A nucleic acid aptamer that specifically binds to IGFBP3 and its application

Abstract

This invention discloses a nucleic acid aptamer that specifically binds to IGFBP3 and its application. It provides multiple nucleic acid aptamers that can specifically bind to IGFBP3 protein. These aptamers have the characteristics of small molecular weight, stable chemical properties, and easy storage and labeling, and can be used to prepare kits to detect the risk of cancer.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, specifically to a nucleic acid aptamer that specifically binds to IGFBP3 and its applications. Background Technology

[0002] The insulin-like growth factor (IGF) signaling system plays a central role in cell growth, differentiation, and proliferation. IGFBP3, the most abundant IGF-binding protein in human serum, is a growth-inhibiting and apoptosis-inducing molecule that exerts its effects through both IGF-dependent and IGF-independent mechanisms. It functions by arresting the cell cycle and inducing apoptosis. IGFBP3 is transported to the nucleus via a protein β-mediated mechanism, in which it has been shown to interact with retinoid X receptor α and possibly other nuclear elements. The antiproliferative signaling pathway of IGFBP3 requires an active transforming growth factor-β (TGF-β) signaling pathway, and IGFBP3 stimulates the phosphorylation of TGF-β signaling intermediates Smad2 and Smad3. IGFBP3 has an IGF-independent effect in inhibiting cancer cell proliferation. In vivo, nuclear transcription factors, retinoid X receptor (RXR)-α, and IGFBP3 functionally interact to reduce prostate tumor growth and prostate-specific antigen (PSA). Several clinical studies have shown that individuals with IGFBP3 levels in the higher-than-normal range may have a reduced risk of certain common cancers, including breast cancer, prostate cancer, colorectal cancer, and lung cancer. Furthermore, IGFBP3 can inhibit glucose uptake by adipocytes under insulin stimulation, independently of IGF. Therefore, the detection of IGFBP3 levels has significant clinical importance. Currently, the main detection method is the ELISA double-antibody sandwich method, which is time-consuming, requires IGFBP3 antibodies, is costly, has low accuracy, and cannot accurately detect protein content.

[0003] Nucleic acid aptamers are DNA or RNA molecules isolated through systematic evolution of ligands using exponential enrichment (SELEX) technology. They can bind with high affinity and specificity to corresponding targets such as proteins, metal ions, small molecules, peptides, and even whole cells, thus showing broad prospects in biochemical analysis, environmental monitoring, basic medicine, and new drug synthesis. Compared with antibodies, nucleic acid aptamers have advantages such as smaller molecular weight, better stability, ease of modification, no immunogenicity, and shorter production cycle, eliminating a series of processes such as animal immunization, feeding, protein extraction, and purification. Currently, there are no reports on using nucleic acid aptamers to detect IGFBP3 levels. Summary of the Invention

[0004] Purpose of the invention: The purpose of this invention is to provide one or more nucleic acid aptamers that specifically bind to the IGFBP3 protein. Another purpose of this invention is to provide a kit containing the aforementioned aptamers. A further purpose of this invention is to provide the application of the aforementioned nucleic acid aptamers in the preparation of in vitro diagnostic kits for the IGFBP3 protein.

[0005] Technical solution: In order to achieve the above-mentioned objective, the present invention provides a nucleic acid aptamer that specifically binds to IGFBP3, selected from any one or more nucleotide sequences with at least 90% homology to the nucleotide sequences shown in SEQ ID NO.1-SEQ ID NO.3, and / or any one or more nucleotide sequences that are complementary to the nucleotide sequences shown in SEQ ID NO.1-SEQ ID NO.3 and have at least 90% homology.

[0006] More specifically, the nucleic acid aptamer is selected from any one or more combinations of the nucleotide sequences shown in SEQ ID NO.1-SEQ ID NO.3, and / or any one or more combinations of nucleotide sequences complementary to the bases of the nucleotide sequences shown in SEQ ID NO.1-SEQ ID NO.3, as shown in the table below:

[0007]

[0008] This invention utilizes the in vitro exponential enrichment ligand phylogenetic evolution (SELEX) technique to screen and obtain nucleic acid aptamers that specifically bind to highly expressed IGFBP3 protein. These aptamers exhibit high specificity, small molecular weight, chemical stability, and ease of storage and labeling. The affinity (KD value) values ​​of these aptamers to the target IGFBP3 protein, as determined by SPR, do not exceed 50 nM. More preferably, the KD value of IGFBP3-61 reaches 26.8 nM.

