CN119570798A - Nucleic acid aptamer combined with GPRIN protein, application and screening method thereof - Google Patents

Abstract

The invention discloses a nucleic acid aptamer binding to GPRI N1 protein, an application and a screening method thereof. By improving the screening conditions, a nucleic acid aptamer with a small molecular weight, stable chemical properties, easy storage and labeling, capable of binding to GPRI N1 protein with high affinity and having very good specificity is obtained through screening. The nucleic acid aptamer can be used for detection, diagnosis, imaging and treatment, and has broad application prospects.

Description

Nucleic acid aptamer combined with GPRIN protein, application and screening method thereof

Technical Field

The invention relates to the technical field of biology, in particular to a nucleic acid aptamer combined with GPRIN protein, application and a screening method thereof.

Background

GRIN1 (Glutamate Ionotropic Receptor NMDA Type Subunit 1) is a novel protein with no substantial homology to known protein domains. It is expressed predominantly in the brain and binds specifically to activated G (z) alpha, G (o) alpha and G (i) alpha through its carboxy terminal region. The carboxy terminus of protein KIAA0514 (GRIN 2) is homologous to GRIN1 and also binds to activated G (o) alpha. GRIN1 and G (o) alpha are both membrane-bound proteins that are enriched in neurite outgrowth cones. Co-expression of GRIN1 or GRIN2 with activated G (o) alpha resulted in the formation of a fine process network in Neuro2a cells, suggesting that these pathways may function downstream of G (o) alpha to control neurite outgrowth.

Diseases associated with GRIN1 include neurodevelopmental disorders with or without hyperactivity and seizures, autosomal dominant and neurodevelopmental disorders with or without hyperactivity and seizures, autosomal recessive inheritance. Related pathways include deregulation of NMDA receptors, glutamate binding and activation and MAPK family signalling cascades.

Aptamer refers to DNA or RNA molecules obtained by screening and separating by an exponential enrichment ligand system evolution (SELEX) technology, 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 wide prospects in biochemical analysis, environmental monitoring, basic medicine, new drug synthesis and the like. Compared with an antibody, the nucleic acid aptamer has the advantages of small molecular weight, better stability, easy transformation and modification, no immunogenicity, short preparation period, capability of being synthesized artificially and the like, and is free of a series of processes of animal immunization, feeding, protein extraction, purification and the like. Therefore, if a nucleic acid aptamer with higher affinity with GPRI N protein and high specificity can be found, the detection of GPRI N protein with high sensitivity and high specificity can be realized, and the drug development targeting GPRI N protein can be facilitated.

Therefore, it is highly desirable to find a nucleic acid aptamer which has high binding affinity for GPRI N protein, good specificity, high binding affinity after accurate truncation, easy modification and artificial synthesis, good stability and convenient use.

Disclosure of Invention

In order to overcome at least one of the defects described in the prior art, the invention provides a nucleic acid aptamer binding to GPRI N protein, application and a screening method thereof. Can solve the problems.

The invention adopts the technical proposal for solving the problems that:

A nucleic acid aptamer binding to GPRI N protein, the nucleic acid aptamer having a nucleotide sequence as shown in SEQ ID NO.1, or having at least 30% homology with SEQ ID NO.1 and binding to GPRI N protein, or an RNA sequence transcribed from the nucleotide sequence as shown in SEQ ID NO. 1.

The inventor designs and synthesizes a random single-stranded DNA library and a corresponding primer based on a SELEX technology, and is used for screening a nucleic acid aptamer which has small molecular weight, stable chemical property, easy preservation and marking and can be combined with GPRI N protein with high affinity, so that the nucleic acid aptamer which is combined with GPRI N protein with high affinity is screened and obtained, and the nucleic acid aptamer has higher affinity with GPRI N protein for GRI N1-19 (SEQ ID NO. 1) and higher specificity.

It will be appreciated that nucleotide sequences having at least 30%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 92%, at least 94%, at least 96%, at least 98% or at least 99% homology to the nucleic acid aptamers provided herein and which bind to GPRI N protein, e.g. a portion of the nucleotide sequence shown in any of the above nucleic acid aptamers, or an added portion of the nucleotide sequence, may be deleted, still have a high affinity for GPRI N protein, and remain within the scope of the present invention.

