CN110791504A - Technology for screening extracellular vesicle surface protein specific aptamer based on immunomagnetic bead method - Google Patents
Technology for screening extracellular vesicle surface protein specific aptamer based on immunomagnetic bead method Download PDFInfo
- Publication number
- CN110791504A CN110791504A CN201911099384.6A CN201911099384A CN110791504A CN 110791504 A CN110791504 A CN 110791504A CN 201911099384 A CN201911099384 A CN 201911099384A CN 110791504 A CN110791504 A CN 110791504A
- Authority
- CN
- China
- Prior art keywords
- screening
- extracellular vesicles
- extracellular
- positive
- surface protein
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
- C12N15/115—Aptamers, i.e. nucleic acids binding a target molecule specifically and with high affinity without hybridising therewith ; Nucleic acids binding to non-nucleic acids, e.g. aptamers
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
- C12N15/1034—Isolating an individual clone by screening libraries
- C12N15/1065—Preparation or screening of tagged libraries, e.g. tagged microorganisms by STM-mutagenesis, tagged polynucleotides, gene tags
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2310/00—Structure or type of the nucleic acid
- C12N2310/10—Type of nucleic acid
- C12N2310/16—Aptamers
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Genetics & Genomics (AREA)
- Engineering & Computer Science (AREA)
- Chemical & Material Sciences (AREA)
- Biomedical Technology (AREA)
- Organic Chemistry (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Biotechnology (AREA)
- General Engineering & Computer Science (AREA)
- Molecular Biology (AREA)
- Microbiology (AREA)
- Plant Pathology (AREA)
- Biophysics (AREA)
- Physics & Mathematics (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- Crystallography & Structural Chemistry (AREA)
- Bioinformatics & Computational Biology (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention discloses a technology for screening extracellular vesicle surface protein specific aptamers based on an immunomagnetic bead method, which comprises the following steps: 1) synthesizing upstream and downstream primers and a random library; 2) culturing a target cell and obtaining a cell culture supernatant; 3) extracting the extracellular vesicles by a particle size selection method; 4) the surface of the CD63 antibody-modified immunomagnetic beads captures extracellular vesicles; 5) positive screening to obtain library sequences capable of being combined with target extracellular vesicles; 6) positive and negative alternate screening to obtain specific target aptamer; 7) the enrichment of the library was examined by flow cytometry. The invention adopts a particle size selection method to extract the extracellular vesicles, is simple and rapid, has high recovery rate of the extracellular vesicles, uses immunomagnetic beads with the surface modified with CD63 antibody to capture exosomes, can rapidly separate the extracellular vesicles, further can improve the screening efficiency of target aptamer through positive screening and positive-negative alternate screening, and can carry out liquid biopsy and treatment of diseases with extracellular vesicle surface protein as a target by using the screened aptamer.
Description
Technical Field
The invention belongs to the field of molecular biology, and particularly relates to a technology for screening extracellular vesicle surface protein specific aptamers based on an immunomagnetic bead method.
Background
Extracellular Vesicles (EVs) are vesicle-like bodies with a double-layer membrane structure that are shed from cell membranes or secreted by cells, have diameters varying from 40nm to 1000nm, are released from cells to the Extracellular environment, and are present in body fluids such as blood, saliva, amniotic fluid, breast milk, urine, and the like. EVs contain specific substances in the cells from which they are derived, including proteins (e.g., cytokines, membrane receptor proteins, cytoskeletal proteins, heat shock proteins, etc.), nucleic acids (e.g., DNA, mRNA, small interfering RNA, long non-coding RNA, etc.) and lipids, and when different physiological conditions and diseases occur, proteins and nucleic acids in EV are differentially expressed, so that isolated and purified EVs can be identified based on the specificity of protein composition. Therefore, EVs provide important basis for early diagnosis, treatment and prognosis of tumors.
The EVs separation method comprises ultracentrifugation, gel exclusion chromatography, filtration, precipitation and the like, but the traditional methods of ultracentrifugation, gel exclusion chromatography and the like for separation based on physical characteristics such as size, density, surface charge and the like have the problems of low flux, complexity, no standardization method, low recovery rate and low purity; the commercial kit adopting the precipitation separation method has the problem that non-exosome material pollution is easy to occur, and the separation of EVs is still a big problem which limits related research and application of EVs at present.
The novel particle size selection method is characterized in that macromolecular substances such as cell fragments and the like in cell culture supernatant, micromolecular substances and small-particle-size trapped extracellular vesicles are sequentially filtered in a membrane-passing mode twice, the extracellular vesicles with uniform particle sizes, complete structures and complete composition can be obtained, the method is simple to operate, the extracellular vesicles can be rapidly separated, the selected nanofiltration membrane has no specific adsorption, and the recovery rate of the extracellular vesicles is high.
The immunomagnetic bead separation technology is a biological technology which uses magnetic microspheres with superparamagnetism as carriers to capture and enrich target substances. The immunomagnetic beads are also called immunomagnetic microspheres, and are formed by taking nano magnetic materials as solid phase carriers and adding a macromolecule coating layer on the surface of the solid phase carriers to introduce active groups (carboxyl, amino, sulfydryl, aldehyde group and the like) so that the solid phase carriers can be effectively combined with biological groups or biological ligands to realize the specific separation of target substances. The immunomagnetic separation technology comprises the following steps: large specific surface area, good magnetic responsiveness, high separation speed, simple operation, no need of large-scale instruments, low cost, high separation efficiency, good biocompatibility and the like. At present, the immunomagnetic separation technology has good application in the aspects of separating and enriching microorganisms, extracting nucleic acid, purifying protein, sorting cells and the like. In the aspect of aptamer screening, the immunomagnetic beads and extracellular vesicles are incubated, and the extracellular vesicles are identified and captured by specific antibodies connected with the magnetic beads, so that an immunomagnetic bead-extracellular vesicle compound can be formed, the compound can be combined with a target sequence in a ssDNA library, and the unbound sequence can be removed under the action of an external magnetic field, so that specific aptamers are enriched, and the purpose of screening the specific aptamers is finally achieved.
