CN111593053B

CN111593053B - DNA aptamer for identifying human programmed death receptor 1, method and application

Info

Publication number: CN111593053B
Application number: CN202010459988.3A
Authority: CN
Inventors: 章晓联; 肖振
Original assignee: Wuhan University WHU
Current assignee: Wuhan University WHU
Priority date: 2020-05-27
Filing date: 2020-05-27
Publication date: 2022-02-15
Anticipated expiration: 2040-05-27
Also published as: CN111593053A

Abstract

The invention discloses a DNA aptamer for identifying a human programmed death receptor 1, a method and application, and belongs to the field of biochemical detection. Screening aptamers by using a Cell-SELEX technology, transfecting L02 cells with the constructed pDisplay/PD-1N surface display expression plasmid, and carrying out resistance screening by using G418 to obtain a Cell line L02-PD-1N for stably expressing PD-1N. And screening by taking the L02-PD-1N cell as a target, performing repeated multiple rounds of incubation-elution-amplification by using the oligonucleotide library and the cell line, performing reverse screening by using the cell which does not express PD-1N, and finally obtaining the ssDNA (single stranded deoxyribonucleic acid), namely the aptamer, capable of binding the PD-1N with high affinity by identifying the enriched ssDNA sub-library, sequencing and comparing affinity. The invention provides a DNA aptamer for identifying human PD-1N, which takes the aptamer as a core, can utilize biotin, fluorescent markers and the like to identify PD-1 molecules on the surfaces of T lymphocytes in cells or tissues, and has potential application prospects in diagnosis and treatment of tuberculosis, tumors and the like.

Description

DNA aptamer for identifying human programmed death receptor 1, method and application

Technical Field

The invention relates to the technical field of biochemical detection, in particular to a DNA aptamer for identifying a human programmed death receptor 1, a method and application thereof.

Background

Programmed death receptor 1, also known as PD-1 or CD279, is an important adaptive immune molecule on the surface of T cells. PD-1 is a 288 amino acid type I transmembrane protein composed of an immunoglobulin (Ig) superfamily domain, a transmembrane domain, and an intracellular domain, containing an immunoreceptor tyrosine-based inhibitory motif (ITIM) and an immunoreceptor tyrosine-based switching motif (ITSM) (Keir et al, 2008).

PD-1 has two ligand molecules: PD-L1 and PD-L2, which are members of the B7 family. PD-1, as an immune checkpoint molecule, can defend against autoimmunity by two mechanisms: promoting apoptosis of antigen-specific T cells in lymph nodes (programmed cell death) and reducing apoptosis of regulatory T cells (anti-inflammatory, suppressor T cells). Researches show that PD-1 and ligand molecules thereof play a negative regulation role in cell signal transduction, inhibit the function of immune cells and promote immune escape. PD-1 inhibitors block the interaction between PD-1/PD-L1, enhance the immune system, and can be used for treating cancer (button et al, 2007).

The expression level of PD-1 molecules on the surface of T cells in a tumor microenvironment is generally high (Sharpe and Pauken,2018), and many tumor cells and circulating tumor cells generally highly express PD-L1 molecules (Gandini et al, 2016; Syn et al, 2017), and the inhibition of the interaction between PD-1/PD-L1 can enhance T cell response and mediate preclinical antitumor activity (Baumeister et al, 2016).

The screening method of the exponential enrichment ligand phylogenetic evolution (SELEX) refers to the screening of a random library with a large number of sequences for functional aptamers directed against a particular target (Ellington and Szostak, 1990). This technique refers to a stepwise screening process for obtaining desired single-stranded DNA or RNA via repeated rounds of binding, isolation, and amplification, and designates such oligonucleotide sequences with specific binding capacity as "aptamers", i.e., aptamers (Tuerk and Gold, 1990). Aptamers can form specific spatial structures through electrostatic interactions, van der waals forces, hydrogen bonding, hydrophobic interactions, pi-pi stacking, and molecular shape complementation, etc., to bind target molecules with high affinity and specificity, with dissociation constants Kd values typically in the picomolar to nanomolar range (Hermann and Patel, 2000).

