CN109943568B - Single-stranded nucleic acid aptamer and application thereof - Google Patents
Single-stranded nucleic acid aptamer and application thereof Download PDFInfo
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Abstract
The invention discloses a nucleotide chain aptamer and application thereof, belonging to the technical fields of biomedicine and clinical medicine. Wherein the aptamer satisfies: (1) Has a general formula shown as 5'-AGAGACGGACACAGGATGAGC-N40-CCTTCCCCAAGACAGCATCCA-3' formed by sequentially connecting a nucleotide sequence shown as SEQ ID NO. 1, a 40bp random nucleotide sequence and a nucleotide sequence shown as SEQ ID NO. 2; (2) capable of specifically binding to RhD antigen. The adapter can specifically shield the RhD antigen on the surface of the red blood cells, and the RhD positive red blood cells are changed into RhD negative red blood cells. The invention also discloses application of the aptamer in preparation of a modifying agent for modifying RhD positive red blood cells into RhD negative red blood cells. The invention can avoid hemolytic transfusion reaction caused by alloimmunity, and can be used for non-homotypic transfusion of RhD negative patients in emergency.
Description
Technical Field
The invention belongs to the technical fields of biomedicine and clinical medicine. In particular, the invention relates to a single-stranded nucleic acid aptamer and uses thereof.
Background
In clinical transfusion practice, the Rh blood group system is one of the most clinically significant blood group systems, and is also the most complex and polymorphic blood group system among the 36 blood group systems of humans. Blood transfusion that does not match the Rh blood group system can cause blood transfusion adverse reactions such as hemolysis, renal failure and the like and even death due to alloimmunization. Rh blood group system antigens are controlled and encoded by RHD and RHCE genes, and 54 antigens are found. RhD antigen is the most immunogenic antigen in Rh blood group system antigen, and has the most obvious clinical significance, and is also a main factor affecting clinical blood safety.
Previous studies showed that infusion of 30 μl of RhD positive erythrocytes in RhD negative patients resulted in alloanti-D. To ensure transfusion safety, rhD negative patients can only infuse negative blood. However, in Chinese Han population, rhD negative individuals account for only 0.1% -0.4%. With increasing clinical blood volume, there is often a shortage of RhD negative blood supply, which severely affects the daily transfusion treatment of RhD negative patients, especially in the case of acute blood loss, and can threaten patient life. In contrast, the RhD positive blood is quite rich, and the RhD positive red blood cells are changed into negative red blood cells, so that the problem of RhD negative clinical blood supply shortage can be solved.
The advent of cell-targeted exponential enrichment ligand system performance techniques (cell systematic evolution of ligands by exponential enrichment, cell-SELEX) has provided the opportunity to achieve the engineering of RhD universal blood. The aptamer for identifying a target molecule can be obtained through Cell-SELEX screening, has the advantages of strong specificity, high affinity, small volume and the like, can be obtained in large quantity through a gene synthesis or PCR amplification method, and has low cost and no toxicity to human bodies. The aptamer has been widely used in the fields of high-sensitivity detection and analysis, diagnosis, protein interaction, development of new anti-infective drugs, difficult-to-treat viral infectious diseases (such as AIDS, HBV, HSV, rabies virus, etc.), etc.
Disclosure of Invention
The inventor unexpectedly screens out a single-stranded nucleic acid (ssDNA) aptamer which can be combined with RhD antigen with high affinity and high specificity and can shield antigenicity through a ligand system evolution technology of red blood cell subtraction index enrichment, can realize the 'reformation' of RhD blood type to achieve the purpose of blood general use, and can provide red blood cells with 'blood type compatibility' for RhD negative rare blood type patients in emergency.
Thus, the invention provides a single-stranded nucleic acid aptamer which can specifically bind to RhD antigen, shield RhD antigenicity and convert RhD positive erythrocytes into RhD negative erythrocytes. The therapeutic agent can be prepared, and when RhD negative blood cannot be obtained in time, rhD positive red blood cells are changed into negative red blood cells by using the therapeutic agent, so that the therapeutic agent is used for emergency transfusion of RhD negative patients.
