CN107760686B - Aptamer of DKK-1 protein and application thereof - Google Patents
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Abstract
The invention discloses an aptamer for identifying DKK-1 protein or a derivative thereof, wherein the aptamer has a nucleotide sequence shown by any one of SEQ ID No. 1-SEQ ID No. 24. The invention also discloses the application of the aptamer or the derivative thereof. The aptamer of the DKK-1 protein disclosed by the invention can be efficiently and specifically combined with the DKK-1 protein, can be used for capturing the DKK-1 protein from a complex system or realizing in-vitro detection of the DKK-1 protein, is suitable for purification and detection of the DKK-1 protein and diagnosis of DKK-1 related diseases, and has a wide application prospect.
Description
Technical Field
The invention relates to the technical field of biological engineering, in particular to a nucleic acid aptamer of DKK-1 protein and application thereof.
Background
Dickkopf-1(DKK-1) induces apoptosis of various tumor cells in vivo through a Wnt/beta-catenin canonical signaling pathway and a Wnt non-canonical signaling pathway, regulates bone metastasis of various tumor cells, and promotes infiltration and invasion of certain tumor cells, thereby playing an important role in the aspects of occurrence and development of tumors. DKK-1 has been reported as a diagnostic marker for lung and liver cancer.
An aptamer (aptamer) is a single-stranded oligonucleotide of less than 100 bases in length, which is screened from a random library by the SELEX (systematic Evolution of Ligands by amplification) technique. The technology was first reported in 1990, using a library with known sequences at both ends and 15-60 random bases in the middle as a starting point, and then using a PCR amplification technology to exponentially enrich oligonucleotides specifically binding to a target molecule, and then preparing single-stranded DNA or transcribing the single-stranded DNA into RNA, and putting the RNA into the next round of screening. After 6-15 rounds of incubation, elution and amplification, and the final round of library is cloned and sequenced, the aptamer with high affinity with the target and strong specificity can be obtained.
Aptamers have several unique advantages over antibodies. Firstly, the nucleic acid aptamer can specifically recognize various biological molecules and also can recognize non-biological molecules, the recognition target comprises metal ions, organic dyes, medicines, amino acids, proteins, cells and the like, and the target with lower immunogenicity and toxicity can also obtain a corresponding aptamer sequence; secondly, the preparation cost of the aptamer is low, the molecular weight of the oligonucleotide is small, the immunogenicity is low, the oligonucleotide can be chemically synthesized, the modification is easy, and the cost is low; thirdly, the aptamer has stable physicochemical property, good stability, easy storage and insensitivity to high temperature and severe conditions. Therefore, the aptamer has a good application prospect.
Disclosure of Invention
The invention aims to solve the technical problem that an aptamer specifically binding DKK-1 protein is lacked at present, and provides the aptamer of the DKK-1 protein, which can be efficiently and specifically bound with the DKK-1 protein, can be used for capturing the DKK-1 protein from a complex system or realizing in-vitro detection of the DKK-1 protein, and is suitable for purification and detection of the DKK-1 protein and diagnosis of DKK-1 related diseases.
In order to solve the technical problems, the invention is realized by the following technical scheme:
in one aspect of the present invention, there is provided an aptamer recognizing DKK-1 protein, having a nucleotide sequence shown by any one of SEQ ID No.1 to SEQ ID No.24, or a derivative thereof.
X in the SEQ ID NO. 12-22 sequences is a modified base with the following structure:
in the present invention, the derivatives include:
(1) deleting or adding one or more nucleotides in any one of the aptamer sequences shown in SEQ ID NO. 1-SEQ ID NO.24 to obtain the aptamer derivative with the same function as the aptamer;
(2) carrying out base substitution or modification on any one of the aptamer sequences shown in SEQ ID NO. 1-SEQ ID NO.24 to obtain the aptamer derivative with the same function as the aptamer;
(3) modifying the molecular skeleton of any one of the nucleic acid aptamer sequences shown in SEQ ID NO. 1-SEQ ID NO.24 to obtain a nucleic acid aptamer derivative with the same function as the nucleic acid aptamer;
(4) obtaining a nucleic acid aptamer derivative with the same function as the nucleic acid aptamer through the peptide nucleic acid coded by the nucleic acid aptamer shown in SEQ ID NO. 1-SEQ ID NO. 24; or
(5) One end or middle of any one of the nucleic acid aptamer sequences shown in SEQ ID NO. 1-SEQ ID NO.24 is added with a signal molecule and/or an active molecule and/or a functional group to obtain the nucleic acid aptamer derivative with the same function as the nucleic acid aptamer.
