CN105713081A - Aptamers K17 of ray angiogenesis inhibiting factor 1 and screening method and application thereof - Google Patents
Aptamers K17 of ray angiogenesis inhibiting factor 1 and screening method and application thereof Download PDFInfo
- Publication number
- CN105713081A CN105713081A CN201610101408.7A CN201610101408A CN105713081A CN 105713081 A CN105713081 A CN 105713081A CN 201610101408 A CN201610101408 A CN 201610101408A CN 105713081 A CN105713081 A CN 105713081A
- Authority
- CN
- China
- Prior art keywords
- albumen
- ray
- functional areas
- aptamers
- aptamer
- 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.)
- Granted
Links
Classifications
-
- 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
-
- C—CHEMISTRY; METALLURGY
- 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
- C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
- C12N15/1034—Isolating an individual clone by screening libraries
- C12N15/1048—SELEX
-
- C—CHEMISTRY; METALLURGY
- 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
-
- C—CHEMISTRY; METALLURGY
- 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
- C12N2310/00—Structure or type of the nucleic acid
- C12N2310/10—Type of nucleic acid
- C12N2310/16—Aptamers
Abstract
Provided are a set of aptamers capable of recognizing functional area protein of a ray angiogenesis inhibiting factor 1 and a screening method and application of the aptamers. An oligonucleotide sequence comprises SEQ ID No.2-11, and the aptamers have high affinity specificity, and can be used for detecting functional area protein of the ray angiogenesis inhibiting factor 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 technique thereof and application.
Background technology
In the last few years, oligonucleotide aptamer was as the promising replacement 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, building the strand random oligonucleotide library of synthetic, its random sequence length is about 20-100 base.Utilize the characteristic that single stranded oligonucleotide molecular configurations is flexible and changeable, random oligonucleotide library is interacted with target molecule, retain the oligonucleotide being combined with spatial conformation with target molecule, through repeated amplification, screen several circulation, can make to be enriched with the oligonucleotide sequence of this target specific bond, the final specific oligonucleotide aptamer obtaining multiple target molecule, i.e. aptamer.The aptamer that SELEX technology screening obtains is utilized to identify that the pattern of molecule is similar with protein antibodies, but compared with protide antibody, nucleic acid aglucon has more superiority, if do not limited by immune condition and immunogenicity, can external synthetic, degeneration is reversible with renaturation, 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 recognise that the undistinguishable protein molecule of monoclonal antibody.And the target molecule of aptamer is widely, little of dye molecule, big to complete virion and bacterial pathogens, even complete cell can also go out the oligonucleotide aptamer of high-affinity by cutting down SELEX technology screening.
Ray Angiostatin 1 functional areas are hereinafter referred to as ray functional areas, 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: ray Angiostatin 1 significantly inhibits chick chorioallantoic membrane itself and KB cell induction chick chorioallantoic membrane angiogenesis；It is the growth and the transfer that all significantly inhibit nude mouse Lewis lung cancer of lumbar injection or gavage, reduces tumor tissues microvessel density, lower the expression of angiogenic factors VEGF；The transfer of nude mouse Melanoma B16 cell also there is high inhibition effect；Lower the expression of short transfer factor CD44v6 and ErBb2；More remarkable effect during with 5-fluorouracil (5-FU) use in conjunction.The PTS Avastin that the action target spot of ray Angiostatin 1 is developed from U.S. gene technology institute is different.Avastin is gene engineering product, is the antibody of VEGF, and its action target spot is VEGF；Ray Angiostatin 1 is natural product, and it acts not only on VEGF, also acts on bFGF and PDGF.Therefore, carry out specific discriminating for ray Angiostatin 1 functional areas and screening is the consistent pursuit in this area.
Based on considerations above, the present invention is target protein for the purpose of the albumen of ray Angiostatin 1 functional areas, adopting SELEX technology to obtain 10 special aptamer of ray Angiostatin 1 functional areas albumen, what combination application can be rapid, sensitive, special detects ray Angiostatin 1 functional areas albumen.Due to single strand dna oligonucleotide aptamer stable performance, synthesis convenient and cheap, modified after can be directly used for fluorescence or chemiluminescence, chromophoric method detects target bandage, therefore simple to operate, direct.
