CN106520773B - The aptamer WYZ-3 and its screening technique of ovarian mucinous cancer cell 3AO and application - Google Patents
The aptamer WYZ-3 and its screening technique of ovarian mucinous cancer cell 3AO and application Download PDFInfo
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Abstract
The present invention relates to the aptamer WYZ-3 of ovarian mucinous cancer cell 3AO a kind of and its screening technique and applications, the sequence of the acid aptamers WYZ-3 are as follows: 5 '-AGCTGCTATACTAGCTAAAGACCTGAGGTCCTTTGACGTTCCAGGTAGACTTGAAC CGTACCGTAGCGATAGCTAGCTT-3 ', its screening technique is 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 close in vitro At and modification, chemical property is stable, pharmacokinetic property is good, non-immunogenicity the features such as.
Description
Technical field
The present invention relates to the aptamer WYZ-3 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-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 cancer cell 3AO, its sequence are as follows:
5’-AGCTGCTATACTAGCTAAAGACCTGAGGTCCTTTGACGTTCCAGGTAGACTTGAACCGTACCGTA
GCGATAGCTAGCTT-3’
The screening technique of the aptamer WYZ-3 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-3 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-3 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-3 of the ovarian mucinous cancer cell 3AO treats ovarian mucinous in preparation
Application in cancer drug.
The application of the aptamer WYZ-3 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-3 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-3 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-3 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-3 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-3 combination ovarian mucinous cancer cell 3AO is measured using flow cytometry
Curve is drawn from constant.Dissociation constant Kd=30.8 ± 10.6nM.In Fig. 1, abscissa is DNA concentration (nM), and ordinate is
Average fluorescent strength.
Fig. 2 is the offset using flow cytometry aptamer WYZ-3 to ovarian mucinous cancer cell 3AO.?
In Fig. 2, abscissa is fluorescence intensity, and ordinate is number of cells, and solid line WYZ-3, dotted line is unscreened single stranded DNA text
Library.
Fig. 3 is the offset using flow cytometry aptamer WYZ-3 to ovarian serous cancer cell SKOV3.
In Fig. 3, abscissa is fluorescence intensity, and ordinate is number of cells, and solid line WYZ-3, dotted line is unscreened single stranded DNA
Library.
Fig. 4 is the offset using flow cytometry aptamer WYZ-3 to normal ovarian epithelial cell IOSE.?
In Fig. 4, abscissa is fluorescence intensity, and ordinate is number of cells, and solid line WYZ-3, dotted line is unscreened single stranded DNA text
Library.
Fig. 5 is the space structure schematic diagram of aptamer WYZ-3.
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-3 of ovarian mucinous cancer cell 3AO, its sequence are as follows:
5’-AGCTGCTATACTAGCTAAAGACCTGAGGTCCTTTGACGTTCCAGGTAGACTTGAACCGTACCGTA
GCGATAGCTAGCTT-3’
The aptamer WYZ-3 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-3 of the ovarian mucinous cancer cell 3AO, to the 5 ' of 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 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-3 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’-AGCTGCTATACTAGCTAAAGNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNCCGTA
GCGATAGCTAGCTT-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-3:
The screening technique of the aptamer WYZ-3 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-3 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 DNAKit is to library Quality Control；Utilize Quant-iT PicogGreen dsDNA Assay
Kit quantifies library；Using 500 platform of IlluminateNextSeq, bridge-type PCR expansion is carried out by template of single-stranded library
Increase, sequencing primer is annealed, is sequenced in synthesis；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-3 sequence.Analyze secondary structure schematic diagram such as Fig. 5 institute of aptamer WYZ-3 sequence
Show.
Embodiment three: the analysis of aptamer WYZ-3 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-3 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-3 is 30.8 ± 10.6nM, as shown in Figure 1.
The testing result of Fig. 1 shows: aptamer WYZ-3 and the binding ability of target cell ovarian mucinous cancer cell 3AO are strong, dissociation
Constant is in nanomole rank.
Example IV: the analysis of aptamer WYZ-3 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-3 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-3 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-3 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 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 (4)
1. a kind of aptamer WYZ-3 of ovarian mucinous cancer cell 3AO, it is characterised in that: its sequence is as follows:
5’-AGCTGCTATACTAGCTAAAGACCTGAGGTCCTTTGACGTTCCAGGTAGACTTGAACCGTACCGTAGCGA
TAGCTAGCTT-3’。
2. the aptamer WYZ-3 of ovarian mucinous cancer 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 cancer 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 aptamer WYZ-3 of ovarian mucinous cancer cell 3AO described in claim 1-3 any one is detected in preparation
Application in the kit of ovarian mucinous cancer, molecular probe or targeting medium.
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Citations (2)
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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 |
| CN105044322A (en) * | 2015-06-24 | 2015-11-11 | 中国科学院化学研究所 | Application of aptamer in recognition of L selectin and combination with L selectin |
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| Publication number | Priority date | Publication date | Assignee | Title |
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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 |
| CN105044322A (en) * | 2015-06-24 | 2015-11-11 | 中国科学院化学研究所 | Application of aptamer in recognition of L selectin and combination with L selectin |
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| Title |
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| 《An on-chip Cell-SELEX process for automatic selection of high-affinity aptamers specific to differenthistologically classified ovarian cancer cells》;Hung Lien-Yu等;《Lab on a chip》;20141021;第14卷(第20期);第4017-4028页 |
| 《Study of the Molecular Recognition of Aptamers Selected through Ovarian Cancer Cell-SELEX》;Van Simaeys D等;《Plos one》;20101101;第5卷(第11期);第1-7页 |
| 《全细胞的核酸适配体筛选的研究进展》;刘品多等;《色谱》;20160430;第34卷(第4期);第382-388页 |
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