CN115786349B - Aptamer for traceless sorting of killer T lymphocytes in peripheral blood, complementary sequence and application of aptamer - Google Patents
Aptamer for traceless sorting of killer T lymphocytes in peripheral blood, complementary sequence and application of aptamer Download PDFInfo
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Abstract
The invention discloses a nucleic acid aptamer for traceless sorting of killer T lymphocytes in peripheral blood, a complementary sequence and application thereof. The invention has the following advantages: 1) The novel killer T lymphocyte specific nucleic acid aptamer probe is developed, and a peripheral blood killer T lymphocyte traceless sorting method based on the nucleic acid aptamer is developed, so that the method has important clinical significance; 2) The aptamer probe obtained based on the clinical complex sample screening has stronger clinical applicability; 3) The aptamer has the advantages of strong specificity, high affinity, good accuracy and sensitivity and the like for identifying and combining killer T cells; 4) The killer T lymphocyte capture based on the aptamer can realize nondestructive dissociation of the recognition probe and the magnetic beads after capture, realize traceless separation and effectively avoid the interference of exogenous substances on downstream application; 5) The aptamer probe has the advantages of simple production and preparation, good repeatability, good stability and easy storage and transportation, and provides a new method and approach for immunodiagnosis and immunotherapy.
Description
Technical Field
The invention belongs to the technical field of cell separation, and particularly relates to a nucleic acid aptamer for traceless separation of killer T lymphocytes in peripheral blood, a complementary sequence and application thereof.
Background
T lymphocytes are differentiated from lymphocytes in thymus, are the most abundant and complex types of lymphocytes, and can be divided into three subgroups according to the functions: helper T cells, suppressor T cells, and killer T cells. Among them, the killer T lymphocyte is also called cytotoxic T lymphocyte, which is a specific T lymphocyte and secretes various cytokines to participate in immunity. It has killing effect on some virus, tumor cell and other antigen matter, and forms important antiviral and antitumor immunity line with natural killer cell, and is also the main effector cell for chimeric antigen receptor T cell immunotherapy.
Chimeric antigen receptor T cell immunotherapy (CAR-T therapy) is a novel immune cell therapy that is accurate, rapid, efficient, and potentially cures cancer. The basic principle of CAR-T therapy is that T cells are activated in vitro by genetic engineering technology, and a tumor Chimeric Antigen Receptor (CAR) of a positioning navigation device is arranged, so that a common warrior of T lymphocytes is transformed into a super warrior, namely the CAR-T cells, the CAR of the positioning navigation device is utilized to specifically identify tumor cells in vivo, and a large number of various effector factors are released through immune response, so that the tumor cells are effectively killed, and the purpose of treating malignant tumors is achieved. The efficient separation of T lymphocytes in peripheral blood is a precondition for constructing CAR-T cells in vitro, and the in vivo feedback application of the CAR-T cells also puts higher demands on the efficiency, purity, cell activity and exogenous pollution of cell separation.
The most common clinical killer T lymphocyte sorting method is an immunomagnetic bead method based on monoclonal antibodies, and the technical requirements of CAR-T application such as high-efficiency, high-purity and traceless sorting are difficult to meet at the same time. In addition, the complex biological production process of monoclonal antibody molecules brings great safety problems to the in vivo application of CAR-T cells; the differences between production batches of monoclonal antibodies, harsh storage and transport conditions, are also key factors leading to their lower enrichment efficiency. Therefore, development of a cell traceless sorting technology that can achieve high efficiency and purity is a clinical urgent need in the field of immune cell therapy.
The Aptamer (Aptamer) is a single-stranded DNA/RNA sequence which can be screened from a nucleic acid library by utilizing an exponential enrichment ligand system evolution technology (System Evolution of Ligands by Exponential Enrichment; SELEX) and can be combined with a target with high specificity and high affinity. The recognition and binding of nucleic acid aptamers to their target molecules has a specificity and affinity similar to antigen-antibody interactions, and is visually referred to as "antibodies to chemists". In addition, the aptamer has the outstanding advantages of wide target range, easiness in accurate preparation and modification marking, flexible and controllable design, low manufacturing cost and the like, and has wide application and unique advantages in the fields of chemical sensing, molecular medicine and the like.
According to the invention, a clinical peripheral blood mononuclear Cell complex sample is directly taken as a screening target, and the aptamer XD-4 and XD-9 obtained through screening by a Cell-SELEX technology can specifically identify and bind with killer T lymphocytes with high affinity, and are weak in binding with other blood cells in peripheral blood. The aptamer can be used as a high-efficiency molecular recognition probe to specifically recognize and mark the killer T lymphocytes in the peripheral blood, so that the high-efficiency capture of the killer T lymphocytes in the peripheral blood is realized. Meanwhile, complementary sequences of the nucleic acid aptamer are designed, and the formation of functional and active secondary structures of the nucleic acid aptamer is inhibited by utilizing the specific hybridization between the complementary sequences, so that the release of the nucleic acid aptamer probe and the magnetic beads from the cell surface is realized, and the purpose of traceless sorting is achieved. The method effectively reduces the influence of exogenous substances on cell activity, T cell expansion, activation and in-vivo application on the premise of realizing efficient capture of the killer T lymphocytes in peripheral blood, and has wide application prospect in the field of cell immunotherapy.
Disclosure of Invention
The primary purpose of the invention is to provide a peripheral blood killer T lymphocyte aptamer with high specificity and high affinity as a molecular probe.