Summary of the invention
It is an object of the invention to provide and not only there is quick, simple to operate, the stability of detection higher than features such as antibody, and preparation method easily, shorter one group of oligonucleotide sequence that can recognise that ray Angiostatin 1 functional areas albumen of manufacturing cycle and preparation method thereof.
The described oligonucleotide sequence that can recognise that ray Angiostatin 1 functional areas albumen, including SEQIDNo.2-11, and adopts an oligonucleotide sequence therein just can complete the recognition detection to ray Angiostatin 1 functional areas albumen；
The preparation method of described one group of oligonucleotide sequence that can recognise that ray Angiostatin 1 functional areas albumen comprises the following steps:
1, the synthesis ssDNA oligonucleotide library (5 '-TCAGTCGCTTCGCCGTCTCCTTC----for screening
N35----GCACAAGAGGGAGACCCCAGAGGG-3 '), wherein N35 is 35 random oligonucleotides；
2, SELEX screening is carried out after being mixed with ray Angiostatin 1 functional areas albumen respectively by oligonucleotide library, it is thus achieved that aptamer enriched library；
3, after SELEX has screened, the aptamer enriched library obtained is carried out cloning and sequencing；
4, selecting the height copy ssDNA occurred in sequencing result, carry out affine specific checking, screening obtains the oligonucleotide sequence that can recognise that ray Angiostatin 1 functional areas albumen.
Detailed description of the invention
Embodiment 1
1, the preparation of ray Angiostatin 1 functional areas albumen
The mode adopting yeast well known to those skilled in the art recombinant expressed obtains has bioactive ray Angiostatin 1 functional areas identical with ray Angiostatin 1 functional areas albumen albumen, and this protein sequence is such as shown in SEQIDNO:1；The concentration of protein solution is 15mg/ml.
2, the synthesis of library and primer
2.1, the synthesis ssDNA oligonucleotide library (5 '-TCAGTCGCTTCGCCGTCTCCTTC----for screening
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 areas albumen, separate, concrete grammar is as follows:
Take the ssDNA oligonucleotide library 4 μ L of 100 μMs, it is diluted to 100 μ l with 2 × binding buffer liquid, 95 DEG C of degeneration 5min, 100 μ l ray Angiostatin 1 functional areas albumen are added after ice bath 10min, shaking table is in conjunction with 30min, the more centrifugal 5min of 6000rpm, abandons supernatant, then wash precipitation with 1 × binding buffer liquid, abandon supernatant；Precipitation adds 1 × binding buffer liquid 100 μ L, 96 DEG C of heating 5min, the then centrifugal 10min of 15000rpm, take supernatant, precipitation is again heated and is centrifuged, merges supernatant, then separable obtaining has ssDNA level library of affinity with ray Angiostatin 1 functional areas albumen；Described 2 × binding buffer liquid is the solution after 20 × binding buffer liquid distilled water dilutes 10 times, and described 1 × binding buffer liquid is the solution after 20 × binding buffer liquid distilled water dilutes 20 times；Described 20 × binding buffer formula of liquid is 1MNaCl, 50mMKCl, 500mMTris-HCl, 10mMMgCl2, pH7.4.
2.2.2ssDNA with the combination of ray Angiostatin 1 functional areas albumen, separate, concrete grammar is as follows:
What step 2.2.1 separation obtained can with the protein bound ssDNA in ray Angiostatin 1 functional areas, again with 100 μ l ray Angiostatin 1 functional areas albumen shaking tables in conjunction with 30min, subsequent step is with step 2.2.1, then separable to ssDNA the level library having affinity with ray Angiostatin 1 functional areas albumen.
