CN112251442A - Aptamer specifically binding to human connective tissue growth factor, derivative and application thereof - Google Patents
Aptamer specifically binding to human connective tissue growth factor, derivative and application thereof Download PDFInfo
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- CN112251442A CN112251442A CN202011160305.0A CN202011160305A CN112251442A CN 112251442 A CN112251442 A CN 112251442A CN 202011160305 A CN202011160305 A CN 202011160305A CN 112251442 A CN112251442 A CN 112251442A
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- growth factor
- connective tissue
- tissue growth
- aptamer
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- 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
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- 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
- G01N33/74—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving hormones or other non-cytokine intercellular protein regulatory factors such as growth factors, including receptors to hormones and growth factors
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- C—CHEMISTRY; METALLURGY
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- G01N2800/00—Detection or diagnosis of diseases
- G01N2800/10—Musculoskeletal or connective tissue disorders
- G01N2800/101—Diffuse connective tissue disease, e.g. Sjögren, Wegener's granulomatosis
- G01N2800/102—Arthritis; Rheumatoid arthritis, i.e. inflammation of peripheral joints
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Abstract
The invention relates to the technical field of biology and medicine, in particular to a nucleic acid aptamer specifically binding to human connective tissue growth factor, a derivative and an application thereof. The aptamer provided by the invention has the advantages of being easier to synthesize than a protein antibody, having small molecular weight, being capable of modifying and replacing different parts, being more stable, easy to store, having no batch-to-batch difference and the like; the CTGF protein can be combined in a normal-temperature serum environment; in addition, compared with some aptamer of CTGF protein which is published at present, the aptamer provided by the invention has higher affinity to the CTGF protein, has more stable structure, has strong binding in PBS buffer solution, can identify the CTGF protein in a protein spot hybridization experiment, can be used for quickly, quickly and accurately detecting the CTGF protein, and can be combined with the CTGF protein in serum.
Description
Technical Field
The invention relates to the technical field of biology and medicine, in particular to a nucleic acid aptamer specifically binding to human connective tissue growth factor, a derivative and an application thereof.
Background
Rheumatoid Arthritis (RA) is a chronic progressive autoimmune disease with an extremely complex etiology. Early stage shows wandering joint pain and dysfunction, joint stiffness and serious deformation can often occur in late stage, disability rate is high, and the health of the masses is seriously harmed. Early discovery, early diagnosis and early treatment are the main principles for controlling the progress of RA diseases, so that early and accurate diagnosis of RA has great significance for whether patients can accept early intervention so as to delay the progress of the diseases.
At present, clinical diagnosis of RA is mainly based on joint affected conditions, symptom duration, acute-phase reactants, serum indexes such as serum Rheumatoid Factor (RF), anti-citrullinated protein antibody (ACPA) and the like, but the early-phase clinical symptoms of RA are atypical and diverse, and the joint affected is often in a later stage, and the acute-phase reactants are non-specific, so the serological indexes are particularly important for early diagnosis and accurate diagnosis of RA.
However, both RF and ACPA, recommended by current guidelines, have certain limitations. RF and ACPA can be detected in RA patients, and also widely exist in autoimmune patients such as Osteoarthritis (OA), Systemic Lupus Erythematosus (SLE) and the like, so that the sensitivity or specificity is low, namely 69%, 85% and 67% and 95%, respectively, while the sensitivity and specificity of the combined diagnosis of RA of the RF and ACPA are only 78% and 82%, the misdiagnosis rate is as high as 19%, the performance in early RA is not satisfactory, and the positive rate is only 57%, so that the diagnosis index which can carry out early and accurate diagnosis on RA patients and is simple and convenient to operate is urgently needed to be searched in the field. The research has important significance for early discovery, early diagnosis and early treatment in RA patients, thereby slowing down the disease process, controlling clinical symptoms and improving the life quality.
The subject of the invention, human connective tissue growth factor (CTGF/CCN2), belongs to the CCN protein family rich in cysteine, is a secreted peptide closely related to angiogenesis, has a molecular weight of 4.5kb, comprises 4 structural domains, consists of 5 exons and 4 introns, encodes 349 amino acids, and has the number of GENBANK NP _ 001892. The current research suggests that CTGF plays a certain promoting role in cell proliferation and migration, angiogenesis and mitosis, and the interaction with TGF-beta plays an important role in the change of fibrosis. In recent years, more studies have reported the role of CTGF in the pathogenesis of RA, such as affecting the progression of RA diseases by affecting osteoclast metabolism, regulating TSP-1/TGF-beta/CTGF signaling pathways, and the like. (reference: 1. connecting tissue growth factor in tissues with tissue identification algorithm. arthritis Research & Therapy 2009,11.2.Nishimura K, Sugiyama D, Kogata Y, Tsuji G, Nakazawa T, Kawano S, et al. meta-analysis: diagnostic acid of anti-cyclic transformed peptide encoding gene, tissue culture vector for nucleic acid identification medium, D.D.D.C.D.D.C.D.D.C.D.D.D.D.D.D.D.; No. 146(11) GF 797; 3. sub.J.S.D.D.D.D.D.D.C.D.D.D.C.D.D.D.D.D.D.D.D.D.D.D.D.D.D.D.D.D.D. 3. sub.C.D.D.D.D.D.D.D.C.D.D.C.D.D.D.D.C.D.D.D.D.D.C.D.D.D.D.D.C.D.D.D.D.D.D.C.C.D.D.D.D.D.D.D.D.D.D.D.D.C. No. D.D.D.D.D.D.D.D.D.D.D.D.D.D.D.D.D.D.D.D.D.D.D.D.D.D.D.D.D.D.D.D.D.D.D.D.D.D.D.D.D.D.D.D.D.D.D.D.D.D.D.D.D.D.D.D.D.D.D.D.D.D.D.D.D.D.D.D.D.D.D.D.D.D.D.D.D.D.D.D.D.D.D.D.D.D.D.D.D.D.D.D.D The protein which is stably and continuously expressed in the process of RA diseases and can be released into blood by synovial fluid has more outstanding capability for RA diagnosis, combined diagnosis and differential diagnosis.
