CN103642810A - Nucleic acid aptamer and screening method thereof, and application of nucleic acid aptamer in prostate cancer cell strain detection - Google Patents
Nucleic acid aptamer and screening method thereof, and application of nucleic acid aptamer in prostate cancer cell strain detection Download PDFInfo
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Abstract
The present invention discloses a nucleic acid aptamer, wherein the sequence of the nucleic acid aptamer comprises a DNA segment represented by any one sequence selected from a sequence 1, a sequence 2 and a sequence 3. The nucleic acid aptamer can further be various similar sequences with high homology or a derivative obtained from the sequence of the present invention. The invention further discloses a nucleic acid aptamer screening method, which comprises: synthesizing a random single-stranded DNA library and primers, carrying out SELEX screening, carrying out PCR amplification of library, preparing a DNA single strand library, and finally carrying out repeated screening, negative screening and multi-round screening to obtain the nucleic acid aptamer. The nucleic acid aptamer and the derivative thereof can be used in recognition of the prostate cancer cell strain PC-3 or preparation of kits, molecular probes and targeted mediums for prostate cancer detection, and can further be used in design and preparation of prostate cancer treatment drugs. Compared with the protein antibody, the nucleic acid aptamer of the present invention has advantages of high affinity, high specificity, no immunogenicity, capability of being chemically synthesized, small molecular weight, stability, easy storage, easy labeling and the like.
Description
Technical field
The present invention relates to a kind of aptamer and screening thereof and application, relate in particular to a kind of aptamer and screening method and application that can be used for detecting prostate cancer cell.
Background technology
Prostate cancer (prostate cancer, PCa) is one of modal malignant tumour of the American-European countries male sex, and mortality ratio is only second to lung cancer.In recent years, along with the rising of aging population ratio and the change of dietary structure, the sickness rate of China PCa presents obvious ascendant trend, and it is the first that domestic research report shows that District of Shanghai PCa sickness rate has leapt to male urinary system tumour.Due to the concealment of prostate cancer onset, when most of prostate cancers obtain making a definite diagnosis, it has been progressive stage or late period, at present to developing into the prostate cancer in this stage, take endocrine therapy clinically, although endocrine therapy is originally effective to most patients, anticancer completely, patient can final progress be hormone refractory type prostate cancer (hormone-refractory prostate cancer, HRPC), and cancer cells shift.But the early stage limitation surgery for prostate cancer radical cure of clinical discovery is effective, 5 years survival rates of foreign literature report are that 99%, 10 year survival rate is 95%.Therefore, if whether accurate evaluation prostate cancer occurs to shift and invasion and attack in early days, the position of transfer and size, the scope of invasion and attack and degree, have great meaning for the operation of clinician's choose reasonable and treatment plan.Transitivity HRPC treatment is very thorny, becomes the underlying cause of death of prostate cancer.With regard to prostate cancer patients with terminal, treatment means is limited at present, and effect is poor, and its reason is that drug dose is high, and toxic side effect is large, and terms of settlement is except developing new medicine, and targeted therapy becomes topmost method.In addition, the patient for biochemical recurrence after Prostate Cancer after Radical, lacks effective targeting diagnosis measure at present, is difficult to judge position and the position of tumor recurrence, makes clinician select rational treatment plan very difficult.At present, lacking the target medium of high degree of specificity, is the targeting diagnosis of restriction prostate cancer and the bottleneck for the treatment of development.
Aptamer, is and the similar single stranded oligonucleotide molecule of antibody function.Can specific recognition comprise the multiple targets such as metal ion, organic molecule, polypeptide, protein, cell, its avidity and antibody are quite even higher than antibody.And.Aptamers molecule is little, does not carry genetic information, without immune prototype, and stable chemical nature, synthesizing the advantages such as convenience and batch difference are little is that antibody cannot replace.Therefore aptamer is having broad prospects aspect the diagnosis of biological detection, disease and targeted therapy.
Summary of the invention
In order to overcome the deficiencies in the prior art, the technical problem to be solved in the present invention is to provide a kind ofly to be had that high degree of specificity, non-immunogenicity, molecular weight are little, stable chemical nature, is easy to preserve and the aptamer and the derivative thereof that can be used for detecting ptostate cancer PC 3 cell line of mark also corresponding screening method and the application that described aptamer is provided.
