CN109355294B

CN109355294B - Nucleic acid aptamer for specifically recognizing Vimentin and application thereof

Info

Publication number: CN109355294B
Application number: CN201811364334.1A
Authority: CN
Inventors: 周蒙滔; 孙红光; 吴施佳; 张�杰
Original assignee: First Affiliated Hospital of Wenzhou Medical University
Current assignee: First Affiliated Hospital of Wenzhou Medical University
Priority date: 2018-11-16
Filing date: 2018-11-16
Publication date: 2022-06-28
Anticipated expiration: 2038-11-16
Also published as: CN109355294A

Abstract

The invention discloses a nucleic acid aptamer capable of identifying Vimentin and application thereof, wherein the sequence of the nucleic acid aptamer is shown as SEQ ID No. 1. The aptamer AptVim of the invention can specifically bind to Vimentin, and the Kd value is 70.9 +/-12.3 nM. After the AptVim terminal is connected with biotin or fluorescent molecules, the conventional Vimentin antibody can be replaced, the detection of Vimentin protein expressed by cells or tissues can be completed only by one step, and the method has the advantages of simpler required reaction conditions, higher stability and lower cost. In addition, the aptamer of the invention can induce tumor cells to undergo apoptosis after being introduced into the tumor cells by a proper transfection reagent. Not only can be used as an EMT diagnostic reagent for basic experiments and clinical cancers, but also can induce the apoptosis of tumor cells for tumor treatment.

Description

Nucleic acid aptamer for specifically recognizing Vimentin and application thereof

Technical Field

The invention belongs to the technical field of biology, and particularly relates to a nucleic acid aptamer for specifically recognizing Vimentin and application thereof.

Background

Vimentin (Vimentin, Vim) is a midfilament protein that is expressed primarily in mesenchymal cells and in mesoderm-derived cells. Vimentin is highly conserved evolutionarily, involved in a variety of physiological processes: maintain the form of cell skeleton, promote cell adhesion and migration, regulate cell apoptosis, signal transduction, gene regulation, etc. Vimentin is a major secreted protein in the process of epithelial-mesenchymal transition (EMT) of tumor cells, and thus has recently become an important marker for identifying EMT occurrence. A large number of researches show that Vimentin is over-expressed in various epithelial malignant tumors, and participates in EMT and various complex credit pathways, so that the growth of the tumors is accelerated, the infiltration and the metastasis of cancer cells are promoted, and the important role is played in the development and the formation of the cancers. The expression of Vimentin protein related to precancerous lesion of tumor is reduced by DNA methylation at gene level, so that the Vimentin methylation degree can also be used as an index for diagnosis, staging, prognosis and the like of various cancers such as gastric cancer, pancreatic cancer, colon cancer and the like. The research proves that the over-expression of Vimentin can cause the enhancement of the motility and the invasiveness of cancer cells, and the characteristics are weakened after the expression of Vimentin is knocked out, thereby revealing that the Vimentin has clinical treatment value. The Visentin protein is expected to be a target for clinical prediction of cancer, and the Research on the Visentin protein also has important value for cancer treatment (Ko S H, sub S H, Kim B J, et al. expression of the interventional parameter in developing cells as a marker of a systemic therapeutic cell [ J ]. Pancreas,2004,28(2): 121-128; Satelli A, Li S. Visentin protein and its Molecular target for cancer therapy [ J ]. Cellular & Molecular Life Sciences,2011,68(18): 3033-3046; Fuyuhiro Y, Yashim M, Noda S, clinical design of Molecular tissue protein, Molecular tissue vector, No. 13-32; 5. alpha. Molecular tissue, 9. alpha. Molecular tissue, 9. expression of cancer therapy [ 12. expression of Molecular tissue, 9. gamma.: 5243. expression of cancer therapy [ 12. Molecular tissue culture [ K ], 2009,29(1):279-281).

Although the recent Vimentin monoclonal antibody has higher affinity and specificity, the antibody has some defects, such as high immunogenicity, instability, difficult chemical modification, batch-to-batch variability, limited production method, high production cost and the like, so that the Vimentin monoclonal antibody has certain limitations on basic research and clinical application.

