CN103387988B - Aptamer EpCAM Ccut of epithelial cell adhesion molecule and preparation method thereof - Google Patents
Aptamer EpCAM Ccut of epithelial cell adhesion molecule and preparation method thereof Download PDFInfo
- Publication number
- CN103387988B CN103387988B CN201310328256.0A CN201310328256A CN103387988B CN 103387988 B CN103387988 B CN 103387988B CN 201310328256 A CN201310328256 A CN 201310328256A CN 103387988 B CN103387988 B CN 103387988B
- Authority
- CN
- China
- Prior art keywords
- epcam
- aptamer
- cell adhesion
- epithelial cell
- adhesion molecule
- 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.)
- Expired - Fee Related
Links
- 108010066687 Epithelial Cell Adhesion Molecule Proteins 0.000 title claims abstract description 78
- 108091023037 Aptamer Proteins 0.000 title claims abstract description 44
- 102000018651 Epithelial Cell Adhesion Molecule Human genes 0.000 title claims abstract 14
- 238000002360 preparation method Methods 0.000 title abstract description 9
- 210000004027 cell Anatomy 0.000 claims description 20
- 238000001514 detection method Methods 0.000 claims description 4
- 238000003384 imaging method Methods 0.000 claims description 3
- 201000010099 disease Diseases 0.000 claims description 2
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 claims description 2
- 238000003745 diagnosis Methods 0.000 claims 1
- 108020004414 DNA Proteins 0.000 abstract description 40
- 108091034117 Oligonucleotide Proteins 0.000 abstract description 26
- 238000012216 screening Methods 0.000 abstract description 24
- 102000053602 DNA Human genes 0.000 abstract description 23
- 108020004682 Single-Stranded DNA Proteins 0.000 abstract description 18
- 238000000034 method Methods 0.000 abstract description 12
- 108090000623 proteins and genes Proteins 0.000 abstract description 8
- 102000004169 proteins and genes Human genes 0.000 abstract description 7
- 230000015572 biosynthetic process Effects 0.000 abstract description 5
- 231100000956 nontoxicity Toxicity 0.000 abstract description 3
- 102000039446 nucleic acids Human genes 0.000 abstract description 3
- 108020004707 nucleic acids Proteins 0.000 abstract description 3
- 150000007523 nucleic acids Chemical class 0.000 abstract description 3
- 238000003786 synthesis reaction Methods 0.000 abstract description 3
- 238000013461 design Methods 0.000 abstract description 2
- 102100031940 Epithelial cell adhesion molecule Human genes 0.000 description 70
- 206010028980 Neoplasm Diseases 0.000 description 24
- 201000011510 cancer Diseases 0.000 description 10
- 239000007788 liquid Substances 0.000 description 9
- 239000012148 binding buffer Substances 0.000 description 7
- 230000021164 cell adhesion Effects 0.000 description 7
- 210000002919 epithelial cell Anatomy 0.000 description 7
- 238000006243 chemical reaction Methods 0.000 description 6
- 238000000684 flow cytometry Methods 0.000 description 6
- YBJHBAHKTGYVGT-ZKWXMUAHSA-N (+)-Biotin Chemical compound N1C(=O)N[C@@H]2[C@H](CCCCC(=O)O)SC[C@@H]21 YBJHBAHKTGYVGT-ZKWXMUAHSA-N 0.000 description 5
- 238000012408 PCR amplification Methods 0.000 description 5
- 238000010494 dissociation reaction Methods 0.000 description 5
- 230000005593 dissociations Effects 0.000 description 5
- 208000005443 Circulating Neoplastic Cells Diseases 0.000 description 4
- 238000010438 heat treatment Methods 0.000 description 4
- 230000008569 process Effects 0.000 description 4
- 238000004393 prognosis Methods 0.000 description 4
- 108010090804 Streptavidin Proteins 0.000 description 3
- 230000008901 benefit Effects 0.000 description 3
- 238000002983 circular dichroism Methods 0.000 description 3
- 206010073071 hepatocellular carcinoma Diseases 0.000 description 3
- 231100000844 hepatocellular carcinoma Toxicity 0.000 description 3
- 238000011160 research Methods 0.000 description 3
- 238000005406 washing Methods 0.000 description 3
- 101150084967 EPCAM gene Proteins 0.000 description 2
- 101000920667 Homo sapiens Epithelial cell adhesion molecule Proteins 0.000 description 2
- 241000699666 Mus <mouse, genus> Species 0.000 description 2
- 206010060862 Prostate cancer Diseases 0.000 description 2
- 208000000236 Prostatic Neoplasms Diseases 0.000 description 2
- 101150057140 TACSTD1 gene Proteins 0.000 description 2
- 229950009084 adecatumumab Drugs 0.000 description 2
- 229960002685 biotin Drugs 0.000 description 2
- 235000020958 biotin Nutrition 0.000 description 2
- 239000011616 biotin Substances 0.000 description 2
- 239000008280 blood Substances 0.000 description 2
- 210000004369 blood Anatomy 0.000 description 2
- 238000010367 cloning Methods 0.000 description 2
- 238000004925 denaturation Methods 0.000 description 2
- 230000036425 denaturation Effects 0.000 description 2
- 238000011033 desalting Methods 0.000 description 2
- 230000004069 differentiation Effects 0.000 description 2
- 238000013399 early diagnosis Methods 0.000 description 2
- 238000007421 fluorometric assay Methods 0.000 description 2
- 210000004907 gland Anatomy 0.000 description 2
- 230000012447 hatching Effects 0.000 description 2
- 238000000338 in vitro Methods 0.000 description 2
- 238000002347 injection Methods 0.000 description 2
- 239000007924 injection Substances 0.000 description 2