The nucleic acid aptamer is XD-4 and/or XD-9 respectively, wherein the XD-4 sequence is as follows:
5’-ATCCAGAGTGACGCAGCAAGTGTCGTGAGGAGCTTGAAATCCAATCTGCACGGCCAGTGCGGATGGACACGGTGGCTTAGT-3’；
the XD-9 sequence was:
5’-ATCCAGAGTGACGCAGCATTGCGACCCCCTATGCGGTCGGTGAGGAGCTTGAAATCCCAATGATGGACACGGTGGCTTAGT-3’。
further, by adding or deleting bases or base substitution to the nucleic acid aptamer, a nucleic acid aptamer with the same function is obtained; preferably wherein the nucleic acid aptamer XD-4 is truncated to XD-4c, which has the sequence:
5’-CAAGTGTCGTGAGGAGCTTGAAATCCAATCTGCACGGCCAGTGCGGATGGACACGACACTTA-3’；
the nucleic acid aptamer XD-9 is truncated into XD-9a, and the sequence is as follows:
5’-TCCAGAGTGACGCAGCATTGCGACCCCCTATGCGGTCGGTGAGGAGCTTGAAATCCCAATGATGGACACTCTGGC-3’。
further, the aptamer is labeled with a fluorescent substance, a radioactive substance, a therapeutic substance, biotin, or an enzyme-labeled substance, thereby obtaining a aptamer derivative having the same function as the aptamer.
The second object of the invention is to provide a method for identifying and enriching killer T cells in peripheral blood based on aptamer,
the nucleic acid aptamer comprises the nucleic acid aptamer.
A third object of the invention is to provide the use of said aptamer in the preparation of a preparation for specifically recognizing and binding killer T cells in peripheral blood.
The fourth object of the present invention is to provide a method for traceless release of killer T cells in peripheral blood based on complementary sequences, wherein the complementary release sequences are used for releasing killer T cells in peripheral blood recognized and enriched by the nucleic acid aptamer XD-4 and/or XD-9 sequences, and the XD-4 sequences are as follows:
5’-ATCCAGAGTGACGCAGCAAGTGTCGTGAGGAGCTTGAAATCCAATCTGCACGGCCAGTGCGGATGGACACGGTGGCTTAGT-3’。
preferably its complementary release sequence 4-RA1 is as follows:
5’-ACTAAGCCACCGTGTCCATCCGCACTGGCCGTGCAG-3’；
the XD-9 sequence was:
5’-ATCCAGAGTGACGCAGCATTGCGACCCCCTATGCGGTCGGTGAGGAGCTTGAAATCCCAATGATGGACACGGTGGCTTAGT-3’；
preferably its complementary release sequence 9-RA1 is as follows:
5’-ACTAAGCCACCGTGTCCATCATTGGGATTTCAAGCT-3’。
further, by adding or deleting bases or base substitutions to the aptamer or the complementary release sequence, a aptamer or a complementary release sequence having the same function is obtained.
The fifth object of the invention is to provide an application of a complementary release sequence in preparation of a preparation for nondestructively releasing killer T cells in peripheral blood captured by a nucleic acid aptamer, wherein the nucleic acid aptamer XD-4 has the sequence as follows:
5’-ATCCAGAGTGACGCAGCAAGTGTCGTGAGGAGCTTGAAATCCAATCTGCACGGCCAGTGCGGATGGACACGGTGGCTTAGT-3’；
its complementary release sequence 4-RA1 is as follows:
5’-ACTAAGCCACCGTGTCCATCCGCACTGGCCGTGCAG-3’；
the nucleic acid aptamer XD-9 sequence is as follows:
5’-ATCCAGAGTGACGCAGCATTGCGACCCCCTATGCGGTCGGTGAGGAGCTTGAAATCCCAATGATGGACACGGTGGCTTAGT-3’；
its complementary release sequence 9-RA1 is as follows:
5’-ACTAAGCCACCGTGTCCATCATTGGGATTTCAAGCT-3’。
the sixth object of the present invention is to provide a complementary release sequence for non-destructive release of killer T cells in peripheral blood captured by a nucleic acid aptamer, the complementary release sequence 4-RA1 being as follows:
5’-ACTAAGCCACCGTGTCCATCCGCACTGGCCGTGCAG-3’；
the nucleic acid aptamer XD-4 sequence is as follows:
5’-ATCCAGAGTGACGCAGCAAGTGTCGTGAGGAGCTTGAAATCCAATCTGCACGGCCAGTGCGGATGGACACGGTGGCTTAGT-3’；
its complementary release sequence 9-RA1 is as follows:
5’-ACTAAGCCACCGTGTCCATCATTGGGATTTCAAGCT-3’；
the nucleic acid aptamer XD-9 sequence is as follows:
5’-ATCCAGAGTGACGCAGCATTGCGACCCCCTATGCGGTCGGTGAGGAGCTTGAAATCCCAATGATGGACACGGTGGCTTAGT-3’。
the seventh object of the present invention is to provide a preparation for non-destructive release of killer T cells in peripheral blood captured by a nucleic acid aptamer, comprising the complementary release sequence 4-RA1 as follows:
5’-ACTAAGCCACCGTGTCCATCCGCACTGGCCGTGCAG-3’；
the nucleic acid aptamer XD-4 sequence is as follows:
5’-ATCCAGAGTGACGCAGCAAGTGTCGTGAGGAGCTTGAAATCCAATCTGCACGGCCAGTGCGGATGGACACGGTGGCTTAGT-3’；
its complementary release sequence 9-RA1 is as follows:
5’-ACTAAGCCACCGTGTCCATCATTGGGATTTCAAGCT-3’；
the nucleic acid aptamer XD-9 sequence is as follows:
5’-ATCCAGAGTGACGCAGCATTGCGACCCCCTATGCGGTCGGTGAGGAGCTTGAAATCCCAATGATGGACACGGTGGCTTAGT-3’。
the high-specificity and high-affinity peripheral blood killer T lymphocyte aptamer molecular probe provided by the invention is obtained by the following steps:
1) The complex sample of mononuclear cells in clinical peripheral blood is used as a screening target, so that the difference between a cell line and a clinical actual sample is effectively eliminated, and the applicability of the screening nucleic acid aptamer probe in a complex clinical system is enhanced.