In order to achieve the above object, the present invention provides in one aspect a single-stranded nucleic acid aptamer satisfying:
(1) Has a general formula shown as 5'-AGAGACGGACACAGGATGAGC-N40-CCTTCCCCAAGACAGCATCCA-3' formed by sequentially connecting a nucleotide sequence shown as SEQ ID NO. 1, a 40bp random nucleotide sequence and a nucleotide sequence shown as SEQ ID NO. 2;
(2) Can specifically bind to RhD antigen.
In some embodiments of the invention, the random nucleotide sequence is selected from one of SEQ ID NOs 3-14.
In some embodiments of the invention, the random nucleotide sequence is selected from one of SEQ ID NO. 3, SEQ ID NO. 4, SEQ ID NO. 6, SEQ ID NO. 7 and SEQ ID NO. 11.
In a specific embodiment of the present invention, the random nucleotide sequence is selected from one of SEQ ID NO. 3 and SEQ ID NO. 4.
In a second aspect, the invention provides a single stranded nucleic acid aptamer composition comprising at least two single stranded nucleic acid aptamers, any aptamer satisfying:
(1) Has a general formula shown as 5'-AGAGACGGACACAGGATGAGC-N40-CCTTCCCCAAGACAGCATCCA-3' formed by sequentially connecting a nucleotide sequence shown as SEQ ID NO. 1, a 40bp random nucleotide sequence and a nucleotide sequence shown as SEQ ID NO. 2;
(2) Can specifically bind to RhD antigen.
In some embodiments of the invention, the random nucleotide sequence is selected from at least two of SEQ ID NOs 3-14.
In some embodiments of the invention, the random nucleotide sequence is selected from at least two of SEQ ID NO. 3, SEQ ID NO. 4, SEQ ID NO. 6, SEQ ID NO. 7 and SEQ ID NO. 11.
In some embodiments of the invention, the random nucleotide sequences are SEQ ID NO. 3 and SEQ ID NO. 4, respectively.
A third aspect of the invention provides the use of an aptamer according to the first aspect of the invention or an aptamer composition according to the second aspect of the invention in the preparation of an remodelling agent for use in the modification of RhD positive erythrocytes to RhD negative erythrocytes.
In a fourth aspect the present invention provides an engineering agent for engineering RhD-positive erythrocytes to RhD-negative erythrocytes, said engineering agent comprising an aptamer according to the first aspect of the invention or an aptamer composition according to the second aspect of the invention.
The beneficial effects of the invention are that
The single-stranded nucleic acid aptamer is a small molecular substance consisting of 82 bases, can be specifically combined with RhD antigen, can effectively shield erythrocyte RhD antigen, lose bioactivity, block specific combination with alloantibody, avoid hemolytic transfusion reaction caused by alloimmunity, and can be used for non-homotypic transfusion of RhD negative patients in emergency.
Drawings
FIG. 1 is a flow chart of a method for preparing red blood cell depleting SELEX screening of rhD antigen-specific ssDNA nucleic acid aptamers of example 1.
FIG. 2 is an agarose gel electrophoresis chart of the secondary library preparation effect in example 1, which shows that: a Marker;1 is the symmetrical PCR amplification product of the step five; 2 is the asymmetric PCR amplification product of the step six; and 3 is a product of the seventh step, which is purified on the basis of the product of the second step.
FIG. 3 shows the fluorescence intensity changes after screening RhD antigen with different ssDNA full-length sequences in the validation of example 2, wherein the abscissa indicates ssDNA number and the ordinate indicates fluorescence intensity.
FIG. 4 shows the change in fluorescence intensity after binding of RhD-positive erythrocytes to SEQ ID NO. 15 and SEQ ID NO. 16 in the affinity assay of example 2, wherein the abscissa indicates the concentration of the full-length sequence of ssDNA and the ordinate indicates the fluorescence intensity.