In order to solve the problems of the technologies described above, on the one hand, the invention provides a kind of aptamer, the nucleotide sequence of this aptamer comprises the DNA fragmentation shown in any sequence in following sequence 1, sequence 2, sequence 3:
Sequence 1:
5’-ACGCTCGGATGCCACTACAGCTGGTTCGGTTTGGTGACTTCGTTCTTCGTTGTGGTGCTTAGTGGCTCATGGACGTGCTGGTGAC-3’；
Sequence 2:
5’-TGCCACTACAGCTGGTTCGGTTTGGTGACTTCGTTCTTCGTTGTGGTGCTTAGTGGC-3’；
Sequence 3:
5’-TGCCACTAAGCTGGTTCGGTTTGGTGACTTCGTTCTTCGTTGTGGTGCTTAGTGGC-3’。
As the improvement to technique scheme, selectively, a certain position that the nucleotides sequence of above-mentioned aptamer lists is phosphorylated, methylates, amination, sulfhydrylation or isotropic substance.
As the improvement to technique scheme, selectively, the nucleotides sequence of aforementioned nucleic acid aptamers lists and is connected with fluorescent substance, radioactive substance, therapeutic substance, vitamin H, digoxin, nano luminescent material or enzyme labelling.
No matter be to replace or nucleotide sequence after modifying through part above, all there is former aptamer basic identical or similar molecular structure, physico-chemical property and function, all can be used for detecting prostate cancer.
As a total technical conceive, the present invention also provides a kind of aptamer, and the nucleotide sequence of described aptamer comprises any one in following three kinds of sequences:
(1) more than 60% (for example aforementioned nucleic acid aptamers sequence can be deleted or increased the Nucleotide of part complementation) with the homology of the nucleotide sequence of aptamer described in aforementioned all technical schemes;
(2) sequence of hybridizing with the nucleotide sequence of aptamer described in aforementioned all technical schemes;
(3) the RNA sequence of transcribing with the nucleotide sequence of aptamer described in aforementioned all technical schemes.
As a total technical conceive, the present invention also provides a kind of aptamer derivative, described derivative is the phosphorothioate backbone that the skeleton of the nucleotide sequence of aptamer described in aforementioned all technical schemes derives, or the corresponding peptides nucleic acid that described in aforementioned all technical schemes, aptamer is transformed into.
No matter the aptamer deriving above or other derivatives that derive, all have former aptamer basic identical or similar Molecular connectivity and structure properties and function, all can be used for detecting prostate cancer.
On the other hand, it is a kind of as the screening method of aforementioned nucleic acid aptamers that the present invention also provides, and comprises the following steps:
(1) random single-stranded DNA banks and the primer shown in synthetic following sequence:
Random nucleic acid library:
5-ACGCTCGGATGCCACTACAG-（45N）-CTCATGGACGTGCTGGTGAC-3’
5 ' primer: 5 '-Fam-ACGCTCGGATGCCACTACAG-3 ';
3 ' primer: 5 '-Bio-GTCACCAGCACGTCCATGAG-3 ';
(2) aptamer screening: above-mentioned random nucleic acid library and PC-3 cell strain cell are hatched; After having hatched, collect PC-3 cell strain cell; Heat denatured separation and combination, in the aptamers of PC-3 cell strain cell, is the aptamer one-level library of screening gained;
(3) negative screening: step (2) resulting one-level library and non-target cell are hatched, hatched the supernatant after rear collecting cell is hatched, exclude the nucleotide sequence of non-specific binding, obtain enrichment and aptamers group target cell specific binding library;
(4) selectivity, the avidity of each sequence of the resulting aptamers group of difference authentication step (3), finally obtain aptamer.
