CN109355294A - The aptamer of specific recognition Vimentin and its application - Google Patents
The aptamer of specific recognition Vimentin and its application Download PDFInfo
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Abstract
The invention discloses the aptamer of recognizable Vimentin and its applications, and sequence is as shown in SEQ ID No.1.Aptamer AptVim of the invention can specifically bind Vimentin, and Kd value is 70.9 ± 12.3nM.The alternative tradition Vimentin antibody after the end AptVim connects biotin or fluorescent molecule, cell expression or the detection of tissue expression Vimentin albumen can be completed by being usually only necessary to a step, not only required reaction condition is simpler, and stability is higher, and cost is cheaper.In addition, aptamer of the invention, which after suitable transfection reagent imports tumour cell, can induce tumour cell, occurs apoptosis.It can be used as the EMT diagnostic reagent of infrastest and clinical cancer, the apoptosis that also can induce tumour cell is used for oncotherapy.
Description
Technical field
The invention belongs to field of biotechnology, and in particular to the aptamer of specific recognition Vimentin and its answer
With.
Background technique
Vimentin (Vimentin, Vim) is a kind of is mainly expressed in mesenchymal cell and the cell of mesoderma origin
In intermediate filament protein.Vimentin is upper highly conserved in evolution, participates in a variety of physiology processes: cytoskeleton is maintained,
Promote cell adhesion and migration, adjusts Apoptosis, signal transduction, Gene regulation etc..Vimentin is on tumour cell occurs
KDa major secretory protein during skin mesenchymal transformation (EMT), therefore become the important symbol object that identification EMT occurs in recent years.
And a large number of studies show that Vimentin is over-expressed in a variety of epithelial malignancies, by participating in EMT and Various Complex
Credit access accelerates tumour growth, promotes the infiltration and transfer of cancer cell, play an important role to the development and formation of cancer.DNA
It methylates and lowers the expression of tumour precancerous lesion correlation Vimentin albumen, therefore Vimentin methylation in gene level
Also can be used as the kinds cancers such as gastric cancer, cancer of pancreas, colon cancer diagnosis, by stages with the indexs such as prognosis.Research confirms Vimentin
Overexpression can cause cancer cell dynamic role and invasion enhancing, knock out its expression after these characteristics weaken, disclosing it has
Clinical treatment value.Vimentin albumen is expected to become the target spot of cancer clinical prediction, and the research of Vimentin albumen is simultaneously
There is important value (Ko S H, Suh S H, Kim B J, et al.Expression of the to treatment of cancer
intermediate filament vimentin in proliferating duct cells as a marker of
pancreatic precursor cells[J].Pancreas,2004,28(2):121-128；Satelli A,Li
S.Vimentin in cancer and its potential as a molecular target for cancer
therapy[J].Cellular&Molecular Life Sciences,2011,68(18):3033-3046；Fuyuhiro Y,
Yashiro M,Noda S,et al.Clinical significance of vimentin-positive gastric
cancer cells[J].Anticancer Research,2010,30(12):5239-43；Shirahata A,Sakata M,
Sakuraba K,et al.Vimentin methylation as a marker for advanced colorectal
carcinoma.[J].Anticancer Research,2009,29(1):279-281)。
The Vimentin monoclonal antibody developed in recent years although compatibility and specificity with higher, but antibody itself
Possessed some defects, such as high immunogenicity, unstable, chemical modification is difficult, the otherness between batch, production method are limited
And production cost valuableness etc., make it in basic research and clinical application by certain limitation.
