CN106591313B - The aptamer WYZ-1 and its screening technique of ovarian mucinous cancer cell 3AO and application - Google Patents
The aptamer WYZ-1 and its screening technique of ovarian mucinous cancer cell 3AO and application Download PDFInfo
- Publication number
- CN106591313B CN106591313B CN201611081817.1A CN201611081817A CN106591313B CN 106591313 B CN106591313 B CN 106591313B CN 201611081817 A CN201611081817 A CN 201611081817A CN 106591313 B CN106591313 B CN 106591313B
- Authority
- CN
- China
- Prior art keywords
- cell
- cancer cell
- ovarian
- aptamer
- ovarian mucinous
- 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.)
- Active
Links
- 230000002611 ovarian Effects 0.000 title claims abstract description 69
- 206010028980 Neoplasm Diseases 0.000 title claims abstract description 62
- 201000011510 cancer Diseases 0.000 title claims abstract description 59
- 108091023037 Aptamer Proteins 0.000 title claims abstract description 52
- 238000012216 screening Methods 0.000 title abstract description 25
- 238000000034 method Methods 0.000 title abstract description 24
- 238000002360 preparation method Methods 0.000 claims description 8
- MHMNJMPURVTYEJ-UHFFFAOYSA-N fluorescein-5-isothiocyanate Chemical compound O1C(=O)C2=CC(N=C=S)=CC=C2C21C1=CC=C(O)C=C1OC1=CC(O)=CC=C21 MHMNJMPURVTYEJ-UHFFFAOYSA-N 0.000 claims description 4
- 239000000523 sample Substances 0.000 claims description 3
- 238000002626 targeted therapy Methods 0.000 claims description 3
- JWBWJOKTZVXSRT-DWQAGKKUSA-N 5-[(3as,4s,6ar)-2-oxo-1,3,3a,4,6,6a-hexahydrothieno[3,4-d]imidazol-4-yl]-2-aminopentanoic acid Chemical compound N1C(=O)N[C@@H]2[C@H](CCCC(N)C(O)=O)SC[C@@H]21 JWBWJOKTZVXSRT-DWQAGKKUSA-N 0.000 claims description 2
- LTMHDMANZUZIPE-AMTYYWEZSA-N Digoxin Natural products O([C@H]1[C@H](C)O[C@H](O[C@@H]2C[C@@H]3[C@@](C)([C@@H]4[C@H]([C@]5(O)[C@](C)([C@H](O)C4)[C@H](C4=CC(=O)OC4)CC5)CC3)CC2)C[C@@H]1O)[C@H]1O[C@H](C)[C@@H](O[C@H]2O[C@@H](C)[C@H](O)[C@@H](O)C2)[C@@H](O)C1 LTMHDMANZUZIPE-AMTYYWEZSA-N 0.000 claims description 2
- 239000003560 cancer drug Substances 0.000 claims description 2
- 238000007385 chemical modification Methods 0.000 claims description 2
- LTMHDMANZUZIPE-PUGKRICDSA-N digoxin Chemical compound C1[C@H](O)[C@H](O)[C@@H](C)O[C@H]1O[C@@H]1[C@@H](C)O[C@@H](O[C@@H]2[C@H](O[C@@H](O[C@@H]3C[C@@H]4[C@]([C@@H]5[C@H]([C@]6(CC[C@@H]([C@@]6(C)[C@H](O)C5)C=5COC(=O)C=5)O)CC4)(C)CC3)C[C@@H]2O)C)C[C@@H]1O LTMHDMANZUZIPE-PUGKRICDSA-N 0.000 claims description 2
- 229960005156 digoxin Drugs 0.000 claims description 2
- LTMHDMANZUZIPE-UHFFFAOYSA-N digoxine Natural products C1C(O)C(O)C(C)OC1OC1C(C)OC(OC2C(OC(OC3CC4C(C5C(C6(CCC(C6(C)C(O)C5)C=5COC(=O)C=5)O)CC4)(C)CC3)CC2O)C)CC1O LTMHDMANZUZIPE-UHFFFAOYSA-N 0.000 claims description 2
- 239000003068 molecular probe Substances 0.000 claims description 2
- 230000008685 targeting Effects 0.000 claims description 2
- 210000004027 cell Anatomy 0.000 abstract description 89
- 210000002919 epithelial cell Anatomy 0.000 abstract description 10
- 230000015572 biosynthetic process Effects 0.000 abstract description 3
- 230000005847 immunogenicity Effects 0.000 abstract description 3
- 230000009870 specific binding Effects 0.000 abstract description 3
- 238000003786 synthesis reaction Methods 0.000 abstract description 3
- 239000000126 substance Substances 0.000 abstract description 2
- 238000012986 modification Methods 0.000 abstract 1
- 230000004048 modification Effects 0.000 abstract 1
- 108020004414 DNA Proteins 0.000 description 25
- 239000006228 supernatant Substances 0.000 description 20
- 102000053602 DNA Human genes 0.000 description 18
- 108020004682 Single-Stranded DNA Proteins 0.000 description 13
- 239000000872 buffer Substances 0.000 description 10
- 238000012408 PCR amplification Methods 0.000 description 9
- 238000011282 treatment Methods 0.000 description 6
- 238000004458 analytical method Methods 0.000 description 5
- 238000000684 flow cytometry Methods 0.000 description 5
- 238000011534 incubation Methods 0.000 description 5
- 239000000047 product Substances 0.000 description 5
- TWRXJAOTZQYOKJ-UHFFFAOYSA-L Magnesium chloride Chemical compound [Mg+2].[Cl-].[Cl-] TWRXJAOTZQYOKJ-UHFFFAOYSA-L 0.000 description 4
- 238000000703 high-speed centrifugation Methods 0.000 description 4
- 239000005457 ice water Substances 0.000 description 4