In another aspect of the invention, there is provided the use of the above aptamer or derivative thereof in the preparation of a product for capturing and purifying DKK-1 protein.
In another aspect of the invention, there is also provided the use of the above aptamer or derivative thereof for the preparation of a product for the in vitro detection of DKK-1 protein.
In another aspect of the invention, there is also provided the use of the above aptamer or a derivative thereof for the preparation of a product for the diagnosis or treatment of a disease associated with DKK-1. The DKK-1 related diseases comprise lung cancer, liver cancer, esophageal cancer, pancreatic cancer, myeloma, endometrial cancer, cervical cancer, rheumatoid arthritis and the like.
In another aspect of the invention, there is provided a product for purifying or detecting DKK-1 protein in vitro comprising at least one aptamer sequence represented by SEQ ID No.1 to SEQ ID No. 24.
In another aspect of the invention, there is provided a product for diagnosing DKK-1 associated diseases, comprising at least one aptamer sequence represented by SEQ ID No.1 to SEQ ID No. 24.
The product comprises a kit or a detection chip.
The aptamer specifically and efficiently combined with DKK-1 protein can be used for DKK-1 protein capture, in-vitro detection or clinical diagnosis of DKK-1 related diseases, has wide application prospect, can be prepared manually in large quantities, has simple method and low cost, and is beneficial to market popularization.
Drawings
The present invention will be described in further detail with reference to the accompanying drawings and specific embodiments.
FIG. 1 is a graph of the binding of an aptamer to a target of DKK-1 protein of example 4 of the invention;
FIG. 2 is a graph showing the results of verifying the binding specificity of an aptamer to DKK-1 protein according to example 4 of the present invention;
FIG. 3 is a drawing-down result of a DKK-1 protein general base aptamer of the present invention in a serum base in example 4;
FIG. 4 is a graph of DKK-1 protein aptamer to target binding of example 5 of the invention;
FIG. 5 is a graph showing the results of verifying the binding specificity of an aptamer to DKK-1 protein according to example 5 of the present invention;
FIG. 6 is a graph of DKK-1 protein aptamer to target binding of example 6 of the invention;
FIG. 7 is a graph showing the results of verifying the binding specificity of an aptamer to DKK-1 protein in example 6 of the present invention.
Detailed Description
Because the existing aptamer capable of efficiently and specifically binding DKK-1 protein is lacked, a series of aptamers specific to the DKK-1 protein are obtained by screening, and the aptamers can be bound with the DKK-1 protein with high affinity and high specificity, so that the application prospect is good. In order to screen these aptamers, the present invention first synthesizes a DNA library with known sequences at both ends and 30 random bases in the middle. DKK-1 protein is used as target protein, and SELEX technology is adopted to screen DNA, RNA and modified DNA aptamers with high affinity and high specificity. The secondary structure of the aptamer was predicted by structure prediction software mfold analysis. Through enzyme-linked aptamer detection technology (ELASA) and pull down technology, the affinity and specificity of the aptamer to a target protein are identified, a plurality of aptamer sequences with high affinity and strong specificity to DKK-1 protein are obtained, and the aptamers can form respective specific stem-loop structures.
The modified base of the aptamer containing modified base has the following structure:
in the present invention, a nucleic acid containing a modified base is chemically or biologically synthesized, and the modified base to be used is usually a phosphoramidite or triphosphate derivative of IAA-dU or a salt thereof as a starting material.
Preferably, the triphosphate derivative of IAA-dU is 5- [ (3-indolyl) propionamide-N-propenyl ] -2' -deoxyuridylic acid (IAA-dUTP for short), having the following structural formula:
EXAMPLE 1 screening of aptamers to DKK-1 protein by unmodified DNA bases
An initial random library was chemically synthesized with the following sequence:
tagggaagagaaggacatatgat(SEQ ID NO.25)-N30-ttgactagtacatgaccacttga(SEQ ID NO.26)
wherein N30 is 30 random oligonucleotides.