[0009] Furthermore, the nucleic acid aptamers specifically binding to IGFBP3 described in this invention include not only the aforementioned single-stranded DNA specifically binding to the IGFBP3 protein, but also single-stranded DNA that is complementary to any one of the nucleotide sequences in SEQ ID NO.1-SEQ ID NO.3 and specifically binds to the IGFBP3 protein, or RNA transcribed from any one of the nucleotide sequences in SEQ ID NO.1-SEQ ID NO.3 and specifically binding to the IGFBP3 protein. All nucleic acid aptamers target the IGFBP3 protein.

[0010] It should be noted that the nucleic acid aptamer preferably exists in a secondary structure with minimum free energy (MFE), and necessarily includes stem-loop and loop-convex structures, thereby achieving specific binding with the IGFBP3 protein.

[0011] Furthermore, the homology of not less than 90% mentioned in this invention is limited to several base changes without altering the original secondary structure. In order to improve the stability of the secondary structure or increase the affinity with the target, those skilled in the art can make further modifications based on it, including but not limited to any one or more combinations of phosphorylation, methylation, amination, thiolation, replacing oxygen with sulfur, replacing oxygen with selenium, and isotopization.

[0012] Furthermore, the target is derived from human biological samples, including but not limited to any type of biological sample such as saliva, blood, or serum.

[0013] The present invention also provides a kit comprising the nucleic acid aptamers as described above.

[0014] Furthermore, the kit provided by the present invention may also contain reagents for purifying and / or detecting IGFBP3 protein. In addition, the kit provided by the present invention may further contain IGFBP3-expressing cells or tissues; and may further contain cells or exosomes for capturing said expressing cells or tissues.

[0015] In addition to preparing in vitro diagnostic kits as described above, the nucleic acid aptamers provided by this invention can also be used in immunotherapy for tumor treatment, and the kits provided by this invention can be used to detect IGFBP3 protein levels.

[0016] The nucleic acid aptamers provided by this invention are characterized by highly specific binding to IGFBP3 protein, small molecular weight, chemical stability, and ease of storage and labeling. For cancers known to be regulated by IGFBP3 protein levels, such as breast cancer, prostate cancer, colorectal cancer, and lung cancer, the provided kits can help detect the risk of developing these cancers and can also be used in immunotherapy for tumor treatment. Attached Figure Description

[0017] Figure 1 The secondary structure prediction diagram of IGFBP3-61 is shown, with MFE = -10.18 kcal / mol and an octane rating of 30 °C.

[0018] Figure 2 The secondary structure prediction diagram of IGFBP3-35 is shown, with MFE = -18.33 kcal / mol and temperature at 30 °C.

[0019] Figure 3The secondary structure prediction diagram of IGFBP3-48 is shown, with MFE = -12.84 kcal / mol and an octane rating of 30 °C.

[0020] Figure 4 The data (SPR data) are the affinity test data of the 6-round libraries (IG1-IG6) obtained in Example 2 of this invention with IGFBP3 and control proteins (M1, M2).

[0021] Figure 5 The affinity test results between the nucleic acid aptamer IGFBP3-61 and the IGFBP3 protein in Example 3 of this invention are shown.

[0022] Figure 6 The results of the ELISA experiment of nucleic acid aptamer IGFBP3-61 and IGFBP3 protein in Example 4 of this invention are shown. Detailed Implementation

[0023] The present invention will be further described below with reference to specific embodiments. However, those skilled in the art should understand that the following embodiments are intended to facilitate a better understanding of the present invention, and the present invention is not limited to these specific embodiments.

[0024] Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, the experimental materials used in the following examples are conventional biochemical reagents, which can be purchased commercially.

[0025] Example 1: Screening of ssDNA aptamers that specifically bind to IGFBP3 protein

[0026] 1. Design DNA libraries and primers

[0027] Random single-stranded DNA library: 5'-GGGACCAGCACACGCATAAC-36N-GCGTTATGCGTGCTACCGTG-3'; where "36N" represents a sequence composed of 36 arbitrary nucleotide bases. This library was synthesized by Sangon Biotech (Shanghai) Co., Ltd.

