CN101148667A - Preparation and use for affinity human albumin nucleic acid aptamer - Google Patents
Preparation and use for affinity human albumin nucleic acid aptamer Download PDFInfo
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- CN101148667A CN101148667A CNA2006100157395A CN200610015739A CN101148667A CN 101148667 A CN101148667 A CN 101148667A CN A2006100157395 A CNA2006100157395 A CN A2006100157395A CN 200610015739 A CN200610015739 A CN 200610015739A CN 101148667 A CN101148667 A CN 101148667A
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Abstract
The nucleic acid gamete of affiliating human albumin features its DNA molecule in the nucleotide sequence of ATCCGCCTGATTAGCGATACTGGGACTGACTGATACGAAGGCATGATTG GGACACTACTTGAGCAAAATCACCTGCAGGG. It is prepared through a systemic evolution index enriching process for screening human albumin oligonucleotide gamete, and may be used for replacing antibody in detecting, separating and purifying human albumin. The derived RNA sequence has the same function as the oligonucleotide sequence derived through hybridization, deletion, increment, displacement or modification. The present invention has the features of simplicity, high speed, low cost, etc. The oligonucleotide has relatively small molecular weight and may be synthesized chemically in low cost. It has affinity and specificity higher than that of antibody, easy marking optionally in different parts, high repeatability and high stability.
Description
(1) technical field
The present invention relates to biological technical field, particularly a kind of have the preparation and the purposes of the nucleic acid aptamer of high specific and high-affinity binding ability with human albumin.
(2) background technology
Human albumin is generated by liver cell, mainly is present in blood plasma, has biological function widely, the albuminous input of clinical a large amount of needs treatment, and the diagnosis of the auxiliary disease of albuminous detection in the body fluid.As: in the blood albumin concentration increase the plasma extraction that is common in serious dehydration and causes, reduce and then be more common in liver, kidney diseases such as chronic hepatitis, liver cirrhosis, liver cancer, ephritis.In the urine detection of microalbumin to the illness of some ephrosis functional disorders be one than routine urinalysis or the more responsive index of urine total protein inspection, particularly at the chronic kidney injury disease, in lysises such as diabetic nephropathy, hypertension, systemic lupus erythematous, change has more susceptibility to the prompting renal function in the detection of microalbuminuria.Some nearest researchs are thought, microalbuminuria is not only the index of diabetic complication early diagnosis, and the generation of hypertension and cardiovascular disorder, development, prognosis, treatment effectiveness evaluation etc. are also had important references be worth early sign and independent hazard factor that this index positive is the blood vessel extensive injuries.Therefore the range of application of microalbumin uroscopy has expanded the routine inspection project as diseases such as cardiovascular disorder, hypertension to, can find potential pathology by this inspection, for early diagnosis, the early treatment of disease provides Useful Information and lab index.At present, the method for measuring micro-urine protein commonly used is a radioimmunology, and the somebody carries out the development of ELISA measuring reagent kit.
Phyletic evolution index concentration technology (SELEX technology) is a kind of new combinatorial chemistry technique that grows up early 1990s, it uses jumbo random oligonucleotide library, the outer pcr amplification technology of combination, oligonucleotide with index concentration and target molecule specific combination, through multi-turns screen, obtain the oligonucleotide aptamer (aptamers) of avidity height, high specificity.This technology successfully applies to the screening of many target molecules, comprises metal ion, organic dye, medicine, protein, amino acid and various cytokines etc.
(3) summary of the invention
The objective of the invention is to substitute antibody, the human albumin early detection and the separation purification method of a kind of succinct quick, highly sensitive, high specific is provided by the oligonucleotide aptamer of phyletic evolution index concentration technology (SELEX technology) screening human albumin.
Technical scheme of the present invention:
The single stranded DNA random library and the primer that are used for phyletic evolution index concentration technology screening among the present invention, synthetic by U.S. invitrogen company, two ends are fixed sequence program, the centre is the stochastic sequence of 35 bases: 5 '-ATCCGCCTGATTAGCGATACT-(N35)-ACTTGAGCAAAATCACCTGCAGGGG-3 ', storage capacity is 10
14More than;
Primer 1:5 '-ATCCGCCTGATTAGCGATACT-3 ';
Primer 2: 5 '-biotin-CCCCTGCAGGTGATTTTGCTCAAGT-3 ';
Primer 3:5 '-biotin-ATC CGCCTGATTA GCGATACT-3 ';
Primer 4:5 '-CCCCTGCAGGTGATTTTGCTCAAGT-3 '.
Human albumin is purchased in Tianjin China and is given birth to bio tech ltd;
The GelRed nucleic acid dye is purchased the company in Biotium;
Nitrocellulose filter is purchased the company in U.S. Mi Libo (MilliPore);
It is Time Inc. that the purified reagent of oligonucleotide is purchased in sky, Beijing;
PCR test kit and T carrier are purchased the company in U.S. Pu Luomaige (Promega).