- 238000000926 separation method Methods 0.000 description 4
- 238000012163 sequencing technique Methods 0.000 description 4
- 239000000243 solution Substances 0.000 description 4
- 206010033128 Ovarian cancer Diseases 0.000 description 3
- 230000008901 benefit Effects 0.000 description 3
- 230000027455 binding Effects 0.000 description 3
- 230000010261 cell growth Effects 0.000 description 3
- 230000001413 cellular effect Effects 0.000 description 3
- 238000004925 denaturation Methods 0.000 description 3
- 230000036425 denaturation Effects 0.000 description 3
- 238000010494 dissociation reaction Methods 0.000 description 3
- 230000005593 dissociations Effects 0.000 description 3
- 238000005516 engineering process Methods 0.000 description 3
- 238000003384 imaging method Methods 0.000 description 3
- 239000003446 ligand Substances 0.000 description 3
- 230000008569 process Effects 0.000 description 3
- 238000012360 testing method Methods 0.000 description 3
- OZFAFGSSMRRTDW-UHFFFAOYSA-N (2,4-dichlorophenyl) benzenesulfonate Chemical compound ClC1=CC(Cl)=CC=C1OS(=O)(=O)C1=CC=CC=C1 OZFAFGSSMRRTDW-UHFFFAOYSA-N 0.000 description 2
- 239000012591 Dulbecco’s Phosphate Buffered Saline Substances 0.000 description 2
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 2
- LRHPLDYGYMQRHN-UHFFFAOYSA-N N-Butanol Chemical compound CCCCO LRHPLDYGYMQRHN-UHFFFAOYSA-N 0.000 description 2
- 206010061535 Ovarian neoplasm Diseases 0.000 description 2
- 238000000137 annealing Methods 0.000 description 2
- 238000010586 diagram Methods 0.000 description 2
- 239000008103 glucose Substances 0.000 description 2
- 229910001629 magnesium chloride Inorganic materials 0.000 description 2
- 102000039446 nucleic acids Human genes 0.000 description 2
- 108020004707 nucleic acids Proteins 0.000 description 2
- 150000007523 nucleic acids Chemical class 0.000 description 2
- 230000010355 oscillation Effects 0.000 description 2
- 230000004962 physiological condition Effects 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 230000004083 survival effect Effects 0.000 description 2
- 239000011534 wash buffer Substances 0.000 description 2
- 238000005406 washing Methods 0.000 description 2
- 102000002322 Egg Proteins Human genes 0.000 description 1
- 108010000912 Egg Proteins Proteins 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical class CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 1
- 108091034117 Oligonucleotide Proteins 0.000 description 1
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 1
- XSQUKJJJFZCRTK-UHFFFAOYSA-N Urea Chemical compound NC(N)=O XSQUKJJJFZCRTK-UHFFFAOYSA-N 0.000 description 1
- 230000003321 amplification Effects 0.000 description 1
- 238000003149 assay kit Methods 0.000 description 1
- 239000004202 carbamide Substances 0.000 description 1
- 238000002512 chemotherapy Methods 0.000 description 1
- 230000009514 concussion Effects 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 238000003745 diagnosis Methods 0.000 description 1
- 238000002405 diagnostic procedure Methods 0.000 description 1
- 238000004090 dissolution Methods 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 238000001962 electrophoresis Methods 0.000 description 1
- 235000019441 ethanol Nutrition 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 230000002349 favourable effect Effects 0.000 description 1
- 230000006870 function Effects 0.000 description 1
- 238000001502 gel electrophoresis Methods 0.000 description 1
- 238000010438 heat treatment Methods 0.000 description 1
- 238000012165 high-throughput sequencing Methods 0.000 description 1
- 238000000338 in vitro Methods 0.000 description 1
- 238000007689 inspection Methods 0.000 description 1
- 238000013507 mapping Methods 0.000 description 1
- 239000003550 marker Substances 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 230000000877 morphologic effect Effects 0.000 description 1
- 230000009871 nonspecific binding Effects 0.000 description 1
- 238000003199 nucleic acid amplification method Methods 0.000 description 1
- 208000012988 ovarian serous adenocarcinoma Diseases 0.000 description 1
- 201000003709 ovarian serous carcinoma Diseases 0.000 description 1
- 210000001672 ovary Anatomy 0.000 description 1
- 210000004681 ovum Anatomy 0.000 description 1
- 230000007170 pathology Effects 0.000 description 1
- 238000002264 polyacrylamide gel electrophoresis Methods 0.000 description 1
