CN111549034A

CN111549034A - Aptamer specifically binding with chemokine ligand-5 and application thereof

Info

Publication number: CN111549034A
Application number: CN202010437574.0A
Authority: CN
Inventors: 张金阳; 胡腾; 张阿梅; 宋玉竹; 韩芹芹; 夏雪山; 王炳辉; 徐瑞贤
Original assignee: Kunming University of Science and Technology
Current assignee: Kunming University of Science and Technology
Priority date: 2020-05-21
Filing date: 2020-05-21
Publication date: 2020-08-18
Anticipated expiration: 2040-05-21
Also published as: CN111549034B

Abstract

The invention discloses a nucleic acid aptamer specifically combined with chemokine ligand-5, the nucleotide sequence of which is shown as SEQ ID NO. 1, the invention takes chemokine ligand-5 with His label as positive sieve protein, takes fixed medium Ni-NTA as reverse sieve, clones the nucleic acid aptamer secondary library on pMD19T carrier, and obtains the nucleic acid aptamer specifically combined with chemokine ligand-5 through clone sequencing; the experimental result shows that the screened aptamer has an inhibiting effect on the chemotactic effect of the chemokine ligand-5; the invention has potential value in the detection of the chemotactic factor and the treatment of the diseases related to the chemotactic factor ligand CCL 5.

Description

Aptamer specifically binding with chemokine ligand-5 and application thereof

Technical Field

The invention relates to a nucleic acid aptamer specifically bound with chemokine ligand-5 (CCL 5) and application thereof, belonging to the technical field of biomedicine.

Background

Chemokine ligand 5 (CCL 5) is a regulator of many immune, allergic, and inflammatory responses, involved in the migration and accumulation of lymphocytes, monocytes, and eosinophils at sites of inflammatory and pathological injury in tissues and organs, and also a mediator of the angiogenic process. The chemokine CCL5 induces an inflammatory immune response by binding to cell surface chemokine receptors. CCL5 can induce leukocyte infiltration into inflammatory sites and participate in many inflammatory responses. It plays an important role in the pathophysiological processes of many diseases, such as diabetes, multiple sclerosis, rheumatoid arthritis, organ transplant rejection, cardiovascular and cerebrovascular diseases, human immunodeficiency virus, etc. In conclusion, the CCL5 aptamer can be potentially valuable for detecting chemokines and treating diseases related to the chemokine CCL 5.

Disclosure of Invention

The invention aims to provide an aptamer of chemokine ligand-5 (CCL 5) with high specificity and affinity, wherein the nucleotide sequence of the aptamer is shown as SEQ ID NO. 1.

The screening method of the specific CCL5 aptamer is based on the principle of a ligand index Enrichment Evolution system (Systematic Evolution of Ligands by exogenous genetic engineering), recombinant CCL5 protein with a His label is used as a positive screen protein, a fixed medium Ni-NTA is used as a reverse screen, and the aptamer specifically combined with chemokine ligand-5 protein is screened out.

The invention carries out modification including but not limited to FITC, biotin, amino, polyethylene glycol and the like on the 5 'end or the 3' end of the aptamer so as to prolong the in vivo half-life and trace the application.

The invention adopts the following technical scheme to realize the purpose of the invention:

1. screening of aptamers that specifically bind to chemokine ligand-5

The screening method of the mouse chemokine ligand-5 specific aptamer is based on the principle of a ligand index Enrichment Evolution system (Systematic Evolution of Ligands by amplification) and uses the chemokine ligand-5 with a His label as a positive sieve protein and a fixed medium Ni-NTA as a reverse sieve, the obtained aptamer secondary library is cloned on a pMD19T carrier, and the nucleic acid aptamer specifically combined with the chemokine ligand-5 is obtained by cloning and sequencing, and the nucleotide sequence of the nucleic acid aptamer is as follows:

5‘-ATCCAGAGTGACGCAGCACTGAAAGACCGGCAGTTACGCGTGGAGGGGTGGATGGTTATGGACACGGTGGCTTAGT-3’。

2. aptamer secondary structure prediction

The prediction of the secondary structure of the aptamer of the chemokine ligand-5 by using online software MFold shows that the aptamer molecule has a stem-loop structure, the secondary structure of which is shown in figure 1, and Gibbs free energy: Δ G = -4.7421.