The tetraspanin (CD9, CD63 and the like) is common protein and appears in all exosomes, so that the CD63 antibody is selected to modify the magnetic beads, the spectrum of the method is enhanced, and the immunomagnetic beads have the advantages of easiness in capturing extracellular vesicles, easiness in separation, rapidness and simplicity, reduction in interference of other impurities, reduction in cost and the like.
Aptamers are short, single-stranded oligonucleotide molecules (RNA or ssDNA) screened from in vitro synthetic oligonucleotide libraries by the exponential enrichment of ligands phylogenetic technique (SELEX). The aptamer can be efficiently bound to a target substance by folding into a secondary or tertiary structure in such action modes as hydrogen bonding and shape matching. Aptamers affect the biological activity and biological function of a target substance by specifically binding to the target substance, and function similarly to antibodies. Compared with antibodies, the aptamer has the remarkable advantages of no immunogenicity, stable property, easiness in synthesis, easiness in modification, wide target substance range and the like. At present, the target of screening for aptamers is mainly cells, and the Cell-SELEX technology is relatively mature. Based on the Cell-SELEX technology, specific aptamers of various tumor cells have been screened, but extracellular vesicle aptamer screening based on an immunomagnetic bead method is rarely reported.
Disclosure of Invention
The invention aims to provide a technology for screening extracellular vesicle surface protein specific aptamers based on an immunomagnetic bead method, which is characterized in that uniform and complete extracellular vesicles are extracted by a particle size selection method, a random library and immunomagnetic beads capturing the extracellular vesicles are mixed and incubated, after 5-6 rounds of positive screening, positive and negative alternate screening is carried out to screen out aptamers with high specificity, the obtained aptamers are used as new cancer markers, and the aptamers are applied to early liquid biopsy to provide a more accurate and efficient technical scheme.
In order to achieve the technical purpose, the technical scheme adopted by the invention is as follows: the technology for screening the extracellular vesicle surface protein specific aptamer based on the immunomagnetic bead method comprises the following steps:
1) synthesizing upstream and downstream primers and a random library;
2) obtaining a target cell culture supernatant;
3) extracting the extracellular vesicles by a particle size selection method;
4) the surface of the CD63 antibody-modified immunomagnetic beads captures extracellular vesicles;
5) positive screening to obtain library sequences bound to extracellular vesicles;
6) positive and negative alternate screening to obtain specific target aptamer;
7) the library enrichment was monitored by flow cytometry.
Further, the random library in step 1) is a single-stranded oligonucleotide of 80nt, which is synthesized by bio-corporation, the sequence of the single-stranded oligonucleotide is 5 '-GTTGGTGAGGTAACGGCTCA-40 nt-TAGGTGGCAAGCGTTATCCG-3', wherein 40nt represents a random sequence of 40 nucleotides (nt), and the upstream and downstream primer sequences are respectively: upstream primer F (SEQ ID NO: 1): 5 '-FAM-GTTGGTGAGGTAACGGCTCA-3'; downstream primer R (SEQ ID NO: 2): 5 '-biotin-CGGATAACGCTTGCCACCTA-3'; wherein FAM is a fluorescent label, and Biotin is a Biotin label.
Further, the culturing of the target cells and the obtaining of the cell culture supernatant in the step 2) are as follows: and (2) placing the target cells into a cell culture solution containing Fetal Bovine Serum (FBS) for culture, placing the cells into the cell culture solution containing the FBS without the extracellular vesicles for continuous culture when the cells grow to a stable state, and obtaining a cell culture supernatant when the cells grow to 70-80% of the bottom of the dish.
Further, the extraction of the extracellular vesicles by the particle size selection method in the step 3) is as follows: filtering the cell culture supernatant collected in the step 2) through 20-6000 nm filter membranes respectively by a vacuum suction pump, washing the cell culture supernatant for three times by a washing solution, and then resuspending and collecting extracellular vesicles by a PBS (phosphate buffer solution).
Wherein the cleaning solution is prepared from 4.5g/L glucose and 5mM MgCl2·6H2O was dissolved in 0.01M PBS buffer.
Wherein, the extracellular vesicles extracted in the step 3) are subjected to concentration measurement by a Nanosight particle size analyzer.
Further, the step 4) of capturing the extracellular vesicles by the immunomagnetic beads with the surface modified CD63 antibodies is as follows: washing the immunomagnetic beads modified with the CD63 antibody on the surface once by using a separation solution, dissolving the immunomagnetic beads in a proper amount of the separation solution, adding a proper amount of the extracellular vesicles extracted in the step 3), then incubating at 4 ℃ overnight, carrying out magnetic separation by using a magnet, washing three times by using the separation solution, removing the unbound extracellular vesicles, and retaining the extracellular vesicles captured by the immunomagnetic beads.
Wherein the separating medium is 0.1% BSA in 0.01M PBS buffer.
Wherein the appropriate number of extracellular vesicles is about 1X 1010And (4) respectively.
Further, the positive screening in the step 5) comprises the following steps:
5.1) random library with capture extracellular vesicles immunomagnetic beads incubation: dissolving a random library in a binding solution, carrying out high-temperature treatment at 95 ℃ for 5min by using a metal bath, immediately carrying out ice bath for 10min to form a single-chain secondary structure, mixing the single-chain secondary structure with the immunomagnetic beads for capturing the extracellular vesicles in the step 4), and incubating for 90min at 4 ℃;
further, the preparation of the binding solution is as follows: 4.5g/L glucose, 5mM MgCl2·6H2O, 0.1mg/mL yeast tRNA and 1mg/mL BSA in 0.01M PBS buffer;
5.2) separation of extracellular vesicles from free DNA: magnetically separating the immunomagnetic beads in the mixed solution after incubation in the step 5.1) by using a magnet, removing the supernatant, washing the supernatant by using a washing solution, re-suspending the immunomagnetic beads combined with the extracellular vesicles by using a TE buffer solution, boiling the solution at 100 ℃, magnetically separating the immunomagnetic beads, and collecting the supernatant containing the single-chain secondary structure;
5.3) preparation of Secondary libraries:
5.31) PCR amplification: performing PCR amplification by taking the supernatant containing the single-chain secondary structure collected in the step 5.2) as a template and taking the upstream primer and the downstream primer synthesized in the step 1) as a specific primer pair to obtain a PCR amplification product, wherein the PCR amplification conditions are as follows:
an amplification system: upstream and downstream primers F/R (10. mu.M) each 0.5. mu.L, template 20ng, 2 XPromix 10. mu.L, supplemented with ddH2O to a total volume of 20 muL;
and (3) amplification procedure: 5min at 95 ℃, 30s at 58.6 ℃ and 30s at 72 ℃ for 3min in N cycles; storing at 4 ℃. Wherein, the number of N rounds of circulation is determined according to the gel imaging result in the front round of screening, and the first round of amplification is carried out for 8 cycles;
5.32) magnetically separating PCR amplification products: and (3) incubating and combining the PCR amplification product modified with biotin in the step 5.31) with streptavidin immunomagnetic beads, magnetically separating the streptavidin immunomagnetic beads, removing supernatant, washing by using separation liquid, adding sodium hydroxide to prepare single-stranded ssDNA serving as a secondary library, and measuring the concentration of the secondary library by using an ultramicro ultraviolet spectrophotometer.