The aptamer (aptamer) refers to a DNA or RNA molecule obtained by screening and separating by an exponential enrichment ligand system evolution technology (SELEX), and can be combined with other targets such as proteins, metal ions, small molecules, polypeptides and even whole cells with high affinity and specificity, so that the aptamer has a wide prospect in the aspects of biochemical analysis, environmental monitoring, basic medicine, new drug synthesis and the like. Compared with the antibody, the aptamer has the advantages of small molecular weight, better stability, easy modification, no immunogenicity, short preparation period, artificial synthesis and the like, and a series of processes of animal immunization, feeding, protein extraction and purification and the like are omitted. Some researchers have published some aptamer sequences of the CTGF protein they found, however, the aptamer of the CTGF protein has not been available at the level of detection kit or drug. Therefore, there is a need in the art for aptamers with higher binding affinity for CTGF proteins.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provide an aptamer specifically binding to human connective tissue growth factor, a derivative and an application thereof.
The technical scheme adopted by the invention is as follows: an aptamer that specifically binds to human connective tissue growth factor, the aptamer sequence comprising at least one of the following nucleotide sequences in A-D:
A. any one of the DNA sequences shown in SEQ ID Nos.1 to 4;
B. a DNA sequence having a homology of 60% or more with any one of the DNA sequences represented by SEQ ID Nos.1 to 4, wherein the homology may be 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;
C. a DNA sequence which hybridizes with any one of the DNA sequences shown in SEQ ID Nos.1 to 4;
D. an RNA sequence transcribed from any one of the DNA sequences shown in SEQ ID Nos.1 to 4;
wherein, the nucleotide sequences can be specifically combined with the human connective tissue growth factor.
The invention designs and synthesizes a random single-stranded DNA library and corresponding primers, obtains aptamer specifically binding with human connective tissue growth factor by nine rounds of screening by a proper magnetic bead method, and respectively names the aptamer as CTGF-W2-1-76nt, CTGF-W2-1-56nt, CTGF-W2-1-46nt-1 and CTGF-W1-2-56 nt;
the sequence of CTGF-W2-1-76nt is shown in SEQ ID No.1, and the sequence is as follows: 5'-TTCAGCACTCCACGCATAGCAGGCCGGGAGGGTACCCATTGATGGTGTGATGACTGCCTATGCGTGCTACCGTGAA-3', respectively;
the sequence of CTGF-W2-1-56nt is shown in SEQ ID No.2, and the sequence is: 5'-CACGCATAGCAGGCCGGGAGGGTACCCATTGATGGTGTGATGACTGCCTATGCGTG-3', respectively;
the sequence of CTGF-W2-1-46nt-1 is shown in SEQ ID No.3, and the sequence is as follows: 5'-CACGCATAGCAGGCCGGGAGGGTACCCATTGATGGTGTGATGACTG-3', respectively;
the sequence of CTGF-W1-2-56nt is shown in SEQ ID No.4, and the sequence is: 5'-CACGCATAGCGGACTGTGTTAGGTGTACCGCCATCTCTTTGGGATCCCTATGCGTG-3' are provided.
The aptamer sequence is modified, the modification including phosphorylation, methylation, amination, sulfhydrylation, substitution of oxygen with sulfur, substitution of oxygen with selenium, or isotopolyization.
The nucleic acid aptamer sequence is connected with a fluorescent marker, a radioactive substance, a therapeutic substance, biotin, digoxigenin, a nano luminescent material, a small peptide, siRNA or enzyme.
The fluorescent marker, radioactive substance, therapeutic substance, biotin, digoxigenin, nano luminescent material, small peptide, siRNA, and enzyme are commonly used as a marker, a detection substance, a diagnostic substance, or a therapeutic substance.
The modified or connected nucleic acid aptamer has the molecular structure, the physicochemical property and the function which are basically the same as or similar to those of the original nucleic acid aptamer, and can be combined with the human connective tissue growth factor; the aptamer after modification or ligation can maintain or improve the affinity of the aptamer to human connective tissue growth factor, or can improve the stability of the aptamer.
An aptamer derivative that specifically binds to human connective tissue growth factor, said derivative being a phosphorothioate scaffold derived from the scaffold of the sequence of an aptamer that specifically binds to human connective tissue growth factor as described above, or a corresponding locked nucleic acid or peptide nucleic acid modified from an aptamer that specifically binds to human connective tissue growth factor as described above. The aptamer derivatives have substantially the same or similar molecular structural properties and functions as the original aptamer, i.e., specifically bind to human connective tissue growth factor.
Use of an aptamer or derivative thereof that specifically binds to human connective tissue growth factor as described above for the detection of human connective tissue growth factor.
The application of the aptamer or the derivative thereof specifically binding to the human connective tissue growth factor in preparing a kit for detecting the human connective tissue growth factor or a molecular probe for detecting the human connective tissue growth factor.
The aptamer can be used for detecting the concentration of CTGF protein in the serum of a subject, and further judging whether the subject has diseases related to abnormal expression level of CTGF, for example, diseases related to abnormal inflammatory response, such as rheumatoid arthritis and the like; inhibition of CTGF activity is useful for treating diseases associated with CTGF abnormalities, such as rheumatoid arthritis and the like.
Use of an aptamer or derivative thereof that specifically binds to human connective tissue growth factor as described above for purifying human connective tissue growth factor.
