CN105349545A - NT-proBNP nucleic acid aptamer and application thereof - Google Patents
NT-proBNP nucleic acid aptamer and application thereof Download PDFInfo
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Abstract
The invention discloses an NT-proBNP nucleic acid aptamer which belongs to the technical field of NT-proBNP detection and application of the NT-proBNP nucleic acid aptamer. The nucleic acidaptamer comprises any one in nucleotide sequences shown in SEQ ID: 1-15. The NT-proBNP nucleic acid aptamer is oligonucleotide itself, relatively small in molecular weight, can be synthesized chemically, thus the cost is saved, and the affinity and the specificity are higher than an antibody; meanwhile, the nucleic acid aptamer is convenient to mark, the repeatability and the stability are good, and the storage is easy. A NT-proBNP detection kit and an NT-proBNP test strip which are prepared from the aptamer are sensitive in NT-proBNP detection, easy to optimize, and can be applied to heart failure diagnosis, heart failure prognosis and assessment of monitoring, danger classification of acute coronary syndrome or mild cardiac insufficiency diagnosis.
Description
Technical field
The invention belongs to NT-proBNP detection technique field, particularly a kind of NT-proBNP aptamer and application thereof.
Background technology
Brain natriuretic peptide (BNP) is found in pig brain by Sudoh etc. the earliest, is a kind of polypeptide class neurohormone be made up of 32 amino acid peptide chains.The BNP that human heart is initially secreted is a kind of precursor be made up of 132 amino acid, thereafter digested being degraded to has bioactive BNP (32 amino-acid residue compositions) and plasma pro-brain natriuretic peptide levels N-terminal (NT-ProBNP, 76 amino-acid residue compositions).BNP and NT-ProBNP is primarily of ventricle response release after myocardium pressurized, the mark responsive and special to heart function, its concentration is in blood relevant to the severity of cardiac dysfunction, can be used for the severity of more objective appraisal patient disease and the prognosis of patient.Be widely used in kit for diagnosing heart failure, the assessment of heart failure prognosis and supervision, the classification of risks of acute coronary artery syndrome, early stage or slight cardiac insufficiency etc.
NT-ProBNP comparatively BNP has more advantage on evaluation heart function, it is advantageous that: 1, NT-proBNP is made up of 76 amino acid, comparatively BNP long half time, its transformation period is greater than 1h, remove slower in blood, can accumulation higher concentration, more can reflect state and the change of heart function delicately, especially in early days in diagnosis; Sample can be deposited, and namely can centralab detect, and also can detect by bedside, BNP can only detect by bedside at once; 2, due to without physiologically active, thus not by the interference for the treatment of with synthesis BNP; 3, NT-proBNP can use serum and plasma, strong interference immunity, and BNP can only use plasma specimen.4, the quantitative result of NT-proBNP can provide reliable, and has good repeated clinical information, is more suitable for clinical application.Therefore, trend NT-proBNP being replaced BNP is presented gradually in current clinical application.
Current NT-proBNP detection method is mainly immunologic detection method, and one is competitive enzyme-linked immune analysis, as the product of BiomedicaGruppe company; One is double antibody sandwich method, as the double-antibody sandwich chemoluminescence method of RocheDiagnostics, declares patent of invention.At present, based on the method for antibody, there is some superiority, but also have some intrinsic defects.Now, scientist simultaneously attention focusing to molecular biology new technology in the application of biological detection.
SELEX technology (phyletic evolution index concentration technology) is a kind of new combinatorial chemistry technique of development at the beginning of the nineties.Its ultimate principle utilizes Protocols in Molecular Biology exactly, and build the strand random oligonucleotide library of synthetic, wherein the general length of stochastic sequence is at about 20-40bp, and library capacity is 10
14-10
15between.Due to strand random oligonucleotide acid fragment particularly RNA easily form the secondary structure such as hair fastener, pocket, false joint, G-tetrad, therefore can with protein, little peptide, even metal ion combines, and formation has the mixture of very strong bonding force.This method has easy, quick, economic dispatch feature, with other combinatorial chemical libraries as random peptide library, antibody library are compared with phage display libraries, the aptamer filtered out from oligonucleotide library has many advantages: a. itself is oligonucleotide, molecular weight can chemosynthesis, cost-saving; B. there is the specificity higher than antibody and close affinity; C. be convenient to mark, selectively mark at different sites; D. good stability, reproducible, be easy to preserve, to high temperature and drastic conditions insensitive.Therefore, oligonucleotide aptamers has a good application prospect in clinical detection and diagnosis.Related nucleic acid aptamers new detecting technique widespread use biological detection, bio-sensing, in-vivo imaging, protein nucleic acid functional study every field, show tempting prospect.