Further, the positive sieve in the step 5) is continuously subjected to 5-6 rounds, and in the 5-6 rounds of positive sieves, the single chain combined with the extracellular vesicle captured by the immunomagnetic beads in the step 4) in the next round of positive sieves is a product obtained by treating the secondary library prepared in the previous round of positive sieves at a high temperature of 95 ℃ in a metal bath for 5min and immediately performing ice bath for 10 min.
Further, the negative sieve in the step 6) comprises the following steps:
6.1) extracting normal cell extracellular vesicles: obtaining normal extracellular vesicles through a particle size selection method, and analyzing the concentration of the extracted normal extracellular vesicles through a Nanosight particle sizer;
6.2) extracting the aptamer which is not combined with the extracellular vesicle of the normal cell: mixing and incubating the secondary library prepared in the step 5.3) with immunomagnetic beads for capturing normal extracellular vesicles, and collecting supernatant after magnetic separation.
Further, the positive and negative alternate screening is as follows: and (3) carrying out primary positive screening and primary negative screening for one round, carrying out next round of positive screening after one round of negative screening, wherein in the next round of positive screening, the single chain combined with the extracellular vesicles secreted by the cancer captured by the immunomagnetic beads in the step 4) is the supernatant collected in the previous round of negative screening, and carrying out ice bath treatment on the single chain for 10min immediately after carrying out high-temperature treatment on the single chain at 95 ℃ in a metal bath for 5 min.
Further, the total number of positive screening rounds in the step 5) and the total number of positive and negative alternate screening rounds in the step 6) are 10-20 rounds, after the positive screening in the step 5) is continuously performed for 5-6 rounds, the negative screening is introduced for positive and negative alternate screening, the incubation time of the positive screening is gradually shortened in the positive and negative alternate screening, the cleaning force is gradually increased by gradually increasing the cleaning times during separation, the time of the negative screening is gradually increased, and the screening pressure is gradually increased to obtain the specific aptamer.
Further, the flow cytometry detection technology is adopted in the step 7) to detect the fluorescence intensity of the extracellular vesicles mixed and incubated with the secondary library, when the screening reaches a certain number of turns, the detected fluorescence intensity value does not change any more and tends to be stable, which indicates that the secondary library is successfully enriched, the screening reaches a plateau stage, and the screening can be terminated.
The invention has the following beneficial effects:
1) the method for selecting the particle size is adopted to extract the extracellular vesicles from the supernatant of the target cell culture, so that the method is simple and rapid and the recovery rate of the extracellular vesicles is high;
2) the exosome is captured by the immunomagnetic beads with the surface modified with the CD63 antibody, extracellular vesicles can be rapidly separated under the action of a magnetic field, the screening time is shortened, the steps are simple, convenient and rapid, the extracellular vesicles can be further purified by capturing the extracellular vesicles by the immunomagnetic beads, the influence of foreign impurities on the screening is avoided, meanwhile, the screening of nonspecific aptamers aiming at surface protein CD63 with rich surface content is avoided, the screening probability of specific aptamers is enhanced, the use of a filtering membrane is reduced, and the cost can be reduced;
3) the screening efficiency of the target aptamer can be improved by positive screening and positive and negative alternate screening;
4) the fluorescence intensity of the secondary library of two continuous positive and negative alternative screens and the extracellular vesicles after combination is detected by a flow cytometry detection technology and does not change any more and tends to be stable, the termination of screening can be determined, the specificity of the screened target nucleic acid aptamer is further detected by the flow cytometry detection technology, a novel accurate, noninvasive, simple and economic method for screening early cancer liquid biopsy is provided for clinic, and the method has very important social value and clinical significance.
Drawings
FIG. 1 is a technical flow diagram of the present invention;
FIG. 2 is a graph showing the results of the enrichment degree of the library by flow cytometry, wherein: the abscissa represents fluorescence intensity, the ordinate represents cell number, a: the average fluorescence intensity of the surfaces of the Beas-2b extracellular vesicles after mixed incubation with the random library (a), the secondary library screened in round 6 (b), the secondary library screened in round 17 (c) and the secondary library screened in round 18 (d); b: average fluorescence intensity of the extracellular vesicles surface of a549 cells after mixed incubation with the random library (e), the secondary library screened in round 6 (f), the secondary library screened in round 17 (g), and the secondary library screened in round 18 (h);
FIG. 3is a diagram showing the results of flow cytometry for detecting the specificity of the aptamer screened in example 1, wherein: the abscissa represents fluorescence intensity, the ordinate represents cell number, a is control: the Beas-2B extracellular vesicles (a) and the Beas-2B extracellular vesicles are mixed with a fluorescence-labeled random library (B) and a fluorescence-labeled aptamer (SEQ ID NO: 3) Ap4(c) respectively to obtain the average fluorescence intensity of the surfaces of the Beas-2B extracellular vesicles after incubation, and B is an experimental group: and the average fluorescence intensity of the surfaces of the A549 cell extracellular vesicles after the A549 cell extracellular vesicles (d) and the A549 cell extracellular vesicles are respectively mixed and incubated with a fluorescence-labeled random library (e) and a fluorescence-labeled aptamer (SEQ ID No: 3) Ap4(f) obtained by sequencing.