Aptamer (Aptamer) is to be evolved using in-vitro screening technology-based on the Fas lignand system of index concentration
(systematic evolution of ligands by exponential enrichment, SELEX) technology is from large capacity
One section screened in nucleic acid molecule libraries is with the single stranded DNA or RNA widow's core compared with high specific and affinity combination target substance
Nucleotide sequence.Aptamer can match in excellence or beauty antibody, and the advantage for having some antibody no in terms of affinity and specificity: by
In the physicochemical property of its small molecule nucleic acid, aptamer immunogenicity is low, has quick tissue penetration and cell internalizing energy
Power, higher heat/chemical stability；And the screening period of aptamer is short, is easy to chemical modification or is coupled different function
Can group, synthetic technology is simple, and repeatability is strong etc. between batch.Therefore aptamer has in basic research and clinical application
Excellent prospect (Sun H, Zu Y.A Highlight of Recent Advances in Aptamer Technology
and Its Application.Molecules 2015,20(7):11959-80；O,Walter J G,
Shoham Y,et al.Aptamer-based downstream processing of his-tagged proteins
utilizing magnetic beads[J].Biotechnology&Bioengineering 2011,108(10):2371-
9)。
Summary of the invention
It is an object of the present invention to provide a kind of nucleic acid adaptations for specific recognition vimentin (Vimentin)
Body and its core sequence.
The core sequence of aptamer provided by the invention be following a1)-a3) and in it is any:
A1) single strand dna shown in SEQ ID No.1 19-58；
A2) and single strand dna shown in SEQ ID No.1 19-58 has 60% or 60% or more homology, and
Single strand dna with the same function；
A3) one or several nucleotide are deleted or increased to single strand dna shown in SEQ ID No.1 19-58,
And single strand dna with the same function.
The aptamer be following b1)-b3) and in it is any:
B1) single strand dna shown in SEQ ID No.1；
B2) there is with single strand dna shown in SEQ ID No.1 60% or 60% or more homology, and have identical
The single strand dna of function；
B3) one or several nucleotide are deleted or increased to single strand dna shown in SEQ ID No.1, and there is phase
The single strand dna of congenerous.
" homology " refers to the sequence similarity with native sequence nucleic acid." homology " includes suitable with nucleic acid of the invention
Ligand or its core sequence have 60% or higher, have 65% or higher, 70% or higher, 75% or higher, 80% or more
Height, 85% or higher 90% or higher or 95% or more high homology nucleotide sequence.Homology can with the naked eye or
Computer software is evaluated.Using computer software, the homology between two or more sequences can use percentage (%)
It indicates, can be used to evaluate the homology between correlated series.
Above-mentioned aptamer and its core sequence all belong to the scope of protection of the present invention.
It is a further object to provide a kind of derivatives of aptamer for specific recognition vimentin
Object.
It is following (1)-provided by the present invention for identifying and/or in conjunction with the derivative of the aptamer of vimentin
(5) any one:
(1) above-mentioned aptamer or its core sequence are carried out nucleotide to replace or modification, is obtained suitable with the nucleic acid
The derivative of ligand aptamer with the same function；
(2) transform the skeleton of above-mentioned aptamer or its core sequence as phosphorothioate ester skeleton, obtain with it is described
The derivative of aptamer aptamer with the same function；
(3) by one end or the upper signaling molecule of intermediate connection of above-mentioned aptamer or its core sequence and/or activity point
Son and/or functional group, obtain the derivative with aptamer aptamer with the same function；
(4) RNA molecule encoded by above-mentioned aptamer or its core sequence；
(5) peptide nucleic acid encoded by above-mentioned aptamer or its core sequence.
In said derivative, the derivative in (3) can be for mark fluorescent molecule Cy3's or biotin molecule biotin
Aptamer, such as the nucleic acid adaptation in the aptamer 5 ' end mark fluorescent molecule Cy3 or biotin molecule biotin
Body.
It is a still further object of the present invention to provide above-mentioned aptamer or the new applications of said derivative.
The present invention provides above-mentioned aptamers or said derivative in specific recognition and/or to combine in vimentin
Application.
The present invention also provides above-mentioned aptamers or said derivative in preparation specific recognition and/or to combine waveform
Application in the product of albumen.
The present invention also provides above-mentioned aptamers or said derivative to detect or assist in detection waveform albumen
Using.
The present invention also provides above-mentioned aptamers or said derivative in preparation detection or auxiliary detection waveform albumen
Product in application.
The present invention also provides above-mentioned aptamers or said derivative whether to identify or assist identification tumour cell
The application in EMT occurs.
The present invention also provides above-mentioned aptamers or said derivative in preparation identification or auxiliary identification tumour cell
Whether application in the product of EMT is occurred.
In above-mentioned application, the tumour cell is Vimentin positive tumor cell.