NT-proBNP is the polypeptide of 76 amino-acid residues; molecular weight is little; available epitope limitation; and by Roche patent protection, aptamer molecular weight only has 10-20KD, much smaller compared with IgG antibody 150kD; in the detection of setting up sandwich assay; the specific nucleic acid aptamers of multiple antigen site can be obtained, antibody molecule amount can be overcome and cause sterically hindered impact greatly, be easy to the foundation of sandwich-type detection procedures.
Summary of the invention
The present invention is directed to deficiency of the prior art, its object is to provide a kind of NT-proBNP aptamer and application thereof.
To achieve these goals, the technical scheme taked of the present invention is as follows;
A kind of NT-proBNP aptamer, described aptamer is any in the nucleotide sequence shown in SEQID:1-15.
The monoclonal antibody that described aptamer can be prepared with NT-proBNP albumen or NT-proBNP albumen n end aminoacid sequence combines.
The aminoacid sequence of described NT-proBNP albumen n end is GlySerAlaSerAspLeuGluThrSerGlyLeuGlnGluGlnArg.
The application of a kind of NT-proBNP aptamer in preparation NT-proBNP detection kit.
Described NT-proBNP aptamer is by biotin labeling.
The application of a kind of NT-proBNP aptamer in preparation NT-proBNP test strip.
Described NT-proBNP aptamer is by colloid gold label.
Consisting of of described NT-proBNP test strip: be stained with sample pad (2), pad (3), nitrocellulose filter (4) and absorbent pad (5) on PVC backing (1) successively; Described pad (3) is coated with by the NT-proBNP aptamer of colloid gold label; Described nitrocellulose filter is provided with detection line (6) and nature controlling line (7); Wherein, detection line (6) is coated with the amino acid whose monoclonal antibody of NT-proBNP albumen n end, nature controlling line (7) is coated with the reagent be combined with the aptamer of the NT-proBNP of colloid gold label.
The described reagent be combined with the aptamer of the NT-proBNP of colloid gold label is the complementary sequence of NT-proBNP aptamer.
Advantage of the present invention is: described NT-proBNP aptamer itself is oligonucleotide, molecular weight, can chemosynthesis, cost-saving, has the affinity higher than antibody and specificity; NT-proBNP aptamer is simultaneously convenient to mark, repeatability and good stability, is easy to preserve.The NT-proBNP detection kit prepared with described NT-proBNP aptamer and test strip detect sensitive to NT-proBNP, and are easy to optimize.
Accompanying drawing explanation
Fig. 1 is the NT-proBNP gene of synthesis and the order-checking comparison chart of proBNP gene order.
Fig. 2 is the double digestion electrophorogram of pGEX4T-1-NT-ProBNP expression vector.
Fig. 3 is the SDS-PAGE electrophorogram of the GST-NT-proBNP albumen after purifying; Wherein, 1: full bacterium before induction, 2: induced precipitation, 3: induction supernatant, 4:GST column purification penetrates liquid, and 5-9:GST purifying wash-out collects liquid, and 10: Protein Marker.
Fig. 4 is the SDS-PAGE electrophorogram of the 12G4 monoclonal antibody after purifying.
Fig. 5 is that positive colony PCR identifies electrophorogram.
Fig. 6 is NT-proBNP aptamer activity rating column diagram.
Fig. 7 is a kind of structural representation of NT-proBNP test strip.
Embodiment
Below in conjunction with the drawings and specific embodiments, the present invention will be further described.
Embodiment 1: full genome synthesis NT-proBNP gene, builds pGEX4T-1-NT-ProBNP expression vector and purifying GST-NT-proBNP albumen.
(1) proBNP gene order is inquired about by genebank, according to the N terminal sequence sheared after ripe BNP and NT-proBNP sequences Design complete sequence synthetic schemes, undertaken codon optimized by DNAwork2.0 software, do not change aminoacid sequence composition, and make albumen be easier at protokaryon expression in escherichia coli, then the two ends of nucleotide sequence after optimization add EcoRI and SalI restriction enzyme site, be convenient to be connected with expression vector clone, the nucleotide sequence after optimization design is as shown in SEQID:16.Sequence after optimization is delivered Jin Sirui company synthesize, Fig. 1 is the NT-proBNP gene of synthesis and the order-checking comparison chart of proBNP gene order.
