1:5 when liquid is diluted.The body fluid is carried out using buffer
The volume ratio of the body fluid and the buffer further can be 1:1 when dilution.
In the present invention, when the viscosity of the body fluid larger (such as blood plasma, saliva), mentioned using the dilution of the body fluid
Take excretion body.When the viscosity of the body fluid smaller (such as urine), excretion body is extracted using the directly utilization body fluid.
The cultured products of the cell can be small using cell 24-48 described in the culture solution culture for going excretion body serum to configure
When obtained cultured products.It is described to go excretion body serum that obtain for fetal calf serum to be centrifuged 12~16 hours at 110000g
Supernatant.
The cultured products can be the product for being cultivated the cell, can also be the non-thin of the cultured products
Born of the same parents part.The non-cellular fractions can be by being centrifuged to obtain by the cultured products.
In the above method, the body fluid can be blood, serum, urine or saliva;The cell is tumour cell.It is described swollen
Oncocyte concretely cervical cancer cell, liver cancer cells or pancreatic cancer cell etc..
In the above method, the addition extraction material extraction into the liquid to be extracted containing excretion body, which obtains excretion body, to wrap
It includes: the extraction material being added into the liquid to be extracted containing excretion body, obtains extraction mixture;The extraction is mixed
Object is centrifuged, and enters excretion body in precipitating, is collected precipitating, is removed the extraction material in the precipitating, obtain excretion
Body.
The above method may additionally include the extraction material is added into the liquid to be extracted before remove the liquid to be extracted
In dead cell and/or cell residue the step of.Remove dead cell and/or cell residue in the liquid to be extracted can pass through by
The liquid to be extracted is realized through gradient low-speed centrifugal and/or filter membrane.The gradient low-speed centrifugal specifically can in accordance with the following steps according to
Secondary progress: 300g is centrifuged 10 minutes, and 2000g is centrifuged 10 minutes, and 10000g is centrifuged 30 minutes.The filter membrane can be 0.2 μm of filter membrane.
The above method may additionally include the extraction mixture is centrifuged before the extraction mixture is incubated for
The step of.The time of the incubation can be 1~10 minute.Concretely 2~5 minutes time of the incubation (such as 5 minutes).
The incubation can be carried out at 1000~2500rpm.The incubation can specifically carry out at 1500 rpm.The incubation can be in room
It is carried out under warm (25 DEG C) or low temperature.The low temperature can be 4 DEG C.
In the above method, the condition of the centrifugation can be to be centrifuged under 2000g.The time of centrifugation can be 30 seconds.
In the above method, the extraction material can be the metal oxide;Material is extracted described in the extraction mixture
Material meets following a1 with the proportion of the liquid to be extracted), a2), a3) or a4):
A1) liquid to be extracted be the cell culture solution, described in the extraction mixture extraction material with it is described
The proportion of liquid to be extracted is a11) or a12):
A11) 5mg:(10~300) mL;
a12)5mg:10mL;
A2) extracting solution is the dilution of serum or serum, extraction material described in the extraction mixture with it is described
The proportion of liquid to be extracted is a21) or a22):
A21) 5mg:(30~500) μ L;
a22)5mg:100μL;
A3) extracting solution is the dilution of saliva or saliva, extraction material described in the extraction mixture with it is described
The proportion of liquid to be extracted is a31) or a32):
A31) 5mg:(1~125) μ L;
a32)5mg:100μL;
A4) extracting solution is urine, and the proportion of material and the liquid to be extracted is extracted described in the extraction mixture
For a41) or a42):
A41) 5mg:(10~75) mL;
a42)5mg:20mL。
In the above method, the extraction material removed in the precipitating can include: be added and wash into the precipitating
De- liquid, obtains elution mixture, and the eluent is the ammonium hydroxide (ammonium hydroxide of such as 15g/100mL) of (3~20) g/100mL;By institute
It states elution mixture to be centrifuged, enters excretion body in supernatant, collect supernatant, remove impurity in the supernatant i.e.
