CN109580960A - The separation method of extracellular vesica and the method and kit of the extracellular vesicle surface marker of detection - Google Patents
The separation method of extracellular vesica and the method and kit of the extracellular vesicle surface marker of detection Download PDFInfo
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Abstract
The present invention provides the methods and kit of a kind of separation method of extracellular vesica and the extracellular vesicle surface marker of detection, the separation method includes: after pretreated biological sample to be mixed to incubation with phosphatidylserine capture molecule-affinity tag fusion protein, solid phase carrier is added, the extracellular vesicle complexes of carrier-are formed；The extracellular vesicle complexes of carrier-are separated from biological sample again.The detection method includes: that the extracellular vesicle complexes of above-mentioned resulting carrier-are mixed incubation with marker antibody, obtains the extracellular vesica-luciferase compound of carrier-；Extracellular vesica-luciferase the compound of carrier-is cracked, the detection that shines is carried out.This method high, high specificity to the separative efficiency of extracellular vesica, and high sensitivity when detecting.In addition, the luciferase by selecting different substrates or different wave length, is detected while realizing two kinds and the above marker.
Description
Technical field
The present invention relates to biological monitoring fields, separation method and detection in particular to a kind of extracellular vesica
The method and kit of extracellular vesicle surface marker.
Background technique
Extracellular vesica refers to the spherical film property vesica for being wrapped and being formed by lipid bilayer, by cell natural secretion
And enter the small vesica, including excretion body, film particles, microcapsule bubble and apoptotic body etc. of body fluid circulatory.These extracellular vesicas
Surface carry it is many predictive disease or can reflect the protein marker of disease process, be the important inspection for realizing liquid biopsy
Survey object.However, the extracellular half-life period of vesica in blood only has several hours, it is simultaneously from the cell external capsule of pathological tissues
Steep accounting very little, it is therefore desirable to which high sensitive method is detected.
At present frequently with a kind of detection method are as follows: using the extracellular vesica specific antibody such as CD63, CD81, from sample
The extracellular vesica of middle capture, sequentially add the marker antibody of biotin labeling, Streptavidin-horseradish peroxidase and
Substrate carries out enzymatic color developing detection.However, due to capturing cell external capsule from sample using specific antibodies such as CD63, CD81
The limited efficacy of bubble, and the content of extracellular vesicle surface marker and its small, lead to the sensitive of this enzymatic color developing detection
It spends limited, it cannot accurately be detected.
In consideration of it, special propose the application.
Summary of the invention
The first object of the present invention is to provide a kind of separation method of extracellular vesica, and this method is by utilizing phosphatide
The phosphatidylserine of the outer vesica of the combination cell of acyl serine capture molecule specificity captures extracellular vesica, separative efficiency
High, high specificity.
The second object of the present invention is to provide a kind of detection method of extracellular vesicle surface marker, and this method passes through
The quantitative detection of extracellular vesicle surface marker, high sensitivity are realized using the marker antibody that coupling has luciferase.
The third object of the present invention is to provide a kind of for detecting the kit of extracellular vesicle surface marker.
In order to realize above-mentioned purpose of the invention, the following technical scheme is adopted:
A kind of separation method of extracellular vesica comprising:
Pretreated biological sample is mixed into incubation with phosphatidylserine capture molecule-affinity tag fusion protein
Afterwards, adding can consolidate with what the affinity tag in phosphatidylserine capture molecule-affinity tag fusion protein was specifically bound
Phase carrier forms the extracellular vesicle complexes of carrier-；
The extracellular vesicle complexes of carrier-are separated from biological sample again.
A kind of detection method of extracellular vesicle surface marker comprising:
The extracellular vesicle complexes of the carrier-obtained using the separation method of any one of Claims 1 to 5 are had with coupling
The marker antibody of luciferase, which mixes, to be incubated for, and the extracellular vesica-luciferase compound of carrier-is obtained；
Extracellular vesica-luciferase the compound of carrier-is cracked, and the substrate of luciferase is added, detects luciferase
The luminous intensity of catalysis.
It is a kind of for detecting the kit of extracellular vesicle surface marker comprising: luciferase-Antibody Fusion egg
White, phosphatidylserine capture molecule-affinity tag fusion protein and solid phase carrier.
Compared with prior art, the invention has the benefit that
It is captured from sample extracellularly in compared with the prior art using the extracellular vesica specific antibody such as CD63, CD81
The method of vesica, the separation method of this extracellular vesica provided herein, by utilizing phosphatidylserine capture point
The phosphatidylserine of the outer vesica of the combination cell of sub- specificity captures extracellular vesica, due to extracellular vesicle surface
For the content of phosphatidylserine far more than molecules such as CD63, CD81, the separative efficiency of this method of the invention is high, specific
By force.
Compared with the prior art the marker antibody of middle biotin labeling, Streptavidin-horseradish peroxidase and
The method that substrate carries out enzymatic color developing detection, the detection method of this extracellular vesicle surface marker provided by the present application, by
High in the luminous efficiency of luciferase, the efficiency of especially Gaussia luciferase is common firefly luciferase
1000 times, therefore realize the highly sensitive detection for being better than common enzymatic development process.In addition, by selecting different substrates or different wave length
Luciferase, realize two kinds and above marker while detected.
Detailed description of the invention
In order to more clearly explain the embodiment of the invention or the technical proposal in the existing technology, to embodiment or will show below
There is attached drawing needed in technical description to be briefly described.
Fig. 1 is the intermolecular schematic diagram be combineding with each other of the present invention.
Fig. 2 is the lactadherin C1/C2-Fc or non-specificity IgG or CD63 or CD81 used in the present invention, respectively and
Quality (the average value of the extracellular vesica mixing of the 10 μ g luciferases label of purification extracellular vesica isolated after being incubated for
± standard error).
Specific embodiment
Embodiment of the present invention is described in detail below in conjunction with embodiment, but those skilled in the art will
Understand, the following example is merely to illustrate the present invention, and is not construed as limiting the scope of the invention.It is not specified in embodiment specific
Condition person carries out according to conventional conditions or manufacturer's recommended conditions.Reagents or instruments used without specified manufacturer is
The conventional products that can be obtained by commercially available purchase.
In a first aspect, present embodiment provides a kind of separation method of extracellular vesica.
Wherein, extracellular vesica is that cell is secreted, falls off or generate after broken, has lipid membrane structure.Extracellularly
Vesica includes excretion body (40~150nm), microcapsule bubble (100~1000nm), apoptotic body and particle (100~1000nm).This
Biological sample in embodiment include blood plasma, serum, urine, cerebrospinal fluid, saliva, sweat, sperm, tissue hydrops, cell or
Tissue culture supernatant.
The separation method the following steps are included:
Step 1: pretreated biological sample is mixed with phosphatidylserine capture molecule-affinity tag fusion protein
It is incubated for, forms extracellular vesica-phosphatidylserine capture molecule-affinity tag fusion protein compound.