2.2.3 asymmetric PCR amplification ssDNA, concrete grammar is as follows:
Step 2.2.2 being separated ssDNA the level library obtained and carries out asymmetric PCR amplification, the asymmetric PCR amplification system that cumulative volume is 25 μ l is: 10 × PCR buffer: 2 μ l；P1 (10 μMs): 1 μ l；P2 (0.2 μM): 1 μ l；DNTP (each 2.5mM): 0.4 μ l；MgCl2(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 response parameter: 94 DEG C of denaturation 4min, then carries out 94 DEG C of degeneration 30s of 40 circulations, 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 affinity, concrete grammar is as follows:
2.2.4.1 amplification: expanding, with the primer P1 asymmetric PCR with digoxigenin labeled, ssDNA the level library screened, amplification condition is identical with the asymmetric PCR amplification system of step 2.2.3 and parameter with parameter；
2.2.4.2 with protein binding: take step 2.2.4.1 and expand the PCR primer 100 μ L of gained, 95 DEG C of degeneration 5min, add after ice bath 10min in 100 μ L albumen, are sufficiently mixed, at room temperature in conjunction with 30min, then 6000rpm is centrifuged, and separates albumen and supernatant, includes and the ssDNA with digoxigenin labeled of combination in albumen in albumen, supernatant is unconjugated ssDNA, do a blank being not added with ssDNA simultaneously, namely replace PCR primer with 2 × binding buffer liquid, carry out aforesaid operations equally；
2.2.4.3 washing: being washed 1 time by albumen 1 × binding buffer liquid 500 μ L, 6000rpm is centrifuged, and abandons supernatant, takes albumen；
2.2.4.4 be combined with enzyme mark rabbit anti digoxin antibody: in albumen, add the excessive enzyme mark rabbit anti digoxin antibody of 100 μ L1: 900TBS dilutions, after being sufficiently mixed, react 10min, so as to the ssDNA of 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: weigh Tris6.06g, adds distilled water 40ml and dissolves, and drips dense HCl and adjusts pH to 7.6, is settled to 100ml.
2.2.4.5 washing: 6000rpm is centrifuged, and removes supernatant, then washs 3 times with 1 × binding buffer liquid 500 μ L, obtains albumen；
2.2.4.6TMB (tetramethyl benzidine) colour developing: add the 400 μ resuspended albumen of L distilled water, add 200 μ LTMB nitrite ions, after lucifuge colour developing 10min, terminate reaction with 2mol/LH2SO4200 μ L, measure the light absorption value OD450 at 450nm place, namely this value reflects the affinity of the ssDNA being 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 absorbance OD is obtained blank；
Described TMB nitrite ion uses conventional compound method preparation.
2.2.4.7 the molar concentration of DNA in PCR primer is measured: take step 2.2.4.1 and expand the PCR primer of 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 electrophoresis method quantitative assay PCR primer, obtain the molar concentration of corresponding DNA, and then the DNA molal quantity in 100 μ LPCR products can be calculated.
2.2.4.8 the affinity in corresponding library is calculated:
2.3 repeat screening, method particularly includes: using each product screening library as next round taking turns asymmetric PCR, repeat above-mentioned SELEX and screen step 2.2, until affinity no longer rises, eventually pass 16 screenings taken turns and obtain the aptamer enriched library of ssDNA.After asymmetric PCR expands, 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, obtaining 10 and ray Angiostatin 1 functional areas albumen has better affine specific oligonucleotide sequence (aptamer), particular sequence is as follows:
The concrete data of affinity are as follows:
| Aptamer title | Affinity | Aptamer title | Affinity |
| 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 are analyzed
Fluorescently-labeled adaptor sequence and ray Angiostatin 1 functional areas 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 10 high-affinity adaptor sequence being adopted identical experimental implementation, obtaining every is the Kd value configured:
| 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 quickly combined with target protein and Stability Analysis of Structures is not readily separated.
Adopting the secondary structure of 10 aptamers of DNAMAN software building 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
It is respectively adopted BSA, human hemoglobin, ray Angiostatin 1 functional areas albumen and 10 aptamers and carries out specific detection, 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 areas protein binding.
Sequence table
< 110 > opens bravely
The aptamer K17 of < 120 > ray Angiostatin 1 and screening technique 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 areas albumen for aptamer screening, its sequence is shown in SEQIDNO:1.
2. the oligonucleotide sequence identifying ray Angiostatin 1 functional areas albumen, it is characterised in that its sequence is shown in SEQIDNo.9.
