CN109439624B

CN109439624B - Method for identifying or enriching nucleated red blood cells

Info

Publication number: CN109439624B
Application number: CN201811452001.4A
Authority: CN
Inventors: 谭蔚泓; 叶茂; 彭波; 刘超; 张慧
Original assignee: Hunan University
Current assignee: Hunan University
Priority date: 2018-11-30
Filing date: 2018-11-30
Publication date: 2020-12-04
Anticipated expiration: 2038-11-30
Also published as: CN109439624A

Abstract

The present invention provides a method for identifying or enriching nucleated red blood cells. The specificity of the specific aptamer and the nucleated erythrocyte in the peripheral blood is utilized to identify or enrich the nucleated erythrocyte. Compared with the prior art, the method provided by the invention has the remarkable advantages that: (1) provides a new method for capturing or enriching nucleated erythrocytes, and has important significance; (2) the nucleic acid aptamer is used for carrying out specific recognition or capture on the nucleated red blood cells, so that the recognition or capture efficiency of target cells can be effectively improved, and the method has important significance for related research of the nucleated red blood cells; (3) the aptamer has small molecular weight, is chemically synthesized in the preparation method, and has the advantages of easy modification, strong stability, convenience for storage and the like. (4) The aptamer has good specificity to the nucleated red blood cells in the identification process, good affinity, simple operation and high accuracy and sensitivity. Provides a new method and approach for disease screening or prenatal diagnosis of fetuses.

Description

Method for identifying or enriching nucleated red blood cells

Technical Field

The invention belongs to the technical field of nucleated red blood cell detection, and particularly relates to a method for identifying or enriching nucleated red blood cells.

Background

Nucleated red blood cells are not visible in normal adult peripheral blood, and are found in small amounts in the peripheral blood of newborns within 1 week of birth. The appearance of nucleated red blood cells in the peripheral blood of adults is a pathological phenomenon. It can be seen that: proliferation of anemia: most commonly seen in various anemias, acute blood loss anemia, megaloblastic anemia, severe hypopigmented anemia. The appearance of late or intermediate erythroblasts is common. The appearance of nucleated red blood cells in peripheral blood indicates that erythroid hyperplasia in bone marrow is obviously active; (vii) erythroleukemia, erythroleukemia: the abnormal proliferation of immature erythrocyte in marrow and releasing into blood, and the abnormal proliferation of primitive erythrocyte and early erythrocyte is common; ③ extramedullary hematopoiesis: when bone marrow fibrosis occurs, tissues such as spleen, liver and lymph node recover the hematopoietic function of embryonic stage, and because these tissues lack the regulation and control ability to release blood cells, a large amount of immature blood cells enter peripheral blood. The erythroblasts at all developmental stages are visible, and the immature granulocytes and megakaryocytes can be seen; fourthly, other: such as metastatic cancer of the bone marrow, severe hypoxia, etc.

In addition, the nucleated erythrocyte exists in the peripheral blood of pregnant women stably, has short life cycle, disappears in the peripheral blood of women after delivery quickly, and is used for prenatal diagnosis without being interfered by the last pregnancy. In addition, it has a relatively significant increase in pathological conditions, which contributes to noninvasive prenatal diagnosis in the pathological obstetrics. Fetal erythroid cells develop earlier than white blood cells in the early gestation phase, and the ratio of fetal erythroid cells to white blood cells is about 1000:1 in yolk sac and liver hematopoiesis, theoretically, there are more nucleated red blood cells entering maternal peripheral blood in the early gestation phase than other types of fetal cells. The nucleated erythrocyte contains complete genome of fetus, can be used for analyzing and researching genetic diseases of fetus, and theoretically, the analysis and the diagnosis of the genetic diseases of fetus can be carried out by one nucleated erythrocyte. Therefore, there is a great potential to capture nucleated red blood cells in the peripheral blood of mothers for prenatal diagnosis or to analyze and study various possible genetic diseases of fetuses.

The existing methods for capturing or enriching nucleated red blood cells mainly comprise density gradient centrifugation, magnetic activated cell sorting, fluorescence activated cell sorting, micromanipulation separation and microfluidic devices, wherein the density gradient centrifugation and micromanipulation separation are based on the density and morphological characteristics of the nucleated red blood cells, and the magnetic activated cell sorting, fluorescence activated cell sorting and microfluidic devices are used for specific capture through CD71 antigen specifically expressed on the surface of the nucleated red blood cells by using antibodies aiming at CD 71. The existing ligands capable of capturing CD71, including transferrin and CD71 antibody, have corresponding defects. Transferrin and antibody preparation cost is high, uniformity in the preparation process is poor, modification is not easy, and the capture enrichment process is complex to operate.