A kind of affinity human albumin nucleic acid aptamer is characterized in that nucleotides sequence classifies as: the dna molecular shown in ATCCGCCTGATTAGCGATACTGGGACTGACTGATACGAAGGCATGATTGGGACACT ACTTGAGCAAAATCACCTGCAGGG or the ATCCGCCTGATTAGCGATACTGCGGAATTGGATGAATCGGGACCACGCGAGTATAT ACTTGAGCAAAATCACCTGCAGGGG.
A kind of above-mentioned affinity human albumin nucleic acid aptamer is characterized in that: the single stranded DNA sequence is one of the kind of De both shown in the accompanying drawing 1 structure.
A kind of above-mentioned affinity human albumin nucleic acid aptamer is characterized in that: the oligonucleotide aptamer homologous sequence with identical function accounts for more than 60%.
A kind of above-mentioned affinity human albumin nucleic acid aptamer, it is characterized in that: derived RNA sequence has identical functions.
A kind of above-mentioned affinity human albumin nucleic acid aptamer is characterized in that: be the oligonucleotide sequence that can hybridize with dna sequence dna.
A kind of above-mentioned affinity human albumin nucleic acid aptamer is characterized in that: deletion of any position or the resulting oligonucleotide aptamer sequence of increase part oligonucleotide residue at nucleic acid aptamer have identical functions.
A kind of above-mentioned affinity human albumin nucleic acid aptamer is characterized in that: after the displacement of Nucleotide kind and rare base was carried out in any position of nucleic acid aptamer, the oligonucleotide aptamer sequence that obtains had identical functions.
A kind of above-mentioned affinity human albumin nucleic acid aptamer, it is characterized in that: after any position at nucleic acid aptamer was carried out phosphorylation, methylated, amino, sulfydryl, isotropic substance, vitamin H, digoxin, fluorescent substance, nano luminescent material or enzyme labelling modify, the oligonucleotide aptamer sequence that obtains had identical functions.
A kind of above-mentioned affinity human albumin nucleic acid aptamer is characterized in that: be used for the detection and the separation and purification of human albumin.
A kind of preparation method of above-mentioned affinity human albumin nucleic acid aptamer, carry out according to the following steps: 1) single stranded DNA random oligonucleotide library is synthetic; 2) utilize phyletic evolution index concentration method that oligonucleotide library is screened; 3) oligonucleotide of amplification and human albumin specific combination; 4) carry out the next round screening, take turns above screening back through 12 and obtain the purpose oligonucleotide sequence; 5) cloning and sequencing.
Advantage of the present invention is: have easy, quick, economic dispatch characteristics, compare with other combinatorial chemical libraries such as random peptide library, antibody library and phage display library, the many advantages of adaptive sub-tool that from oligonucleotide library, filter out: 1) itself be oligonucleotide, molecular weight is less, can chemosynthesis, save cost; 2) have affinity and the specificity higher than antibody; 3) be convenient to mark and can be at different sites mark selectively; 4) repeatability and good stability, and be easy to preserve, promptly insensitive to high temperature and violent condition.Therefore, phyletic evolution index concentration technology has a good application prospect, particularly in the antibody engineering field.
(4) description of drawings
Fig. 1: affinity human albumin nucleic acid aptamer dna sequence dna and RNA sequential structure figure.
Fig. 2: affinity human albumin nucleic acid aptamer clone's PCR identifies and shows figure.
Fig. 3: the affinity human albumin nucleic acid aptamer gel retardation assay shows figure.
Fig. 4: the test of affinity human albumin nucleic acid aptamer affinity analyzing shows figure.
(5) embodiment
Embodiment 1: the nucleic acid aptamer of in-vitro screening and human albumin specific combination
Utilize primer 1:5 '-ATCCGCCTGATTAGCGATACT-3 ' and primer 2: 5 '-biotin-CCCCTGCAGGTGATTTTGCTCAAGT-3 ' amplification single stranded DNA storehouse, 5 ' end is had biotin labeled double-stranded DNA product to be combined with the streptavidin magnetic bead, at ambient temperature, effect is 30 minutes; Be the NaOH solution of 0.15mol/L then with concentration, reacted 15 minutes, the double-stranded DNA sex change is unwind; The magnetic separation obtains single stranded DNA; Single stranded DNA behind the anti-sieve of empty nitrocellulose filter and salmon sperm dna and albumin are hatched jointly; Single stranded DNA, elutes single stranded DNA on nitrocellulose filter by albumin bound from albumin, as the template of amplification, carry out the next round screening; Carry out 12 altogether and take turns screening, and in the end several the wheel in the screening, adding is lower than the 100ng albumin, so that obtain expecting the oligonucleotide with high-affinity; Taking turns screening gained library with the 12nd is that template is carried out PCR; Reclaim 81bp place segment after the agarose electrophoresis, with T carrier sustained reaction 16～18 hours under 4 ℃ condition, transformed competence colibacillus cell; Coated plate carries out blue hickie screening, and after cultivating 16～18 hours under 37 ℃ of conditions, picking mono-clonal hickie is put ammonia benzyl liquid nutrient medium and shaken bacterium 16～18 hours; Extract plasmid, identify have purpose band person to carry out sequencing through PCR.The target stripe of 81bp as shown in Figure 1.