FIG. 5 shows the effect of masking RhD antigen by the combination of SEQ ID NO. 15 and SEQ ID NO. 16 in example 2, in order from left to right: a is a flow cytometry test result; b is the observation result under a microscope without combined treatment of SEQ ID NO. 15 and SEQ ID NO. 16; the results were observed under a microscope at a concentration of 500pmol of SEQ ID NO:15 and SEQ ID NO:16.
Detailed Description
In order to make the technical problems, technical schemes and beneficial effects solved by the invention more clear, the invention is further described in detail below with reference to the embodiments.
Examples
The following examples are presented herein to demonstrate preferred embodiments of the present invention. It will be appreciated by those skilled in the art that the techniques disclosed in the examples which follow represent techniques discovered by the inventor to function in the practice of the invention, and thus can be considered to constitute preferred modes for its practice. Those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit or scope of the invention.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs, the disclosure of which is incorporated herein by reference as is commonly understood by reference.
Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the claims.
Example 1
The embodiment provides a preparation method of red blood cell subtractive SELEX screening RhD antigen-specific ssDNA aptamer, the preparation flow and main preparation parameters of which are shown in fig. 1, table 1, comprising the following steps:
TABLE 1 erythrocyte reduction SELEX parameters
Step one: negative selection. Into a 1.5mL centrifuge tube, 100. Mu.L of a concentration of 2.0X10 were added in sequence 6 /μL RhD - The red blood cell physiological saline suspension, 950. Mu.L of blood preservation solution III (CPDA-I preservation solution), 15.2. Mu.L of salmon sperm DNA and 100. Mu.L of ssDNA library were incubated at room temperature (25 ℃) for 30min. Red Blood Cells (RBCs) were removed by centrifugation at 5000rpm for 2min and the supernatant transferred to another clean 1.5mL centrifuge tube for forward screening.
The ssDNA library consists of 82bp, a random sequence of 40bp is arranged in the middle of the library, primer binding regions respectively consisting of 21bp are arranged at two sides of the library, and the 5' -end binding region is as follows:
5'-AGAGACGGACACAGGATGAGC-3'(SEQ ID NO:1)
the 3' end binding region is:
5'-CCTTCCCCAAGACAGCATCCA-3'(SEQ ID NO:2)
thereby making up of
5 '-AGAGACGGACACAGGATGAGC-N40-CCTTCCCCAAGACAGCATCCA-3'.
ssDNA libraries were synthesized by TaKaRa corporation. Salmon Sperm DNA was purchased from Life Technologies (Salmon sprm DNA, cat# AM9680, lot # 1502041).
Step two: forward screening. 100. Mu.L of 2.0X10 concentration was added to the negatively screened library 4 /μL RhD + The erythrocyte physiological saline suspension is incubated for 60min at room temperature. Centrifuging to remove supernatant, adding 400 μl of physiological saline and 100 μl of erythrocyte magnetization onto erythrocyte buttonThe red blood cells are resuspended in liquid and placed on a magnetic rack for standing for 2min. The supernatant was aspirated. The washing was repeated 5 times, each time with a tube.
The red blood cell magnetizing solution was purchased from Diago corporation (MagneLys, lot number: 581000).
