CN106520773A - The aptamer WYZ 3 of ovarian mucinous cancerous cell 3AO and its screening technique and application - Google Patents
The aptamer WYZ 3 of ovarian mucinous cancerous cell 3AO and its screening technique and application Download PDFInfo
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- 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
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- C12N15/1034—Isolating an individual clone by screening libraries
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Abstract
The present invention relates to a kind of aptamer WYZ 3 of ovarian mucinous cancerous cell 3AO and its screening technique and application, the sequence of sour aptamers WYZ 3 is：5 ' AGCTGCTATACTAGCTAAAGACCTGAGGTCCTTTGACGTTCCAGGTAGACTTGAAC CGTACCGTAGCGATAGCTAGCTT 3 ', its screening technique is based on SELEX triage techniqueses, using ovarian serous cancerous cell SKOV3 and normal ovarian epithelial cell IOSE as anti-sieve cell, with ovarian mucinous cancerous cell 3AO as positive sieve cell, filter out the aptamer with ovarian mucinous cancerous cell 3AO specific bindings, it has high specific, high-affinity, can synthesize in vitro and modify, stable chemical nature, pharmacokinetic property is good, the features such as non-immunogenicity.
Description
Technical field
The present invention relates to a kind of aptamer WYZ-3 of ovarian mucinous cancerous cell 3AO and its screening technique and application.
Background technology
Sensitivity difference and treatment outcome Chang Bujia of the ovarian mucinous cancer to chemotherapeutics.Accordingly, it would be desirable to using more
It is many targetedly to study to seek more preferably to improve the Therapeutic Method of ovarian mucinous cancer survival.In the diagnosis of ovarian cancer
In, the main method of employing includes B ultrasonic, CT, MRI, laparoscopy and blood serum tumor markers inspection, these diagnostic methods master
If being based on morphological criteria, lack the specificity marker related to ovarian mucinous cancer.In addition, all ovarian cancers are generally adopted
The Comprehensive Treatment strategy of identical Therapeutic Method, i.e. operative treatment combined chemotherapy, these Therapeutic Method less pertinences are preferable.Base
In monoclonal antibody technique, the targeted therapy strategy for developing in recent years represents in terms of ovarian mucinous cancer survival is improved
Go out unique advantage, but some defects that antibody itself has, such as immunogenicity, diversity between unstability, batch etc.,
There is certain limitation in making its practical application clinically.In sum, find the special of ovarian mucinous cancerous cell
Property identification molecule, tumor diagnosis and treatment in it is significant.
The content of the invention
It is an object of the invention to provide a kind of ovarian mucinous cancerous cell 3AO with high specific and high-affinity
Aptamer WYZ-3 and its screening technique and application.
The purpose of the present invention is achieved through the following technical solutions：
A kind of aptamer WYZ-3 of ovarian mucinous cancerous cell 3AO, its sequence are as follows：
5’-AGCTGCTATACTAGCTAAAGACCTGAGGTCCTTTGACGTTCCAGGTAGACTTGAACCGTACCGTAG
CGATAGCTAGCTT-3’
The screening technique of the aptamer WYZ-3 of described ovarian mucinous cancerous cell 3AO, based on aptamer
External SELEX triage techniqueses (the phyletic evolution technology of index concentration part), with ovarian serous cancerous cell SKOV3 and normal ovum
Nest epithelial cell IOSE, is filtered out and ovarian mucinous with ovarian mucinous cancerous cell 3AO as positive sieve cell as anti-sieve cell
The aptamer of cancerous cell 3AO specific bindings.
The application of the aptamer WYZ-3 of described ovarian mucinous cancerous cell 3AO, is preparing detection ovarian mucinous
Application in the test kit of cancer, molecular probe or targeting medium.
The application of the aptamer WYZ-3 of described ovarian mucinous cancerous cell 3AO, is preparing treatment ovarian mucinous
Application in the targeted therapy probe of cancer.
The application of the aptamer WYZ-3 of described ovarian mucinous cancerous cell 3AO, is preparing treatment ovarian mucinous
Application in cancer drug.