There are various methods for purifying human connective tissue growth factor, such as: incubating the aptamer or the aptamer derivative with a sample solution containing CTGF protein to ensure that the aptamer and the aptamer are specifically combined to form a compound, recovering the compound, and eluting the combined CTGF protein by high salt or other methods, namely purifying to obtain the CTGF protein;
or, firstly, the aptamer is fixed on a solid phase matrix, a sample solution containing the CTGF protein slowly flows through the solid phase matrix, the sample solution and the sample solution can be specifically combined and can not be combined with other unrelated proteins, then the solid phase matrix is washed by buffer solution, the unbound unrelated proteins are removed, and the combined CTGF is eluted and collected by high salt or other methods, namely the CTGF 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 has the following beneficial effects: the aptamer provided by the invention has the advantages of being easier to synthesize than a protein antibody, having small molecular weight, being capable of modifying and replacing different parts, being more stable, easy to store, having no batch-to-batch difference and the like; the CTGF protein can be combined in a normal-temperature serum environment; in addition, compared with some aptamer of CTGF protein which is published at present, the aptamer provided by the invention has higher affinity to the CTGF protein, has more stable structure, has strong binding in PBS buffer solution, can identify the CTGF protein in a protein spot hybridization experiment, can be used for quickly, quickly and accurately detecting the CTGF protein, and can be combined with the CTGF protein in serum.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and it is within the scope of the present invention for those skilled in the art to obtain other drawings based on the drawings without inventive exercise.
FIG. 1 shows the results of the affinity SPR measurements of the libraries obtained in the fifth, seventh and ninth rounds of example 1 on human connective tissue growth factor.
FIG. 2 shows the result of SPR detection of the affinity of the DNA sequence shown in SEQ ID Nos.1-4 with human connective tissue growth factor in PBS buffer.
FIG. 3 shows the result of SPR measurement of the binding KD values of the DNA sequences shown in SEQ ID Nos.1 to 4 to CTGF.
FIG. 4 shows the results of detection of human connective tissue growth factor by protein dot blot using the DNA sequence shown in SEQ ID Nos. 1-4.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention will be described in further detail with reference to the accompanying drawings.
An aptamer that specifically binds to human connective tissue growth factor, the aptamer sequence comprising at least one of the following nucleotide sequences in A-D:
A. any one of the DNA sequences shown in SEQ ID Nos.1 to 4;
B. a DNA sequence having a homology of 60% or more with any one of the DNA sequences represented by SEQ ID Nos.1 to 4, wherein the 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 is a nucleotide partially complementary to any one of the DNA sequences represented by SEQ ID Nos.1 to 4;
C. a DNA sequence which hybridizes with any one of the DNA sequences shown in SEQ ID Nos.1 to 4;
D. an RNA sequence transcribed from any one of the DNA sequences shown in SEQ ID Nos.1 to 4;
wherein, the nucleotide sequences can be specifically combined with the human connective tissue growth factor.
The invention designs and synthesizes a random single-stranded DNA library and corresponding primers, obtains aptamer specifically binding with human connective tissue growth factor by nine rounds of screening by a proper magnetic bead method, and respectively names the aptamer as CTGF-W2-1-76nt, CTGF-W2-1-56nt, CTGF-W2-1-46nt-1 and CTGF-W1-2-56 nt;
the sequence of CTGF-W2-1-76nt is shown in SEQ ID No.1, and the sequence is as follows: 5'-TTCAGCACTCCACGCATAGCAGGCCGGGAGGGTACCCATTGATGGTGTGATGACTGCCTATGCGTGCTACCGTGAA-3', respectively;
the sequence of CTGF-W2-1-56nt is shown in SEQ ID No.2, and the sequence is: 5'-CACGCATAGCAGGCCGGGAGGGTACCCATTGATGGTGTGATGACTGCCTATGCGTG-3', respectively;
the sequence of CTGF-W2-1-46nt-1 is shown in SEQ ID No.3, and the sequence is as follows: 5'-CACGCATAGCAGGCCGGGAGGGTACCCATTGATGGTGTGATGACTG-3', respectively;
the sequence of CTGF-W1-2-56nt is shown in SEQ ID No.4, and the sequence is: 5'-CACGCATAGCGGACTGTGTTAGGTGTACCGCCATCTCTTTGGGATCCCTATGCGTG-3' are provided.
The aptamer sequence is modified, the modification including phosphorylation, methylation, amination, sulfhydrylation, substitution of oxygen with sulfur, substitution of oxygen with selenium, or isotopolyization.
The nucleic acid aptamer sequence is connected with a fluorescent marker, a radioactive substance, a therapeutic substance, biotin, digoxigenin, a nano luminescent material, a small peptide, siRNA or enzyme.
The fluorescent marker, radioactive substance, therapeutic substance, biotin, digoxigenin, nano luminescent material, small peptide, siRNA, and enzyme are commonly used as a marker, a detection substance, a diagnostic substance, or a therapeutic substance.
The modified or connected nucleic acid aptamer has the molecular structure, the physicochemical property and the function which are basically the same as or similar to those of the original nucleic acid aptamer, and can be combined with the human connective tissue growth factor; the aptamer after modification or ligation can maintain or improve the affinity of the aptamer to human connective tissue growth factor, or can improve the stability of the aptamer.
An aptamer derivative that specifically binds to human connective tissue growth factor, said derivative being a phosphorothioate scaffold derived from the scaffold of the sequence of an aptamer that specifically binds to human connective tissue growth factor as described above, or a corresponding locked nucleic acid or peptide nucleic acid modified from an aptamer that specifically binds to human connective tissue growth factor as described above. The aptamer derivatives have substantially the same or similar molecular structural properties and functions as the original aptamer, i.e., specifically bind to human connective tissue growth factor.
Use of an aptamer or derivative thereof that specifically binds to human connective tissue growth factor as described above for the detection of human connective tissue growth factor.
The application of the aptamer or the derivative thereof specifically binding to the human connective tissue growth factor in preparing a kit for detecting the human connective tissue growth factor or a molecular probe for detecting the human connective tissue growth factor.