Since extracellular vesicle surface has a large amount of phosphatidylserine, biological sample and phosphatidylserine are captured
When molecule-affinity tag fusion protein mixing is incubated for, the phosphatidyl of phosphatidylserine capture molecule and extracellular vesicle surface
Serine specific binding, so that affinity tag is coupled at extracellular vesicle surface, forms extracellular vesica-phosphatidyl silk ammonia
Sour capture molecule-affinity tag fusion protein compound is convenient for subsequent solid phase carrier separating step.
Further, the phosphatidylserine capture in the phosphatidylserine capture molecule-affinity tag fusion protein
Molecule includes that lactadherin (Lactadherin, also referred to as MFGE8), the polypeptide of C1 and/or C2 structural domain containing lactadherin, film are viscous
At least one of albumen-V (Annexin V).Preferably, which is the C1 of source of people lactadherin
And/or the polypeptide or film mucoprotein-V of C2 structural domain, these three capture molecules can be with phosphatidylserine specific efficient knots
It closes, to improve the separative efficiency of extracellular vesica.
Further, affinity tag includes IgG Fc sequence, Avi-tag, GST, hexahistine.Preferably, using IgG
Fc sequence or Avi-tag, the wherein amino acid sequence of Avi-tag are as follows: GLNDIFEAQKIEWHE.
Further, phosphatidylserine capture molecule-affinity tag fusion protein is prepared using conventional genetic engineering
Method, it is preferred that use escherichia expression system, be conducive to mass production.
Further, the preprocess method of biological sample includes: that biological sample is filtered or is centrifuged, to remove sample
In cell, cell fragment and aggregate, obtain biological sample supernatant.Preferably, 0.2~0.8 μm of filter is selected when filtering
Film or 0.3~0.7 μm of filter membrane or 0.4~0.6 μm of filter membrane.Preferably, it is centrifuged at 10000~16000g, 4~25 DEG C
10~30min, or to be centrifuged 15~25min at 12000~14000g, 3~7 DEG C.
Further, mixing temperature when being incubated for phosphatidylserine capture molecule-affinity tag in biological sample is 4
~37 DEG C, incubation time be 10min~2h.
Step 2: adding can be special with the affinity tag in phosphatidylserine capture molecule-affinity tag fusion protein
Property combine solid phase carrier, formed the extracellular vesicle complexes of carrier-.
Further, the surface coupling of the solid phase carrier has affinity molecule, which can be special with affinity tag
Property combine, so that the extracellular vesica formed in step 1-phosphatidylserine capture molecule-affinity tag fusion protein be answered
It closes object to be coupled on solid phase carrier, that is, forms the extracellular vesicle complexes of carrier-, be convenient for later separation.
Wherein, at least one of affinity molecule, including Protein A/G, Avidin, glutathione and Ni ion.It is excellent
Selection of land, affinity molecule are Protein A/G or Avidin.
Further, solid phase carrier includes at least one of magnetic bead, carbon nanotube and polymer microballoon.Preferably, Gu
Phase carrier is magnetic bead.It is further preferable that solid phase carrier is the magnetic bead that surface coupling has Protein A/G.
Step 3: again separating the extracellular vesicle complexes of carrier-from biological sample.
Further, when the extracellular vesicle complexes of carrier-being separated from biological sample, using magnetic suck method, centrifugation
Method or filtration method.
It is not using in the prior art for vesicle surface wide spectrum in this separation method that present embodiment provides
Property antigen antibody, but use can with phosphatidylserine specifically bind lactadherin C1 and/or C2 structural domain, film glue egg
The molecules such as white V.Since phosphatidylserine is the important component of extracellular vesica phospholipid bilayer, content considerably beyond
The surfaces such as CD63, CD81, CD9 broad spectrum activity antigen, therefore this method is higher to the separative efficiency of extracellular vesica.
Second aspect, present embodiment provide a kind of detection method of extracellular vesicle surface marker.
The detection method includes:
Step S1: the extracellular vesicle complexes of the carrier-obtained by above-mentioned separation method and coupling there is into luciferase
Marker antibody mix and be incubated for, obtain the extracellular vesica-luciferase compound of carrier-.
Further, when the extracellular vesicle complexes of carrier-mix incubation with the marker antibody that coupling has luciferase
Temperature be 4~37 DEG C, incubation time is 1~12h.
Further, the marker antibody that coupling has luciferase includes expressing luciferase and marker resisting simultaneously
Luciferase-antibody fusion protein of body (front-rear position of luciferase and antibody on amino acid sequence can be interchanged)；When
So, the marker antibody which has luciferase also includes will be after luciferase and marker antibody coupling by chemical method
Product.
Further, luciferase-antibody fusion protein is prepared using the conventional method in genetic engineering.Preferably,
Using escherichia expression system, it is conducive to mass production.
Further, marker antibody is the small molecular antibody that can identify extracellular vesicle surface marker, marker
Antibody includes single-chain antibody, nano antibody or Fab segment.
Further, in luciferase-antibody fusion protein, connected between luciferase and marker antibody by flexible small peptide
It connects；Preferably, the amino acid sequence of flexible small peptide is (GGGGS)2、(GGGGS)3、G(SGGGG)2S、(GGS)4、(GSG)4Or A
(EAAAK)2A。
Further, more preferred luciferase-antibody fusion protein is " luciferase-flexibility peptide-single-chain antibody "
Fusion protein.
Luciferase belongs to from sea pansy, extra large firefly, Gaussia, cream puts Daphnia, long abdomen Daphnia and Oplophorus
At least one of belong to.Preferably, luciferase is Gluc, is preferably expressed with the coupling mode of antibody
" luciferase-flexibility peptide-single-chain antibody " fusion protein.The efficiency of Gluc is common firefly luciferase
1000 times, the sensitivity of detection can be greatly improved.
It in the detection method, is resisted using common fluorogen, alkaline phosphatase, the complete of luciferase coupling
Body, but use Gluc and single-chain antibody (single chain variable fragment, ScFv) or receive
The recombination fusion protein of meter Kang Ti (nanobody) (being connected between the two by flexible small peptide).It is well known that the hair of luciferase
Light efficiency is greater than fluorogen (FITC, rhodamine etc.), and the luminous efficiency of Gluc is usual firefly luciferin again
1000 times of enzyme, and molecular weight is smaller.Compared with complete antibody, single-chain antibody or nano antibody molecular weight are smaller, are easy to and resist
Original combines, and structure is simple, is easy to recombinantly express.Gluc and single-chain antibody or nano antibody are passed through into flexibility
Small peptide connects into fusion protein recombinant expression, and the more common first production antibody of production procedure is coupled the mode of fluorogen more again
Simply.
Step S2: the extracellular vesica-luciferase compound of cracking carrier-, and the substrate of luciferase is added, it detects glimmering
The luminous intensity of light element enzymatic.