2) The complex sample of mononuclear cells in clinical peripheral blood is used for competitive screening, so that in-vitro screening pressure capable of meeting clinical requirements is provided, and the specificity of screening sequences is greatly improved by enriching sequences of specific binding killer T lymphocytes in the complex sample.
3) The nucleotide aptamer probes XD-4 and XD-9 specific to the killer T lymphocyte are successfully obtained through high-throughput sequencing and bioinformatics analysis, wherein the sequence of XD-4 is as follows:
5’-ATCCAGAGTGACGCAGCAAGTGTCGTGAGGAGCTTGAAATCCAATCTGCACGGCCAGTGCGGATGGACACGGTGGCTTAGT-3’。
the XD-9 sequence was:
5’-ATCCAGAGTGACGCAGCATTGCGACCCCCTATGCGGTCGGTGAGGAGCTTGAAATCCCAATGATGGACACGGTGGCTTAGT-3’。
4) The nucleic acid aptamer XD-4 is truncated into XD-4c, and the sequence is as follows:
5’-CAAGTGTCGTGAGGAGCTTGAAATCCAATCTGCACGGCCAGTGCGGATGGACACGACACTTA-3’；
the nucleic acid aptamer XD-9 is truncated into XD-9a, and the sequence is as follows:
5’-TCCAGAGTGACGCAGCATTGCGACCCCCTATGCGGTCGGTGAGGAGCTTGAAATCCCAATGATGGACACTCTGGC-3’。
the traceless sorting method for the killer T lymphocytes in the peripheral blood based on the aptamer has the advantages of high sensitivity, strong specificity and simplicity and convenience in operation. The specific embodiment is as follows:
1. peripheral blood collection and pretreatment
1) EDTA-K 2 The vacuum anticoagulation blood collection tube collects 10mL of peripheral blood of a patient and sends the peripheral blood to a laboratory within 12 h.
2) Peripheral Blood Mononuclear Cells (PBMCs) are obtained by removing a large number of mature erythrocytes by density gradient centrifugation or erythrocyte selective lysis.
2. Aptamer synthesis and modification
The 5' -terminal biotin-modified aptamer sequences (designated XD-4-biotin and XD-9-biotin) were synthesized by an automated solid phase synthesis technique.
3. Peripheral blood killer T lymphocyte enrichment
1) Taking appropriate amount of XD-4-biotin or XD-9-biotin aptamer, heating at 95deg.C for 5min, and cooling at 4deg.C for 10min.
2) PBMCs cells are resuspended by Binding Buffer, and a proper amount of prepared XD-4-biotin and XD-9-biotin nucleic acid aptamer are added for incubation at 4 ℃ for 30min with shaking, so that the nucleic acid aptamer is fully combined with killer T lymphocytes.
3) Excess unbound aptamer probe was removed by centrifugation and washed twice by Washing Buffer centrifugation.
4) The incubation is performed by adding anti-biotin antibody (or streptavidin) labeled magnetic beads, so that the aptamer-bound cells are labeled with magnetic beads, and the killer T lymphocytes in the enriched PBMCs are isolated by magnetic separation.
4. Nondestructive release of captured killer T lymphocytes
Adding a proper amount of complementary sequences into the cells enriched by magnetic separation, standing for 30min, and destroying the secondary structure of the aptamer through the base complementary pairing action between nucleic acid molecules, thereby realizing the nondestructive release of the captured killer T lymphocytes.
5. Analysis and characterization of traceless sorted T cells
1) And (3) adopting multi-marker immunofluorescence labeling, and characterizing the efficiency, purity and the like of sorting the killer T lymphocytes through a flow cytometry and a laser confocal fluorescence microscope.
2) Cell viability was characterized by trypan blue staining for traceless sorted killer T lymphocytes.
The invention has the advantages over the prior art: 1) The peripheral blood killer T lymphocyte capturing aptamer probe with excellent performance is provided, a novel peripheral blood killer T lymphocyte capturing or enriching method based on the aptamer is established, and the method has wide application potential; 2) Complex clinical samples are adopted as screening targets and competitive screening is introduced, so that the specificity and clinical applicability of the obtained nucleic acid aptamer are greatly improved; 3) The aptamer has the application advantages of easy accurate synthesis and modification, controllable quality, small batch-to-batch difference, stable property, easy storage and transportation, low preparation cost, large-scale production and the like; 4) A lossless cell release method based on a complementary sequence is developed, so that the damage to cells and the influence on downstream analysis in the capturing process are greatly reduced; 5) Provides a new method and a new way for developing immune cell therapy based on killer T lymphocytes, and has important clinical significance.
Drawings
FIG. 1 flow and confocal characterization of nucleic acid aptamers XD-4 and XD-9 binding to CD8+ T cells in peripheral blood.
FIG. 1A is a flow chart of the binding of nucleic acid aptamers XD-4 and XD-9 to CD8+ T cells and CD 8-cells in peripheral blood.
FIG. 1B is a confocal analysis of the binding of aptamers XD-4 and XD-9 to CD8+ T cells in peripheral blood.
FIG. 2 analysis of T lymphocyte subpopulations in peripheral blood bound by nucleic acid aptamers XD-4 and XD-9.
FIG. 2A is a flow chart of the binding of the nucleic acid aptamers XD-4, XD-9 to CD3+/CD4+ T cells and CD3+/CD8+ T cells in peripheral blood.