In some embodiments, as an improvement over the above-described embodiments, a position on the nucleotide sequence of the above-described aptamer may be modified, e.g., phosphorylated, methylated, aminated, sulfhydrylated, substituted with sulfur, substituted with selenium, isotopically substituted with oxygen, etc., provided that the thus-modified aptamer sequence has desirable properties, e.g., may have an affinity for binding GPRIN1 protein equal to or greater than the parent aptamer sequence prior to modification, or may have greater stability, although the affinity is not significantly improved.

Thus, in some embodiments, the nucleotide sequence of the nucleic acid aptamer is modified and the modified nucleic acid aptamer specifically binds to GPRIN protein, the modification selected from at least one of phosphorylation, methylation, amination, sulfhydrylation, substitution of oxygen with sulfur, substitution of oxygen with selenium, and isotopicization, while remaining within the scope of the invention.

Further, the nucleic acid aptamer has a sequence which is at least 30% homologous to any one of SEQ ID NO.1 and which binds to the nucleotide sequence of GPRIN protein and is modified.

Further, the retrofitting includes at least one of the following retrofitting methods:

(1) Attaching a fluorescent label to the aptamer;

(2) Ligating a radioactive substance to the aptamer;

(3) Ligating a therapeutic substance to the aptamer;

(4) Ligating biotin to the aptamer;

(5) Ligating digoxin to the aptamer;

(6) Connecting a nano luminescent material on the nucleic acid aptamer;

(7) Ligating a small peptide to the aptamer;

(8) The siRNA is linked to the aptamer.

The conjugate of the aptamer disclosed by the invention refers to a conjugate formed by connecting other groups to the aptamer, such as fluorescent markers with marking effect, such as FAM, radioactive substances, therapeutic substances, biotin, digoxin, nano luminescent materials, small peptides, siRNA or enzyme markers, and the like, so that the modified aptamer sequence has desirable properties, such as affinity for binding GPRIN protein, which is equal to or higher than that of the parent aptamer sequence before modification, or higher stability although the affinity is not obviously improved.

In other words, the above nucleic acid aptamer, whether partially substituted or modified, has substantially the same or similar molecular structure, physicochemical properties and functions as the original nucleic acid aptamer, and can be used for binding to GPRIN protein.

The invention also provides a derivative of the nucleic acid aptamer, which adopts the nucleic acid aptamer combined with GPRIN protein, wherein the derivative of the nucleic acid aptamer comprises a phosphorothioate framework or peptide nucleic acid combined with GPRIN protein, which is modified by a nucleotide sequence framework of the nucleic acid aptamer or a conjugate of the nucleic acid aptamer.

The term "phosphorothioate backbone" as used herein has the meaning generally understood by those of ordinary skill in the art and refers to a phosphodiester backbone of RNA and DNA nucleic acid aptamers in which the non-bridging oxygen atoms may be replaced with one or two sulfur atoms, resulting in a phosphorothioate backbone having phosphorothioate or phosphorodithioate linkages, respectively. Such phosphorothioate backbones are known to have increased binding affinity for their targets, as well as increased resistance to nuclease degradation.

The term "peptide nucleic acid" as used herein has a meaning generally understood by those of ordinary skill in the art and refers to an analogue of an artificially synthesized DNA molecule, which was first reported by Nielsen et al in 1991. An oligonucleotide mimetic linked by peptide bonds, called a peptide nucleic acid, was synthesized using N-2- (aminoethyl) -glycine (N- (2-aminoethyl) -glycine) units instead of the sugar-phosphate backbone as the repeating structural unit. Since Peptide Nucleic Acids (PNAs) do not have a phosphate group as on DNA or RNA, the phenomenon of electrical repulsion between PNAs and DNA is lacking, resulting in a greater binding strength between the two than between DNA and DNA.

The invention also provides a nucleic acid aptamer and application of the nucleic acid aptamer derivative, wherein the nucleic acid aptamer comprises at least one of the following components:

(1) Quantitatively or qualitatively detecting GPRIN proteins;

(2) Purifying GPRIN protein;

(3) Imaging GPRIN protein 1;

(4) Preparing a drug targeting GPRIN proteins;

(5) Preparing reagents or medicaments for diagnosing and treating GPRIN a1 expression abnormality;

the invention also provides a screening method of nucleic acid aptamer combined with GPRIN protein, which comprises the following steps:

S1, synthesizing a random single-stranded DNA library and a primer;

S2, screening by a magnetic bead method, namely performing at least 7 rounds of reverse screening and screening, and adding serum at the beginning of round 4.