- 230000003993 interaction Effects 0.000 description 2
- 238000002372 labelling Methods 0.000 description 2
- 239000011159 matrix material Substances 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 239000002773 nucleotide Substances 0.000 description 2
- 125000003729 nucleotide group Chemical group 0.000 description 2
- 238000012163 sequencing technique Methods 0.000 description 2
- 239000000243 solution Substances 0.000 description 2
- 210000000130 stem cell Anatomy 0.000 description 2
- 238000012546 transfer Methods 0.000 description 2
- 238000011282 treatment Methods 0.000 description 2
- 210000004881 tumor cell Anatomy 0.000 description 2
- 208000010507 Adenocarcinoma of Lung Diseases 0.000 description 1
- 206010006187 Breast cancer Diseases 0.000 description 1
- 208000026310 Breast neoplasm Diseases 0.000 description 1
- 101710147220 Ent-copalyl diphosphate synthase, chloroplastic Proteins 0.000 description 1
- 101100518501 Mus musculus Spp1 gene Proteins 0.000 description 1
- 241000699670 Mus sp. Species 0.000 description 1
- 206010033128 Ovarian cancer Diseases 0.000 description 1
- 206010061535 Ovarian neoplasm Diseases 0.000 description 1
- 101100163901 Rattus norvegicus Asic2 gene Proteins 0.000 description 1
- 201000000582 Retinoblastoma Diseases 0.000 description 1
- 238000005411 Van der Waals force Methods 0.000 description 1
- 229930003756 Vitamin B7 Natural products 0.000 description 1
- 230000003321 amplification Effects 0.000 description 1
- 230000027455 binding Effects 0.000 description 1
- 230000004071 biological effect Effects 0.000 description 1
- 208000035269 cancer or benign tumor Diseases 0.000 description 1
- 230000012292 cell migration Effects 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 238000001142 circular dichroism spectrum Methods 0.000 description 1
- 210000005266 circulating tumour cell Anatomy 0.000 description 1
- 230000004087 circulation Effects 0.000 description 1
- 210000001072 colon Anatomy 0.000 description 1
- 230000002596 correlated effect Effects 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 230000007812 deficiency Effects 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 210000000981 epithelium Anatomy 0.000 description 1
- 208000021045 exocrine pancreatic carcinoma Diseases 0.000 description 1
- 201000006585 gastric adenocarcinoma Diseases 0.000 description 1
- 229910052739 hydrogen Inorganic materials 0.000 description 1
- 239000001257 hydrogen Substances 0.000 description 1
- 230000002209 hydrophobic effect Effects 0.000 description 1
- 230000036039 immunity Effects 0.000 description 1
- 238000009169 immunotherapy Methods 0.000 description 1
- 230000006698 induction Effects 0.000 description 1
- 201000007270 liver cancer Diseases 0.000 description 1
- 208000014018 liver neoplasm Diseases 0.000 description 1
- 230000003211 malignant effect Effects 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
- 230000001404 mediated effect Effects 0.000 description 1
- 239000003068 molecular probe Substances 0.000 description 1
- 230000009871 nonspecific binding Effects 0.000 description 1
- 231100000252 nontoxic Toxicity 0.000 description 1
- 230000003000 nontoxic effect Effects 0.000 description 1
- 238000003199 nucleic acid amplification method Methods 0.000 description 1
- 238000011275 oncology therapy Methods 0.000 description 1
- 230000010355 oscillation Effects 0.000 description 1
- 230000001575 pathological effect Effects 0.000 description 1
- 230000003449 preventive effect Effects 0.000 description 1
- 238000011321 prophylaxis Methods 0.000 description 1
- 210000000664 rectum Anatomy 0.000 description 1
- 230000001105 regulatory effect Effects 0.000 description 1
- 230000004044 response Effects 0.000 description 1
- 239000000523 sample Substances 0.000 description 1
- 230000019491 signal transduction Effects 0.000 description 1
- 230000004083 survival effect Effects 0.000 description 1
- 230000002194 synthesizing effect Effects 0.000 description 1
- 239000013077 target material Substances 0.000 description 1
- 238000002626 targeted therapy Methods 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 239000011735 vitamin B7 Substances 0.000 description 1
- 235000011912 vitamin B7 Nutrition 0.000 description 1
Landscapes
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The aptamer EpCAM of epithelial cell adhesion molecule? Ccut and preparation method thereof, relates to a kind of nucleic acid.The aptamer of the epithelial cell adhesion molecule EpCAM with high specific and high-affinity and preparation method thereof and application are provided.The aptamer of described epithelial cell adhesion molecule EpCAM has G tetramer structure or loop-stem structure, preparation method comprises: design and synthesize single stranded DNA random oligonucleotide storehouse, screening object oligonucleotide sequence, and identify its specificity be combined with target protein and avidity by flow cytometer showed method.The aptamer nontoxicity that screening obtains, molecular weight is little, good penetrability, be easy to synthesis and mark, a specific recognition EpCAM albumen, and specific recognition expresses the clone of EpCAM albumen, does not have recognition function to the clone not expressing this albumen.