Primer P1: TAGGGAAGAGAAGGACATATGAT (SEQ ID NO.27)
Primer P2: 5' phosphorylation-TCAAGTGGTCATGTACTAGTCAA (SEQ ID NO.28)
50pmol of DKK-1 protein containing 6 XHis tag was added to the 1nmol library, and incubated at 25 ℃ for 30 min. Subsequently 50ul His-Mag beads were added and incubated at 25 ℃ for 30 min. WB for magnetic beads (137mM NaCl, 2.7mM KCl,10mM Na)2HPO4,2mM KH2PO4,5mM MgCl25mM imidazole, 0.02% tween-20) were washed three times with 1ml each time. Magnetic bead50ul of 500mM imidazole was added thereto, allowed to stand at room temperature for 1min, and the supernatant was aspirated. The product was pre-amplified for 6 cycles in a 500ul PCR system. Then, the optimal cycle number of amplification is determined by a cycle number gradient experiment, and 6cycle, 8cycle, 10cycle, 12cycle and 14cycle are amplified respectively by using a pre-amplification product as a template and a 5-tube 50ul PCR system. And (4) performing electrophoresis on the product, selecting the most appropriate cycle number and amplifying the screened product to prepare the double strand. The amplification product was purified and quantified using Nanodrop. 5ul 10 Xdigestion buffer, 1ul lambda exonuclease was added to each 2ug of product and made up to 50ul with water, and digestion was carried out at 37 ℃ for 30min to obtain a secondary library for the next round of screening. Screening was performed for a total of six rounds.
Through screening, the following sequence of common base DNA aptamers is obtained:
example 2 screening of aptamers to DKK-1 protein by modification of DNA base
An initial random library was chemically synthesized with the following sequence:
ATCCAGAGTGACGCAGCA(SEQ ID NO.29)-40N-TGGACACGGTGGCTTAGT(SEQ ID NO.30)
wherein N40 is 40 random oligonucleotides.
Primer P3: 5' phosphorylation-ATCCAGAGTGACGCAGCA (SEQ ID NO.31)
Primer P4: 5' biotin-ACTAAGCCACCGTGTCCA (SEQ ID NO.32)
A library of modified bases is prepared. The preparation method takes a common DNA single strand of 5' -end modified biotin as a template, and comprises the following steps: 10 buffer 25ul, P4(100uM)24ul, dA, dG, dC (2 mM each) 10ul, IAA-dUTP (5- [ (3-indolyl) propionamide-N-propenyl ] -2' -deoxyuridylic acid) 10ul, KOD enzyme 10ul, water 71ul, template (20uM)100 ul. 95 ℃ for 1min, 55 ℃ for 1min and 72 ℃ for 1 h. The product was added to 300ul of SA agarose bead and shaken at room temperature for 10 min. After three WB washes, 700ul of 150mM NaOH was added and shaken at room temperature for 5 min. 640ul of supernatant was aspirated and neutralized with 160ul of 600mM HCl. Using water as a control, Nanodrop measures the single stranded DNA concentration. The concentration range of the product is 20-40 ng/ul, and the A260/A280 range is 1.60-1.80.
After quantification of the library, 50pmol of DKK-1 protein containing 6 XHis tag was added to 1nmol of the product, and the product was incubated at 25 ℃ for 30 min. Subsequently 50ul His-Mag magnetic beads (GE) were added and incubated at 25 ℃ for 30 min. WB for magnetic beads (137mM NaCl, 2.7mM KCl,10mM Na)2HPO4,2mM KH2PO4,5mM MgCl25mM imidazole, 0.02% tween-20) were washed three times with 1ml each time. 50ul of 500mM imidazole was added, and the mixture was allowed to stand at room temperature for 1min, and the supernatant was aspirated. The product was pre-amplified for 6 cycles in a 500ul PCR system. Then, the optimal cycle number of amplification is determined by a cycle number gradient experiment, and 6cycle, 8cycle, 10cycle, 12cycle and 14cycle are amplified respectively by using a pre-amplification product as a template and a 5-tube 50ul PCR system. And (4) performing electrophoresis on the product, selecting the most appropriate cycle number and amplifying the screened product to prepare the double strand. Preparing a single chain from the amplification product by a magnetic bead method, adding 50ul of streptavidin magnetic beads, shaking for 10min, and cleaning WB for three times, wherein each time is 1 ml. The beads were then neutralized with 25ul300mM HCl by adding 50ul of 150mM NaOH to elute single strands. The product is extended to prepare a modified chain and recovered for quantification, 5ul 10 Xenzyme digestion buffer solution and 1ul lambda exonuclease are added into every 2ug of the product, and water is used for complementing to 50ul for enzyme digestion, so as to obtain a secondary library for the next round of screening. Six rounds of modified base selection were performed.