Detailed Description
In order to make the technical solutions of the present invention better understood by those skilled in the art, the present invention is further described below by using specific examples.
The experimental procedures used in the following examples are conventional unless otherwise specified, and the reagents used therein are commercially available without further specification.
Detailed description of the preferred embodiment 1
The method for screening the human lung adenocarcinoma cell A549 cell extracellular vesicle surface protein specific aptamer based on an immunomagnetic bead method comprises the following steps:
1) synthesis of upstream and downstream primers and random library
Specific primer pair F/R and random library sequences are synthesized by Shanghai biological Limited, and the sequence of the upstream primer F is shown as SEQ ID NO: 1, the sequence of the downstream primer R is shown as SEQ ID NO: 2, the sequence of the random library is shown as SEQ ID NO: 3, showing:
upstream primer F (SEQ ID NO: 1): 5 '-FAM-GTTGGTGAGGTAACGGCTCA-3'
Downstream primer R (SEQ ID NO: 2): 5 '-biotin-CGGATAACGCTTGCCACCTA-3';
random library: 35 '-GTTGGTGAGGTAACGGCTCA-40 nt-TAGGTGGCAAGCGTTATCCG-3';
further, the random library in the step 1) is a single-stranded oligonucleotide of 80nt, the middle 40nt of the single-stranded oligonucleotide is 40 random base sequences, the 5 'end of the upstream primer is modified by a fluorescent label (FAM), and the 5' end of the downstream primer is labeled by Biotin (Biotin).
2) Culture of a549 cells and harvest of cell culture supernatant: placing human lung adenocarcinoma cell A549 cell from ATCC into RPMI 1640 culture solution containing 10% Fetal Bovine Serum (FBS) for culturing, observing cell growth to a stable state under a microscope, replacing the culture solution, continuously culturing the cell in RPMI 1640 culture medium containing 10% fetal bovine serum (fetal bovine serum without extracellular vesicles after special treatment) for 24-48h, and collecting cell supernatant when the cell abundance reaches 80%;
3) extracting the A549 cell extracellular vesicles by a particle size selection method: filtering the culture supernatant of the A549 cells collected in the step 2) by a vacuum suction pump through a 600 nm-aperture polycarbonate membrane-track etching membrane and a 20 nm-aperture porous anodic alumina membrane in sequence, washing the supernatant for three times, then resuspending and collecting the extracellular vesicles by using PBS (phosphate buffer solution), and measuring the concentration of the extracted extracellular vesicles by using a Nanosight particle sizer;
further, the washing solution was 4.5g/L glucose and 5mM MgCl2·6H2O was dissolved in 0.01M PBS buffer.
4) The method for capturing extracellular vesicles by using the immunomagnetic beads with the surface modified with CD63 antibodies specifically comprises the following steps: washing the immunomagnetic beads modified with the CD63 antibody on the surface once by using a separation solution, dissolving the immunomagnetic beads in a proper amount of the separation solution, adding a proper amount of the extracellular vesicles extracted in the step 3), then incubating at 4 ℃ overnight, carrying out magnetic separation by using a magnet, washing three times by using the separation solution, removing the unbound extracellular vesicles, and retaining the extracellular vesicles captured by the immunomagnetic beads.
Wherein the separating medium is 0.1% BSA in 0.01M PBS buffer.
Wherein the appropriate amount of extracellular vesicles is about 1 × 1010And (4) respectively.
Among them, immunomagnetic beads (Exosome-Human CD63Isolation/detection reagent) (from cell culture media) surface-modified with CD63 antibody were purchased from seimer heschel technologies (china) ltd.
5) The positive screening is used for obtaining a library sequence combined with the extracellular vesicles, and the method specifically comprises the following steps:
5.1) random library with capture extracellular vesicles immunomagnetic beads incubation: dissolving a random library in a binding solution, carrying out high-temperature treatment at 95 ℃ for 5min by using a metal bath, immediately carrying out ice bath for 10min to form a single-chain secondary structure, and then mixing and incubating the A549 cell extracellular vesicles captured by the immunomagnetic beads in the step 4), the 4 Xbinding solution and the single-chain secondary structure for 90min at a ratio of 5:2: 1;
wherein the binding solution is 4.5g/L glucose, 5mM MgCl2·6H2O, 0.1mg/mL yeast tRNA and 1mg/mL BSA were dissolved in 0.01M PBS buffer.
5.2) separation of extracellular vesicles from free DNA: magnetically separating immunomagnetic beads from the mixed solution after incubation in the step 5.1) by using a magnet, removing supernatant, cleaning by using a cleaning solution, re-suspending the immunomagnetic beads combined with extracellular vesicles by using TE buffer solution, boiling at 100 ℃, magnetically separating the immunomagnetic beads, and collecting supernatant containing single-chain secondary structures
5.3) preparation of Secondary libraries:
5.31) PCR amplification: performing PCR amplification by taking the supernatant containing the single-chain secondary structure collected in the step 5.2) as a template and taking the upstream and downstream primers (F/R) synthesized in the step 1) as a specific primer pair to obtain a PCR amplification product, wherein the PCR amplification conditions are as follows:
an amplification system: upstream and downstream primers F/R (10. mu.M) each 0.5. mu.L, template 20ng, 2 XPromix 10. mu.L, supplemented with ddH2O to a total volume of 20 muL;
and (3) amplification procedure: 5min at 95 ℃, 30s at 58.6 ℃ and 30s at 72 ℃ for 3min in N cycles; 4 ℃ and infinity.