The Aptamer (Aptamer) is prepared by using in vitro screening technology-ligand system based on exponential enrichmentA section of single-stranded DNA or RNA oligonucleotide sequence which is obtained by screening a large-capacity nucleic acid molecule library by the systematic evolution of ligands by expression engineering (SELEX) technology and binds a target substance with higher specificity and affinity. Aptamers are comparable to antibodies in terms of affinity and specificity, and have some advantages not found in antibodies: due to the physicochemical property of small molecular nucleic acid, the aptamer has low immunogenicity, rapid tissue penetration and cell internalization capacity and higher thermal/chemical stability; and the screening period of the aptamer is short, different functional groups are easy to chemically modify or couple, the synthesis technology is simple, the repeatability among batches is strong, and the like. Thus, nucleic acid aptamers have promising prospects in basic research and clinical applications (Sun H, Zu Y.A high height of Recent Advances in Aptamer Technology and Its applications. 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)。

Disclosure of Invention

An object of the present invention is to provide an aptamer for specifically recognizing Vimentin (Vimentin) and a core sequence thereof.

The core sequence of the aptamer provided by the invention is any one of a1) -a 3):

a1) a single-stranded DNA molecule as shown in SEQ ID No.1 at positions 19-58;

a2) a single-stranded DNA molecule which has homology of 60 percent or more than 60 percent with the single-stranded DNA molecule shown in the 19 th to 58 th sites of SEQ ID No.1 and has the same function;

a3) a single-stranded DNA molecule with the same function, wherein one or more nucleotides are deleted or added in the single-stranded DNA molecule shown in the 19 th to 58 th positions of SEQ ID No. 1.

The aptamer is any one of the following b1) -b 3):

b1) a single-stranded DNA molecule shown as SEQ ID No. 1;

b2) single-stranded DNA molecules which have homology of 60 percent or more than 60 percent with the single-stranded DNA molecules shown in SEQ ID No.1 and have the same functions;

b3) a single-stranded DNA molecule with the same function, wherein one or more nucleotides are deleted or added in the single-stranded DNA molecule shown in SEQ ID No. 1.

The "homology" refers to sequence similarity to a native nucleic acid sequence. "homology" includes a nucleotide sequence having 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, or 90% or more, or 95% or more homology to the aptamer of the present invention or a core sequence thereof. Homology can be assessed visually or by computer software. Using computer software, homology between two or more sequences can be expressed as a percentage (%), which can be used to assess homology between related sequences.

The aptamer and the core sequence thereof belong to the protection scope of the invention.

It is another object of the present invention to provide a derivative of an aptamer for specifically recognizing vimentin.

The derivative of the aptamer for identifying and/or binding vimentin provided by the invention is any one of the following (1) to (5):

(1) carrying out nucleotide substitution or modification on the aptamer or a core sequence thereof to obtain a derivative of the aptamer with the same function as the aptamer;

(2) the skeleton of the aptamer or the core sequence of the aptamer is transformed into a phosphorothioate skeleton, so that a derivative of the aptamer with the same function as the aptamer is obtained;

(3) connecting a signal molecule and/or an active molecule and/or a functional group to one end or the middle of the aptamer or the core sequence thereof to obtain a derivative of the aptamer with the same function as the aptamer;

(4) an RNA molecule encoded by the aptamer or a core sequence thereof;

(5) a peptide nucleic acid encoded by the above-mentioned nucleic acid aptamer or a core sequence thereof.

Among the above derivatives, the derivative in (3) can be an aptamer labeled with a fluorescent molecule Cy3 or biotin molecule biotin, such as an aptamer labeled with a fluorescent molecule Cy3 or biotin molecule biotin at the 5' end of the aptamer.

It is still another object of the present invention to provide a novel use of the above aptamer or the above derivative.

The invention provides the application of the aptamer or the derivative in specific recognition and/or binding of vimentin.

The invention also provides the application of the aptamer or the derivative in preparing products for specifically recognizing and/or binding vimentin.

The invention also provides the application of the aptamer or the derivative in detection or auxiliary detection of vimentin.

The invention also provides application of the aptamer or the derivative in preparation of products for detecting or assisting in detecting vimentin.

The invention also provides the application of the aptamer or the derivative in identifying or assisting in identifying whether the tumor cells have EMT.

The invention also provides the application of the aptamer or the derivative in preparing products for identifying or assisting in identifying whether tumor cells generate EMT.

In the application, the tumor cell is a Vimentin positive tumor cell.

