CN105541986A - Aptamers K9 of skate angiogenesis inhibitor 1 and screening method and application of aptamers K9 - Google Patents
Aptamers K9 of skate angiogenesis inhibitor 1 and screening method and application of aptamers K9 Download PDFInfo
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- CN105541986A CN105541986A CN201610099682.5A CN201610099682A CN105541986A CN 105541986 A CN105541986 A CN 105541986A CN 201610099682 A CN201610099682 A CN 201610099682A CN 105541986 A CN105541986 A CN 105541986A
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- aptamers
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- albumen
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- 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
- C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
- C07K14/461—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from fish
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- 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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- C12N2310/00—Structure or type of the nucleic acid
- C12N2310/10—Type of nucleic acid
- C12N2310/16—Aptamers
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- C12N2320/10—Applications; Uses in screening processes
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- C12N2320/00—Applications; Uses
- C12N2320/10—Applications; Uses in screening processes
- C12N2320/13—Applications; Uses in screening processes in a process of directed evolution, e.g. SELEX, acquiring a new function
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- C12N2330/00—Production
- C12N2330/30—Production chemically synthesised
- C12N2330/31—Libraries, arrays
Abstract
The invention provides a set of aptamers capable of recognizing protein in a functional zone of a skate angiogenesis inhibitor 1 and a preparation method of the aptamers. Oligonucleotide sequences comprise SEQ ID No.2-11. The aptamers have high affinity specificity and can be used for detecting the protein in the functional zone of the skate angiogenesis inhibitor 1.
Description
Technical field
The invention belongs to biological technical field, specifically, the present invention relates to a kind of ray Angiostatin 1 aptamer and screening method thereof and application.
Background technology
In the last few years, oligonucleotide aptamer was as the promising alternative molecule of antibody molecule, and its research is comparatively noticeable.Oligonucleotide aptamer is obtained by SELEX technology (Systematicevolutionofligandsbyexponentialenrichment) biological libraries technology screening, the principle of this technology utilizes Protocols in Molecular Biology exactly, build the strand random oligonucleotide library of synthetic, its stochastic sequence length is about 20-100 base.Utilize the characteristic that single stranded oligonucleotide molecular configurations is flexible and changeable, random oligonucleotide library and target molecule are interacted, retain the oligonucleotide be combined with target molecule with spatial conformation, through repeated amplification, screen several circulation, can make to obtain enrichment with the oligonucleotide sequence of this target specific combination, specific oligonucleotide acid aptamer, the i.e. aptamer of the multiple target molecule of final acquisition.Pattern and the protein antibodies of the aptamer identification molecule utilizing SELEX technology screening to obtain are similar, but compared with protein antibody, nucleic acid aglucon has more superiority, if do not limited by immune condition and immunogenicity, can external synthetic, Denaturation and Renaturation is reversible, can modify and be conducive to long-term preservation and room temperature transport etc.The more important thing is, aptamer has higher specificity than antibody, even can identify the undistinguishable protein molecule of monoclonal antibody.And the target molecule of aptamer widely, little of dye molecule, large to complete virion and bacterial pathogens, even complete cell also can go out the oligonucleotide aptamer of high-affinity by abatement SELEX technology screening.
Ray Angiostatin 1 functional zone are hereinafter referred to as ray functional zone, and ray Angiostatin 1 is the protein (molecular weight 42KD) that we separate first from a kind of ray tissue of South China Sea.Result of study shows: that ray Angiostatin 1 significantly suppresses chick chorioallantoic membrane itself and that KB cell is induced chick chorioallantoic membrane vasculogenesis; Be growth and the transfer that abdominal injection or gavage all significantly suppress nude mouse Lewis lung cancer, reduce tumor tissues microvessel density, lower the expression of angiogenic factors VEGF; Also high inhibition effect is had to the transfer of nude mouse Melanoma B16 cell; Lower the expression of short transfer factor CD44v6 and ErBb2; With more remarkable effect during 5 FU 5 fluorouracil (5-FU) combined utilization.The action target spot of ray Angiostatin 1 is different from the PTS Avastin that gene engineering institute of the U.S. develops.Avastin is gene engineering product, and be the antibody of VEGF, its action target spot is VEGF; Ray Angiostatin 1 is natural product, and it not only acts on VEGF, also acts on bFGF and PDGF.Therefore, carrying out specific discriminating and screening for ray Angiostatin 1 functional zone is the consistent pursuit in this area.