In the embodiment, the number of cycles N is determined by optimization according to the gel imaging result in the previous screening, 8 cycles of first-round amplification are carried out, and 2, 4, 6, 8, 10, 12, 14, 16, 18 and 20 rounds of random libraries are determined by optimization of the number of cycles of cyclic amplification respectively before experiments;
5.32) magnetically separating PCR amplification products: and (3) incubating and combining the PCR amplification product modified with biotin in the step 5.31) with streptavidin-modified magnetic beads, magnetically separating the streptavidin-modified magnetic beads, removing supernatant, washing by using separation liquid, adding sodium hydroxide to prepare single-stranded ssDNA serving as a secondary library, and measuring the concentration of the secondary library by using an ultra-micro ultraviolet spectrophotometer.
Wherein the streptavidin modified magnetic beads are self-prepared for the laboratory.
Further, the positive sieve in the step 5) is continuously subjected to 5-6 rounds, and in the 5-6 rounds of positive sieves, the single chain combined with the extracellular vesicle captured by the immunomagnetic beads in the step 4) in the next round of positive sieves is a product obtained by treating the secondary library prepared in the previous round of positive sieves at a high temperature of 95 ℃ in a metal bath for 5min and immediately performing ice bath for 10 min.
6) Introducing a negative sieve, and screening alternately to obtain a specific target aptamer, wherein the negative sieve comprises the following steps:
6.1) extraction of normal cell extracellular vesicles: obtaining normal extracellular vesicles through a particle size selection method, and analyzing the concentration of the extracted normal extracellular vesicles through a Nanosight particle sizer;
6.2) extraction of aptamers not bound to normal extracellular vesicles: mixing and incubating immunomagnetic beads capturing normal extracellular vesicles, 4 x binding solution and the secondary library prepared in the step 5.32) for 30min at a ratio of 5:2:1, performing magnetic separation, and collecting supernatant.
Further, the positive and negative alternate screening is as follows: and (3) carrying out primary positive screening and primary negative screening for one round, carrying out next round of positive screening after one round of negative screening, wherein in the next round of positive screening, the single chain combined with the A549 cell extracellular vesicles captured by the immunomagnetic beads in the step 4) is the supernatant collected in the previous round of negative screening, and carrying out ice bath treatment on the single chain for 10min immediately after carrying out high-temperature treatment on the single chain at 95 ℃ for 5min by using a metal bath.
Further, the total number of positive screening rounds in the step 5) and the total number of positive and negative alternate screening rounds in the step 6) are 10-20 rounds, after the positive screening in the step 5) is continuously performed for 5-6 rounds, the negative screening is introduced for positive and negative alternate screening, the incubation time of the positive screening is gradually shortened in the positive and negative alternate screening, the cleaning force is gradually increased by gradually increasing the cleaning times during separation, the time of the negative screening is gradually increased, and the screening pressure is gradually increased to obtain the specific aptamer.
7) Monitoring library enrichment by flow cytometry:
the method comprises the following specific steps:
7.1) amplifying the secondary libraries of each round of screening (the secondary library of round 6 screening, the secondary library of round 17 screening and the secondary library of round 18 screening) respectively, and preparing FAM fluorescence modified single-stranded libraries;
7.2) carrying out metal bath treatment at the high temperature of 95 ℃ for 5min on the FAM fluorescence modified single-chain library, and then immediately carrying out ice bath for 10 min;
7.3) mixing the A549 cell extracellular vesicles, 4 multiplied by binding solution and the single chains processed in the step 7.2) according to the proportion of 5:2:1, incubating for 1h, filtering the mixed solution through a 20nm filter membrane, washing the washing solution, and then resuspending and collecting the vesicles trapped outside the 20nm filter membrane by PBS to serve as an experimental group; mixing Beas-2b extracellular vesicles, 4 Xbinding solution and the single chains processed in the step 7.2) at a ratio of 5:2:1, incubating for 1h, filtering the mixed solution through a 20nm filter membrane, washing the washing solution, and then resuspending and collecting the extracellular vesicles trapped on the 20nm filter membrane by PBS to serve as a control group.
Detecting the surface average fluorescence intensity of the Beas-2b extracellular vesicles or the A549 cell extracellular vesicles after mixed incubation with the secondary library by a flow cytometer, as shown in FIG. 2A, compared with the random library (a), the surface average fluorescence intensity of each round of screened secondary libraries (the secondary library (b) screened in the 6 th round, the secondary library (c) screened in the 17 th round and the secondary library (d) screened in the 18 th round) after being combined with the Beas-2b extracellular vesicles is basically unchanged along with the increase of the number of screening rounds, which indicates that the screened aptamer is not combined with the Beas-2b extracellular vesicles; as shown in fig. 2B, as compared with the random library (e), the average fluorescence intensity of the surface of the secondary libraries screened in each round (the secondary library screened in round 6 (f), the secondary library screened in round 17 (g) and the secondary library screened in round 18 (h)) after binding to the extracellular vesicles of a549 cells is significantly increased, and when the number of rounds of screening reaches round 18 (h), the extracellular vesicles do not increase any more than that of round 17 (g), indicating that the secondary libraries are successfully enriched, and when the number of rounds of screening reaches round 17, the screening reaches a plateau stage, and the screening can be terminated;
8) detecting the specificity of the screened target aptamer by using a flow cytometry technology: amplifying the screened product of the 17 th round by fluorescent PCR, sending the amplified product to Shanghai Biometrics Limited company for clone sequencing, combining the aptamer sequences (SEQ ID No: 3) obtained by random library and sequencing with Beas-2b human normal lung epithelial cell extracellular vesicles and A549 human lung adenocarcinoma cell extracellular vesicles respectively, incubating for 15min, washing for 3 times by PBS buffer solution, and performing specificity detection on the aptamer sequence (SEQ ID No: 3) combined with extracellular vesicle surface protein by using a Beckman flow cytometer;
the method comprises the following specific steps:
8.1) synthesizing FAM modified single chains from the random library and the screened specific aptamers in a biological company respectively;
8.2) dissolving the single chain of which the 5' -end is modified with FAM fluorescence by using sterile water, carrying out high-temperature treatment on the single chain at 95 ℃ for 5min by using a metal bath, and immediately carrying out ice bath for 10min to obtain a library;
8.3) mixing Beas-2b extracellular vesicles with 4 multiplied by binding solution and a random library treated in 8.2) according to the proportion of 5:2:1, incubating for 1h, filtering the mixed solution through a 20nm filter membrane, washing the mixed solution, then resuspending and collecting the extracellular vesicles trapped on the 20nm filter membrane by PBS; mixing Beas-2b extracellular vesicles with 4 multiplied by binding solution and the specific aptamer treated in 8.2) according to the ratio of 5:2:1, incubating for 1h, filtering the mixed solution through a 20nm filter membrane, washing the washing solution, and then resuspending and collecting the extracellular vesicles trapped on the 20nm filter membrane by PBS; mixing the A549 cell extracellular vesicles with 4 multiplied by binding solution and a random library treated in 8.2) according to the ratio of 5:2:1, incubating for 1h, filtering the mixed solution through a 20nm filter membrane, washing the washed solution, and then resuspending and collecting the vesicles trapped on the 20nm filter membrane extracellular vesicles by PBS; mixing the A549 cell extracellular vesicles with 4 multiplied by binding solution and the specific aptamer treated in 8.2) according to the ratio of 5:2:1, incubating for 1h, filtering the mixed solution through a 20nm filter membrane, washing the washing solution, and then resuspending and collecting the vesicles trapped on the 20nm filter membrane extracellular vesicles by PBS;
wherein, the extraction of Beas-2b extracellular vesicles refers to step 2).