As the improvement to technique scheme, selectively, it is a kind of as the screening method of aforementioned nucleic acid aptamers that the present invention also provides, and comprises the following steps:
(1) random single-stranded DNA banks and the primer shown in synthetic following sequence:
Random nucleic acid library:
5’-ACGCTCGGATGCCACTACAG-（45N）-CTCATGGACGTGCTGGTGAC-3’；
5 ' primer: 5 '-Fam-ACGCTCGGATGCCACTACAG-3 ';
3 ' primer: 5 '-Bio-GTCACCAGCACGTCCATGAG-3 ';
(2) the elementary screening of aptamer: above-mentioned random nucleic acid library and PC-3 cell strain cell are hatched; After having hatched, collect PC-3 cell strain cell washing; Heat denatured separation and combination, in the aptamers of PC-3 cell strain cell, is the aptamer one-level library of screening gained;
(3) pcr amplification one-level library: conventional pcr amplification is carried out in the one-level library that screening in step (2) is obtained, and obtains amplified production;
(4) prepare DNA single chain library: with the resulting amplified production of Streptavidin dextran microspheres separating bio element markers step (3); Then utilize elutriant that double-stranded DNA sex change is unwind, collect the DNA single chain library of mark;
(5) instead sieve: step (4) resulting DNA single chain library and non-target cell are hatched, hatched the supernatant after rear collecting cell is hatched, exclude the nucleotide sequence of non-specific binding, obtain the aptamers group library with target cell specific binding;
(6) just sieve: resulting supernatant and the target cell that contains fit group of library of specificity of step (5) hatched, and elution of bound is in the high-affinity aptamers group on target cell surface, after pcr amplification, obtain enrichment and aptamers group target cell specific binding library;
(7) aptamer Cycle Screening: with the resulting amplified production of enrichment aptamers group library step of replacing (3) of described step (6) gained, and the screening process of repeating step (4)～(6), until the avidity selectivity in the aptamers group library obtaining no longer rises;
(8) the resulting aptamers group of cloning and sequencing step (7) library, identifies respectively and selectivity, the avidity of each sequence finally obtains aptamer.
As the further improvement to technique scheme, selectively, the present invention also provides a kind of screening method of aptamer, comprises the following steps:
(1) random single-stranded DNA banks and the primer shown in synthetic following sequence:
Random nucleic acid library:
5’-ACGCTCGGATGCCACTACAG-（45N）-CTCATGGACGTGCTGGTGAC-3’；
5 ' primer: 5 '-Fam-ACGCTCGGATGCCACTACAG-3 ';
3 ' primer: 5 '-Bio-GTCACCAGCACGTCCATGAG-3 ';
(2) the elementary screening of aptamer: above-mentioned random nucleic acid library and PC-3 cell strain cell are hatched; After having hatched, collect PC-3 cell strain cell washing; Heat denatured separation and combination, in the aptamers of PC-3 cell strain cell, is the aptamer one-level library of screening gained;
(3) pcr amplification one-level library: conventional pcr amplification is carried out in the one-level library that screening in step (2) is obtained, and obtains amplified production;
(4) prepare DNA single chain library: with the resulting amplified production of Streptavidin dextran microspheres separating bio element markers step (3); Then utilize elutriant that double-stranded DNA sex change is unwind, collect the DNA single chain library of mark;
(5) instead sieve: step (4) resulting DNA single chain library and non-target cell are hatched, hatched the supernatant after rear collecting cell is hatched, exclude the nucleotide sequence of non-specific binding, obtain the aptamers group library with target cell specific binding;
(6) just sieve: resulting supernatant and the target cell that contains fit group of specificity of step (5) hatched to fit group of the high-affinity of elution of bound on target cell surface;
(7) pcr amplification aptamers group library: conventional pcr amplification is carried out in the aptamers group library that screening in step (6) is obtained, and obtains amplified production;
(8) prepare aptamers group strand library: with the resulting amplified production of Streptavidin dextran microspheres separating bio element markers step (7); Then utilize elutriant that double-stranded DNA sex change is unwind, collect the aptamers group strand library of mark;
(9) aptamer Cycle Screening: with the resulting DNA single chain of aptamers group strand library alternative steps (4) library of described step (8) gained, and the screening process of repeating step (5)～(8), until the avidity selectivity in the aptamers group library obtaining no longer rises;
(10) the resulting aptamers group of cloning and sequencing step (9) library, identifies respectively and selectivity, the avidity of each sequence finally obtains aptamer.
The third aspect, the present invention also provides a kind of aforementioned nucleic acid aptamers or in preparation, has detected the application in prostate gland cancer reagent kit, molecular probe or target medium as aptamer derivative.
Fourth aspect, the present invention also provides a kind of aforementioned nucleic acid aptamers or the purposes of aptamer derivative in design and preparation treatment prostate cancer medicine.
Compared with prior art, the invention has the advantages that: the aptamer that the present invention obtains by screening has than the higher avidity of protein antibodies and specificity; Non-immunogenicity; Can external chemosynthesis, molecular weight is little, can different sites be modified and be replaced, and sequence is stable, is easy to preserve; Be convenient to mark (not needing two of mark to resist) etc.While adopting aptamer of the present invention to carry out the detection of prostate cancer cell, operate more simply, rapidly, and the synthetic cost of aptamer of the present invention is low compared with antibody preparation cost, and the cycle is short, favorable reproducibility.