Description
Technical field
The present invention relates to a kind of nucleic acid, particularly the aptamer preparation method of EpCAM (Epithelialcelladhesionmolecule) epithelial cell adhesion molecule and the application in tumour early detection, treatment and prognosis thereof.
Background technology
EpCAM (Epithelialcelladhesionmolecule) epithelial cell adhesion molecule belongs to adhesion molecule family, also referred to as 17-A, ESA, EGP40, Trop-1, KSA, CD326, TACSTD1, CO17-1A, GA733-2 etc.EpCAM a kind of to be correlated with Ca2+ oscillations tranducin 11 (tumor-associatedcalciumsignaltransducer1 by tumour, TACSTD1) the single pass transmembrane albumen of a molecular weight 30-40kDa of genes encoding, participate in regulating the function such as cell adhesion, mediated signal transduction, cell migration, propagation and differentiation (1, KurtzJE, DufourP.Adecatumumab:ananti-EpCAMmonoclonalantibody, fromthebenchtothebedside ［ J ］ .ExpertOpinBiolTher, 2010,10 (6): 951-958; 2, TrzpisM, McLaughlinPM, deLeijLM, etal.Epithelialcelladhesionmolecule.Morethanacarcinomama rkerandadhesionmolecule ［ J ］ .AmJPathol, 2007,171 (2): 386-395).
EpCAM expresses on people's part normal epithelium cell and most of malignant epithelial cell surface, generally believes that it plays an important role to the biological characteristics of tumour at present.Under pathologic condition, EpCAM is almost expressed in all gland cancer, comprise Colon and rectum gland cancer, adenocarcinoma of stomach, mammary cancer, ovarian cancer, adenocarcinoma of lung, prostate cancer, carcinoma of the pancreas and hepatocellular carcinoma and retinoblastoma (1, KurtzJE, DufourP.Adecatumumab:ananti-EpCAMmonoclonalantibody, fromthebenchtothebedside ［ J ］ .ExpertOpinBiolTher, 2010,10 (6): 951-958).Research finds that EpCAM is also the marker of some tumor stem cells, the research of Kimura etc. find the EpCAM positive liver-cancer stem cell can induction of immunity defect mouse generation tumour (3, KimuraO, TakahashiT, IshiiN, etal.Characterizationoftheepithelialcelladhesionmolecule (EpCAM)+cellpopulationinhepatocellularcarcinomacelllines ［ J ］ .CancerSci, 2010,101 (10): 2145-2155.).The hepatocellular carcinoma EpCAM positive and negative sub-population cell are expelled in the Mice Body of immune deficiency respectively, the cancer cells number EpCAM positive of result display formation needed for tumour is far fewer than EpCAM negative patient, and the tumour that the mouse of injection EpCAM positive cancer cell produces is larger than injection EpCAM negative patient, illustrates that the cancer cells tumorigenicity of the EpCAM positive is larger than the cancer cells of EpCAM feminine gender.In hepatocellular carcinoma cells system, the subgroup clone speed of the EpCAM positive is far longer than the subgroup of EpCAM feminine gender.Research also finds, the cancer cells of the EpCAM positive can be divided into the positive and negative cell of EpCAM, and the cancer cells of EpCAM feminine gender can only be divided into the cell of EpCAM feminine gender, illustrates that the cancer cells of the EpCAM positive has the potential of multinomial differentiation.
The Preventive of tumour is the major cause that its mortality ratio remains high, and circulating tumour cell is then the root of tumor recurrence, transfer.Timeliness coverage circulating tumor cells, prophylaxis of tumours can recur and transfer, improves survival, improve prognosis.EpCAM antibody is combined with chip technology for separating of the tumour cell in blood of cancer patients, and result successfully isolates the tumour cell (comprising all 7 routine early prostate cancer patients) that quantity does not wait in the blood sample of 99.1% (115/116); And the disease process highlights correlations of the change of circulating tumor cell number and imaging examination (4, NagratbS, SequistLV, MabeswaranS, eta1.Isolationofrarecirculatingtumourcellsincancerpatien tsbymicrochiptechnology.Nature, 2007,450 (7173): 1235-1239).But antibody, due to shortcomings such as molecular weight is comparatively large, steric hindrance is large, the easy degradeds of difficult storage, largely inhibits the detection of circulating tumor cell.