- 238000003908 quality control method Methods 0.000 description 1
- 238000004064 recycling Methods 0.000 description 1
- 238000007789 sealing Methods 0.000 description 1
- 239000013049 sediment Substances 0.000 description 1
- 230000035945 sensitivity Effects 0.000 description 1
- 210000002966 serum Anatomy 0.000 description 1
- 239000002002 slurry Substances 0.000 description 1
- 239000013595 supernatant sample Substances 0.000 description 1
- 230000002194 synthesizing effect Effects 0.000 description 1
- 238000011269 treatment regimen Methods 0.000 description 1
- 238000002604 ultrasonography Methods 0.000 description 1
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
- C12N15/1048—SELEX
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
- G01N33/57407—Specifically defined cancers
- G01N33/57449—Specifically defined cancers of ovaries
-
- 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)
- Engineering & Computer Science (AREA)
- Genetics & Genomics (AREA)
- Chemical & Material Sciences (AREA)
- Biomedical Technology (AREA)
- Molecular Biology (AREA)
- Biotechnology (AREA)
- Organic Chemistry (AREA)
- General Engineering & Computer Science (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Wood Science & Technology (AREA)
- Zoology (AREA)
- Physics & Mathematics (AREA)
- Immunology (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- Microbiology (AREA)
- Biophysics (AREA)
- Plant Pathology (AREA)
- Urology & Nephrology (AREA)
- Hematology (AREA)
- Oncology (AREA)
- Bioinformatics & Computational Biology (AREA)
- Cell Biology (AREA)
- Hospice & Palliative Care (AREA)
- Crystallography & Structural Chemistry (AREA)
- Food Science & Technology (AREA)
- Medicinal Chemistry (AREA)
- Analytical Chemistry (AREA)
- General Physics & Mathematics (AREA)
- Pathology (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The present invention relates to the aptamer WYZ-1 of ovarian mucinous cancer cell 3AO a kind of and its screening technique and applications, the sequence of the aptamer WYZ-1 are as follows: 5 '-AGCTGCTATACTAGCTAAAGCCTTAGCTAGCTCAATAACGCGATATAGCATGCGCG ATGCCCGTAGCGATAGCTAGCTT-3 ', its screening technique are as follows: be based on SELEX screening technique, using ovarian serous cancer cell SKOV3 and normal ovarian epithelial cell IOSE as counter-selection cell, it is positive sieve cell with ovarian mucinous cancer cell 3AO, filter out the aptamer with ovarian mucinous cancer cell 3AO specific binding, it has high specific, high-affinity, it can body Outer synthesis and modification, the features such as chemical property is stable, pharmacokinetic property is good, non-immunogenicity.
Description
Technical field
The present invention relates to the aptamer WYZ-1 of ovarian mucinous cancer cell 3AO a kind of and its screening technique and applications.
Background technique
Sensibility difference and treatment outcome Chang Bujia of the ovarian mucinous cancer to chemotherapeutics.Therefore, it is necessary to using more
More targetedly researchs are to seek the treatment method for more preferably improving ovarian mucinous cancer survival.In the diagnosis of oophoroma
In, the main method of use includes B ultrasound, CT, MRI, celioscopy and blood serum tumor markers inspection, these diagnostic methods master
If being based on morphological criteria, lack specificity marker relevant to ovarian mucinous cancer.In addition, all oophoromas generally use
Identical treatment method, i.e. the complex treatment strategy of operative treatment combined chemotherapy, these treatment method less pertinences are ideal.Base
In monoclonal antibody technique, the targeted therapy strategy developed in recent years shows in terms of improving ovarian mucinous cancer survival
Unique advantage out, but some defects possessed by antibody itself, such as the otherness between immunogenicity, unstability, batch,
Make that there is certain limitation in its practical application clinically.In conclusion finding the special of ovarian mucinous cancer cell
Property identification molecule, be of great significance in the diagnosing and treating of tumour.
Summary of the invention
The purpose of the present invention is to provide a kind of ovarian mucinous cancer cell 3AO's with high specific and high-affinity
Aptamer WYZ-1 and its screening technique and application.