Step three: is combined with RhD + ssDNA extraction of erythrocytes. To the red blood cells from which the supernatant had been removed, 200. Mu.L of nuclease-free water was added at 95℃for 5 minutes. 300. Mu.L of Lysis/Binding Buffer was added. The centrifuge tube is inverted for 4 to 6 times and incubated for 5 minutes at room temperature. 150. Mu.L of isopropanol (isopanol) was added. A further 50. Mu.L (2 mg) of the suspended magnetic beads was added. Incubate on Mixer for 10min at room temperature. Placing on a magnetic rack, standing for 2min, and sucking supernatant. 850. Mu.L of Washing Buffer 1 (isopropanol was added as indicated before use), the tube was inverted 3-4 times and the beads were resuspended. Standing on a magnetic rack for 1min, and removing the supernatant. After repeated Washing, 450. Mu.L of Washing Buffer 2 was added and the bead suspension was transferred to another clean centrifuge tube. Standing on a magnetic rack for 2min, and removing supernatant. The wash was repeated once without further tube transfer. After the residual liquid is completely absorbed, the magnetic beads are dried on a magnetic rack, so that the ethanol is completely volatilized. It takes about 10-15 min at room temperature. mu.L of the solution Buffer was added, and the beads were resuspended with a sample addition at 70℃for 3min. Removing the magnetic beads, and obtaining the supernatant as ssDNA.
The ssDNA extraction reagent was purchased from Life Technologies (Dynabeads SILANE ivral NA, lot number: 37011D).
Step four: and quantitatively detecting the concentration of ssDNA. The ssDNA concentration in the supernatant was detected by fluorescence quantification. The reaction system is as follows: 8 mu L H 2 O, PCR grad, 10. Mu.L Master Mix, 1. Mu.L ssDNA template, 1. Mu.L concentration of 10 pmol/. Mu.L upstream and downstream Mix primers. Amplification conditions were strictly according to the kit instructions.
The ssDNA fluorescent quantitative detection reagent was purchased from Roche company (LightCycler 480 SYBR Green I Master, lot number: 04707516001).
Step five: amplification and purification of the secondary library. The ssDNA library is amplified by PCR using biotin-labeled upstream and downstream primers to obtain dsDNA carrying biotin, and a sufficient template is provided for asymmetric PCR.
The PCR reaction system is as follows:
the Taq enzyme is selected from a kit of model M1665S of Promega company.
The biotin-labeled upstream primer is as follows:
5’-biotin-AGAGACGGACACAGGATGAGC-3’；
the biotin-labeled downstream primer is:
5’-biotin-TGGATGCTGTCTTGGGGAAGG-3’；
wherein, biotin is biotin, and the primer is synthesized by TaKaRa company.
Amplification conditions:
the first stage:
94℃ 5min
and a second stage: (11 cycles)
94℃ 30s
59℃ 30s
72℃ 30s
And a third stage:
72℃ 5min
preserving at 4 ℃ to obtain the dsDNA amplification product carrying biotin.
Step six: and (3) carrying out asymmetric PCR amplification by taking the dsDNA amplification product with biotin obtained in the step (V) as a template and using an upstream primer without biotin mark and a downstream primer with biotin mark, wherein the mass ratio of the upstream primer to the downstream primer is 20:1. The dsDNA and byproducts obtained after asymmetric PCR amplification all carry biotin, while the ssDNA does not contain biotin.
The AmpliTaq Gold Fast PCR Master Mix (2×) was from the 4390939 kit of Life Technologies company.
The upstream primer is 5'-AGAGACGGACACAGGATGAGC-3';
the biotin-marked downstream primer is 5'-biotin-TGGATGCTGTCTTGGGGAAGG-3';
wherein biotin is biotin, and the primer is synthesized by TaKaRa company.
Amplification was performed according to the following conditions:
the first stage:
95℃ 10min
and a second stage: (30 cycles)
96℃ 3s
59℃ 3s
68℃ 3s
And a third stage:
72℃ 10s
preserving at 4℃to obtain an amplification product comprising dsDNA and byproducts both carrying biotin, and ssDNA containing no biotin.
Step seven: finally, adding streptavidin magnetic beads into the amplification product in the step six, and removing all dsDNA and byproducts carrying biotin in the amplification product, thereby obtaining high-purity and high-concentration ssDNA.