The expression of EpCAM is relevant to the prognosis of tumour, can be used as a target site of neoplasm targeted therapy.But at present because the immunotherapy of EpCAM is due to reasons such as antibodies specific are poor, bonding force is low, clinical test results is not satisfactory.Therefore the molecular probe designing the identification epithelial cell adhesion factor EpCAM of a kind of high-affinity and highly selective will have great importance to the early diagnosis of tumour, treatment and prognosis.
Aptamer to refer to from the single stranded DNA/RNA library of synthetic that screening obtains can with high specificity with the single stranded oligonucleotide that is combined with target molecules of high-affinity ground.Aptamer forms special three-dimensional structure by weak force between hydrogen bond, Van der Waals force, hydrophobic interaction equimolecular, as hair clip, false knot, bulge loop, the G-tetramer etc., thus identifies that target material even affects its biological activity specifically.The distinctive biochemical characteristic of aptamer itself makes it have many advantages in biomedical applications field, and as wide in target molecule scope, avidity and high specificity, synthetic modification fast and easy, molecular weight, good bio-compatibility, nontoxic, vitro stability is good.Aptamer can pass through part index concentration phyletic evolution technology (systematicevolutionofligandsbyexponentialenrichment, SELEX) screening obtains, its ultimate principle is that artificial chemistry synthesizes an oligonucleotide library in vitro, comprise RNA and ssDNA of RNA, ssDNA or modification, by the interaction of oligonucleotide library and target molecule, in conjunction with the oligonucleotide of the exponential amplification of round pcr and target molecule specific combination, take turns through several or tens ofly take turns screening enrichment process, obtaining the aptamer of high-affinity and high specific.
Summary of the invention
The first object of the present invention is to provide the aptamer EpCAMCcut of the epithelial cell adhesion molecule with high specific and high-affinity.
The second object of the present invention is to provide the nucleus of the expression epithelial cell adhesion molecule with high specific and high-affinity fit.
The third object of the present invention is the preparation method of the aptamer providing epithelial cell adhesion molecule.
Aptamer called after EpCAMA, EpCAMB, EpCAMC, EpCAMD and the EpCAMCcut respectively of described epithelial cell adhesion molecule EpCAM (Epithelialcelladhesionmolecule), its sequence is as follows:
The aptamer of described epithelial cell adhesion molecule EpCAM and prescind based on said structure, extend, the aptamer of epithelial cell adhesion molecule EpCAM that number of base is replaced, the aptamer of described epithelial cell adhesion molecule EpCAM has G tetramer structure or loop-stem structure.
The preparation method of the aptamer of described epithelial cell adhesion molecule EpCAM, comprises the following steps:
1) synthesizing single-stranded DNA random oligonucleotide storehouse; With Ni microballon for matrix, the nonspecific binding moieties in screening removing DNA random oligonucleotide storehouse, obtains the nucleotide sequence with epithelial cell adhesion molecule EpCAM specific combination with the screening of epithelial cell adhesion molecule EpCAM microballon;
2) nucleotide sequence of step 1) gained and epithelial cell adhesion molecule EpCAM specific combination is carried out pcr amplification, pcr amplification product with streptavidin microballon for matrix is separated, unwind by alkaline denaturation, filter, purifying, obtain the single-stranded DNA banks for next round screening, form time one-level nucleic acid library;
3) by step 2) single-stranded DNA banks of gained carries out next round screening, after 12 take turns screening, obtain object oligonucleotide sequence; Object oligonucleotide sequence described in cloning and sequencing, and identify its specificity be combined with target protein and avidity by flow cytometer showed method.
In step 1), the two ends in described single stranded DNA random oligonucleotide storehouse are fixed sequence program, and centre is the stochastic sequence of 40 bases, is 5'-AGCGTCGAATACCACTACAG-40nt-CTAATGGAGCTCGTGGTCAG-3', and storage capacity is 10
15above.
In step 2) in, the primer of described pcr amplification is:
Primer 1:5'-FAM-AGCGTCGAATACCACTACAG-3';
Primer 2: 5'-Biotin-CTGACCACGAGCTCCATTAG-3';
Described pcr amplification product is the double-stranded DNA library that 3 ' end band has biotin labeling, 5 ' end band has FAM to mark.
The aptamer of described epithelial cell adhesion molecule EpCAM as with the aptamer of clone of expressing EpCAM albumen.
The preparation method of the aptamer of epithelial cell adhesion molecule EpCAM of the present invention comprises: design and synthesize single stranded DNA random oligonucleotide storehouse, screening object oligonucleotide sequence, and identify its specificity be combined with target protein and avidity by flow cytometer showed method.The aptamer nontoxicity that screening obtains, molecular weight is little, good penetrability, be easy to synthesis and mark, a specific recognition EpCAM albumen, and specific recognition expresses the clone of EpCAM albumen, does not have recognition function to the clone not expressing this albumen.