The purpose of the present invention is achieved through the following technical solutions:
A kind of aptamer WYZ-1 of ovarian mucinous cancer cell 3AO, its sequence are as follows:
5’-AGCTGCTATACTAGCTAAAGCCTTAGCTAGCTCAATAACGCGATATAGCATGCGCGATGCCCGTA
GCGATAGCTAGCTT-3’
The screening technique of the aptamer WYZ-1 of the ovarian mucinous cancer cell 3AO, based on aptamer
External SELEX screening technique (the phyletic evolution technology of index concentration ligand), with ovarian serous cancer cell SKOV3 and normal ovum
Nest epithelial cell IOSE is positive sieve cell as counter-selection cell with ovarian mucinous cancer cell 3AO, is filtered out and ovarian mucinous
The aptamer of cancer cell 3AO specific binding.
The application of the aptamer WYZ-1 of the ovarian mucinous cancer cell 3AO detects ovarian mucinous in preparation
Application in the kit of cancer, molecular probe or targeting medium.
The application of the aptamer WYZ-1 of the ovarian mucinous cancer cell 3AO treats ovarian mucinous in preparation
Application in the targeted therapy probe of cancer.
The application of the aptamer WYZ-1 of the ovarian mucinous cancer cell 3AO treats ovarian mucinous in preparation
Application in cancer drug.
The application of the aptamer WYZ-1 of the ovarian mucinous cancer cell 3AO is thin in research ovarian mucinous cancer
Application in cellular surface albumen.
The application of the aptamer WYZ-1 of the ovarian mucinous cancer cell 3AO is contaminated in ovarian carcinoma pathology
In color, separation ovarian mucinous cancer cell in and the application in the relevant internal molecular imaging of ovarian neoplasm.
For the prior art, the present invention has the advantages that
1. aptamer WYZ-1 has the advantages that high specific, its specific recognition ovarian mucinous cancer cell
3AO does not have other ovarian cancer cells, normal ovarian epithelial cell etc. or with weaker identification function.
2. aptamer WYZ-1 also have high-affinity, can synthesize and modify in vitro, chemical property is stable, medicine generation is dynamic
The features such as mechanical property is good, non-immunogenicity.
3. aptamer is in cellular biology of tumor, clinical trial diagnostics, molecular imaging new technology development and target
It has broad application prospects and important learning value in terms for the treatment of.
4. the synthesis cost of aptamer WYZ-1 is low compared with the cost of Antibody preparation, and the period is short, favorable reproducibility.
Detailed description of the invention
Fig. 1 is the solution that aptamer WYZ-1 combination ovarian mucinous cancer cell 3AO is measured using flow cytometry
Curve is drawn from constant.Dissociation constant Kd=21.7 ± 7.7nM.In Fig. 1, abscissa is DNA concentration (nM), and ordinate is flat
Equal fluorescence intensity.
Fig. 2 is the offset using flow cytometry aptamer WYZ-1 to ovarian mucinous cancer cell 3AO.?
In Fig. 2, abscissa is fluorescence intensity, and ordinate is number of cells, and solid line WYZ-1, dotted line is unscreened single stranded DNA text
Library.
Fig. 3 is the offset using flow cytometry aptamer WYZ-1 to ovarian serous cancer cell SKOV3.
In Fig. 3, abscissa is fluorescence intensity, and ordinate is number of cells, and solid line WYZ-1, dotted line is unscreened single stranded DNA
Library.
Fig. 4 is the offset using flow cytometry aptamer WYZ-1 to normal ovarian epithelial cell IOSE.?
In Fig. 4, abscissa is fluorescence intensity, and ordinate is number of cells, and solid line WYZ-1, dotted line is unscreened single stranded DNA text
Library.
Fig. 5 is the space structure schematic diagram of aptamer WYZ-1.
Specific embodiment
The content of present invention is described in detail with embodiment with reference to the accompanying drawings of the specification:
A kind of aptamer WYZ-1 of ovarian mucinous cancer cell 3AO, its sequence are as follows:
5’-AGCTGCTATACTAGCTAAAGCCTTAGCTAGCTCAATAACGCGATATAGCATGCGCGATGCCCGTA
GCGATAGCTAGCTT-3’
The aptamer WYZ-1 of the ovarian mucinous cancer cell 3AO, at 37 DEG C, 0.137M Na+, 0.001M
Mg2+Physiological condition under, with characteristic loop-stem structure, space structure is as follows:
The aptamer WYZ-1 of the ovarian mucinous cancer cell 3AO, to the 5 ' of the aptamer WYZ-1
End or 3 ' ends carry out FITC, amino, biotin or digoxin chemical modification.