The application of the aptamer WYZ-3 of described ovarian mucinous cancerous cell 3AO is thin in research ovarian mucinous cancer
Application in cellular surface albumen.
The application of the aptamer WYZ-3 of described ovarian mucinous cancerous cell 3AO, contaminates in ovarian carcinoma pathology
Application in color, in separating ovarian mucinous cancerous cell and in the related internal molecular imaging of ovarian tumor.
For than prior art, it is an advantage of the current invention that：
1. aptamer WYZ-3 has the advantages that high specific, its specific recognition ovarian mucinous cancerous cell
3AO, does not have to other ovarian cancer cells, normal ovarian epithelial cell etc. or with weaker identification function.
2. aptamer WYZ-3 also have high-affinity, can synthesize in vitro and modify, stable chemical nature, medicine generation it is dynamic
Mechanical property is good, non-immunogenicity the features such as.
3. aptamer is in cellular biology of tumor, clinical experiment diagnosticss, molecular imaging new technology development and target
To having broad application prospects and important learning value in terms for the treatment of.
4. low cost of the synthesis cost of aptamer WYZ-3 compared with Antibody preparation, and cycle is short, favorable reproducibility.
Description of the drawings
Fig. 1 is to determine solutions of the aptamer WYZ-3 with reference to ovarian mucinous cancerous cell 3AO using flow cytometry
Curve is drawn from constant.Dissociation constant Kd=30.8 ± 10.6nM.In FIG, abscissa is DNA concentration (nM), and vertical coordinate is
Average fluorescent strength.
Fig. 2 is the skew using flow cytometry aptamer WYZ-3 to ovarian mucinous cancerous cell 3AO.
In Fig. 2, abscissa is fluorescence intensity, and vertical coordinate is number of cells, and solid line is WYZ-3, and dotted line is unscreened single stranded DNA text
Storehouse.
Fig. 3 is the skew using flow cytometry aptamer WYZ-3 to ovarian serous cancerous cell SKOV3.
In figure 3, abscissa is fluorescence intensity, and vertical coordinate is number of cells, and solid line is WYZ-3, and dotted line is unscreened single stranded DNA
Library.
Fig. 4 is the skew using flow cytometry aptamer WYZ-3 to normal ovarian epithelial cell IOSE.
In Fig. 4, abscissa is fluorescence intensity, and vertical coordinate is number of cells, and solid line is WYZ-3, and dotted line is unscreened single stranded DNA text
Storehouse.
Space structure schematic diagrams of the Fig. 5 for aptamer WYZ-3.
Specific embodiment
Present invention is described in detail with reference to Figure of description and embodiment：
A kind of aptamer WYZ-3 of ovarian mucinous cancerous cell 3AO, its sequence are as follows：
5’-AGCTGCTATACTAGCTAAAGACCTGAGGTCCTTTGACGTTCCAGGTAGACTTGAACCGTACCGTAG
CGATAGCTAGCTT-3’
The aptamer WYZ-3 of described ovarian mucinous cancerous cell 3AO, at 37 DEG C, 0.137M Na+, 0.001M
Mg2+Physiological condition under, which has distinctive loop-stem structure, and its space structure is as follows：
The aptamer WYZ-3 of described ovarian mucinous cancerous cell 3AO, 5 ' to the aptamer WYZ-3
End or 3 ' ends carry out FITC, amino, biotin or Digoxin chemical modification.
The screening technique of the aptamer WYZ-3 of described ovarian mucinous cancerous cell 3AO, based on aptamer
External SELEX triage techniqueses, using ovarian serous cancerous cell SKOV3 and normal ovarian epithelial cell IOSE as anti-sieve cell,
With ovarian mucinous cancerous cell 3AO as positive sieve cell, filter out suitable with the nucleic acid of ovarian mucinous cancerous cell 3AO specific bindings
Part.
It is screening target cell using ovarian mucinous cancerous cell 3AO, with corresponding ovarian serous cancerous cell
SKOV3 and normal ovarian epithelial cell IOSE as anti-sieve cell, screening remove in DNA oligonucleotide libraries with ovary serosity
Property cancerous cell, the part of normal ovarian epithelial cell non-specific binding, it is ensured that the aptamer that obtains of screening can be special
The combination ovarian mucinous cancerous cell 3AO of property.