Based on above consideration, the present invention is target protein for the purpose of the albumen of ray Angiostatin 1 functional zone, adopt SELEX technology to obtain the special aptamer of 10 ray Angiostatin 1 functional zone albumen, Combination application can rapidly, sensitivity, special ray Angiostatin 1 functional zone albumen detected.Due to the stable performance of single strand dna oligonucleotide aptamer, synthesis convenient and cheap, modified after can be directly used in fluorescence or chemoluminescence, chromophoric method detects target bandage, therefore simple to operate, direct.
Summary of the invention
The object of the present invention is to provide not only to have and detect quick, simple to operate, stability higher than features such as antibody, and preparation method easily, shorter one group of preparation cycle can identify oligonucleotide sequence of ray Angiostatin 1 functional zone albumen and preparation method thereof.
The described oligonucleotide sequence that can identify ray Angiostatin 1 functional zone albumen, comprises SEQIDNo.2-11, and an employing oligonucleotide sequence wherein just can complete the recognition detection to ray Angiostatin 1 functional zone albumen;
Described one group can identify that the preparation method of the oligonucleotide sequence of ray Angiostatin 1 functional zone albumen comprises the following steps:
1, the ssDNA oligonucleotide library (5 '-TCAGTCGCTTCGCCGTCTCCTTC----for screening is synthesized
N35----GCACAAGAGGGAGACCCCAGAGGG-3 '), wherein N35 is 35 random oligonucleotides;
2, carry out SELEX screening after being mixed with ray Angiostatin 1 functional zone albumen respectively by oligonucleotide library, obtain aptamer enriched library;
3, after SELEX has screened, cloning and sequencing is carried out to the aptamer enriched library obtained;
4, select the height copy ssDNA occurred in sequencing result, carry out affine specific checking, screening obtains the oligonucleotide sequence that can identify ray Angiostatin 1 functional zone albumen.
Embodiment
Embodiment 1
1, the preparation of ray Angiostatin 1 functional zone albumen
Adopt the recombinant expressed mode of yeast well known to those skilled in the art to obtain and have bioactive ray Angiostatin 1 functional zone identical with ray Angiostatin 1 functional zone albumen albumen, this protein sequence is as shown in SEQIDNO:1; The concentration of protein solution is 15mg/ml.
2, the synthesis of library and primer
2.1, the ssDNA oligonucleotide library (5 '-TCAGTCGCTTCGCCGTCTCCTTC----for screening is synthesized
N35----GCACAAGAGGGAGACCCCAGAGGG-3 '), wherein N35 is 35 random oligonucleotides;
Primer P1:TCAGTCGCTTCGCCGTCTCCTTC;
Primer P2:CCCTCTGGGGTCTCCCTCTTGTGC.
2.2, the SELEX screening of aptamer, concrete grammar is as follows:
2.2.1ssDNA with the combination of ray Angiostatin 1 functional zone albumen, be separated, concrete grammar is as follows:
Get the ssDNA oligonucleotide library 4 μ L of 100 μMs, 100 μ l are diluted to 2 × binding buffer liquid, 95 DEG C of sex change 5min, 100 μ l ray Angiostatin 1 functional zone albumen are added after ice bath 10min, shaking table is in conjunction with 30min, the more centrifugal 5min of 6000rpm, abandons supernatant, then use 1 × binding buffer liquid to wash precipitation, abandon supernatant; 1 × binding buffer liquid 100 μ L is added again, 96 DEG C of heating 5min, the then centrifugal 10min of 15000rpm in precipitation, get supernatant liquor, to precipitation heating centrifugal again, merge supernatant liquor, then separablely obtain ssDNA the level library having avidity with ray Angiostatin 1 functional zone albumen; Described 2 × binding buffer liquid is that 20 × binding buffer liquid distilled water dilutes the solution after 10 times, and described 1 × binding buffer liquid is that 20 × binding buffer liquid distilled water dilutes the solution after 20 times; Described 20 × binding buffer liquid formula is 1MNaCl, 50mMKCl, 500mMTris-HCl, 10mMMgCl2, pH7.4.
2.2.2ssDNA with the combination of ray Angiostatin 1 functional zone albumen, be separated, concrete grammar is as follows:
By step 2.2.1 be separated obtain can with the protein bound ssDNA in ray Angiostatin 1 functional zone, again with 100 μ l ray Angiostatin 1 functional zone albumen shaking tables in conjunction with 30min, subsequent step is with step 2.2.1, then separable have ssDNA level library of avidity to ray Angiostatin 1 functional zone albumen.