Aptamers, also known as "chemoantibodies," are used functionally as antagonists, agonists, or targeting ligands. Aptamers have unique advantages over antibodies: such as ease of chemical synthesis, small size, low molecular weight and non-immunogenicity, and can be modified as required (Zhou and Rossi, 2017). Based on the advantages that the aptamer has great application potential in the biomedical field such as diagnosis and treatment, research projects of the aptamer are increasing, and the obtained aptamer can be widely used for biological diagnosis, biomarker discovery, molecular imaging, targeted therapy, drug delivery and the like. In 2004, the american Food and Drug Administration (FDA) approved the first aptamer drug pegaptanib (trade name Macugen) for the treatment of age-related macular degeneration new Angiogenesis (AMD), which marks the formal entry of aptamer drugs into clinical treatment.

Disclosure of Invention

An object of the present invention is to provide a DNA aptamer capable of specifically recognizing human PD-1N.

Another object of the present invention is to provide a method for screening and identifying the DNA aptamers produced as described above.

The invention also aims to provide application of the DNA aptamer, and the DNA aptamer (aptamer) has potential application prospects in clinical diagnosis and treatment. The DNA aptamer (nucleic acid aptamer) is used as a core, can be used for detecting PD-1 molecules in cells or tissues by utilizing labeling or polyvalent modification such as biotin/fluorescence/enzyme labeling/chemiluminescence and the like, and has potential application prospects for diagnosis and treatment of tuberculosis and tumor patients.

The specific technical scheme is as follows:

in a first aspect, the present invention provides a method for screening a DNA aptamer that recognizes an extracellular domain (PD-1N) of human programmed death receptor 1(PD-1), comprising: namely, a screening method for producing a DNA aptamer recognizing human PD-1N; comprises the following steps:

(1) transfecting L02 cells by using a pDisplay/PD-1N recombinant plasmid, and screening by using G418 to obtain an L02-PD-1N cell line, which comprises the following steps:

l02 cells are transfected by pDisplay/PD-1N recombinant plasmids, G418 antibiotics are used for resistance screening, and then the expression of PD-1N protein and genes is verified by western blotting, flow cytometry and RT-qPCR technology, so that a stably transfected L02-PD-1N cell line is obtained.

(2) Repeatedly incubating, eluting and amplifying the DNA oligonucleotide library and the L02-PD-1N cell line to obtain each round of enriched sub-libraries, which specifically comprise the following steps:

and (3) repeatedly incubating, eluting and amplifying the synthesized oligonucleotide sequence library and the L02-PD-1N stable cell line, and obtaining ssDNA capable of specifically binding PD-1N protein by identifying the enriched ssDNA sub-library after twelve rounds of screening.

Preferably, the method further comprises the step (3): obtaining the enriched ssDNA sub-libraries of each round, determining the enriched sub-library with the best affinity through an enzyme-linked oligonucleotide adsorption test and a flow cytometry, and then screening several candidate aptamers with better specificity and affinity by identifying the affinity of a single aptamer, wherein the specific steps are as follows:

starting from the third round, a negative screen was performed using an L02 cell line that does not express PD-1N to remove non-specifically bound ssDNA sequences; detecting the affinity of the ssDNA sub-library obtained after each round of screening and PD-1N by using an enzyme-linked oligonucleotide adsorption test (ELONA) to obtain an ssDNA sub-library with the best affinity; constructing the ssDNA sub-library with the optimal affinity to a TSV-007VST vector by a molecular cloning technology to obtain a single ssDNA sequence, identifying the affinity and the specificity of the ssDNA sequence by using a flow cytometry and confocal microscope technology, and finally identifying the optimal aptamer.

Further, the identification method of the candidate DNA aptamer screened in step (3) is as follows:

the optimal DNA aptamer is identified by detecting the dissociation constant kd value and the specific binding condition of the aptamer by using flow cytometry and confocal microscopy. The DNA aptamer against human PD-1N, produced by the above process, was designated P13.

In a second aspect, the present invention provides a DNA aptamer that specifically recognizes human programmed death receptor 1(PD-1), comprising: the DNA aptamer is prepared by the method of claim 3; the sequence of the DNA aptamer is shown as SEQ ID NO. 1.