The invention has the advantages that: first, by the albumen of the identification epithelial cell adhesion factor EpCAM of the His label of eukaryotic expression, and then by the compatible reaction of Hid and Ni, target protein is fixed on microballon, avoid in traditional method and be coupled to the impact on protein conformation on microballon by covalent reaction, ensure that the native conformation of albumen, ensure that screening the aptamer obtained can identify the albumen of film being expressed epithelial cell adhesion factor EpCAM.Secondly, the screening method that microballon-flow cytometry assay combines makes screening more fast easy with detection.And the aptamer nontoxicity that screening obtains, molecular weight is little, good penetrability, is easy to synthesis and mark.By the aptamer specific recognition Epithelial Cell Adhesion Molecule EpCAM that the screening of SELEX method obtains, not there is recognition function to other homologous proteins.The powerful that above-mentioned advantage makes described aptamer become Epithelial Cell Adhesion Molecule EpCAM to detect, identifies the early diagnosis of aptamer in tumour of epithelial cell adhesion factor EpCAM, the catching of circulating tumor cell, organizes, has great importance in living imaging and cancer therapy.
Accompanying drawing explanation
Fig. 1 be in flow cytomery screening process DNA random oligonucleotide storehouse to the enrichment figure of epithelial cell adhesion factor EpCAM.In FIG, X-coordinate is fluorescence intensity, and ordinate zou is microballon number, and curve A is initial DNA random oligonucleotide storehouse, and curve B is the 5th take turns DNA random oligonucleotide storehouse, and curve C is the 12nd take turns DNA random oligonucleotide storehouse.
Fig. 2 be in flow cytomery screening process DNA random oligonucleotide storehouse for the enrichment figure of Ni microballon.In fig. 2, X-coordinate is fluorescence intensity, and ordinate zou is microballon number, and curve A is initial DNA random oligonucleotide storehouse, and curve B is the 5th take turns DNA random oligonucleotide storehouse, and curve C is the 12nd take turns DNA random oligonucleotide storehouse.
Fig. 3 is that cells were tested by flow cytometry gained the 12nd takes turns DNA random oligonucleotide storehouse to the dissociation constant of epithelial cell adhesion factor EpCAM.In figure 3, X-coordinate is DNA concentration (nM), and ordinate zou is average fluorescent strength, ● represent initial DNA random oligonucleotide storehouse, ▽ represents the 12nd and takes turns DNA random oligonucleotide storehouse.
Fig. 4 is that cells were tested by flow cytometry gained aptamer EpCAMA, EpCAMB, EpCAMC, EpCAMD are to the skew of epithelial cell adhesion molecule EpCAM albumen.In the diagram, X-coordinate is fluorescence intensity, and ordinate zou is microballon number.Curve a is 0th; B is 12th; C is EpCAMA; D is pCAMB; E is pCAMC; F is pCAMD.
Fig. 5 and 6 is that cells were tested by flow cytometry gained aptamer EpCAMA, EpCAMB, EpCAMC, EpCAMD are to the skew of various clone.In figs. 5 and 6, X-coordinate is fluorescence intensity, and ordinate zou is microballon number; Curve a is RS; B is EpCAMA; C is EpCAMB; D is EpCAMC; F is EpCAMD.
Fig. 7 is that cells were tested by flow cytometry gained aptamer EpCAMA is to the dissociation constant of HEK-293T and MDA-MB-231 clone.In the figure 7, X-coordinate is DNA concentration (nmol/L), and ordinate zou is average fluorescent strength, ● represent MDA-MB-231 clone, 〇 represents HEK-293T clone.
Fig. 8 is that cells were tested by flow cytometry gained aptamer EpCAMC is to the dissociation constant of HEK-293T and MDA-MB-231 clone.In fig. 8, X-coordinate is DNA concentration (nmol/L), and ordinate zou is average fluorescent strength, and 〇 represents MDA-MB-231 clone, ● represent HEK-293T clone.
Fig. 9 forms parallel G tetramer structure for being detected gained EpCAMA, EpCAMB, EpCAMD aptamer by circular dichroism detector.In fig .9, X-coordinate is wavelength (nm), and ordinate zou is actual measurement ovality (mdeg); Curve a is EpCAMA; B is EpCAMB; C is pCAMD.
Embodiment
The aptamer of embodiment 1 in-vitro screening and epithelial cell adhesion molecule EpCAM specific combination
1) synthetic 5nmol single stranded DNA nucleic acid storehouse is dissolved in binding buffer liquid (12mmol/LPBS, 0.55mmol/LMgCl
2) in, heat-treat: 95 DEG C of heating 5min, place 10min on ice, then ambient temperatare puts 10min;
2) the single stranded DNA nucleic acid storehouse handled well and Ni microballon are hatched, collect the liquid be not combined with Ni microballon;
3) 40min is hatched at the liquid be not combined with Ni microballon and EpCAMNi microballon one being arised from 37 DEG C;
4) the EpCAMNi microballon after using the washing of binding buffer liquid to hatch, then the EpCAMNi microballon combining oligonucleotide is done PCR reaction;
PCR response procedures is: 94 DEG C of denaturation 3min, 94 DEG C of 30s, 53 DEG C of 30s, 68 DEG C of 30s, 10 circulations of increasing, and last 68 DEG C of ends extend 5min,
Primer 1:5'-FAM-AGCGTCGAATACCACTACAG-3';
Primer 2: 5'-Biotin-CTGACCACGAGCTCCATTAG-3';
5), after PCR reaction terminates, product is that 3 ' end band has biotin labeling, the double-stranded DNA that 5 ' end band has FAM to mark, add streptavidin microballon, reaction 30min, then carries out single stranded with 0.1mol/LNaOH, namely obtains the single-stranded DNA banks for next round screening through desalting column purifying;
6) after, often wheel uses the single-stranded DNA banks of 200pmol, and progressively increases washing times to strengthen proof strength.Carry out 12 altogether and take turns screening, then by the enrichment condition of flow cytomery single-stranded DNA banks, storehouse is taken turns in result display the 12nd and target protein EpCAM has obvious combination (see Fig. 1), and is not combined (see Fig. 2) with Ni albumen, finally takes turns storehouse the 12nd and sends to cloning and sequencing.
