CN105541990B - Aptamers K15 of skate angiogenesis inhibitor 1 and screening method and application thereof - Google Patents
Aptamers K15 of skate angiogenesis inhibitor 1 and screening method and application thereof Download PDFInfo
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- CN105541990B CN105541990B CN201610102329.8A CN201610102329A CN105541990B CN 105541990 B CN105541990 B CN 105541990B CN 201610102329 A CN201610102329 A CN 201610102329A CN 105541990 B CN105541990 B CN 105541990B
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- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
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- C12N15/09—Recombinant DNA-technology
- C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
- C12N15/115—Aptamers, i.e. nucleic acids binding a target molecule specifically and with high affinity without hybridising therewith ; Nucleic acids binding to non-nucleic acids, e.g. aptamers
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Abstract
A group of aptamers capable of identifying sea purse angiogenesis inhibiting factor 1 functional domain protein and a preparation method thereof. The oligonucleotide sequences comprise SEQ ID No.2-11, have high affinity specificity respectively, and can be used for detecting the functional region protein of the sea purse angiogenesis inhibiting factor 1.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a skate angiogenesis inhibitor 1 aptamer, a screening method and application thereof.
Background
In recent years, oligonucleotide aptamers have attracted attention as promising alternative molecules to antibody molecules. The oligonucleotide aptamers are obtained by screening through SELEX technology (Systematic evolution of ligands by hybridization) biological library technology, and the principle of the technology is to construct an artificially synthesized single-stranded random oligonucleotide library by utilizing molecular biology technology, wherein the length of a random sequence of the artificially synthesized single-stranded random oligonucleotide library is about 20-100 bases. By utilizing the flexible and changeable characteristics of single-stranded oligonucleotide molecule conformation, the random oligonucleotide library and the target molecule are interacted, the oligonucleotide combined with the target molecule by a space conformation is reserved, and the oligonucleotide sequence specifically combined with the target molecule can be enriched by repeated amplification and screening for a plurality of cycles, and finally the specific oligonucleotide aptamers of various target molecules, namely aptamers, are obtained. The aptamer recognition molecule obtained by screening through SELEX technology has a mode similar to that of a protein antibody, but compared with the protein antibody, the nucleic acid ligand has more superiority, such as the restriction of immune conditions and immunogenicity, in-vitro artificial synthesis, reversible denaturation and renaturation, modification, long-term storage, room-temperature transportation and the like. More importantly, aptamers have higher specificity than antibodies and even recognize protein molecules that cannot be distinguished by monoclonal antibodies. Moreover, the aptamer target molecules are very wide and are as small as dye molecules, and are as large as complete virus particles and bacterial pathogens, even complete cells, so that the high-affinity oligonucleotide aptamers can be screened by the subtractive SELEX technology.
The functional region of the sea purse angiogenesis inhibitor 1 is called the sea purse functional region, and the sea purse angiogenesis inhibitor 1 is a protein (with the molecular weight of 42KD) which is firstly separated from a sea purse tissue in a south sea area. The research result shows that: the sea purse angiogenesis inhibiting factor 1 can obviously inhibit the chicken embryo chorioallantoic membrane and the chicken embryo chorioallantoic membrane angiogenesis induced by human nasopharyngeal carcinoma cells; the growth and the metastasis of the Lewis lung cancer of a nude mouse can be obviously inhibited no matter the nude mouse is injected into the abdominal cavity or is perfused into the stomach, the density of tumor tissue capillaries is reduced, and the expression of angiogenesis promoting factors VEGF is down-regulated; the transfer of melanoma B16 cells of a nude mouse is also strongly inhibited; down-regulating the expression of the transfer-promoting factors CD44v6 and ErBb 2; when the composition is combined with 5-fluorouracil (5-FU), the effect is more remarkable. The action target of the sea purse angiogenesis inhibiting factor 1 is different from the new anti-cancer drug Avastin developed by the American gene technology research institute. Avastin is a gene engineering product, is an antibody of VEGF, and has an action target of VEGF; sea purse angiogenesis inhibitor 1 is a natural product, and not only acts on VEGF, but also acts on bFGF and PDGF. Therefore, the identification and screening of the specificity of the functional domain of sea purse angiogenesis inhibitor 1 is a consistent pursuit in the art.