Further, in the step S2, the serum is added after the 4 th round, and the specific process is that the 5 th round of serum is added, the 20% of serum is added in the 5 th round, and the serum is normal human serum.

The conventional condition of aptamer screening is to carry out in an ion buffer, and serum with a certain concentration is gradually added under the screening condition, so that on one hand, because the serum contains abundant proteins, the serum can be competitively combined with the proteins on the surface of the magnetic beads to bind with the library, and therefore, sequences with weak binding capacity with the target GPRI N1 proteins or only adsorbed on the target GPRI N proteins are removed. On the other hand, the aptamer binds to a target in a serum environment, so that the actual detection environment of the application based on the development of the aptamer in the later period can be better met.

Research proves that in the last rounds of screening, the aptamer with higher affinity and better specificity can be obtained by adopting serum with higher concentration.

In summary, the nucleic acid aptamer combined with GPRI N protein, the application and the screening method thereof provided by the invention have the following technical effects:

1. By improving the screening conditions, screening to obtain a nucleic acid aptamer which has small molecular weight, stable chemical property, easy preservation and marking and can be combined with GPRI N protein with high affinity and high specificity;

2. The structure is relatively stable and simple, is easy to modify, can be artificially synthesized in a short period, has stable chemical properties, and is easy to store and mark;

3. The method can be used for detection, diagnosis, imaging, treatment and other aspects, such as purifying or detecting GPRI N protein with high sensitivity and high specificity, preparing medicines for targeting GPRI N protein, diagnosing and treating GPRI N expression abnormality reagents or medicines and the like, and has wide application prospect.

Drawings

FIG. 1 is a schematic diagram showing the binding ability of several rounds of enrichment library obtained by screening 1-7 rounds of QPCR (fluorescence quantitative PCR) detection and target protein in example 1;

FIG. 2 is a schematic diagram showing the dissolution curves of several rounds of enrichment library and target protein obtained by screening in rounds 1-7 of detection using QPCR (fluorescent quantitative PCR) in example 1;

FIG. 3 is a schematic diagram showing the retention rate of the positive screen library obtained by screening in the 1 st to 7 th rounds of example 1;

FIG. 4 is a graph showing the data of affinity detection of the nucleic acid aptamers GRIN1-19 and GRIN1 protein in example 2;

FIG. 5 is a graph showing the affinity detection data for the aptamer GRIN1-19 and B-Amyloid of example 3;

FIG. 6 is a graph showing affinity detection data for the aptamer GRIN1-19 with TF in example 3;

FIG. 7 is a graph showing affinity detection data for the aptamer GRIN1-19 and SNCA in example 3;

FIG. 8 is a graph showing affinity detection data for the aptamer GRIN1-19 and ADP7 of example 3;

FIG. 9 is a schematic diagram showing the results of detection GPRIN protein specificity by a spot hybridization assay of the aptamer of example 4.

FIG. 10 is a schematic representation of the results of the detection GPRIN protein concentration gradients in a spot hybridization assay of the aptamer of example 4.

Detailed Description

For a better understanding and implementation, the technical solutions of the embodiments of the present invention will be clearly and completely described and discussed below in conjunction with the accompanying drawings, and it is apparent that what is described herein is only a part, but not all, of the examples of the present invention, and all other embodiments obtained by those skilled in the art without making any inventive effort based on the embodiments of the present invention are within the scope of protection of the present invention.

For the purpose of facilitating an understanding of the embodiments of the present invention, reference will now be made to the drawings, by way of example, of specific embodiments, and the various embodiments should not be construed to limit the embodiments of the invention.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used herein in the description of the invention is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention.

Example 1 screening of ssDNA nucleic acid aptamers that bind to GPRIN protein

The method of screening ssDNA nucleic acid aptamers that bind GPRIN protein of this example comprises the steps of:

1. random single stranded DNA libraries and primers shown in the following sequences were synthesized:

random single-stranded DNA library:

5’-TTCAGCACTCCACGCATAGC(36N)CCTATGCGTGCTACCGTG AA-3’(SEQ ID NO.2);

Wherein "36N" represents a sequence of 36 arbitrary nucleotide bases joined together. The library was synthesized by the division of biological engineering (Shanghai).

Primer information is shown in Table 1 and is synthesized by Nanjing Jinsri Biotechnology Co.