Embodiment 2 detects the binding ability of gained single stranded DNA and epithelial cell adhesion molecule EpCAM with flow cytometer showed method
First the fluorescently-labeled single stranded DNA of pcr amplification band, use primer 2: 5'-Biotin-CTGACCACGAGCTCCATTAG-3' and primer 3:5'-FAM-AGCGTCGAATACCACTACAG-3', PCR primer is that 5 ' end band has FAM and 3 ' end band has the double-stranded DNA of vitamin H, add streptavidin microballon, reaction 30min, then carry out single stranded with 0.1mol/LNaOH, namely obtain the single stranded DNA marked for the band FAM of flow cytometer showed through desalting column purifying.
The single stranded DNA of 0nmol/L, 5nmol/L, 10nmol/L, 20nmol/L, 50nmol/L, 100nmol/L, 200nmol/L concentration gradient and target protein EpCAMNi microballon is used to measure dissociation constant (Kd=22.8 ± 6.0).With the DNA solution of the above-mentioned each concentration of 200 μ l binding buffer liquid configuration, 95 DEG C of heating 5min, place 10min on ice, then ambient temperatare puts 10min.Add the EpCAM microballon of 155nmol/L, at 37 DEG C, hatch 40min.Use binding buffer liquid washing microballon 3 times, then microballon is resuspended in 250 μ L binding buffer liquid.The initial DNA random oligonucleotide storehouse arranged without screening compares.
Use the FACSAria flow cytometer of BD company to carry out fluorometric assay to microballon, then with the mapping of sigmaplot software, calculate the dissociation constant (see Fig. 3,7 and 8) of gained aptamer.
Embodiment 3 screening obtains aptamer and various clone specific combination
1) synthetic 5nmol single stranded DNA nucleic acid is dissolved in binding buffer liquid (12mmol/LPBS, 0.55mmol/LMgCl
2) in, heat-treat: 95 DEG C of heating 5min, place 10min on ice, then ambient temperatare puts 10min;
2) single stranded DNA nucleic acid handled well and 10000 cell kinds are hatched in 24 orifice plate 24h, at hatching 30min or 4 DEG C at 37 DEG C, hatch 40min.
3) after hatching, go buffered soln to wash twice, then cell is scraped off be dissolved in 200 μ L buffered soln, use the FACSAria flow cytometer of BD company to carry out fluorometric assay (see Fig. 5 and 6) to microballon.
Embodiment 4 measures gained aptamer EpCAMA with circular dichroism detector and forms parallel G tetramer structure
Prepare 1 μm of ol/L aptamer EpCAMA solution with binding buffer liquid, heat-treat: 95 DEG C of heating 5min, place 10min on ice, then ambient temperatare puts 10min.Then use circular dichroism spectrometer take 0.1nm as the CD spectrum of step scan 400nm to 200nm, multiple scanning 8 times, result locates formation negative peak and posivtive spike at 240nm and 260nm respectively, this peak type and document (5, SattanathanParamasivan, IulianRujan, PhilipH.Bolton.CirculardichroismofquadruplexDNAs:Applica tionstostructure, cationeffectsandligandbinding, 2007, 43:324-331.), the tetrameric characteristic peak of parallel G of report coincide, can judge that obtained aptamer EpCAMA has parallel G tetramer structure (see Fig. 9) thus.
Claims (3)
1. the aptamer of epithelial cell adhesion molecule EpCAM, it is characterized in that the aptamer called after EpCAMCcut of described epithelial cell adhesion molecule EpCAM, its sequence is as follows:
Cactacagaggttgcgtctgtcccacgttgtcatggggggttggcctg。
2. the aptamer to epithelial cell adhesion molecule EpCAM as claimed in claim 1, is characterized in that the aptamer of described epithelial cell adhesion molecule EpCAM has G tetramer structure or loop-stem structure.