Obtain excretion body.
The condition of the centrifugation can be centrifuged 30 seconds for 2000g.
The method, which may additionally include, is added before the eluent the step of washing to the precipitating to the precipitating.
Washing buffer used in the washing can be PBS.
The impurity removed in the supernatant can include: the supernatant is centrifuged, enters excretion body heavy
In shallow lake, collects precipitating and obtain excretion body.The condition of the centrifugation can be 2000g centrifugation 30 seconds.
Following X1) or method X2), also belong to protection scope of the present invention:
X1) the extracting method of excretion body protein, comprising: it extracts to obtain excretion body using the extracting method of the excretion body,
The excretion body is cracked, excretion body lysate is obtained;It is extracted from the excretion body lysate and obtains excretion body protein;
X2) the analysis method of excretion body protein, comprising: excretion body protein is obtained using X1) the method, then to described
Excretion body protein is analyzed, and the analysis of excretion body protein is completed.
The cracking excretion body can be carried out using lysate.The lysate can for 4g/100mL SDS aqueous solution,
8M aqueous solution of urea, dithiothreitol (DTT) and/or RIPA lysate.The cracking can also be carried out by ultrasonic wave added.The ultrasound
Condition can for 100W ultrasound 20 minutes.
Described analyze the excretion body protein can be carried out using SDS-PAGE or LC-MS/MS.
Excretion body extracts kit or excretion body protein extracts kit also belong to protection scope of the present invention, the reagent
Box includes the extraction material.
The kit can contain only the extraction material, can also as the extraction material and extract excretion body needed for its
He forms reagent.
Other described reagents can be the washing buffer and/or the eluent.
Following Y1)-Y4) in any application, also belong to protection scope of the present invention:
Y1) the application for extracting material in the extraction of excretion body protein;
Y2) the application for extracting material in excretion body protein is analyzed or identified;
Y3) application of the kit in the extraction of excretion body protein;
Y4) application of the kit in excretion body protein is analyzed or identified.
It is demonstrated experimentally that excretion in the culture solution to human body fluid or cell may be implemented in excretion body extracting method of the invention
The high efficiency extraction of body, extraction time is short, and the excretion body purity is high of acquisition can obtain complete excretion by elution
Body, and the recyclable recycling of extraction material used;In addition extracting gained excretion body protein can find, gained excretion body protein
Type is more, and compared with conventional method, and gained excretion body protein and excretion body function are closer.The present invention has following beneficial to effect
Fruit:
1. by double using passing through between the phosphate radical and metal oxide of excretion body membrane phospholipid bilayer surface exposure
The chemical action of coordination combines, and extracts to realize to the high specific of excretion body, easy to operate, extraction rate is fast, the rate of recovery
It is high.
2. the desorption to the excretion body of extraction is realized by the condition for changing eluent, and obtaining has complete vesica
The excretion body of structure, and elute later material and can reuse.
3. directly subsequent point can be carried out to the excretion body ingredient of extraction by way of directly cracking to excretion body
Analysis, such as protein, RNA etc..
Detailed description of the invention
Fig. 1 is the testing result of marker protein TSG101 and CD9 in excretion body.
Fig. 2 is the excretion body protein matter Mass Spectrometric Identification result that three kinds of methods are extracted.Wherein, metal oxide method is the present invention
Excretion body extracting method;U1, U2, U3 respectively indicate supercentrifugation extract excretion body parallel laboratory test three times, K1, K2,
K3 respectively indicates the parallel laboratory test three times that commercial kit method extracts excretion body, and T1, T2, T3 respectively indicate metal oxide method
Extract the three repeated experiments of excretion body.
Fig. 3 is that the extracting method (a), supercentrifugation (b) and commercial kit (c) of excretion body of the invention are extracted
Excretion body protein matter Gene Ontology analyzes Cellular Component result.
Fig. 4 is the extraction of excretion body in cell culture fluid.