[0028] Primer information is shown in Table 1, and the primers were synthesized by General Biotech (Anhui) Co., Ltd.

[0029] Table 1. IGFBP3 primers and their sequences

[0030]

[0031] In this primer, S represents the forward primer, A represents the reverse primer, the 25 A's in the sequence represent a polyA tail composed of 25 adenosine nucleotides (A), and "Spacer 18" represents an 18-atom hexaethylene glycol intermediate arm. The structural formula of "Spacer 18" used in the above 3' primers is shown in formula (I).

[0032]

[0033] Primers were prepared into 100 μM stock solutions using DPBS buffer (DPBS: 100 mM, NaCl: 150 mM, KCl: 1 mM, MgCl2: 1 mM, CaCl2: 1 mM; pH 6.0, 25 °C) and stored at -20 °C for later use.

[0034] 2. Magnetic bead screening method

[0035] The magnetic bead method was used for screening, and a total of six rounds of screening were conducted. The screening process for each round is shown in Table 2.

[0036] Table 2. Screening process for IGFBP3 protein aptamers

[0037]

[0038] The specific filtering method is as follows:

[0039] 1) Carboxyl magnetic beads immobilize IGFBP3 (pI:) 8.38

[0040] Take 50 μl of carboxylated magnetic beads (Invitrogen, Dynabeads) TM MyOne TM Carboxylic Acid (#65012) was washed four times with 200 μl of ultrapure water. The magnetic beads were then hooked with a magnet, and the supernatant was discarded. Equal volumes of prepared NHS (N-hydroxysuccinimide; 0.1 M aqueous solution) and EDC (1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride; 0.4 M aqueous solution) were mixed and added to the magnetic beads. The mixture was incubated at 25°C for 20 minutes to activate the carboxyl groups on the surface of the magnetic beads. The beads were then washed twice with DPBS buffer and set aside for later use.

[0041] Take 20 μl of IGFBP3 protein (concentration of 0.5 mg / ml), add 30 μl of sodium acetate solution at pH 4.5 and mix well. Then add the mixture to the activated magnetic beads. Incubate at 25°C on a vertical mixer for 50 min. The IGFBP3 protein will couple to the surface of the magnetic beads through the amino groups on its surface.

[0042] After coupling, place the coupling tube on a magnetic rack, discard the supernatant, and add 100 μl of 1M ethanolamine (pH 8.5) to the magnetic beads. Incubate at 25°C on a vertical mixer for 10 min to block unreacted activation sites on the surface of the magnetic beads. Place the tube on a magnetic rack and discard the blocking solution. Wash the magnetic beads four times with 200 μl of DPBS and label them as MB-IGFBP3.

[0043] 2) Reverse screening

[0044] Back-screening was performed using magnetic beads conjugated with BSA protein (labeled MB-BSA). The method for conjugating BSA protein was the same as that for conjugating IGFBP3 protein. The BSA protein concentration was 0.5 mg / ml, diluted with 10 mM NaAC solution at pH 4.0. Before each round of positive screening targeting IGFBP3 protein, back-screening with BSA-conjugated magnetic beads was performed. The back-screening steps were as follows: After renaturation, the prepared single-stranded nucleotide library was incubated with 50 μl of MB-BSA magnetic beads at 25°C for 60 min on a vertical rotator. The library was then placed on a magnetic rack, and the supernatant was collected. This supernatant was used as the single-stranded nucleotide library for positive screening with MB-IGFBP3 magnetic beads.

[0045] 3) Magnetic bead screening

[0046] Take a 1 OD random single-stranded nucleotide library, centrifuge at 14000 rpm for 10 minutes, and centrifuge to the bottom of the tube. Dissolve the library in DPBS buffer to 10 μM and aliquot into PCR tubes for renaturation. The process is as follows: Set the PCR instrument program to 95°C for 10 minutes to unfold the strands, then incubate at 4°C for 5 minutes, and then at 25°C for 5 minutes. Add the processed library to 50 μL of MB-BSA magnetic beads, mix well, and incubate at 25°C for 30 minutes on a vertical mixer. Place on a magnetic rack, collect the supernatant, and label it "pool-". Use the supernatant as the single-stranded nucleotide library for positive screening with MB-IGFBP3 magnetic beads.