FIG. 2B is a statistical analysis of aptamer XD-4, XD-9 binding to CD3+/CD4+ T cells and CD3+/CD8+ T cells in peripheral blood.
FIG. 3 Universal and specific analysis of aptamer XD-4 and XD-9 binding to T lymphocyte subsets in peripheral blood.
FIG. 3A is a flow chart of the binding of aptamer XD-4, XD-9 to lymphocytes, monocytes and granulocytes in peripheral blood.
FIG. 3B is a statistical analysis of aptamer XD-4, XD-9 binding to lymphocytes, monocytes and granulocytes in peripheral blood.
FIG. 3C is a flow chart of the binding of aptamer XD-4, XD-9 to mature red blood cells in peripheral blood.
FIG. 3D is a statistical analysis of the binding of aptamer XD-4, XD-9 to mature red blood cells in peripheral blood.
FIG. 4 apparent dissociation constants (Kd) of CD8+ T cells in peripheral blood for binding of nucleic acid aptamers XD-4 and XD-9.
FIG. 4A is an analysis of apparent dissociation constants of CD8+ T cells in XD-4-conjugated peripheral blood.
FIG. 4B is an analysis of apparent dissociation constants of CD8+ T cells in XD-9-conjugated peripheral blood.
FIG. 5 truncation and optimization analysis of nucleic acid aptamer XD-4 and XD-9 sequences.
FIG. 5A is a simulation of the secondary structures of nucleic acid aptamers XD-4 and XD-4 c.
FIG. 5B is an analysis of binding of nucleic acid aptamers XD-4 and XD-4c to peripheral blood lymphocytes.
FIG. 5C is a nucleic acid aptamer XD-4 and XD-4C binding peripheral blood lymphocyte target uniformity assay.
FIG. 5D is a simulation of the secondary structures of nucleic acid aptamers XD-9 and XD-9 a.
FIG. 5E is an analysis of binding of nucleic acid aptamers XD-9 and XD-9a to peripheral blood lymphocytes.
FIG. 5F is an analysis of nucleic acid aptamer XD-9 and XD-9a binding to peripheral blood lymphocyte target identity.
FIG. 6 analysis of CD8+ T cell efficiency, purity and cell activity in peripheral blood captured by nucleic acid aptamers XD-4 and XD-9.
FIG. 6A is an analysis of the efficiency of nucleic acid aptamers XD-4 and XD-9 in capturing CD8+ T cells in peripheral blood.
FIG. 6B is an analysis of purity of nucleic acid aptamers XD-4 and XD-9 trap cells.
FIG. 6C is an activity assay of nucleic acid aptamers XD-4 and XD-9 trap cells.
FIG. 7 analysis of nucleic acid aptamer XD-4 and XD-9 complementary sequences for traceless release efficiency, purity and cell activity.
FIG. 7A is a trace-free release efficiency analysis of the complementary sequences 4-RA1 and 9-RA 1.
FIG. 7B is a purity analysis of complementary sequence 4-RA1 and 9-RA1 traceless released cells.
FIG. 7C is an activity assay of complementary sequences 4-RA1 and 9-RA1 traceless releasing cells.
Detailed Description
The following examples are intended to further illustrate the invention, but not to limit it.
Reagents used in the present invention:
SEPMATE TM TUBE (STEMCELL), lymphoprep,250mL (STEMCELL), PBMC high efficiency centrifuge tubes, 15mL (TBD), CD8 microblads, human (Miltenyi), LS Separation columns (Miltenyi), anti-Biotin Microbeads (Miltenyi), hu CD3 BV421 SK7 (BD), hu CD8 PE HIT8a (BD), PE Mouse FITC Anti-human CD4 (Biolegend).
The preparation method comprises the following steps:
washing Buffer: 0.5082g of magnesium chloride hexahydrate, 2.25g of glucose were weighed, 30mL of DPBS was added, dissolved, added to the remaining 470mL of DPBS, and stored at 4 ℃.
Binding Buffer: 0.01g BSA was weighed, 10mL Washing buffer and 100. Mu.L tRNA (100 mg/mL) were added, vortexed, dissolved well and stored at 4 ℃.
10 xACK Lysis Buffer: 40.118g NH was weighed 4 Cl，5g KHCO 3 ，0.186g EDTA-Na 2 30mL of sterile water was added, dissolved, and then 470mL of sterile water was added, and the pH was adjusted to 7.20,4 ℃for storage. When in use, the mixture is directly diluted into 1X ACK Lysis Buffer by using sterile water.
The instrument used in the invention:
AL204 electronic balance (Mettler Toledo), centrifuge 5418R desk Centrifuge (Eppendorf), dxP Athena TM Flow cytometry (Cytek), nikon confocal two-photon microscope (Nikon).
Example 1: streaming and confocal characterization of nucleic acid aptamers XD-4 and XD-9 binding to CD8+ T cells in peripheral blood.
1) Collecting peripheral blood 4-8mL, K of patient from hospital by professional medical staff 2 EDTA anticoagulation and inversion mixing to prevent blood clotting and transportation to laboratory at ambient temperature.
2) PBMCs in peripheral blood are obtained through density gradient centrifugation, a proper amount of sample diluent (DPBS, containing 1% FBS) is added to a final volume of 10mL, after the mixture is fully inverted and uniformly mixed, cells are precipitated through centrifugation (300 g, 5min and 25 ℃), the supernatant is discarded, 10mL of sample diluent is added to wash for one time, and the PBMCs are collected and placed on ice for standby.
3) Cd8+ T lymphocytes and CD 8-cells in PBMCs were obtained separately using magnetic sorting of CD8 microbeds, after which both cells were resuspended and counted using Binding Buffer.