The aptamer is ssDNA or ssRNA which is obtained by screening through a systematic evolution of ligands by exogenous evolution (SELEX) technology of in vitro exponential enrichment and can be combined with a target molecule with high affinity and high specificity. Compared with an antibody, the aptamer has the advantages of small molecular weight (5-15kD), chemical synthesis, easy modification, strong stability, convenient storage and the like.

We found that a nucleic acid aptamer TY8, which can recognize CD71, can act as a potent ligand of CD71 and can recognize in peripheral blood for disease diagnosis, or captured nucleated red blood cells can be used for research.

Disclosure of Invention

The invention aims to provide a method for identifying or enriching nucleated red blood cells, which has high sensitivity, strong specificity and simple and convenient operation.

The purpose of the invention is realized by adopting the following technical scheme.

A method for identifying or enriching nucleated red blood cells, which utilizes the specific combination of aptamer and nucleated red blood cells in peripheral blood to identify or enrich nucleated red blood cells, wherein the sequence of the aptamer is as follows: 5 '-ACTCATAGGGTTAGGGGCTGCTGGCCAGATACTCAGATGGTAGGGTTACTATGAGC-3', SEQ NO. 1.

The method for identifying or enriching nucleated red blood cells can also obtain the aptamer with the same function by adding or deleting bases or substituting the bases for the aptamer.

The method for identifying and enriching the nucleated red blood cells can also connect the aptamer with a fluorescent substance, a radioactive substance, a therapeutic substance, biotin or an enzyme-labeled substance to obtain the aptamer derivative which has the same ability of combining the nucleated red blood cells with the aptamer.

The method for identifying and enriching nucleated red blood cells comprises the following steps:

(1) and peripheral blood pretreatment: obtaining a mononuclear cell layer by density gradient centrifugation;

(2) and aptamer pretreatment: connecting biotin;

(3) and an enrichment process: incubating the aptamer and peripheral blood, washing to remove redundant aptamer, adding streptavidin-modified magnetic beads for incubation, and finally capturing nucleated red blood cells under the action of a magnet.

The nucleic acid aptamer of the invention is used for capturing and enriching nucleated red blood cells and comprises the following detailed steps:

1. collecting peripheral blood sample, placing in heparin anticoagulation vacuum blood collection tube, and sending to laboratory within 4 h;

2. blood samples were diluted in equal volumes with DPBS containing 5% BSA and 4% EDTA;

3. adding the ficoll lymphocyte separation solution with the density of 1.077g/ml into a centrifuge tube, and then slowly adding blood into the upper layer of the ficoll lymphocyte separation solution to ensure that the interface is clear;

4. centrifuging with centrifuge density gradient at 400rcf for 30min to obtain layered blood sample: the top layer is yellow clear serum, the middle layer is white transparent lymph separation liquid, the bottom is red mature red blood cell sediment, and a white flocculent ring layer between the top layer and the middle layer is obtained, and the white flocculent ring layer is a monocyte layer;

5. adding proper amount of DPBS, rotating at 250rcf for 10min, centrifuging at normal temperature to obtain cell precipitate, and repeating the steps;

6. taking a proper amount of TY8 with biotin labels, heating at 95 ℃ for 5min, and then cooling at 4 ℃ for 10 min;

7. resuspending the cells by using a Binding buffer, adding a biotin-labeled aptamer TY8, and placing the mixture in a shaking table at 4 ℃ for incubation for 40min to ensure that the aptamer can identify the nucleated red blood cells in the blood;

8. adding equal volume of Washing buffer, mixing uniformly, centrifuging at 250rcf, 5min and 4 ℃ to obtain cell sediment, and Washing repeatedly once according to the cell sediment;

9. adding a proper amount of magnetic beads modified with streptavidin into the cell sediment, and incubating for 20min at 4 ℃ in a Washing buffer solution environment containing 0.1% of Tween, so that the streptavidin and the biotin are fully combined;

10. adding the system onto a magnet, standing for 1 min to make the magnetic beads adsorbed by the magnet, removing the solution part, adding Washing buffer containing 0.1% Tween, and Washing repeatedly;

11. the captured nucleated red blood cells were observed in confocal.

After the nucleated red blood cells are captured by the method, the nucleated red blood cells not only can be researched, but also can be used for researching or diagnosing related diseases, and if the nucleated red blood cells in the peripheral blood of pregnant women are captured, the method can be used for researching the nucleated red blood cells of fetuses or screening the diseases.