Taking 1.5 times volume of streptavidin magnetic beads of the amplified product in the step six
Paramagnetic Particles, promega, Z5482), washed 3 times with PBS-Tween (pH 7.4,with 0.02%Tween-20), attached to a magnet rack for 30s, and the supernatant removed. The ssDNA amplification product was transferred to a magnetic bead tube and incubated for 20min at room temperature with shaking. The supernatant was aspirated on a magnetic rack, which was the secondary library of ssDNA required.
And (3) result detection: the purity of ssDNA was checked using high resolution agarose gel electrophoresis and the results are shown in fig. 2.
Example 2
This example provides a method for screening single stranded nucleic acid aptamers capable of specifically binding to and masking the antigenicity of RhD antigen by the ssDNA secondary library of example 1:
step one: sequencing of the screened ssDNA sequence pool
Sequencing adapter sequences were added to both ends of the ssDNA screening library obtained in example 1:
CCATCTCATCCCTGCGTGTCTCCGACTCAG；
CCTCTCTATGGGCAGTCGGTGAT。
sequencing was performed after PCR amplification using a Ion Torrent Personal Genome Machine semiconductor sequencer (done by sea division on Life Technologies, U.S.A.). According to the sequencing result, we screen out ssDNA random sequences which are dominant in the sequence library by taking the sequence which is provided with the target sequence in the middle and the connectors at the two ends and is completely matched with the connectors as verification objects, see table 2, and the screening standards are as follows: a sequence with copy number >1000 in ssDNA sequence library and variable region length of 40 bp.
TABLE 2 dominant ssDNA aptamer variable region base sequences
| Sequence numbering | Random fragment base sequence (5 '. Fwdarw.3') |
| SEQ ID NO:3 | GGCCTGGTCTGTTAGCCGGGTAGCAGCCCCGGCACCTATT |
| SEQ ID NO:4 | GGGTAGCAGCCCCGCGGAGGGTCGGCTATAAGAACCAAGA |
| SEQ ID NO:5 | CTATTCCCCACGTCACTTTTCCCGTAGGTTGGACTCGACC |
| SEQ ID NO:6 | GGTGCAGGGGGGTCGGAGAAGAGGTTGAGGGGAGAGGGGT |
| SEQ ID NO:7 | ACGGCCTCTGTATAATGCTGGCCTTGACGCTTGTCCCTTG |
| SEQ ID NO:8 | ACGGCCTCTGTACAATGCTGGCCTTGACGCTTGTCCCTTG |
| SEQ ID NO:9 | TACACCAATCTCCCCCCTACATTCTCCCACCAGCACTCCA |
| SEQ ID NO:10 | GGGTAGCAGCCCCGCGGAGGGTCGGCTATAAGAACTAGGA |
| SEQ ID NO:11 | GGGTAGCAGCCCCGCGGAGGGTCGGCTATAAGAACCAGGA |
| SEQ ID NO:12 | GGGTAGCAGCCCCGCGGAGGGTCAGCTATAAGAACCAGGA |
| SEQ ID NO:13 | GGGTAGCAGCCCCGCGGAGGGTCGGCTATAGGAACCAGGA |
| SEQ ID NO:14 | CTATTCCCCACGTCACTTTTCCCGTAGGCTGGACTCGACC |
Step two: performing multiple index verification on the random sequence of the ssDNA screened in the step one
1. Validity verification (ability verification of screening RhD antigenicity)
In a microplateRhD positive erythrocytes (4X 10) were added 6 Red blood cells/well), 500pmol of the full-length sequence of the screened monospecific ssDNA was added, respectively, and incubated at room temperature (25 ℃) for 60min. Then 50. Mu.L of labeled monoclonal anti-D [ 128-fold dilution with a titer of 512 (1024, score 109) was added]Incubate for 30min at room temperature. After washing 3 times with physiological saline, 100. Mu.L of 1000-fold diluted FITC-labeled goat anti-human IgG (F (ab') 2 fluorescent secondary antibody) was added, incubated at room temperature for 15min, and then washed 3 times with physiological saline, 200. Mu.L of physiological saline was added, and the mixture was homogenized, and the fluorescence intensity of erythrocytes was measured by flow cytometry.