The aptamer can be used for detecting the concentration of CTGF protein in the serum of a subject, and further judging whether the subject has diseases related to abnormal expression level of CTGF, for example, diseases related to abnormal inflammatory response, such as rheumatoid arthritis and the like; inhibition of CTGF activity is useful for treating diseases associated with CTGF abnormalities, such as rheumatoid arthritis and the like.
Use of an aptamer or derivative thereof that specifically binds to human connective tissue growth factor as described above for purifying human connective tissue growth factor.
There are various methods for purifying human connective tissue growth factor, such as: incubating the aptamer or the aptamer derivative with a sample solution containing CTGF protein to ensure that the aptamer and the aptamer are specifically combined to form a compound, recovering the compound, and eluting the combined CTGF protein by high salt or other methods, namely purifying to obtain the CTGF protein;
or, firstly, the aptamer is fixed on a solid phase matrix, a sample solution containing the CTGF protein slowly flows through the solid phase matrix, the sample solution and the sample solution can be specifically combined and can not be combined with other unrelated proteins, then the solid phase matrix is washed by buffer solution, the unbound unrelated proteins are removed, and the combined CTGF is eluted and collected by high salt or other methods, namely the CTGF 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 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 of aptamers that specifically bind to human connective tissue growth factor
1. Synthesizing a random single-stranded DNA library and primers shown in the following sequences:
random single-stranded DNA library:
5’-TTCAGCACTCCACGCATAGC-36N-CCTATGCGTGCTACCGTGAA-3’
wherein "36N" represents a sequence in which 36 arbitrary nucleotide bases are linked, and the library is synthesized by Biotechnology engineering (Shanghai) GmbH; the primers were synthesized by Nanjing Kingsrei Biotech, Inc., and the primer information is shown in Table 1,
TABLE 1 primers and sequences thereof
Remarking: LibP1S1 is a forward primer; LibP1A2 is a reverse primer; LibP1S1-FAM is a forward primer of a fluorescent label; LibP1A2-ployA is a reverse primer connected with a polyA tail, wherein the polyA is a polyA tail consisting of 19 adenylate (A), and the 'Spacer 18' is an 18-atom hexaethylene glycol Spacer arm, and the structural formula is as follows:
random single-stranded DNA library and primers are respectively prepared into 100 mu M stock solution by PBS buffer solution ((KCl:0.2g/L, KH2PO4:0.24g/L, NaCl:8g/L, Na2HPO4:1.44 g/L; PH7.2, 25 ℃), and the stock solution is sequentially marked as random single-stranded DNA library stock solution, LibP1S1 stock solution, LibP1A2 stock solution, LibP1S1-FAM stock solution and LibP1A2-ployA stock solution and stored at-20 ℃ for later use.
2. Screening by magnetic bead method
2.1 immobilization of target protein by Carboxylic bead (CTGF protein):
(1) 50. mu.l of carboxyl magnetic beads (Invitrogen, Dynabeads) were takenTMMyOneTMCarboxylic Acid, #65012), using 200. mu.l of PBS buffer (KCl:0.2g/L, KH)2PO4:0.24g/L，NaCl:8g/L，Na2HPO41.44g/L) for 4 times, fishing magnetic beads by a magnet, and removing supernatant;
(2) taking 50 mu l of prepared NHS (N-hydroxysuccinimide; 0.1M aqueous solution) and EDC (1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride; 0.4M aqueous solution), mixing in equal volume, adding into the magnetic beads cleaned in the previous step, incubating for 20min at room temperature to activate the carboxyl on the surfaces of the magnetic beads, cleaning the magnetic beads for 1 time by using PBS buffer solution to obtain the activated magnetic beads, and using the activated magnetic beads in the next step as soon as possible;
(3) 20 mul of CTGF protein (cargo number: 9190-CC-050; R & D Systems) with the concentration of 50ug/ml is taken, 80 mul of 10mM NaAC solution with the pH value of 4.5 is added to the CTGF protein, the mixture is mixed evenly, then the mixture is added to the activated magnetic beads, the mixture is incubated for 60min at room temperature by a shaking table (if the magnetic beads need shaking evenly when aggregation is not constant), so that the CTGF protein is coupled to the surfaces of the magnetic beads through amino groups on the surfaces of the protein, the magnetic beads are magnetically fished, and the supernatant is removed to obtain the magnetic beads with fixed targets;
(4) adding 100 mu l of 1M ethanolamine with the pH value of 8.5 into a magnetic bead for fixing a target, incubating for 10min at room temperature by using a shaking table, sealing unreacted activation sites on the surface of the magnetic bead, fishing the magnetic bead by using a magnet, removing a supernatant, and washing for 4 times by using 200 mu l of PBS buffer solution, wherein the magnetic bead is marked as MB-CTGF magnetic bead for later use;
2.2 Carboxylic bead immobilization of BSA protein:
the method is the same as 2.1, replacing CTGF protein with BSA protein, and obtaining magnetic beads which are marked as MB-BSA magnetic beads for standby application under the same other conditions;
2.3 magnetic bead screening Process:
2.3.1 library renaturation treatment: taking a 1OD random single-stranded DNA library as an initial library, centrifuging at 14000rpm for 10min, centrifuging library powder to the bottom of a tube, dissolving the library powder to 5 mu M by using PBS buffer solution, and subpackaging the library powder into PCR tubes for renaturation treatment, wherein the renaturation treatment process comprises the following steps: setting a program of a PCR instrument, keeping the program at 95 ℃ for 10min, taking out the PCR tube, immediately carrying out ice-water bath for 5min, then placing the PCR tube at room temperature, balancing the temperature to room temperature to obtain a renaturation treated library, wherein the renaturation treatment aims to untie a folded chain and then fold the chain into a certain conformation;