TABLE 1 primers and sequences thereof

Primer name	Sequence (5 '-3')
		Lib13S1	TTCAGCACTCCACGCATAGC(SEQ ID NO.3)
Lib13-FAM-S1	FAM-TTCAGCACTCCACGCATAGC(SEQ ID NO.4)
		Lib13-Biotin-A2	Biotin-TTCACGGTAGCACGCATAGG(SEQ ID NO.5)
Lib13A2	TTCACGGTAGCACGCATAGG(SEQ ID NO.6)

Wherein S in the primer name represents the forward primer, and A in the primer name represents the reverse primer.

The primers were prepared with DPBS buffer (0.1 g/L of calcium chloride, 0.2g/L of potassium dihydrogen phosphate, 0.1g/L of magnesium chloride hexahydrate, 8g/L of sodium chloride, 2.8915g/L of disodium hydrogen phosphate dodecahydrate; pH7.4,25 ℃) to give 100. Mu.M stock solutions, and stored at-20℃for further use.

2. Magnetic bead method screening

The screening was carried out by a magnetic bead method for 7 rounds in total, and the screening flow of each round is shown in Table 2.

Table 2, GPRIN protein nucleic acid aptamer screening procedure

The specific screening process is as follows:

1) Carboxyl magnetic bead immobilized GPRIN protein

Mu.l of carboxyl magnetic beads (Jiangsu Biotechnology Co., ltd., product number: FM 2221) were taken, washed with 200. Mu.l of ultrapure water for 4 times, and the magnetic beads were magnet-fished to remove the supernatant. Taking prepared NHS (N-hydroxysuccinimide; 0.1M aqueous solution) and EDC (1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride; 0.4M aqueous solution) which are 100 mu L each, mixing in equal volume, adding into magnetic beads, incubating at 25 ℃ for 20 minutes to activate carboxyl groups on the surfaces of the magnetic beads, and washing the magnetic beads with DPBS buffer for 2 times for later use.

Mu.l GPRIN protein (P03428 PA, available from Eboson science and technology Co., ltd., concentration 1mg/ml after dissolution) was taken, and 468. Mu.l of 10mM sodium acetate, pH5.5, was added to the activated magnetic beads and mixed well. Incubation was performed on a vertical mixer for 60 minutes at 25 ℃, and GPRIN protein was coupled to the surface of the magnetic beads via the amino groups on the protein surface.

After the coupling, the coupling tube was placed on a magnetic rack, the supernatant was aspirated off, 500. Mu.l of 1M ethanolamine pH8.5 was added to the beads, incubated on a 25℃vertical mixer for 10 minutes, and unreacted activation sites on the surface of the beads were blocked. Placing on a magnetic rack, and absorbing and discarding the sealing liquid. The beads were washed 4 times with 250 μl DPBS, labeled MB-GPRIN1.

2) Reverse screening and screening

The preparation of the anti-sieve magnetic beads comprises the step of coupling His small peptide with the magnetic beads, wherein the His small peptide is chemically synthesized by Nanjing Jinsri biotechnology limited company, and the step of coupling the His small peptide is the same as the step of coupling GPRIN protein. His small peptide concentration is 10mg/ml, with pH4.5 10mM NaAC solution dilution, specifically, 10 u lHis small peptide, adding 90 u l pH4.5 10mM NaAc solution mixing. The rest steps are the same. The coupled magnetic beads were labeled MB-His.

Library dissolution and renaturation treatment, namely, 1OD random single-stranded nucleotide library is taken, centrifuged at 14000rpm for 5 minutes, the library is centrifuged to the bottom of a tube, and is dissolved to 10 mu M by using DPBS buffer solution, and the mixture is split into PCR tubes for renaturation treatment after uniform mixing. The procedure is as follows, the PCR instrument is programmed to hold at 95 ℃ for 10 minutes, the purpose of this step is to unwind the folded strand, then hold at 4 ℃ for 5 minutes, and then equilibrate to room temperature. The treated library was added to 50. Mu.l of MB-His magnetic beads, mixed and incubated on a vertical mixer for a period of time at room temperature. Placing the mixture on a magnetic rack, collecting supernatant, marking the supernatant as pool-, and performing positive screening on the supernatant serving as a single-stranded nucleic acid library and MB-GPRIN1 magnetic beads. For each round of magnetic bead screening, the MB-His was used for the reverse screening prior to the forward screening targeting GPRIN protein, and the reverse screening supernatant was used as a single-stranded nucleotide library for the forward screening with MB-GPRIN magnetic beads. Specifically, the library pool after reverse screening was added to 50. Mu.l MB-GPRIN1 magnetic beads and incubated at 25℃for 40 minutes on a vertical mixer. The supernatant was removed and the beads were retained and washed 4 times with 200 μl DPBS. Finally, 200 mu lDPBS of the magnetic beads after washing was added, the mixture was bathed in boiling water for 10 minutes, and the supernatant was collected and labeled elution-GPRIN1.