In a third aspect, the present invention provides a use of the DNA aptamer specifically recognizing human programmed death receptor 1(PD-1) as described above, characterized in that: the DNA aptamer is taken as a core, and biotin/fluorescence/enzyme labeling/chemiluminescence labeling or multivalent modification is utilized to prepare a reagent for detecting PD-1 molecules on the surfaces of T lymphocytes in tuberculosis and tumor tissues.

The aptamer can be combined with PD-1 molecules on the surfaces of T lymphocytes in tissues of tuberculosis and tumor patients through fluorescent labeling, and has application potential of molecular diagnosis or targeted therapy.

The invention has the following advantages and beneficial effects:

(1) the aptamer P13 provided by the invention has a dissociation constant in a nanomolar level (Kd 89.5 +/-24.4 nM) determined by flow cytometry, and has strong affinity; confocal experiments demonstrated that the aptamer was able to specifically bind to PD-1N.

(2) The aptamer P13 provided by the invention can identify PD-1 molecules infiltrating the surface of T lymphocytes in tissues of tuberculosis and tumor patients through fluorescent labeling, and the aptamer has application potential of molecular diagnosis or targeted therapy.

Drawings

FIG. 1 Western blot assay for detecting expression of PD-1N in L02-PD-1N cell line

FIG. 2 RT-qPCR assay for mRNA expression levels of PD-1N from L02-PD-1N cell line.

FIG. 3 flow cytometry detection of PD-1N expression on the surface of L02-PD-1N cell line.

FIG. 4. detection of ssDNA sub-library affinity. The ELONA measures the affinity of each ssDNA sub-library.

FIG. 5 detection of affinity of a single aptamer. ELONA measures the affinity of individual aptamers.

FIG. 6 measurement of dissociation constant Kd value of aptamer. Flow cytometry determines the dissociation constant of the aptamer.

FIG. 7. specificity identification of aptamer P8. Cellular immunofluorescence assays identify the specificity of the aptamer.

FIG. 8. specificity identification of aptamer P13. Cellular immunofluorescence assays identify the specificity of the aptamer.

FIG. 9 aptamer secondary structure analysis. The secondary structure of aptamer P13 with the best affinity and specificity was analyzed by software DNAMAN (note: the four deoxynucleotides in the structural prediction are shown as A, G, C, T).

FIG. 10 aptamer P13 binds to PD-1 molecules on the surface of T lymphocytes in the tissues of tuberculosis patients.

FIG. 11 aptamer P13 binds to PD-1 molecules on the surface of T lymphocytes in tumor patient tissues.

Figure 12 aptamer P13 does not bind T lymphocytes in inflammatory granulomatous tissue.

FIG. 13 expression analysis of T lymphocyte surface PD-1 in inflammatory granuloma tissue.

(in FIG. 10-13, the green fluorescence of FITC was changed to white)

Detailed Description

The invention is described below with reference to the figures and the specific embodiments.

Unless otherwise specified, the reagents used in the following examples are commercially available and the procedures are conventional.

EXAMPLE 1 construction of cell line L02-PD-1N stably expressing PD-1N

Firstly, constructing a stable cell line expressing PD-1N as a screening vector of cell-SELEX.

Construction of PD-1N recombinant plasmid: according to a coding sequence (NM-005018.3) and a protein structure of a human PDCD1 gene, amplifying a PD-1N sequence of an extracellular segment of the protein, cloning the sequence to a pDisplay surface display eukaryotic expression vector, transfecting an L02 cell with a pDisplay/PD-1N recombinant plasmid, and performing resistance screening by using G418 to obtain a cell line capable of stably expressing PD-1N.

2.RT-qPCR, flow cytometry and Western blotting are used for verifying the expression of PD-1 in the cell line, and an L02-PD-1N stable transfer cell line is obtained and is used as a screening vector for a SELEX test.

L02-PD-1N cells were grown in 10% fetal bovine serum DMEM medium as positive selection cells for cell-SELEX; l02 cells were used as negative selection cells to remove oligonucleotide sequences that were not specifically bound during SELEX.