2.3.2 reverse screening: adding the renaturation treated library into MB-BSA magnetic beads obtained in the step 2.2, incubating for 60min at 25 ℃ on a vertical mixer, fishing the magnetic beads by a magnet, and collecting supernatant pool-for the positive screening step; adding the incubated magnetic beads into 200 mu l of PBS buffer solution for washing for 4 times, adding the washed magnetic beads into 200 mu l of PBS buffer solution, carrying out boiling water bath for 10min, fishing the magnetic beads by using a magnet, collecting the boiled supernatant and marking the supernatant as an resolution-BSA for later use, and discarding the magnetic beads;
2.3.3 Positive Sieve: adding the supernatant into 2.1 MB-CTGF magnetic beads, incubating for 60min in a table at room temperature, absorbing and discarding the supernatant after finishing, keeping the magnetic beads, washing the magnetic beads for 4 times by PBS buffer solution, 200 mu l each time, absorbing and discarding the supernatant, adding 200 mu l of PBS buffer solution into the washed magnetic beads, carrying out boiling water bath for 10min, fishing the magnetic beads by a magnet, and recovering the supernatant to be marked as resolution-CTGF;
2.3.4 calculate the library retention for the positive and negative screens: taking Roche 8 tandem PCR tubes, adding 28 mu l Q-PCRmix into each hole, then respectively adding 2 mu l of each of the solution-BSA and solution-CTGF, and carrying out fluorescent quantitative PCR, wherein the procedure is as follows: pre-denaturation at 95 ℃ for 2 min; denaturation at 95 deg.C for 0.5min, annealing at 60 deg.C for 0.5min, and extension at 72 deg.C for 0.5min for 25 cycles;
then, according to the number of the take-off cycles of Q-PCR and a standard curve, respectively calculating the number of molecules in the solution-CTGF and the solution-BSA, and respectively calculating the ratio of the number of the molecules to the total number of molecules of the input initial library to obtain the library retention rates of the positive sieve and the negative sieve, so that the effect of each round of screening can be effectively monitored;
wherein, the formula of the Q-PCR mix is shown in the table 2:
TABLE 2 formulation of Q-PCR mix
| Reagent | Volume (μ l) |
| ddH2O | 826 |
| 10 pfu |
100 |
| dNTPmix(10mM) | 20 |
| |
5 |
| |
5 |
| |
4 |
| |
40 |
The conditions and library retention for each round of screening are shown in table 3:
TABLE 3 conditions for each round of screening, library retention for each round
2.4 emulsion PCR (e-PCR): using nucleic acid molecule in elusion-CTGF as template, and using emulsion PCR (ePCR) to amplify, the method is as follows: adding all the template solution-CTGF into 2ml of PCR mix, mixing uniformly, adding ePCR micro-droplets with 4 times of volume to generate oil, and performing vortex preparation to obtain emulsion; and subpackaging the emulsion into PCR tubes for amplification, wherein the amplification conditions are as follows, and the volume of the PCR tubes is 100 mu l/tube: pre-denaturation at 95 deg.C for 2min, denaturation at 95 deg.C for 60s, annealing at 60 deg.C for 60s, and extension at 72 deg.C for 60s, for 30 cycles, and storing at 4 deg.C;
the ePCR microdroplet generating oil was purchased from Oncupotumy (Aptamy) Biotech Ltd (product number: EPO100) of Anhui province, and the PCR mix formulation is shown in Table 4.
TABLE 4 formulation of PCR mix
2.5 preparation of Single Strand: collecting all ePCR products in a 15ml pointed-bottom centrifuge tube, adding n-butanol with 2 times of volume, and oscillating on a vortex mixer to fully mix uniformly; centrifuging at 9000rpm at 25 deg.C for 10 min; the upper phase (n-butanol and oil) was removed and concentrated PCR amplification product was obtained, in a volume ratio of 1:1 adding TBE urea denaturation buffer solution, boiling for denaturation for 15min to denature DNA, then carrying out ice bath for 1min, carrying out urea denaturation polyacrylamide gel electrophoresis on all samples, and carrying out electrophoresis at 400V until bromophenol blue reaches the bottom of gel, so that FAM-labeled single-stranded DNA is separated from lengthened reverse single-stranded DNA with polyA. Wherein, the TBE urea modified buffer solution and the urea modified polyacrylamide gel are purchased from Onpotummeh Biotechnology, Inc., and the product numbers are TLB-5 and DDP-100 respectively;
2.6 gel cutting to recover FAM-labeled single-stranded DNA: the gel was removed and placed on a plastic film, ex (nm): 495, em (nm): detecting FAM marked single-stranded DNA under 517 conditions; the target band was cut off directly with a clean blade, the strips were transferred to a 1.5ml EP tube and triturated, 1ml ddH was added2And (3) transferring the FAM-labeled single-stranded DNA in the gel into the solution in boiling water bath for 10min after O, centrifuging to remove gel fragments, and leaving a supernatant, wherein the supernatant is purified by n-butyl alcohol, and the purification method comprises the following steps: adding n-butanol with 2 times of volume, and shaking on a vortex mixer to fully mix; centrifuging at 9000rpm at 25 deg.C for 5 min; removing the upper phase (n-butanol) to obtain concentrated FAM-labeled single-stranded DNA, dialyzing overnight with 3.5KD dialysis bag to obtain secondary library, wherein the dialysate is PBS buffer solution;
3. and (3) multi-round screening: 9 rounds of repeated screening are carried out, each round of operation is carried out by taking the secondary library obtained in the previous 2.6 steps as an initial library, the SPR is used for detecting the change of the identification capacity of the secondary library of each round on the CTGF protein in the screening process (the SPR result of the affinity detection of the secondary library obtained by the fifth round, the seventh round and the ninth round and the CTGF protein is shown in figure 1, the specific operation steps are shown in example 2), when the identification capacity of the secondary library on the CTGF protein meets the requirement (namely the binding capacity of the secondary library and a target is higher than that of the random single-stranded DNA library input in the first round), after the obtained secondary library is subjected to clone sequencing analysis, a plurality of sequences are selected and synthesized by Beijing Openhei department biotechnology company for detecting the affinity, and after the affinity detection and the sequence optimization, the strong binding capacity of the 4 DNA sequences and the CTGF is finally determined (see examples 3-5), namely the aptamer which is specifically combined with the human connective tissue growth factor and is respectively named as CTGF-W2-1-76nt, CTGF-W2-1-56nt, CTGF-W2-1-46nt-1 and CTGF-W1-2-56 nt;