Fig. 5 is reversible elution experiments result.
Fig. 6 is the excretion body result directly extracted in cell culture fluid using metal oxide.Arrow show excretion body.
Fig. 7 is that excretion body extracts in urine.
Specific embodiment
The present invention is further described in detail With reference to embodiment, and the embodiment provided is only for explaining
The bright present invention, the range being not intended to be limiting of the invention.Experimental method in following embodiments is unless otherwise specified
Conventional method.Material as used in the following examples, reagent, instrument etc., are commercially available unless otherwise specified.
Quantitative test in following embodiment, is respectively provided with three repeated experiments, and results are averaged.
DMEM buffer in following embodiments is Gibico Products, article No. 31053-028.
PBS in following embodiments is Corning Products, article No.: 21-040-CVR.
The present invention provides a kind of extracting methods of excretion body, this method comprises: into the liquid to be extracted containing excretion body
Extraction material extraction is added and obtains excretion body;Extracting material is metal oxide or the function material for being modified with the metal oxide
Material;Double coordinations can occur with phosphate anion for metal oxide.
It is below TiO with metal oxide2, liquid to be extracted be human serum, cell culture fluid and urine for be specifically described
The extracting method of excretion body.Centrifugation in following embodiments is carried out at 4 DEG C unless otherwise specified.
Embodiment 1, excretion body extract
One, the LC-MS/MS of the extraction of excretion body and its protein is analyzed in human serum
Step 1: serum sample pretreatment
100 μ L human serums (through the people's informed consent) are taken, the dilution of DMEM buffer is added into serum by 1:1 by volume,
Obtain serum dilution;Serum dilution is subjected to gradient density centrifugal and removes dead cell and/or cell residue, centrifugal condition
Are as follows: 300g is centrifuged 10 minutes, and 2000g is centrifuged 10 minutes, and 10000g is centrifuged 30 minutes, takes supernatant, and supernatant is filtered through 0.2 μm
After film filtering, filtrate, as pretreated serum sample are collected.
Step 2: excretion body extracts in serum sample
Weigh 5mg TiO2, pure water and the rinse of DMEM buffer 2 times, the TiO after being cleaned are used respectively2;To after cleaning
TiO2The middle 200 μ L of pretreated serum sample that step 1 is added and obtains, obtains extraction mixture;By extraction mixture at 4 DEG C
Lower concussion (1500rpm) is incubated for after five minutes, is centrifuged 30 seconds at 2000g, enters excretion body in precipitating, collects precipitating;Xiang Chen
Washing buffer is added in shallow lake, and ((washing is heavy by being centrifuged collection in 30 seconds at 2000g for PBS) concussion washing precipitating for the buffer
Form sediment and complete), the precipitating after being washed;Eluent is added into the precipitating after washing, and (eluent is the ammonia of 10g/100mL
Water), obtain elution mixture;Elution mixture is centrifuged 30 seconds at 2000g, enters excretion body in supernatant, in collection
Clear liquid to get arrive excretion body extracting solution;Obtained excretion body extracting solution is centrifuged 30 seconds at 2000g, enters excretion body heavy
In shallow lake, precipitating is collected to get excretion body is arrived.
Excretion body protein is extracted, detects its excretion body marker protein TS G101 and CD9 using western-blot, as a result such as
Shown in Fig. 1, the results show that containing TSG101 and CD9 in obtained excretion body.