The present invention also provides above-mentioned aptamers or said derivative as the application in EMT diagnostic reagent.
The present invention also provides the application of above-mentioned aptamer or said derivative in induction tumor death.
The present invention also provides above-mentioned aptamers or said derivative in the product for preparing inducing apoptosis of tumour cell
In application.
The present invention also provides the application of above-mentioned aptamer or said derivative in the drug of exploitation oncotherapy.
In above-mentioned application, the tumour can be common tumour, such as gastric cancer, cancer of pancreas, colon cancer.
It is a still further object of the present invention to provide a kind of detection methods of the vimentin of cell or tissue expression.
The detection method of the vimentin of cell or tissue provided by the invention expression include with above-mentioned aptamer or
The step of said derivative is detected.
It is described with above-mentioned aptamer or above-mentioned to spread out in the detection method of the vimentin of above-mentioned cell or tissue expression
The method that biology is detected can be conventional method in the prior art, such as immunohistochemistry (IHC), Western Blot, streaming
Experiment and Dot Blot experiment etc..
In the detection method of the vimentin of above-mentioned cell or tissue expression, the aptamer end connects biotin
Or fluorescent molecule, such as Cy3 or biotin.
Final object of the present invention is to provide a kind of method of inducing apoptosis of tumour cell.
The method of inducing apoptosis of tumour cell provided by the invention includes leading above-mentioned aptamer or said derivative
Enter the step in tumour cell.
In the method for above-mentioned inducing apoptosis of tumour cell, it is thin that the aptamer imports tumour after transfection reagent wraps up
In born of the same parents.
Further, final concentration of 1-2 μM in tumour cell system of the aptamer, such as 1 μM, 1.5 μM or 2
μM。
Further, the transfection reagent can be common transfection reagent, such as Turbofect.
In the method for above-mentioned inducing apoptosis of tumour cell, the tumour cell can be common tumour cell, such as colon cancer
Cell, pancreatic cancer cell, stomach cancer cell etc..The pancreatic cancer cell concretely Panc-1 cell or BxPC-3 cell.
Using Vimentin as target molecule, obtained using SELEX technology screening specific recognition and can be combined the present invention
Aptamer AptVim, the Kd value of Vimentin can reach 70.9 ± 12.3nM.Aptamer AptVim of the invention
The identification that can be used for the special target spot Vimentin of EMT, compared to traditional Vimentin antibody, aptamer AptVim is not only steady
Qualitative higher, cost is cheaper, and the reaction condition needed for it is simpler, as incubation time is shorter, reaction temperature more
Close to room temperature, cell can be completed by being usually only necessary to a step after the end aptamer AptVim connects biotin or fluorescent molecule
Expression or the detection of tissue expression Vimentin albumen.In addition, aptamer AptVim of the invention is through suitable transfection examination
After agent (such as Turbofect) imports tumour cell, it can induce tumour cell and apoptosis occur.Therefore aptamer of the invention
AptVim can be used as the EMT diagnostic reagent of infrastest and clinical cancer, also can induce the apoptosis of tumour cell for swelling
Tumor treatment.Further research based on aptamer AptVim of the invention is expected to generate new targeted drug or control with improving
Treatment method provides new tool for treatment of cancer.
Detailed description of the invention
Fig. 1 is the streaming experimental result picture that SELEX screens amplifying nucleic acid aptamers enrichment condition.Scheming a is the 4th, 7,8 wheel screenings
The combination situation of obtained ssDNA product, random library and Vimentin albumen coupling agarose nickel bead；Scheme b (negative control
Group) it is ssDNA product, random library and the combination situation without coupling agarose nickel bead that the 4th, 7,8 wheel screenings obtain.
Fig. 2 is the Dot blot experimental result picture that SELEX screens amplifying nucleic acid aptamers enrichment condition.Random library is feminine gender
Control.
Fig. 3 is the secondary structure prediction figure of aptamer AptVim.
Fig. 4 is the streaming experiment of the combination situation of aptamer AptVim and Vimentin albumen coupling agarose nickel bead
Result figure.Figure a is the combination situation of aptamer AptVim, random library and Vimentin albumen coupling agarose nickel bead；
Scheming b (negative control) is the combination feelings that aptamer AptVim, random library and 6X histidine polypeptide are coupled agarose nickel bead
Condition.