The aptamer of identifying ptostate cancer PC 3 cell line of the present invention all can specificity and the identification ptostate cancer PC 3 cell line of high-affinity, and binding ability is strong.Utilize aptamer of the present invention to the danger of tumour, to carry out layering from molecule angle; It is to the specific recognition of neoplasm metastasis, combination, the evaluation to target, and the diagnosis and the treatment that can be metastasis provide good means, and this early detection for prostate cancer is significant.
Accompanying drawing explanation
Fig. 1 is the enrichment process in aptamers library in embodiment of the present invention screening process: peak shape represents that PC-3 cell takes turns with fluorescein isothiocyanate (FITC) mark library and the 1st, 5 takes turns, 10 takes turns, 15 takes turns the situation that screening product is combined.
Fig. 2 is the restraining effect result of 2 years Zorubicin aptamers of sequence to different clones in the embodiment of the present invention.
Fig. 3 is the flow cytometer detection result of sequence 2 aptamers and target cell and non-target cell binding ability in the embodiment of the present invention.
Fig. 4 is the Confocal image of sequence 2 aptamers and target cell and part non-target cell binding ability in the embodiment of the present invention.
Fig. 5 is matched curve and the dissociation constant (Kd) that in the embodiment of the present invention, sequence 2 and sequence 3 aptamers are combined with target cell.
Fig. 6 is sequence 2 aptamers and hyperplastic prostate tissue (BPH) in the embodiment of the present invention, the comparison of prostate cancer fluorescent staining strength difference.
Embodiment
Following embodiment is convenient to better understand the present invention, but is not limited to the present invention.Experimental technique in following examples if no special instructions, is ordinary method.In subordinate embodiment experiment material used if no special instructions, be from routine biochemistry reagent shop, buy resulting.
Cell derived: this tests all cells used all from Chinese Academy of Sciences's Shanghai cell bank and Union Medical College cell bank.
The present invention utilizes the in-vitro screening technology SELEX technology of aptamer, with ptostate cancer PC 3 cell line for just sieving target, take multiple normal cell strain or other JEG-3 as anti-sieve target, from external synthetic random oligo DNA library, filter out the aptamer with described ptostate cancer PC 3 cell line specific combination.
In the present invention, described aptamer sequence can be selected from the sequence of natural existence or synthetic, or the same sequence in any other source.
In the present invention, described aptamer sequence contains all identical sequences with the Nucleotide of described characteristic sequence.
In the present invention, can aptamer be modified or be transformed, obtain the derivative of described aptamer, the derivative of described aptamer can be:
A), by described aptamer deletion or increase the complementary Nucleotide of part, what obtain has the derivative of the aptamer of identical function with described aptamer.
B) described aptamer is carried out to Nucleotide replacement or part is modified, what obtain has the aptamer derivative of identical function with described aptamer.
C) transform the skeleton of described aptamer as phosphorothioate backbone, what obtain has the aptamer derivative of identical function with described aptamer.
D) transform aptamer as peptide nucleic acid(PNA), what obtain has the derivative of the aptamer of identical function with described aptamer.
E) above-mentioned aptamer is connected after the materials such as upper fluorescent substance, radioactive substance, therapeutic substance, vitamin H, enzyme labelling, what obtain has the aptamer derivative of identical function with described aptamer.
The target of sequence of the present invention is not being expressed with normal prostate tissue, and has compared with high expression level in cancerous tissue.Target in the embodiment of the present invention is ptostate cancer PC 3 cell line.
Embodiment 1: the screening of prostate gland cancer cell specificity aptamer
1. the design in random nucleic acid library is with synthetic
The synthetic two ends of design comprise 20 Nucleotide (primer), and centre comprises that the nucleotide sequence library of 45 Nucleotide stochastic sequences is as follows:
5’-ACGCTCGGATGCCACTACAG-（45N）-CTCATGGACGTGCTGGTGAC-3’。
2.Cell-SELEX screening obtains aptamer
2.1 hatch: with binding buffer solution (PBS, the glucose containing 0.45%, 5mM MgCl
2, 0.1mg/mL herring sperm dna) and dissolve above-mentioned random nucleic acid library, 95 ℃ of constant temperature oscillation 5min, put into rapidly ice; Then with cultivate the ptostate cancer PC 3 cell line good with pre-treatment and on ice, hatch 1 hour.
2.2 dissociate: after having hatched, pour out the liquid of hatching in culturing bottle, with lavation buffer solution (PBS, the glucose containing 0.45%, 5mM MgCl
2) wash and hatch the cell in culturing bottle; By 1mL sterilized water scraping, hatch culturing bottle inner cell, be placed in EP pipe boiling water bath heating 10min, heat denatured separation and combination is in the aptamers of PC-3 cell surface; 12000rmp centrifuging and taking supernatant, is the specific nucleic acid aptamers library of screening gained PC-3 cell.