It is a second object of the present invention to provide a specific use of the above-mentioned nucleic acid aptamer, which comprises:

the nucleic acid aptamer is applied to the preparation of a reagent for identifying or enriching nucleated red blood cells.

The nucleic acid aptamer is applied to preparing a reagent combined with protein CD71 on the surface of nucleated red blood cells.

Compared with the prior art, the method provided by the invention has the remarkable advantages that: (1) provides a new method for capturing or enriching nucleated erythrocytes, and has important significance; (2) the nucleic acid aptamer is used for carrying out specific recognition and capture on the nucleated red blood cells, so that the recognition and capture efficiency of target cells can be effectively improved, and the method has important significance for related research of the nucleated red blood cells; (3) the aptamer has small molecular weight (5-15kD), is chemically synthesized by a preparation method, and has the advantages of easy modification, strong stability, convenient storage and the like; (4) the aptamer has good specificity to the nucleated red blood cells in the capture process, good affinity, simple operation and high accuracy and sensitivity. (5) Provides a new method and approach for disease screening or prenatal diagnosis of fetuses.

Drawings

FIG. 1 shows the binding of aptamer TY8 to a human erythroleukemia cell line;

FIG. 2 shows the binding of aptamer TY8 to hematopoietic stem cell-induced nucleated red blood cells;

a is streaming data and B is confocal data;

FIG. 3 shows the binding of the aptamer TY8 to nucleated red blood cells in cord blood;

FIG. 4 shows capture of nucleated red blood cells in peripheral blood by aptamer TY8 coupled to magnetic beads;

FIG. 5 shows the binding of the aptamer TY8 to a cell membrane protein;

FIG. 6 shows the SDS-PAGE analysis of aptamer TY8 binding protein;

FIG. 7 shows the co-localization of fluorescent confocal results of aptamer TY8 with an antibody against CD 71;

FIG. 8 is a graph showing the results of western blotting with CD71 demonstrating that the nucleic acid aptamer TY8 binds;

in the lowermost horizontal frame is a luminescence picture of incubation with CD71 antibody after membrane transfer;

FIG. 9 shows the dissociation constant calculation for binding of aptamer TY8 to CD71 protein.

Detailed Description

The following examples are intended to further illustrate the invention without limiting it.

Reagents formulated in the examples of the invention:

washing buffer: the PBS solution contained 5mM MgCl₂4.5g/L glucose;

binding buffer: the PBS solution contained the following: 5mM MgCl₂4.5g/L glucose, 0.1mg/mL yeast tRNA,1mg/mL BSA and 20% FBS.

Anticoagulation buffer solution: DPBS + 0.5% BSA + 0.4% EDTA.

Hypotonic buffer: 25.2mL of Washing buffer was put into a 50mL centrifuge tube, and 3mL of 10 XCocktail, 1.5mL of 1M Tris-HCl, and 300. mu.L of 100 XPMSF were added thereto, mixed by shaking, and stored at 4 ℃.

Membrane protein lysate: 5mL of hypotonic buffer was placed in a 15mL centrifuge tube, 50. mu.L of Triton-X-100 was added to the tube, and the tube was stored at 4 ℃.

Reagents purchased:

the ELISA kit is purchased from BD company, and comprises: coating buffer, assay solvent, SAv-HRP (streptavidin-coated HRP reagent), substrate solution, stop solution, and the like.

DPBS, BSA, EDTA, hydroxyethyl starch, 1.077g/ml Ficoll lymphocyte isolate, cytokine: IL3, scf, epo, fbs; sfem medium, trypsin, proteinase K, DNA blocking solution, etc. were purchased from sequoyinfei.

cocktail, PMSF, Triton-X-100 is available from sigma.

The loading buffer, acryl gel, Tris-HCl, SDS, APS, TEMED, bromophenol blue, Coomassie brilliant blue staining solution and the like were purchased from Biyun Tian.

Streptavidin-coated sepharose beads were purchased from GE.

PL45 cells and the erythroleukemia cell line HEL were obtained from the Shanghai cell Bank.

Example 1: TY8 binding to erythroleukemia cell line experiments

(1) Culturing an erythroleukemia cell line HEL until logarithmic growth phase, and washing with DPBS for three times;

(2) counting, obtaining 30 ten thousand cells, and resuspending the cells by 200 microliter Binding buffer;

(3) adding cy5 fluorescently labeled TY8 to a final concentration of 250 nM;

(4) incubating at 4 deg.C on a shaker at 80rpm for 40 min;

(5) washing with 500. mu.l Washing buffer three times at 1000rpm, 4 ℃ for 5 min;

(6) resuspend with 500. mu.l Washing buffer and analyze by up-flow.