The invention also provides the application of the aptamer or the derivative as an EMT diagnostic reagent.

The invention also provides the application of the aptamer or the derivative in inducing tumor apoptosis.

The invention also provides the application of the aptamer or the derivative in preparing a product for inducing tumor cell apoptosis.

The invention also provides the application of the aptamer or the derivative in the development of drugs for treating tumors.

In the above application, the tumor may be common tumor, such as gastric cancer, pancreatic cancer, colon cancer, etc.

It is still another object of the present invention to provide a method for detecting vimentin expressed by cells or tissues.

The method for detecting vimentin expressed by cells or tissues according to the present invention comprises a step of detecting the vimentin with the aptamer or the derivative.

In the method for detecting vimentin expressed by the above-mentioned cells or tissues, the method for detecting the vimentin using the above-mentioned aptamer or the above-mentioned derivative may be a conventional method in the prior art, such as Immunohistochemistry (IHC), Western Blot, flow assay, Dot Blot assay, and the like.

In the method for detecting vimentin expressed by cells or tissues, biotin or a fluorescent molecule, such as Cy3 or biotin, is linked to the end of the aptamer.

It is a final object of the present invention to provide a method of inducing apoptosis in tumor cells.

The method for inducing apoptosis of a tumor cell according to the present invention comprises a step of introducing the aptamer or the derivative into a tumor cell.

In the above method for inducing apoptosis of tumor cells, the aptamer is encapsulated by a transfection reagent and then introduced into the tumor cells.

Further, the aptamer may be present in the tumor cell system at a final concentration of 1-2. mu.M, such as 1. mu.M, 1.5. mu.M or 2. mu.M.

Further, the transfection reagent may be a common transfection reagent, such as Turbofect.

In the above method for inducing apoptosis of tumor cells, the tumor cells may be common tumor cells, such as colon cancer cells, pancreatic cancer cells, gastric cancer cells, and the like. The pancreatic cancer cells can be specifically Panc-1 cells or BxPC-3 cells.

The invention takes Vimentin as a target molecule, and obtains the aptamer AptVim which can specifically identify and combine with the Vimentin by utilizing SELEX technology screening, wherein the Kd value of the aptamer AptVim can reach 70.9 +/-12.3 nM. The aptamer AptVim can be used for identifying an EMT specific target point Vimentin, compared with a traditional Vimentin antibody, the aptamer AptVim has higher stability and lower cost, the required reaction conditions are simpler, such as shorter incubation time and reaction temperature closer to room temperature, and the detection of cell expression or tissue expression Vimentin protein can be usually completed only by one step after the tail end of the aptamer AptVim is connected with biotin or fluorescent molecules. In addition, the aptamer AptVim of the invention can induce tumor cells to undergo apoptosis after being introduced into the tumor cells by a suitable transfection reagent (such as Turbofect). Therefore, the aptamer AptVim can be used as an EMT diagnostic reagent for basic experiments and clinical cancers and can also be used for inducing apoptosis of tumor cells for tumor treatment. Further research and improvement based on the aptamer AptVim of the invention are expected to generate new targeted drugs or treatment methods, and provide new tools for cancer treatment.

Drawings

FIG. 1 is a diagram showing the results of a flow experiment of the enrichment of aptamers in SELEX screening. FIG. a shows the binding of ssDNA products, random libraries and Vimentin protein-coupled agarose nickel beads from rounds 4, 7 and 8; panel b (negative control) shows the binding of ssDNA products, random libraries and uncoupled agarose nickel beads from round 4, 7 and 8 screens.

FIG. 2 is a graph showing the Dot blot experimental results of the aptamer enrichment in SELEX screening. Random libraries were negative controls.

FIG. 3 is a diagram showing the secondary structure prediction of the aptamer AptVim.

FIG. 4 is a diagram showing the results of flow experiments on the binding of aptamer AptVim to Vimentin protein-coupled agarose nickel beads. FIG. a shows the binding of the aptamer AptVim, a random library to Vimentin protein-coupled agarose nickel beads; panel b (negative control) shows the binding of aptamer AptVim, a random library to 6 Xhistidine polypeptide-conjugated agarose nickel beads.

FIG. 5 is a graph showing the result of Dot blot experiment on the binding condition of aptamer AptVim and Vimentin protein. Random libraries were negative controls.