3. carry out pcr amplification library
Get the PC-3 cell-specific aptamer library of 100 μ L screening gained and carry out conventional pcr amplification, amplimer is 5 ' Fam-ACGCTCGGATGCCACTACAG3 ', 5 ' Bio-GTCACCAGCACGTCCATGAG3 '; Amplification condition is: 94 ℃ of 3min, and 94 ℃ of 30sec, 58.5 ℃ of 30sec, 72 ℃ of 30sec, through suitable circulation wheel number, 72 ℃ of 3min; After first round screening, need 10 circulations of the pre-amplification in the PC-3 cell-specific aptamer library of full income, then carry out the amplification of this step, obtain amplified production, use biotin labeling amplified production.
4. prepare DNA single chain library
The 200 μ L rifle heads by the 60 μ L inputs of Streptavidin dextran microspheres with filter membrane, make micro-wash-out post, 150 μ L PBS washed twice, by the double-stranded DNA of pcr amplification gained in step 3 post that pressurizeed completely, the affinity interaction by the vitamin H on double-stranded DNA and the Streptavidin in agarose microbeads fully captures agarose microbeads surface by double-stranded DNA; PBS washed twice, then adds the 200mM NaOH solution 100 μ L double-stranded DNA that dissociates, and makes the strand of flag F ITC with elutriant, flow out and collect completely; Except salt plug is with after the washing of 10mL sterilized water, collect the elutriant obtaining after adding alkaline denaturation, naturally drip off.Add 1mL sterilized water, collect the solution dripping, this is DNA single chain library.
5. instead sieve
After the DNA single chain library dissolving that step 4 screening is obtained, constant temperature oscillation, on ice, hatch 1 hour with non-target cell after pretreatment (as human liver cancer cell SMMC-7721), hatch the supernatant after rear collecting cell is hatched, discarded the nucleotide sequence of being combined with non-target cell.
6. just sieve
With the random nucleic acid library in the resulting supernatant liquor alternative steps 2 of step 5, and the operation of repeating step 2,3,4, obtain the specific nucleic acid aptamers strand library of PC-3 cell.
7. multi-turns screen
The DNA single chain library that alternative steps 4 screenings in the resulting specific nucleic acid aptamers strand of step 6 library are obtained, continue to repeat the operating process of above-mentioned steps 5～6, before each operation all, in single job, that obtain and nucleotide sequence target cell specific combination are initial nucleic acid library, until the avidity selectivity in the aptamers group library obtaining no longer rises.
In described screening method, but each take turns screening and progressively increase screening pressure, to promote the enrichment degree of screening aptamer.Described increase screening pressure can linearly reduce incubation time and the corresponding linear incubation time that increases nucleotide sequence library and non-target cell of nucleotide sequence library and target cell.
8. the aptamer that analysis and identification obtains after repeatedly screening
Products therefrom, through cloning and sequencing analysis, after sequencing result finishing analysis, finally can be obtained to three sequences that enrichment is high, and these three sequences are the aptamer that can be used for detecting ptostate cancer PC 3 cell line of above-mentioned the present embodiment.
Sequence 1:
5’-ACGCTCGGA
TGCCACTACAGCTGGTTCGGTTTGGTGACTTCGT TCTTCGTTGTGGTGCTTAGTGGCTCATGGACGTGCTGGTGAC-3’
Sequence 2:
5’-
TGCCACTACAGCTGGTTCGGTTTGGTGACTTCGTTCTTCGTTGT GGTGCTTAGTGGC-3’
Sequence 3:
5’-
TGCCACTAAGCTGGTTCGGTTTGGTGACTTCGTTCTTCGTTGTG GTGCTTAGTGGC-3’
The aptamer obtaining is made to molecular probe at 5 ' end mark fluorescent molecule, get respectively the molecular probe solution of 0nM, 1nM, 2nM, 4nM, 8nM, 16nM, 32nM, 64nM, 128nM, 256nM, with 5 * 10
5individual PC-3 cell is hatched 20min on ice, then uses lavation buffer solution washed twice, by the fluorescence intensity of cells were tested by flow cytometry cell surface.As the aptamer of cell energy combined with fluorescent mark, with fluorescence intensity, concentration and probe concentration is mapped, use formula Y=B
maxx/(K
d+ X) calculate the equilibrium dissociation constant K of aptamer
d.Result shows, the equilibrium dissociation constant of aptamer is all within the scope of nmole.
