CN104031137B - The aptamer of ray Angiostatin 1 and screening technique thereof and application - Google Patents
The aptamer of ray Angiostatin 1 and screening technique thereof and application Download PDFInfo
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- CN104031137B CN104031137B CN201410249737.7A CN201410249737A CN104031137B CN 104031137 B CN104031137 B CN 104031137B CN 201410249737 A CN201410249737 A CN 201410249737A CN 104031137 B CN104031137 B CN 104031137B
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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
- C12N15/11—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
-
- 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
- C12N2320/00—Applications; Uses
- C12N2320/10—Applications; Uses in screening processes
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- 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
- 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
-
- 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
- C12N2330/00—Production
- C12N2330/30—Production chemically synthesised
- C12N2330/31—Libraries, arrays
Abstract
The aptamer of ray Angiostatin 1 and screening technique thereof and application.One group of aptamer that can identify ray Angiostatin 1 functional areas albumen and preparation method thereof.Oligonucleotide sequence includes SEQ ID No.2～11, and it is respectively provided with higher affine specificity, may be used for the detection of ray Angiostatin 1 functional areas albumen.
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 the most noticeable.Oligonucleotide aptamer is obtained by SELEX technology (Systematic evolution of ligands by exponential enrichment) 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 utilizing SELEX technology screening to obtain identifies 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 identify the undistinguishable protein molecule of monoclonal antibody.And the target molecule of aptamer is widely, little to dye molecule, greatly to complete virion and bacterial pathogens, the most complete cell can also go out the oligonucleotide aptamer of high-affinity by abatement SELEX technology screening.
Hereinafter referred to as ray functional areas, ray Angiostatin 1 functional areas, ray Angiostatin 1 is the protein (molecular weight 42KD) that we separate first from South China Sea a kind of ray tissue.Result of study shows: that ray Angiostatin 1 significantly inhibits chick chorioallantoic membrane itself and that KB cell is induced chick chorioallantoic membrane angiogenesis；Either lumbar injection or gavage all significantly inhibit growth and the transfer of nude mouse Lewis lung cancer, reduce tumor tissues microvessel density, under wither the expression of angiogenic factors VEGF；Transfer to nude mouse Melanoma B16 cell also has high inhibition effect；Lower the expression promoting transfer factor CD44v6 and ErBb2；More notable with effect during 5-fluorouracil (5-FU) use in conjunction.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, using 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, the most 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 identify ray Angiostatin 1 functional areas albumen of manufacturing cycle and preparation method thereof.
The described oligonucleotide sequence that can identify ray Angiostatin 1 functional areas albumen, including SEQ ID No.2-11, and uses 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 identify ray Angiostatin 1 functional areas albumen comprises the following steps:
1, synthesis is for the ssDNA oligonucleotide library (5 '-TCA GTC GCT TCG CCG TCT CCT TC----of screening
N35----GCA CAA GAG GGA GAC CCC AGA GGG-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 high copy ssDNA occurred in sequencing result, carry out affine specific checking, screening obtains the oligonucleotide sequence that can identify 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 using 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 as shown in SEQ ID NO:1；The concentration of protein solution is 15mg/ml.
2, library and the synthesis of primer
2.1, synthesis is for the ssDNA oligonucleotide library (5 '-TCA GTC GCT TCG CCG TCT CCT TC----of screening
N35----GCA CAA GAG GGA GAC CCC AGA GGG-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, with 2 × combine buffer and be diluted to 100 μ l, 95 DEG C of degeneration 5min, 100 μ l ray Angiostatin 1 functional areas albumen are added after ice bath 10min, shaking table combines 30min, then 6000rpm is centrifuged 5min, abandons supernatant, then with 1 × combine buffer and wash precipitation, abandon supernatant；Adding 1 in precipitation × combine buffer 100 μ L, 96 DEG C of heating 5min, then 15000rpm is centrifuged 10min, take supernatant, precipitation again being heated and is centrifuged, merges supernatant, the most separable obtaining has ssDNA level library of affinity with ray Angiostatin 1 functional areas albumen；Described 2 × combine buffer be 20 × combine the solution after buffer distilled water dilutes 10 times, described 1 × combine buffer be 20 × combine the solution after buffer distilled water dilutes 20 times；Described 20 × to combine buffer formulation be 1M NaCl, 50mM KCl, 500mM Tris-HCl, 10mM MgCl2, pH7.4.