In order to determine whether TY8 bound nucleated erythrocytes, the erythroleukemia cell line HEL was first used, and the aptamers and random libraries were incubated with this cell line, respectively, followed by up-flow assay, which demonstrated that TY8 was able to specifically bind erythroleukemia cell lines, see fig. 1, the cell type of which was nucleated erythrocytes.

Example 2: TY8 nucleated erythrocyte experiment combining hemopoietic stem cell to induce differentiation

(1) The umbilical cord blood is taken, added with a proper amount of heparin for anticoagulation and is processed in time.

(2) Adding an anticoagulation buffer solution in a ratio of 1:1, diluting and uniformly mixing, and then adding the diluted umbilical cord blood: anticoagulation buffer solution: 6% hydroxyethyl starch was added at a ratio of 2:2:1 and allowed to stand at room temperature for 1 hour.

(3) Aspirate the supernatant into a new centrifuge tube, approximately 40 ml/tube. Centrifuging at 1500rpm for 10min, and removing supernatant.

(4) The cell pellet was resuspended in anticoagulation buffer, and then a 15ml centrifuge tube was added with 1.077g/ml of Ficoll lymphocyte separation medium at about 3 ml/tube, and the cell fluid was added to the upper layer of the Ficoll lymphocyte separation medium at about 7 ml/tube, centrifuged at 1800rpm for 30 min.

(5) And (4) sucking the second layer of the mononuclear cells into a new centrifuge tube, adding an anticoagulation buffer solution to the full volume, rotating at 1500rpm for 15min, and centrifuging to remove the supernatant. Adding anticoagulation buffer solution to resuspend the cells, counting the cells, then 1500rpm, 15min, and centrifuging to remove the supernatant.

(6) Adding 300 μ l of anticoagulation buffer solution for resuspending cells, then adding 100 μ l of blocking solution, standing at room temperature in the dark for 5min, then adding an appropriate amount of magnetic beads, incubating at room temperature in the dark for 30min, then adding an appropriate amount of anticoagulation buffer solution for washing, 300rpm, 10min, and centrifuging to remove supernatant.

(7) Resuspending the cells in the anticoagulation buffer solution again, placing the separation column on a magnetic plate, adding 2ml of the anticoagulation buffer solution to clean the separation column, then adding the cell resuspension solution, adding 2ml of the anticoagulation buffer solution to wash the separation column, and dropwise adding the cell suspension along the wall of the column during column passing.

(8) The column was removed and placed in a new 15ml centrifuge tube, 2ml of anticoagulation buffer was added and the cells were blown down.

(9) Then, culturing, centrifuging once again before cell culture at 300rpm for 10min, discarding the anticoagulation buffer solution, re-suspending the cells by using a prepared culture medium, and adding the cells into a culture dish until day 6, wherein the culture medium is an sfem culture medium containing IL 310 ng/ml, scf 50ng/ml, epo 1u/ml and 10% fbs; on day 6, the medium was changed to sfem medium containing 30% fbs, 3u/ml epo, and the culture was continued until day 14.

(10) Adding a proper amount of DPBS into the cells cultured on the 8 th day, centrifuging and washing twice at 1000rpm for 5min, adding TY8 marked with cy5 and CD71 marked with PE, incubating for 40min in a dark place on ice, adding a proper amount of precooled DPBS, and centrifuging and washing twice at 1000rpm for 5 min;

(11) by confocal and flow analysis, it was observed that both aptamer TY8 and antibody CD71 bound to the induced nucleated red blood cells and that co-localization was present.

To further determine whether TY8 bound nucleated erythrocytes, differentiated nucleated erythrocytes induced by CD34 positive cells were used, labeled with CD71 antibody and labeled with the aptamer TY8, and the consistency of flow results and co-localization effect of co-focusing results were observed, see fig. 2.