[0047] The filtered library was added to 50 μl of MB-IGFBP3 magnetic beads and incubated at 25 °C for 60 min on a vertical mixer. The mixture was then placed on a magnetic rack, the supernatant was discarded, and the magnetic beads were retained. The beads were washed four times with 200 μl of DPBS. After washing, the magnetic beads were added to 200 μl of DPBS and boiled in a water bath for 10 min. The supernatant was collected and labeled elution-IGFBP3.

[0048] Using the nucleic acid molecules in elution-IGFBP3 as templates, emulsion PCR (ePCR) was performed for amplification: all elution-IGFBP3 template was added to 2 ml of PCR mix and mixed well. Four volumes of ePCR microdroplet generating oil were added, and the mixture was vortexed to prepare an emulsion. The emulsion was divided into 100 μl portions and added to PCR tubes. The amplification conditions were as follows: 95℃ pre-denaturation for 2 minutes, 95℃ denaturation for 60 seconds, 60℃ annealing for 60 seconds, and 72℃ extension for 60 seconds, for a total of 35 cycles. The ePCR microdroplet generating oil was purchased from Anhui Aptamy Biotechnology Co., Ltd. (Catalog No.: EPO100). The PCR mix formulation is shown in Table 3.

[0049] Table 3 ePCR mix formulation

[0050] <![CDATA[ddH2O]]> 866μl 10*pfu enzyme buffer 100μl dNTPmix(10mM) 20μl Forward primer Lib1S1-ployA-FAM (100 μM) 5μl Reverse primer Lib1A2 (100 μM) 5μl Pfu enzyme 4μl (500U)

[0051] Purification of amplification products with n-butanol: Collect all ePCR products in 15ml conical centrifuge tubes, add 2 volumes of n-butanol, and vortex to mix thoroughly; centrifuge at 9000 rpm for 10 minutes at 25°C using a benchtop centrifuge; discard the upper phase (n-butanol) to obtain concentrated PCR amplification products, add TBE / urea denaturing buffer at a volume ratio of 1:1, boil for 15 minutes to denature the DNA, then incubate on ice for 1 minute, and perform urea-denaturing polyacrylamide gel electrophoresis on all samples at 300V until bromophenol blue reaches the bottom of the gel, separating the elongated FAM-labeled strands from the reversed strands. The formulation of 7M urea-denaturing polyacrylamide gel is shown in Table 4.

[0052] Table 4. Formulation of Modified Polyacrylamide Gel

[0053] Urea 3.78g 40% polyacrylamide 1.8ml 5*TBE 1.8ml <![CDATA[ddH2O]]> 2.25ml 10% APS 60μl TEMED 15μl

[0054] Gel extraction and recovery of FAM-tagged strands: Remove the gel and place it on a plastic membrane. Ex (nm): 495, Em (nm): 517 to detect the desired FAM-tagged ssDNA. Use a clean blade to cut the target band directly. Transfer the gel strip to a 1.5 ml EP tube and crush it. Add 1 ml ddH2O and boil in a water bath for 10 minutes to transfer the ssDNA from the gel to a solution. Centrifuge to remove gel fragments and retain the supernatant. Purify the supernatant with n-butanol using the same method as in section 2.4. Dialyze the obtained DNA single strands overnight using a 3KD dialysis bag; this can then be used as the library for the next round of screening.

[0055] The magnetic bead method was repeated for 6 rounds of screening. Each operation used the secondary library obtained in the previous operation as the starting nucleic acid library. During the screening process, SPR was used to detect the change in the recognition ability of the DNA single-stranded library for IGFBP3 protein. When the recognition ability of the DNA single-stranded library for IGFBP3 protein met the requirements, that is, the binding ability of the screened DNA single-stranded library to the target was higher than that of the library used in the initial screening, the obtained product was cloned and sequenced to obtain the nucleic acid aptamer.

[0056] In the screening method, the screening pressure can be increased round by round to improve the enrichment of nucleic acid aptamers and shorten the screening process. Increasing the screening pressure includes reducing the amount of single-stranded DNA library used, the amount of target protein used, and the incubation time for both; increasing the washing time; increasing the number of washing cycles; and increasing the amount of reverse screening magnetic beads used.