2.2.3 asymmetric PCR amplification ssDNA, concrete grammar is as follows:
Be separated to step 2.2.2 ssDNA the level library obtained and carry out asymmetric PCR amplification, to be the asymmetric PCR amplification system of 25 μ l be cumulative volume: 10 × PCR damping fluid: 2 μ l; P1 (10 μMs): 1 μ l; P2 (0.2 μM): 1 μ l; DNTP (each 2.5mM): 0.4 μ l; MgCl
2(25mM): 1.2 μ l; SsDNA template (0.2 μ g/ μ l): 2 μ l; Taq DNA polymerase (5u/ μ l): 0.2 μ l; DdH2O:17.2 μ l; PCR reaction parameter: 94 DEG C of denaturation 4min, then carries out 40 circulations, 94 DEG C of sex change 30s, 58 DEG C of annealing 30s, and 72 DEG C extend 20s, and last 72 DEG C extend 7min;
2.2.4 the mensuration of avidity, concrete grammar is as follows:
2.2.4.1 increase: to increase ssDNA the level library screened with the primer P1 asymmetric PCR with digoxigenin labeled, amplification condition is identical with parameter with the asymmetric PCR amplification system of step 2.2.3 with parameter;
2.2.4.2 with protein binding: get step 2.2.4.1 and to increase the PCR primer 100 μ L of gained, 95 DEG C of sex change 5min, add after ice bath 10min in 100 μ L albumen, fully mix, at room temperature in conjunction with 30min, then 6000rpm is centrifugal, protein isolate and supernatant liquor, includes and the ssDNA of band digoxigenin labeled that combines in albumen in albumen, unconjugated ssDNA in supernatant liquor, do the blank that does not add ssDNA simultaneously, namely use 2 × binding buffer liquid to replace PCR primer, carry out aforesaid operations equally;
2.2.4.3 wash: albumen 1 × binding buffer liquid 500 μ L is washed 1 time, and 6000rpm is centrifugal, abandons supernatant, gets albumen;
2.2.4.4 be combined with enzyme mark rabbit anti digoxin antibody: add the excessive enzyme mark rabbit anti digoxin antibody that 100 μ L1: 900TBS dilute in albumen, fully after mixing, reaction 10min, the ssDNA making it the digoxigenin labeled in albumen is combined;
Described TBS is 0.5MTris-NaCl solution, and compound method is: first water-soluble 8.5 ~ 9gNaCl, then adds Tris-HCl (0.5M, pH7.6) solution 100ml, finally adds water and is settled to 1L; 0.5MTris-HCl (pH7.6,100ml) solution preparation method: take Tris6.06g, adds distilled water 40ml and dissolves, and drips dense HCl and adjusts pH to 7.6, be settled to 100ml.
2.2.4.5 wash: 6000rpm is centrifugal, removes supernatant, then uses 1 × binding buffer liquid 500 μ L to wash 3 times, obtains albumen;
2.2.4.6TMB (tetramethyl benzidine) colour developing: add the resuspended albumen of 400 μ L distilled water, add 200 μ LTMB nitrite ions again, after lucifuge colour developing 10min, with 2mol/LH2SO4200 μ L termination reaction, measure the light absorption value OD450 at 450nm place, namely this value reflects the avidity of the ssDNA be combined with bacterium, namely OD combines, and blank carries out above-mentioned steps 2.2.4.3,2.2.4.4 equally, 2.2.4.5 and 2.2.4.6, blank corresponding absorbancy OD is obtained blank;
Described TMB nitrite ion uses conventional compound method preparation.
2.2.4.7 the volumetric molar concentration of DNA in PCR primer is measured: the PCR primer of getting step 2.2.4.1 amplification gained, with the initial ssDNA library of concentration known gradient for standard substance, with Bandscan software as image analysis software, adopt the DNA content in ethidium bromide agarose gel electrophoretic method quantitative assay PCR primer, obtain the volumetric molar concentration of corresponding DNA, and then the DNA mole number in 100 μ LPCR products can be calculated.