Example 3: TY8 binding nucleated red blood cell experiments in umbilical cord blood

(1) Collecting umbilical cord blood, placing the umbilical cord blood in a heparin anticoagulant vacuum blood collection tube, and delivering the umbilical cord blood to a laboratory within 4 hours;

(2) diluting a blood sample with an anticoagulation buffer solution in equal volume, adding 4ml of ficoll lymphocyte separation solution with the density of 1.077g/ml into a 15ml clean centrifugal tube, and slowly adding the blood into the centrifugal tube to ensure that an interface is clear;

(3)400rcf, 30min density gradient centrifugation to obtain stratified blood samples: the top layer is yellow clear serum, the middle layer is white transparent lymph separation liquid, the bottom is red mature red blood cell sediment, a white flocculent ring layer between the top layer and the middle layer is sucked by a pipette and is placed in a new 15ml centrifuge tube, and the white flocculent ring layer is a monocyte layer;

(4) adding a proper amount of DPBS into cells of the monocyte layer, centrifuging at normal temperature for 10min at 250rcf to obtain cell sediment, then adding a proper amount of DPBS for resuspension, and centrifuging at normal temperature for 10min at 250rcf to obtain cell sediment;

(5) resuspending with a proper amount of Binding buffer, adding corresponding CD71 antibody, GPA antibody and Cy5 fluorescence-labeled TY8 aptamer, incubating, labeling, and incubating at 4 ℃ for 40 min;

(6) adding equal volume of Washing buffer, mixing uniformly, centrifuging at 250rcf, 5min and 4 ℃ to obtain cell sediment, and Washing repeatedly according to the cell sediment;

(7) finally, resuspending with a proper amount of Washing buffer, detecting in an up-flow manner to obtain cells with double positive signals, namely nucleated red blood cells, and analyzing the cell group labeled with the aptamer TY 8.

The clinical sample umbilical cord blood is adopted to carry out TY8 combined examination of fetal nucleated red blood cells, firstly, the umbilical cord blood is subjected to density gradient centrifugation to obtain a mononuclear cell layer, the mononuclear cell layer is subjected to antibody fluorescent labeling of nucleated red blood cell markers CD71 and GPA, and meanwhile, the mononuclear cell layer is respectively incubated with TY8 and a random library, then, flow analysis is carried out, and TY8 can be specifically identified in nucleated red blood cell populations with CD71 and GPA double positive, as shown in figure 3.

Example 4: TY8 coupled magnetic bead capture to nucleated red blood cell experiment

(1) Collecting peripheral blood sample, placing in heparin anticoagulation vacuum blood collection tube, and sending to laboratory within 4 hours;

(2) diluting the blood sample with DPBS containing 5% BSA and 4% EDTA in equal volume, adding 4ml of ficoll lymphocyte separation solution with the density of 1.077g/ml into a 15ml clean centrifuge tube, and slowly adding the blood into the centrifuge tube to ensure that the interface is clear;

(3)400rcf, 30min density gradient centrifugation to obtain stratified blood samples: the top layer is yellow clear serum, the middle layer is white transparent lymph separation liquid, the bottom is red mature red blood cell sediment, a white flocculent ring layer between the top layer and the middle layer is absorbed by a pipette and is placed in a new 15ml centrifugal tube, and the white flocculent ring layer is a mononuclear cell layer;

(5) resuspending the cells with a Binding buffer, adding a proper amount of CD71 antibody labeled with PE dye and a nucleic acid aptamer TY8 with biotin, and placing on ice for 40min to make the nucleic acid aptamer recognize nucleated red blood cells in blood;

(7) adding a proper amount of magnetic beads marked with streptavidin and a nuclear dye hoechst33342, and incubating for 20min at 4 ℃ in a Washing buffer solution environment containing 0.1% of Tween to ensure that the streptavidin and the biotin are fully combined;

(8) adding the system onto a magnet, standing for 1 min to make the magnetic beads adsorbed by the magnet, removing the solution part, adding Washing buffer containing 0.1% Tween, and repeating for three times;

(9) finally, resuspend with Washing buffer of 0.1% tween and observe the capture of nucleated erythrocytes by magnetic beads in confocal manner, see fig. 4.

Example 5: aptamer TY8 type identification assay for binding targets

The experimental procedure was as follows:

(1) preparation of ssDNA: taking aptamer TY8 and a random library chain, denaturing at 95 ℃ for 5min, then renaturing on ice for 10min, centrifuging at 4 ℃ and 5000rpm for 3min, adding a precooling Binding buffer to enable the DNA concentration to be 250nM, and placing on ice for standby.

(2) Preparation of cells: PL45 cells were cultured in four dishes to logarithmic phase, the medium was discarded, washed twice with 2mL DPBS, two dishes were digested at room temperature for 10min with 200. mu.l 0.25% trypsin and 200. mu.l 0.1mg/mL proteinase K, respectively, and the other two dishes were digested under the same conditions with 0.2% EDTA, respectively, followed by addition of 500. mu.l complete medium to stop the digestion, centrifugation at 800rpm for 4min, and the supernatant was aspirated off. Washing twice with Washing buffer, and counting with cell counting plate to obtain cell number of 3 × 10⁵And (4) respectively.

