CN113122544A - Aptamer specifically binding with TIMP1 protein and application thereof - Google Patents
Aptamer specifically binding with TIMP1 protein and application thereof Download PDFInfo
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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
- 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
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
- G01N33/57484—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites
-
- 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
The invention discloses an aptamer specifically binding TIMP1 protein and application thereof, wherein the aptamer sequence comprises at least one of the following four nucleotide sequences: A. a DNA sequence shown as SEQ ID No. 1-8; B. a DNA sequence having more than 60% homology with the DNA sequence shown in SEQ ID No. 1-8; C. a DNA sequence which hybridizes with the DNA sequences shown in SEQ ID Nos. 1-8; D. an RNA sequence transcribed from the DNA sequence shown in SEQ ID Nos. 1 to 8; wherein, the four nucleotide sequences can be specifically combined with TIMP1 protein; the invention also discloses a nucleic acid aptamer derivative; the invention also discloses an application of the aptamer or the aptamer derivative. The aptamer disclosed by the invention can be specifically combined with TIMP1 protein, and has the advantages of small molecular weight, stable chemical property, easiness in synthesis, short production time, low cost, easiness in storage and marking, no batch-to-batch difference and the like.
Description
Technical Field
The invention relates to the technical field of biology, in particular to a nucleic acid aptamer specifically binding TIMP1 protein and application thereof.
Background
The TIMP1 protein is a glycoprotein that can be expressed from various tissues of an organism. The protein is an inhibitory enzyme of matrix metalloprotease. TIMP-1 major secretory cells are a variety of connective tissue cells, thought to be the major regulator of MMPs activity in tissues, and can form a 1: 1, thereby exerting its inhibitory effect. Both maintain extracellular matrix homeostasis. At present, a plurality of studies show that the TIMP1 protein is related to the occurrence and development of various cancers such as colorectal cancer, lung cancer, pancreatic cancer and the like and tumors, so that the TIMP1 protein can be used as a diagnostic marker of the related tumors and cancers and a target of tumor treatment medicines.
Aptamers are single-stranded DNA or RNA sequences similar to antibodies that specifically recognize specific targets, and are generally obtained by SELEX technology. Has been widely used in the identification, detection and treatment of various types of targets.
The TIMP1 aptamer can be used for developing various types of detection kits and tumor treatment medicines.
Sequence information for aptamers directed to TIMP1 has not been reported so far, and there is a need in the art for aptamers with high affinity for TIMP 1.
Disclosure of Invention
The main purpose of the present application is to provide an aptamer specifically binding to TIMP1 protein and its application.
In order to achieve the above purpose, the invention provides the following technical scheme:
based on the technical problems in the background art, the invention provides the aptamer specifically binding TIMP1 and the application thereof, and the aptamer has high specificity to TIMP1, small molecular weight, stable chemical property, easy storage and easy marking.
The invention provides an aptamer specifically binding TIMP1 protein, wherein the aptamer sequence comprises at least one of the following nucleotide sequences:
A. any one of the DNA sequences shown in SEQ ID Nos. 1 to 8, wherein:
the sequence of SEQ ID No.1 is:
5’-GAACGACACCATACACTGCCAGTGAGACGAGATGAA-3’；
the sequence of SEQ ID No.2 is:
5’-CACGCACGCTACAGATTCCAACGTATTAATACGTGC-3’；
the sequence of SEQ ID No.3 is:
5’-CGAGTGACACAAGTCGATAAAATCGCATCTAACTGT-3’；
the sequence of SEQ ID No.4 is:
5’-TCACAACACATCGTGTTCCTGTTCAGTCATATCTCAAG-3’；
the sequence of SEQ ID No.5 is:
5’-ACCAAACCTCTCCTATGATTCATCGAGTCACGTGT-3’；
the sequence of SEQ ID No.6 is:
5’-TTCACTCACATGGCACATCACACGACTCTCACTTTT-3’；
the sequence of SEQ ID No.7 is:
5’-CCTATGTCTACCTTGCTCTTTCGTTGTCTGCTCACT-3’；
the sequence of SEQ ID No.8 is:
5’-GGATATACTTGGCGTGTGTCGATACGAATGTCTCGG-3’；
B. a DNA sequence having a homology of 60% or more with any of the DNA sequences shown in SEQ ID Nos. 1 to 8;
C. a DNA sequence which hybridizes with any one of the DNA sequences shown in SEQ ID Nos. 1 to 8;
D. an RNA sequence transcribed from any of the DNA sequences shown in SEQ ID Nos. 1 to 8;
wherein, the nucleotide sequences can be specifically combined with TIMP1 protein.