Fig. 5 is the Dot blot experimental result picture of aptamer AptVim and Vimentin protein binding situation.At random
Library is negative control.
Fig. 6 is the combination of the agarose nickel bead of aptamer AptVim and the Vimentin albumen coupling under various concentration
Situation.Negative control is the agarose nickel bead of 6X histidine polypeptide coupling.
Fig. 7 is aptamer AptVim, Vimentin antibody respectively to the Western of Vimentin Protein Detection
Blot result.
Fig. 8 be aptamer AptVim, Vimentin antibody respectively with liver cancer tissue Vimentin is protein bound exempts from
Epidemic disease group result.
Fig. 9 is the apoptosis rate statistics that different experiments group induces Vimentin positive cell.Vimentin positive cell strain
Panc-1 and BxPC-3 carry out different experiments processing respectively, experimental setup include control group (NC), Turbofect processing group,
Turbofect+ random library processing group, AptVim processing group, Turbofect+AptVim processing group.The longitudinal axis is the early stage of cell
Apoptosis rate (Annexin V+/PI-Cell proportion).
Specific embodiment
Embodiment below facilitates a better understanding of the present invention, but does not limit the present invention.Experiment in following embodiments
Method is unless otherwise specified conventional method.Test material as used in the following examples is unless otherwise specified certainly
What routine biochemistry reagent shop was commercially available.Quantitative test in following embodiment is respectively provided with three repeated experiments, as a result makes even
Mean value.
Experimental material source as used in the following examples is as follows: recombined human Vimentin albumen (6X histidine tag)
Purchased from Divine Land Yi Qiao, Beijing company；NC film, Ni-Sepharose Beads, NAP-5 desalting column are purchased from GE Lifesciences；
6X histidine polypeptide (purity > 95%) is purchased from Chinese Peptide Co., Ltd.；DPBS (contains Ca2+And Mg2+)、Streptavidin
Agarose Beads, tRNA, Turbofect transfection reagent, non-enzymatic digestive juice are purchased from Thermofisher Scientific；
BSA (no protease) is purchased from Sigma-Aldrich；RIPA lysate, BCA protein quantification kit, Tween-20, DEPC water,
ECL luminescence reagent box, immunohistochemistry related reagent are purchased from green skies biotech company；Sodium hydroxide, agarose are purchased from Shanghai
Sheng Gong bioengineering Co., Ltd；HS Taq PCR kit is purchased from TAKARA；Human Vimentin Biotinylated
Antibody、Humanβ-actin Antibody、HRP-conjugated Anti-mouse IgG Secondary
Antibody, TACS Annexin V-FITC apoptosis kit, Streptavidin-HRP are purchased from R&D systems；Cancer of pancreas
Cell strain Panc-1 (Vimentin+, number SCSP-535) and BxPC-3 (Vimentin-, number is TCHu 12) and it is purchased from
Institute of section cell bank uses DMEM/10%FBS culture medium culture.
SELEX initial libraries sequence involved in following embodiments: 5 '-ATCCAGAGTGACGCAGCA-N(40)-TGGACACGGTGGCTTAGT- 3 ', upstream primer sequence: 5 '-ATCCAGAGTGACGCAGCA, downstream primer sequence: 5 '-
ACTAAGCCACCGTGTCCA, special nucleic acid aptamer sequence, primer sequence and its 5 ' end Cy3 or biotin modifications are by upper
Hai Shenggong biosynthesis is simultaneously purified through HPLC.
The screening of embodiment 1, aptamer
One, the design and synthesis of random library
Designing and synthesizing both ends includes 18 fixed nucleotides, the intermediate random library including 40 nucleotide: 5 '-ATCCAGAGTGACGCAGCA-N(40)-TGGACACGGTGGCTTAGT-3'；N (40) represents the stochastic kernel of 40 A, T, C or G
Nucleotide sequence.