(3) Incubation of ssDNA with cells: and (2) taking 200 mu l of the random library chain into EDTA digested cells, respectively taking 200 mu l of TY8 into the cells digested in the other three modes, gently blowing and uniformly mixing heavy suspension cells, incubating for 1h in a shaking table at 4 ℃ in a dark place, adding Washing buffer for centrifugation and Washing twice after incubation, adding 500 mu l of Washing buffer, passing the prepared sample to be detected through a membrane to disperse the sample into single cells, and detecting the fluorescence intensity of the cells by a flow cytometer. As shown in fig. 5, TY8 could target and recognize EDTA-treated PL45 cells, but not proteinase K-and trypsin-treated PL45 cells, indicating that the type of target to which TY8 binds was a protein.

Example 6: aptamer target mass spectrum identification experiment

(one) extraction of cell membrane proteins

(1) Culturing PL45 (pancreatic cancer cell line) to logarithmic phase, removing old culture medium, cleaning DPBS twice, adding EDTA for digestion at room temperature, collecting cells, and Washing twice by Washing buffer centrifugation;

(2) adding appropriate amount of hypotonic buffer solution, shaking and mixing, and shaking at 4 deg.C for 30 min; centrifuging at 4000rpm for 10min, and removing the supernatant; washing with hypotonic buffer solution for 3 times;

(3) adding appropriate amount of membrane protein lysate into centrifuge tube, shaking, mixing, lysing at 4 deg.C for 30min, centrifuging, and retaining supernatant at 4000rpm,4 deg.C for 10min. The remaining supernatant, i.e., the protein sample, was stored at-80 ℃.

(II) preparation of PAGE collagen samples

(1) Blocking of streptavidin-coated sepharose beads: three 1.5ml EP tubes were loaded with 100. mu.l agarose gel beads and centrifuged at 2500rpm for 3min to label them as blank, library and TY8 samples, respectively. To each EP tube, 1mL of 5% BSA was added, mixed well with shaking, and blocked on a shaker at 4 ℃ for 1h.

(2) And (3) sealing membrane protein, namely adding 3ml of DNA sealing solution into the collected protein sample, shaking uniformly, incubating for 1h at 4 ℃, and taking out a proper amount of DNA sealing solution to be used as a whole protein sample group after sealing.

(3) After blocking the Sepharose beads and membrane proteins, the gel beads were washed 5 times with Washing buffer at 2500rpm 4 ℃ for 3min and placed on ice until use.

(4) Sepharose beads, library, aptamer and membrane protein incubation the blocked membrane proteins were evenly divided into 3 groups and each was added to an EP tube of 3 blocked Sepharose beads, and biotin-modified random libraries and TY8 were added according to the respective labels. Shaking and mixing evenly, putting into a shaking table at 4 ℃ and incubating for 1h.

(5) After incubation, Washing buffer washes were centrifuged 5 times at 2500rpm for 3min at 4 ℃, and the supernatant was discarded after centrifugation.

(6) Protein denaturation: adding 2 × loading buffer equal volume to the agarose gel beads, denaturing at 100 deg.C for 10min, and storing on ice for 10min at-80 deg.C.

(III) SDS-PAGE

(1) Preparation of PAGE gel 8% separation gel and 5% concentrated gel 10% SDS-PAGE protein separation gel (5mL) in a small beaker, 2mL ddH was added in order₂O,1.6mL 30% acryloyl gel, 1.25mL 1.5M Tris-HCl (pH8.8),0.05mL 10% SDS,0.05mL 10% APS, 0.002mL TEMEED, mixed well and added to the gel plate. Standing at room temperature, and adding 5% SDS-PAGE protein concentrated gel after the gel is solidified. Sequentially suck 3.4mL of ddH₂O,0.85mL of 30% acrylamide, 0.625mL of 1.0M Tris-HCl (pH6.8),0.05mL of 10% SDS,0.05mL of 10% APS,0.005mL of TE.

(2) And (2) electrophoresis, namely, correctly filling the prepared SDS-PAGE gel into an electrophoresis tank, adding 1X electrophoresis solution (one gel is 750mL), adding the prepared sample into a hole at a voltage of 60V for electrophoresis according to the sequence of a marker, a blank bead sample, a library group bead sample and a nucleic acid aptamer bead sample, raising the voltage to 120V for continuous electrophoresis after the bromophenol blue band is transferred to the lower layer separation gel, and finishing the electrophoresis until the bromophenol blue band is transferred to a gel substrate.