Preferably, the DNA sequence having a homology of 60% or more with any of the DNA sequences represented by SEQ ID Nos. 1 to 8 may have a homology of 60% or more, 70% or more, 80% or more, 85% or more, 90% or more, 92% or more, 94% or more, 96% or more, 98% or more, or 99% or more.
Preferably, the aptamer sequence is modified, the modification comprising phosphorylation, methylation, amination, carboxylation, hydroformylation, sulfhydrylation, substitution of oxygen with sulfur, substitution of oxygen with selenium, or isotopolication.
Preferably, the aptamer sequence is linked to a fluorescent label, quencher, azide, alkynyl, radioactive, therapeutic, biotin, digoxigenin, maleimide, nanomaterial, polyethylene glycol, small peptide, siRNA or enzyme.
The fluorescent marker, radioactive substance, therapeutic substance, biotin, digoxigenin, maleimide, nanomaterial, small peptide, siRNA, or enzyme is a commonly used marker, detection, diagnosis, or therapeutic substance.
The modified or linked aptamer has the molecular structure, the physicochemical property and the function which are basically the same as or similar to those of the original aptamer and can be combined with TIMP1 protein; the modified or linked aptamer can maintain or improve the affinity of the aptamer to the TIMP1 protein, or can improve the stability of the aptamer.
The present invention also provides an aptamer derivative which is a phosphorothioate backbone sequence derived from the backbone of the aptamer sequence, or a peptide nucleic acid modified from the aptamer by replacing a base in the aptamer sequence as claimed in any one of claims 1 to 3 with a nucleic acid variant (e.g. locked nucleic acid, artificial base, mirror nucleic acid, etc.).
In other words, the partially substituted or modified aptamer sequence has substantially the same or similar molecular structure, physicochemical properties and functions as the original aptamer, and binds to TIMP1 protein.
The phosphorothioate backbone sequences, nucleic acid variant sequences and peptide nucleic acids described above can be prepared using aptamers according to methods routine in the art.
The invention also provides the application of the aptamer or the aptamer derivative in preparing anti-tumor or anti-inflammation medicaments.
The purification method of TIMP1 is various, such as: incubating the aptamer or aptamer derivative with a sample solution containing TIMP1 protein to allow specific binding of the aptamer and the sample solution to form a complex, recovering the complex, and eluting the bound TIMP1 protein by high salt or other methods, thereby obtaining TIMP1 protein after purification;
or, the aptamer or the aptamer derivative is firstly fixed on a solid phase matrix, a sample solution containing the TIMP1 protein slowly flows through the solid phase matrix, the sample solution and the solid phase matrix are specifically combined and are not combined with other unrelated proteins, then the solid phase matrix is washed by a buffer solution, the unbound unrelated proteins are removed, and the combined TIMP1 protein is eluted by high salt or other methods and collected, namely the TIMP1 protein is obtained by purification. The person skilled in the art will be able to select an appropriate purification method depending on the actual requirements.
The invention also provides application of the aptamer or the aptamer derivative in a detection kit for purifying TIMP1 protein and TIMP1 protein and a detection probe for TIMP1 protein.
The invention also provides a detection kit comprising the aptamer or the aptamer derivative.
The invention also provides a detection probe, which comprises the aptamer or the aptamer derivative.