In addition, using the Cell-SELEX screening techniques of standard, keeping the form and activity of screening cell intact, it is ensured that thin
Cellular surface biomacromolecule is closer to biological internal molecular conformation.
The screening technique of the aptamer WYZ-3 of described ovarian mucinous cancerous cell 3AO, it comprises the following steps：
(1) single-stranded DNA banks shown in following sequence are prepared：
Single-stranded DNA banks：
5’-AGCTGCTATACTAGCTAAAGNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNCCGTAG
CGATAGCTAGCTT-3’；
(2) SKOV3 cells are counter sieves：The library that step (1) is prepared and SKOV3 cell incubations, collect after the completion of incubation thin
Supernatant after born of the same parents' incubation；
(3) IOSE cells are counter sieves：By supernatant obtained by step (2) and IOSE cell incubations, cell is collected after the completion of incubation and is incubated
Supernatant after educating；
(4) 3AO cells are just sieved：By the supernatant obtained by step (3) and 3AO cell incubations, after the completion of incubation, supernatant is abandoned；
Washing 3AO cells after 3AO cells are scraped, carry out heat denatured in being transferred to EP pipes, and supernatant is collected in centrifugation afterwards；
(5) PCR enrichments：Supernatant obtained by step (4) enters performing PCR amplification, obtains pcr amplification product；Wherein PCR is expanded
Primer used is：
Primers F up:5’-FAM-AGCTGCTATACTAGCTAAAG-3’；
Primer Ldown:5’-TTTTTTTTTTTTTTTTTTTTTTT-spacer C9-AAGCTAGCTATCGCTACGG-
3’；
TTTTTTTTTTTTTTTTTTTTTTT-spacer C9 make the chain length of two primers different, are easy to PCR amplifications to produce
The denaturing electrophoretic of thing is separated.
(6) single stranded DNA purification：Pcr amplification product is carried out after degeneration unwinds, denaturing electrophoretic is separated, purified pool FAM marks
The ssDNA of note；
(7) aptamer Cycle Screening：The secondary library that ssDNA obtained by step (6) is screened as next round, and
The screening process of repeat step (2)～(6).
Embodiment one：The screening of aptamer WYZ-3：
The screening technique of the aptamer WYZ-3 of described ovarian mucinous cancerous cell 3AO, it comprises the following steps：
(1) screen the preparation in library：
The synthesizing single-stranded DNA library of student on commission's work biological engineering limited company (5 '-
AGCTGCTATACTAGCTAAAGNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNCCGTAGCGATAGCTAGC
TT-3’)；Take the single-stranded DNA banks of 1 pipe 1OD, high speed centrifugation 2min；Lid is gently opened in ventilated chamber, 300 μ L knots are added
Close buffer (glucose containing 4.5g/L, 100mg/L yeast tRNA, 1g/L BSA, 5mM MgCl in DPBS2), vortex oscillation is molten
Solution；Centrifugation lid, 95 DEG C of water-bath 5min, rapid ice-water bath 2min are sealed with sealed membrane；High speed centrifugation 2min, it is standby.
(2) SKOV3 cells are counter sieves：The library suction SKOV3 cell growth coverages that step (1) is prepared reach about 60%
In the diameter 100mm culture dishs 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 cells are counter sieves：Obtained by aspiration step (2), supernatant reaches about 60% or so to IOSE cell growth coverages
Diameter 100mm culture dishs in；4 DEG C of shaking tables vibrate 1h；Supernatant 1000rpm is centrifuged into 10min, supernatant is collected；
(4) 3AO cells are just sieved：Supernatant obtained by aspiration step (3) reaches about 60% or so to 3AO cell growth coverages
Diameter 60mm culture dishs in, 4 DEG C of shaking tables vibrate 1h；Abandon supernatant；With lavation buffer solution (glucose containing 4.5g/L in DPBS,
5mM MgCl2) washing 3AO cells three times；Scraped with cell and 3AO cells are scraped, 3AO cells are shifted with 1mL lavation buffer solutions
To in EP pipes, 95 DEG C of water-bath 5min, rapid ice-water bath 2min；10000rpm is centrifuged 5min, leaves and takes supernatant.