3. Affinity and specificity detection of CCL-5 protein specific aptamers

Synthesizing a biotin-labeled chemokine ligand-5 specific aptamer, and detecting the affinity and specificity of the aptamer by an enzyme-linked oligonucleotide detection method; the result shows that Apta-mCCL5-21 has high specificity and affinity.

4. Inhibition of cell chemotaxis by chemokine ligand-5 specific aptamers

Placing the recombinant chemokine ligand-5 protein and the synthesized nucleic acid aptamer without a mark in RAW264.7 cell culture solution, and detecting cell migration through a transwell experiment; the results show that chemokine ligand-5 specific aptamers are capable of inhibiting the chemotaxis of chemokine ligand-5.

Another objective of the invention is to apply the aptamer binding specifically to chemokine ligand-5 in the preparation of a reagent or kit for detecting chemokine ligand-5.

Another objective of the present invention is to use the aptamers that specifically bind to chemokine ligand-5 as described above in the preparation of formulations that inhibit the chemotactic activity of chemokine ligand-5.

Another object of the present invention is to use the above-mentioned aptamers that specifically bind to chemokine ligand-5 as molecular probes.

Compared with the prior art, the invention has the advantages that:

1. the aptamer of the invention has the advantage of high specificity, only has the capability of recognizing the chemokine mCCL5 protein, and has no or weaker recognition power for other proteins and the like;

2. the aptamer has the characteristics of high affinity, capability of in vitro synthesis and modification, stable chemical property, good pharmacokinetic property, no immunogenicity and the like;

3. compared with the preparation of an antibody, the synthesis of the aptamer has lower cost; the period is short, and the stability and the repeatability are good;

4. the aptamer has wide application prospect in the aspects of clinical laboratory diagnostics and clinical targeted therapy.

Drawings

FIG. 1 is a schematic diagram of the secondary structure of an aptamer of the invention;

FIG. 2 is a graph plotting dissociation constants of aptamers using the ELONA method; the abscissa of the graph is the concentration of the biotin-labeled aptamer, and the ordinate is the absorbance value at 450 nm;

FIG. 3 shows the result of the specificity analysis of the aptamer by the ELONA method, in which the abscissa, from left to right, represents the blank control BSA, the recombinant rabies virus L protein (RABV-L), the recombinant ZIKV-PrM protein, etc., and the recombinant chemokine ligand-5;

FIG. 4 shows the result of using transwell experiment to inhibit chemotactic effect of chemokine mCCL5 on aptamer, wherein a and d are negative controls; b. panel e is a positive control treated with 20 μ g of recombinant chemokine mCCL 5; c. panel f is experimental group, 20. mu.g recombinant chemokine ligand-5 and 200. mu. mol/L aptamer together;

FIG. 5 shows the cell count results of each experimental group in example 5;

in the above-mentioned figure, Apta #21 is aptamer Apta-mCCL 5-21.

Detailed Description

The present invention is further illustrated by the following figures and examples, without limiting the scope of the invention thereto, wherein the process is carried out in a conventional manner unless otherwise specified, and wherein reagents are used, such as reagents used or formulated in a conventional manner, unless otherwise specified.

Example 1: construction of prokaryotic expression vector of mouse chemotactic factor CCL5 and expression and purification of recombinant protein

The chemokine ligand-5 prokaryotic expression vector construction and recombinant protein expression and purification method refers to the prokaryotic expression and purification of murine CCL5 protein, university of Kunming technology, 2019, 44 (2): 97-104 ".

Example 2: screening of murine chemokine ligand-5 specific aptamers

1. Coupling of recombinant CCL-5 protein to Ni-NTA

The recombinant CCL-5 protein is fixed in a Ni-NTA medium, and the specific steps are as follows: 1mL of renatured CCL-5 protein is taken and placed at 4 ℃ to melt the recombinant protein for later use; putting 1mL of Ni-NTA into a chromatographic column, discarding a preservation solution contained in a fixed medium, and washing the fixed medium by using deionized water with 5 times of column volume to remove residual preservation solution; transferring the thawed recombinant protein solution to a chromatographic column containing a Ni-NTA medium; the mixture was placed in a shaker at room temperature and shaken slowly at 200 rpm for about 4 h.