Wherein Beas-2b extracellular vesicles (a) and Beas-2b extracellular vesicles are mixed with a fluorescence-labeled random library (b) and a fluorescence-labeled aptamer Ap4(c) obtained by sequencing to serve as a control group (A); taking the A549 cell extracellular vesicles (d) and the A549 cell extracellular vesicles as an experimental group (B), wherein the A549 cell extracellular vesicles are respectively mixed and incubated with a fluorescence-labeled random library (e) and a fluorescence-labeled aptamer Ap4(f) obtained by sequencing;
as shown in FIG. 3, the average fluorescence intensity of the extracellular vesicle surfaces of the cells of the control group (A) and the experimental group (B) is that the aptamer Ap4 has strong binding capacity with the extracellular vesicle secreted by the target cell A549 (FIG. 3B-f) and does not substantially bind with the extracellular vesicle secreted by the Beas-2B cell (FIG. 3A-c), which indicates that the aptamer obtained by sequencing specifically binds with the extracellular vesicle of the A549 human lung adenocarcinoma cell and non-specifically binds with the extracellular vesicle of the Beas-2B human normal lung epithelial cell, and indicates that the A549 cell extracellular vesicle surface protein specific aptamer can be screened by the extracellular vesicle aptamer screening technology based on the immunomagnetic bead method.
The above is only a preferred embodiment of the present invention, and the protection scope of the present invention is not limited to the above-mentioned embodiments, and all technical solutions belonging to the idea of the present invention belong to the protection scope of the present invention. It should be noted that modifications and embellishments within the scope of the invention may be made by those skilled in the art without departing from the principle of the invention.
Sequence listing
<110> Nanjing drum building hospital
<120> extracellular vesicle surface protein specific aptamer screening technology based on immunomagnetic bead method
<160>3
<170>SIPOSequenceListing 1.0
<210>1
<211>20
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>1
gttggtgagg taacggctca 20
<210>2
<211>20
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>2
cggataacgc ttgccaccta 20
<210>3
<211>80
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>3
gttggtgagg taacggctca gttgtggggg ggtgggtggt ttctgggttt acttgggcgc 60
taggtggcaa gcgttatccg 80
Claims (11)
1. The technology for screening the extracellular vesicle surface protein specific aptamer based on the immunomagnetic bead method is characterized by comprising the following steps:
1) synthesizing upstream and downstream primers and a random library;
2) obtaining a target cell culture supernatant;
3) extracting the extracellular vesicles by a particle size selection method;
4) the surface of the CD63 antibody-modified immunomagnetic beads captures extracellular vesicles;
5) positive screening to obtain library sequences bound to extracellular vesicles;
6) positive and negative alternate screening to obtain specific target aptamer;
7) the library enrichment was monitored by flow cytometry.
2. The method for screening extracellular vesicle surface protein-specific aptamers according to claim 1, wherein the extracellular vesicle surface protein-specific aptamers are selected from the group consisting of: the random library in the step 1) is a single-stranded oligonucleotide of 80nt, the sequence of the single-stranded oligonucleotide is 5 '-GTTGGTGAGGTAACGGCTCA-40 nt-TAGGTGGCAAGCGTTATCCG-3', wherein 40nt represents a random sequence of 40 nucleotide bases (nt), and the sequences of the upstream primer and the downstream primer are respectively as follows: upstream primer F (SEQ ID NO: 1): 5 '-FAM-GTTGGTGAGGTAACGGCTCA-3'; downstream primer R (SEQ ID NO: 2): 5 '-biotin-CGGATAACGCTTGCCACCTA-3'; wherein FAM is a fluorescent label, and Biotin is a Biotin label.
3. The method for screening extracellular vesicle surface protein-specific aptamers according to claim 1, wherein the extracellular vesicle surface protein-specific aptamers are selected from the group consisting of: the step 2) of obtaining the target cell culture supernatant comprises the following steps: and (2) placing the target cells into Fetal Bovine Serum (FBS) for culture, placing the cells into the Fetal Bovine Serum (FBS) without extracellular vesicles for continuous culture when the cells grow to a stable state, and obtaining cell culture supernatant when the cells grow to 70% -80% of the bottom of the dish.
4. The method for screening extracellular vesicle surface protein-specific aptamers according to claim 3, wherein the extracellular vesicle surface protein-specific aptamers are selected from the group consisting of: the extraction of the extracellular vesicles by the particle size selection method in the step 3) comprises the following steps: filtering the cell culture supernatant collected in the step 2) through a vacuum suction pump according to the needs by filter membranes with the pore sizes of 20nm-5000nm respectively, washing for three times by a washing liquid, then using PBS buffer solution to resuspend and collect extracellular vesicles, and measuring the concentration of the extracted extracellular vesicles by a Nanosight particle sizer.