Embodiment 2: detect cell-specific
Selected altogether 12 kinds of cell strains to carry out specificity analyses: wherein prostate cancer cell subbreed is three kinds, is respectively PC-3, LNCap, DU-145,22RV-1; Immortalization normal prostatic epithelial cell RWPE-1; Liver cancer cell SMMC-7721; Cervical cancer cell HeLa, lung cell A549, MCF-7; Colorectal carcinoma ubcellular is LoVo; Leukemia cell Jurkat, K562.
Each cell strain all carries out following steps (1) and step (2) is processed, in triplicate.
1. by cell counting, get respectively 5 * 10
5individual cell is scattered in binding buffer liquid, then adds the aptamer of mark, final concentration 0.25 μ M.
2. the mixed solution of cell and aptamer is hatched on ice 20 minutes, after twice of centrifuge washing, utilize flow cytometer to detect the combination situation of aptamer and above-mentioned different cell strain cells.After the random library of cell and FITC mark is hatched, do flow cytometer and detect, setting one fluorescence intensity level is greater than 95% cell fluorescence intensity as flow cytometer background fluorescence.The cell of usining is combined rear fluorescence intensity and is surpassed the percentage of cells of this threshold value (0:< 15% as the power of weighing aptamer and Cell binding ability with aptamer; +: 15-30%; ++: < 30-65%; +++: 65-85%; ++++: > 85%).
The cells were tested by flow cytometry result that different cell strain cells are combined with aptamer as shown in Figure 3.Different cell strain cells are combined and are the results are shown in Table 1 with aptamer.
Table 1: the avidity of sequence 2 aptamers to different tumour cells
| Cell category | In conjunction with situation |
| PC-3 | ++++ |
| LNCap | - |
| DU-145 | - |
| 22RV-1 | - |
| RWPE-1 | - |
| SMMC-7721 | - |
| HeLa | - |
| LoVo | - |
| HCT-116 | - |
| HCT-8 | - |
| MCF-7 | - |
| A549 | - |
| Jurkat | - |
| K562 | - |
Embodiment 3: carry Zorubicin aptamers to cell-targeting restraining effect
2000 cells of every hole inoculation in 96 orifice plates, cultivate after 24 hours, the every Zorubicin of 2 μ M and 0.5 μ M of adding for 4 hours carries Zorubicin aptamers (1 molecular sequences 2 aptamers can be carried 4 molecule Zorubicins) 100 μ L, hatch after 15 minutes, extract pastille substratum out, by cell rinsing once, then add 100 μ L fresh cultures, totally 4 times.Continue to cultivate after 48 hours, every hole adds 10 μ L cck-8, hatches after 40 minutes for 37 ℃, in microplate reader, with wavelength 450nm, measures absorption value (Fig. 4).
As shown in Figure 4, medicine carrying aptamers is to target cell PC-3(1) increment suppress obviously to strengthen, and to HeLa(2) and SMMC-7721(3) the inhibition enhancement of cell is not obvious.
Embodiment 4: aptamer application testing
Application aptamer, to patients with prostate cancer tissue section strain, carries out the prediction of postoperative recurrence risk.By fluorescein isothiocyanate (FITC) flag sequence 2 aptamers sequences, and with the random nucleic acid library in fluorescein isothiocyanate (FITC) mark embodiment 1.
(1) tissue slice dewaxing aquation
1. bake sheet: cut into slices in 60 ℃ of oven for baking 20min;
2. be placed at once the first cylinder dimethylbenzene 15min, then insert in the second cylinder dimethylbenzene 15 minutes;
3. be placed into successively 10min in dehydrated alcohol, 95% ethanol 5min, 70% ethanol 5min;
4. tap water rinses in the basin of 5min(sluggish flow), distilled water rinse one time.
(2) section statining microwave thermal antigen retrieval
1. microwave thermal reparation: get appropriate TE buffer (EDTA0.292g, Tris-base6.05g, be dissolved in 1000mL distilled water, pH=8.0), section is put into the container that fills repair liquid, put in microwave oven and be heated to boiling, stop heating, liquid in container temperature is declined, remain between 95 ℃～98 ℃ and continue 15 minutes.Taking-up container, naturally cools to room temperature, takes out section, after distilled water flushing, then uses PBS(0.01M pH7.4) soak 5 minutes * 3 times (that guarantee immersion for the first time is the PBS newly joining).