4) Setting sample groups, each sample cell number being 3×10 5 The system was 125. Mu.L.
5) To the samples there were added 1.25. Mu.L of CD3, 2. Mu.L of CD4, 10. Mu.L of CD8 anti-body analysis magnetic separation obtained CD8+ T lymphocytes and CD 8-cell subsets. Meanwhile, XD-4 (cy 5) and XD-9 (cy 5) with final concentration of 100nM were added to other cell samples, and the mixture was gently shaken and mixed, placed in an ice box, and incubated for 35-40min on a 200rpm horizontal shaker, and the cells were gently shaken and mixed every 15 min.
6) After the incubation, 200. Mu.L of Washing Buffer was added and washed 1 time, 300g,25℃and the supernatant was centrifuged off for 5 min. Then, 400. Mu.L of wash Buffer was added to the suspension, and the suspension was transferred to a flow tube, and the fluorescence intensity of the cells was measured by flow.
Flow results As shown in FIG. 1, on the one hand, peripheral blood PBMCs mainly contain granulocytes, monocytes and lymphocytes, while killer T lymphocytes (CD8+ T cells) are a subset of lymphocytes, and CD8 protein is a specific marker for killer T lymphocytes. Therefore, commercial killer T lymphocyte magnetic separation magnetic beads (CD 8 microblades) can specifically isolate killer T lymphocytes in enriched PBMCs. On the other hand, CD3 is a total T lymphocyte marker in lymphocytes, CD8 is a killer T lymphocyte marker, and CD4 is a helper T lymphocyte marker, useful for flow analysis of T lymphocyte subpopulations composition. Therefore, CD8 microblades were used here to successfully isolate CD8+ T cells of high purity, while CD8+ T cells were hardly contained in magnetically sorted outgoing cells (CD 8-cells). Meanwhile, the nucleic acid aptamers XD-4 and XD-9 specifically bound to CD8+ T cells, but not CD 8-cells. Furthermore, the result of the confocal was also shown that the nucleic acid aptamers XD-4 and XD-9 specifically bound CD3+CD8+CD4-T lymphocytes.
Example 2: nucleic acid aptamers XD-4 and XD-9 were analyzed in combination with T lymphocyte subpopulations in peripheral blood.
1) Collecting peripheral blood 4-8mL, K of patient from hospital by professional medical staff 2 EDTA anticoagulation and inversion mixing to prevent blood clotting and transportation to laboratory at ambient temperature.
2) PBMCs in peripheral blood are obtained through density gradient centrifugation, a proper amount of sample diluent (DPBS, containing 1% FBS) is added to a final volume of 10mL, after the mixture is fully inverted and uniformly mixed, cells are precipitated through centrifugation (300 g, 5min and 25 ℃), the supernatant is discarded, 10mL of sample diluent is added to wash for one time, and the PBMCs are collected and placed on ice for standby.
3) Setting sample groups, each sample cell number being 4×10 5 The system was 125. Mu.L.
4) 1.25. Mu.L of CD3, 2. Mu.L of CD4, 10. Mu.L of CD8 Antibody were added to the group samples, and the mixture was gently shaken and mixed with 100nM final concentrations of XD-4 (cy 5) and XD-9 (cy 5), placed in an ice box, and incubated for 35-40min on a 200rpm horizontal shaker, and gently shaken and mixed with cells every 15 min.
5) After the incubation, 200. Mu.L of Washing Buffer was added and washed 1 time, 300g,25℃and the supernatant was centrifuged off for 5 min. Then, 400. Mu.L of wash Buffer was added to the suspension, and the suspension was transferred to a flow tube, and the fluorescence intensity of the cells was measured by flow.
Flow results As shown in FIG. 2, on the one hand, peripheral blood PBMCs contain mainly granulocytes, monocytes and lymphocytes, while lymphocytes mainly include T cells, B cells, NK cells, etc. Wherein killer T lymphocytes (cd8+ T cells) and helper T lymphocytes (cd4+ T cells) are the two major subpopulations of T lymphocytes. On the other hand, CD3 is a total T lymphocyte marker in lymphocytes, CD8 is a killer T lymphocyte marker, and CD4 is a helper T lymphocyte marker, useful for flow analysis of T lymphocyte subpopulations composition. Here, the proportion of killer T lymphocytes (CD3CD8+ T cells) and helper T lymphocytes (CD3CD4+ T cells) in PBMCs was first analyzed using CD3 (BV 421), CD4 (FITC), CD8 (PE). Meanwhile, nucleic acid aptamers XD-4 (cy 5) and XD-9 (cy 5) were added to analyze the proportion of cell subsets targeted by the nucleic acid aptamers, namely (XD-4+cell and XD-9+cell). Finally, the overlapping proportion of the cell subsets targeted by the aptamer XD-4 and XD-9 and the CD3CD8+ T cells or the CD3CD4+ T cells is analyzed through four-color flow, so that the T lymphocyte subset combined by the aptamer is judged. Thus, it can be seen from the flow results that XD-4 and XD-9 specifically bound CD3CD8+ T cells in PBMCs, but not CD3CD4+ T cells. The statistical analysis results also further showed that the ratio of XD-4+ cells, XD-9+ cells and CD3+CD8+ T cells were highly coincident, that is, the nucleic acid aptamers XD-4 and XD-9 specifically bound to killer T lymphocytes (CD3CD8+ T cells).
Example 3: universal and specific analysis of aptamer XD-4 and XD-9 binding to peripheral blood T lymphocyte subpopulations.
1) Collecting peripheral blood 4-8mL, K of multiple patients randomly from hospital by professional medical staff 2 EDTA anticoagulation and inversion mixing to prevent blood clotting and transportation to laboratory at ambient temperature.