By screening, a modified base aptamer having the following sequence, wherein X ═ IAA-dU:
example 3 screening of aptamers to DKK-1 protein by RNA bases
An initial random library was chemically synthesized with the following sequence:
ATCCAGAGTGACGCAGCA(SEQ ID NO.29)-40N-TGGACACGGTGGCTTAGT(SEQ ID NO.30)
wherein N40 is 40 random oligonucleotides.
Primer P5: ATCCAGAGTGACGCAGCA (SEQ ID NO.31)
Primer P6: GATAATACGACTCACTATAGGGACTAAGCCACCGTGTCCA (SEQ ID NO.33)
An RNA library was prepared. The single strand of the DNA library was used as a template, and PCR amplification was performed using P5 and P6. After the double-stranded product is purified and quantified, T7RNA polymerase is added for transcription, and the temperature is 42 ℃ for 3 h. The DNA template was then digested by adding DNase I and the RNA was purified using a purification column. Nanodrop measures RNA concentration.
After quantification of the library, 50pmol of DKK-1 protein containing 6 XHis tag was added to 1nmol of the library, and the library was incubated at 25 ℃ for 30 min. Subsequently 50ul His-Mag magnetic beads (GE) were added and incubated at 25 ℃ for 30 min. WB for magnetic beads (137mM NaCl, 2.7mM KCl,10mM Na)2HPO4,2mM KH2PO4,5mM MgCl25mM imidazole, 0.02% tween-20) were washed three times with 1ml each time. 200ul DEPC water was added to the beads, and the supernatant was aspirated at 95 ℃ for 5 min. After the supernatant is reversely transcribed into DNA, the optimum cycle number of amplification is determined by a cycle number gradient experiment, and 6cycle, 8cycle, 10cycle, 12cycle and 14cycle are respectively amplified by taking a reverse transcription product as a template and matching a 5-tube 50ul PCR system. And (4) performing electrophoresis on the product, selecting the most appropriate cycle number and amplifying the screened product to prepare the double strand. The amplified product is subjected to transcription reaction to obtain a secondary library for the next round of screening. RNA screening was performed for a total of six rounds.
Through screening, obtain the following sequence of RNA aptamer:
example 4 specific binding of the obtained common base aptamer to DKK-1 protein
1. Enzyme-linked aptamer adsorption assay (ELASA)
DKK-1 protein is diluted in PBS buffer solution (pH7.4, 0.22um filter membrane filtration preservation), and the final concentration is 200 ng/mL; 50uL per well were coated in a half volume ELISA microplate, sealed and incubated overnight at 4 ℃ in a 96 well plate. Abandoning the liquid in the holes, filling the holes with washing liquid, standing for 10 seconds, throwingDry, patted dry on absorbent paper after repeating four times. Let 8(9) concentrations (highest concentration set according to DKK-1 aptamer affinity respectively) be set, biotin-labeled DKK-1 aptamer was diluted in binding buffer in multiple ratios, and duplicate wells were made for each concentration. Concentration dilutions were added to each well at 50uL volumes per well and blank control wells and BSA control wells were set. Seal plates and incubate at 25 ℃ for 2 hours. After repeating the manual plate washing step, 50uL of SA-HRP enzyme-labeled reaction solution is added into each hole, the plates are sealed, and the mixture is incubated for 1 hour at 25 ℃. The plates were washed manually, 50uL of TMB chromogenic mix was added to each well, the plates were closed and incubated at 37 ℃ for 20 minutes. Each hole is added with 50uL 1N H2SO4And (5) stopping the solution, and mixing uniformly. The OD of each well was read with a microplate reader at a wavelength of 450nm (reference wavelength of 630 nm).