This method Repair strength is greater than boiling water bath, but is less than Pressure method.
(3) aptamers is hatched staining procedure
1. binding buffer solution incubated at room 60min first and containing 20% FBS and 1mg/ml herring sperm dna cuts into slices;
2. then contain the binding buffer solution incubated at room 60min of 0.25 μ M FITC flag sequence 2 aptamers with 200 μ l, contrast dyeing process is identical, and blank does not dye;
3. with lavation buffer solution washing three times;
4. dry, anti-cancellation mountant mounting, observes.
As seen from Figure 5: sequence 2 aptamers have the specific binding capacity to prostate cancer cell PC-3, through the matched curve of different concns sequence bonding strength, calculating dissociation constant is 73.59nM, and avidity is stronger.And itself and other non-target cell does not almost have binding ability.The image of flow cytometer detected result and confocal is consistent.
In the detection of clinical practice sample section, (Fig. 6) can see that sequence 2 aptamers have good recognition detection ability equally to prostate cancer, can distinguish hyperplasia of prostate (benign lesion) and tumor tissues (malignant change).Hyperplastic prostate tissue can not be combined with aptamers substantially, prostate cancer tissue can with the stronger combination of sequence 2 aptamers.
Claims (10)
1. an aptamer, the nucleotide sequence of described aptamer comprises the DNA fragmentation shown in any sequence in following sequence 1, sequence 2, sequence 3:
Sequence 1:
5’-ACGCTCGGATGCCACTACAGCTGGTTCGGTTTGGTGACTTCGTTCTTCGTTGTGGTGCTTAGTGGCTCATGGACGTGCTGGTGAC-3’；
Sequence 2:
5’-TGCCACTACAGCTGGTTCGGTTTGGTGACTTCGTTCTTCGTTGTGGTGCTTAGTGGC-3’；
Sequence 3:
5’-TGCCACTAAGCTGGTTCGGTTTGGTGACTTCGTTCTTCGTTGTGGTGCTTAGTGGC-3’。
2. aptamer as claimed in claim 1, is characterized in that: a certain position that the nucleotides sequence of described aptamer lists is phosphorylated, methylates, amination, sulfhydrylation or isotropic substance.
3. aptamer as claimed in claim 1 or 2, is characterized in that: the nucleotides sequence of described aptamer lists and is connected with fluorescent substance, radioactive substance, therapeutic substance, vitamin H, digoxin, nano luminescent material or enzyme labelling.
4. an aptamer, is characterized in that: the nucleotide sequence of described aptamer comprises any one in following three kinds of sequences:
(1) with the homology of nucleotide sequence of aptamer described in claim 1 or 2 or 3 more than 60%;
(2) sequence of hybridizing with the nucleotide sequence of aptamer described in claim 1 or 2 or 3;
(3) the RNA sequence that described in claim 1 or 2 or 3, the nucleotide sequence of aptamer is transcribed.
5. an aptamer derivative, it is characterized in that: described derivative is the phosphorothioate backbone that the skeleton of the nucleotide sequence of aptamer described in claim 1 or 2 or 3 derives, or the corresponding peptides nucleic acid that described in claim 1 or 2 or 3, aptamer is transformed into.
6. a screening method for aptamer as claimed in claim 1, comprises the following steps:
(1) random single-stranded DNA banks and the primer shown in synthetic following sequence:
Random nucleic acid library:
5-ACGCTCGGATGCCACTACAG-（45N）-CTCATGGACGTGCTGGTGAC-3’；
5 ' primer: 5 '-Fam-ACGCTCGGATGCCACTACAG-3 ';
3 ' primer: 5 '-Bio-GTCACCAGCACGTCCATGAG-3 ';
(2) aptamer screening: above-mentioned random nucleic acid library and PC-3 cell strain cell are hatched; After having hatched, collect PC-3 cell strain cell; Heat denatured separation and combination, in the aptamers of PC-3 cell strain cell, is the aptamer one-level library of screening gained;
(3) negative screening: step (2) resulting one-level library and non-target cell are hatched, hatched the supernatant after rear collecting cell is hatched, exclude the nucleotide sequence of non-specific binding, obtain enrichment and aptamers group target cell specific binding library;
(4) selectivity, the avidity of each sequence of the difference resulting aptamers group of authentication step (3) library, finally obtain aptamer.