the sequence of CTGF-W2-1-76nt is shown in SEQ ID No.1, and the sequence is as follows: 5'-TTCAGCACTCCACGCATAGCAGGCCGGGAGGGTACCCATTGATGGTGTGATGACTGCCTATGCGTGCTACCGTGAA-3', respectively;
the sequence of CTGF-W2-1-56nt is shown in SEQ ID No.2, and the sequence is: 5'-CACGCATAGCAGGCCGGGAGGGTACCCATTGATGGTGTGATGACTGCCTATGCGTG-3', respectively;
the sequence of CTGF-W2-1-46nt-1 is shown in SEQ ID No.3, and the sequence is as follows: 5'-CACGCATAGCAGGCCGGGAGGGTACCCATTGATGGTGTGATGACTG-3', respectively;
the sequence of CTGF-W1-2-56nt is shown in SEQ ID No.4, and the sequence is: 5'-CACGCATAGCGGACTGTGTTAGGTGTACCGCCATCTCTTTGGGATCCCTATGCGTG-3', respectively;
in the screening method, the screening pressure can be increased by turns so as to improve the enrichment degree of the screened aptamer and shorten the screening process. The increase of the screening pressure comprises the reduction of the amount of the initial library and the amount of the target protein, the shortening of the incubation time of the initial library and the target protein, and the increase of the magnetic bead washing time and the washing times (the detailed parameters of the screening are shown in the table 3).
Example 2
Surface Plasmon Resonance (SPR) was performed to examine the affinity of the CTGF protein and the secondary library obtained in the fifth, seventh, and ninth rounds of screening in example 1.
Solution preparation:
the random single-stranded DNA library obtained in the first round of screening, the secondary library obtained in the fifth round of screening, the secondary library obtained in the seventh round of screening, and the secondary library obtained in the ninth round of screening in example 1 were diluted with PBS to a solution having a concentration of 500nM, which was designated as solution pool0, solution pool5, solution pool7, and solution pool9, respectively.
The operation is as follows:
coupling CTGF protein to a2 nd channel on the surface of a CM5 chip (GE Healthcare product number: BR100399) and marking as a CTGF channel, wherein the 1 st channel is not coupled with any protein and only activated and closed to be marked as a contrast channel;
the CTGF channel coupling process is as follows: firstly, washing the chip by 50mM NaOH aqueous solution, injecting 20 mul of sample with the flow rate of 10 mul/min, then injecting 50 mul of sample with the mixed solution of EDC and NHS with equal volume, and activating the chip with the flow rate of 5 mul/min; diluting CTGF protein with 10mM sodium acetate with pH4.5 to a final concentration of 20 mug/mL, injecting sample with a sample volume of 50 mug, a flow rate of 5 mug/min and a CTGF protein coupling amount of 2500 Ru; then ethanolamine is used for sealing the chip, the flow rate is 5 mu L/min, and the sample injection is 50 mu L;
control channel activation and blocking procedures were as follows: firstly, washing the chip by 50mM NaOH aqueous solution, injecting 20 mul of sample with the flow rate of 10 mul/min, then injecting 50 mul of sample with the mixed solution of EDC and NHS with equal volume, and activating the chip with the flow rate of 5 mul/min; then ethanolamine is used for sealing the chip, the flow rate is 5 mu L/min, and the sample injection is 50 mu L;
the EDC and NHS are the same as those in 2.1 of example 1.
And (3) detection: setting detection parameters by using Biacore T200, and carrying out sample injection at the flow rate of 30 mu L/min for 3min under the dissociation condition of 30 mu L/min for 5 min; in all the processes, the buffer solution for running the instrument is PBS buffer solution;
sequentially and respectively injecting a CTGF channel and a control channel into a solution pool0, a solution pool5, a solution pool7 and a solution pool9, and detecting results are shown in figure 1, wherein figure 1 is an affinity SPR (surface plasmon resonance) detecting result of the library obtained in the fifth round, the seventh round and the ninth round and the human connective tissue growth factor in example 1;
as can be seen from FIG. 1, the secondary libraries obtained from the fifth, seventh and ninth rounds of screening all have obvious binding with the target human connective tissue growth factor, and meet the requirement of sequencing.
Example 3
Surface Plasmon Resonance (SPR) detection of affinity of aptamers CTGF-W2-1-76nt, CTGF-W2-1-56nt, CTGF-W2-1-46nt-1 and CTGF-W1-2-56nt protein in PBS buffer
Solution preparation:
and diluting CTGF-W2-1-76nt, CTGF-W2-1-56nt, CTGF-W2-1-46nt-1 and CTGF-W1-2-56nt into solutions with the concentration of 500nM by using a PBS buffer solution respectively, and recording the solutions as solution 1, solution 2, solution 3 and solution 4 in sequence.
The operation is as follows: the CTGF protein was coupled to the surface of a CM5 chip (GE Healthcare cat # BR100399) and the CTGF channel coupling procedure was performed as described in example 2.
And (3) detection: a surface plasmon resonance (GE Healthcare, model: Biacore T200) is used for setting detection parameters, 30 mu L/min 3min of sample injection is carried out, 30 mu L/min 5min of dissociation is carried out, solution 1, solution 2, solution 3 and solution 4 are sequentially sample injection, the detection result is shown in figure 2, and figure 2 is the detection result of the affinity SPR of the DNA sequence shown in SEQ ID Nos.1-4 and the human connective tissue growth factor in PBS buffer solution.