EXAMPLE 2 screening of PD-1N aptamers

And (3) repeatedly incubating, eluting and amplifying with a laboratory synthesized oligonucleotide sequence library (ssDNA library) by using the obtained L02-PD-1N cell line as a screening carrier to finally obtain a proper oligonucleotide sequence.

Identification of ssDNA libraries: ssDNA is amplified to dsDNA using symmetric PCR, followed by non-denaturing SDS-PAGE gel electrophoresis experiments, and the ssDNA library is correct in size. The library information was 5 '-GCGGAATTCAACAGTCCGAGCC-N30-GGGTCAATGCGTCATA-3' (68nt)(N represents A, T, C or G four bases), SEQ ID NO. 2. The two ends of the library are fixed sequences, and are used for binding templates of aptamer amplification primers, and the upstream primer P1 of the library: 5' -GCGGAATTCAACAGTCCGAGCC-3' (22nt) (underlined is an EcoRI cleavage site) SEQ ID NO.3, the reverse primer P2: 5' -GCGGGATCCTATGACGCATTGACCC-3' (25nt) (underlined is a BamHI cleavage site), SEQ ID NO. 4. Both libraries and primers were synthesized by Oncorhynchus bio.

2. L02-PD-1N cells of sufficient growth status were collected, incubated with the ssDNA library denatured in a water bath at 95 ℃ for 1h at 37 ℃ and subsequently centrifuged to collect cells, and the ssDNA sub-library was obtained by ethanol precipitation and PCR techniques for the next round of screening. After twelve rounds of repeated incubation-elution-amplification screening, wherein the anti-screening cells in the tenth round are human PBMC cells, the ssDNA sub-library with the best affinity, namely the ssDNA sub-library after the eleventh round of screening, is obtained by using biotin labeling ssDNA sub-library and enzyme-linked oligonucleotide adsorption assay (ELONA).

3. Constructing the ssDNA sub-library with the best affinity to a T vector, obtaining 26 candidate aptamers through monoclonal screening and sequencing, and constructing a phylogenetic tree of the aptamers by utilizing MEGA-X through an NJ algorithm.

4. Aptamers are labeled with biotin and FAM fluorescent groups through asymmetric PCR, and several aptamers with the best affinity are screened by using enzyme-linked oligonucleotide adsorption assay (ELONA) and Flow Cytometry (FCM).

TABLE 1 Cell-SELEX screening conditions

Example 3 identification of PD-1N aptamer specificity

1. The pDisplay/PD-1N recombinant plasmid was transfected into 293T cells, and the expression of PD-1N was verified by Western blotting and flow cytometry, followed by specificity verification of the aptamer.

2. The laser confocal cell culture dish is pre-balanced, 1mL of cell culture solution is added into the dish, then the dish is placed into a cell culture box for 5min, and a liquid transfer machine removes the cell culture solution in the dish.

3. Taking a proper amount of cells, resuspending the cells with a cell culture solution, adding the cell culture solution into a laser confocal cell culture dish, wherein the total amount of the cell suspension is 1mL, and the cell amount is controlled to be 1 multiplied by 10⁵When the cells cover about 80% of the bottom of the dish.

4. The bottom of the dish was covered with 200-300. mu.L of 4% paraformaldehyde, the cells were fixed at room temperature for 15min, the fixative was removed and rinsed 3 times with PBS, each for 3min (the fixed cells could be stored at 4 ℃ for future use).

5. PBS in the confocal cell culture dish was removed, then 200ul of 3% BSA blocking solution was added dropwise, the bottom of the dish was covered, and the dish was blocked at room temperature for 30 min.

6. The blocking solution was removed by a pipette, and then a suitable amount of FITC-labeled aptamer was added dropwise to the dish, the cells at the bottom of the dish were covered, the dish was gently shaken to make the liquid cover uniformly, and the dish was covered and incubated overnight at 4 ℃ in the dark (total amount of aptamer was about 200pmol, total volume was about 300. mu.L).

7. Removing liquid in the confocal culture dish, dropwise adding 200 mu LDAPI (1 mu g/mL) dye solution, covering cells at the bottom of the dish, incubating at room temperature in a dark place for 5min, rinsing with PBST for 3 times, rinsing for 5min each time, removing the liquid in the confocal cell culture dish by using absorbent paper, and storing at 4 ℃ in a dark place.