7. a screening method for aptamer as claimed in claim 1, comprises the following steps:
(1) random single-stranded DNA banks and the primer shown in synthetic following sequence:
Random nucleic acid library:
5’-ACGCTCGGATGCCACTACAG-（45N）-CTCATGGACGTGCTGGTGAC-3’；
5 ' primer: 5 '-Fam-ACGCTCGGATGCCACTACAG-3 ';
3 ' primer: 5 '-Bio-GTCACCAGCACGTCCATGAG-3 ';
(2) the elementary screening of aptamer: above-mentioned random nucleic acid library and PC-3 cell strain cell are hatched; After having hatched, collect PC-3 cell strain cell washing; Heat denatured separation and combination, in the aptamers of PC-3 cell strain cell, is the aptamer one-level library of screening gained;
(3) pcr amplification one-level library: conventional pcr amplification is carried out in the one-level library that screening in step (2) is obtained, and obtains amplified production;
(4) prepare DNA single chain library: with the resulting amplified production of Streptavidin dextran microspheres separating bio element markers step (3); Then utilize elutriant that double-stranded DNA sex change is unwind, collect the DNA single chain library of mark;
(5) instead sieve: step (4) resulting DNA single chain library and non-target cell are hatched, hatched the supernatant after rear collecting cell is hatched, exclude the nucleotide sequence of non-specific binding, obtain the aptamers group library with target cell specific binding;
(6) just sieve: resulting supernatant and the target cell that contains fit group of library of specificity of step (5) hatched, and elution of bound is in the high-affinity aptamers group on target cell surface, after pcr amplification, obtain enrichment and aptamers group target cell specific binding library;
(7) aptamer Cycle Screening: with the resulting amplified production of enrichment aptamers group library step of replacing (3) of described step (6) gained, and the screening process of repeating step (4)～(6), until the avidity selectivity in the aptamers group library obtaining no longer rises;
(8) the resulting aptamers group of cloning and sequencing step (7) library, identifies respectively and selectivity, the avidity of each sequence finally obtains aptamer.
8. screening method as claimed in claim 1, is characterized in that:
(1) random single-stranded DNA banks and the primer shown in synthetic following sequence:
Random nucleic acid library:
5’-ACGCTCGGATGCCACTACAG-（45N）-CTCATGGACGTGCTGGTGAC-3’；
5 ' primer: 5 '-Fam-ACGCTCGGATGCCACTACAG-3 ';
3 ' primer: 5 '-Bio-GTCACCAGCACGTCCATGAG-3 ';
(2) the elementary screening of aptamer: above-mentioned random nucleic acid library and PC-3 cell strain cell are hatched; After having hatched, collect PC-3 cell strain cell washing; Heat denatured separation and combination, in the aptamers of PC-3 cell strain cell, is the aptamer one-level library of screening gained;
(3) pcr amplification one-level library: conventional pcr amplification is carried out in the one-level library that screening in step (2) is obtained, and obtains amplified production;
(4) prepare DNA single chain library: with the resulting amplified production of Streptavidin dextran microspheres separating bio element markers step (3); Then utilize elutriant that double-stranded DNA sex change is unwind, collect the DNA single chain library of mark;
(5) instead sieve: step (4) resulting DNA single chain library and non-target cell are hatched, hatched the supernatant after rear collecting cell is hatched, exclude the nucleotide sequence of non-specific binding, obtain the aptamers group library with target cell specific binding;
(6) just sieve: resulting supernatant and the target cell that contains fit group of specificity of step (5) hatched to fit group of the high-affinity of elution of bound on target cell surface;
(7) pcr amplification aptamers group library: conventional pcr amplification is carried out in the aptamers group library that screening in step (6) is obtained, and obtains amplified production;
(8) prepare aptamers group strand library: with the resulting amplified production of Streptavidin dextran microspheres separating bio element markers step (7); Then utilize elutriant that double-stranded DNA sex change is unwind, collect the aptamers group strand library of mark;
(9) aptamer Cycle Screening: with the resulting DNA single chain of aptamers group strand library alternative steps (4) library of described step (8) gained, and the screening process of repeating step (5)～(8), until the avidity selectivity in the aptamers group library obtaining no longer rises;
(10) the resulting aptamers group of cloning and sequencing step (9) library, identifies respectively and selectivity, the avidity of each sequence finally obtains aptamer.
9. aptamer or the application of aptamer derivative as claimed in claim 5 in test kit, molecular probe or the target medium of preparation detection prostate cancer as described in any one in claim 1～4.
10. aptamer or the aptamer derivative as claimed in claim 5 as described in any one in claim 1～4 treated the purposes in prostate cancer medicine in design and preparation.
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