2. In vitro screening of CCL-5 aptamers

In order to obtain a nucleic acid aptamer having high specificity and high affinity, stringent screening conditions can be appropriately used. In the screening process, the incubation time of ssDNA and recombinant protein is gradually shortened, the concentration of the recombinant protein in the screening process is reduced, the washing times are properly increased, and negative screening steps are added, and the SELEX scheme for screening the CCL-5 protein aptamer is specifically as follows:

TABLE 1 CCL-5 protein aptamer SELEX screening protocol

Note: (+) indicates the addition of a negative selection step at the initial step of selection; (-) indicates no negative selection step was added at the start of the selection.

3. Negative screening: the designed nucleic acid aptamer library has extremely high abundance of nucleic acid sequences, and sequences capable of being combined with an immobilization medium may exist, so that the situation that nonspecific combination, namely the sequences capable of being combined with the immobilization medium and having low affinity with a target molecule, is avoided in subsequent screening. Prior to incubation of the recombinant protein with the library, the nucleic acid library is incubated with Ni-NTA media which is not immobilized with the recombinant protein. The efficiency of target molecule screening and the specificity of the aptamer are improved by removing part of the nucleic acid which is compatible with the medium; the negative screening comprises the following steps:

preparation of single-stranded library: uniformly mixing a 20 mu L100 mu M single-chain aptamer library in 100 mu L PBS buffer solution; heating at 95 deg.C for 10min, and cooling at 4 deg.C for use, wherein the aptamer library is synthesized by Shanghai Bioengineering Co., Ltd. (5 '-ATCCAGAGTGACGCAGCANNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNTGGACACGGTGGCTTAGT-3');

washing Ni-NTA: putting fresh Ni-NTA medium into the assembled chromatographic column, and removing the preservation solution; washing the Ni-NTA medium with 5 times of deionized water to remove redundant preservation solution;

③ incubation of the single-stranded library with Ni-NTA: mixing the prepared single-chain library with the washed Ni-NTA medium, and adding 2mL of coupling buffer solution to completely suspend the Ni-NTA; oscillating for 2 hours at room temperature at 20 rpm;

(iv) single-stranded library collection: collecting a flow liquid from the chromatographic column, washing the Ni-NTA medium by using 2mL of coupling buffer solution in a grading way, and collecting the flow liquid which is used as a screening initial library;

4. aptamer forward screening based on Ni-NTA affinity chromatography column

Firstly, a clean chromatographic column is assembled for standby; closing the outlet of the chromatographic column, adding the recombinant protein-Ni-NTA conjugate into the chromatographic column, opening the outlet, and washing the chromatographic column for multiple times by using a coupling buffer solution;

the first round of screening directly uses the aptamer library synthesized by Shanghai bioengineering technology company; preparing the obtained secondary library into a single-chain library and uniformly mixing the single-chain library with the conjugate for the rest screening rounds, and incubating for 1h at room temperature;

washing of non-specific nucleic acid aptamers: multiple washes with 10mM PBS buffer to remove non-specifically bound single-stranded library;

④ eluting the single-chain DNA-recombinant protein complex with three to four times of column volume of elution buffer solution (50 mM, EDTA; 10mM PBS solution) and recovering the ssDNA in the collected liquid, wherein the recovery step comprises a, adding isopropanol into all the collected liquid to make the final volume fraction of the collected liquid 20%, the purpose of the step is to increase the recovery rate of the single-chain DNA, transferring the mixed liquid to an adsorption column in batches, centrifuging at 12000rpm for 1min, then discarding the waste liquid in the collection tube, b, adding 700 muL rinsing solution of absolute ethyl alcohol into the collection tube in advance, centrifuging at 12000rpm for 1min, discarding the waste liquid in the collection tube, washing once again, the step is to remove impurities such as protein in the sample, c, centrifuging at 12000rpm for 2min to remove the redundant rinsing solution, placing the adsorption column at room temperature, standing for 10-15min, completely airing, d, transferring the adsorption column to a clean centrifuge tube, suspending and adding 30-80 muL of preheated sterile ddH into the center of the adsorption column₂And O, standing at room temperature for 10min, centrifuging at 12000rpm for 2min, and collecting the single-stranded library (the obtained liquid can be added into the center of the adsorption membrane again, and the centrifugation and recovery can be repeated, so that the recovery amount of ssDNA in the collected liquid can be increased).