Positive and blank controls were simultaneously set, and 3 parallel wells were made. The positive control was prepared by substituting physiological saline for ssDNA in the above test. Red blood cells after incubation of RhD positive red blood cells with FITC-sheep anti-human IgG (F (ab') 2 served as a blank.
The above monoclonal anti-D was purchased from Shanghai blood Biomedicine Limited (lot number: 20160725); the Goat anti-human IgG (F (ab ') 2 fluorescent antibody was purchased from Jackson (Goat F (ab') 2 Anti Human IgG:FITC, lot number: 125089), the flow cytometer used for the flow assay was FACSCanto II from BD Co., USA, and the full-length sequence of the screened monospecific ssDNA was synthesized by Shanghai Biotechnology Co., ltd.
Analysis of results:
the degree of decrease in fluorescence intensity of the test group was observed with reference to the positive control fluorescence intensity. Using IBM SPSS Statistics software, one-Way Anova (One-Way Anova) was used to compare whether the mean differences were significant, with p <0.05 being the difference statistically significant.
Fig. 3 shows the change in fluorescence intensity after masking of RhD antigen by the full-length sequence of each ssDNA to block anti-D binding to RhD antigen, as can be seen from the figure: the ssDNA full-length sequence composed of SEQ ID NO. 3 and SEQ ID NO. 4 has obvious effect of shielding RhD antigen, and the statistical difference is obvious compared with the positive control (p values are all < 0.05).
Conclusion:
2 effective ssDNA random sequences were obtained by screening, the full-length sequences of the ssDNA are shown in Table 3:
TABLE 3 screening for potent ssDNA full-length sequences
2. Verification of affinity
100. Mu.L of 0.2% RhD was added to a 96-well plate + The red blood cell physiological saline suspension is added with Alexa Fluor 488 labeled monospecific ssDNA Seq 1 and Seq 2 respectively in different holes to make the final concentration of each ssDNA reach 200nmol/L, 400nmol/L, 800nmol/L, 1000nmol/L and 1200nmol/L. Incubation was performed at 37℃with shaking for 60min.4500rcf (Labofuge 400R,Heraeus Instruments), and centrifuged for 5min. The liquid in the plate is thrown off. After washing 3 times with physiological saline and removing the supernatant by centrifugation for the last time, 200. Mu.L of physiological saline was added, and the fluorescence intensity of erythrocytes was measured by flow cytometry.
The Alexa Fluor 488-labeled monospecific ssDNA described above was synthesized by Shanghai Biotechnology Co., ltd.
Detection result:
FIG. 4 shows the change in fluorescence intensity after binding of SEQ ID NO. 15, SEQ ID NO. 16 to RhD-positive erythrocytes, the dissociation constants were calculated using GraphPad Prims V6 software based on the change in fluorescence intensity (dissociation constant, K d ). Data analysis complex nonlinear fit analysis requires that Kd values be calculated using the following formula:
bmax is the maximum binding rate, X is the concentration of the full-length sequence of each ssDNA, and Y is the fluorescence intensity value corresponding to the X value. The Kd values of SEQ ID NO:15 and SEQ ID NO:16 are calculated as follows: 580.5 + -142.0 nM and 737.7 + -161.8 nM.
Conclusion:
the affinity is as follows from high to low: SEQ ID NO. 15, SEQ ID NO. 16.
3. Dose-response relationship of ssDNA shielding RhD antigen
The dose-response relationship of ssDNA-masked RhD antigen was analyzed by the flow method and the indirect anti-globulin assay (IAT), respectively.