Two, SELEX is screened
SELEX screening process is divided into positive screening and negative sense screens two parts.Specific step is as follows:
1, positive screening
By 10 μ g recombined human Vimentin albumen (6X histidine tag) and appropriate agarose nickel bead (Ni-Sepharose
Beads it) is incubated at room temperature 30 minutes, is coupled to it on agarose nickel bead, and Binding Buffer I (1%BSA, 0.1%
Tween-20,0.2mg/mL tRNA, DPBS, pH7.4) spare, the unbonded object of washing removing is resuspended, as forward direction screening
Target.By 95 DEG C of random library in 10OD (about 14nmol) step 1 denaturation, and after being slowly cooled to room temperature, above-mentioned system is added
It is incubated at room temperature 1 hour in the agarose nickel bead for the Vimentin albumen coupling got ready.DEPC water will be used after the washing of agarose nickel bead
It is resuspended, 95 DEG C are denaturalized 10 minutes, and supernatant is collected after centrifugation as template and carries out PCR amplification (95 DEG C of 30s；56.3℃30s；72℃
30s).The primer sequence of PCR amplification is as follows: upstream primer sequence: 5 '-ATCCAGAGTGACGCAGCA；Downstream primer sequence:
5'-ACTAAGCCACCGTGTCCA.PCR product is identified by 3% agarose electrophoresis, and the highest of no non-specific band is selected to expand
Increase condition of the recurring number as a large amount of preparation PCR products, PCR cycle number in every wheel screening is specifically optimized with this.
2, prepared by the library ssDNA
5 ' end Cy3 modification the libraries ssDNA the preparation method is as follows: with 5 ' end Cy3 modification upstream primer and 5 ' end
The downstream primer of biotin modification carries out PCR amplification, and the PCR product of biotinization is coupled to Streptavidin sepharose 4B
On (Streptavidin Agarose Beads), 200mM NaOH dissociation upstream is single-stranded, through NAP-5 column desalting and purifying, as
The library ssDNA of 5 ' end Cy3 modifications；5 ' end Biotin modification the libraries ssDNA the preparation method is as follows: with 5 ' end biotin repair
The upstream primer of decorations and without label downstream primer amplification, the PCR product of biotinization is coupled to Streptavidin agarose
Pearl, abandoning downstream is single-stranded after 200mM NaOH dissociation, and 50 μ L 0.1%SDS are added, and 100 DEG C are boiled 5min, takes supernatant mistake after centrifugation
NAP-5 column desalting and purifying, the library ssDNA of as 5 ' end biotin modifications.It is carried out by template of the library ssDNA of above-mentioned preparation
Next round screening, and gradually decrease Vimentin albumen, Vimentin albumen coupling agarose nickel bead dosage and ssDNA
Library concentration gradually shortens screening time.8 wheels are repeated in positive screening process.
3, negative sense screening process
For the enrichment for reducing non-specific nucleic acid sequence in screening library, negative sense screening is introduced while forward direction screening.It is negative
It is essentially identical to screening and positive screening process, the difference is that using the agarose nickel bead that is coupled 6X histidine polypeptide as sieving
Target is selected, library is the library ssDNA of last round of positive screening preparation, and the library ssDNA is gradually decreased in screening process
Concentration increases the dosage for being coupled the agarose nickel bead of 6X histidine polypeptide, and extends incubation time.Negative sense is screened in the 4th, 7,8
It is carried out after the positive screening of wheel.
Three, streaming experiment detects each wheel with Dot Blot experiment and screens library enrichment degree
Streaming experiment is respectively adopted and detects each wheel screening library enrichment degree with Dot Blot experiment.Specific step is as follows:
1, streaming is tested
Agarose nickel bead and 1 μ g Vimentin albumen after taking 1 μ L washing to be resuspended are incubated at room temperature 30 minutes, and washing removes
Unbonded object.After 95 DEG C of the library ssDNA (or aptamer) denaturation of Cy3 label is cooled to room temperature, above-mentioned preparation is added
In the agarose nickel bead of good Vimentin albumen coupling (final concentration 200nM), it is incubated at room temperature 1 hour.Streaming is used after washing
Cell instrument (BD bioscience, FACSCalibur) detects the enrichment degree of the library ssDNA (or aptamer).