After the extracellular vesica of carrier-- luciferase compound cracking, luciferase is set to be in free state, and pass through
Magnetic suck method, centrifugal process or filtration method remove solid phase carrier, and the solid phase carrier in fluorescence detection is avoided to produce fluorescence signal
Raw interference, to improve the accuracy of detection.
Further, when cracking carrier-extracellularly vesica-luciferase compound, 0.1%~1% dodecane is added
Base sodium sulphate (SDS) is incubated at room temperature 10-30min.
In the detection method, by the way that coupling is had the marker antibody of luciferase and combines consolidating for extracellular vesica
The mixing of phase carrier is incubated for, and so that luciferase-antibody is would be incorporated into the extracellular vesica on carrier, and elute unbonded fluorescein
Enzyme；Finally, lysate is added, make luciferase separate out.After removing carrier, supernatant is taken, fluorogenic substrate is added, detection shines
Intensity, and compared with standard curve, obtain the quantitative result of marker on extracellular vesica.Due to the luminous efficiency of luciferase
High, the luminous efficiency of especially Gluc is 1000 times of common firefly luciferase, therefore realizes and be better than
The highly sensitive detection of common enzymatic development process.Furthermore, it is possible to which the luciferase by selecting different substrates or different wave length, real
It is detected while showing two kinds and the above marker.
The third aspect, present embodiment also provide it is a kind of for detecting the kit of extracellular vesicle surface marker, should
Kit can be commercialized, and be mass produced.
The kit, comprising: luciferase-antibody fusion protein, phosphatidylserine capture molecule-affinity tag fusion
Albumen and solid phase carrier.
Further, luciferase-antibody fusion protein is luciferase-flexibility peptide-single-chain antibody；
Preferably, luciferase is Gluc；
Preferably, the amino acid sequence of flexible peptide is (GGGGS)3；
Further, in phosphatidylserine capture molecule-affinity tag fusion protein, phosphatidylserine capture molecule
The polypeptide or film mucoprotein-V of C1 and/or C2 structural domain including source of people lactadherin；Affinity tag include IgG Fc sequence or
Avi-tag。
Further, the surface coupling of solid phase carrier has affinity molecule, and solid phase carrier includes magnetic bead, carbon nanotube and polymerization
At least one of object microballoon；Affinity molecule, including at least one in Protein A/G, Avidin, glutathione and Ni ion
Kind.Preferably, solid phase carrier is the magnetic bead that surface coupling has Protein A/G.
Feature and performance of the invention are described in further detail with reference to embodiments:
Embodiment 1
The present embodiment provides a kind of separation methods of extracellular vesica comprising:
A. one section of DNA sequence for successively expressing lactadherin C1/C2 structural domain and IgG Fc is obtained by being commercialized gene chemical synthesis
(DNA sequence dna is for encoding lactadherin C1/C2-Fc albumen, amino acid sequence for column (codon optimization is Bacillus coli expression)
As shown in SEQ ID NO.1), and be building up in pET-His plasmid using conventional molecule clone technology, obtain expression vector.
By conventional escherichia expression system, lactadherin C1/C2-Fc fusion protein is obtained through Ni column purification；
B. by the lung cancer patient blood of EDTA anticoagulation in 1000g centrifugal force, be centrifuged 15min at room temperature, remove blood
Red blood cell, leucocyte, blood platelet in liquid etc., obtain blood plasma.It is 0.8 μm of membrane filtration that blood plasma is further used to aperture, is gone
Except cell fragment therein, albumen aggregate and the vesica being relatively large in diameter, filtered fluid is obtained；
C. 1mL filtered fluid is mixed with 2 μ g lactadherin C1/C2-Fc fusion proteins, is placed on rotation vortex mixer, in room temperature
After lower incubation 30min, lactadherin C1/C2-Fc fusion protein is in conjunction with extracellular vesica.Add 50 μ g Protein A/G-
Magnetic bead is placed on rotation vortex mixer, is incubated for 30min at room temperature, it is compound to form extracellular vesica-lactadherin C1/C2- magnetic bead
Object.The extracellular vesica of magnetic-adsorption-lactadherin C1/C2- bead complexes discards supernatant liquid, and it is compound that 1mL PBS resuspension is added
Object, in triplicate to get extracellular vesica-lactadherin C1/C2- bead complexes.
Embodiment 2
The present embodiment provides a kind of detection methods of extracellular vesicle surface marker, wherein and the marker is PD-L1,
The biological sample of detection is blood plasma.
The detection method includes:
A. one section of successively expression Gluc, (GGGGS) is obtained by being commercialized gene chemical synthesis3Link peptide,
(DNA sequence dna is for encoding Gaussia fluorescence for the DNA sequence dna (codon optimization is Bacillus coli expression) of PD-L1 single-chain antibody
Plain enzyme-PD-L1 single-chain antibody, amino acid sequence is as shown in SEQ ID NO.2), and using conventional molecule clone technology structure
It is built in pET-His plasmid, obtains expression vector.By conventional escherichia expression system, obtained through Ni column purification
Gluc-PD-L1 scfv fusion protein；
B. in the separated obtained extracellular vesica-lactadherin C1/C2- bead complexes of embodiment 1,0.5 μ g is added
Gluc-PD-L1 single-chain antibody is placed on rotation vortex mixer, after being incubated for 1h at room temperature, Gaussia fluorescein
Enzyme-PD-L1 single-chain antibody is integrated on the PD-L1 of extracellular vesicle surface.Magnetic-adsorption Gluc-PD-L1 is single
Extracellular vesica-lactadherin C1/C2- the bead complexes of chain antibody-, discard supernatant liquid, 1mL PBS are added, compound, weight is resuspended
Again three times；
C. the extracellular vesica-lactadherin C1/C2- magnetic bead of magnetic-adsorption Gluc-PD-L1 single-chain antibody-is multiple
Object is closed, liquid is discarded supernatant, 100 μ L 0.5%SDS lysates are added, precipitating is resuspended, stands 20min at room temperature；
D. magnetic-adsorption magnetic bead takes 50 μ L supernatants to be added in 96 orifice plates, is placed in the microplate reader with syringe pump to be measured.If
Set microplate reader parameter: 50 μ L coelenterazines (200ng/mL), waiting time 2s, time for exposure 10s is added in every hole.With reference sample ratio
It is right, obtain the content situation of extracellular vesicle surface PD-L1 in 1mL blood plasma.