Marking the recovered library, and performing PCR amplification by using the collected library as a template to prepare a secondary screening library, wherein the RCR reaction system is as follows, and the primer sequences are as follows:

a forward primer: ATCCAGAGTG ACGCAGCA, respectively;

reverse primer: ACTAAGCCAC CGTGTCCA, respectively;

TABLE 2 Single-stranded DNA library amplification System

And carrying out PCR reaction after the 20 mu L system is configured, wherein the reaction procedure is as follows: denaturation at 94 deg.C for 5 min; wherein 35 cycles comprise denaturation at 94 ℃ for 30s, annealing at 55 ℃ for 30s, and extension at 72 ℃ for 20 s; finally extending for 5 min; the reaction was terminated at 16 ℃.

Carrying out agarose gel recovery on the library subjected to PCR amplification to obtain a pure library fragment; putting dsDNA in the recovery solution in a metal bath, heating at 95 ℃ for 10min, and completely cooling in a refrigerator; obtaining single-stranded DNA for next screening;

sixthly, repeating the steps and carrying out 10 rounds of screening; after the chromatographic column is assembled, adding Ni-NTA coupled with CCL-5 protein, washing the coupled complex for multiple times by using a coupling buffer solution, adding 10 mu L of prepared secondary library for incubation, in the process, adding one-step negative screening every three rounds, gradually reducing the concentration of the recombinant protein and properly increasing the number of times of elution to obtain the aptamer with high specificity and high affinity, wherein a detailed screening system is shown in Table 1;

seventhly, after the 10 th round of SELEX screening is carried out, agarose gel recovery is carried out on the obtained library amplification products, and the obtained library is recovered for subsequent clone sequencing.

Example 2: analysis of the aptamer library after screening

After 10 rounds of screening, Shanghai workers are entrusted to analyze an aptamer library after the tenth round of product screening by using a high-throughput sequencing technology, Illumina bridge PCR compatible primer is introduced through PCR, the DNA is accurately quantified by using the Qubit2.0, and Illuminate Next sequence 500 platform is used to perform bridge PCR amplification by using the library as a template, and the sequencing primer is annealed and synthesized and sequenced at the same time; and comparing and analyzing the sequencing results.

Repeated sequences appear in the sequencing result through analysis, which indicates that the sequences are enriched in the SELEX screening process to obtain 1 repeated aptamer sequence Apta-mCCL5-21, and the nucleotide sequence is shown as SEQ ID NO. 1.

Example 3: aptamer Apta-mCCL5-21 affinity assay

1. Coating of antigen: diluting chemokine ligand-5 with 50mmol/L carbonate coating buffer solution to a concentration of 10 μ g/mL, adding 100 μ L/well into 96-well enzyme label plate, and standing at 4 deg.C overnight;

2. the coating solution is discarded the next day, the mixture is washed three times by PBST, and 150 mu L of 1% BSA is added into each hole for blocking for 2 h;

3. PBST is washed for three times, aptamer with different concentrations are added into each hole, the concentrations are respectively 500, 250, 200, 100, 5, 2.5, 1.25 and 1 nmol/L, and incubation is carried out for 2h at 37 ℃;

4. PBST washing three times, each hole adding 100 u L1: 500 diluted SM-HRP secondary antibody, 37 degrees C were incubated for 1h;

5. PBST was washed 5 times, 100. mu.L of TMB substrate was added, and color development was carried out for 20min in the dark, followed by addition of 2mol/L H₂SO₄The reaction was terminated, the absorbance was measured at 450nm, and the measurement result was analyzed using software Prism 8;

the result is shown in FIG. 2, from which it can be seen that Apta-mCCL5-21 has a dissociation equilibrium constant of KD =7.379nmol/L, indicating that the aptamer Apta-mCCL5-21 has higher affinity with the recombinant protein.