The results are shown in fig. 1, the aptamer with common bases obtained by screening has high affinity with DKK-1 protein, for example, the affinity constant of DKK14 is 17.956 ± 2.160 nM; DKK58 has an affinity constant of 15.492 ± 1.741 pM; DKK10 had an affinity constant of 1.277 ± 0.092 nM. Moreover, the binding of these aptamers to DKK-1 protein has high specificity, and hardly binds to other proteins such as BSA protein (see fig. 2).
DKK-1 protein common base aptamer pull-down experiment
And (4) pretreatment of the serum matrix. Mixing 100ng DKK-1 pure protein and 50uL of human serum, incubating at room temperature for 1 hour, and diluting with binding buffer solution to a final volume of 200 uL; and (3) sucking 20uL streptavidin magnetic beads, washing for 2 times by 1mL binding buffer solution, adding the serum sample into the pre-washed magnetic beads, placing the pre-washed magnetic beads in air at 25 ℃, performing rotary incubation for 1 hour, separating the magnetic beads in a magnetic field, and taking the supernatant for later use. The magnetic beads are coated with aptamers. 20uL of streptavidin magnetic beads are absorbed, 1mL of binding buffer solution is washed for 2 times, then 200uL of binding buffer solution is added, 20pmol of common nucleotide aptamer (negative control group is empty magnetic beads without coating aptamer) synthesized by the manufacturer is added, the mixture is placed in air at 25 ℃ for rotary incubation for 1 hour, magnetic beads are collected by a magnetic field, 1mL of binding buffer solution is washed, and the washing is carried out for 3 times. The pretreated serum substrate is incubated with magnetic beads coated with a common nucleotidic aptamer. And mixing the serum and the magnetic beads uniformly, placing the mixture in air at 25 ℃, performing rotary incubation for 1 hour, collecting the magnetic beads through a magnetic field, and washing the mixture for 3 minutes in 1mL of washing buffer solution by oscillation for 4 times. And (3) sucking 20uL of 1X SDS loading buffer solution, mixing the collected magnetic beads uniformly, and performing denaturing elution in a water bath at 95 ℃ for 5 min. The obtained sample is subjected to 8% SDS-PAGE gel, and finally silver staining detection is carried out.
The results are shown in fig. 3, and DKK-1 common base aptamers as capture probes in pull-down experiments can specifically bind to target proteins in serum matrix. In FIG. 3, lanes 1-7 are: fermentas pre-staining protein marker SM 0671; 2. negative control, pure protein X (50 ng); 3. negative control: empty magnetic beads and diluted serum substrate pull-down added with DKK-1 pure protein (100ng) are used for obtaining results; 4,5. respectively coating the magnetic beads of the protein X aptamer and mixing with diluted serum substrate pull-down result of X pure protein (100 ng); DKK-1 pure protein (50 ng); 7. the result was obtained by mixing magnetic beads coated with Dkk1 protein common base aptamers with a diluted serum substrate pull-down mixed with DKK-1 pure protein (100 ng).
Example 5 screening of the obtained modified base aptamer specifically binds to DKK-1 protein
Enzyme-linked aptamer adsorption assay (ELASA)
DKK-1 protein is diluted in PBS buffer solution (pH7.4, 0.22um filter membrane filtration preservation), and the final concentration is 200 ng/mL; 50uL per well were coated in a half volume ELISA microplate, sealed and incubated overnight at 4 ℃ in a 96 well plate. Abandoning the liquid in the holes, filling the holes with washing liquid, standing for 10 seconds, spin-drying, repeating for four times, and then patting on absorbent paper to dry. And (4) setting 8(9) concentrations (respectively setting the highest concentration according to the affinity of the DKK-1 modified base aptamer), diluting the biotin-labeled DKK-1 modified base aptamer in a binding buffer solution in a multiplying ratio, and making a plurality of holes at each concentration. Concentration dilutions were added to each well at 50uL volumes per well and blank control wells and BSA control wells were set. Seal plates and incubate at 25 ℃ for 2 hours. After repeating the manual plate washing step, 50uL of SA-HRP enzyme-labeled reaction solution is added into each hole, the plates are sealed, and the mixture is incubated for 1 hour at 25 ℃. The plates were washed manually, 50uL of TMB chromogenic mix was added to each well, the plates were closed and incubated at 37 ℃ for 20 minutes. Each hole is added with 50uL 1N H2SO4And (5) stopping the solution, and mixing uniformly. The OD of each well was read with a microplate reader at a wavelength of 450nm (reference wavelength of 630 nm).