5. The method for screening extracellular vesicle surface protein-specific aptamers according to claim 4, wherein the extracellular vesicle surface protein-specific aptamers are selected from the group consisting of: the step 4) of capturing the extracellular vesicles by the immunomagnetic beads with the surface modified with the CD63 antibody comprises the following steps: washing the immunomagnetic beads modified with the CD63 antibody on the surface once by using a separation solution, dissolving the immunomagnetic beads in the separation solution, adding a proper amount of the extracellular vesicles extracted in the step 3), then incubating overnight at 4 ℃, carrying out magnetic separation by using a magnet, washing three times by using the separation solution, removing the unbound extracellular vesicles, and retaining the immunomagnetic beads for capturing the extracellular vesicles.
6. The method for screening extracellular vesicle surface protein-specific aptamers according to claim 5, wherein the extracellular vesicle surface protein-specific aptamers are selected from the group consisting of: the positive screening in the step 5) comprises the following steps:
5.1) random library with capture extracellular vesicles immunomagnetic beads incubation: dissolving a random library in a binding solution, carrying out high-temperature treatment at 95 ℃ for 5min by using a metal bath, immediately carrying out ice bath for 10min to form a single-chain secondary structure, mixing the single-chain secondary structure with the immunomagnetic beads for capturing the extracellular vesicles in the step 4), and incubating for 90min at 4 ℃;
5.2) separation of extracellular vesicles from free DNA: magnetically separating the immunomagnetic beads in the mixed solution incubated in the step 5.1) by using a magnet, removing the supernatant, washing the supernatant by using a washing solution, re-suspending the immunomagnetic beads combined with the extracellular vesicles by using a TE buffer solution, magnetically separating the immunomagnetic beads after boiling at 100 ℃, and collecting the supernatant containing the single-chain secondary structure;
5.3) preparation of the Secondary library
5.31) PCR amplification: performing PCR amplification by taking the supernatant containing the single-chain secondary structure collected in the step 5.2) as a template and taking the upstream primer and the downstream primer synthesized in the step 1) as a specific primer pair to obtain a PCR amplification product, wherein the PCR amplification conditions are as follows:
an amplification system: upstream and downstream primers F/R (10. mu.M) each 0.5. mu.L, template 20ng, 2 XPromix 10. mu.L, supplemented with ddH2O to a total volume of 20 muL;
and (3) amplification procedure: 5min at 95 ℃, 30s at 58.6 ℃ and 30s at 72 ℃ for 3min in N cycles; storing at 4 deg.C;
wherein, the number of N rounds of circulation is determined according to the gel imaging result in the front round of screening, and the first round of amplification is carried out for 8 cycles;
5.32) magnetically separating PCR amplification products: and (3) incubating and combining the PCR amplification product modified with biotin in the step 5.31) with streptavidin immunomagnetic beads, magnetically separating the streptavidin immunomagnetic beads, removing supernatant, washing by using separation liquid, adding sodium hydroxide to prepare single-stranded ssDNA serving as a secondary library, and measuring the concentration of the secondary library by using an ultramicro ultraviolet spectrophotometer.
7. The method for screening extracellular vesicle surface protein-specific aptamers according to claim 6, wherein the extracellular vesicle surface protein-specific aptamers are selected from the group consisting of: and (3) continuously carrying out 5-6 rounds of positive screening in the step 5), wherein in the 5-6 rounds of positive screening, the single chain combined with the extracellular vesicles captured by the immunomagnetic beads in the step 4) in the next round of positive screening is a product obtained by treating the secondary library prepared in the previous round of positive screening at the high temperature of 95 ℃ in a metal bath for 5min and immediately carrying out ice bath for 10 min.
8. The method for screening extracellular vesicle surface protein-specific aptamers according to claim 7, wherein the extracellular vesicle surface protein-specific aptamers are selected from the group consisting of: the negative sieve in the step 6) comprises the following steps:
6.1) extracting normal cell extracellular vesicles: filtering by a particle size selection method to obtain normal extracellular vesicles, and analyzing the concentration of the extracted normal extracellular vesicles by a Nanosight particle sizer;
6.2) isolating aptamers that do not bind to normal extracellular vesicles: mixing and incubating the secondary library prepared according to the method in the step 5.3) with normal cell extracellular vesicles captured by immunomagnetic beads, and collecting supernatant through a magnetic separation method.
9. The method for screening extracellular vesicle surface protein-specific aptamers according to claim 8, wherein the extracellular vesicle surface protein-specific aptamers are selected from the group consisting of: and 6) performing positive and negative alternate screening in the step 6), namely performing one round of screening by positive screening and negative screening, performing the next round of positive screening after one round of negative screening, wherein in the next round of positive screening, the single chain combined with the extracellular vesicles captured by the immunomagnetic beads in the step 4) is the supernatant collected in the previous round of negative screening, and the single chain is treated by ice bath for 10min immediately after being treated at the high temperature of 95 ℃ in a metal bath for 5 min.
10. The method for screening extracellular vesicle surface protein-specific aptamers according to claim 9, wherein the extracellular vesicle surface protein-specific aptamers are selected from the group consisting of: the total number of positive screening rounds of positive and negative alternate screening in the step 5) and the total number of positive and negative alternate screening rounds in the step 6) are 10-20 rounds, after the positive screening in the step 5) is continuously carried out for 5-6 rounds, the negative screening is introduced for positive and negative alternate screening, the incubation time of the positive screening is gradually shortened in the positive and negative alternate screening, the cleaning force is gradually increased by gradually increasing the cleaning times during separation, the negative screening time is gradually increased, and the screening pressure is gradually increased to obtain the specific nucleic acid aptamer.