The aptamer or the aptamer derivative can be used for detecting the concentration of TIMP1 protein in the serum of a subject, and further judging whether the subject has diseases such as inflammatory diseases, tumors and the like related to abnormal TIMP1 expression level; the purified TIMP1 can be used for treating diseases related to TIMP1 protein abnormality, such as regulation of metalloprotease activity.
The aptamer or the aptamer derivative can be used for detecting the content of TIMP1 in blood, tissues and cells of a subject.
The inventor obtains aptamers which are specifically combined with TIMP1 from a random single-stranded DNA library through seven rounds of screening by a proper magnetic bead method, and the aptamers are named as TIMP1-04, TIMP1-11, TIMP1-18, TIMP1-20, TIMP1-21, TIMP1-49, TIMP1-58 and TIMP1-61 respectively;
the sequence of TIMP1-04 is shown in SEQ ID No.1, and the sequence is:
5’-GAACGACACCATACACTGCCAGTGAGACGAGATGAA-3’；
the sequence of TIMP1-11 is shown in SEQ ID No.2, and the sequence is:
5’-CACGCACGCTACAGATTCCAACGTATTAATACGTGC-3’；
the sequence of TIMP1-18 is shown in SEQ ID No.3, and the sequence is:
5’-CGAGTGACACAAGTCGATAAAATCGCATCTAACTGT-3’；
the sequence of TIMP1-20 is shown in SEQ ID No.4, and the sequence is:
5’-TCACAACACATCGTGTTCCTGTTCAGTCATATCTCAAG-3’；
the sequence of TIMP1-21 is shown in SEQ ID No.5, and the sequence is:
5’-ACCAAACCTCTCCTATGATTCATCGAGTCACGTGT-3’；
the sequence of TIMP1-49 is shown in SEQ ID No.6, and the sequence is:
5’-TTCACTCACATGGCACATCACACGACTCTCACTTTT-3’；
the sequence of TIMP1-58 is shown in SEQ ID No.7, and the sequence is:
5’-CCTATGTCTACCTTGCTCTTTCGTTGTCTGCTCACT-3’；
the sequence of TIMP1-61 is shown in SEQ ID No.8, and the sequence is:
5’-GGATATACTTGGCGTGTGTCGATACGAATGTCTCGG-3’。
compared with the prior art, the invention has the advantages that:
compared with a protein antibody, the invention has the advantages of easy synthesis, small molecular weight, capability of modifying and replacing different parts, more stability, easy storage, no batch-to-batch difference and the like; the invention can combine TIMP1 protein in the serum environment at normal temperature; in addition, the invention relates to the first publication of aptamer sequences of TIMP1 protein, which is not published yet for the aptamer sequences of TIMP1 protein.
Drawings
FIG. 1 is a screening process by a magnetic bead method;
FIG. 2 shows the SPR detection of the binding of TIMP1-04, TIMP1-11, TIMP1-18, TIMP1-20, TIMP1-21, TIMP1-49, TIMP1-58, TIMP1-61 and TIMP1 proteins;
FIG. 3 is a photograph of an image showing the binding specificity of Nitrocellulose filter binding assay verifying TIMP1-04, TIMP1-11, TIMP1-18, TIMP1-20, TIMP1-21, TIMP1-49, TIMP1-58, TIMP1-61 and TIMP1 proteins;
FIG. 4 is data for quantification of the imaged photograph of FIG. 3 using ImageLab software;
FIG. 5 shows the use of the aptamers of the invention in a chemiluminescent assay kit;
FIG. 6 shows the application of the aptamer of the invention in ELISA detection kit.
Detailed Description
In order to make the technical solutions in the embodiments of the present application better understood, the technical solutions in the embodiments of the present application will be clearly and completely described below with reference to examples, and it is obvious that the described embodiments are only some embodiments of the present application, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present application.