2) First, 100. Mu.L of whole blood, i.e., a sample of mature red blood cells, was retained. The rest peripheral blood is separated by density gradient centrifugation to obtain PBMCs (including lymphocytes, monocytes, granulocytes and the like) in the peripheral blood, a proper amount of sample diluent (DPBS, containing 1% FBS) is added to a final volume of 10mL, after fully reversing and uniformly mixing, the cells are precipitated by centrifugation (300 g, 5min, 25 ℃), the supernatant is discarded, 10mL of sample diluent is added to wash for one time, and the PBMCs are collected and placed on ice for standby.
3) Setting sample groups comprising whole blood samples and PBMCs, each sampleThe number of cells was 5X 10 5 The system was 125. Mu.L.
4) Adding 100nM final concentration of XD-4 (FAM) and XD-9 (FAM) into the group samples, gently shaking and mixing, placing into ice box, horizontally shaking at 200rpm, incubating for 35-40min, and gently shaking and mixing cells every 15 min.
5) After the incubation, 200. Mu.L of Washing Buffer was added and washed 1 time, 300g,25℃and the supernatant was centrifuged off for 5 min. Then, 400. Mu.L of wash Buffer was added to the suspension, and the suspension was transferred to a flow tube, and the fluorescence intensity of the cells was measured by flow.
In examples 1 and 2, the nucleic acid aptamers XD-4 and XD-9 have been demonstrated to specifically bind to CD8+ T cells, where the nucleic acid aptamers were further analyzed for clinical universality and specificity. Flow results As shown in FIG. 3, XD-4 and XD-9 specifically bound CD8+ T cells in lymphocytes, but not other blood cells, including monocytes, granulocytes and mature erythrocytes, for different patient peripheral blood samples collected at random.
Example 4: nucleic acid aptamers XD-4 and XD-9 were assayed for apparent dissociation constant (Kd) of CD8+ T cells in human peripheral blood.
1) Collecting peripheral blood 4-8mL, K of patient from hospital by professional medical staff 2 EDTA anticoagulation and inversion mixing to prevent blood clotting and transportation to laboratory at ambient temperature.
2) PBMCs in peripheral blood are obtained through density gradient centrifugation, a proper amount of sample diluent (DPBS, containing 1% FBS) is added to a final volume of 10mL, after the mixture is fully inverted and uniformly mixed, cells are precipitated through centrifugation (300 g, 5min and 25 ℃), the supernatant is discarded, 10mL of sample diluent is added to wash for one time, and the PBMCs are collected and placed on ice for standby.
3) Cd8+ T lymphocytes in PBMCs were obtained separately using magnetic sorting of CD8 microbeds, resuspended and counted using Binding Buffer.
4) Setting sample groups, each group of samples having a concentration gradient of 0, 25, 50, 100, 150, 250, 400, 600nM for aptamer, and 3 samples having a cell count of 3×10 for each concentration gradient 5 The system was 200. Mu.L.
5) Respectively adding XD-4 and XD-9 with corresponding concentrations into each group of cell samples, gently shaking and mixing, placing into an ice box, horizontally shaking at 200rpm, incubating for 60min, and gently shaking and mixing cells every 15 min.
6) After the incubation, 200. Mu.L of Washing Buffer was added and washed 1 time, 300g,25℃and the supernatant was centrifuged off for 5 min. Then 400. Mu.L of Washing Buffer resuspended cells were transferred to a flow tube, the fluorescence intensity of the cells was measured by flow assay and Kd value was calculated.
The results in FIG. 4 show that the nucleic acid aptamers XD-4, XD-9 have a relatively high affinity with CD8+ T cells, with Kd values of 4.54.+ -. 2.08nM and 4.66.+ -. 0.93nM, respectively.
Example 5: truncating and optimizing the nucleic acid aptamer XD-4 and XD-9 sequences.
1) The truncations and optimizations of the nucleic acid aptamer XD-4 and XD-9 sequences are based on the secondary structure (NUPACK) formed by folding, and the primer sequences at the two ends are subjected to appropriate truncations and base substitution optimizations. Here, the nucleic acid aptamer XD-4 (81 nt) was truncated and optimized to XD-4c (62 nt), the nucleic acid aptamer XD-9 (81 nt) was truncated and optimized to XD-9a (75 nt), and FAM fluorescence was synthesized and labeled for verifying the binding capacity to CD8+ T cells in peripheral blood, while verifying whether the sequence after the truncated and optimized still binds to the target identical to the original sequence.
2) Collecting peripheral blood 4-8mL, K of patient from hospital by professional medical staff 2 EDTA anticoagulation and inversion mixing to prevent blood clotting and transportation to laboratory at ambient temperature.
3) PBMCs in peripheral blood are obtained through density gradient centrifugation, a proper amount of sample diluent (DPBS, containing 1% FBS) is added to a final volume of 10mL, after the mixture is fully inverted and uniformly mixed, cells are precipitated through centrifugation (300 g, 5min and 25 ℃), the supernatant is discarded, 10mL of sample diluent is added to wash for one time, and the PBMCs are collected and placed on ice for standby.
4) Setting sample groups, each sample cell number being 3×10 5 The system was 200. Mu.L.
5) Respectively adding XD-4 (FAM), XD-4c (FAM), XD-9 (FAM) and XD-9a (FAM) with 50nM concentration into each group of cell samples, gently shaking and mixing, placing into an ice box, horizontally shaking at 200rpm for 35-40min, and gently shaking and mixing cells every 15 min.