Step 3: the cracking of excretion body and its LC-MS/MS analysis of protein
Material (precipitating after washing) obtained by step 2 containing excretion body is placed on ice, 18 μ L4g/ are added thereto
The SDS aqueous solution of 100mL, then ultrasonic (ultrasound condition is carried out at 100W) cracks 20 minutes, obtains ultrasonic product;It will be ultrasonic
Product is centrifuged 5 minutes at 16000g, collects whole supernatants;82 μ L 8M aqueous solution of urea and two sulphur are added into supernatant
Threitol obtains lysate, and the concentration of dithiothreitol (DTT) is 20mmol/L in the lysate;The lysate is incubated for 4 at 37 DEG C
Hour, obtain pyrolysis product;Pyrolysis product is transferred in FASP pipe, washed with 8M aqueous solution of urea (FASP pipe be it is a kind of by
The pipe of molecular weight retention, washing process are completed by centrifugation, and centrifugal condition is that 14000g is centrifuged 10 minutes, after centrifugation,
Most solution and small-molecule substance are centrifuged to lower layer and are received in pipe, and excretion body is trapped in the FASP pipe on upper layer) 2
It is secondary;Then iodoacetamide (final concentration 50mmol/L) is added, is protected from light at room temperature 1 hour, with 50mM ammonium bicarbonate aqueous solution
Washing 3 times, be added 1 μ g trypsase, 37 DEG C digestion 12 hours, 45 DEG C of heat of acquired solution are done, with 0.1% (v/v) formic acid water
Solution constant volume obtains excretion body protein extracting solution, excretion body protein extracting solution loading is carried out LC-MS/MS analysis, applied sample amount is
1μg。
Then acquisition mass spectrometric data searches target data library searching using MAXQUANT.Target database used is RefSeq
human database.Retrieval parameter is set as trypsase holoenzyme and cuts, and sets 2 protease leakage enzyme sites, and protein is fixed
Carbamidomethyl (C) is chosen in modification, variable to be modified to Oxidation (M).Mass spectrum first quality error be 15ppm, two
Grade quality error is 0.6Da, and false positive rate is set as 1%.
The results show that being 384 using the Identification of Fusion Protein number of serum excretion body protein obtained by the above method, hence it is evident that better than super
228) and commercial kit fast centrifugal process (carries out the identification of protein using identical method, gained Identification of Fusion Protein number is
(MinuteTMHi-Efficiency Exosome Precipitation Reagent, company: Invent
Biotechnologies, Inc, article No.: CAT#EI-O27) (identification of protein, gained albumen are carried out using identical method
Identify that number extracts the Identification of Fusion Protein number of same serum sample for 252) (Fig. 2).The Gene Ontology analysis present invention extracts outer
Secrete the cell component of body protein matter, the results showed that protein obtained by this method and excretion body function are more closely (Fig. 3).Show benefit
Extracting excretion body with method of the invention has better extraction effect.
Wherein, the step of extracting serum excretion body using supercentrifugation is as follows: 200 microlitres of serum are taken, it is isometric to be added
After PBS dilution, 2000g is centrifuged 30 minutes at 4 DEG C, takes supernatant to be centrifuged 45 minutes with 12000g again, by 0.2 μm of filter membrane mistake of supernatant
After filter, 110000g be centrifuged 70 minutes, abandon supernatant, after bottom sediment part is cleaned with PBS, then through 110000g be centrifuged 70 minutes,
Abandon supernatant, serum excretion body obtained by sediment fraction, that is, supercentrifugation.
2, excretion body extracts in cell culture fluid
2.1 supercentrifugations extract excretion body
After culture solution culture HeLa cell 24-48 hours of the fetal calf serum configuration of no excretion body, culture solution is collected;It will
Culture solution is first centrifuged through 300g at 4 DEG C and removes within 10 minutes cell therein, then therein through 2000g centrifugation removal in 10 minutes
Dead cell, then be centrifuged 30 minutes through 10000g, organelle therein etc. is removed, supernatant is collected, supernatant 110000g is centrifuged
70 minutes, excretion body is contained in precipitating, after precipitating is cleaned with PBS, then through 110000g centrifugation 70 minutes, precipitating is collected, will sink
It forms sediment after partially being cleaned again with PBS, then is centrifuged 70 minutes through 110000g, collect precipitating and obtains the excretion body in cell culture fluid.