Embodiment 3
The present embodiment provides a kind of separation methods of extracellular vesica comprising:
A. one section of DNA sequence dna (password for successively expressing film mucoprotein-V and IgG Fc is obtained by being commercialized gene chemical synthesis
Son is optimized for Bacillus coli expression) (DNA sequence dna is for encoding film mucoprotein-V-IgG Fc, amino acid sequence such as SEQ ID
Shown in NO.3), and be building up in pET-His plasmid using conventional molecule clone technology, obtain expression vector.Pass through routine
Escherichia expression system obtains film mucoprotein-V-Fc fusion protein through Ni column purification；
B. it is 0.8 μm of membrane filtration by Serum of Patients With Breast Cancer aperture, removes cell fragment therein, albumen is reunited
Object and the vesica being relatively large in diameter, obtain filtered fluid；
C. 1mL filtered fluid is mixed with 2 μ g film mucoprotein-V-Fc fusion proteins, is placed on rotation vortex mixer, at room temperature
After being incubated for 30min, film mucoprotein-V-Fc fusion protein is in conjunction with extracellular vesica.50 μ g Protein A/G- carbon are added to receive
Mitron is placed on rotation vortex mixer, is incubated for 30min at room temperature, it is multiple to form extracellular vesica-film mucoprotein-V- carbon nanotube
Close object.After centrifugation, liquid is discarded supernatant, 1mL PBS is added, compound is resuspended, glue egg in triplicate to get extracellular vesica-film
White-V- carbon mano-tube composite.
Embodiment 4
The present embodiment provides a kind of detection methods of extracellular vesicle surface marker, wherein the marker is HER2, inspection
The biological sample of survey is serum.
The detection method includes:
A. one section of successively expression Gluc, G (SGGGG) is obtained by being commercialized gene chemical synthesis2S link peptide,
The DNA sequence dna (codon optimization is Bacillus coli expression) of HER2 single-chain antibody, (DNA sequence dna is for encoding Gaussia fluorescence
Plain enzyme-HER2 single-chain antibody, amino acid sequence is as shown in SEQ ID NO.4), and using conventional molecule clone technology building
Into pET-His plasmid, expression vector is obtained.By conventional escherichia expression system, Gaussia is obtained through Ni column purification
Luciferase-HER2 scfv fusion protein；
B. in the separated obtained extracellular vesica of embodiment 1-film mucoprotein-V- carbon mano-tube composite, it is added 0.5
μ g Gluc-HER2 single-chain antibody is placed on rotation vortex mixer, after being incubated for 1h at room temperature, Gaussia fluorescence
Plain enzyme-HER2 single-chain antibody is integrated on the HER2 of extracellular vesicle surface.After centrifugation, it is glimmering to get Gaussia to discard supernatant liquid
Extracellular vesica-film mucoprotein-V- the carbon mano-tube composite of light element enzyme-HER2 single-chain antibody-, it is compound to be added 1mL PBS resuspension
Object, in triplicate；
C. in the extracellular vesica-lactadherin-carbon mano-tube composite of Gluc-HER2 single-chain antibody-,
100 μ L 0.5%SDS lysates are added, precipitating is resuspended, stands 20min at room temperature；
D. centrifugation removal carbon nanotube, take 50 μ L supernatants be added 96 orifice plates in, be placed in the microplate reader with syringe pump to
It surveys.Microplate reader parameter is arranged: 50 μ L coelenterazines (200ng/mL), waiting time 2s, time for exposure 10s is added in every hole.With reference
Sample compares, and obtains the content situation of extracellular vesicle surface HER2 in 1mL serum.
Embodiment 5
The present embodiment provides a kind of separation methods of extracellular vesica comprising:
A. obtaining one section by commercialization gene chemical synthesis, successively (codon is excellent for the DNA sequence dna of expression lactadherin and Avi-tag
Turn to Bacillus coli expression), (DNA sequence dna is for encoding lactadherin-Avi-tag, amino acid sequence such as SEQ ID NO.5
It is shown), and be building up in pET-His plasmid using conventional molecule clone technology, obtain expression vector.Pass through conventional large intestine
Bacillus expression system obtains lactadherin-Avi-tag fusion protein through Ni column purification.Wherein Avi-tag is by biotin ligase
BirA institute biotinylation；
B. by bladder cancer's urine in 1000g centrifugal force, be centrifuged 15min at room temperature, remove cell in urine, compared with
Big particulate matter etc..It is further 0.8 μm of membrane filtration with aperture, removes cell fragment therein, albumen aggregate and straight
The biggish vesica of diameter, obtains filtered fluid；
C. 10mL filtered fluid is concentrated to 1mL with 100kD super filter tube, is mixed with 2 μ g lactadherin-Avi-tag fusion proteins,
It is placed on rotation vortex mixer, after being incubated for 30min at room temperature, lactadherin-Avi-tag fusion protein is in conjunction with extracellular vesica.
50 μ g Avidins-polymer microballoon is added, is placed on rotation vortex mixer, is incubated for 30min at room temperature, form cell external capsule
Bubble-lactadherin-polymer microballoon compound.After centrifugation, liquid is discarded supernatant, 1mL PBS is added, compound is resuspended, in triplicate,
Up to extracellular vesica-lactadherin-polymer microballoon compound.
Embodiment 6
The present embodiment provides a kind of detection methods of extracellular vesicle surface marker, wherein and the marker is CTLA-4,
The biological sample of detection is urine.
The detection method includes:
A. by be commercialized gene chemical synthesis obtain one section successively express CTLA-4 nano antibody, (GGS) 4 link peptide,
The DNA sequence dna (codon optimization is Bacillus coli expression) of Gluc, (DNA sequence dna is anti-for CTLA-4 nanometers
Body-Gluc, amino acid sequence is as shown in SEQ ID NO.6), and using conventional molecule clone technology structure
It is built in pET-His plasmid, obtains expression vector.By conventional escherichia expression system, CTLA- is obtained through Ni column purification
4 nano antibodies-Gluc fusion protein；
B. in the separated obtained extracellular vesica-lactadherin-polymer microballoon compound of embodiment 1,0.5 μ g is added
CTLA-4 nano antibody-Gluc is placed on rotation vortex mixer, and after being incubated for 1h at room temperature, CTLA-4 nanometers anti-
Body-Gluc is integrated on the CTLA-4 of extracellular vesicle surface.After centrifugation, liquid is discarded supernatant to get polymer
Extracellular vesica-CTLA-4 nano antibody-Gluc the compound of microballoon-lactadherin-, is added 1mL PBS and is resuspended again
Object is closed, in triplicate；
C. in the extracellular vesica-CTLA-4 nano antibody-Gluc compound of polymer microballoon-lactadherin-
In, 100 μ L 0.5%SDS lysates are added, precipitating is resuspended, stands 20min at room temperature；
D. centrifugation removal polymer microballoon, takes 50 μ L supernatants to be added in 96 orifice plates, is placed in the microplate reader with syringe pump
It is to be measured.Microplate reader parameter is arranged: 50 μ L coelenterazines (200ng/mL), waiting time 2s, time for exposure 10s is added in every hole.With ginseng
Sample comparison is examined, obtains the content situation of extracellular vesicle surface CTLA-4 in 10mL urine.
Experimental example 1
This experimental example is provided the present invention and provides extracellular vesica separation method and resisted in the prior art using CD63, CD81
The comparison of the efficiency of body separation.