6) Simultaneously, XD-4c (FAM) with a final concentration of 50nM and XD-4 (unmodified) with a final concentration of 1. Mu.M, and XD-9a (FAM) with a final concentration of 50nM and XD-9 (unmodified) with a final concentration of 1. Mu.M were added to the remaining group samples, mixed with gentle shaking, placed in an ice box, incubated for 35-40min with a horizontal shaker at 200rpm, and mixed with gentle shaking at 15min intervals. Through competitive binding experiments, it was verified whether XD-4c and XD-9a bound to targets identical to the original sequences.
7) After the incubation, 200. Mu.L of Washing Buffer was added and washed 1 time, 300g,25℃and the supernatant was centrifuged off for 5 min. Then, 400. Mu.L of wash Buffer was added to the suspension, and the suspension was transferred to a flow tube, and the fluorescence intensity of the cells was measured by flow.
FIG. 5 shows that the nucleic acid aptamers XD-4c and XD-9a remained comparable in binding ability to the original sequence. Meanwhile, nucleic acid aptamers XD-4c and XD-9a bind to targets identical to the original sequences.
Example 6: analysis of CD8+ T cell efficiency, purity and cell Activity in peripheral blood captured by nucleic acid aptamers XD-4 and XD-9.
1) Collecting peripheral blood 4-8mL, K of patient from hospital by professional medical staff 2 EDTA anticoagulation and inversion mixing to prevent blood clotting and transportation to laboratory at ambient temperature.
2) PBMCs in peripheral blood are obtained through density gradient centrifugation, a proper amount of sample diluent (DPBS, containing 1% FBS) is added to a final volume of 10mL, after the mixture is fully inverted and uniformly mixed, cells are precipitated through centrifugation (300 g, 5min and 25 ℃), the supernatant is discarded, 10mL of sample diluent is added to wash for one time, and the PBMCs are collected and placed on ice for standby.
3) Setting sample groups, respectively adding corresponding aptamer to each group of cell samples to a final concentration of 50nM, gently shaking and mixing, placing in an ice box, incubating for 35-40min by a horizontal shaking table at 200rpm, and gently shaking and mixing cells every 15 min. At the same time, positive control samples were set, i.e., CD8+ T cells from PBMCs were isolated using CD8 microblades.
4) After the incubation is finished, 25 ℃ and 300g; centrifuging for 5min, discarding the supernatant, washing the cells for 1 time, adding Anti-biotin MicroBeads, gently shaking, mixing, placing in an ice box, incubating for 30min with a horizontal shaker at 200rpm, and gently shaking, mixing the cells every 15 min.
5) After the incubation is finished, capturing each part of separated cells by magnetic separation to obtain the aptamer and the CD8 anti-body, simultaneously carrying out multi-marker immunofluorescence Antibody to specifically mark the T lymphocytes in the cells, and analyzing the capturing efficiency and the capturing purity.
6) Simultaneously analyzing the cellular activity of the aptamer and the CD8 anti-body capture isolated cells by trypan blue staining.
The results in FIG. 6 show that the nucleic acid aptamers XD-4 and XD-9 can achieve high-efficiency and high-purity capture and separation of CD8+ T cells, the capture efficiency, purity and cell activity are all above 90%, and the separation performance equivalent to that of CD8 anti-body is shown.
Example 7: nucleic acid aptamer XD-4 and XD-9 complementary sequences were analyzed for traceless release efficiency, purity and cell activity.
1) Collecting peripheral blood 4-8mL, K of patient from hospital by professional medical staff 2 EDTA anticoagulation and inversion mixing to prevent blood clotting and transportation to laboratory at ambient temperature.
2) PBMCs in peripheral blood are obtained through density gradient centrifugation, a proper amount of sample diluent (DPBS, containing 1% FBS) is added to a final volume of 10mL, after the mixture is fully inverted and uniformly mixed, cells are precipitated through centrifugation (300 g, 5min and 25 ℃), the supernatant is discarded, 10mL of sample diluent is added to wash for one time, and the PBMCs are collected and placed on ice for standby.
3) Setting sample groups, respectively adding nucleic acid aptamer XD-4 and XD-9 to the final concentration of 50nM in each group of cell samples, gently shaking and mixing, placing in an ice box, horizontally shaking at 200rpm, incubating for 35-40min, and gently shaking and mixing cells every 15 min.
4) After the incubation is finished, 25 ℃ and 300g; centrifuging for 5min, discarding the supernatant, washing the cells for 1 time, adding Anti-biotin MicroBeads, gently shaking, mixing, placing in an ice box, incubating for 30min with a horizontal shaker at 200rpm, and gently shaking, mixing the cells every 15 min.
5) After the incubation, the nucleic acid aptamers XD-4 and XD-9 captured T lymphocytes were enriched by magnetic sorting. Thereafter, complementary sequences 4-RA1 and 9-RA1 (500. Mu.L) were added to the column at 50-fold concentrations, respectively, and incubated at room temperature for 35-40min. 9mL Washing Buffer,3mL times were added to the separation column, and T lymphocytes that were competitively released by the complementary sequences were washed, while the cells retained and shed by the separation column were collected, and analyzed for traceless release efficiency by counting. In addition, the activity of traceless released cells was analyzed by trypan blue staining.
6) At the same time, the released cells were labeled CD3, CD4, CD8 anti by flow analysis for purity of the released cells.
The results in FIG. 7 show that the complementary sequences 4-RA1 and 9-RA1 can effectively release nucleic acid aptamers XD-4 and XD-9 to capture CD8+ T cells, and the release efficiencies of the nucleic acid aptamers XD-4 and XD-9 can reach 48.9% and 60.6%, respectively. The purity of the released cells is over 95%, and the cell activities are 91.9% and 94.4%, respectively.