(5) PCR enrichments：The Supernatant samples obtained by 50 μ L steps (4) are taken, is added in 1mL PCRmix；Vortex oscillation is mixed
After even, enter performing PCR amplification by 50 μ L subpackages of every pipe, amplification condition is：After 95 DEG C of heating denaturations 10min, 94 DEG C of degeneration 30s,
60 DEG C of annealing 30s, 72 DEG C of extension 30s, 25 circulations.
Contain in wherein 1mL PCRmix：H2O 871μL；10 × PCR buffer, 100 μ L；
Primers F up:5 '-FAM-AGCTGCTATACTAGCTAAAG-3 ' and Ldown:5’-
The each 3 μ L of TTTTTTTTTTTTTTTTTTTTTTT-spacer C9-AAGCTAGCTATCGCTACGG-3 '；3 μ L of pfu enzymes；dNTP
20μL.Primers F up and the equal student on commission's work biological engineering limited company synthesis of primer Ldown.
(6) single stranded DNA purification：Pcr amplification product is mixed, adds the n-butyl alcohol of 6 times of volumes, vortex concussion to mix,
14000rpm, room temperature centrifugation 10min；About 50 μ L of suction foot sample, are mixed with TBE/7M Urea buffer equal-volumes, 95 DEG C
Water-bath 10min, rapid ice-water bath 2min；(400V voltages) is separated with the urea-denatured polyacrylamide gel electrophoresis of 7M；Electrophoresis is tied
Shu Hou, in fluoroscopic imaging systems, cuts the ssDNA bands of glue reclaim FAM labelling；Gel is shredded, 500 μ L ddH are added2O,
10min is boiled, high speed centrifugation collects supernatant；2.5 times of volume ethanols, room temperature are added to place 15min, high speed centrifugation abandons supernatant, sinks
Form sediment and dissolved with 100 μ L combination buffers.
(7) aptamer Cycle Screening：The secondary library that ssDNA obtained by step (6) is screened as next round, and
The screening process of repeat step (2)～(6).
Embodiment two：The analysis of aptamer WYZ-3 sequences：
After 12 wheel screenings, reclaim 3AO cells and just sieving supernatant, and entrust the limited public affairs of the gloomy promise biotechnology share of the upper Shanghai's style
Department is analyzed to library sequence using high throughput sequencing technologies, and analysis process is：PCR expands enriched library, and plus sequencing
Joint and Index parts；Purified library is selected by gel electrophoresiss；Passed through using Agilent 2100Bioanalyzer
Agilent High Sensitivity DNAKit are to library Quality Control；Using Quant-iT PicogGreen dsDNA Assay
Kit is carried out quantitatively to library；Using 500 platforms of IlluminateNextSeq, the expansion of bridge-type PCR is carried out by template of single-stranded library
Increasing, sequencing primer are annealed, are sequenced in synthesis；And sequencing result is compared and analysis is enriched with.
Using the UNAFold network platforms analyze in physiological conditions (37 DEG C, 0.137M Na+, 0.001M Mg2+), the core
The secondary structure of sour aptamers WYZ-3 sequence.Analyze secondary structure schematic diagram such as Fig. 5 institutes of aptamer WYZ-3 sequences
Show.
Embodiment three：The analysis of aptamer WYZ-3 and ovarian mucinous cancerous cell 3AO binding abilities：
1. target cell 3AO is inoculated in in 24 orifice plates (50,000/hole), in 37 DEG C, 5%CO2Culture 24h.
2. the single stranded DNA of FITC labellings (is included into aptamer WYZ-3 and unscreened single-stranded 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. with combination buffer washed cell twice, the single stranded DNA solution of the above-mentioned gradient concentrations of 200 μ L is added per hole, and
It is placed in.