Embodiment 2: detect every screening gained single stranded DNA of taking turns with the gel blocking method
With every take turns sieved for the single stranded DNA template, utilize
Primer 3:5 '-biotin-ATC CGCCTGATTA GCGATACT-3 ' and
Primer 4:5 '-CCCCTGCAGGTGATTTTGCTCA AGT-3 ' carries out asymmetric PCR, hatch as detection probes and albumin, with the mixture of hatching through the non-denaturing polyacrylamide electrophoresis, through the GelRed dyeing, observe the band situation, as shown in Figure 2.Because the formation of the adaptive sub-mixture of protein-nucleic acid causes the retardance of gel band, shows the affine specificity of nucleic acid aptamer and albumin bound.
Embodiment 3: the nucleic acid aptamer of employing biotin modification is used for the detection of human albumin
Synthetic and with the nucleic acid aptamer of biotin modification mark, the dot blot method is measured the affinity characteristic of adaptive son of purpose and human albumin: is that quality is respectively with albumin with a series of amounts: 0,0.1,0.2,0.3,0.4 μ g point is on nitrocellulose filter, after waiting to absorb fully, film was sealed 1～1.5 hour at ambient temperature with 3% bovine serum albumin (BSA) and 100 μ l salmon sperm dnas; Then the adaptive son of the DNA of synthesizing biotinylated mark is diluted to the 1ml volume with TBS damping fluid (Tris-HCl: concentration 0.05M, pH 7.4), hybridizes with film; Through 1 hour, after the TBS buffer solution for cleaning, hatch luminous tracing with the avidin of horseradish enzyme labelling under the room temperature.The result produce at contrast place immaculate, and the albumin place presents black splotch, shows the specificity of nucleic acid aptamer and albumin bound, can be used for albuminous detection as shown in Figure 3.
Embodiment 4: nucleic acid aptamer is used for the separation and purification of human albumin as affinity ligand
The nucleic acid aptamer that obtains is coupled on the magnetic bead as affinity ligand; Mix with human serum and to hatch; Separate magnetic bead with magnetic separating device,, can obtain the pure product of human albumin, reach the separation and purification of human albumin the albumin process wash-out that is adsorbed on the magnetic bead.
Claims (10)
1. affinity human albumin nucleic acid aptamer is characterized in that nucleotides sequence classifies as:
ATCCGCCTGATTAGCGATACTGGGACTGACTGATACGAAGG
CATGATTGGGACACTACTTGAGCAAAATCACCTGCAGGG or
ATCCGCCTGATTAGCGATACTGCGGAATTGGATGAATCGGG
ACCACGCGAGTATATACTTGAGCAAAATCACCTGCAGGGG
Shown dna molecular.
2. affinity human albumin nucleic acid aptamer according to claim 1 is characterized in that: the single stranded DNA sequence is one of the kind of De both shown in the accompanying drawing 1 structure.
3. affinity human albumin nucleic acid aptamer according to claim 1 is characterized in that: the oligonucleotide aptamer homologous sequence with identical function accounts for more than 60%.
4. affinity human albumin nucleic acid aptamer according to claim 1, it is characterized in that: derived RNA sequence has identical functions.
5. affinity human albumin nucleic acid aptamer according to claim 1 is characterized in that: be the oligonucleotide sequence that can hybridize with dna sequence dna.
6. affinity human albumin nucleic acid aptamer according to claim 1 is characterized in that: deletion of any position or the resulting oligonucleotide aptamer sequence of increase part oligonucleotide residue at nucleic acid aptamer have identical functions.
7. affinity human albumin nucleic acid aptamer according to claim 1 is characterized in that: after the displacement of Nucleotide kind and rare base was carried out in any position of nucleic acid aptamer, the oligonucleotide aptamer sequence that obtains had identical functions.
8. affinity human albumin nucleic acid aptamer according to claim 1, it is characterized in that: after any position at nucleic acid aptamer was carried out phosphorylation, methylated, amino, sulfydryl, isotropic substance, vitamin H, digoxin, fluorescent substance, nano luminescent material or enzyme labelling modify, the oligonucleotide aptamer sequence that obtains had identical functions.
9. according to any described affinity human albumin nucleic acid aptamer among the claim 1-8, it is characterized in that: be used for the detection and the separation and purification of human albumin.