Based on the consideration, the invention uses the sea purse angiogenesis inhibitor 1 functional domain protein as the target protein, obtains 10 sea purse angiogenesis inhibitor 1 functional domain protein specific aptamers by adopting SELEX technology, and can quickly, sensitively and specifically detect the sea purse angiogenesis inhibitor 1 functional domain protein by combined application. The single-stranded DNA oligonucleotide aptamer has stable performance, convenient synthesis and low cost, and can be directly used for detecting a target binding band by a fluorescence or chemiluminescence and color development method after being modified, so the operation is simple and direct.
Disclosure of Invention
The invention aims to provide a group of oligonucleotide sequences capable of identifying sea purse angiogenesis inhibiting factor 1 functional domain proteins and a preparation method thereof, wherein the oligonucleotide sequences have the characteristics of high detection speed, simple operation, higher stability than an antibody and the like, are easy to prepare and have a short preparation period.
The oligonucleotide sequence capable of identifying the sea purse angiogenesis inhibiting factor 1 functional region protein comprises SEQ ID No.2-11, and identification and detection of the sea purse angiogenesis inhibiting factor 1 functional region protein can be completed by adopting one oligonucleotide sequence;
the preparation method of the oligonucleotide sequence capable of identifying the sea purse angiogenesis inhibiting factor 1 functional region protein comprises the following steps:
1. synthesis of ssDNA oligonucleotide library for screening (5' -TCA GTC GCT TCG CCG TCT CCTTC-)
N35- - -GCA CAA GAG GGA GAC CCC AGA GGG-3'), wherein N35 is 35 random oligonucleotides;
2. respectively mixing the oligonucleotide library with the sea purse angiogenesis inhibiting factor 1 functional domain protein, and then carrying out SELEX screening to obtain an aptamer enrichment library;
3. after SELEX screening is completed, cloning and sequencing the obtained aptamer enrichment library;
4. and selecting high-copy ssDNA (single-stranded deoxyribonucleic acid) appearing in a sequencing result, verifying affinity specificity, and screening to obtain an oligonucleotide sequence capable of identifying the functional region protein of the sea purse angiogenesis inhibiting factor 1.
Detailed Description
Example 1
1. Preparation of sea purse angiogenesis inhibiting factor 1 functional zone protein
The method for obtaining the sea purse angiogenesis inhibiting factor 1 functional domain protein with the same biological activity as the sea purse angiogenesis inhibiting factor 1 functional domain protein by adopting a yeast recombinant expression mode well known by a person skilled in the art, wherein the protein sequence is shown as SEQ ID NO: 1 is shown in the specification; the concentration of the protein solution was 15 mg/ml.
2. Synthesis of libraries and primers
2.1 Synthesis of ssDNA oligonucleotide library for screening (5' -TCA GTC GCT TCG CCG TCT CCTTC-)
N35- - -GCA CAA GAG GGA GAC CCC AGA GGG-3'), wherein N35 is 35 random oligonucleotides;
primer P1: TCAGTCGCTTCGCCGTCTCCTTC, respectively;
primer P2: CCCTCTGGGGTCTCCCTCTTGTGC are provided.
2.2, SELEX screening of the aptamers, wherein the specific method comprises the following steps:
2.2.1 binding and separating of ssDNA and sea purse angiogenesis inhibitor 1 functional domain protein, the specific method is as follows:
taking 4 mu L of a 100 mu M ssDNA oligonucleotide library, diluting to 100 mu L with 2 multiplied by binding buffer solution, denaturing at 95 ℃ for 5min, adding 100 mu L of ray angiogenesis inhibiting factor 1 functional domain protein after ice bath for 10min, binding by a shaking table for 30min, centrifuging at 6000rpm for 5min, discarding supernatant, washing precipitate with 1 multiplied by binding buffer solution, and discarding supernatant; adding 100 mu L of 1 Xbinding buffer solution into the sediment, heating at 96 ℃ for 5min, then centrifuging at 15000rpm for 10min, taking supernatant, heating and centrifuging the sediment again, and combining the supernatant to obtain a ssDNA secondary library with affinity to the protein of the functional region of the sea purse angiogenesis inhibitor 1; the 2 × binding buffer solution is a solution obtained by diluting the 20 × binding buffer solution by 10 times with double distilled water, and the 1 × binding buffer solution is a solution obtained by diluting the 20 × binding buffer solution by 20 times with double distilled water; the 20 × binding buffer formulation was 1M NaCl, 50mM KCl, 500mM Tris-HCl, 10mM MgCl2, pH 7.4.