The nucleic acid molecules elution-GPRIN1 were used as templates for amplification by conventional PCR. The method comprises adding all templates elution-GPRIN1 into 2ml PCR mix, mixing, dividing the mixture of templates and PCR mix into 100 μl/tube, adding into PCR tube, and preserving at 95deg.C for 2 min, 95deg.C for 60 s, 60 s for annealing at 60deg.C, 72 s for 25 cycles, and 4 deg.C. PCR mix was formulated from dNTPs purchased from Norflua (P031-02) and rtaq enzyme purchased from Baozhen (R500Z).

The amplified products were purified using commercially available world and SA magnetic beads (SM 017100) to prepare a secondary library for the next round of screening. 2mL of the PCR product was added with 1/5 volume of 4M sodium chloride, 160uL of SA magnetic beads washed with DPBS and the supernatant was removed, and after 30min incubation at room temperature in a shaker, the supernatant of the PCR product was removed. The beads were washed three times with DPBS containing 0.02% Tween20, and after removal of the supernatant, 100uL of 40mM sodium hydroxide solution was added, and after three minutes incubation, the beads were removed by magnetic attraction. Then 4uL of 1M hydrochloric acid is added into the supernatant to neutralize the single chain, then 104uL of 2 xDPBS is added for salt concentration dilution and neutralization, and finally 208uL of secondary library dissolved in 1 xDPBS is obtained, and the secondary library can be used as a library for the next round of screening.

The magnetic bead method was repeated for 7 rounds, each run was performed using the secondary library obtained in the previous run as the starting nucleic acid library, and after the library was subjected to the variegation treatment, it was incubated with MB-GPRIN1 magnetic beads. In the screening process, SPR is used for detecting the change of the recognition capability of the DNA single-chain library to GPRIN th round protein, when the recognition capability of the DNA single-chain library to GPRIN th protein meets the requirement, namely, the binding capability of the screened DNA single-chain library to target protein is higher than that of the library (figure 1) which is input by the screening initiation, in figure 1, p2, p3, p4 and p6 respectively represent libraries obtained by screening in rounds 1, 2,3, 4 and 6, the affinity of library targets obtained in round 5 and 6 is high, and p6 is much higher than that of p1 and p2, thereby meeting the sequencing requirement, and the obtained library is subjected to high-throughput sequencing analysis.

3. Analyzing and identifying the aptamer obtained after screening, namely, after high-throughput sequencing analysis of the obtained enriched library product, selecting a plurality of sequences to be synthesized by Jin Weizhi biological (Jiangsu) technology Co., ltd, and detecting the affinity.

In the subsequent detection, from the 100 sequences obtained in the final round 7, 1 sequence with the strongest binding capacity was determined, and the aptamer having the nucleotide sequence shown as SEQ ID NO.1 was designated GRIN1-19, and the specific sequence of SEQ ID NO.1 was TTCAGCACTCC ACGCATAGCACGGTCTGTGCCTGCGGTTGTTCAAGGGCTGTATTGCC TATGCGTGCTACCGTGAA.

Example 2 Surface Plasmon Resonance (SPR) detection of the affinity of GPRIN protein nucleic acid aptamer to GPRIN1 protein

The aptamer GRIN1-19 (SEQ ID NO. 1) was synthesized by Suzhou gold intellectual Biotechnology Co., ltd and diluted to 500nM with DPBS buffer.