An aptamer that specifically binds to TIMP1 protein, the aptamer sequence comprising at least one of the following four nucleotide sequences:
A. any one of the DNA sequences shown in SEQ ID Nos. 1 to 8, wherein:
the sequence of SEQ ID No.1 is:
5’-GAACGACACCATACACTGCCAGTGAGACGAGATGAA-3’；
the sequence of SEQ ID No.2 is:
5’-CACGCACGCTACAGATTCCAACGTATTAATACGTGC-3’；
the sequence of SEQ ID No.3 is:
5’-CGAGTGACACAAGTCGATAAAATCGCATCTAACTGT-3’；
the sequence of SEQ ID No.4 is:
5’-TCACAACACATCGTGTTCCTGTTCAGTCATATCTCAAG-3’；
the sequence of SEQ ID No.5 is:
5’-ACCAAACCTCTCCTATGATTCATCGAGTCACGTGT-3’；
the sequence of SEQ ID No.6 is:
5’-TTCACTCACATGGCACATCACACGACTCTCACTTTT-3’；
the sequence of SEQ ID No.7 is:
5’-CCTATGTCTACCTTGCTCTTTCGTTGTCTGCTCACT-3’；
the sequence of SEQ ID No.8 is:
5’-GGATATACTTGGCGTGTGTCGATACGAATGTCTCGG-3’；
B. a DNA sequence having a homology of 60% or more with any of the DNA sequences shown in SEQ ID Nos. 1 to 8;
C. a DNA sequence which hybridizes with any one of the DNA sequences shown in SEQ ID Nos. 1 to 8;
D. an RNA sequence transcribed from any of the DNA sequences shown in SEQ ID Nos. 1 to 8;
wherein, all the four nucleotide sequences can be specifically combined with TIMP1 protein.
The aptamer sequence is modified, the modification comprises phosphorylation, methylation, amination, carboxylation, hydroformylation, sulfhydrylation, oxygen substitution by sulfur, oxygen substitution by selenium or isotopic functionalization; the nucleic acid aptamer sequence is connected with a fluorescent marker, a quenching group, an azide group, an alkynyl group, a radioactive substance, a therapeutic substance, biotin, digoxin, maleimide, a nano material, polyethylene glycol, a small peptide, siRNA or enzyme.
An aptamer derivative that specifically binds to TIMP1 protein, said aptamer derivative having a phosphorothioate backbone sequence derived from the backbone of said aptamer sequence, or having a nucleic acid variant replacing a base in said aptamer sequence, or a peptide nucleic acid modified from said aptamer.
The application of the aptamer or the aptamer derivative in preparing anti-tumor or anti-inflammation medicines; the application of the aptamer or the aptamer derivative in purifying a detection kit of TIMP1 protein and TIMP1 protein and a detection probe of TIMP1 protein; preferably, the detection kit is radioimmunoassay, enzyme-linked immunoassay, immunofluorescence assay, immunohistochemistry, chemiluminescence, flow-through fluorescence method assay, PET imaging, cell, tissue or in vivo imaging.
The invention also provides a detection kit, which comprises the aptamer or the aptamer derivative; the invention also provides a detection probe comprising the aptamer or the aptamer derivative.
The technical solution of the present invention will be described in detail below with reference to specific examples.
The experimental methods described in the following examples are all conventional methods unless otherwise specified; the experimental materials used are all conventional biochemical reagents and are commercially available, unless otherwise specified.