2.2.4.8 the avidity in corresponding library is calculated:
2.3 repeat screening, concrete grammar is: the screening library of product as next round of taking turns asymmetric PCR using each, repeat above-mentioned SELEX and screen step 2.2, until avidity no longer rises, eventually pass the aptamer enriched library that 16 screenings taken turns obtain ssDNA.After asymmetric PCR amplification, condition is with step above, clone and check order, obtain 20 effective ssDNA that copy number is the highest, 20 aptamers are carried out affine specificity verification respectively, obtain 10 and have better affine specific oligonucleotide sequence (aptamer) to ray Angiostatin 1 functional zone albumen, concrete sequence is as follows:
The concrete data of avidity are as follows:
| Aptamer title | Avidity | Aptamer title | Avidity |
| 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, specificity and the affinity of 20 aptamers is analyzed
Fluorescently-labeled adaptor sequence and ray Angiostatin 1 functional zone albumen are hatched, carry out flow cytometry detection, wherein 10 sequences show high fluorescent, GraphPadPrism5.0 software is used to do nonlinear regression curve for saturation curve, respectively identical experimental implementation is adopted to 10 high-affinity adaptor sequence, obtains the Kd value that every bar is configuration:
| Aptamer title | Kd value (nM) | Aptamer title | 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 |
Wherein the Kd value of K20 is minimum, illustrates can be combined fast with target protein and Stability Analysis of Structures is not easily separated.
Adopt the secondary structure of DNAMAN software building 10 aptamers and calculate their minimum free energy, its structure minimum free energy is also all less, and structure is also relatively stable.
2.5 aptamer specificity analyses
BSA, human hemoglobin, ray Angiostatin 1 functional zone albumen and 10 aptamers are adopted to carry out specific detection respectively, find through binding tests, these 10 sequences do not combine with BSA or human hemoglobin, and only keep higher specificity with ray Angiostatin 1 functional zone protein binding.
Sequence table
< 110 > opens bravely
The aptamer K9 of < 120 > ray Angiostatin 1 and screening method thereof and application
〈160〉13
〈210〉1
〈211〉225
〈212〉PRT
< 213 > ray
〈400〉1
TLDIYKQLRDKETPSGFTLDDVIQTGVDNPGHPFIMTVGCVAGDEESYEVFKALFDPVIQ60
DRHGGYKPTDKHKTDLNHENLKGGDDLDPNYVLSSRVRTGRSIKGIALPPHCSRGERRLV120
EKLCLEGLATLTGEFQGKYYPLTTMSDAEQQQLIDDHFLFDKPVSPLLLASGMARDWPDA180
RGIWHNNDKTFLVWVNEEDHLRVISMQKGGNMKEVFRRFCVGLKK225
〈210〉2
<211〉84
〈212〉DNA
< 213 > artificial sequence
〈400〉K2
1TCAGTCGCTTCGCCGTCTCCTTCATGATCGCGCTGACAAATTAGGCCATTCAATCAGAGC
61ACAAGAGGGAGACCCCAGAGGG
〈210〉3
<211〉84
〈212〉DNA
< 213 > artificial sequence
〈400〉K6
1TCAGTCGCTTCGCCGTCTCCTTCCCGTGATGAATTGCTGATGAGCGCAGCATGGAGCTGC
61ACAAGAGGGAGACCCCAGAGGG
〈210〉4
<211〉84
〈212〉DNA
< 213 > artificial sequence
〈400〉K9
1TCAGTCGCTTCGCCGTCTCCTTCTGACGCATTCGGATCCAAGTTAATTAAATAACTGCGC
61ACAAGAGGGAGACCCCAGAGGG
〈210〉5
<211〉84
〈212〉DNA
< 213 > artificial sequence
〈400〉K10
1TCAGTCGCTTCGCCGTCTCCTTCATTGCAACCTGAGGCCATGGGACAGACCATGATAGGC
61ACAAGAGGGAGACCCCAGAGGG
〈210〉6
<211〉84
〈212〉DNA
< 213 > artificial sequence
〈400〉K14
1TCAGTCGCTTCGCCGTCTCCTTCAACTTGGACCCTTGAGCGATGAAGTAACGGTTTACGC
61ACAAGAGGGAGACCCCAGAGGG
〈210〉7
<211〉84
〈212〉DNA
< 213 > artificial sequence
〈400〉K15
1TCAGTCGCTTCGCCGTCTCCTTCGCACTGCTACCGATATTACATATATGGAGATACAGGC
61ACAAGAGGGAGACCCCAGAGGG
〈210〉8
<211〉84
〈212〉DNA
< 213 > artificial sequence
〈400〉K16
1TCAGTCGCTTCGCCGTCTCCTTCGGCCGAGTAACAGATTGGAACCCAACTGAGTGAGAGC
61ACAAGAGGGAGACCCCAGAGGG
〈210〉9
<211〉84
〈212〉DNA
< 213 > artificial sequence
〈400〉K17
1TCAGTCGCTTCGCCGTCTCCTTCTTATGGACGAGTAGAGGTACGATGACCCAATGATTGC
61ACAAGAGGGAGACCCCAGAGGG