1. The GPRIN protein was coupled to the 2 nd channel on the CM5 chip surface by washing the chip with 50mM NaOH at a flow rate of 10. Mu.l/min, and then mixing two reagents of equal volumes of EDC (1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride; 0.4M aqueous solution) and NHS (N-hydroxysuccinimide; 0.1M aqueous solution) and feeding 50. Mu.l of the activated chip at a flow rate of 5. Mu.l/min. GPRIN1 protein was diluted with 10mM sodium acetate, pH5.5, to a final concentration of 20. Mu.g/mL, and the sample was introduced at a volume of 50. Mu.L, at a flow rate of 5. Mu.L/min, and at a GPRIN1 protein coupling amount of 3359Ru. After the sample injection was completed, the chip was blocked with ethanolamine at a flow rate of 10. Mu.L/min and 100. Mu.L was injected. Channel 1 was treated as described above, the His protein coupling step, the activation and blocking steps were identical and served as control channels.

2. The detection comprises the steps of setting detection parameters by using a surface plasmon resonance instrument (GE HEALTHCARE, model: biacore 8K), sequentially flowing diluted 1 aptamer sample through the 1 channel and the 2 channel, wherein the procedure of each aptamer comprises the steps of sampling 30 mu L/min, time for 2 min, dissociating 30 mu L/min, time for 2 min, regenerating 1M NaCl 30 mu L/min, time for 30 seconds, and sequentially sampling diluted 1 aptamer.

The affinity detection data of the aptamer GRIN1-19 and GPRIN protein are shown in FIG. 4, and KD values are shown in Table 3 below, which illustrate the binding capacity of the corresponding aptamer to the target protein GPRIN1 protein. These data demonstrate that these nucleic acid aptamers all detected strong binding to GPRIN protein with the SPR instrument.

Table 3, affinity of aptamer to GPRIN protein (smaller KD value means greater affinity)

Nucleic acid aptamer	Affinity KD (nM) with GPRIN protein
		GRIN1-19(SEQ ID NO.1)	8.90

As can be seen from Table 3, the affinity of 1 nucleic acid aptamer (SEQ ID NO. 1) obtained by screening from 100 sequences to GPRIN proteins was particularly high.

EXAMPLE 3 specificity Studies of nucleic acid aptamer

In this example, the protein B-Amyloid, TF, SNCA, ADP was used in place of the protein GPRIN, and the protein B-Amyloid, TF, SNCA, ADP was coupled to the 2 nd channels of the four Channal CM5 chips in the same manner as in example 4 in which the protein GPRIN was immobilized on the SPR chip, with coupling amounts of 322RU, 2268RU, 1413RU, and 3824RU, respectively. And sequentially injecting 1 piece of diluted GRIN1-19 aptamer.

Affinity detection data of the aptamer GRIN1-19 and B-Amyloid are shown in FIG. 5, affinity detection data of TF and SNCA are shown in FIG. 6, and affinity detection data of ADP7 and ADP7 are shown in FIG. 8;

it can be seen that the aptamer GRIN1-19 has very good specificity.

Example 4 detection of GPRIN protein by nucleic acid aptamer-based Spot hybridization experiments

The procedure of the spot hybridization experiment performed in this example for 1 aptamer GRIN1-19 with high affinity and high specificity is as follows:

1.2 nitrocellulose membranes (available from Millipore) 8 cm. Times.2 cm were used, GPRIN protein was diluted to 1mg/ml with DPBS, and control protein B-Amyloid, TF, SNCA, ADP7 was also diluted to 1mg/ml, respectively. Spotting 2ul onto nitrocellulose membrane, and naturally air-drying for 40 min.

2. After drying, the mixture was blocked with 10% BSA at room temperature for 1 hour, and after the blocking, the mixture was washed 3 times with DPBST (DPBS contains 0.5 th of a tween 20), and blotted.

3. After the cellulose membrane was washed clean, the aptamer synthesized by the gold-only biosynthesis in example 3 was diluted to 0.5uM with biotin modification, and then the diluted aptamer and the protein on the nitrocellulose membrane were incubated for 30min at room temperature on a shaker. After incubation, DPBST was used three times, and the solution was placed on a shaker for 5 minutes each.

4. The test strip was then transferred to a new centrifuge tube, and HRP-strepitavidin (purchased from Beyotime Biotech mg/mL) was diluted in a ratio of DPBS buffer=1:2000, 3mL was added to wet it out, and incubated at room temperature for 30min in the absence of light. After the incubation, DPBS-T is used for washing for 2min, and the washing is repeated for 3 times, and the washing is sucked.

5. The test strip was transferred to clean PE glove, ECL chromogenic kit (purchased from Beyotime Biotech), 100uL of solution A and 100uL of solution B were mixed, the surface of the test strip was soaked, and incubated in the dark for 5min.