Claims (9)
1. A nucleic acid aptamer for traceless sorting of killer T lymphocytes in peripheral blood, characterized by being capable of specifically recognizing and binding to killer T lymphocytes in peripheral blood; the nucleic acid aptamer is XD-4 and/or XD-9 respectively,
wherein the XD-4 sequence is:
5’-ATCCAGAGTGACGCAGCAAGTGTCGTGAGGAGCTTGAAATCCAATCTGCACGGCCAGTGCGGATGGACACGGTGGCTTAGT-3’;
the XD-9 sequence was:
5’-ATCCAGAGTGACGCAGCATTGCGACCCCCTATGCGGTCGGTGAGGAGCTTGAAATCCCAATGATGGACACGGTGGCTTAGT-3’。
2. the aptamer of claim 1, wherein the aptamer XD-4 is truncated to XD-4c, the XD-4c sequence being:
5’-CAAGTGTCGTGAGGAGCTTGAAATCCAATCTGCACGGCCAGTGCGGATGGACACGACACTTA-3’；
the aptamer XD-9 is truncated into XD-9a, and the sequence of the XD-9a is as follows:
5’-TCCAGAGTGACGCAGCATTGCGACCCCCTATGCGGTCGGTGAGGAGCTTGAAATCCCAATGATGGACACTCTGGC-3’。
3. the aptamer of claim 1 or 2, wherein the aptamer is labeled with a fluorescent substance, a radioactive substance, a therapeutic substance, biotin, or an enzyme-labeled substance.
4. A method for identifying and enriching killer T cells in peripheral blood based on a nucleic acid aptamer, wherein the nucleic acid aptamer comprises the nucleic acid aptamer of any one of claims 1 to 3.
5. Use of a nucleic acid aptamer according to any one of claims 1-3 for the preparation of a preparation that specifically recognizes and binds killer T cells in peripheral blood.
6. A method for traceless release of killer T cells in peripheral blood based on complementary sequences, which is characterized in that the complementary release sequences are utilized to release the killer T cells in the peripheral blood identified and enriched by the nucleic acid aptamer XD-4 and/or XD-9 sequences,
the XD-4 sequence was:
5’-ATCCAGAGTGACGCAGCAAGTGTCGTGAGGAGCTTGAAATCCAATCTGCACGGCCAGTGCGGATGGACACGGTGGCTTAGT-3’；
its complementary release sequence 4-RA1 is as follows:
5’-ACTAAGCCACCGTGTCCATCCGCACTGGCCGTGCAG-3’；
the XD-9 sequence was:
5’-ATCCAGAGTGACGCAGCATTGCGACCCCCTATGCGGTCGGTGAGGAGCTTGAAATCCCAATGATGGACACGGTGGCTTAGT-3’；
its complementary release sequence 9-RA1 is as follows:
5’-ACTAAGCCACCGTGTCCATCATTGGGATTTCAAGCT-3’。
7. the application of complementary release sequence in preparing preparation for non-destructive release of killer T cells in peripheral blood captured by aptamer is characterized in that,
the complementary release sequence 4-RA1 is as follows:
5’-ACTAAGCCACCGTGTCCATCCGCACTGGCCGTGCAG-3’；
the nucleic acid aptamer XD-4 has the sequence as follows:
5’-ATCCAGAGTGACGCAGCAAGTGTCGTGAGGAGCTTGAAATCCAATCTGCACGGCCAGTGCGGATGGACACGGTGGCTTAGT-3’；
the complementary release sequence 9-RA1 is as follows:
5’-ACTAAGCCACCGTGTCCATCATTGGGATTTCAAGCT-3’；
the nucleic acid aptamer XD-9 has the sequence as follows:
5’-ATCCAGAGTGACGCAGCATTGCGACCCCCTATGCGGTCGGTGAGGAGCTTGAAATCCCAATGATGGACACGGTGGCTTAGT-3’。
8. a complementary release sequence for nondestructively releasing killer T cells in peripheral blood captured by a nucleic acid aptamer is characterized in that,
the complementary release sequence 4-RA1 is as follows:
5’-ACTAAGCCACCGTGTCCATCCGCACTGGCCGTGCAG-3’；
the nucleic acid aptamer XD-4 sequence is as follows:
5’-ATCCAGAGTGACGCAGCAAGTGTCGTGAGGAGCTTGAAATCCAATCTGCACGGCCAGTGCGGATGGACACGGTGGCTTAGT-3’；
and/or
The complementary release sequence 9-RA1 is as follows:
5’-ACTAAGCCACCGTGTCCATCATTGGGATTTCAAGCT-3’；
the nucleic acid aptamer XD-9 sequence is as follows:
5’-ATCCAGAGTGACGCAGCATTGCGACCCCCTATGCGGTCGGTGAGGAGCTTGAAATCCCAATGATGGACACGGTGGCTTAGT-3’。
9. a preparation for nondestructively releasing killer T cells in peripheral blood captured by a nucleic acid aptamer, which is characterized in that,
the complementary release sequence 4-RA1 is included as follows:
5’-ACTAAGCCACCGTGTCCATCCGCACTGGCCGTGCAG-3’；
the nucleic acid aptamer XD-4 sequence is as follows:
5’-ATCCAGAGTGACGCAGCAAGTGTCGTGAGGAGCTTGAAATCCAATCTGCACGGCCAGTGCGGATGGACACGGTGGCTTAGT-3’；
and/or
The complementary release sequence 9-RA1 is as follows:
5’-ACTAAGCCACCGTGTCCATCATTGGGATTTCAAGCT-3’；
the nucleic acid aptamer XD-9 sequence is as follows:
5’-ATCCAGAGTGACGCAGCATTGCGACCCCCTATGCGGTCGGTGAGGAGCTTGAAATCCCAATGATGGACACGGTGGCTTAGT-3’。
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