2.2.2 binding and separating of ssDNA and sea purse angiogenesis inhibitor 1 functional domain protein, the specific method is as follows:
the ssDNA which is obtained by separation in the step 2.2.1 and can be combined with the protein of the sea purse angiogenesis inhibiting factor 1 functional area is combined with 100 mu l of the protein of the sea purse angiogenesis inhibiting factor 1 functional area for 30min in a shaking table, and the subsequent step is synchronous with the step 2.2.1, so that a ssDNA secondary library which has affinity with the protein of the sea purse angiogenesis inhibiting factor 1 functional area can be separated.
2.2.3 asymmetric PCR amplification of ssDNA, the specific procedure is as follows:
and (3) carrying out asymmetric PCR amplification on the ssDNA secondary library obtained by separation in the step 2.2.2, wherein the total volume of the asymmetric PCR amplification system is 25 mul: 10 × PCR buffer: 2 mu l of the solution; p1(10 μ M): 1 mul; p2(0.2 μ M): 1 mul; dNTPs (2.5 mM each): 0.4 μ l; MgCl2(25 mM): 1.2 μ l; ssDNA template (0.2. mu.g/. mu.l): 2 mu l of the solution; taq DNA polymerase (5 u/. mu.l): 0.2 μ l; ddH 2O: 17.2 μ l; PCR reaction parameters: pre-denaturation at 94 ℃ for 4min, 40 cycles of denaturation at 94 ℃ for 30s, annealing at 58 ℃ for 30s, extension at 72 ℃ for 20s, and final extension at 72 ℃ for 7 min;
2.2.4 determination of affinity, the specific method is as follows:
2.2.4.1 amplification: using primer P1 with digoxin label to do asymmetric PCR to amplify the screened ssDNA secondary library, wherein the amplification conditions and parameters are the same as those of the asymmetric PCR amplification system and parameters in step 2.2.3;
2.2.4.2 binding to proteins: taking 100 mu L of PCR product obtained by amplification in the step 2.2.4.1, carrying out denaturation at 95 ℃ for 5min, carrying out ice bath for 10min, then adding the PCR product into 100 mu L of protein, fully mixing, combining the protein at room temperature for 30min, then centrifuging at 6000rpm, separating protein and supernatant, wherein the protein contains ssDNA (single-stranded deoxyribonucleic acid) which is combined with the protein and is provided with a digoxin label, the supernatant is unbound ssDNA, and meanwhile, a blank without ssDNA is made, namely 2 × binding buffer solution is used for replacing the PCR product, and the operation is carried out in the same way;
2.2.4.3 washing: washing the protein with 1 × 500 μ L of binding buffer solution for 1 time, centrifuging at 6000rpm, removing supernatant, and collecting protein;
2.2.4.4 binding to enzyme-labeled rabbit anti-digoxin antibody: adding 100 μ L of enzyme-labeled rabbit anti-digoxin antibody diluted by 1: 900TBS into protein, mixing thoroughly, reacting for 10min to combine with digoxin-labeled ssDNA in protein;
the TBS is 0.5M Tris-NaCl solution, and the preparation method comprises the following steps: dissolving 8.5-9 g of NaCl in water, adding 100ml of Tris-HCl (0.5M, pH7.6) solution, and finally adding water to a constant volume of 1L; preparation method of 0.5M Tris-HCl (pH7.6, 100ml) solution: weighing 6.06g of Tris, adding 40ml of double distilled water for dissolution, dropwise adding concentrated HCl to adjust the pH value to 7.6, and fixing the volume to 100 ml.
2.2.4.5 washing: centrifuging at 6000rpm, removing supernatant, and washing with 1 × 500 μ L buffer solution for 3 times to obtain protein;
2.2.4.6 TMB (tetramethylbenzidine) color development: adding 400 mu L of double distilled water resuspended protein, then adding 200 mu L of TMB color development solution, after color development for 10min in a dark place, terminating the reaction by 2mol/L H2SO4200 mu L, measuring the absorbance OD450 at 450nm, wherein the absorbance OD450 reflects the affinity of ssDNA bound with bacteria, namely OD binding, and carrying out the steps 2.2.4.3, 2.2.4.4, 2.2.4.5 and 2.2.4.6 in the blank to obtain the blank corresponding to the absorbance OD blank;
the TMB color developing solution is prepared by using a conventional preparation method.