2, Dot Blot is tested
Will on Vimentin albumen gradation point sample to NC film, after drying at room temperature with Binding Buffer II (3%BSA,
0.1%Tween-20,0.2mg/mL tRNA, DPBS, pH7.4) closing.After the denaturation cooling of 95 DEG C of the library ssDNA, use
Binding Buffer I adjusts final concentration of 200nM, is incubated at room temperature 1 hour with the NC film closed.By Streptavidin-
HRP and NC film react at room temperature 45 minutes, and ECL exposes after washing.
The streaming that SELEX screens the enrichment of amplifying nucleic acid aptamers is tested with Dot Blot experimental result picture respectively such as Fig. 1 and Fig. 2
It is shown.The result of streaming experiment and Dot Blot experiment shows that the 7th has taken turns the library ssDNA obtained with the positive screening of the 8th wheel
Obvious enrichment, and each library is not reacted with the agarose nickel bead of 6X histidine polypeptide coupling.
Four, high-flux sequence obtains aptamer AptVim
By the upstream and downstream primer amplification of the library the ssDNA not tape label of the positive screening enrichment of the 8th wheel, (PCR condition is same
On), PCR product serves the raw work biology in sea and carries out high-flux sequence (NGS), and analyzes the rich of each nucleic acid aptamer sequence in library
Degree, obtains the nucleic acid aptamer sequence of a high abundance (abundance 12.4%), complete sequence is as follows: 5 '-ATCCAGAGTGAC
GCAGCACGGGGGTTTAGGCAGACACCTGACGCGTTCTGGAAAGTGCTGGACACGGTGGCTTAGT-3’(SEQ ID
No.1), which is named as AptVim, wherein (lower stroke of sequence shown in SEQ ID No.1 the 19th~58
The sequence of line part) be aptamer AptVim core region sequence.
Five, the secondary structure analysis of aptamer AptVim
With the secondary structure of Mfold software prediction aptamer AptVim, pre- geodesic structure is as shown in Figure 3.
The binding ability of embodiment 2, aptamer AptVim and Vimentin albumen
One, the combination situation of streaming experiment and Dot Blot experiment detection aptamer AptVim and Vimentin albumen
5 ' end Cy3 modification AptVim sequences and 5 ' end biotin modifications are respectively synthesized according to the method in embodiment 1
The combination situation that streaming experiment detects itself and Vimentin albumen with Dot blot experimental method is respectively adopted in AptVim sequence.
Specific detection method is the same as the method in 1 step 3 of embodiment.
Flow cytometer detection result is distinguished as shown in Figure 4 and Figure 5 with Dot Blot testing result.The results show that aptamer
AptVim is obviously combined with Vimentin albumen, without in conjunction with 6X histidine polypeptide.
Two, the affinity determination of aptamer AptVim
For the affinity for measuring aptamer AptVim and Vimentin albumen, 5 ' end Cy3 are modified into AptVim most
Whole working concentration is adjusted to 400nM, 200nM, 100nM, 50nM, 25nM, 12.5nM, 6.25nM, 3.125nM, uses fluidic cell
Instrument (method is the same) measure its under various concentration with the protein bound mean fluorescent intensity of Vimentin (MFI), and with formula Y
=Bmax X/ (Kd+X) calculates affinity (Kds).
The protein bound streaming result of AptVim and Vimentin under various concentration is as shown in fig. 6, be computed, nucleic acid adaptation
Body AptVim and the protein bound affinity of Vimentin are 70.9 ± 12.3nM.
Three, Western Blot detects the combination of AptVim and cell Vimentin albumen
Pancreatic cancer cell Panc-1 (Vimentin+) and BxPC-3 (Vimentin-) after RIPA is cracked, supernatant passes through
BCA method protein quantification takes 40 μ g total protein of cell after 12%SDS-PAGE is separated by electrophoresis, is transferred to NC film, Binding
Buffer II is closed 1 hour, spare after washing.
AptVim detection group: AptVim concentration to 200nM and NC film is adjusted with Binding Buffer I and is incubated for.
Vimentin antibody test group (positive control): the Vimentin antibody of biotinization is used without tRNA
Binding Buffer I is diluted by 1:2000, is incubated for NC film.NC film is then incubated for 45 points with Streptavidin-HRP
Clock, ECL exposes after washing.