The screening technique of the aptamer WYZ-1 of the ovarian mucinous cancer cell 3AO, based on aptamer
External SELEX screening technique, using ovarian serous cancer cell SKOV3 and normal ovarian epithelial cell IOSE as counter-selection cell,
It is positive sieve cell, is filtered out suitable with the nucleic acid of ovarian mucinous cancer cell 3AO specific binding with ovarian mucinous cancer cell 3AO
Ligand.
It is screening target cell using ovarian mucinous cancer cell 3AO, with corresponding ovarian serous cancer cell
SKOV3 and normal ovarian epithelial cell IOSE as counter-selection cell, screening remove in DNA oligonucleotide library with ovary slurries
Property cancer cell, normal ovarian epithelial cell non-specific binding part, guarantee that the obtained aptamer of screening can be special
The combination ovarian mucinous cancer cell 3AO of property.
In addition, keeping the form of screening cell and activity intact using the Cell-SELEX screening technique of standard, guarantee thin
Cellular surface large biological molecule is closer to the intracorporal molecular conformation of biology.
The screening technique of the aptamer WYZ-1 of the ovarian mucinous cancer cell 3AO, it the following steps are included:
(1) prepare single-stranded DNA banks shown in following sequence:
Single-stranded DNA banks:
5’-AGCTGCTATACTAGCTAAAGNNNNNNNNNNNNNNNNNNNNNNNN NNNNNNNNNNNNNNNNCCGT
AGCGATAGCTAGCTT-3'；
(2) SKOV3 cell counter-selection: the library that step (1) is prepared and SKOV3 cell incubation are collected thin after the completion of being incubated for
Supernatant after born of the same parents' incubation；
(3) IOSE cell counter-selection: by supernatant obtained by step (2) and IOSE cell incubation, cell is collected after the completion of being incubated for and is incubated
Supernatant after educating；
(4) 3AO cell just sieves: by step (3) resulting supernatant and 3AO cell incubation, after the completion of incubation, abandoning supernatant；
It washs 3AO cell and by after under 3AO cell scraper, is transferred in EP pipe and carries out heat denatured, supernatant is collected in centrifuge separation later；
(5) PCR is enriched with: the resulting supernatant of step (4) carries out PCR amplification, obtains pcr amplification product；Wherein PCR amplification
Primer used are as follows:
Primers F up:5 '-FAM-AGCTGCTATACTAGCTAAAG-3 '；
Primer Ldown:5 '-TTTTTTTTTTTTTTTTTTTTTTT-spacer
C9-AAGCTAGCTATCGCTACGG-3'；
TTTTTTTTTTTTTTTTTTTTTTT-spacer C9 keeps the chain length of two primers different, produces convenient for PCR amplification
The denaturing electrophoretic of object separates.
(6) single stranded DNA purifies: after pcr amplification product is carried out denaturation unwinding, denaturing electrophoretic separation, and purified pool FAM mark
The ssDNA of note；
(7) aptamer Cycle Screening: the secondary library that step (6) resulting ssDNA is screened as next round, and
Repeat the screening process of step (2)~(6).
Embodiment one: the screening of aptamer WYZ-1:
The screening technique of the aptamer WYZ-1 of the ovarian mucinous cancer cell 3AO, it the following steps are included:
(1) preparation in library is screened:
Synthesizing single-stranded DNA library (the 5 '-AGCTGCTATACTAGCTAAAGN of student on commission's work bioengineering limited liability company
NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNCCGTAGCGATAGCTAGCTT-3')；Take that 1 pipe 1OD's is single-stranded
DNA library, high speed centrifugation 2min；Pipe lid is gently opened in vent cabinet, and 300 μ L combination buffers are added and (contain 4.5g/ in DPBS
L glucose, 100mg/L yeast tRNA, 1g/L BSA, 5mM MgCl2), vortex oscillation dissolution；Seal with sealing film centrifuge tube
Lid, 95 DEG C of water-baths 5min, rapid ice-water bath 2min；High speed centrifugation 2min, it is spare.
(2) the library sucking SKOV3 cell growth coverage that step (1) prepares SKOV3 cell counter-selection: is reached about 60%
In the diameter 100mm culture dish of left and right, 1mL is supplied with combination buffer；4 DEG C of shaking tables vibrate 1h；By supernatant 1000rpm from
Heart 10min collects supernatant；
(3) IOSE cell counter-selection: supernatant obtained by aspiration step (2) to IOSE cell growth coverage reaches about 60% or so
Diameter 100mm culture dish in；4 DEG C of shaking tables vibrate 1h；Supernatant 1000rpm is centrifuged 10min, collects supernatant；
(4) 3AO cell just sieves: aspiration step (3) resulting supernatant to 3AO cell growth coverage reaches about 60% or so
Diameter 60mm culture dish in, 4 DEG C of shaking tables vibrate 1h；Abandon supernatant；With washing buffer (glucose containing 4.5g/L in DPBS,
5mM MgCl2) washing 3AO cell three times；With cell scraper by under 3AO cell scraper, 3AO cell is shifted with 1mL washing buffer
Into EP pipe, 95 DEG C of water-baths 5min, rapid ice-water bath 2min；10000rpm is centrifuged 5min, leaves and takes supernatant.