2.2.4.7 determination of the molar concentration of DNA in the PCR product: taking the PCR product obtained by the amplification in the step 2.2.4.1, taking an initial ssDNA library with a known concentration gradient as a standard, taking Bandscan software as image analysis software, quantitatively measuring the DNA content in the PCR product by ethidium bromide agarose gel electrophoresis to obtain the molar concentration of the corresponding DNA, and further calculating the DNA mole number in 100 mu L of the PCR product.
2.2.4.8 calculation of the affinity of the corresponding library:
2.3 repeated screening, the specific method is as follows: and (3) taking the product of each round of asymmetric PCR as a screening library of the next round, repeating the SELEX screening step 2.2 until the affinity is not increased any more, and finally obtaining the aptamer enrichment library of the ssDNA through 16 rounds of screening. After asymmetric PCR amplification, under the same conditions as the previous steps, cloning and sequencing to obtain 20 effective ssDNAs with the highest copy number, and respectively carrying out affinity specificity verification on 20 aptamers to obtain 10 oligonucleotide sequences (aptamers) with better affinity specificity to the functional region protein of the sea purse angiogenesis inhibitor 1, wherein the specific sequences are as follows:
the affinity data are as follows:
| aptamer name | Affinity of | Aptamer name | Affinity of |
| K2 | 0.62 | K15 | 0.43 |
| K6 | 0.53 | K16 | 0.51 |
| K9 | 0.50 | K17 | 0.59 |
| K10 | 0.47 | K19 | 0.62 |
| K14 | 0.60 | K20 | 0.49 |
2.4 analysis of specificity and affinity of 20 aptamers
Incubating the fluorescence-labeled aptamer sequences with the functional domain protein of the sea purse angiogenesis inhibiting factor 1, performing flow cytometry detection, wherein 10 sequences show high fluorescence intensity, performing a nonlinear regression curve on a saturation curve by using GraphPad prism5.0 software, and performing the same experimental operation on 10 high-affinity aptamer sequences respectively to obtain Kd values each of which is configured:
| aptamer name | Kd value (nM) | Aptamer name | Kd value (nM) |
| K2 | 35.27 | K15 | 59.23 |
| K6 | 51.73 | K16 | 38.97 |
| K9 | 43.81 | K17 | 44.83 |
| K10 | 50.46 | K19 | 34.57 |
| K14 | 35.89 | K20 | 47.81 |
The Kd value of K20 is the smallest, which indicates that the protein can be combined with the target protein rapidly and has stable structure and is not easy to separate.
The DNAMAN software is adopted to construct the secondary structures of 10 aptamers and calculate the minimum free energy of the aptamers, the minimum free energy of the structures is small, and the structures are relatively stable.
2.5 aptamer specificity assay
The specificity detection is respectively carried out by adopting BSA, human hemoglobin, ray angiogenesis inhibiting factor 1 functional zone protein and 10 aptamers, and the combination test shows that the 10 sequences are not combined with the BSA or the human hemoglobin, but are only combined with the ray angiogenesis inhibiting factor 1 functional zone protein to keep higher specificity.