Example 1
Screening for aptamers that specifically bind to TIMP1 protein
1. Screening ssDNA sequences specifically binding with TIMP1 from the ssDNA library by using a paramagnetic particle method to obtain an enriched library, wherein the protein screening kit is purchased from Ongpu Toolmai (Aptamy) Biotech limited, Anhui province (product number: SEP-201903);
a. activating the carboxyl magnetic beads, and then respectively coupling with a positive sieve target substance and a negative sieve target substance to prepare positive sieve magnetic beads and negative sieve magnetic beads;
b. incubating and separating the anti-screening magnetic beads and the random nucleic acid library to obtain supernatant fluid;
c. b, incubating the supernatant obtained in the step b with the positive sieve magnetic beads, and then washing and eluting to obtain an eluent;
d. taking the eluent to carry out PCR amplification, and then preparing a DNA single chain to obtain a primary library;
e. the a-d is one-round screening, the random nucleic acid library in the a is replaced by the DNA single-stranded library obtained from the previous round of screening, the next round of screening is carried out, and the enrichment library of the target sequence is obtained through multiple rounds of screening;
2. performing high-throughput sequencing analysis on the enriched library obtained in the step 1;
3. verifying and detecting the affinity and specificity of the aptamer sequence obtained in the step 2 to obtain a aptamer which is targeted to bind with TIMP1 protein;
the sequence of the screened aptamer which is targeted to bind with the TIMP1 protein is shown as SEQ ID NO.1-8 in the sequence table.
Example 2
Surface Plasmon Resonance (SPR) detection of TIMP1 aptamer binding to TIMP1 protein
1. The nucleic acid aptamer SEQ ID No: 1-8 are respectively diluted to 1 mu M by DPBS buffer solution;
2. coupling TIMP1 protein and 8His polypeptide to channels 1 and 2 on the surface of a CM5 chip respectively: the chip was washed with 50mM NaOH and injected at 30. mu.L/min, then 50. mu.L of activated chip was injected after mixing equal volumes of two reagents, EDC (1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride; 0.4M aqueous solution) and NHS (N-hydroxysuccinimide; 0.1M aqueous solution), at 10. mu.L/min. The TIMP1 protein and the 8His polypeptide are respectively diluted by 10mM sodium acetate with pH 4.5 to a final concentration of 50 mu g/mL and then injected, the injection volume is 50 mu L, the flow rate is 10 mu L/min, the coupling amount of the TIMP1 protein is 5500Ru, and the coupling amount of the 8His polypeptide is 600 Ru. After the sample injection is finished, ethanolamine is added to seal the chip, the flow rate is 10 mu L/min, and the sample injection is 50 mu L;
3. and (3) detection: the detection parameters were set using Biacore T200, and the nucleic acid aptamer SEQ ID No: 1-8) sampling samples in sequence. The detection data are shown in FIG. 2, and the data indicate that TIMP1-04, TIMP1-11, TIMP1-18, TIMP1-20, TIMP1-21, TIMP1-49, TIMP1-58 and TIMP1-61 are combined with the target protein TIMP-1.
Example 3
Verification of specificity of binding of nucleotide sequences shown in SEQ ID Nos. 1-8 and TIMP1 protein by Nitrocellulose filter binding assay
1. Respectively diluting TIMP-1 protein, AFP protein, CA199 protein, CEA protein, BSA protein and 8His polypeptide to 25 μ g/mL by using PBS solution;
2. fluorescence modification of FAM to aptamers TIMP1-04, TIMP1-11, TIMP1-18, TIMP1-20, TIMP1-21, TIMP1-49, TIMP1-58 and TIMP1-61 were respectively diluted to 1 μ M using PBS solution, and renaturation treatment was performed: denaturation at 95 deg.C for 10min, ice-cooling for 5min, and balancing at room temperature for 10 min;
3. respectively mixing the renatured aptamer with TIMP-1 protein, AFP protein, CA199 protein, CEA protein, BSA protein and 8His polypeptide in equal volume, and incubating for 1h at room temperature;
4. filtering the incubated mixed solution through a nitrocellulose membrane (using a Bio-Dot SF microfiltration device), photographing by using an imaging system, and quantifying the fluorescence intensity of the strip by using ImageLab software;
5. the results are shown in FIGS. 3 and 4, which show that the FAM fluorescence-modified aptamers TIMP1-04, TIMP1-11, TIMP1-18, TIMP1-20, TIMP1-21, TIMP1-49, TIMP1-58, TIMP1-61 all bind to TIMP1 protein and bind to 5 control proteins: AFP, CA199, CEA, BSA, 8His polypeptides were weakly bound, indicating that the binding of TIMP1-04, TIMP1-11, TIMP1-18, TIMP1-20, TIMP1-21, TIMP1-49, TIMP1-58, TIMP1-61 and TIMP1 proteins has very good specificity.