3. the oligonucleotide sequence shown in claim 2 is for screening the application of ray Angiostatin 1 functional areas albumen.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610101408.7A CN105713081B (en) | 2014-06-06 | 2014-06-06 | The aptamer K17 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 |
| CN201610101408.7A CN105713081B (en) | 2014-06-06 | 2014-06-06 | The aptamer K17 and its screening technique of ray Angiostatin 1 and application |
Related Parent Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201410249737.7A Division CN104031137B (en) | 2014-06-06 | 2014-06-06 | The aptamer of ray Angiostatin 1 and screening technique thereof and application |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN105713081A true CN105713081A (en) | 2016-06-29 |
| CN105713081B CN105713081B (en) | 2019-04-12 |
Family
ID=56156969
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201610101408.7A Active CN105713081B (en) | 2014-06-06 | 2014-06-06 | The aptamer K17 and its screening technique of ray Angiostatin 1 and application |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN105713081B (en) |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101260151A (en) * | 2007-03-05 | 2008-09-10 | 广东海洋大学 | Separation and primary structure of sea purse blood vessel generation inhibitive factor 1, and application thereof in preparing blood vessel generation inhibiting medicament and antineoplastic medicam |
| 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 |
-
2014
- 2014-06-06 CN CN201610101408.7A patent/CN105713081B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101260151A (en) * | 2007-03-05 | 2008-09-10 | 广东海洋大学 | Separation and primary structure of sea purse blood vessel generation inhibitive factor 1, and application thereof in preparing blood vessel generation inhibiting medicament and antineoplastic medicam |
| 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 |
|---|---|
| CN105713081B (en) | 2019-04-12 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN103966224B (en) | A kind of aptamer and screening technique thereof and application | |
| CN104109194B (en) | The aptamer and its screening technique of SFRP1 albumen and application | |
| ES2393056T3 (en) | Nucleic Acid Synthesis Procedure | |
| Huang et al. | Selection and characterization of DNA aptamers against Staphylococcus aureus enterotoxin C1 | |
| US9316647B2 (en) | Aptamers and diagnostic methods for detecting the EGF receptor | |
| CN104293793A (en) | Oligonucleotide aptamer specifically recognizing T-2 toxin | |
| CN108754000A (en) | Drug resistant gene mcr-4/5/8 fluorescent quantitative PCR detection methods | |
| CN102154528A (en) | Primer, probe and kit for detecting rotavirus and Norovirus liquid phase chips | |
| CN104031137B (en) | The aptamer of ray Angiostatin 1 and screening technique thereof and application | |
| CN102010865B (en) | Nucleic acid aptamer capable of specifically recognizing Listeria monocytogenes, screening method and application thereof | |
| CN107058329B (en) | Aptamer specifically binding to Newcastle disease virus and screening method and application thereof | |
| EP3292217B1 (en) | Formation of hairpins in situ using force-induced strand invasion | |
| CN102766693A (en) | Nucleic acid aptamer for detecting human hepatoma cell line SMMC-7721 as well as screening method and application thereof | |
| JP2017500020A5 (en) | ||
| CN107858357B (en) | Bovine respiratory syncytial G protein specific aptamer | |
| CN106191319B (en) | A kind of multi-fluorescence immunoassay method of 6 kinds of fowl respiratory pathogens of quick differentiation | |
| CN105713081A (en) | Aptamers K17 of ray angiogenesis inhibiting factor 1 and screening method and application thereof | |
| CN105669851A (en) | Aptamers K6 of ray angiogenesis inhibitor 1 and screening method and application of aptamers | |
| CN105646695A (en) | Aptamer K16 of angiogenesis inhibiting factor 1 of skates as well as screening method and application of aptamer K16 | |
| CN105669852B (en) | The aptamer K10 and its screening technique of ray Angiostatin 1 and application | |
| KR101362725B1 (en) | DNA Aptamer Specifically Binding to Surface of Living Cell of Vibrio parahemolyticus and Uses Thereof | |
| CN104278036B (en) | Set of oligonucleotide aptamers for specifically recognizing Sh.sonnei | |
| CN104818277A (en) | A group of oligonucleotide aptamers specifically recognizing staphylococcus aureus subsp.aureus | |
| CN106636329A (en) | Specific method for detecting trace circulating tumor cells in mammal blood | |
| JP2011502487A5 (en) |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| TA01 | Transfer of patent application right | ||
| TA01 | Transfer of patent application right |
Effective date of registration: 20190304 Address after: 314113 D7 Building, 555 Pioneer Road, Dayun Town, Jiashan County, Jiaxing City, Zhejiang Province (Residence Declaration) Applicant after: Zhejiang Pharmaceutical Garden Biotechnology Co., Ltd. Address before: 310027 Zhejiang University, 38 Xihu District Zhejiang Road, Zhejiang University, Zhejiang Applicant before: Zhang Yong |
|
| GR01 | Patent grant | ||
| GR01 | Patent grant |