(5) PCR is enriched with: being taken 50 μ L step (4) resulting Supernatant samples, is added in 1mL PCRmix；Vortex oscillation is mixed
After even, dispensed by every 50 μ L of pipe and carry out PCR amplification, amplification condition are as follows: after 95 DEG C of heating initial denaturation 10min, 94 DEG C of denaturation 30s,
60 DEG C of annealing 30s, 72 DEG C of extension 30s, 25 recycle.
Wherein contain in 1mL PCRmix: H2O 871μL；10 × PCR buffer, 100 μ L；
Primers F up:5 '-FAM-AGCTGCTATACTAGCTAAAG-3 ' and Ldown:5 '-
Each 3 μ L of TTTTTTTTTTTTTTTTTTTTTTT-spacer C9-AAGCTAGCTATCGCTACGG-3 '；3 μ L of pfu enzyme；dNTP
20μL.The equal student on commission's work bioengineering limited liability company synthesis of the primers F up and primer Ldown.
(6) single stranded DNA purifies: pcr amplification product is mixed, the n-butanol of 6 times of volumes is added, vortex concussion mixes,
14000rpm, room temperature are centrifuged 10min；About 50 μ L of suction foot sample, mixes, 95 DEG C in equal volume with TBE/7M Urea buffer
Water-bath 10min, rapid ice-water bath 2min；With the urea-denatured polyacrylamide gel electrophoresis separation (400V voltage) of 7M；Electrophoresis knot
Shu Hou, in fluoroscopic imaging systems, the ssDNA band of gel extraction FAM label；Gel is shredded, 500 μ L ddH are added2O,
10min is boiled, high speed centrifugation collects supernatant；2.5 times of volume ethanols are added, are placed at room temperature for 15min, high speed centrifugation abandons supernatant, sinks
It forms sediment and is dissolved with 100 μ L combination buffers.
(7) aptamer Cycle Screening: the secondary library that step (6) resulting ssDNA is screened as next round, and
Repeat the screening process of step (2)~(6).
Embodiment two: the analysis of aptamer WYZ-1 sequence:
After 12 wheel screenings, recycling 3AO cell just sieves supernatant, and entrusts the upper gloomy limited public affairs of promise biotechnology share of the Shanghai's style
Department analyzes library sequence using high throughput sequencing technologies, analytic process are as follows: PCR amplification enriched library, and plus sequencing
Connector and the part Index；Purified library is selected by gel electrophoresis；Passed through using Agilent 2100Bioanalyzer
Agilent High Sensitivity DNA Kit is to library Quality Control；Utilize Quant-iT PicogGreen dsDNA
Assay Kit quantifies library；Using IlluminateNextSeq500 platform, bridge-type is carried out by template of single-stranded library
PCR amplification, sequencing primer annealing, the sequencing of the side Bian Hecheng；And analysis is compared and is enriched with to sequencing result.
Using the UNAFold network platform analysis in physiological conditions (37 DEG C, 0.137M Na+, 0.001M Mg2+), the core
The secondary structure of sour aptamers WYZ-1 sequence.Analyze secondary structure schematic diagram such as Fig. 5 institute of aptamer WYZ-1 sequence
Show.
Embodiment three: the analysis of aptamer WYZ-1 and ovarian mucinous cancer cell 3AO binding ability:
1. target cell 3AO is inoculated in 24 orifice plates (50,000/hole), in 37 DEG C, 5%CO2Culture is for 24 hours.
2. single stranded DNA (including the aptamer WYZ-1 and unscreened single-stranded for being marked FITC with combination buffer
DNA library) it is configured to the solution that concentration is 0nM, 10nM, 50nM, 100nM, 200nM；95 DEG C of water-bath 5min, rapid ice-water bath
2min。
3. washing cell twice with combination buffer, the single stranded DNA solution of the 200 above-mentioned gradient concentrations of μ L is added in every hole, and
It is placed in and is incubated for 30min on ice.
4. after being incubated for, abandoning supernatant, being swept cell with cell scraper；It is washed twice with combination buffer, cell is resuspended, body
Product is 250 μ L.
5. the FACSAria flow cytometer using BD company carries out fluoremetry to cell；Measurement result GraphPad
The mapping of 6 software of Prism, the dissociation constant for calculating gained aptamer WYZ-1 is 21.7 ± 7.7nM, as shown in Figure 1.Figure
1 testing result shows: aptamer WYZ-1 and the binding ability of target cell ovarian mucinous cancer cell 3AO are strong, and dissociation is normal
Number is in nanomole rank.