(3) Protein fixation: taking out SDS-PAGE gel, removing concentrated gel, placing separation gel in stationary liquid for 2 hr, and rinsing with ultrapure water for 15min each time for 3 times.

(4) The PAGE gel was transferred to Coomassie Brilliant blue stain overnight, and the Coomassie Brilliant blue stained gel was washed 4 times with ultrapure water for 15min each time. Protein bands are clearly visible on the gel.

(5) The scanner scans the SDS-PAGE gel and the results are shown in FIG. 6.

(IV) cutting the gel for mass spectrometry

In order to identify the differential proteins shown in the SDS-PAGE gel, the proteins were extracted by digestion and identified by mass spectrometry. As shown in table 1, the highest CD71 score among the co-detected proteins in the differential bands was 2088.63 on the first row, which was much higher than the Keratin on the second row (a common hair and skin-derived protein, which may occur due to improper handling), and the band coverage reached 70.13%. Given that CD71 is a transmembrane protein, we speculated that CD71 protein is likely to be a target for TY8 binding.

Table 1: mass spectrometry results for TY8 binding proteins

Example 7: co-localization experiments with TY8 and CD71 antibodies

(1) Preparation of FITC-TY8 and PE-anti CD 71: the FITC-modified TY 895 ℃ was denatured for 5 minutes, cooled on ice for 10 minutes, and added to Binding buffer to prepare 250nM DNA solution. Mu.l of PE-modified CD71 antibody was removed and 190. mu.l of Binding buffer was added and placed on ice until use.

(2) Preparation of cells: PL45 cells were seeded into optical petri dishes for culture. The medium was discarded and cells were washed twice with DPBS for future use.

(3) Incubation of TY8 and CD71 antibodies with cells: mu.l of FITC-modified TY8 and 200. mu.l of PE-modified CD71 antibody were added to the same PL45 cell culture dish, mixed gently and incubated on a shaker at 4 ℃ for 1h.

(4) After incubation, Washing buffer was washed twice, and 1mL of Washing buffer was added to the petri dish and photographed under a confocal laser microscope, as shown in FIG. 7 for TY8 co-localization with CD71 antibody.

Example 8: aptamer-pull down experiment

(one) extraction of cell membrane proteins

(1) Culturing PL45 to logarithmic phase, removing old culture medium by suction, cleaning DPBS twice, adding EDTA for digestion at room temperature, collecting cells, and Washing twice by Washing buffer centrifugation;

(II) preparation of PAGE collagen samples

(III) SDS-PAGE

(1) Preparation of PAGE gel 8% separation gel and 5% concentrated gel 10% SDS-PAGE protein separation gel (5mL) in a small beaker, 2mL of ddH was added in order₂O,1.6mL of 30% acryl gel, 1.25mL of 1.5M Tris-HCl (pH8.8),0.05mL of 10% SDS,0.05mL of 10% APS, 0.002mL of TEMED, and mixing well and adding to the gel plate. Standing at room temperature, and adding 5% SDS-PAGE protein concentrated gel after the gel is solidified. Sequentially draw 3.4ml of ddH₂O,0.85mL of 30% acrylamide, 0.625mL of 1.0M Tris-HCl (pH6.8),0.05mL of 10% SDS,0.05mL of 10% APS,0.005mL of TEMED.

(IV) luminescence of the rotating film

(1) Preparing a membrane conversion buffer solution in advance, and standing at 4 ℃ for later use.

(2) After SDS-PAGE electrophoresis is finished, taking out the gel, removing the upper concentrated gel, assembling the gel according to the sequence of the anode splint, the sponge, the filter paper, the NC membrane, the gel, the filter paper, the sponge and the splint, and placing the gel into a membrane transferring tank for transferring 300mA for 90 min. When the film is rotated, the film rotating groove needs to be placed in ice.

(3) The NC membrane was removed, stained with ponceau red stain, and the positions of CD71 protein and GAPDH protein (control protein) were cut and blocked with 5% milk at room temperature for 1h.

(4) Protein bands were incubated with the respective primary antibodies overnight at 4 ℃.

(5) The protein bands were washed 3 times with TBST for 10min each.

(6) Protein bands were incubated with the corresponding anti-antibody for 1h at room temperature, followed by washing 3 times with TBST, 10min each.