Sequence listing
110 Zhang Yong
Aptamer K15 of < 120 > ray angiogenesis inhibitor 1, and screening method and application thereof
〈160〉13
〈210〉1
〈211〉225
〈212〉PRT
Ray (213)
〈400〉1
TLDIYKQLRD KETPSGFTLD DVIQTGVDNP GHPFIMTVGC VAGDEESYEV FKALFDPVIQ 60
DRHGGYKPTD KHKTDLNHEN LKGGDDLDPN YVLSSRVRTG RSIKGIALPP HCSRGERRLV 120
EKLCLEGLAT LTGEFQGKYY PLTTMSDAEQ QQLIDDHFLF DKPVSPLLLA SGMARDWPDA 180
RGIWHNNDKT FLVWVNEEDH LRVISMQKGG NMKEVFRRFC VGLKK 225
〈210〉2
<211〉 84
〈212〉DNA
Artificial sequence of < 213 >
〈400〉K2
1 TCAGTCGCTT CGCCGTCTCC TTCATGATCG CGCTGACAAA TTAGGCCATT CAATCAGAGC
61 ACAAGAGGGA GACCCCAGAG GG
〈210〉3
<211〉 84
〈212〉DNA
Artificial sequence of < 213 >
〈400〉K6
1 TCAGTCGCTT CGCCGTCTCC TTCCCGTGAT GAATTGCTGA TGAGCGCAGC ATGGAGCTGC
61 ACAAGAGGGA GACCCCAGAG GG
〈210〉4
<211〉 84
〈212〉DNA
Artificial sequence of < 213 >
〈400〉K9
1 TCAGTCGCTT CGCCGTCTCC TTCTGACGCA TTCGGATCCA AGTTAATTAA ATAACTGCGC
61 ACAAGAGGGA GACCCCAGAG GG
〈210〉5
<211〉 84
〈212〉DNA
Artificial sequence of < 213 >
〈400〉K10
1 TCAGTCGCTT CGCCGTCTCC TTCATTGCAA CCTGAGGCCA TGGGACAGAC CATGATAGGC
61 ACAAGAGGGA GACCCCAGAG GG
〈210〉6
<211〉 84
〈212〉DNA
Artificial sequence of < 213 >
〈400〉K14
1 TCAGTCGCTT CGCCGTCTCC TTCAACTTGG ACCCTTGAGC GATGAAGTAA CGGTTTACGC
61 ACAAGAGGGA GACCCCAGAG GG
〈210〉7
<211〉 84
〈212〉DNA
Artificial sequence of < 213 >
〈400〉K15
1 TCAGTCGCTT CGCCGTCTCC TTCGCACTGC TACCGATATT ACATATATGG AGATACAGGC
61 ACAAGAGGGA GACCCCAGAG GG
〈210〉8
<211〉 84
〈212〉DNA
Artificial sequence of < 213 >
〈400〉K16
1 TCAGTCGCTT CGCCGTCTCC TTCGGCCGAG TAACAGATTG GAACCCAACT GAGTGAGAGC
61 ACAAGAGGGA GACCCCAGAG GG
〈210〉9
<211〉 84
〈212〉DNA
Artificial sequence of < 213 >
〈400〉K17
1 TCAGTCGCTT CGCCGTCTCC TTCTTATGGA CGAGTAGAGG TACGATGACC CAATGATTGC
61 ACAAGAGGGA GACCCCAGAG GG
〈210〉10
<211〉 84
〈212〉DNA
Artificial sequence of < 213 >
〈400〉K19
1 TCAGTCGCTT CGCCGTCTCC TTCCGATTGA GGGAGATTAC GCATATGAGT ACAACTGAGC
61 ACAAGAGGGA GACCCCAGAG GG
〈210〉11
<211〉 84
〈212〉DNA
Artificial sequence of < 213 >
〈400〉K20
1 TCAGTCGCTT CGCCGTCTCC TTCTTTAGAC CCGATAATGT TGTTTTGGTG ACCGAATTGC
61 ACAAGAGGGA GACCCCAGAG GG
〈210〉12
<211〉23
〈212〉DNA
Artificial sequence of < 213 >
〈400〉P1
TCAGTCGCTT CGCCGTCTCC TTC
〈210〉13
<211〉 24
〈212〉DNA
Artificial sequence of < 213 >
〈400〉P2
CCCTCTGGGG TCTCCCTCTT GTGC
Claims (2)
1. An oligonucleotide sequence for identifying a functional region protein of sea purse angiogenesis inhibiting factor 1 is characterized in that the sequence is shown as SEQ ID No. 7.