2.2.2ssDNA with the combination of ray Angiostatin 1 functional areas albumen, separate, concrete grammar is as follows:
By step 2.2.1 isolated can be with the protein bound ssDNA in ray Angiostatin 1 functional areas, 30min is combined again with 100 μ l ray Angiostatin 1 functional areas albumen shaking tables, subsequent step is with step 2.2.1, the most 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, cumulative volume is that the asymmetric PCR amplification system of 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 archaeal dna polymerase (5u/ μ l): 0.2 μ l；DdH2O:17.2 μ l；PCR response parameter: 94 DEG C of denaturations 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 the PCR primer 100 μ L of step 2.2.4.1 amplification gained, 95 DEG C of degeneration 5min, add after ice bath 10min in 100 μ L albumen, are sufficiently mixed, at room temperature combine 30min, then 6000rpm is centrifuged, and separates albumen and supernatant, includes and the ssDNA of the band digoxigenin labeled of combination in albumen in albumen, supernatant is unconjugated ssDNA, do a blank being not added with ssDNA simultaneously, i.e. with 2 × combine buffer and replace PCR primer, carry out aforesaid operations equally；
2.2.4.3 washing: by albumen with 1 × combine buffer 500 μ L and wash 1 time, 6000rpm is centrifugal, abandons supernatant, takes albumen；
2.2.4.4 being combined with enzyme mark rabbit anti digoxin antibody: add the enzyme mark rabbit anti digoxin antibody of the excess of 100 μ L1: 900TBS dilutions in albumen, after being sufficiently mixed, reaction 10min, the ssDNA of the digoxigenin labeled being allowed in albumen are combined；
Described TBS is 0.5M Tris-NaCl solution, and compound method is: first water-soluble 8.5～9g NaCl, then adds Tris-HCl (0.5M, pH7.6) solution 100ml, finally adds water and is settled to 1L；0.5M Tris-HCl (pH7.6,100ml) solution compound 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 with 1 × combine buffer 500 μ L washing 3 times, obtain albumen；
2.2.4.6TMB (tetramethyl benzidine) colour developing: add the 400 μ resuspended albumen of L distilled water, add 200 μ L TMB nitrite ions, after lucifuge colour developing 10min, terminate reaction with 2mol/L H2SO4200 μ L, measure the light absorption value OD450 at 450nm, this value i.e. reflects the affinity of the ssDNA being combined with bacterium, i.e. 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 the PCR primer of step 2.2.4.1 amplification gained, with the initial ssDNA library of concentration known gradient as standard substance, with Bandscan software as image analysis software, use the DNA content in ethidium bromide agarose gel electrophoresis method quantitative determination PCR primer, obtain the molar concentration of corresponding DNA, and then the DNA molal quantity in 100 μ L PCR primer 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 have preferable affine specific oligonucleotide sequence (aptamer) to ray Angiostatin 1 functional areas albumen, particular sequence is as follows:
The concrete data of affinity are as follows:
2.4, specificity and the affinity of 20 aptamers are analyzed
Fluorescently-labeled adaptor sequence is hatched with ray Angiostatin 1 functional areas albumen, carry out flow cytometry detection, wherein 10 sequences show high fluorescent, GraphPad Prism5.0 software is used to do nonlinear regression curve for saturation curve, respectively 10 high-affinity adaptor sequence being used identical experimental implementation, having obtained 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.
Using the secondary structure of 10 aptamers of DNAMAN software building and calculate their minimum free energy, its structure minimum free energy is the least, and structure is the most 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.
Claims (2)
1. the oligonucleotide sequence identifying ray Angiostatin 1 functional areas albumen, it is characterised in that sequence
It is classified as shown in SEQ ID No.2.
2. the oligonucleotide sequence shown in claim 1 is used for screening ray Angiostatin 1 functional areas albumen
Application.
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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 |
| CN201610101224.0A CN105541988B (en) | 2014-06-06 | 2014-06-06 | Aptamers K14 of skate angiogenesis inhibitor 1 and screening method and application thereof |
| CN201610101377.5A CN105567698B (en) | 2014-06-06 | 2014-06-06 | The aptamer K19 and its screening technique of ray Angiostatin 1 and application |
| CN201610101402.XA CN105669852B (en) | 2014-06-06 | 2014-06-06 | The aptamer K10 and its screening technique of ray Angiostatin 1 and application |
| 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 |
| CN201610098919.8A CN105669851B (en) | 2014-06-06 | 2014-06-06 | The aptamer K6 and its screening technique of ray Angiostatin 1 and application |
| CN201610101615.2A CN105646695B (en) | 2014-06-06 | 2014-06-06 | The aptamer K16 and its screening technique of ray Angiostatin 1 and application |
| CN201610102089.1A CN105541989B (en) | 2014-06-06 | 2014-06-06 | Aptamers K20 of skate angiogenesis inhibitor 1 and screening method and application thereof |
| CN201610101408.7A CN105713081B (en) | 2014-06-06 | 2014-06-06 | The aptamer K17 and its screening technique of ray Angiostatin 1 and application |
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| CN201610101377.5A Division CN105567698B (en) | 2014-06-06 | 2014-06-06 | The aptamer K19 and its screening technique of ray Angiostatin 1 and application |
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| CN201610102329.8A Division 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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| CN201610101615.2A Division CN105646695B (en) | 2014-06-06 | 2014-06-06 | The aptamer K16 and its screening technique of ray Angiostatin 1 and application |
| CN201610101408.7A Division CN105713081B (en) | 2014-06-06 | 2014-06-06 | The aptamer K17 and its screening technique of ray Angiostatin 1 and application |
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