11. The method for screening extracellular vesicle surface protein-specific aptamers based on immunomagnetic bead method according to claim 10, wherein: and 7) detecting the fluorescence intensity of the extracellular vesicles mixed and incubated with the secondary library by adopting a flow cytometry detection technology, wherein when the screening reaches a certain number of turns, the detected fluorescence intensity value does not change any more and tends to be stable, which indicates that the secondary library is successfully enriched, the screening reaches a plateau stage, and the screening can be terminated.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201911099384.6A CN110791504A (en) | 2019-11-12 | 2019-11-12 | Technology for screening extracellular vesicle surface protein specific aptamer based on immunomagnetic bead method |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201911099384.6A CN110791504A (en) | 2019-11-12 | 2019-11-12 | Technology for screening extracellular vesicle surface protein specific aptamer based on immunomagnetic bead method |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN110791504A true CN110791504A (en) | 2020-02-14 |
Family
ID=69444006
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201911099384.6A Pending CN110791504A (en) | 2019-11-12 | 2019-11-12 | Technology for screening extracellular vesicle surface protein specific aptamer based on immunomagnetic bead method |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN110791504A (en) |
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112695037A (en) * | 2020-11-24 | 2021-04-23 | 江苏为真生物医药技术股份有限公司 | EGFR-specific nucleic acid aptamer and application thereof |
| WO2021137999A1 (en) * | 2020-01-02 | 2021-07-08 | Ohio State Innovation Foundation | Immunomagnetic sequential ultrafiltration platform for enrichment and purification of extracellular vesicles from biofluids |
| CN113583954A (en) * | 2021-07-16 | 2021-11-02 | 武汉大学 | Circulating extracellular vesicle in-situ labeling and rapid separation method |
| CN113897419A (en) * | 2021-11-08 | 2022-01-07 | 浙江大学 | Kit and method for extracellular vesicle capture or extracellular vesicle quantitative analysis or extracellular vesicle content quantitative analysis |
| CN114859055A (en) * | 2022-04-10 | 2022-08-05 | 武汉大学 | Specific subtype extracellular vesicle separation and detection kit and use method thereof |
| CN116515844A (en) * | 2023-06-30 | 2023-08-01 | 四川大学华西医院 | Migration body aptamer and screening method and application thereof |
-
2019
- 2019-11-12 CN CN201911099384.6A patent/CN110791504A/en active Pending
Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2021137999A1 (en) * | 2020-01-02 | 2021-07-08 | Ohio State Innovation Foundation | Immunomagnetic sequential ultrafiltration platform for enrichment and purification of extracellular vesicles from biofluids |
| CN112695037A (en) * | 2020-11-24 | 2021-04-23 | 江苏为真生物医药技术股份有限公司 | EGFR-specific nucleic acid aptamer and application thereof |
| CN113583954A (en) * | 2021-07-16 | 2021-11-02 | 武汉大学 | Circulating extracellular vesicle in-situ labeling and rapid separation method |
| CN113583954B (en) * | 2021-07-16 | 2023-11-28 | 珈泌生物科技(武汉)有限责任公司 | In-situ labeling and rapid separation method for circulating extracellular vesicles |
| CN113897419A (en) * | 2021-11-08 | 2022-01-07 | 浙江大学 | Kit and method for extracellular vesicle capture or extracellular vesicle quantitative analysis or extracellular vesicle content quantitative analysis |
| CN114859055A (en) * | 2022-04-10 | 2022-08-05 | 武汉大学 | Specific subtype extracellular vesicle separation and detection kit and use method thereof |
| CN116515844A (en) * | 2023-06-30 | 2023-08-01 | 四川大学华西医院 | Migration body aptamer and screening method and application thereof |
| CN116515844B (en) * | 2023-06-30 | 2023-09-08 | 四川大学华西医院 | Migration body aptamer and screening method and application thereof |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN110791504A (en) | Technology for screening extracellular vesicle surface protein specific aptamer based on immunomagnetic bead method | |
| JP2020202860A (en) | Methods for isolating microvesicles, and methods for extracting nucleic acids from biological samples | |
| DK2542689T3 (en) | Method for isolating target cells | |
| US9829483B2 (en) | Methods of isolating extracellular vesicles | |
| CN110191962A (en) | The sequencing and analysis of allochthon associated nucleic acid | |
| CN110954703A (en) | Method for simultaneously detecting protein in exosome, RNA and exosome membrane protein | |
| WO2019214063A1 (en) | Method for detecting circulating tumor cells | |
| WO2017136430A1 (en) | Exosome-total-isoloation-chip (exotic) device for isolation of exosome-based biomarkers | |
| US20220112555A1 (en) | Profiling microvesicle nucleic acids and uses thereof as signatures in diagnosis of renal transplant rejection | |
| CN109161547B (en) | Nucleic acid aptamer and application thereof in detection of pathogenic vibrio alginolyticus | |
| CN110229820A (en) | The special aptamers screening technique of extracellular vesicle surface albumen based on partial size back-and-forth method | |
| CN114959046A (en) | Kit for detecting lncRNA-NEAT1 in gastric cancer exosomes and application of kit in gastric cancer diagnosis | |
| CN116515844B (en) | Migration body aptamer and screening method and application thereof | |
| CN113528531B (en) | Aptamer for detecting human lung cancer cell strain A549 extracellular vesicles and application thereof | |
| CN111172112B (en) | Full transcriptome expression profile of exosome and construction method and application thereof | |
| CN114990124B (en) | Aptamer of membrane protein target CD44, screening method and application thereof | |
| CN109266653B (en) | Reagent, device and method for capturing drug-resistant heterogeneous circulating tumor cells and performing gene analysis | |
| CN114657184B (en) | Multivalent aptamer functionalized DNA nanostructure probe and preparation method and application thereof | |
| CN114574498B (en) | Nucleic acid aptamer for targeting tumor-associated fibroblasts and application thereof | |
| CN111363749A (en) | Aptamer for detecting Chinese softshell turtle iridovirus and construction method and application thereof | |
| CN114990061B (en) | Culture method for inducing expansion of central memory T cells | |
| CN115197914A (en) | Method for separating and detecting circulating tumor cells | |
| CN117987416A (en) | Aptamer capable of specifically recognizing gastric cancer cells and application thereof | |
| CN115932259A (en) | Detection method combining immunomagnetic separation with real-time fluorescence quantitative PCR and application of detection method in rapid detection of food-borne pathogenic bacteria | |
| CN116410928A (en) | DNA nano structure for capturing circulating tumor cells and preparation and application thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| RJ01 | Rejection of invention patent application after publication | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20200214 |