Aptamer AptVim, Vimentin antibody are respectively to the Western Blot result of Vimentin Protein Detection
As shown in Figure 7.The results show that aptamer AptVim is identical as the detection effect of Vimentin antibody, can specifically with
Vimentin protein hybridization in Panc-1 cell, and develop the color in the BxPC-3 cell for not expressing Vimentin without band.
Four, the combination of immunohistochemistry (IHC) detection AptVim and cancerous tissue Vimentin albumen
The specific hepatocellular carcinoma paraffin-embedded tissue slice of pathological diagnosis is spare after dimethylbenzene dewaxes aquation.
AptVim dyeing group: histotomy is received (pH 6.0) solution through 10mM citric acid and is acted on 30 minutes under the conditions of 37 DEG C
Antigen retrieval is carried out, through 3%H after washing2O2Room temperature acts on 20 minutes closing endogenous peroxydases, 0.1% Streptavidin
(Streptavidin) room temperature acts on 30 minutes closing endogenous biotin.Histotomy and AptVim (final concentration 200nM) exist
It reacts after twenty minutes, is incubated at room temperature 20 minutes with Streptavidin-HRP (1:800 dilution) at room temperature, DAB colour developing.
Vimentin antibody dyeing group: its dyeing flow and AptVim dyeing group are essentially identical, the difference is that antigen is repaired
Multiple to carry out under the conditions of 95 DEG C, Vimentin antibody (Human Vimentin Biotinylated Antibody) is cut with tissue
The incubation time of piece is 90 minutes.
Aptamer AptVim and Vimentin antibody respectively with the protein bound immune group of liver cancer tissue Vimentin
It is as shown in Figure 8 to change result.It, equally can be with the results show that although the dyeing condition of aptamer AptVim is more simple
Specific stain clearly is carried out to the Vimentin albumen in liver cancer tissue, reaches and dyes same effect with Vimentin antibody
Fruit.
Apoptosis occurs for embodiment 3, aptamer AptVim induction Vimentin positive cell
Panc-1 cell is inoculated in 6 orifice plates respectively with BxPC-3 cell and is incubated overnight, according to the difference of reagent treatment
It is divided into following each group:
AptVim+Turbofect processing group: aptamer AptVim (adjusts aptamer with serum-free DMEM
AptVim concentration is to 1 μM) after Turbofect is wrapped up, it handles cell 24 hours；
Turbofect processing group: Turbofect is handled cell 24 hours；
Turbofect+ random library processing group: random library (serum-free DMEM adjusts random library concentration to 1 μM) warp
After Turbofect package, handle cell 24 hours；
AptVim processing group: wrapping up without Turbofect, directly handled with aptamer AptVim cell 24 hours,
Final concentration of 1 μM of aptamer AptVim.
NC control group: unused aptamer AptVim and Turbofect processing.
Each experimental group through non-enzymatic digestive juice dissociated cell, then by cell after the processing of each processing group respectively with FITC-
Annexin V antibody (1:100) and PI dyestuff (1:10) are incubated at room temperature 30 minutes, then through flow cytometer after being washed with DPBS
(FACS Calibur, BD Biosciences) detects Annexin V+/PI-Cell proportion obtains the Annexin of each experimental group
V+/PI-Early stage apoptosis rate.
Different experiments group induces the early apoptosis rate statistical result of Vimentin positive cell as shown in Figure 9.The results show that
Aptamer AptVim can effectively induce Vimentin positive expression cell Panc-1 to occur bright after Turbofect is wrapped up
Aobvious apoptosis (apoptosis rate 18.4%, P < 0.01 * *), and other processing groups and Vimentin feminine gender expression cell BxPC-3 are not
Obvious apoptosis occurs.
Sequence table
<110>The First Affiliated Hospital of Wenzhou Medical University
<120>aptamer of specific recognition Vimentin and its application
<160>1
<170>PatentIn version 3.5
<210>1
<211>76
<212>DNA
<213>artificial sequence (Artificial Sequence)
<400>1
atccagagtg acgcagcacg ggggtttagg cagacacctg acgcgttctg gaaagtgctg 60
gacacggtgg cttagt 76
Claims (10)
1. a kind of aptamer for specific recognition vimentin is any in following a1)-a3):
A1) single strand dna shown in SEQ ID No.1 19-58；
A2) and single strand dna shown in SEQ ID No.1 19-58 has 60% or 60% or more homology, and has
The single strand dna of identical function；
A3) one or several nucleotide are deleted or increased to single strand dna shown in SEQ ID No.1 19-58, and have
There is the single strand dna of identical function.