(7) And preparing a luminescent liquid, uniformly adding the luminescent liquid on the protein strip, and placing the protein strip into a gel imager for imaging.

The results are shown in FIG. 8, demonstrating that aptamer TY8 binds to CD 71.

Example 9: ELISA experiments

(1) An elisa plate was taken for a total of eight wells. A new 1.5ml EP tube was filled with coating buffer followed by CD71 pure protein to a final concentration of 0.3. mu.M. Then added to the elisa well and incubated overnight at 4 ℃ with shaking.

(2) The next day, the coating buffer was aspirated and washed three times with Washing buffer.

(3) Add 250. mu.l of assay solution to each well and incubate with shaker at room temperature for 1h.

(4) The solution was aspirated and washed three times with Washing buffer.

(5) According to the experimental dosage, the aptamer TY 895 ℃ is denatured for 5min in advance, and the nucleic acid aptamer is renatured for 10min on ice. Different concentrations of biotin-TY8 were made up with Binding buffer:

5 '-biotin-TTTTTTACTCATAGGGTTAGGGGCTGCTGGCCAGATACTCAGATGGTAGGGTTACTATGAGC-3', SEQ NO.2, with the concentration of each well being 0, 0.025, 0.05, 0.1, 0.15, 0.25, 0.5, 0.75. mu.M, the stepwise dilution method is adopted for preparing aptamers with different concentrations, and the aptamers are incubated for 1h at 4 ℃ in a shaking table.

(6) The solution was aspirated and washed four times with Washing buffer.

(7) Add 100. mu.l of SAv-HRP to each well and incubate for 1h at RT.

(8) The solution was aspirated and washed five times with Washing buffer.

(9) Mu.l of substrate solution was added to each well and incubated with shaking light for 30min at room temperature.

(10) Add 50. mu.l of stop solution per well.

(11) And (3) detecting the absorption values of A450nm and A570nm by a microplate reader to obtain a difference value, and paying attention to light shielding operation in the detection process.

(12) Kd curves were generated using Graphpad software based on the absorbance measured.

The affinity of TY8 for transferrin receptor 1(CD71 protein) was examined and the dissociation constant of the aptamer from the target was calculated using GraphPad software (see figure 9). Directly incubating CD71 pure protein with different concentrations of biotin-TY8 labeled nucleic acid aptamers, adding SAv-HRP, detecting by using an enzyme-labeling instrument, and calculating to obtain the dissociation constant Kd of the TY8 combined with the CD71 pure protein, which is 50.48 nM. Kd values were in nM range, which demonstrates the strong affinity of TY8 for CD71 protein.

Sequence listing

<110> university of Hunan

<120> A method for identifying or enriching nucleated red blood cells

<160> 2

<170> SIPOSequenceListing 1.0

<210> 1

<211> 56

<212> DNA

<213> Unknown (Unknown)

<400> 1

actcataggg ttaggggctg ctggccagat actcagatgg tagggttact atgagc 56

<210> 2

<211> 62

<212> DNA

<213> Unknown (Unknown)

<400> 2

ttttttactc atagggttag gggctgctgg ccagatactc agatggtagg gttactatga 60

gc 62

Claims

1. A method for enriching nucleated red blood cells, which is characterized in that the nucleated red blood cells are enriched by utilizing the specific combination of aptamer and the nucleated red blood cells in peripheral blood, and the sequence of the aptamer is as follows:

5＇

-ACTCATAGGGTTAGGGGCTGCTGGCCAGATACTCAGATGGTAGGGTTACTATGAGC-3＇。

2. the method according to claim 1, wherein said aptamer is conjugated with a fluorescent substance, a radioactive substance, biotin, or an enzyme-labeled substance to obtain a derivative of said aptamer having the same ability to bind to nucleated red blood cells as said aptamer.

3. Method for the enrichment of nucleated red blood cells according to claim 1 or 2, characterized in that it comprises the following steps:

(2) and aptamer pretreatment: connecting biotin;

4. The application of the aptamer in preparing the reagent for enriching the nucleated red blood cells in the peripheral blood is characterized in that the sequence of the aptamer is as follows:

5＇

-ACTCATAGGGTTAGGGGCTGCTGGCCAGATACTCAGATGGTAGGGTTACTATGAGC-3＇。

5. the use according to claim 4, wherein said aptamer is conjugated with a fluorescent substance, a radioactive substance, biotin, or an enzyme-labeled substance to obtain a derivative of said aptamer having the same ability to bind to nucleated red blood cells as said aptamer.