(2) by synthetic gene NT-proBNP from PUC57 carrier after double digestion glue reclaim, and by pGex4T-1 carrier double digestion, reclaim carrier segments, gene fragment and carrier segments will be reclaimed through T
4dNA ligase connects, and is built into pGEX4T-1-NT-ProBNP expression vector, and is transformed in bacillus coli DH 5 alpha competent cell, is coated with dull and stereotyped, and cultivate 16h for 37 degree, picking mono-clonal carries out liquid culture and extract plasmid carrying out double digestion qualification.Fig. 2 is the double digestion electrophorogram of pGEX4T-1-NT-ProBNP expression vector, from the known expression vector establishment success of figure.
(3) be converted in e. coli bl21 (DE3) competent cell by the pGEX4T-1-NT-ProBNP expression vector built, picking mono-clonal carries out IPTG abduction delivering, selects the high mono-clonal of expression amount and preserves as bacterial classification.Amplify spawn culture, collect thalline after inducing, ultrasonication, adopt GST label affinity column to carry out affinity purification, collect elution peak and carry out SDS-PAGE electrophoretic analysis, as shown in Figure 3.The GST-NT-proBNP purity of protein of purifying reaches more than 95%, can meet ensuing experiment demand.
Embodiment 2: the amino acid whose monoclonal antibody of preparation NT-proBNP albumen n end.
Analyze the epitope of NT-proBNP albumen, select N to hold dominant antigen epi-position, the N that people synthesizes NT-proBNP albumen altogether holds 15 amino acid polypeptide (GlySerAlaSer
aspleuGluThrSerGlyLeuGlnGluGlnArg), immunity is carried out by after the polypeptide of synthesis and bovine serum albumin (BSA) covalent coupling, after serum titer reaches requirement, carry out splenocyte separation, and with SP/20 myeloma cell fusion, carry out single cell clone, single cell clone high for antibody titer is carried out subclone, as shown in table 1: 5G4,5E7 and 12G4 monoclonal antibody activity is higher, by active for this 3 strain higher monoclonal antibody preparation ascites, and carry out purifying by proteinA affinity chromatography.Fig. 4 is the SDS-PAGE electrophorogram of the 12G4 monoclonal antibody after purifying, is shown as heavy chain, light chain two band.
Table 1: the screening (OD450nm absorbancy) of monoclonal antibody
Embodiment 3: the NT-proBNP aptamer that screening is matched with the monoclonal antibody obtained.
Screening step is as follows:
1, silicon bag magnetic bead is through the process of APTES (2%) Aminosilylation, makes it be with active amino, forms amino magnetic bead;
2, by the NT-proBNP monoclonal antibody (5G4) of 8ug/ml, through EDC/NHS (4mg/ml/11mg/ml) activated carboxyl at 37 DEG C, soak time is 15min; Then get the monoclonal antibody after 1ml activation and 1mg amino magnetic bead coupling 1h at 37 DEG C, form coupled antibody magnetic bead;
3, clean coupled antibody magnetic bead 5 times with PBS, then close with 1%BSA and spend the night;
4, the GST-NT-proBNP albumen adding 100ul (2mg/ml), in the coupled antibody magnetic bead of 1ml, hatches 2h, forms the coupled antibody magnetic bead in conjunction with GST-NT-proBNP albumen;
5,5 times are washed with SHMCK (0.2%Tween-20);
6, by the ssDNA library of synthetic, (two ends are fixed sequence program, centre is the stochastic sequence of 35bp: GCATCACTACAGTCATTACGC-35bp-ATCGTGTGAAGTGCTGTCCC) 95 DEG C of heat denatured 5min in SHMCK, then cool in frozen water; Incubated at room temperature 20min, adds salmon sperm dna 10ul, tRNA5ul, 1%BSA;
7, then joined in coupled antibody magnetic bead and hatched that 1h is counter to be sieved;
8, Magneto separate, absorption supernatant joins in the coupled antibody magnetic bead in conjunction with GST-NT-proBNP albumen, incubated at room 1h;
9,5 times are washed with SHMCK (0.2%Tween-20);
10, add 200ul pure water 95 DEG C and hatch 5min, Magneto separate, draw supernatant;
11, supernatant is through 1/10 volume 3M sodium acetate (pH3.5), 4ul settling agent, 2.5 times of volume dehydrated alcohol precipitations, the centrifugal 15min of 12000rpm;
12, precipitate through water dissolution, pcr amplification becomes 100uldsDNA double-strand (Tu-1), and amplification system is:
13, PCR primer preserves the template as next round library after Qiagen test kit concentrates.