[0057] 3. Aptamer screening and affinity testing

[0058] After cloning and sequencing analysis of the enriched library products, several sequences were selected and synthesized by Shanghai Sangon Biotech. Affinity was then tested; the affinity testing method is detailed in Example 2. Three sequences were identified as having strong binding ability and, after verification, ideal affinity for binding IGFBP3 protein. These sequences were named IGFBP3-61 (SEQ ID NO.1), IGFBP3-35 (SEQ ID NO.2), and IGFBP3-48 (SEQ ID NO.3), respectively. The predicted secondary structure diagrams of these three nucleic acid aptamers are shown below. Figures 1 to 3 As shown. Figure 4 The study further demonstrated the change in affinity of IGFBP3-61 during the screening process, showing that as the screening rounds progressed, the affinity-rich libraries were continuously enriched, and the screening effect was good.

[0059] Example 2: Surface plasmon resonance (SPR) detection of affinity between nucleic acid aptamers and IGFBP3 protein.

[0060] Experimental methods:

[0061] 1) Take a CM5 chip (purchased from GE, part number BR100530), clean the chip twice with 1% SDS NaOH at a flow rate of 10 μl / min for 180 s, and clean it once with 400 μl 50 mM NaOH at a flow rate of 10 μl / min for 180 s;

[0062] 2) Chip activation, coupling, and blocking. (FC1 was activated and then blocked; FC2 was coupled with IGFBP3 protein 2800Ru; FC3 was coupled with control protein M1 (BSA protein) 2000Ru; FC4 was coupled with control protein M2 2600Ru).

[0063] 3) Activate channels 1, 2, 3 and 4 with a mixture of 0.1M NHS and 0.4M EDC (1:1) for 600 s at a flow rate of 5 μl / min;

[0064] 4) IGFBP3 protein was diluted with sodium acetate solution at pH 4.0 at a ratio of 1:10. The solution was injected into two channels at a time of 600 s and a flow rate of 5 μl / min, resulting in a 2800 RU eluent on the chip surface. Similarly, M1 protein was coupled to channel 3, resulting in a 2000 RU eluent on the chip surface. Control protein M2 was coupled to channel 3, resulting in a 2600 RU eluent on the chip surface. Channel 1 was left untreated.

[0065] 5) Block channels 1, 2, 3, and 4 with ethanolamine solution at pH 8.5 for 600 s at a flow rate of 5 μl / min;

[0066] 6) Affinity test: The screened library was diluted with DPBS to 500 nM * 100 μl and injected. The program was set as follows: channels 1, 2, 3, and 4; injection time 240 s, flow rate 30 μl / min, dissociation time 60 s; regeneration was performed with 1 M NaCl under the following conditions: injection time 60 s, flow rate 30 μl / min.

[0067] Experimental results: such as Figure 4 As shown, the library affinity increases with each round of screening, resulting in good screening performance. Furthermore, the library does not bind to the blank channel or control protein, indicating good specificity.

[0068] Example 3: Surface plasmon resonance detection of the affinity between nucleic acid aptamers and IGFBP3 protein.

[0069] Anhui General Biotechnology was commissioned to synthesize nucleic acid aptamer IGFBP3-61 (SEQ ID NO:1) and diluted with DPBS buffer to: 0.03125, 0.0625, 0.125, 0.25, 0.5, and 1 uM.

[0070] To conjugate IGFBP3 protein to the CM5 chip surface: First, clean the chip with 50 mM NaOH, then inject 20 μL at a flow rate of 10 μL / min. Next, activate the chip by injecting 50 μL of a mixture of equal volumes of EDC and NHS aqueous solution at a flow rate of 5 μL / min. Dilute the IGFBP3 protein to a final concentration of 50 μg / mL with 10 mM sodium acetate at pH 4.0, then inject 50 μL at a flow rate of 5 μL / min. The IGFBP3 protein conjugation amount is 5000 Ru. After injection, block the chip with ethanolamine at a flow rate of 5 μL / min, injecting 50 μL.

[0071] Detection: The kinetic detection parameters were set using a surface plasmon resonance spectrometer (GE Healthcare, model: Biacore T200). The injection rate was 30 μL / min for 3 min, the dissociation rate was 30 μL / min for 5 min, and the regeneration rate was 30 μL / min for 0.5 min with 1M NaCl. The diluted nucleic acid aptamers IGFBP3-61 were then injected.