As can be seen from FIG. 2, the aptamers CTGF-W2-1-76nt, CTGF-W2-1-56nt, CTGF-W2-1-46nt-1 and CTGF-W1-2-56nt all have very high affinity with the target human connective tissue growth factor in PBS buffer, and the increased RU values are all very high.
Example 4
Surface Plasmon Resonance (SPR) detection of bound KD values of aptamers CTGF-W2-1-76nt, CTGF-W2-1-56nt, CTGF-W2-1-46nt-1 and CTGF-W1-2-56nt proteins
Solution preparation:
diluting CTGF-W2-1-76nt with PBS buffer solution to obtain a series of solutions with different concentrations: 600nM, 300nM, 150nM, 75nM, 37.5nM, 18.75nM, 9.375 nM.
Diluting CTGF-W2-1-56nt with PBS buffer solution to obtain a series of solutions with different concentrations: 600nM, 300nM, 150nM, 75nM, 37.5nM, 18.75nM, 9.375 nM.
CTGF-W2-1-46nt-1 is diluted into a series of solutions with different concentrations by PBS buffer solution, and the concentrations are as follows: 600nM, 300nM, 150nM, 75nM, 37.5nM, 18.75nM, 9.375 nM.
Diluting CTGF-W1-2-56nt with PBS buffer solution to obtain a series of solutions with different concentrations: 600nM, 300nM, 150nM, 75nM, 37.5nM, 18.75nM, 9.375 nM.
The operation is as follows:
the CTGF protein was coupled to the surface of a CM5 chip (GE Healthcare cat # BR 100399): CTGF protein was diluted with 10mM sodium acetate pH4.5 to a final concentration of 20. mu.g/mL, and the sample volume was 20. mu.L, flow rate was 5uL/min, the CTGF protein coupling amount was 1500Ru, and the other CTGF channel coupling procedure was as described in example 2.
And (3) detection: using a surface plasma resonance instrument (GE Healthcare, model: Biacore T200) to set detection parameters, injecting 10 muL/min 3min, dissociating 10 muL/min 5min, and regenerating: 1M NaCl 30 muL/min 30s, the buffer solution operated by the instrument is PBS buffer solution, the serial solutions matched with the nucleic acids are sampled from low concentration to high concentration in sequence, and the detection result is shown in figure 4; FIG. 4 shows the results of SPR measurement of the binding KD values of the DNA sequences shown in SEQ ID Nos.1 to 4 to human connective tissue growth factor.
As can be seen from FIG. 4, the binding KD of CTGF-W2-1-76nt to human connective tissue growth factor is 1.17E-8M; the binding KD value of the CTGF-W2-1-56nt and the human connective tissue growth factor is as follows: 1.96E-8M; the binding KD value of the CTGF-W2-1-46nt-1 and the human connective tissue growth factor is as follows: 4.57E-8M, CTGF-W1-2-56nt bound to human connective tissue growth factor with KD values: 7.44E-8M; KD values are all above nM level, and the affinity of the DNA sequence shown in SEQ ID Nos.1-4 and human connective tissue growth factor is very high
Example 5
Protein dot hybridization experiment for detecting interaction of biotin-modified aptamers CTGF-W2-1-76nt, CTGF-W2-1-56nt, CTGF-W2-1-46nt-1 and CTGF-W1-2-56nt protein
Solution preparation:
CTGF protein series solutions: CTGF protein is diluted into solutions with the concentrations of 0.5mg/ml, 0.25mg/ml, 0.125mg/ml, 0.0625mg/ml and 0.03125mg/ml by PBS buffer respectively.
Control protein solution: the control protein TNF-alpha was diluted with PBS buffer to a concentration of 0.5 mg/ml.
10% BSA solution: BSA protein was diluted with PBS buffer to a 10 wt% solution.
PBST buffer: tween20 was diluted with PBS buffer to 0.5% o by volume.
Renaturation of aptamer solution: CTGF-W2-1-76nt, CTGF-W2-1-56nt, CTGF-W2-1-46nt-1 and CTGF-W1-2-56nt are respectively diluted into 1 mu M solution by PBS buffer solution, and the solution is respectively treated by renaturation (the temperature is kept at 95 ℃ for 10min, then the solution is immediately ice-cooled for 5min, and then the solution is balanced at room temperature for 10min) to obtain the renaturated nucleic acid aptamer solution corresponding to each solution.
HRP-labeled streptavidin solution: HRP-labeled streptavidin (purchased from boaosen, cat # bs-0437P-HRP) was added to PBST buffer according to 1: 5000 (v/v).
Color development liquid:solution A: solution B is 1:1(v/v), (Pierce)TMECL Western Blotting Substrate, available from Saimer Feishale, cat # 32106, and lane 1 and 2 are kit-in-band solutions).
The operation is as follows:
taking 1 mu L of each of the CTGF protein series solution and the control protein solution, respectively spotting the CTGF protein series solution and the control protein solution on a nitrocellulose membrane (purchased from Millipore, the specification is 15cm multiplied by 20cm), naturally drying, then sealing for 2h at room temperature by using 10% BSA solution, then washing for 3 times by using PBST buffer solution, and completely sucking; 4 parallel samples are made;
respectively incubating the 4 renaturation aptamer solutions with a protein shaking table on a nitrocellulose membrane at room temperature for 2h, washing with PBST buffer solution for three times, and placing on the shaking table for 10min each time during washing; adding HRP-labeled streptavidin solution, incubating for 30min in a shaking table at room temperature, washing with PBST buffer solution for three times, and placing on the shaking table for 10min each time during washing; color development is performed at room temperature by adding a color development liquid, and the result of photographing is observed by using an instrument (an integrated chemiluminescence imaging system of Shanghai Qixiang scientific instruments Co., Ltd.), and the result is shown in FIG. 4, wherein FIG. 4 is the result of detecting the human connective tissue growth factor by protein dot hybridization of the DNA sequence shown in SEQ ID Nos. 1-4.