〈210〉10
<211〉84
〈212〉DNA
< 213 > artificial sequence
〈400〉K19
1TCAGTCGCTTCGCCGTCTCCTTCCGATTGAGGGAGATTACGCATATGAGTACAACTGAGC
61ACAAGAGGGAGACCCCAGAGGG
〈210〉11
<211〉84
〈212〉DNA
< 213 > artificial sequence
〈400〉K20
1TCAGTCGCTTCGCCGTCTCCTTCTTTAGACCCGATAATGTTGTTTTGGTGACCGAATTGC
61ACAAGAGGGAGACCCCAGAGGG
〈210〉12
<211〉23
〈212〉DNA
< 213 > artificial sequence
〈400〉P1
TCAGTCGCTTCGCCGTCTCCTTC
〈210〉13
<211〉24
〈212〉DNA
< 213 > artificial sequence
〈400〉P2
CCCTCTGGGGTCTCCCTCTTGTGC
Claims (3)
1., for a ray Angiostatin 1 functional zone albumen for aptamer screening, its sequence is for shown in SEQIDNO:1.
2. identify an oligonucleotide sequence for ray Angiostatin 1 functional zone albumen, it is characterized in that its sequence is for shown in SEQIDNo.4.
3. the oligonucleotide sequence shown in claim 2 is for screening the application of ray Angiostatin 1 functional zone albumen.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610099682.5A CN105541986B (en) | 2014-06-06 | 2014-06-06 | The aptamer K9 and its screening technique of ray Angiostatin 1 and application |
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410249737.7A CN104031137B (en) | 2014-06-06 | 2014-06-06 | The aptamer of ray Angiostatin 1 and screening technique thereof and application |
| CN201610099682.5A 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 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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| CN201610102329.8A Active CN105541990B (en) | 2014-06-06 | 2014-06-06 | Aptamers K15 of skate angiogenesis inhibitor 1 and screening method and application thereof |
| 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 |
| CN201610099682.5A Active CN105541986B (en) | 2014-06-06 | 2014-06-06 | The aptamer K9 and its screening technique of ray Angiostatin 1 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 |
| CN201610101224.0A Active CN105541988B (en) | 2014-06-06 | 2014-06-06 | Aptamers K14 of skate angiogenesis inhibitor 1 and screening method and application thereof |
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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 |
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| CN201610101224.0A Active CN105541988B (en) | 2014-06-06 | 2014-06-06 | Aptamers K14 of skate angiogenesis inhibitor 1 and screening method and application thereof |
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| CN105693849A (en) * | 2014-07-13 | 2016-06-22 | 马海龙 | Aptamer NKXA5 specifically for Neutrokine-alpha protein and application of aptamer NKXA5 |
| 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 |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| 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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| US20090105172A1 (en) * | 2005-03-07 | 2009-04-23 | Diener John L | Stabilized Aptamers to PSMA and Their Use as Prostate Cancer Therapeutics |
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- 2014-06-06 CN CN201610101377.5A patent/CN105567698B/en active Active
- 2014-06-06 CN CN201410249737.7A patent/CN104031137B/en active Active
- 2014-06-06 CN CN201610099682.5A patent/CN105541986B/en active Active
- 2014-06-06 CN CN201610102089.1A patent/CN105541989B/en not_active Expired - Fee Related
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
|---|---|
| CN105567698A (en) | 2016-05-11 |
| CN105541988B (en) | 2020-05-19 |
| CN105541989A (en) | 2016-05-04 |
| CN105541988A (en) | 2016-05-04 |
| CN105541986B (en) | 2019-04-12 |
| CN105541989B (en) | 2020-06-02 |
| CN105541990B (en) | 2020-05-15 |
| CN105541990A (en) | 2016-05-04 |
| CN104031137A (en) | 2014-09-10 |
| CN105567698B (en) | 2018-08-31 |
| CN104031137B (en) | 2016-08-24 |
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