3. the aptamer of epithelial cell adhesion molecule EpCAM as claimed in claim 1 is for the application in the detection of epithelial cell adhesion molecule EpCAM positive cell line, imaging, but is not used in the diagnosis of disease.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201310328256.0A CN103387988B (en) | 2012-09-24 | 2012-09-24 | Aptamer EpCAM Ccut of epithelial cell adhesion molecule and preparation method thereof |
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201210362899.2A CN102827845B (en) | 2012-09-24 | 2012-09-24 | Nucleic acid aptamer of epithelial cell adhesion molecule and preparation method thereof |
| CN201310328256.0A CN103387988B (en) | 2012-09-24 | 2012-09-24 | Aptamer EpCAM Ccut of epithelial cell adhesion molecule and preparation method thereof |
Related Parent Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201210362899.2A Division CN102827845B (en) | 2012-09-24 | 2012-09-24 | Nucleic acid aptamer of epithelial cell adhesion molecule and preparation method thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN103387988A CN103387988A (en) | 2013-11-13 |
| CN103387988B true CN103387988B (en) | 2016-01-06 |
Family
ID=47331156
Family Applications (5)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201310328291.2A Expired - Fee Related CN103387989B (en) | 2012-09-24 | 2012-09-24 | Aptamer EpCAM D of epithelial cell adhesion molecule and preparation method thereof |
| CN201310328301.2A Expired - Fee Related CN103387990B (en) | 2012-09-24 | 2012-09-24 | Aptamer EpCAM B of epithelial cell adhesion molecule and preparation method thereof |
| CN201210362899.2A Active CN102827845B (en) | 2012-09-24 | 2012-09-24 | Nucleic acid aptamer of epithelial cell adhesion molecule and preparation method thereof |
| CN201310328257.5A Expired - Fee Related CN103409427B (en) | 2012-09-24 | 2012-09-24 | Aptamer EpCAM C of epithelial cell adhesion molecule and preparation method thereof |
| CN201310328256.0A Expired - Fee Related CN103387988B (en) | 2012-09-24 | 2012-09-24 | Aptamer EpCAM Ccut of epithelial cell adhesion molecule and preparation method thereof |
Family Applications Before (4)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201310328291.2A Expired - Fee Related CN103387989B (en) | 2012-09-24 | 2012-09-24 | Aptamer EpCAM D of epithelial cell adhesion molecule and preparation method thereof |
| CN201310328301.2A Expired - Fee Related CN103387990B (en) | 2012-09-24 | 2012-09-24 | Aptamer EpCAM B of epithelial cell adhesion molecule and preparation method thereof |
| CN201210362899.2A Active CN102827845B (en) | 2012-09-24 | 2012-09-24 | Nucleic acid aptamer of epithelial cell adhesion molecule and preparation method thereof |
| CN201310328257.5A Expired - Fee Related CN103409427B (en) | 2012-09-24 | 2012-09-24 | Aptamer EpCAM C of epithelial cell adhesion molecule and preparation method thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (5) | CN103387989B (en) |
Families Citing this family (14)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103343127B (en) * | 2013-07-24 | 2015-06-03 | 厦门大学 | Aptamer LXL-2 for breast cancer cell MDA-MB-231 and applications of aptamer |
| KR101641920B1 (en) * | 2014-09-05 | 2016-07-22 | 한국생명공학연구원 | Nucleic acid aptamer specifically binding to EpCAM and their uses |
| CN104458375B (en) * | 2014-12-08 | 2018-05-29 | 中南大学湘雅医院 | Application of aptamer and derivative thereof and rapid imaging method for frozen tissue section |
| ES2796504T3 (en) * | 2015-02-11 | 2020-11-27 | Univ Deakin | EpCAM aptamers and conjugates thereof |
| CN106011235B (en) * | 2016-05-16 | 2019-10-29 | 南京大学 | A kind of memebrane protein analysis method based on the amplification of DNA molecular cascade signal |
| CN106668874A (en) * | 2016-12-08 | 2017-05-17 | 上海纳米技术及应用国家工程研究中心有限公司 | Method for carrying drugs or nano particles into specific target cells based on aptamer and cell-penetrating peptide |
| TWI831792B (en) * | 2018-06-12 | 2024-02-11 | 合一生技股份有限公司 | Nucleic acid aptamers targeting lymphocyte activation gene 3 (lag-3) and uses thereof |
| CN109576272A (en) * | 2018-07-02 | 2019-04-05 | 广西医科大学 | A kind of DKK-1 aptamer and its application |
| CN109266606A (en) * | 2018-10-09 | 2019-01-25 | 安徽科技学院 | It is a kind of extract blood plasma excretion body kit and its application |
| CN109513011A (en) * | 2018-12-25 | 2019-03-26 | 福州大学 | A kind of preparation method and applications of the self-assembled nanometer grain with target function |
| CN110004147B (en) * | 2019-03-05 | 2021-01-08 | 厦门大学 | Aptamer of epithelial cell adhesion molecule EpCAM screened from human plasma and preparation method and application thereof |
| CN110592093B (en) * | 2019-09-10 | 2023-08-25 | 上海交通大学医学院附属仁济医院 | Aptamer capable of recognizing EpCAM protein, and preparation method and application thereof |