2. a kind of aptamer for specific recognition vimentin is any in following b1)-b3):
B1) single strand dna shown in SEQ ID No.1；
B2) there is with single strand dna shown in SEQ ID No.1 60% or 60% or more homology, and there is identical function
Single strand dna；
B3) one or several nucleotide are deleted or increased to single strand dna shown in SEQ ID No.1, and there is identical function
The single strand dna of energy.
3. a kind of derivative of the aptamer for specific recognition vimentin, for following (1)-(5) any one:
(1) by aptamer described in claim 1 or aptamer as claimed in claim 2 carry out nucleotide replace or
Modification, and the derivative with aptamer aptamer with the same function；
(2) skeleton of aptamer described in claim 1 or aptamer as claimed in claim 2 is transform as thio
Phosphate ester skeleton, and the derivative with aptamer aptamer with the same function；
(3) by one end or intermediate connection of aptamer described in claim 1 or aptamer as claimed in claim 2
Upper signaling molecule and/or bioactive molecule and/or functional group, and be adapted to aptamer nucleic acid with the same function
The derivative of body；
(4) RNA molecule encoded by aptamer described in claim 1 or aptamer as claimed in claim 2；
(5) peptide nucleic acid encoded by aptamer described in claim 1 or aptamer as claimed in claim 2.
4. aptamer described in claim 1 or aptamer as claimed in claim 2 or core as claimed in claim 3
The derivative of sour aptamers identifies and/or combines the application in vimentin in specific recognition；
Or, aptamer described in claim 1 or aptamer as claimed in claim 2 or as claimed in claim 3
Application of the derivative of aptamer in the product of preparation specific recognition and/or combination vimentin.
5. aptamer described in claim 1 or aptamer as claimed in claim 2 or core as claimed in claim 3
The derivative of sour aptamers is detecting or is assisting the application in detection waveform albumen；
Or, aptamer described in claim 1 or aptamer as claimed in claim 2 or as claimed in claim 3
Application of the derivative of aptamer in the product of preparation detection or auxiliary detection waveform albumen.
6. aptamer described in claim 1 or aptamer as claimed in claim 2 or core as claimed in claim 3
Application of the derivative of sour aptamers in induction tumor death；
Or, aptamer described in claim 1 or aptamer as claimed in claim 2 or as claimed in claim 3
Application of the derivative of aptamer in the product for preparing inducing apoptosis of tumour cell.
7. aptamer described in claim 1 or aptamer as claimed in claim 2 or core as claimed in claim 3
The derivative of sour aptamers is as the application in EMT diagnostic reagent；
Or, aptamer described in claim 1 or aptamer as claimed in claim 2 or as claimed in claim 3
The derivative of aptamer is identifying or is assisting whether identification tumour cell occurs the application in EMT；
Or, aptamer described in claim 1 or aptamer as claimed in claim 2 or as claimed in claim 3
Application of the derivative of aptamer in the product whether preparation identification or auxiliary identification tumour cell occur EMT.
8. aptamer described in claim 1 or aptamer as claimed in claim 2 or core as claimed in claim 3
Application of the derivative of sour aptamers in the drug of exploitation oncotherapy.
9. a kind of detection method of the vimentin of cell or tissue expression, including with aptamer described in claim 1
Or the derivative of aptamer as claimed in claim 2 or aptamer as claimed in claim 3 the step of being detected.
10. a kind of method of inducing apoptosis of tumour cell, including by aptamer described in claim 1 or claim 2
The derivative of the aptamer or aptamer as claimed in claim 3 imports the step in tumour cell.
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| CN113564172A (en) * | 2021-06-30 | 2021-10-29 | 优葆优保健康科技(宁波)有限公司 | Liquid biopsy tumor cell DNA aptamer |
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