8. Images were observed and collected under a confocal laser microscope. The aptamer with the best affinity and specificity was identified and named P13, and its nucleic acid sequence was: 5'-GGATCCTATGACGCATTGACCCGCTGCCTCTACTGAGGCTGTGTCAGTGTGCGGCTCGGACTGTTGAATTC-3' (71nt), SEQ ID NO. 1.

Example 4 use of aptamers (P13) for the identification of PD-1 molecules on the surface of infiltrating T lymphocytes in tissues of tuberculosis and tumor patients

1. Paraffin section dewaxing and rehydration: the paraffin sections were placed in a 60 ℃ oven for 30 minutes. Xylene I, II was dewaxed for 5 minutes, and was put into 100%, 95%, 90%, 80%, 70% methanol for 2 minutes each, and then put into distilled water for 5 minutes.

2. Antigen retrieval: sodium citrate buffer (10mM sodium citrate, 0.05% Tween-20, pH6.0) is added into a beaker and placed into a water bath to be heated until boiling. Putting the slices into a slide holder, putting the slices into a beaker, and boiling the slices for 30 minutes in a water-isolated manner. Taking the beaker out of the water bath kettle, and naturally cooling to room temperature.

Sections were washed twice with PBS (1mL), then blocked with 10% normal goat serum (0.5% PBST dilution), incubated at room temperature for 30 minutes, removed and excess liquid was blotted with a paper towel.

5. Diluted CD3 antibody primary antibody (murine) was added, and the humidified chamber was covered with a sealing film and incubated at 37 ℃ for 1 hour, followed by 3 washes with 0.025% PBST for 5 minutes each. A fluorescently labeled secondary antibody was added and the procedure was as above.

6. Diluted fluorescently labeled aptamer: 300pmol of primary antibody 100. mu.L was placed on the section, and the humidified chamber was covered with a sealing film and incubated at 37 ℃ for 1 hour.

7.0.025% PBST was washed 3 times for 5 minutes each. During each wash, excess liquid was removed with a paper towel.

8. The slides were mounted with an anti-fluorescent shatter-mounted tablet (Google Bio, G1401) and sections were scanned by fluorescence microscopy.

The technical features of the embodiments described above may be arbitrarily combined, and for the sake of brevity, all possible combinations of the technical features in the embodiments described above are not described, but should be considered as being within the scope of the present specification as long as there is no contradiction between the combinations of the technical features.

The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is specific and detailed, but not to be construed as limiting the scope of the 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.

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Claims

1. A DNA aptamer that specifically recognizes human programmed death receptor 1, comprising: the DNA aptamer is prepared by the following method; the sequence of the DNA aptamer is shown as SEQ ID NO. 1;

the method is a screening method for generating a DNA aptamer capable of recognizing human PD-1N, and comprises the following steps:

(1) transfecting L02 cells by using a pDisplay/PD-1N recombinant plasmid, and screening by using G418 to obtain an L02-PD-1N cell line;

(2) performing repeated incubation, elution and amplification on the DNA oligonucleotide library and an L02-PD-1N cell line to obtain each round of enriched sub-libraries;

further comprising the step (3): obtaining each round of enriched ssDNA sub-libraries, determining an enriched sub-library with the best affinity through an enzyme-linked oligonucleotide adsorption test and a flow cytometry, and then screening several candidate DNA aptamers with better specificity and affinity through identifying the affinity of a single DNA aptamer;

the identification method of the candidate DNA aptamer screened in the step (3) is as follows: the optimal DNA aptamer is identified by detecting the dissociation constant kd value and the specific binding condition of the DNA aptamer by using flow cytometry and confocal microscopy.

2. Use of a DNA aptamer according to claim 1, which specifically recognizes human programmed death receptor 1, wherein: the DNA aptamer is taken as a core, and biotin/fluorescence/enzyme labeling/chemiluminescence labeling or multivalent modification is utilized to prepare a reagent for detecting PD-1 molecules on the surfaces of T lymphocytes in tuberculosis and tumor tissues.