Example IV: the analysis of aptamer WYZ-1 and ovarian mucinous cancer cell 3AO binding specificity:
1. count cell to be measured to be simultaneously inoculated in 24 orifice plates (50,000/hole), in 37 DEG C, 5%CO2Culture is for 24 hours.
2. wash cell twice with combination buffer, the single stranded DNA of 200 μ L FITC label is added in every hole, and (including nucleic acid is suitable
Ligand WYZ-1 and unscreened single-stranded DNA banks), it is placed in and is incubated for 30min on ice.
3. after being incubated for, abandoning supernatant, being swept cell with cell scraper；It is washed twice with combination buffer, cell is resuspended, body
Product is 250 μ L.
4. the FACSAria flow cytometer using BD company carries out fluoremetry to cell.
The cell to be measured is respectively ovarian mucinous cancer cell 3AO, ovarian serous cancer cell SKOV3 and normal
Ovarian epithelial cell IOSE, wherein ovarian serous cancer cell SKOV3 and normal ovarian epithelial cell IOSE is that control is thin
Born of the same parents, testing result is as shown in figs. 2 to 4.It is proved by the testing result of the flow cytometry of Fig. 2~4, aptamer of the invention
WYZ-1 has stronger specific recognition capability to ovarian mucinous cancer cell 3AO, and thin to control cell ovarian serous carcinoma
Born of the same parents SKOV3 and normal ovarian epithelial cell IOSE are without identification.
SEQUENCE LISTING
<110>Wu's winter
<120>the aptamer WYZ-1 of ovarian mucinous cancer cell 3AO and its screening technique and application
<160> 3
<210> 1
<211> 79
<212> DNA
<213>artificial sequence
<400> 1
agctgctata ctagctaaag ccttagctag ctcaataacg cgatatagca 50
tgcgcgatgc ccgtagcgat agctagctt 79
<210> 2
<211> 20
<212> DNA
<213>artificial sequence
<400> 2
agctgctata ctagctaaag 20
<210> 3
<211> 19
<212> DNA
<213>artificial sequence
<400> 3
aagctagcta tcgctacgg 19
Claims (5)
1. a kind of aptamer WYZ-1 of ovarian mucinous cancer cell 3AO, it is characterised in that: its sequence is as follows:
5’-AGCTGCTATACTAGCTAAAGCCTTAGCTAGCTCAATAACGCGATATAGCATGCGCGATGCCCGTAGCGA
TAGCTAGCTT-3'；And it is at 37 DEG C, 0.137M Na+, 0.001M Mg2+Under the conditions of its space structure it is as follows:
2. the aptamer WYZ-1 of ovarian mucinous cancer cell 3AO according to claim 1, it is characterised in that: to institute
5 ' the ends or 3 ' ends for stating aptamer WYZ-1 carry out FITC, amino, biotin or digoxin chemical modification.
3. the aptamer WYZ-1 of ovarian mucinous cancer cell 3AO described in claim 1-2 any one is detected in preparation
Application in the kit of ovarian mucinous cancer, molecular probe or targeting medium.
4. the aptamer WYZ-1 of ovarian mucinous cancer cell 3AO described in claim 1-2 any one is treated in preparation
Application in the targeted therapy probe of ovarian mucinous cancer.
5. the aptamer WYZ-1 of ovarian mucinous cancer cell 3AO described in claim 1-2 any one is treated in preparation
Application in ovarian mucinous cancer drug.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201611081817.1A CN106591313B (en) | 2016-11-30 | 2016-11-30 | The aptamer WYZ-1 and its screening technique of ovarian mucinous cancer cell 3AO and application |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201611081817.1A CN106591313B (en) | 2016-11-30 | 2016-11-30 | The aptamer WYZ-1 and its screening technique of ovarian mucinous cancer cell 3AO and application |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN106591313A CN106591313A (en) | 2017-04-26 |
| CN106591313B true CN106591313B (en) | 2019-03-08 |
Family
ID=58593961
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201611081817.1A Active CN106591313B (en) | 2016-11-30 | 2016-11-30 | The aptamer WYZ-1 and its screening technique of ovarian mucinous cancer cell 3AO and application |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN106591313B (en) |
Families Citing this family (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111944819B (en) * | 2020-01-10 | 2022-03-11 | 湖南大学 | Ovarian cancer aptamer capable of being rapidly screened and application of ovarian cancer aptamer in preparation of ovarian cancer detection preparation |
| CN113621615B (en) * | 2021-07-30 | 2023-08-11 | 福建博奥医学检验所有限公司 | Aptamer HPV3101 of HPV31 virus particle and application thereof |
| CN113584040B (en) * | 2021-08-11 | 2023-08-01 | 吴冬 | Aptamer HPV3301 of HPV33 virus particle and application thereof |
| CN113462695B (en) * | 2021-08-11 | 2023-08-01 | 吴冬 | Aptamer HPV3501 of HPV35 virus particle and application thereof |