The results are shown in fig. 4, the modified base aptamer obtained by screening has high affinity with DKK-1 protein, for example, the affinity constant of DKK28 is 84.480 ± 23.523 nM; DKK32 had an affinity constant of 30.244 ± 2.459 nM; DKK34 had an affinity constant of 19.731 ± 0.805 nM. Moreover, the binding of these aptamers to DKK-1 protein has high specificity, and hardly binds to other proteins (see fig. 5).
Example 6 specific binding of RNA aptamers obtained by screening to DKK-1 protein
Enzyme-linked aptamer adsorption assay (ELASA)
DKK-1 protein is diluted in PBS buffer solution (pH7.4, 0.22um filter membrane filtration preservation), and the final concentration is 200 ng/mL; 50uL per well were coated in a half volume ELISA microplate, sealed and incubated overnight at 4 ℃ in a 96 well plate. Abandoning the liquid in the holes, filling the holes with washing liquid, standing for 10 seconds, spin-drying, repeating for four times, and then patting on absorbent paper to dry. The RNA aptamers of the biotin-labeled DKK-1 were diluted in binding buffer in multiple ratios, each concentration being duplicate wells, with 8(9) concentrations (highest concentration was set according to the RNA aptamer affinity of DKK-1, respectively). Concentration dilutions were added to each well at 50uL volumes per well and blank control wells and BSA control wells were set. Seal plates and incubate at 25 ℃ for 2 hours. After repeating the manual plate washing step, 50uL of SA-HRP enzyme-labeled reaction solution is added into each hole, the plates are sealed, and the mixture is incubated for 1 hour at 25 ℃. The plates were washed manually, 50uL of TMB chromogenic mix was added to each well, the plates were closed and incubated at 37 ℃ for 20 minutes. Each hole is added with 50uL 1N H2SO4And (5) stopping the solution, and mixing uniformly. The OD of each well was read with a microplate reader at a wavelength of 450nm (reference wavelength of 630 nm).
The results are shown in fig. 6, the RNA aptamer obtained by screening has high affinity with DKK-1 protein, e.g., the affinity constant of DKK40 is 15.627 ± 1.156 nM; DKK43 had an affinity constant of 8.582 ± 0.544 nM. Moreover, the binding of these aptamers to DKK-1 protein has high specificity, and hardly binds to other proteins (see fig. 7).
The above-mentioned embodiments only express the embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the present invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.
Claims (8)
1. An aptamer recognizing DKK-1 protein, the aptamer having a nucleotide sequence shown as any one of SEQ ID nos. 4, 9 and 11.
2. Use of the aptamer of claim 1 for the preparation of a product for capturing and purifying DKK-1 protein.
3. Use of the aptamer of claim 1 for the preparation of a product for the in vitro detection of DKK-1 protein.
4. Use of the aptamer of claim 1 for the preparation of a product for diagnosing a DKK-1 associated disease.
5. The use of claim 4, wherein the DKK-1 associated disease comprises lung cancer, liver cancer, esophageal cancer, pancreatic cancer, myeloma, endometrial cancer, cervical cancer, rheumatoid arthritis.
6. A product for purifying or detecting DKK-1 protein in vitro comprising at least one aptamer sequence according to SEQ ID No.4, 9 or 11.
7. A product for diagnosing a DKK-1 related disease comprising at least one aptamer sequence represented by SEQ ID No.4, 9 or 11.
8. The product of claim 6 or 7, wherein the product comprises a kit or a detection chip.
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Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CA2446582A1 (en) * | 2001-05-17 | 2002-11-21 | Genome Therapeutics Corporation | Reagents and methods for modulating dkk-mediated interactions |
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Non-Patent Citations (2)
Title |
---|
Dickkopf-1: As a Diagnostic and Prognostic Serum Marker for Early Hepatocellular Carcinoma;Hao Yang等;《Int J Biol Markers》;20130927;第286-297页 * |
DKK-1、DKK-2和GPC3蛋白在肝细胞癌组织中的表达及其临床意义;赵衡等;《肿瘤》;20100531;第428-432页 * |
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