One, experimentation:
A. the stable transfection pDisplay-Gluc in HEK293T cell, to express Gaussia fluorescence on cell membrane
Plain enzyme, then extracellular vesica is separated from the supercentrifugation of cell culture fluid goldstandard, obtain Gluc label
Extracellular vesica standard items, and carry out protein quantification.
B. using 1 μ g lactadherin C1/C2-Fc of the present invention or 1 μ g CD63 antibody or 1 μ g CD81 antibody or 1 μ g
Non-specific IgG (as control), mixes with the extracellular vesica standard items of 10 μ g Glucs label, is placed in rotation
Turn on vortex mixer, is incubated for 30min at room temperature.It is separately added into 50 μ g Protein A/G- magnetic beads again, is placed in rotation vortex mixer
On, it is incubated for 30min at room temperature, forms extracellular vesica-bead complexes.Extracellular vesica-the bead complexes of magnetic-adsorption,
Liquid is discarded supernatant, 1mL PBS is added, compound is resuspended, in triplicate.100 μ L 0.5%SDS lysates are added, precipitating is resuspended,
20min is stood at room temperature.
C. magnetic-adsorption magnetic bead takes 50 μ L supernatants to be added in 96 orifice plates, is placed in the microplate reader with syringe pump to be measured.If
Set microplate reader parameter: 50 μ L coelenterazines (200ng/mL), waiting time 2s, time for exposure 10s is added in every hole.With standard sample ratio
It is right, obtain the amount of separated extracellular vesica.
Two, experimental result
As a result as shown in Fig. 2, the amount for the extracellular vesica isolated using CD63, CD81 antibody is respectively 5 μ g and 3.8 μ g
Left and right, and technology provided by the invention is used, it is left to be up to 8.5 μ g with the amount of the lactadherin C1/C2-Fc extracellular vesica isolated
It is right.It can be seen that separating extracellular vesica using lactadherin C1/C2-Fc, separation effect more better than the prior art can be obtained
Rate.
Experimental example 2
This experimental example provides extracellular vesica detection method provided by the invention and uses biotin mark in the prior art
Marker antibody, Streptavidin-horseradish peroxidase and the substrate of note carry out the comparison of enzymatic color developing detection method.
One, experimental method
The breast cancer disease human plasma for collecting the HER2 positive, the membrane filtration for being 0.8 μm with aperture.By 1mL filtered fluid and 2 μ g
The mixing of lactadherin C1/C2-Fc fusion protein is placed on rotation vortex mixer, after being incubated for 30min at room temperature, lactadherin C1/C2-
Fc fusion protein is in conjunction with extracellular vesica.50 μ g Protein A/G- magnetic beads are added, are placed on rotation vortex mixer, in room
Temperature is lower to be incubated for 30min, forms extracellular vesica-lactadherin C1/C2- bead complexes.Extracellular vesica-the lactadherin of magnetic-adsorption
C1/C2- bead complexes discard supernatant liquid, 1mL PBS are added, compound is resuspended, in triplicate to get extracellular vesica-cream
Viscous element C1/C2- bead complexes.
Then using method of the invention and commercially available HER2ELISA detection kit to the extracellular vesicle surface
HER2 content is detected:
A. method of the invention detection: being added 0.5 μ g Gluc-HER2 single-chain antibody, and it is mixed to be placed in rotation
On even device, after being incubated for 1h at room temperature, Gluc-HER2 single-chain antibody is integrated to extracellular vesicle surface
On HER2.After centrifugation, liquid is discarded supernatant, 1mL PBS is added, compound is resuspended, in triplicate.100 μ L 0.5%SDS are added to split
Liquid is solved, precipitating is resuspended, stands 20min at room temperature.Magnetic-adsorption magnetic bead takes 50 μ L supernatants to be added in 96 orifice plates, is placed in band note
It penetrates to be measured in the microplate reader of pump.Microplate reader parameter is arranged: 50 μ L coelenterazines (200ng/mL) are added in every hole, and waiting time 2s exposes
10s between light time.
The detection of b.ELISA kit: it uses commercially available HER2ELISA detection kit (production of Hangzhou Lian Ke biotech firm)
Compare experiment.To extracellular vesica-bead complexes obtained in a, the biotin labeling that kit includes is added
After being washed three times with the cleaning solution that kit includes, Streptavidin-horseradish peroxidase that kit includes is added in HER2 antibody
Enzyme.After washing three times, substrate is added, carries out enzymatic colour developing.
Two, experimental result:
Using method provided by the invention, extracellular vesicle surface HER2 content is detected, by with standard sample
It compares, show that the extracellular vesicle surface HER2 content in patient's blood plasma is 3.2 ± 1.7ng/mL.And it uses commercially available
HER2ELISA detection kit, which is detected, (uses marker antibody, the Streptavidin-horseradish peroxide of biotin labeling
Compound enzyme and substrate carry out enzymatic color developing detection method), result readings is very low after carrying out enzymatic colour developing, with standard curve ratio
It is right, show that HER2 content is less than 0.1ng/mL.It can be seen that this detection method provided by the present application is aobvious compared to common enzymatic
Color method, sensitivity significantly improve.
Although illustrate and describing the present invention with specific embodiment, it will be appreciated that without departing substantially from of the invention
Many other change and modification can be made in the case where spirit and scope.It is, therefore, intended that in the following claims
Including belonging to all such changes and modifications in the scope of the invention.