2. The use of the oligonucleotide sequence of claim 1 for screening ray angiogenesis inhibitor 1 domain proteins.
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| CN201610102329.8A CN105541990B (en) | 2014-06-06 | 2014-06-06 | Aptamers K15 of skate angiogenesis inhibitor 1 and screening method and application thereof |
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| CN201610102329.8A CN105541990B (en) | 2014-06-06 | 2014-06-06 | Aptamers K15 of skate angiogenesis inhibitor 1 and screening method and application thereof |
| CN201410249737.7A CN104031137B (en) | 2014-06-06 | 2014-06-06 | The aptamer of ray Angiostatin 1 and screening technique thereof and application |
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| CN201410249737.7A Division CN104031137B (en) | 2014-06-06 | 2014-06-06 | The aptamer of ray Angiostatin 1 and screening technique thereof and application |
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| CN201610101377.5A Active CN105567698B (en) | 2014-06-06 | 2014-06-06 | The aptamer K19 and its screening technique of ray Angiostatin 1 and application |
| CN201410249737.7A Active CN104031137B (en) | 2014-06-06 | 2014-06-06 | The aptamer of ray Angiostatin 1 and screening technique thereof and application |
| CN201610102089.1A Expired - Fee Related CN105541989B (en) | 2014-06-06 | 2014-06-06 | Aptamers K20 of skate angiogenesis inhibitor 1 and screening method and application thereof |
| CN201610099682.5A Active CN105541986B (en) | 2014-06-06 | 2014-06-06 | The aptamer K9 and its screening technique of ray Angiostatin 1 and application |
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| CN201410249737.7A Active CN104031137B (en) | 2014-06-06 | 2014-06-06 | The aptamer of ray Angiostatin 1 and screening technique thereof and application |
| CN201610102089.1A Expired - Fee Related CN105541989B (en) | 2014-06-06 | 2014-06-06 | Aptamers K20 of skate angiogenesis inhibitor 1 and screening method and application thereof |
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| CN105693847A (en) * | 2014-07-13 | 2016-06-22 | 马海龙 | Aptamer NKXA9 specifically for Neutrokine-alpha protein and application of aptamer NKXA9 |
| CN105693848A (en) * | 2014-07-13 | 2016-06-22 | 马海龙 | Aptamer NKXA7 specifically for Neutrokine-alpha protein and application of aptamer NKXA7 |
| CN105693849A (en) * | 2014-07-13 | 2016-06-22 | 马海龙 | Aptamer NKXA5 specifically for Neutrokine-alpha protein and application of aptamer NKXA5 |
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|---|---|---|---|---|
| WO2006096754A2 (en) * | 2005-03-07 | 2006-09-14 | Archemix Corp. | Stabilized aptamers to psma and their use as prostate cancer therapeutics |
| CN101724631A (en) * | 2008-11-03 | 2010-06-09 | 广东海洋大学 | Preparation of functional area of sea purse blood vessel growth inhibition factor 1 and use of the functional area of sea purse blood vessel growth inhibition factor 1 in medicaments for preventing and curing tumors |
| CN103060326A (en) * | 2012-12-17 | 2013-04-24 | 集美大学 | Six oligonucleotide sequences and application thereof |
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- 2014-06-06 CN CN201610102089.1A patent/CN105541989B/en not_active Expired - Fee Related
- 2014-06-06 CN CN201610099682.5A patent/CN105541986B/en active Active
- 2014-06-06 CN CN201610102329.8A patent/CN105541990B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2006096754A2 (en) * | 2005-03-07 | 2006-09-14 | Archemix Corp. | Stabilized aptamers to psma and their use as prostate cancer therapeutics |
| CN101724631A (en) * | 2008-11-03 | 2010-06-09 | 广东海洋大学 | Preparation of functional area of sea purse blood vessel growth inhibition factor 1 and use of the functional area of sea purse blood vessel growth inhibition factor 1 in medicaments for preventing and curing tumors |
| CN103060326A (en) * | 2012-12-17 | 2013-04-24 | 集美大学 | Six oligonucleotide sequences and application thereof |
Also Published As
| Publication number | Publication date |
|---|---|
| CN105541986A (en) | 2016-05-04 |
| CN105541989A (en) | 2016-05-04 |
| CN105541988B (en) | 2020-05-19 |
| CN104031137A (en) | 2014-09-10 |
| CN105567698B (en) | 2018-08-31 |
| CN105567698A (en) | 2016-05-11 |
| CN105541986B (en) | 2019-04-12 |
| CN105541989B (en) | 2020-06-02 |
| CN104031137B (en) | 2016-08-24 |
| CN105541988A (en) | 2016-05-04 |
| CN105541990A (en) | 2016-05-04 |
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