Example 4: specific detection of aptamer Apta-mCCL5-21

1. Coating of antigen: dissolving chemotactic factor mCCL5 and other antigens (ZIKV-PrM and RABV-L) by using 50mmol/L carbonate coating buffer solution until the concentration is 10 mu g/mL, adding the solution into a 96-hole enzyme label plate according to the amount of 100 mu L/hole, and standing overnight at 4 ℃;

2. the coating solution is discarded the next day, washed three times with PBST, and 150 μ L of 1% BSA is added to each well for blocking for 2 h;

3. PBST is washed for three times, aptamer with the concentration of 200nmol/L is added into each hole, and incubation is carried out for 2h at 37 ℃;

4. PBST was washed 3 times, 100. mu.L of 1:500 diluted secondary SM-HRP antibody was added to each well, and incubated at 37 ℃ for 1 h.

5. PBST was washed 5 times, 100. mu.L of TMB substrate was added, and color development was carried out for 20min in the dark, followed by addition of 2mol/L H₂SO₄The reaction is terminated at 45Absorbance was measured at 0nm and the measurement was analyzed using software Prism 8;

the results are shown in FIG. 3, both the ZIKV-PrM and the recombinant rabies virus L protein contain histidine tags, and the aptamer has no cross reaction to the histidine tags, so that the aptamer Apta-mCCL5-21 can specifically recognize the recombinant CCL5 protein.

Example 5: transwell assay for determination of inhibition of chemokine ligand-5 chemotaxis by aptamer Apta-mCCL5-21

1. RAW264.7 cells were inoculated in 10% FBS 1640 medium at 37 ℃ with 5% CO_２Culturing until the state is good;

2. taking a bottle of RAW264.7 cells, discarding supernatant, adding 3mL of serum-free 1640 culture medium, scraping with a cell scraper, beating, mixing uniformly, and counting about 3.25 × 10⁶A cell;

3. mu.L of cell culture fluid was mixed with 284. mu.L of serum-free 1640 medium (total of 5 × 10)⁵Individual cells) were added to the upper chamber of the transwell;

4. adding 750 mu L of 10% FBS 1640 culture medium into the lower layer, and respectively setting a negative control group containing no recombinant mCCL5 protein, a positive control group containing 20 mu g of recombinant chemokine mCCL5, and an experimental group containing 20 mu g of recombinant protein and 200 mu mol/L aptamer; the recombinant protein is renatured unfiltered aseptic protein and is filtered by a 0.2 mu m filter; at 37 ℃ with 5% CO₂Culturing in an incubator;

5. after 18h of culture, gently rubbing the cells in the upper layer of the transwell chamber with a cotton swab; fixing with methanol for 15min, dyeing with 0.1% crystal violet, and observing;

the results are shown in FIGS. 4 and 5, and it can be seen that aptamer Apta-mCCL5-21 can inhibit the chemotactic activity of CCL 5.

Sequence listing

<110> university of Kunming science

<120> aptamer specifically binding to chemokine ligand-5 and use thereof

<160>4

<170>SIPOSequenceListing 1.0

<210>1

<211>76

<212>DNA

<213> Artificial sequence (Artificial)

<400>1

atccagagtg acgcagcact gaaagaccgg cagttacgcg tggaggggtg gatggttatg 60

gacacggtgg cttagt 76

<210>2

<211>18

<212>DNA

<213> Artificial sequence (Artificial)

<400>2

atccagagtg acgcagca 18

<210>3

<211>18

<212>DNA

<213> Artificial sequence (Artificial)

<400>3

actaagccac cgtgtcca 18

<210>4

<211>76

<212>DNA

<213> Artificial sequence (Artificial)

<400>4

atccagagtg acgcagcann nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnntg 60

gacacggtgg cttagt 76

Claims

1. An aptamer that specifically binds to chemokine ligand-5, wherein: the nucleotide sequence is shown as SEQ ID NO. 1.

2. Use of the aptamer according to claim 1, which specifically binds to chemokine ligand-5, in the preparation of a reagent or kit for detecting chemokine ligand-5.

3. Use of the aptamer according to claim 1, which specifically binds to chemokine ligand-5, for the preparation of a preparation for inhibiting the chemotactic activity of chemokine ligand-5.

4. Use of an aptamer according to claim 1 which specifically binds to chemokine ligand-5 as a molecular probe.