4., after being incubated, supernatant is abandoned, is scraped with cell and cell is swept；Washed twice with combination buffer, cell is resuspended, body
Product is 250 μ L.
5. fluoremetry is carried out to cell using the FACSAria flow cytometers of BD companies；Measurement result GraphPad
6 softwares of Prism are mapped, and the dissociation constant for calculating gained aptamer WYZ-3 is 30.8 ± 10.6nM, as shown in Figure 1.
The testing result of Fig. 1 shows：The binding ability of aptamer WYZ-3 and target cell ovarian mucinous cancerous cell 3AO is strong, dissociation
Constant is in nanomole rank.
Example IV：The analysis of aptamer WYZ-3 and ovarian mucinous cancerous cell 3AO binding specificities：
1. count cell to be measured and be inoculated in 24 orifice plates (50,000/hole), in 37 DEG C, 5%CO2Culture 24h.
2. with combination buffer washed cell twice, add the single stranded DNA of 200 μ L FITC labellings (suitable including nucleic acid per hole
Part WYZ-3 and unscreened single-stranded DNA banks), it is placed in being incubated 30min on ice.
3., after being incubated, supernatant is abandoned, is scraped with cell and cell is swept；Washed twice with combination buffer, cell is resuspended, body
Product is 250 μ L.
4. fluoremetry is carried out to cell using the FACSAria flow cytometers of BD companies.
Described cell to be measured is respectively ovarian mucinous cancerous cell 3AO, ovarian serous cancerous cell SKOV3 and normal
Ovarian epithelial cell IOSE, wherein, ovarian serous cancerous cell SKOV3 and normal ovarian epithelial cell IOSE is thin for control
Born of the same parents, testing result is as shown in figs. 2 to 4.Proved by the testing result of Fig. 2～4 flow cytometry, the aptamer of the present invention
WYZ-3 has stronger specific recognition capability to ovarian mucinous cancerous cell 3AO, and thin to compared with control cells ovarian serous carcinoma
Born of the same parents SKOV3 and normal ovarian epithelial cell IOSE are without identification.
SEQUENCE LISTING
<110>Wu Dong
<120>The aptamer WYZ-3 of ovarian mucinous cancerous cell 3AO and its screening technique and application
<160> 3
<210> 1
<211> 79
<212> DNA
<213>Artificial sequence
<400> 1
agctgctata ctagctaaag acctgaggtc ctttgacgtt ccaggtagac 50
ttgaaccgta 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 (10)
1. a kind of aptamer WYZ-3 of ovarian mucinous cancerous cell 3AO, it is characterised in that：Its sequence is as follows：
5’-AGCTGCTATACTAGCTAAAGACCTGAGGTCCTTTGACGTTCCAGGTAGACTTGAACCGTACCGTAGCGAT
AGCTAGCTT-3’。
2. the aptamer WYZ-3 of ovarian mucinous cancerous cell 3AO according to claim 1, it is characterised in that：37
DEG C, 0.137M Na+, 0.001M Mg2+Under the conditions of its space structure it is as follows：
3. the aptamer WYZ-3 of ovarian mucinous cancerous cell 3AO according to claim 1, it is characterised in that：To institute
5 ' the ends or 3 ' ends for stating aptamer WYZ-3 carry out FITC, amino, biotin or Digoxin chemical modification.
4. the screening of the aptamer WYZ-3 of the ovarian mucinous cancerous cell 3AO according to claim 1-3 any one
Method, it is characterised in that：Based on the external SELEX triage techniqueses of aptamer, with ovarian serous cancerous cell SKOV3 and just
Often ovarian epithelial cell IOSE, is filtered out viscous with ovary with ovarian mucinous cancerous cell 3AO as positive sieve cell as anti-sieve cell
The aptamer of fluidity cancerous cell 3AO specific bindings.