2.2 standard excretion bodies evaluate metal oxide concentration effect
Step 2.1 gained excretion body is divided into several parts and is used as standard excretion body, every part corresponding outer comprising 2 μ g protein contents
Secrete body.Take 2mg TiO2, it is resuspended in 100 μ L DMEM buffers, 1 part of standard excretion body is then added thereto, at 4 DEG C,
1500rpm concussion is incubated for 5 minutes, and gained suspension 2000g is centrifuged 30 seconds after being then incubated for, and sediment fraction cleans 3 with PBS
It is secondary, 2000g is washed every time and is centrifuged separation in 30 seconds, is finally collected sediment fraction, that is, is enriched with the TiO of excretion body2。
By scanning electron microscope and transmission electron microscope to the TiO for being enriched with excretion body2Characterized, as a result as shown in figure 4, its
In, a is the TiO for not being enriched with excretion body2Scanning electron microscope phenogram, b is the TiO for being enriched with excretion body2Scanning electron microscope characterization
Figure, c and d are the TiO for being enriched with excretion body2Transmission electron microscope phenogram, d be c magnified partial view.The results show that TiO2It can be with
Successfully it is enriched to excretion body.
2.3 reversible elution experiments (material is reusable)
The TiO of excretion body will be enriched with obtained by step 2.22It is cleaned with the ammonium hydroxide of 100 μ L 10g/100mL, 2000g centrifugation
After 30 seconds, collect supernatant as in FASP pipe, after cleaning 2 times with 100 μ L PBS, respectively to after cleaning material and elution
Supernatant is characterized, as a result as shown in figure 5, wherein a is the later TiO of ammonia scrubbing2The scanning electron microscope phenogram of material, b
For the supernatant transmission electron microscope phenogram of elution.Show the TiO for being enriched with excretion body2On excretion body can be eluted, obtain
TiO2It is reusable.
2.3 directly extract the excretion body in cell culture fluid using metal oxide
By 5mg TiO2It is added in the culture solution obtained to 10mL step 2.1, after rocking incubation 30 minutes with head-shaking machine,
2000g is centrifuged 30 seconds, after precipitating is cleaned 2 times with PBS, is characterized by scanning electron microscope to it, as a result as shown in Figure 6.Knot
Fruit shows that metal oxide can be with the excretion body in enrichment of cell culture solution.
3, excretion body extracts in urine
Urina sanguinis 20mL is collected, is successively centrifuged 10 minutes through 2000g, after 10000g is centrifuged 30 minutes, supernatant is filtered with 0.2 μm
After film filtering, 5mg TiO is added2Material, after rocking incubation 30 minutes with head-shaking machine, 2000g is centrifuged 30 seconds, by precipitating PBS
After cleaning 2 times, it is characterized by scanning electron microscope, as a result as shown in Figure 7.The results show that metal oxide can be enriched with
Excretion body in urine.
Claims (10)
1. the extracting method of excretion body, comprising: extraction material extraction is added in the liquid to be extracted of Xiang Hanyou excretion body and obtains excretion
Body;The material that extracts is metal oxide or the functional material for being modified with the metal oxide;
Double coordinations can occur with phosphate anion for the metal oxide.
2. according to the method described in claim 1, it is characterized by: the metal oxide is TiO2、ZrO2、Al(OH)3、
Ga2O3、Fe3O4、Fe2O3、Nb2O3、SnO2、HfO2And/or Ta2O5;
The functional material for being modified with the metal oxide is the graphene oxide for being modified with the metal oxide, magnetism
Nano particle or silicon ball;
And/or the liquid to be extracted is the body fluid, the dilution of the body fluid or the cultured products of cell from human body.
3. according to the method described in claim 2, it is characterized by: the body fluid is blood, serum, urine or saliva;It is described
Cell is tumour cell.
4. method according to claim 1 to 3, it is characterised in that: described into the liquid to be extracted containing excretion body
It includes: that the extraction material is added into the liquid to be extracted containing excretion body that extraction material extraction, which is added, and obtains excretion body,
Obtain extraction mixture;The extraction mixture is centrifuged, enters excretion body in precipitating, collects and precipitates, described in removing
The extraction material in precipitating, obtains excretion body.