SEQUENCE LISTING
<110>Zhou Ming, Ma Yingcong, Fan Song, Li Jingsong
<120>method and kit of the separation method of extracellular vesica and the extracellular vesicle surface marker of detection
<160> 6
<170> PatentIn version 3.5
<210> 1
<211> 556
<212> PRT
<213>artificial sequence
<400> 1
Lys Cys Val Glu Pro Leu Gly Leu Glu Asn Gly Asn Ile Ala Asn Ser
1 5 10 15
Gln Ile Ala Ala Ser Ser Val Arg Val Thr Phe Leu Gly Leu Gln His
20 25 30
Trp Val Pro Glu Leu Ala Arg Leu Asn Arg Ala Gly Met Val Asn Ala
35 40 45
Trp Thr Pro Ser Ser Asn Asp Asp Asn Pro Trp Ile Gln Val Asn Leu
50 55 60
Leu Arg Arg Met Trp Val Thr Gly Val Val Thr Gln Gly Ala Ser Arg
65 70 75 80
Leu Ala Ser His Glu Tyr Leu Lys Ala Phe Lys Val Ala Tyr Ser Leu
85 90 95
Asn Gly His Glu Phe Asp Phe Ile His Asp Val Asn Lys Lys His Lys
100 105 110
Glu Phe Val Gly Asn Trp Asn Lys Asn Ala Val His Val Asn Leu Phe
115 120 125
Glu Thr Pro Val Glu Ala Gln Tyr Val Arg Leu Tyr Pro Thr Ser Cys
130 135 140
His Thr Ala Cys Thr Leu Arg Phe Glu Leu Leu Gly Cys Glu Leu Asn
145 150 155 160
Gly Cys Ala Asn Pro Leu Gly Leu Lys Asn Asn Ser Ile Pro Asp Lys
165 170 175
Gln Ile Thr Ala Ser Ser Ser Tyr Lys Thr Trp Gly Leu His Leu Phe
180 185 190
Ser Trp Asn Pro Ser Tyr Ala Arg Leu Asp Lys Gln Gly Asn Phe Asn
195 200 205
Ala Trp Val Ala Gly Ser Tyr Gly Asn Asp Gln Trp Leu Gln Val Asp
210 215 220
Leu Gly Ser Ser Lys Glu Val Thr Gly Ile Ile Thr Gln Gly Ala Arg
225 230 235 240
Asn Phe Gly Ser Val Gln Phe Val Ala Ser Tyr Lys Val Ala Tyr Ser
245 250 255
Asn Asp Ser Ala Asn Trp Thr Glu Tyr Gln Asp Pro Arg Thr Gly Ser
260 265 270
Ser Lys Ile Phe Pro Gly Asn Trp Asp Asn His Ser His Lys Lys Asn
275 280 285
Leu Phe Glu Thr Pro Ile Leu Ala Arg Tyr Val Arg Ile Leu Pro Val
290 295 300
Ala Trp His Asn Arg Ile Ala Leu Arg Leu Glu Leu Leu Gly Cys Gly
305 310 315 320
Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Lys Thr His Thr Cys Pro
325 330 335
Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe
340 345 350
Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val
355 360 365
Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe
370 375 380
Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro
385 390 395 400
Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr
405 410 415
Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val
420 425 430
Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala
435 440 445
Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg
450 455 460
Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly
465 470 475 480
Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro
485 490 495
Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser
500 505 510
Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln
515 520 525
Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His
530 535 540
Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys
545 550 555
<210> 2
<211> 417
<212> PRT
<213>artificial sequence
<400> 2
Lys Pro Thr Glu Asn Asn Glu Asp Phe Asn Ile Val Ala Val Ala Ser
1 5 10 15
Asn Phe Ala Thr Thr Asp Leu Asp Ala Asp Arg Gly Lys Leu Pro Gly
20 25 30
Lys Lys Leu Pro Leu Glu Val Leu Lys Glu Met Glu Ala Asn Ala Arg
35 40 45
Lys Ala Gly Cys Thr Arg Gly Cys Leu Ile Cys Leu Ser His Ile Lys
50 55 60
Cys Thr Pro Lys Met Lys Lys Phe Ile Pro Gly Arg Cys His Thr Tyr
65 70 75 80
Glu Gly Asp Lys Glu Ser Ala Gln Gly Gly Ile Gly Glu Ala Ile Val
85 90 95
Asp Ile Pro Glu Ile Pro Gly Phe Lys Asp Leu Glu Pro Met Glu Gln
100 105 110
Phe Ile Ala Gln Val Asp Leu Cys Val Asp Cys Thr Thr Gly Cys Leu
115 120 125
Lys Gly Leu Ala Asn Val Gln Cys Ser Asp Leu Leu Lys Lys Trp Leu
130 135 140
Pro Gln Arg Cys Ala Thr Phe Ala Ser Lys Ile Gln Gly Gln Val Asp
145 150 155 160
Lys Ile Lys Gly Ala Gly Gly Asp Gly Gly Gly Gly Ser Gly Gly Gly
165 170 175
Gly Ser Gly Gly Gly Gly Ser Leu Thr Gln Pro Ser Ser Val Ser Ala
180 185 190
Asn Leu Gly Gly Thr Val Lys Ile Thr Cys Ser Gly Gly Ser Gly Ser
195 200 205
Tyr Gly Trp Tyr Gln Gln Lys Ala Pro Gly Ser Ala Pro Val Ser Leu
210 215 220
Ile Tyr Asp Asn Thr Asn Arg Pro Ser Asp Ile Pro Ser Arg Phe Ser
225 230 235 240
Gly Ala Leu Ser Gly Ser Thr Ala Thr Leu Thr Ile Thr Gly Val Gln
245 250 255
Ala Glu Asp Glu Ala Val Tyr Tyr Cys Gly Ser Arg Asp Ser Ser Asn
260 265 270
Ala Gly Ser Val Phe Gly Ala Gly Thr Thr Leu Thr Val Leu Gly Gly
275 280 285
Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Ala Leu Thr
290 295 300
Leu Asp Glu Ser Gly Gly Gly Leu Gln Thr Pro Gly Gly Ala Leu Ser
305 310 315 320
Leu Val Cys Lys Ala Ser Gly Phe Thr Phe Ser Asp Arg Gly Met His
325 330 335
Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Gly Ala Ile
340 345 350
Ser Arg Arg Gly Ser Thr Thr Thr Tyr Ala Pro Ala Val Lys Gly Arg
355 360 365
Ala Thr Ile Thr Arg Asp Asn Gly Gln Ser Thr Val Arg Leu Gln Leu
370 375 380
Asn Asn Leu Thr Ala Glu Asp Thr Ala Thr Tyr Phe Cys Ala Lys Asn
385 390 395 400
Asp Asp Ser Val Gly Ile Val Thr Thr Ser Thr Ile Asp Ala Trp Gly
405 410 415
His
<210> 3
<211> 557
<212> PRT
<213>artificial sequence
<400> 3
Met Ala Gln Val Leu Arg Gly Thr Val Thr Asp Phe Pro Gly Phe Asp
1 5 10 15
Glu Arg Ala Asp Ala Glu Thr Leu Arg Lys Ala Met Lys Gly Leu Gly
20 25 30
Thr Asp Glu Glu Ser Ile Leu Thr Leu Leu Thr Ser Arg Ser Asn Ala
35 40 45
Gln Arg Gln Glu Ile Ser Ala Ala Phe Lys Thr Leu Phe Gly Arg Asp
50 55 60
Leu Leu Asp Asp Leu Lys Ser Glu Leu Thr Gly Lys Phe Glu Lys Leu
65 70 75 80
Ile Val Ala Leu Met Lys Pro Ser Arg Leu Tyr Asp Ala Tyr Glu Leu
85 90 95
Lys His Ala Leu Lys Gly Ala Gly Thr Asn Glu Lys Val Leu Thr Glu
100 105 110
Ile Ile Ala Ser Arg Thr Pro Glu Glu Leu Arg Ala Ile Lys Gln Val
115 120 125
Tyr Glu Glu Glu Tyr Gly Ser Ser Leu Glu Asp Asp Val Val Gly Asp
130 135 140
Thr Ser Gly Tyr Tyr Gln Arg Met Leu Val Val Leu Leu Gln Ala Asn