[0072] The affinity assay results between the nucleic acid aptamer IGFBP3-61 and the IGFBP3 protein are as follows: Figure 5 As shown in Table 5, it can be seen that the value of aptamer-bound protein also increases with the continuous increase of aptamer concentration, and there is a good linear relationship. These data indicate that the IGFBP3-61 SPR instrument detected a strong binding to IGFBP3 protein. After systematic fitting, the final KD value was 26.8 nM.

[0073] Table 5 Affinity Parameters

[0074] ka(1 / Ms) Binding rate constant 4.093e4 Kd(1 / s) Dissociation rate constant 1.097e-3 KD(M) Affinity 2.680e-8

[0075] Example 4: ELISA verification of the affinity between nucleic acid aptamers and IGFBP3 protein.

[0076] Take a new microplate. Coat wells A1-A5 with 10 mg / ml BSA protein (diluted with pH 9.6 coating buffer, 0.05 M Na2CO3 / NaHCO3 buffer, pH 9.6), 100 μl per well, and incubate overnight at 4°C. Coat wells B1-C4 with 10 μg / ml IGFBP3 protein (diluted with pH 9.6 coating buffer), 100 μl per well, and incubate overnight.

[0077] After all proteins were coated, the BSA protein was diluted to 10 mg / ml with 1× pH 9.6 carbonate coating buffer, and 100 μl was added to each well to block any excess sites on the plate. The time was 24 h. The supernatant was removed, and the plate was washed once with DPBS (containing 0.05% Tween 20) and then spun dry.

[0078] Add 100 μl of 1 μg / ml IGFBP3 protein antibody (antibody purchased from Sinocare, biotin modified by ourselves, biotin-modifying reagent from Thermo Fisher) to wells A1-A2; add 100 μl of 500 nM IGFBP3-61 monoclonal antibody (biotin-modified) to wells A3-A4; add 100 μl of DPBS to well A5; add 100 μl of 500 nM IGFBP3-61 monoclonal antibody (biotin-modified) to wells B1-B12; add 1 μg / ml IGFBP3 protein antibody (biotin-modified) to wells C1-C4. Incubate at room temperature for 1 hour. After incubation, wash three times with DPBS (containing 0.05% Tween 20), placing the well on a shaker for 10 minutes each time, and then spin dry after each wash. Add SA-HRP (purchased from Beyotime, catalog number A0303) to DPBS at a ratio of 1:20000 (v / v) and incubate on a shaker at room temperature for 30 minutes. Wash three times with DPBS (containing 0.05% Tween 20), placing the container on a shaker for 10 minutes each time, and shake dry after each wash. Add 100 μl of TMB chromogenic buffer to each well and develop the color at room temperature for 1 minute, observing the color change visually.

[0079] The results are as follows Figure 6 As shown, A1-A5 are the blank groups, where neither the antibody nor the IGFBP3-61 monoclonal antibody binds to the BSA protein. B1-B12 are the monoclonal experimental groups, where the color change is obvious, indicating that the monoclonal antibody binds significantly to the IGFBP3 protein. C1-C4 are the antibody experimental groups, where the antibody binds significantly to the IGFBP3 protein. Comparing the monoclonal antibody and the antibody, the effects are not significantly different, both binding well to the IGFBP3 protein.

[0080] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A nucleic acid aptamer that specifically binds to IGFBP3, characterized in that, The nucleotide sequence of the nucleic acid aptamer is shown in SEQ ID NO.

1.

2. The nucleic acid aptamer that specifically binds to IGFBP3 according to claim 1, characterized in that: The target of the nucleic acid aptamer is the IGFBP3 protein.

3. The nucleic acid aptamer that specifically binds to IGFBP3 according to claim 2, characterized in that: The target was derived from human biological samples.

4. A reagent kit, characterized in that: Including the nucleic acid aptamer as described in claim 1.

5. The reagent kit according to claim 4, characterized in that: It also includes reagents for purifying and / or detecting IGFBP3 protein.

6. The use of the nucleic acid aptamer that specifically binds to IGFBP3 as described in claim 1 in the preparation of an in vitro diagnostic kit for IGFBP3 protein.

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