5. the screening technique of the aptamer WYZ-3 of ovarian mucinous cancerous cell 3AO according to claim 4, which is special
Levy and be：It comprises the following steps：
(1) single-stranded DNA banks shown in following sequence are prepared：
Single-stranded DNA banks：
5’-AGCTGCTATACTAGCTAAAGNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNCCGTAGCGAT
AGCTAGCTT-3’；
(2) SKOV3 cells are counter sieves：The library that step (1) is prepared and SKOV3 cell incubations, collect cell and incubate after the completion of incubation
Supernatant after educating；
(3) IOSE cells are counter sieves：By supernatant obtained by step (2) and IOSE cell incubations, after collecting cell incubation after the completion of incubation
Supernatant；
(4) 3AO cells are just sieved：By the supernatant obtained by step (3) and 3AO cell incubations, after the completion of incubation, supernatant is abandoned；Washing
3AO cells after 3AO cells are scraped, carry out heat denatured in being transferred to EP pipes, and supernatant is collected in centrifugation afterwards；
(5) PCR enrichments：Supernatant obtained by step (4) is entered into performing PCR amplification, pcr amplification product is obtained；Wherein PCR expands institute
Primer is：
Primers F up:5’-FAM-AGCTGCTATACTAGCTAAAG-3’；
Primer Ldown:5’-TTTTTTTTTTTTTTTTTTTTTTT-spacer
C9-AAGCTAGCTATCGCTACGG-3’；
(6) single stranded DNA purification：Pcr amplification product is carried out after degeneration unwinds, denaturing electrophoretic is separated, purified pool FAM labellings
ssDNA；
(7) aptamer Cycle Screening：The secondary library that ssDNA obtained by step (6) is screened as next round, and repeat
The screening process of step (2)～(6).
6. the aptamer WYZ-3 of the ovarian mucinous cancerous cell 3AO according to claim 1-5 any one should
With, it is characterised in that：Application in test kit, molecular probe or the targeting medium for preparing detection ovarian mucinous cancer.
7. the aptamer WYZ-3 of the ovarian mucinous cancerous cell 3AO according to claim 1-5 any one should
With, it is characterised in that：Application in the targeted therapy probe for preparing treatment ovarian mucinous cancer.
8. the aptamer WYZ-3 of the ovarian mucinous cancerous cell 3AO according to claim 1-5 any one should
With, it is characterised in that：Application in treatment ovarian mucinous cancer drug is prepared.
9. the aptamer WYZ-3 of the ovarian mucinous cancerous cell 3AO according to claim 1-5 any one should
With, it is characterised in that：Application in research ovarian mucinous cancer cell surfaces albumen.
10. the aptamer WYZ-3 of the ovarian mucinous cancerous cell 3AO according to claim 1-5 any one should
With, it is characterised in that：In ovarian carcinoma pathological staining, in separating ovarian mucinous cancerous cell and in ovarian tumor phase
Application in the internal molecular imaging for closing.
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| CN107312779A (en) * | 2017-03-24 | 2017-11-03 | 华东医药（杭州）基因科技有限公司 | For separate trophocyte aptamer, separate trophocyte method and chromosomal copy number analysis of variance method |
| CN113481205A (en) * | 2021-07-30 | 2021-10-08 | 吴冬 | Nucleic acid aptamer HPV1801 of HPV18 virus particles and application thereof |
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| CN104988154A (en) * | 2015-06-24 | 2015-10-21 | 中国科学院化学研究所 | Application of nucleic acid aptamers in identifying and being bound with integrin alpha4 |
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| CN104988154A (en) * | 2015-06-24 | 2015-10-21 | 中国科学院化学研究所 | Application of nucleic acid aptamers in identifying and being bound with integrin alpha4 |
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Cited By (3)
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| CN107312779A (en) * | 2017-03-24 | 2017-11-03 | 华东医药（杭州）基因科技有限公司 | For separate trophocyte aptamer, separate trophocyte method and chromosomal copy number analysis of variance method |
| CN113481205A (en) * | 2021-07-30 | 2021-10-08 | 吴冬 | Nucleic acid aptamer HPV1801 of HPV18 virus particles and application thereof |
| CN113481205B (en) * | 2021-07-30 | 2023-11-03 | 杭州凡泰生物科技有限公司 | Aptamer HPV1801 of HPV18 virus particle and application thereof |
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