| CN112843261B (en) * | 2021-03-10 | 2022-11-01 | 中国人民解放军空军军医大学 | EpCAM-targeted radioactive complex and preparation method thereof |
| CN114657184B (en) * | 2022-02-10 | 2023-09-12 | 南京邮电大学 | Multivalent aptamer functionalized DNA nanostructure probe and preparation method and application thereof |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1976951A (en) * | 2004-02-13 | 2007-06-06 | 米克罗麦特股份公司 | Anti-EpCAM immunoglobulins |
| CN102549017A (en) * | 2009-06-09 | 2012-07-04 | 奥菲技术科学研究院 | Anti-EpCAM antibodies |
-
2012
- 2012-09-24 CN CN201310328291.2A patent/CN103387989B/en not_active Expired - Fee Related
- 2012-09-24 CN CN201310328301.2A patent/CN103387990B/en not_active Expired - Fee Related
- 2012-09-24 CN CN201210362899.2A patent/CN102827845B/en active Active
- 2012-09-24 CN CN201310328257.5A patent/CN103409427B/en not_active Expired - Fee Related
- 2012-09-24 CN CN201310328256.0A patent/CN103387988B/en not_active Expired - Fee Related
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1976951A (en) * | 2004-02-13 | 2007-06-06 | 米克罗麦特股份公司 | Anti-EpCAM immunoglobulins |
| CN102549017A (en) * | 2009-06-09 | 2012-07-04 | 奥菲技术科学研究院 | Anti-EpCAM antibodies |
Non-Patent Citations (1)
| Title |
|---|
| RNA aptamer against a cancer stem cell marker epithelial cell adhesion molecule;Sarah Shigdar，et al;《Cancer Science》;20110314;第102卷(第5期);第993页左栏第一段，图2 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN103387989B (en) | 2016-05-18 |
| CN103387990B (en) | 2016-01-06 |
| CN103387989A (en) | 2013-11-13 |
| CN103409427B (en) | 2016-05-18 |
| CN103409427A (en) | 2013-11-27 |
| CN103387988A (en) | 2013-11-13 |
| CN103387990A (en) | 2013-11-13 |
| CN102827845A (en) | 2012-12-19 |
| CN102827845B (en) | 2014-05-07 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN103387988B (en) | Aptamer EpCAM Ccut of epithelial cell adhesion molecule and preparation method thereof | |
| Zhang et al. | Rapid capture and nondestructive release of extracellular vesicles using aptamer-based magnetic isolation | |
| Saenz-Antoñanzas et al. | Liquid biopsy in glioblastoma: opportunities, applications and challenges | |
| Jeong et al. | Integrated magneto–electrochemical sensor for exosome analysis | |
| Souza et al. | 3D Cell-SELEX: Development of RNA aptamers as molecular probes for PC-3 tumor cell line | |
| Liu et al. | Differentiating breast cancer molecular subtypes using a DNA aptamer selected against MCF-7 cells | |
| JP5603933B2 (en) | Nucleic acid aptamer capable of specifically binding to pancreatic cancer cells or tissues and use thereof | |
| CN104293794B (en) | Nucleic acid aptamers specifically combined with beta-amyloid precursor protein lyase 1 and application of aptamers | |
| CN101538570B (en) | Aptamer for typing different subtypes of non-small cell lung cancer and method for screening the same | |
| CN108034658A (en) | A kind of aptamer for detecting people's uveal melanoma cells | |
| CN104711263A (en) | Sequence of aptamer used for targeting human nasopharyngeal carcinoma cell and application thereof | |
| CN103205431A (en) | Nucleic acid aptamer and derivatives thereof, screening method of nucleic acid aptamer, application of nucleic acid aptamer and derivatives in detecting human biliary duct carcinoma cell line | |
| CN109576274A (en) | A kind of Endoglin aptamer and application based on CRISPR/Cas9 | |
| Alsop et al. | A novel tissue atlas and online tool for the interrogation of small RNA expression in human tissues and biofluids | |
| CN109837281A (en) | Specifically bind aptamer and its screening, identification and application of S100P albumen | |
| CN103333896B (en) | Nucleic acid aptamer LXL-1 of breast cancer cell MDA-MB-231 and application thereof | |
| CN103343127B (en) | Aptamer LXL-2 for breast cancer cell MDA-MB-231 and applications of aptamer | |
| CN108866064A (en) | A kind of aptamer of targeted metastatic human breast cancer cell and its application | |
| CN103343128B (en) | Aptamer LXL-3 for breast cancer cell MDA-MB-231 and applications of aptamer | |
| CN103333895B (en) | Nucleic acid aptamer specifically binding to human epidermal growth factor acceptor III type mutant and applications thereof | |
| Chen et al. | Extracellular vesicles as biomarkers in head and neck squamous cell carcinoma: from diagnosis to disease-free survival | |
| CN106282194B (en) | Breast cancer lines specific nucleic acid aptamers and its application in preparation detection, diagnosing and treating human breast carcinoma preparation | |
| Han et al. | A Multiple Targeting Nanoprobe for Identifying Cancer Metastatic Sites Based on Detection of Various mRNAs in Circulating Tumor Cells | |
| CN113930482A (en) | Three-dimensional DNA walker and application thereof in tumor exosome detection | |
| CN104374918B (en) | Heat shock protein 47 glioma associated tumor antigen and application |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20160106 |