As can be seen from FIG. 4, FIG. 4 includes the case of gradient change of each protein concentration and the case of the control protein, it can be seen that biotin-modified CTGF-W2-1-76nt, CTGF-W2-1-56nt, CTGF-W2-1-46nt-1, and CTGF-W1-2-56nt developed color significantly to the CTGF protein, and as the concentration of the CTGF protein decreased, the color of the developed spot became lighter, but not to the TNF-alpha protein, which indicates that biotin-modified CTGF-W2-1-76nt, CTGF-W2-1-56nt, CTGF-W2-1-46nt-1, and CTGF-W1-2-56nt can be used for detecting the membrane hybridization CTGF protein, and the sensitivity was higher.
The above description is only for the preferred embodiment of the present invention, but the scope of the present invention is not limited thereto, and any person skilled in the art should be considered to be within the technical scope of the present invention, and the technical solutions and the inventive concepts thereof according to the present invention should be equivalent or changed within the scope of the present invention.
The above disclosure is only for the purpose of illustrating the preferred embodiments of the present invention, and it is therefore to be understood that the invention is not limited by the scope of the appended claims.
Sequence listing
<110> Wenzhou university of medical science
<120> an aptamer specifically binding to human connective tissue growth factor, its derivatives and uses
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Claims (10)
1. An aptamer that specifically binds human connective tissue growth factor, wherein: the aptamer sequence comprises at least one of the following nucleotide sequences A-D:
A. any one of the DNA sequences shown in SEQ ID Nos.1 to 4;
B. a DNA sequence having a homology of 60% or more with any one of the DNA sequences represented by SEQ ID Nos.1 to 4;
C. a DNA sequence which hybridizes with any one of the DNA sequences shown in SEQ ID Nos.1 to 4;
D. an RNA sequence transcribed from any one of the DNA sequences shown in SEQ ID Nos.1 to 4;
wherein, the nucleotide sequences can be specifically combined with the human connective tissue growth factor.
2. The aptamer according to claim 1, which specifically binds to human connective tissue growth factor, wherein: the aptamer sequence is modified, the modification including phosphorylation, methylation, amination, sulfhydrylation, substitution of oxygen with sulfur, substitution of oxygen with selenium, or isotopolyization.
3. The aptamer according to claim 1, wherein the aptamer sequence is linked to a fluorescent marker, a radioactive substance, a therapeutic substance, biotin, digoxigenin, a nano-luminescent material, a small peptide, siRNA or an enzyme.
4. An aptamer derivative that specifically binds to human connective tissue growth factor, characterized in that: the derivative is a phosphorothioate backbone derived from the backbone of the sequence of an aptamer that specifically binds to human connective tissue growth factor as defined in any one of claims 1 to 3, or the corresponding locked nucleic acid or peptide nucleic acid modified from an aptamer that specifically binds to human connective tissue growth factor as defined in any one of claims 1 to 3.
5. Use of an aptamer according to any of claims 1 to 3 that specifically binds to human connective tissue growth factor for the detection of human connective tissue growth factor.
6. Use of the aptamer derivative according to claim 4, which specifically binds to human connective tissue growth factor, for detecting human connective tissue growth factor.
7. Use of the aptamer according to any one of claims 1 to 3, which specifically binds to human connective tissue growth factor, for preparing a kit for detecting human connective tissue growth factor or a molecular probe for detecting human connective tissue growth factor.
8. Use of the aptamer derivative according to claim 4, which specifically binds to human connective tissue growth factor, for preparing a kit for detecting human connective tissue growth factor or a molecular probe for detecting human connective tissue growth factor.
9. Use of an aptamer according to any of claims 1 to 3 that specifically binds to human connective tissue growth factor for the purification of human connective tissue growth factor.
10. Use of the aptamer derivative specifically binding to human connective tissue growth factor according to claim 4 for purification of human connective tissue growth factor.
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| CN104745587A (en) * | 2015-01-05 | 2015-07-01 | 西安交通大学医学院第一附属医院 | Nucleic acid aptamers for identifying connective tissue growth factor (CTGF) and application of nucleic acid aptamers |
| CN109402127A (en) * | 2018-09-29 | 2019-03-01 | 复旦大学附属眼耳鼻喉科医院 | One group of high affinity nucleic acid aptamers and its application specifically bound with Connective Tissue Growth Factor |
| CN111676225A (en) * | 2020-06-10 | 2020-09-18 | 安徽省昂普拓迈生物科技有限责任公司 | Aptamer specifically binding to PD-1 protein and application thereof |
| CN111979247A (en) * | 2020-05-11 | 2020-11-24 | 广东省药品检验所（广东省药品质量研究所、广东省口岸药品检验所） | Aptamer of beclomethasone as well as screening method and application thereof |
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2020
- 2020-10-27 CN CN202011160305.0A patent/CN112251442A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104745587A (en) * | 2015-01-05 | 2015-07-01 | 西安交通大学医学院第一附属医院 | Nucleic acid aptamers for identifying connective tissue growth factor (CTGF) and application of nucleic acid aptamers |
| CN109402127A (en) * | 2018-09-29 | 2019-03-01 | 复旦大学附属眼耳鼻喉科医院 | One group of high affinity nucleic acid aptamers and its application specifically bound with Connective Tissue Growth Factor |
| CN111979247A (en) * | 2020-05-11 | 2020-11-24 | 广东省药品检验所（广东省药品质量研究所、广东省口岸药品检验所） | Aptamer of beclomethasone as well as screening method and application thereof |
| CN111676225A (en) * | 2020-06-10 | 2020-09-18 | 安徽省昂普拓迈生物科技有限责任公司 | Aptamer specifically binding to PD-1 protein and application thereof |
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