FIG. 6 shows the binding of the aptamer AptVim to Vimentin protein-coupled agarose nickel beads at different concentrations. The negative control was 6 × histidine polypeptide-conjugated agarose nickel beads.

FIG. 7 shows the Western Blot results of the detection of the Visentin protein by the aptamer AptVim and the Visentin antibody, respectively.

FIG. 8 shows the immunohistochemical results of the binding of the aptamer AptVim and Vimentin antibodies to the Vimentin protein of liver cancer tissue.

FIG. 9 is a statistic of apoptosis rate induced by Vimentin positive cells in different experimental groups. The Vimentin positive cell strains Panc-1 and BxPC-3 are respectively subjected to different experimental treatments, and the experimental settings comprise a control group (NC), a Turbofect treatment group, a Turbofect + random library treatment group, an AptVim treatment group, and a Turbofect + AptVim treatment group. The longitudinal axis represents the early apoptosis rate of the cell (Annexin V)⁺/PI^-Cell fraction).

Detailed Description

The following examples are given to facilitate a better understanding of the invention, but do not limit the invention. The experimental procedures in the following examples are conventional unless otherwise specified. The test materials used in the following examples were purchased from a conventional biochemical reagent store unless otherwise specified. The quantitative tests in the following examples, all set up three replicates and the results averaged.

The sources of experimental materials used in the following examples are as follows: recombinant human Vimentin protein (6X histidine tag) was purchased from beijing yinqiao china; NC membranes, Ni-Sepharose Beads, NAP-5 desalting columns from GE Lifesciences; 6X histidine polypeptide (purity) >95%) from medium peptide biochemistry, ltd; DPBS (containing Ca)²⁺And Mg²⁺) Streptavidin Agarose Beads, tRNA, Turbofect transfection reagent, non-enzymatic digest from Thermofisoher Scientific; BSA (protease free) was purchased from Sigma-Aldrich; RIPA lysate, BCA protein quantitative kit, Tween-20, DEPC water, ECL luminescence kit and immunohistochemical related reagents are purchased from Biyuntian biotechnology company; sodium hydroxide and agarose were purchased from Shanghai Biotechnology engineering, Inc.; HS Taq PCR kit was purchased from TAKARA; human Vimentin BiotinylatedAntibodies, Human beta-actin antibodies, HRP-conjugated Anti-mouse IgG Secondary antibodies, TACS Annexin V-FITC apoptosis kit, Streptavidin-HRP from R&D systems; pancreatic cancer cell strain Panc-1 (Vimentin)⁺Number SCSP-535) and BxPC-3 (Vimentin)^-Accession number TCHu 12) were purchased from the cell bank of the department of chinese academy and cultured using DMEM/10% FBS medium.

SELEX initial library sequences referred to in the following examples: 5' -ATCCAGAGTGACGCAGCA-N(40)-TGGACACGGTGGCTTAGT-3', upstream primer sequence: 5' -ATCCAGAGTGACGCAGCA, downstream primer sequence: 5 '-ACTAAGCCACCGTGTCCA, the specific aptamer sequence, the primer sequence and its 5' Cy3 or biotin modification were all biosynthesized and purified by HPLC.

Example 1 screening of aptamers

First, design and Synthesis of random libraries

A random library comprising 18 fixed nucleotides at both ends and 40 nucleotides in the middle was designed and synthesized: 5' -ATCCAGAGTGACGCAGCA-N(40)-TGGACACGGTGGCTTAGT-3'; n (40) represents 40 random nucleotide sequences of A, T, C or G.

Second, SELEX screening

The SELEX screening process is divided into two parts, positive screening and negative screening. The method comprises the following specific steps:

1. forward screening

Mu.g of recombinant human Vimentin protein (6X histidine tag) was incubated with an appropriate amount of agarose nickel Beads (Ni-Sepharose Beads) at room temperature for 30 minutes to couple it to the agarose nickel Beads, and Binding Buffer I (1% BSA, 0.1% Tween-20, 0.2mg/mL tRNA, DPBS, pH7.4) was resuspended for use and washed to remove unbound material, which was used as the forward screening target. 10OD (about 14nmol) step one random library at 95 ℃ denaturation, and slow cooling to room temperature, adding the prepared Vimentin protein coupled agarose nickel beads at room temperature for 1 hours. Agarose nickel beads were washed, resuspended in DEPC water, denatured at 95 ℃ for 10 minutes, centrifuged and the supernatant collected as template for PCR amplification (95 ℃ 30 s; 56.3 ℃ 30 s; 72 ℃ 30 s). The primer sequences for PCR amplification were as follows: the sequence of the upstream primer is as follows: 5' -ATCCAGAGTGACGCAGCA; the sequence of the downstream primer is as follows: 5' -ACTAAGCCACCGTGTCCA. PCR products were identified by 3% agarose electrophoresis, and the highest number of amplification cycles without non-specific bands was selected as a condition for preparing PCR products in large quantities, thereby specifically optimizing the number of PCR cycles in each round of screening.

2. ssDNA library preparation

The preparation method of the 5' Cy3 modified ssDNA library is as follows: performing PCR amplification by using an upstream primer modified by Cy3 at the 5 ' end and a downstream primer modified by biotin at the 5 ' end, coupling a biotinylated PCR product to Streptavidin Agarose Beads (Streptavidin Agarose Beads), dissociating an upstream single chain by 200mM NaOH, and desalting and purifying by using an NAP-5 column to obtain a ssDNA library modified by Cy3 at the 5 ' end; the preparation method of the 5' Biotin-modified ssDNA library is as follows: amplifying by using an upstream primer modified by 5 'end biotin and a non-tag downstream primer, coupling a biotin-treated PCR product to streptavidin agarose beads, dissociating by 200mM NaOH, discarding a downstream single chain, adding 50 muL 0.1% SDS, boiling for 5min at 100 ℃, taking a supernatant after centrifugation, and desalting and purifying by using a NAP-5 column to obtain a 5' end biotin-modified ssDNA library. The ssDNA library prepared above is used as a template to carry out the next round of screening, and the usage amount of Vimentin protein, Vimentin protein coupled agarose nickel beads and the concentration of the ssDNA library are gradually reduced, so that the screening time is gradually shortened. The forward screening process was repeated for 8 rounds.

3. Negative selection process

In order to reduce the enrichment of non-specific nucleic acid sequences in the screened library, negative screening is introduced at the same time of positive screening. The negative screening process and the positive screening process are basically the same, except that the agarose nickel beads coupled with the 6X histidine polypeptide are used as a screening target, the library is the ssDNA library prepared by the previous round of positive screening, the concentration of the ssDNA library is gradually reduced, the using amount of the agarose nickel beads coupled with the 6X histidine polypeptide is increased, and the incubation time is prolonged. Negative selection was performed after 4, 7, and 8 rounds of positive selection.

Thirdly, detecting the enrichment degree of the library screened in each round by a flow type experiment and a Dot Blot experiment

And detecting the enrichment degree of the screened library by adopting a flow test and a Dot Blot test respectively. The method comprises the following specific steps:

1. flow assay

mu.L of washed resuspended Sepharose nickel beads was incubated with 1. mu.g Vimentin protein for 30 minutes at room temperature and washed to remove unbound material. The Cy 3-labeled ssDNA library (or aptamer) was denatured at 95 ℃ and cooled to room temperature, then added to the prepared Vimentin protein-coupled agarose nickel beads (final concentration 200nM) and incubated at room temperature for 1 hour. After washing, the enrichment of ssDNA libraries (or aptamers) was examined using a flow cytometer (BD bioscience, FACSCalibur).

2. Dot Blot experiment

Vimentin protein was spotted onto NC membranes in portions, dried at room temperature and then blocked with Binding Buffer II (3% BSA, 0.1% Tween-20, 0.2mg/mL tRNA, DPBS, pH 7.4). After cooling the ssDNA library at 95 ℃ for denaturation, the final concentration was adjusted to 200nM using Binding Buffer I and incubated with the blocked NC membrane for 1 hour at room temperature. Streptavidin-HRP was reacted with NC membrane for 45 min at room temperature, washed and ECL exposed.

The flow test and Dot Blot test results of nucleic acid aptamer enrichment in SELEX screening are shown in FIGS. 1 and 2, respectively. The results of both the flow and Dot Blot experiments showed that ssDNA libraries obtained from the forward screening of rounds 7 and 8 were significantly enriched and that none of the libraries reacted with agarose nickel beads coupled with 6X histidine polypeptides.