145 150 155 160
Arg Asp Pro Asp Ala Gly Ile Asp Glu Ala Gln Val Glu Gln Asp Ala
165 170 175
Gln Ala Leu Phe Gln Ala Gly Glu Leu Lys Trp Gly Thr Asp Glu Glu
180 185 190
Lys Phe Ile Thr Ile Phe Gly Thr Arg Ser Val Ser His Leu Arg Lys
195 200 205
Val Phe Asp Lys Tyr Met Thr Ile Ser Gly Phe Gln Ile Glu Glu Thr
210 215 220
Ile Asp Arg Glu Thr Ser Gly Asn Leu Glu Gln Leu Leu Leu Ala Val
225 230 235 240
Val Lys Ser Ile Arg Ser Ile Pro Ala Tyr Leu Ala Glu Thr Leu Tyr
245 250 255
Tyr Ala Met Lys Gly Ala Gly Thr Asp Asp His Thr Leu Ile Arg Val
260 265 270
Met Val Ser Arg Ser Glu Ile Asp Leu Phe Asn Ile Arg Lys Glu Phe
275 280 285
Arg Lys Asn Phe Ala Thr Ser Leu Tyr Ser Met Ile Lys Gly Asp Thr
290 295 300
Ser Gly Asp Tyr Lys Lys Ala Leu Leu Leu Leu Cys Gly Glu Asp Asp
305 310 315 320
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Lys Thr His Thr Cys
325 330 335
Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu
340 345 350
Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu
355 360 365
Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys
370 375 380
Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys
385 390 395 400
Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu
405 410 415
Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys
420 425 430
Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys
435 440 445
Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser
450 455 460
Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys
465 470 475 480
Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln
485 490 495
Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly
500 505 510
Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln
515 520 525
Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn
530 535 540
His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys
545 550 555
<210> 4
<211> 422
<212> PRT
<213>artificial sequence
<400> 4
Lys Pro Thr Glu Asn Asn Glu Asp Phe Asn Ile Val Ala Val Ala Ser
1 5 10 15
Asn Phe Ala Thr Thr Asp Leu Asp Ala Asp Arg Gly Lys Leu Pro Gly
20 25 30
Lys Lys Leu Pro Leu Glu Val Leu Lys Glu Met Glu Ala Asn Ala Arg
35 40 45
Lys Ala Gly Cys Thr Arg Gly Cys Leu Ile Cys Leu Ser His Ile Lys
50 55 60
Cys Thr Pro Lys Met Lys Lys Phe Ile Pro Gly Arg Cys His Thr Tyr
65 70 75 80
Glu Gly Asp Lys Glu Ser Ala Gln Gly Gly Ile Gly Glu Ala Ile Val
85 90 95
Asp Ile Pro Glu Ile Pro Gly Phe Lys Asp Leu Glu Pro Met Glu Gln
100 105 110
Phe Ile Ala Gln Val Asp Leu Cys Val Asp Cys Thr Thr Gly Cys Leu
115 120 125
Lys Gly Leu Ala Asn Val Gln Cys Ser Asp Leu Leu Lys Lys Trp Leu
130 135 140
Pro Gln Arg Cys Ala Thr Phe Ala Ser Lys Ile Gln Gly Gln Val Asp
145 150 155 160
Lys Ile Lys Gly Ala Gly Gly Asp Gly Ser Gly Gly Gly Gly Ser Gly
165 170 175
Gly Gly Gly Ser Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val
180 185 190
Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Asn
195 200 205
Ile Lys Asp Thr Tyr Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly
210 215 220
Leu Glu Trp Val Ala Arg Ile Tyr Pro Thr Asn Gly Tyr Thr Arg Tyr
225 230 235 240
Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys
245 250 255
Asn Thr Ala Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala
260 265 270
Val Tyr Tyr Cys Ser Arg Trp Gly Gly Asp Gly Phe Tyr Ala Met Asp
275 280 285
Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly
290 295 300
Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile Gln Met Thr
305 310 315 320
Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile
325 330 335
Thr Cys Arg Ala Ser Gln Asp Val Asn Thr Ala Val Ala Trp Tyr Gln
340 345 350
Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr Ser Ala Ser Phe
355 360 365
Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Arg Ser Gly Thr
370 375 380
Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr
385 390 395 400
Tyr Tyr Cys Gln Gln His Tyr Thr Thr Pro Pro Thr Phe Gly Gln Gly
405 410 415
Thr Lys Val Glu Ile Lys
420
<210> 5
<211> 402
<212> PRT
<213>artificial sequence
<400> 5
Met Pro Arg Pro Arg Leu Leu Ala Ala Leu Cys Gly Ala Leu Leu Cys
1 5 10 15
Ala Pro Ser Leu Leu Val Ala Leu Asp Ile Cys Ser Lys Asn Pro Cys
20 25 30
His Asn Gly Gly Leu Cys Glu Glu Ile Ser Gln Glu Val Arg Gly Asp
35 40 45
Val Phe Pro Ser Tyr Thr Cys Thr Cys Leu Lys Gly Tyr Ala Gly Asn
50 55 60
His Cys Glu Thr Lys Cys Val Glu Pro Leu Gly Leu Glu Asn Gly Asn
65 70 75 80
Ile Ala Asn Ser Gln Ile Ala Ala Ser Ser Val Arg Val Thr Phe Leu
85 90 95
Gly Leu Gln His Trp Val Pro Glu Leu Ala Arg Leu Asn Arg Ala Gly
100 105 110
Met Val Asn Ala Trp Thr Pro Ser Ser Asn Asp Asp Asn Pro Trp Ile
115 120 125
Gln Val Asn Leu Leu Arg Arg Met Trp Val Thr Gly Val Val Thr Gln
130 135 140
Gly Ala Ser Arg Leu Ala Ser His Glu Tyr Leu Lys Ala Phe Lys Val
145 150 155 160
Ala Tyr Ser Leu Asn Gly His Glu Phe Asp Phe Ile His Asp Val Asn
165 170 175
Lys Lys His Lys Glu Phe Val Gly Asn Trp Asn Lys Asn Ala Val His
180 185 190
Val Asn Leu Phe Glu Thr Pro Val Glu Ala Gln Tyr Val Arg Leu Tyr
195 200 205
Pro Thr Ser Cys His Thr Ala Cys Thr Leu Arg Phe Glu Leu Leu Gly
210 215 220
Cys Glu Leu Asn Gly Cys Ala Asn Pro Leu Gly Leu Lys Asn Asn Ser
225 230 235 240
Ile Pro Asp Lys Gln Ile Thr Ala Ser Ser Ser Tyr Lys Thr Trp Gly
245 250 255
Leu His Leu Phe Ser Trp Asn Pro Ser Tyr Ala Arg Leu Asp Lys Gln
260 265 270
Gly Asn Phe Asn Ala Trp Val Ala Gly Ser Tyr Gly Asn Asp Gln Trp
275 280 285
Leu Gln Val Asp Leu Gly Ser Ser Lys Glu Val Thr Gly Ile Ile Thr
290 295 300
Gln Gly Ala Arg Asn Phe Gly Ser Val Gln Phe Val Ala Ser Tyr Lys
305 310 315 320
Val Ala Tyr Ser Asn Asp Ser Ala Asn Trp Thr Glu Tyr Gln Asp Pro
325 330 335
Arg Thr Gly Ser Ser Lys Ile Phe Pro Gly Asn Trp Asp Asn His Ser
340 345 350
His Lys Lys Asn Leu Phe Glu Thr Pro Ile Leu Ala Arg Tyr Val Arg