A flow method: 100. Mu.L of RhD was added to the microplate + Physiological saline suspension (4×10) 6 Red blood cells/well), SEQ ID NO 15 and SEQ ID NO 16 were added in a gradient of 200pmol, 300pmol, 400pmol, 500pmol, respectively, and incubated at 37℃for 60min. Washing with physiological saline 3 times, adding 100 μl of humanized anti-D, incubating at 37deg.C for 30min, washing with physiological saline 3 times, adding 100 μl of 1000-fold diluted FITC-labeled goat anti-human IgG (F (ab') 2 secondary fluorescent antibody, incubating at room temperature for 15min, washing with physiological saline 3 times, adding 200 μl of physiological saline, mixing well, and detecting fluorescence intensity of erythrocytes by flow cytometry.
Indirect anticoccipital method: 100. Mu.L of RhD was added to the microplate + Physiological saline suspension (4×10) 6 Red blood cells/well), SEQ ID NO 15 and SEQ ID NO 16 were added in a gradient of 200pmol, 300pmol, 400pmol, 500pmol, respectively, and incubated at 37℃for 60min. Washing with physiological saline 3 times, adding 100. Mu.L of humanized anti-D, incubating at 37℃for 30min, washing with physiological saline 3 times, adding 100. Mu.L of an anti-globulin reagent, centrifuging for 15s at 1000g, dropping the resulting mixture after resuspension, and observing and recording the results under a microscope (Olympus BX43 type microscope of Olympus Co., japan). A positive control and a blank control are simultaneously set, and the positive control is prepared by replacing ssDNA in the test by physiological saline. The blank was prepared by substituting physiological saline for human anti-D in the above test.
The above mentioned human anti-D is provided by volunteers who produce anti-D; the above-mentioned anti-globulin reagent was purchased from Shanghai blood Biomedicine Limited (lot number: 20141203). The Goat anti-human IgG (F (ab ') 2 fluorescent antibody was purchased from Jackson (Goat F (ab') 2 Anti Human IgG:FITC, lot number: 125089), the flow cytometer used for the flow assay was FACSCanto II from BD Co., USA, and the full-length sequence of the screened monospecific ssDNA was synthesized by Shanghai Biotechnology Co., ltd.
Detection result:
the results of the above test are shown in Table 4 and FIG. 5.
TABLE 4 Table 4
As can be seen from Table 4, FIG. 5 shows that the RhD antigen was completely masked when both SEQ ID NO:15 and SEQ ID NO:16 reached 500pmol with increasing ssDNA concentration, making the test result negative.
Conclusion:
when the combined use of SEQ ID NO. 15 and SEQ ID NO. 16, the red blood cell RhD antigen can be completely shielded when the concentration reaches 500pmol, so that the IAT test result is changed from positive to negative before treatment.
5. Single layer test of monocytes (MMA)
Extracting 6 parts of peripheral blood mononuclear cells by density gradient centrifugation, washing with RPMI 1640 for 1 time, mixing 6 parts of mononuclear cells, and regulating cell concentration to 5×10 6 Individual cells/mL. Add 50. Mu.L of mixed monocytes to chamber slide in CO 2 Incubating for 60min at 37deg.C in incubator to adhere cells. The non-adherent cells were gently rinsed off with physiological saline and 100. Mu.L of 1X 10 cells were added to each well 7 Test, positive and negative control erythrocytes per cell/mL in CO 2 Incubate for 90min at 37℃in incubator. After the slide glass was washed 3 times with physiological saline, it was stained with Rayleigh-giemsa. 500 monocytes were observed under a microscope and each group of test results was judged with a 3% phagocytosis/adhesion rate as the positive limit.
The red blood cells of the test group are RhD positive red blood cells, which are incubated with SEQ ID NO. 15 and SEQ ID NO. 16 at 500pmol each for 60min at 37 ℃, washed 3 times with physiological saline, added with 2 times of volume of humanized anti-D, mixed and incubated for 60min at 37 ℃. After washing 5 times with physiological saline, the cell concentration was adjusted to 1X 10 with RPMI 1640 7 Individual cells/mL. The positive control group was prepared by substituting physiological saline for the above SEQ ID NO. 15 and SEQ ID NO. 16. The negative control was the replacement of the above RhD positive erythrocytes with RhD negative erythrocytes.