| CN113502290B (en) * | 2021-08-19 | 2023-08-11 | 福建博奥医学检验所有限公司 | Aptamer HPV3901 of HPV39 virus particle and application thereof |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103160513A (en) * | 2011-12-16 | 2013-06-19 | 中国医学科学院基础医学研究所 | MUC1 protein nucleic acid aptamer, complex, composition and purposes thereof |
-
2016
- 2016-11-30 CN CN201611081817.1A patent/CN106591313B/en active Active
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103160513A (en) * | 2011-12-16 | 2013-06-19 | 中国医学科学院基础医学研究所 | MUC1 protein nucleic acid aptamer, complex, composition and purposes thereof |
Non-Patent Citations (2)
| Title |
|---|
| Identification of Epithelial Ovarian Tumor-Specific Aptamers;Gregory Benedetto et al.;《Nucleic Acid Therapeutics》;20151231;第25卷(第3期);第163页左栏第3段-第164页左栏第1段，表1-2 |
| Study of the Molecular Recognition of Aptamers Selected through Ovarian Cancer Cell-SELEX;Dimitri Van Simaeys et al.;《Plos One》;20101101;第5卷(第11期);第1-7页 |
Also Published As
| Publication number | Publication date |
|---|---|
| CN106591313A (en) | 2017-04-26 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN106591313B (en) | The aptamer WYZ-1 and its screening technique of ovarian mucinous cancer cell 3AO and application | |
| Sarkozy et al. | The prognostic value of clonal heterogeneity and quantitative assessment of plasma circulating clonal IG-VDJ sequences at diagnosis in patients with follicular lymphoma | |
| CN109806275B (en) | Application of DNA tetrahedron in preparation of nerve repair promoting medicine | |
| JP2015524054A5 (en) | ||
| CN110117595B (en) | Aptamer specifically binding to PDL1 and application thereof | |
| CN106520773B (en) | The aptamer WYZ-3 and its screening technique of ovarian mucinous cancer cell 3AO and application | |
| Koczkodaj et al. | Examination of clonal evolution in chronic lymphocytic leukemia | |
| CN106970224A (en) | A kind of kit of application CD45 immunofluorescences joint CEP probe identification circulating tumor cells and its application | |
| CN106957906A (en) | A kind of primer combination and kit that T cell Minimal Residual Disease of Leukemia is detected applied to high-flux sequence | |
| CN106754936B (en) | The aptamer WYZ-2 and its screening technique of ovarian mucinous cancer cell 3AO and application | |
| CN108342478B (en) | Circulating tumor cell metabolism typing marker and application thereof | |
| JP2024023284A (en) | Methods of using giant cell nucleic acid characterization in cancer screening, diagnostics, treatment and recurrence | |
| US20220325271A1 (en) | Droplet-based single extracellular vesicle sequencing | |
| CN112877333A (en) | Aptamer for specifically recognizing integrin beta 3 subunit protein and application thereof | |
| EP1448792A1 (en) | Diagnosis kit, dna chip, and methods for diagnosing or supervising the treatment of testicular cancer | |
| CN106754935B (en) | The aptamer WYZ-5 and its screening technique of ovarian mucinous cancer cell 3AO and application | |
| CN106591314B (en) | The aptamer WYZ-4 and its screening technique of ovarian mucinous cancer cell 3AO and application | |
| CN105755135B (en) | A kind of No. 17 chromosome centromere probe reagent boxes of people and its preparation method and application | |
| CN108265055A (en) | Specifically bind DNA aptamers and its application of human breast cancer cell line Bcap-37 | |
| Popovic | Routine and novel methods for isolation of extracellular vesicles | |
| CN109266653B (en) | Reagent, device and method for capturing drug-resistant heterogeneous circulating tumor cells and performing gene analysis | |
| WO2022242285A1 (en) | Method for separating placental trophoblast cells from exfoliated cervical cells of pregnant woman | |
| CN105483246A (en) | Application of differential expression of gene in oral cancer diagnosis | |
| CN104651522B (en) | The new opplication of 1 chain of type i collagen α | |
| CN105219842B (en) | The method and kit of tumor susceptibility gene in multistage PCR detections gynaecology swab sample |
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 | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant | ||
| TR01 | Transfer of patent right | ||
| TR01 | Transfer of patent right |
Effective date of registration: 20211117 Address after: 257091 building 10, No. 38, Huanghe Road, economic development zone, Dongying City, Shandong Province Patentee after: DONGYING UNIVERSITY SCIENCE PARK DEVELOPMENT CO.,LTD. Address before: 350200 room 103, No. 2, Nanshan business building, Changle City, Fuzhou City, Fujian Province Patentee before: Wu Dong |