10. the preparation method of an affinity human albumin nucleic acid aptamer, the nucleic acid aptamer of in-vitro screening and human albumin specific combination is characterized in that implementation step is as follows: utilize
Primer 1:5 '-ATCCGCCTGATTAGCGATACT-3 ' and
Primer 2: 5 '-biotin-CCCCTGCAGGTGATTTTGCTCAAGT-3 ' amplification single stranded DNA storehouse, 5 ' end is had biotin labeled double-stranded DNA product combine with the streptavidin magnetic bead, at ambient temperature, act on 30 minutes; Be the NaOH solution of 0.15mol/L then with concentration, reacted 15 minutes, the double-stranded DNA sex change is unwind; The magnetic separation obtains single stranded DNA; Single stranded DNA behind the anti-sieve of empty nitrocellulose filter and salmon sperm dna and albumin are hatched jointly; Single stranded DNA, elutes single stranded DNA on nitrocellulose filter by albumin bound from albumin, as the template of amplification, carry out the next round screening; Carry out 12 altogether and take turns screening, and in the end several the wheel in the screening, adding is lower than the 100ng albumin, so that obtain expecting the oligonucleotide with high-affinity; Taking turns screening gained library with the 12nd is that template is carried out PCR;
Reclaim 81bp place segment after the agarose electrophoresis, with T carrier sustained reaction 16～18 hours under 4 ℃ condition, transformed competence colibacillus cell; Coated plate carries out blue hickie screening, and after cultivating 16～18 hours under 37 ℃ of conditions, picking mono-clonal hickie is put ammonia benzyl liquid nutrient medium and shaken bacterium 16～18 hours; Extract plasmid, identify have purpose band person to carry out sequencing through PCR.The target stripe of 81bp as shown in Figure 1.
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| CN102226185A (en) * | 2011-05-09 | 2011-10-26 | 华中科技大学同济医学院附属同济医院 | RNA aptamer of targeting hepatocyte and nucleotide sequence thereof |
| CN102559917A (en) * | 2012-02-27 | 2012-07-11 | 南昌大学 | Application method for diagnosing aptamer based on conventional gel electrophoresis |
| CN103966224A (en) * | 2014-05-12 | 2014-08-06 | 华国光 | Aptamer as well as screening method and application thereof |
| CN105334328A (en) * | 2015-11-01 | 2016-02-17 | 李倩 | Kit and method for detecting ophthalmic diseases |
| CN105334327A (en) * | 2015-11-01 | 2016-02-17 | 李倩 | Kit and method for detecting ophthalmopathy |
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| CN107815453A (en) * | 2017-10-23 | 2018-03-20 | 中山大学中山眼科中心 | A kind of nucleic acid aptamer and application thereof |
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| CN100375750C (en) * | 2003-05-15 | 2008-03-19 | 中国疾病预防控制中心病毒病预防控制所 | Oligopolynucleotide of inhibiting activity of necrosin in human tumor |
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| CN101880313A (en) * | 2008-12-25 | 2010-11-10 | 国家纳米技术与工程研究院 | Method for separating and purifying antibodies |
| CN102226185A (en) * | 2011-05-09 | 2011-10-26 | 华中科技大学同济医学院附属同济医院 | RNA aptamer of targeting hepatocyte and nucleotide sequence thereof |
| CN102559917A (en) * | 2012-02-27 | 2012-07-11 | 南昌大学 | Application method for diagnosing aptamer based on conventional gel electrophoresis |
| CN103966224A (en) * | 2014-05-12 | 2014-08-06 | 华国光 | Aptamer as well as screening method and application thereof |
| WO2016150370A1 (en) * | 2015-03-23 | 2016-09-29 | 北京莱盟麦迪医疗技术有限公司 | Nucleic acid aptamer capable of detecting enox2 protein within cancer patient serum, and application of nucleic acid aptamer |
| WO2016210448A1 (en) * | 2015-06-26 | 2016-12-29 | University Of Utah Research Foundation | Depletion of abundant serum proteins to facilitate biomarker discovery |
| CN105334328A (en) * | 2015-11-01 | 2016-02-17 | 李倩 | Kit and method for detecting ophthalmic diseases |
| CN105334327A (en) * | 2015-11-01 | 2016-02-17 | 李倩 | Kit and method for detecting ophthalmopathy |
| CN107815453A (en) * | 2017-10-23 | 2018-03-20 | 中山大学中山眼科中心 | A kind of nucleic acid aptamer and application thereof |
| CN110579590A (en) * | 2018-06-08 | 2019-12-17 | 首都师范大学 | Urine microalbumin detection method based on DNA aptamer and kit thereof |
| CN110579590B (en) * | 2018-06-08 | 2022-09-30 | 首都师范大学 | Detection method based on DNA aptamer and kit thereof |
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