Example 4
Chemiluminescence method TIMP1 protein detection kit
1. Modifying an amino-labeled TIMP1 aptamer or TIMP1 antibody on magnetic particles by using an amino coupling method to prepare a magnetic bead reagent;
2. modifying an amino-labeled TIMP1 aptamer or TIMP1 antibody onto an acridinium ester using an amino coupling method to prepare an acridinium ester reagent;
3. incubating the magnetic bead reagent with a sample (e.g., blood sample, etc.) containing TIMP1 for 10 min;
4. after the incubation is finished, the sample solution is removed by magnetic attraction, and is washed for 3 times by PBS buffer solution;
5. after being cleaned, the mixture is incubated with an acridinium ester reagent for 10 min;
6 after the incubation is finished, magnetically removing the acridinium ester reagent, and washing for 6 times by using PBS buffer solution;
7. adding an excitation liquid and a pre-excitation liquid, and detecting a luminous value;
8. the detection results are shown in FIG. 5, and the results show that the luminescence value is increased along with the increase of the concentration of TIMP1 in the sample, which indicates that the aptamer obtained by the invention can be used for chemiluminescence detection of TIMP1, and has higher sensitivity and wider linear range.
Example 5
TIMP1 protein detection ELISA kit
1. With NaCO at pH9.63Buffer solution TIMP1 aptamer or antibody diluted to 10 u g/ml, each hole 100 u l loading to ELISA plate, coating overnight;
2. the coating solution was removed from the plate and each well was washed 3 times with 200 μ lpH 7.4.4 PBS buffer for 3 minutes each;
3. BSA protein was diluted to 10mg/ml with PBS buffer pH7.4, 100. mu.l was added to each well, incubated for 2 hours, and the ELISA plates were blocked;
4. removing the protein blocking solution in the hole, and washing for 3 times with pH7.4PBS buffer solution, 3 minutes each time;
5. samples containing TIMP1 protein were then added to each well and incubated for 1 hour; removing the sample solution from the wells, and then washing with PBS buffer for 2 times, 3 minutes each time;
6. biotin-labeled TIMP1 aptamer or antibody was added to each well and incubated for 1 hour;
7. removing liquid in the wells, and washing with PBS buffer for 3min for 2 times;
8. then adding 20000 times diluted SA-HRP into each hole, and incubating for 30 minutes;
9. after the incubation, remove the SA-HRP in the wells, wash the wells 3 times with DPBS with 0.05% Tween 20 for 5 minutes each time;
10. adding 100 mul of TMB color developing agent into each hole, and adding 3MHCl to stop the reaction after 30 minutes;
11. the absorbance of the solution in each well was measured, and the results are shown in fig. 6, which shows that the absorbance increases with the increase of the concentration of TIMP1 in the sample, indicating that the aptamer obtained by the present invention can be used for the ELISA detection of TIMP1, and has high sensitivity and a wide linear range.
Example 6
Application of TIMP1 aptamer in antitumor drug
1. Coupling TIMP1 aptamer with drug molecules (or nano-carriers loaded with the drug molecules, such as liposome and the like) to prepare an aptamer drug conjugate;
2. the aptamer drug conjugate is administered into a human body by injection, oral administration or respiratory tract;
3. because the TIMP1 aptamer can be specifically combined with TIMP1 protein in a human body, the target drug delivery treatment of tumors can be realized;
4. because the TIMP1 aptamer can be specifically combined with TIMP1 protein, the aptamer drug conjugate can also achieve the purpose of treatment by regulating the activity of TIMP1 protein.