Fourthly, obtaining the aptamer AptVim by high-throughput sequencing

Amplifying the ssDNA library enriched by the 8 th round forward screening by using the upstream and downstream primers without the tags (the PCR conditions are the same as above), sending the PCR product to the Shanghai organism for high-throughput sequencing (NGS), and analyzing the abundance of each aptamer sequence in the library to obtain a high-abundance (12.4 percent of abundance) aptamer sequence, wherein the complete sequence is as follows: 5' -ATCCAGAGTGACGCAGCACGGGGGTTTAGGCAGACACCTGACGCGTTCTGGAAAGTGCTGGACACGGTGGCTTAGT-3' (SEQ ID No.1), and the aptamer sequence is named AptVim, wherein the sequence shown in the 19 th to 58 th positions of the SEQ ID No.1 (the underlined sequence) is the core region sequence of the aptamer AptVim.

Fifth, analysis of the Secondary Structure of the aptamer AptVim

The secondary structure of the aptamer AptVim was predicted by using the MFold software, and the predicted structure is shown in FIG. 3.

Example 2 binding Capacity of the aptamer AptVim to Vimentin protein

First, flow-type experiment and Dot Blot experiment are used for detecting the combination condition of aptamer AptVim and Vimentin protein

Respectively synthesizing a 5 'end Cy3 modified AptVim sequence and a 5' end biotin modified AptVim sequence according to the method in the embodiment 1, and respectively detecting the combination condition of the sequences and the Vimentin protein by adopting a flow type experiment and a Dot blot experiment method. The specific detection method is the same as that in step three of example 1.

The streaming detection result and the Dot Blot detection result are shown in fig. 4 and 5, respectively. The results show that the aptamer AptVim binds significantly to the Vimentin protein, but not to the 6X histidine polypeptide.

Second, affinity determination of aptamer AptVim

To determine the affinity of the aptamer AptVim for Vimentin protein, the final working concentrations of 5' Cy3 modified AptVim were adjusted to 400nM, 200nM, 100nM, 50nM, 25nM, 12.5nM, 6.25nM, and 3.125nM, the mean fluorescence densities (MFI) of the modified aptvimentin protein at different concentrations were determined by flow cytometry (as before), and the affinity (Kds) was calculated using the formula Y Bmax/(Kd + X).

The flow results of AptVim binding to Vimentin proteins at different concentrations are shown in FIG. 6, and the calculated affinity of the aptamer AptVim binding to Vimentin proteins is 70.9 + -12.3 nM.

Third, Western Blot detects the combination of AptVim and cell Vimentin protein

Pancreatic cancer cell Panc-1 (Vimentin)⁺) And BxPC-3 (Vimentin)^-) After RIPA lysis, the supernatant was subjected to protein quantification by BCA method, 40. mu.g of total cellular protein was separated by 12% SDS-PAGE electrophoresis, transferred to NC membrane, blocked by Binding Buffer II for 1 hour, and washed for future use.

AptVim test panel: AptVim concentration was adjusted to 200nM with Binding Buffer I and incubated with NC membrane.

Vimentin antibody detection group (positive control): biotin-modified Vimentin antibody was diluted 1:2000 with tRNA-free Binding Buffer I and incubated with NC membrane. The NC membrane was then incubated with Streptavidin-HRP for 45 minutes, washed and ECL exposed.

Western Blot results of aptamer AptVim and Vimentin antibody on detection of Vimentin protein are shown in FIG. 7. The result shows that the aptamer AptVim has the same detection effect with the Vimentin antibody, can specifically hybridize with the Vimentin protein in Panc-1 cells, and has no band color in BxPC-3 cells which do not express Vimentin.

Fourthly, detection of AptVim binding to cancer tissue Vimentin protein by Immunohistochemistry (IHC)

The paraffin embedded tissue section of hepatocellular carcinoma with definite pathological diagnosis is deparaffinized and hydrated by dimethylbenzene for standby.

AptVim staining group: the tissue section is treated with 10mM sodium citrate (pH 6.0) solution at 37 deg.C for 30 min for antigen retrieval, washed and then treated with 3% H₂O₂Endogenous peroxidase was blocked at room temperature for 20 minutes, and endogenous biotin was blocked at 0.1% Streptavidin (Streptavidin) at room temperature for 30 minutes. After the tissue sections were reacted with AptVim (final concentration 200nM) at room temperature for 20 minutes, they were incubated with Streptavidin-HRP (1: 800 dilution) at room temperature for 20 minutes, and DAB color was developed.