Detection result:
the test group had a monocyte phagocytosis/adhesion rate of 2.5%, the positive control group of 19.5%, and the negative control group of 2.2%. The positive detection limit was 3%.
Conclusion:
when SEQ ID NO. 15 and SEQ ID NO. 16 are used in combination, red blood cell RhD antigen can be completely shielded when the concentration reaches 500pmol, so that MMA test results are limited. It is believed that RhD positive erythrocytes treated with SEQ ID NO. 15 and SEQ ID NO. 16 will not cause hemolytic transfusion adverse reactions due to alloimmunization after infusion into RhD negative patients.
All documents mentioned in this application are incorporated by reference as if each were individually incorporated by reference. Further, it will be appreciated that various changes and modifications may be made by those skilled in the art after reading the above teachings, and such equivalents are intended to fall within the scope of the claims appended hereto.
Sequence listing
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gggtagcagc cccgcggagg gtcagctata agaaccagga 40
<210> 13
<211> 40
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 13
gggtagcagc cccgcggagg gtcggctata ggaaccagga 40
<210> 14
<211> 40
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 14
ctattcccca cgtcactttt cccgtaggct ggactcgacc 40
<210> 15
<211> 82
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 15
agagacggac acaggatgag cggcctggtc tgttagccgg gtagcagccc cggcacctat 60
tccttcccca agacagcatc ca 82
<210> 16
<211> 82
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 16
agagacggac acaggatgag cgggtagcag ccccgcggag ggtcggctat aagaaccaag 60
accttcccca agacagcatc ca 82
Claims (3)
1. A single-stranded nucleic acid aptamer composition comprises two single-stranded nucleic acid aptamers, and is characterized in that the nucleotide sequences of the two single-stranded nucleic acid aptamers are respectively shown as SEQ ID NO. 15 and SEQ ID NO. 16.
2. Use of the aptamer composition of claim 1 in the preparation of an remodelling agent for remodelling RhD positive erythrocytes to RhD negative erythrocytes.
3. A remodelling agent for remodelling RhD positive erythrocytes to RhD negative erythrocytes, characterized in that the remodelling agent comprises the aptamer composition of claim 1.
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Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN104818280A (en) * | 2015-04-24 | 2015-08-05 | 深圳市血液中心 | Single-stranded aptamer and application thereof |
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| CN104818280A (en) * | 2015-04-24 | 2015-08-05 | 深圳市血液中心 | Single-stranded aptamer and application thereof |
Non-Patent Citations (7)
| Title |
|---|
| RhD抗体ssDNA适配体的筛选与鉴定;庄乃保等;《中国输血杂志》;20160810;第29卷(第06期);第592-595页 * |
| Single-stranded DNA aptamer targeting and neutralization of anti-D alloantibody_ a potential therapeutic strategy for haemolytic diseases caused by Rhesus alloantibody;Yinze Zhang等;《Blood Transfus.》;20161111;第184-192页 * |
| ssDNA适配体阻断FcγR介导巨噬细胞吞噬作用的研究;李桢等;《中国输血杂志》;20170925(第09期);第981-983页 * |
| 基于RhD蛋白Aptamer筛选技术的随机ssDNA次级文库制备条件的优化;张印则等;《中国输血杂志》;20141225;第27卷(第12期);第1293页、第1295页"3 讨论" * |
| 张印则等.基于RhD蛋白Aptamer筛选技术的随机ssDNA次级文库制备条件的优化.《中国输血杂志》.2014,第27卷(第12期),第1293-1296页. * |
| 红细胞RhD膜蛋白提取与固相化方法的研究;章昊等;《中国输血杂志》;20170925(第09期);第975-977页 * |
| 隐蔽红细胞RhD抗原遮盖效果研究;李执如等;《现代预防医学》;20090210;第36卷(第03期);第578-579页 * |
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