Example 7
Application in medical image diagnosis
1. The TIMP1 aptamer is coupled with radioisotope such as 68Ga or 18F and the like to prepare a PET contrast agent probe, and the probe is specifically combined with TIMP1 protein after entering a human body, thereby realizing PET imaging of a living body;
2. the TIMP1 aptamer is coupled with a fluorescent dye to prepare a fluorescent probe, and the probe is specifically bound to TIMP1 protein after entering a human body, so that the fluorescence imaging of a living body is realized.
The above description is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, several modifications and additions can be made without departing from the method of the present invention, and these modifications and additions should also be regarded as the protection scope of the present invention.
Claims (8)
1. An aptamer that specifically binds to TIMP1 protein, wherein the aptamer sequence comprises at least one of the following four nucleotide sequences:
A. any one of the DNA sequences shown in SEQ ID Nos. 1 to 8, wherein:
the sequence of SEQ ID No.1 is:
5’－GAACGACACCATACACTGCCAGTGAGACGAGATGAA－3’；
the sequence of SEQ ID No.2 is:
5’－CACGCACGCTACAGATTCCAACGTATTAATACGTGC－3’；
the sequence of SEQ ID No.3 is:
5’－CGAGTGACACAAGTCGATAAAATCGCATCTAACTGT－3’；
the sequence of SEQ ID No.4 is:
5’－TCACAACACATCGTGTTCCTGTTCAGTCATATCTCAAG－3’；
the sequence of SEQ ID No.5 is:
5’－ACCAAACCTCTCCTATGATTCATCGAGTCACGTGT－3’；
the sequence of SEQ ID No.6 is:
5’－TTCACTCACATGGCACATCACACGACTCTCACTTTT－3’；
the sequence of SEQ ID No.7 is:
5’－CCTATGTCTACCTTGCTCTTTCGTTGTCTGCTCACT－3’；
the sequence of SEQ ID No.8 is:
5’－GGATATACTTGGCGTGTGTCGATACGAATGTCTCGG－3’；
B. a DNA sequence having a homology of 60% or more with any of the DNA sequences shown in SEQ ID Nos. 1 to 8;
C. a DNA sequence which hybridizes with any one of the DNA sequences shown in SEQ ID Nos. 1 to 8;
D. an RNA sequence transcribed from any of the DNA sequences shown in SEQ ID Nos. 1 to 8;
wherein, all the four nucleotide sequences can be specifically combined with TIMP1 protein.
2. The aptamer of claim 1, wherein the aptamer sequence is modified, wherein the modification comprises phosphorylation, methylation, amination, carboxylation, aldehyde-alkylation, sulfhydrylation, substitution of oxygen with sulfur, substitution of oxygen with selenium, or isotopolyization.
3. The aptamer of claim 1, wherein the aptamer sequence is linked to a fluorescent label, a quencher, an azide, an alkynyl, a radioactive substance, a therapeutic substance, biotin, digoxigenin, maleimide, a nanomaterial, polyethylene glycol, a small peptide, an siRNA, or an enzyme.
4. An aptamer derivative that specifically binds to TIMP1 protein, wherein the aptamer derivative comprises a phosphorothioate backbone sequence derived from the backbone of the aptamer sequence of any one of claims 1 to 3, or wherein a nucleic acid variant is used to replace a base in the aptamer sequence of any one of claims 1 to 3, or a peptide nucleic acid modified from the aptamer of any one of claims 1 to 3.
5. Use of an aptamer according to any of claims 1 to 3 or a derivative of an aptamer according to claim 4 for the preparation of a medicament for the treatment of tumors or inflammation.
6. Use of the aptamer according to any one of claims 1 to 3 or the aptamer derivative according to claim 4 for purification of TIMP1 protein, a detection kit for TIMP1 protein, or a detection probe for TIMP1 protein.
7. A detection kit comprising the aptamer according to any one of claims 1 to 3 or the aptamer derivative according to claim 4.
8. A detection probe comprising the aptamer according to any one of claims 1 to 3 or the aptamer derivative according to claim 4.
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