14, lower whorl library preparation: asymmetric PCR, 600ul, amplification system is:
15, Qiagen test kit reclaims the PCR primer of 600ul, ssDNA, carries out second and takes turns screening;
Second take turns screening step as follows:
1, instead sieve: antibody-coupled magnetic beads;
2, screen: GST-NT-proBNP binding antibody coupled bead, adds salmon sperm dna 10ul, tRNA5ul, 1%BSA;
3, wash: use HMCK (0.2%Tween-20) 10 times;
4, wash-out: hatch 5min at 95 DEG C with pure water;
5, increase: 100uldsDNA (tu-3);
6, asymmetric PCR, 600ulssDNA (Tu-4);
7, precipitate PCR primer: 600ulssDNA+60ul (3M sodium acetate)+6ul (settling agent)+1650ul dehydrated alcohol, centrifugal 15min, 70% ethanol is washed once, dries, and adds 400ulSHMCK and dissolves, and prepares lower whorl screening library system.
Take turns screening through 15, the dsDNA prepared by pcr amplification is connected to carrier T, is transformed in bacillus coli DH 5 alpha competent cell, and coated plate is also cloned, and extracts plasmid, identifies positive colony, as shown in Figure 5 through PCR.According to the positive colony situation of PCR qualification, positive bacteria is sent to and checks order and identify that it is active, obtain NT-proBNP aptamer.
Embodiment 4:NT-proBNP aptamer activity rating.
1, the preparation of reagent:
(1), the preparation of NT-proBNP aptamer: SELEX (IgG) aptamer plasmid 1ul asymmetric PCR 100ul (biotinP1:P2=100:1).Amplification 42 circulation, PCR primer SHMCK dilutes 10 times, and 95 DEG C of heat denatured 5min, cool in frozen water, incubated at room 20min; Add salmon sperm dna 10ul, tRNA5ul, 1%BSA, for subsequent use.
(2), wrap and prepared by plate: the NT-proBNP monoclonal antibody (5G4) of 8ug/ml is added enzyme plate, 37 DEG C of bags are by 2h; PBS washes 5 times, and 1%BSA closes and spends the night, after washing, for subsequent use;
(3), antigen prepares: GST-NT-proBNP antigen protein concentration dilution is to 1ug/ml;
(4), washings: SHMCK (0.2%Tween-20);
(5), HRP labelled streptavidin;
(6), TMB nitrite ion.
Activity rating method:
(1), in the enzyme plate being coated with NT-proBNP monoclonal antibody (5G4) NT-ProBNP antigen is added, incubated at room 1h;
(2), SHMCK (0.2%Tween-20) washes 3 times;
(3), the NT-ProBNP aptamer of preparation, incubated at room 1h is added;
(4), SHMCK (0.2%Tween-20) washes 3 times;
(5), the labelled streptavidin (1:1000) of horseradish peroxidase (HRP), incubated at room 1h is added;
(6), SHMCK (0.2%Tween-20) washes 3 times;
(7), TMB develop the color 15min, microplate reader 450nm measure absorbancy; Relative to blank, the proof of its absorbancy >0.15 is better active, and be applicable to detect needs, measurement result as shown in Figure 6.
As we know from the figure, the activity of the aptamer of screening is all relatively good, meets the requirement of assembling test kit.
The composition of embodiment 6:NT-proBNP detection kit and detection method.