355 360 365
Ile Leu Pro Val Ala Trp His Asn Arg Ile Ala Leu Arg Leu Glu Leu
370 375 380
Leu Gly Cys Gly Leu Asn Asp Ile Phe Glu Ala Gln Lys Ile Glu Trp
385 390 395 400
His Glu
<210> 6
<211> 297
<212> PRT
<213>artificial sequence
<400> 6
Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Ser Val Gln Ala Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Thr Ala Ser Gly Phe Gly Val Asp Gly Thr
20 25 30
Asp Met Gly Trp Tyr Arg Gln Ala Pro Gly Asn Glu Cys Glu Leu Val
35 40 45
Ser Ser Ile Ser Ser Ile Gly Ile Gly Tyr Tyr Ser Glu Ser Val Lys
50 55 60
Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr Leu
65 70 75 80
Gln Met Asn Ser Leu Arg Pro Asp Asp Thr Ala Val Tyr Tyr Cys Gly
85 90 95
Arg Arg Trp Ile Gly Tyr Arg Cys Gly Asn Trp Gly Arg Gly Thr Gln
100 105 110
Val Thr Val Ser Ser Gly Gly Ser Gly Gly Ser Gly Gly Ser Gly Gly
115 120 125
Ser Lys Pro Thr Glu Asn Asn Glu Asp Phe Asn Ile Val Ala Val Ala
130 135 140
Ser Asn Phe Ala Thr Thr Asp Leu Asp Ala Asp Arg Gly Lys Leu Pro
145 150 155 160
Gly Lys Lys Leu Pro Leu Glu Val Leu Lys Glu Met Glu Ala Asn Ala
165 170 175
Arg Lys Ala Gly Cys Thr Arg Gly Cys Leu Ile Cys Leu Ser His Ile
180 185 190
Lys Cys Thr Pro Lys Met Lys Lys Phe Ile Pro Gly Arg Cys His Thr
195 200 205
Tyr Glu Gly Asp Lys Glu Ser Ala Gln Gly Gly Ile Gly Glu Ala Ile
210 215 220
Val Asp Ile Pro Glu Ile Pro Gly Phe Lys Asp Leu Glu Pro Met Glu
225 230 235 240
Gln Phe Ile Ala Gln Val Asp Leu Cys Val Asp Cys Thr Thr Gly Cys
245 250 255
Leu Lys Gly Leu Ala Asn Val Gln Cys Ser Asp Leu Leu Lys Lys Trp
260 265 270
Leu Pro Gln Arg Cys Ala Thr Phe Ala Ser Lys Ile Gln Gly Gln Val
275 280 285
Asp Lys Ile Lys Gly Ala Gly Gly Asp
290 295
Claims (10)
1. a kind of separation method of extracellular vesica, characterized in that it comprises:
After pretreated biological sample is mixed incubation with phosphatidylserine capture molecule-affinity tag fusion protein, then
The solid phase that can be specifically bound with the affinity tag in the phosphatidylserine capture molecule-affinity tag fusion protein is added
Carrier forms the extracellular vesicle complexes of carrier-；
The extracellular vesicle complexes of the carrier-are separated from the biological sample again.
2. the separation method of extracellular vesica according to claim 1, which is characterized in that the phosphatidylserine capture
Phosphatidylserine capture molecule in molecule-affinity tag fusion protein includes lactadherin, C1 and/or C2 containing lactadherin
At least one of the polypeptide of structural domain, film mucoprotein-V.
3. the separation method of extracellular vesica according to claim 1, which is characterized in that the surface of the solid phase carrier is even
It is associated with the affinity molecule that can be specifically bound with the affinity tag, the affinity molecule include Protein A/G, affine
At least one of element, glutathione and Ni ion.
4. the separation method of extracellular vesica according to claim 3, which is characterized in that the solid phase carrier includes magnetic
At least one of pearl, carbon nanotube and polymer microballoon.
5. the separation method of extracellular vesica according to claim 1, which is characterized in that the affinity tag includes IgG
Any one in Fc, Avi-tag, GST and hexahistine.
6. a kind of detection method of extracellular vesicle surface marker, characterized in that it comprises:
The extracellular vesicle complexes of the carrier-obtained using the separation method of any one of Claims 1 to 5 are had with coupling
The marker antibody of luciferase, which mixes, to be incubated for, and the extracellular vesica-luciferase compound of carrier-is obtained；
Extracellular vesica-luciferase the compound of the carrier-is cracked, and the substrate of the luciferase is added, described in detection
The luminous intensity of luciferase catalysis.
7. the detection method of extracellular vesicle surface marker according to claim 6, which is characterized in that the coupling has
The marker antibody of luciferase include can simultaneously expressing luciferase and marker antibody luciferase-Antibody Fusion egg
It is white；
Preferably, the luciferase is Gluc.
8. the detection method of extracellular vesicle surface marker according to claim 7, which is characterized in that the fluorescein
In enzyme-antibody fusion protein, connected between the luciferase and marker antibody by flexible small peptide；Preferably, the flexibility
The amino acid sequence of small peptide is (GGGGS)2、(GGGGS)3、G(SGGGG)2S、(GGS)4、(GSG)4With A (EAAAK)2Appointing in A
It anticipates one kind.
9. the detection method of extracellular vesicle surface marker according to claim 6, which is characterized in that the marker
Antibody is the small molecular antibody that can identify the extracellular vesicle surface marker, and the marker antibody includes single-stranded anti-
Body, nano antibody or Fab segment.
10. a kind of for detecting the kit of extracellular vesicle surface marker, characterized in that it comprises: luciferase-is anti-
Body fusion protein, phosphatidylserine capture molecule-affinity tag fusion protein and solid phase carrier.
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| CN111849903A (en) * | 2020-05-26 | 2020-10-30 | 武汉生之源生物科技股份有限公司 | Kit for separating exosome from cell supernatant and using method thereof |
| WO2021088214A1 (en) * | 2019-11-08 | 2021-05-14 | 天津医科大学 | Short peptide exp and short peptide-based drug delivery system and extracellular vesicle recovery kit |
| CN114650831A (en) * | 2019-06-11 | 2022-06-21 | 伊夫罗生物科学公司 | Processed microbial extracellular vesicles |
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