Vimentin antibody staining group: the staining procedure was essentially the same as that of the AptVim staining group, except that antigen retrieval was performed at 95 ℃ and incubation time of Vimentin Antibody (Human Vimentin Biotinylated Antibody) with tissue sections was 90 minutes.

The immunohistochemical results of the combination of the aptamer AptVim and Vimentin antibodies with the Vimentin protein of the liver cancer tissue are shown in FIG. 8. The result shows that although the staining condition of the aptamer AptVim is simpler, the aptamer AptVim can clearly stain the Vimentin protein in the liver cancer tissue specifically, and the effect similar to that of the staining of the Vimentin antibody is achieved.

Example 3 aptamer AptVim induces apoptosis in Vimentin-positive cells

Panc-1 cells and BxPC-3 cells were inoculated into 6-well plates and cultured overnight, and were divided into the following groups according to the treatment reagents:

AptVim + turbolect treatment group: aptamer AptVim (serum-free DMEM is used for adjusting the concentration of the aptamer AptVim to 1 mu M), and cells are treated for 24 hours after being wrapped by Turbofect;

turbofect treatment group: cells were treated with Turbofect for 24 hours;

turbofect + random library treatment group: treating cells for 24 hours after the random library (serum-free DMEM adjusted to 1 μ M) was coated with Turbofect;

AptVim treatment group: cells were directly treated with aptamer AptVim for 24 hours without Turbofect encapsulation, with a final aptamer AptVim concentration of 1. mu.M.

NC control group: no treatment with aptamers AptVim and Turbofect.

The cells of each experimental group were dissociated by non-enzymatic digestion, and then the treated cells of each treatment group were incubated with FITC-Annexin V antibody (1:100) and PI dye (1: 10) for 30 minutes at room temperature, washed with DPBS, and then assayed for Annexin V by flow cytometry (FACS Calibur, BD Biosciences)⁺/PI^-Cell ratio to obtain Annexin V of each experimental group⁺/PI^-Early apoptosis rate.

The statistics of early apoptosis rate of Vimentin positive cells induced by different experimental groups are shown in FIG. 9. The result shows that after the aptamer AptVim is wrapped by Turbofect, the Vimentin positive expression cell Panc-1 can be effectively induced to generate obvious apoptosis (the apoptosis rate is 18.4%,. times. P <0.01), and the other treatment groups and the Vimentin negative expression cell BxPC-3 do not generate obvious apoptosis.

Sequence listing

<110> first Hospital affiliated with Wenzhou medical university

<120> nucleic acid aptamer capable of specifically recognizing Vimentin and application thereof

<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

1. An aptamer for specifically recognizing vimentin is a single-stranded DNA molecule shown in SEQ ID No. 1.

2. A derivative of an aptamer for specifically recognizing vimentin, which is any one of the following (1) to (3):

(1) a derivative of the aptamer according to claim 1, wherein the aptamer has a signal molecule and/or an active molecule and/or a functional group attached to one end or the middle of the aptamer and has the same function as the aptamer;

(2) an RNA molecule encoded by the aptamer of claim 1;

(3) a peptide nucleic acid encoded by the nucleic acid aptamer of claim 1.

3. Use of the aptamer of claim 1 or the derivative of the aptamer of claim 2 for the preparation of a product that specifically recognizes and/or binds vimentin.

4. Use of the aptamer of claim 1 or the derivative of the aptamer of claim 2 in the preparation of a product for detecting or aiding in the detection of vimentin.

5. Use of the aptamer of claim 1 or the derivative of the aptamer of claim 2 for the preparation of a product for inducing apoptosis in tumor cells.

6. Use of the aptamer of claim 1 or the derivative of the aptamer of claim 2 for the preparation of a product to identify or assist in identifying whether a tumor cell is undergoing EMT.

7. Use of the aptamer of claim 1 or the derivative of the aptamer of claim 2 for the development of a medicament for the treatment of tumors.

8. A method for detecting vimentin expressed by a cell or tissue, comprising the step of detecting with the aptamer of claim 1 or the derivative of the aptamer of claim 2; the methods are for non-disease diagnostic and therapeutic purposes.