1, test kit composition
(1) 96 orifice plates of monoclonal antibody bag quilt
(2) NT-proBNP antigen standard
(3) biotin labeled NT-proBNP aptamer
(4) Streptavidin that marks of horseradish peroxidase (HRP)
(5) TMB nitrite ion
(6) stop buffer
(7) washings
2. detection method
(1) antigen prepares: GST-BNP antigen protein concentration dilution is to 1ug/ml;
(2) washings: SHMCK (0.2%Tween-20);
(3) Streptavidin of HRP mark;
(4) TMB nitrite ion;
(5) in the enzyme plate being coated with antibody, NT-ProBNP antigen is added, incubated at room 1h;
(6) SHMCK (0.2%Tween-20) washes 3 times;
(7) the NT-ProBNP aptamer of preparation is added, incubated at room 1h;
(8) SHMCK (0.2%Tween-20) washes 3 times;
(9) HRP labelled streptavidin (1:1000) is added, incubated at room 1h;
(10) SHMCK (0.2%Tween-20) washes 3 times;
(11) TMB colour developing 15min, stop buffer stops, and microplate reader 450nm measures absorbancy.
The composition of embodiment 7:NT-proBNP test strip and detection method.
1, the composition of test strip
PVC backing 1 is stained with sample pad 2, pad 3, nitrocellulose filter 4 and absorbent pad 5 successively; Pad 3 is coated with by the aptamer of the NT-proBNP of colloid gold label; Nitrocellulose filter is provided with detection line 6 and nature controlling line 7, detection line 6 is coated with the monoclonal antibody of NT-proBNP, nature controlling line 7 is can the aptamer of the NT-proBNP of colloid gold label is combined on pad 3 reagent, and the test strip be assembled into as shown in Figure 7.
2, detection method
Sample (blood plasma) joins in sample pad 2, liquid at the flows by action of absorbent pad 5 on pad 3, combine with the aptamer of the NT-proBNP of the colloid gold label in pad 3, under liquid stream drives, continue to move ahead, at detection line 6, place is combined with the monoclonal antibody of NT-proBNP, form mixture, Radioactive colloidal gold is assembled at this, Radioactive colloidal gold develops the color, unnecessary Radioactive colloidal gold continues the complementary sequence moved ahead in nature controlling line 7 and is combined, all the other liquid are to absorbent pad 5, two developed band are had to be positive after detection terminates, and the content of NT-proBNP is proportionate in the colored intensity of Radioactive colloidal gold and sample.
Claims (9)
1. a NT-proBNP aptamer, is characterized in that, described aptamer is any in the nucleotide sequence shown in SEQID:1-15.
2. a kind of NT-proBNP aptamer according to claim 1, is characterized in that, the monoclonal antibody that described aptamer can be prepared with NT-proBNP albumen or NT-proBNP albumen n end aminoacid sequence combines.
3. a kind of NT-proBNP aptamer according to claim 1, is characterized in that, the aminoacid sequence of described NT-proBNP albumen n end is GlySerAlaSerAspLeuGluThrSerGlyLeuGlnGluGlnArg.
4. the application of a kind of NT-proBNP aptamer described in any one of claim 1-3 in preparation NT-proBNP detection kit.
5. application according to claim 4, is characterized in that, described NT-proBNP aptamer is by biotin labeling.
6. the application of a kind of NT-proBNP aptamer described in any one of claim 1-3 in preparation NT-proBNP test strip.
7. application according to claim 6, is characterized in that, described NT-proBNP aptamer is by colloid gold label.
8. application according to claim 6, it is characterized in that, consisting of of described NT-proBNP test strip: on PVC backing (1), be stained with sample pad (2), pad (3), nitrocellulose filter (4) and absorbent pad (5) successively; Described pad (3) is coated with by the NT-proBNP aptamer of colloid gold label; Described nitrocellulose filter is provided with detection line (6) and nature controlling line (7); Wherein, detection line (6) is coated with the amino acid whose monoclonal antibody of NT-proBNP albumen n end, nature controlling line (7) is coated with the reagent be combined with the aptamer of the NT-proBNP of colloid gold label.
9. a kind of NT-proBNP test strip according to claim 8, is characterized in that, the described reagent be combined with the aptamer of the NT-proBNP of colloid gold label is the complementary sequence of NT-proBNP aptamer.
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| CN111487222B (en) * | 2019-01-28 | 2023-11-14 | 上海乐合生物科技有限公司 | Preparation method of SPR chip for detecting BNP |
| CN110095443A (en) * | 2019-05-09 | 2019-08-06 | 重庆医科大学 | A kind of fluorescent method detecting brain natriuretic peptide based on graphene oxide/aptamer |
| CN111778255A (en) * | 2020-05-29 | 2020-10-16 | 扬州大学 | Screening and identifying method of pepsin ssDNA aptamer |
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