5. according to the method described in claim 4, it is characterized by: the condition of the centrifugation is to be centrifuged under 2000g.
6. according to claim 4 or 5 or the method, it is characterised in that: the extraction material is the metal oxide;Institute
The proportion for stating described in extraction mixture extraction material and the liquid to be extracted meets following a1), a2), a3) or a4):
A1) liquid to be extracted is the culture solution of the cell, and extraction material described in the extraction mixture is with described wait mention
The proportion for taking liquid is a11) or a12):
A11) 5mg:(10~300) mL;
a12)5mg:10mL;
A2) extracting solution is the dilution of serum or serum, and extraction material described in the extraction mixture is with described wait mention
The proportion for taking liquid is a21) or a22):
A21) 5mg:(30~500) μ L;
a22)5mg:100μL;
A3) extracting solution is the dilution of saliva or saliva, and extraction material described in the extraction mixture is with described wait mention
The proportion for taking liquid is a31) or a32):
A31) 5mg:(1~125) μ L;
a32)5mg:100μL;
A4) extracting solution is urine, and the proportion that material and the liquid to be extracted are extracted described in the extraction mixture is
A41) or a42):
A41) 5mg:(10~75) mL;
a42)5mg:20mL。
7. according to the method any in claim 4-6, it is characterised in that: the extraction removed in the precipitating
Material includes: that eluent is added into the precipitating, obtains elution mixture, and the eluent is the ammonia of (3~20) g/100mL
Water;The elution mixture is centrifuged, enters excretion body in supernatant, supernatant is collected, removes in the supernatant
Impurity obtain excretion body.
8. following X1) or method X2):
X1) the extracting method of excretion body protein, comprising: extract to obtain excretion using the method any in claim 1-8
Body cracks the excretion body, obtains excretion body lysate;It is extracted from the excretion body lysate and obtains excretion body protein;
X2) the analysis method of excretion body protein, comprising: excretion body protein is obtained using X1) the method, then to the excretion
Body protein is analyzed or is identified, the analysis or identification of excretion body protein are completed.
9. excretion body extracts kit or excretion body protein extracts kit, including material is extracted described in claims 1 or 2.
10. following Y1)-Y4) in any application:
Y1) application of the extraction material described in claims 1 or 2 in the extraction of excretion body protein;
Y2) application of the extraction material described in claims 1 or 2 in excretion body protein is analyzed or identified;
Y3) application of the kit described in claim 9 in the extraction of excretion body protein;
Y4) application of the kit described in claim 9 in excretion body protein is analyzed or identified.
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CN111366654A (en) * | 2020-04-01 | 2020-07-03 | 上海中科新生命生物科技有限公司 | Protein component analysis method for enriching blood exosomes based on differential centrifugation |
CN113024673A (en) * | 2020-07-21 | 2021-06-25 | 上海交通大学 | Silicon dioxide microsphere modified by phosphatidylserine polypeptide ligand |
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CN113528423A (en) * | 2021-07-06 | 2021-10-22 | 温州医科大学附属眼视光医院 | Method for separating plasma lipoprotein and exosome |
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CN114164203A (en) * | 2022-02-11 | 2022-03-11 | 北京同创正业生物科技有限公司 | Extracellular vesicle purification material and purification method |
CN114164203B (en) * | 2022-02-11 | 2022-05-10 | 北京同创正业生物科技有限公司 | Extracellular vesicle purification material and purification method |
CN115992092A (en) * | 2023-02-18 | 2023-04-21 | 浙江洛兮医学检验实验室有限公司 | Method for extracting exosomes based on transition metal oxyhydroxide |
CN115992092B (en) * | 2023-02-18 | 2023-11-17 | 湖州科元生物科技有限公司 | Method for extracting exosomes based on transition metal oxyhydroxide |
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