6. Imaging system observing and photographing, using the instrument as ImageQuantTMLAS digital imaging system of GE medical life department.

The results are shown in FIG. 9, which is a graph showing the results of specific detection using GRIN 1-19.

As can be seen from FIG. 9, the biotin-modified aptamer GRIN1-19 can be used for detection of the membrane hybridization GPRIN protein, and the GPRIN protein is apparent compared with the spots of the control protein B-Amyloid, TF, SNCA, ADP7, which indicates that the aptamer can be used for accurately detecting GPRIN protein. Thus, the aptamer GRIN1-19 was used for the detection of GPRIN protein.

In this example, the concentration gradient of GRIN1-19 was used for GPRIN protein detection, and the GRIN1-19 was diluted with DPBS to 1mg/mL, 0.50mg/mL, 0.250mg/mL, 0.125mg/mL, 0.063mg/mL, and 0.032mg/mL, and then the spot hybridization was performed according to the above method, and as shown in FIG. 10, the aptamer GRIN1-19 was used for accurate detection of GPRIN protein at 0.032 mg/mL.

Therefore, GRIN1-19 is most preferable, and the sensitivity and accuracy of the detection result are the highest.

The technical means disclosed by the scheme of the invention is not limited to the technical means disclosed by the embodiment, and also comprises the technical scheme formed by any combination of the technical features. It should be noted that modifications and adaptations to the invention may occur to one skilled in the art without departing from the principles of the present invention and are intended to be within the scope of the present invention.

Claims (8)

1. A nucleic acid aptamer binding to GPRIN protein 1, which is characterized in that the nucleic acid aptamer has a nucleotide sequence shown as SEQ ID NO.1, or has at least 30% homology with SEQ ID NO.1 and binds to GPRIN protein, or is an RNA sequence transcribed from the nucleotide sequence shown as SEQ ID NO. 1.

2. A nucleic acid aptamer that binds to a GPRIN protein according to claim 1, wherein the nucleotide sequence of the nucleic acid aptamer is modified by a modification method comprising one or more of phosphorylation, methylation, amination, sulfhydrylation, substitution of oxygen with sulfur, substitution of oxygen with selenium and isotopicization.

3. The aptamer of claim 2, wherein the aptamer has a sequence that is at least 30% homologous to any one of SEQ ID No.1 and that binds to the nucleotide sequence of GPRIN protein and is engineered to bind to the protein GPRIN.

4. A nucleic acid aptamer that binds to GPRIN protein according to claim 3, wherein said engineering comprises at least one of the following engineering methods:

(1) Attaching a fluorescent label to the aptamer;

(2) Ligating a radioactive substance to the aptamer;

(3) Ligating a therapeutic substance to the aptamer;

(4) Ligating biotin to the aptamer;

(5) Ligating digoxin to the aptamer;

(6) Connecting a nano luminescent material on the nucleic acid aptamer;

(7) Ligating a small peptide to the aptamer;

(8) The siRNA is linked to the aptamer.

5. A nucleic acid aptamer derivative employing a nucleic acid aptamer according to any one of claims 1-4 that binds to GPRIN a protein, wherein the nucleic acid aptamer derivative comprises a phosphorothioate backbone or peptide nucleic acid that binds to GPRIN a protein engineered from the nucleotide sequence backbone of the nucleic acid aptamer or conjugate of the nucleic acid aptamer.

6. The aptamer of any one of claims 1 to 4 and the use of the aptamer derivative of claim 5, comprising at least one of the following:

(1) Quantitatively or qualitatively detecting GPRIN proteins;

(2) Purifying GPRIN protein;

(3) Imaging GPRIN protein 1;

(4) Preparing a drug targeting GPRIN proteins;

(5) Reagents or medicaments for diagnosing and treating GPRIN a1 expression abnormalities are prepared.

7. A method for screening a nucleic acid aptamer that binds to GPRIN protein, comprising the steps of:

S1, synthesizing a random single-stranded DNA library and a primer;

S2, screening by a magnetic bead method, namely performing at least 7 rounds of reverse screening and screening, and adding serum at the beginning of round 4.

8. The method for screening nucleic acid aptamer binding to GPRIN protein according to claim 7, wherein in step S2, serum is added after round 4, specifically, 5% serum is added in round 4, 20% serum is added in round 5, and the serum is normal human serum.