CN104109674A - Aptamer of glycosylated hemoglobin and preparation method thereof - Google Patents

Abstract

The invention discloses an aptamer with high specificity and affinity of glycosylated hemoglobin GHb and a preparation method of the aptamer. The nucleotide sequence of the aptamer of the glycosylated hemoglobin GHb comprises any one of sequences from SEQ ID NO: 1 to SEQ ID NO: 4. The preparation method comprises the following steps of: constructing a random sequence oligonucleotide library, preparing a glycosylated hemoglobin-micro magnetic bead composite, initially screening a target material-nucleic acid composite, preparing a secondary single stranded DNA oligonucleotide library, screening and identifying the aptamer of the glycosylated hemoglobin. The prepared aptamer is free from toxicity, small in molecular weight, and liable to synthesize and mark. The aptamer only specifically identifies the glycosylated hemoglobin GHb, and does not identify other homologous non-glycosylated hemoglobin Hb, so that the aptamer can be taken as a molecular beacon for detecting the content of the glycosylated hemoglobin GHb.

Description

Aptamer of a kind of glycolated hemoglobin and preparation method thereof

The application is dividing an application of " aptamer of a kind of glycolated hemoglobin and preparation method thereof ", and the applying date of this original application is that November 14, application number in 2012 are 2012104568467, invention and created name " aptamer of a kind of glycolated hemoglobin and preparation method thereof ".

Technical field

The invention belongs to protein detection technology field, relate to a kind of nucleic acid, particularly relate to the aptamer and preparation method thereof of the glycolated hemoglobin GHb of high specific and high-affinity.

Background technology

Diabetes are worldwide public health problems of human health, and diabetes are a kind of lifelong property diseases, and its complication is to residual major cause till death, so people wish to find as early as possible and to treat diabetes.Traditional diabetes diagnosis and treatment monitoring adopt fasting plasma glucose, postprandial blood sugar and oral glucose tolerance test etc., moment glucose level when but blood sugar parameter only represents blood drawing, and glycolated hemoglobin (GHb) is as the gold standard of the long-term glucose level of reflection, it is also the important indicator of monitoring treating diabetes.GHb be in red corpuscle oxyphorase and glucose slowly, continue and irreversibly carry out the product of non-enzymatic albumen saccharification react.After forming two weeks, be difficult for separately.When the concentration of glucose in blood is higher, the formed GHb content of human body also can be relatively high. and under normal physiological conditions, the growing amount of non-enzymatic saccharification react product is directly proportional to the concentration of reactant.Because protein concn keeps relative stability, saccharification level depends mainly on glucose concn, and the time length also contacting with glucose with protein is relevant.GHb can reflect the average blood sugar level of nearest 2 ~ 3 months.ADA in 2002 clearly stipulates regularly to detect GHb, and the gold mark using it as monitoring diabetes glycemic control.Universal Diabetes Mellitus Knowledge, upgrades treatment theory, monitors and keeps GHb up to standard, more early, more reasonably uses the pharmacological agenies such as Regular Insulin, particularly important for the generation development of controlling diabetic complication.

The detection method of at present conventional glycolated hemoglobin has two large classes: high-pressure liquid phase method (HPLC) and immunization method.The instrument that high-pressure liquid phase method (HPLC) is used is expensive, is difficult in hospital and the laboratory of comparing basic unit universal; Microtrabeculae method manual steps is loaded down with trivial details, and the quality of chromatography time and microtrabeculae is wayward, easily produces operative technique error, and repeatability is not good enough; And interfering factors is a lot, especially the sensitive to pH value and temperature, HbF and variation oxyphorase (HbS, HbC, HbE etc.) disturb result, and annoyance level is determined according to the separating power of post, and examine collection of illustrative plates, so this method can not get generally adopting.Immunization is measured its accuracy of glycolated hemoglobin and repeatability is all weak, and measures the interference that is subject to the materials such as high concentration glucose, high triglyceride, high bilirubin, because the turbidity of sample can make photometric determination produce mistake.In addition, antibody is obtained by animal immune, and cost is high and the screening cycle is long, there are differences between batches; Antibody is responsive to temperature humidity, and volatility is difficult to ensure the shortcoming such as deposits, and also can not get being widely used.

Aptamer is the novel identification molecule of a class that developed recently gets up, and is subject in recent years scientist's extensive concern, a large amount of out screened for the aptamer of important physiologically active molecule; Various analytical procedure and technology based on aptamer are in the news; Aptamer medicine " Macugen " was also gone on the market by FDA official approval in 2005.The oligonucleotide sequence that SELEX technology screening obtains is called as aptamer, and domestic its is translated as aptamer, nucleic acid aptamer, aptamer or aptamer etc.SELEX technology refers to that applied chemistry method synthesizes jumbo random oligonucleotide (being comprised of the fixed sequence program at two ends and middle stochastic sequence) library, by applying selective pressure (in conjunction with target, the process of elutriation and target high special binding fragment), and in conjunction with Amplification Technologies, through the too much circulation selective enrichment of wheel, obtaining the oligonucleotide molecules of being combined with target material high special, can be that RNA can be also DNA, and length is generally 25 ~ 60 Nucleotide.

Aptamer is combined presented hypersensitivity and high specific with target material, it is had a good application prospect in medical diagnosis on disease, although ripe clinical application report is less at present, but the research of applying fit detection target protein is on the increase, the detection new technology based on fit also constantly occurs.Patent CN201010580661.8, name is called aptamer of Protein-tyrosine-phosphatase SHP2 and preparation method thereof, announce the sequence signature of the aptamer of Protein-tyrosine-phosphatase SHP2, by designing the steps such as synthesizing single-stranded DNA random oligonucleotide storehouse, screening object oligonucleotide sequence, flow cytometer showed evaluation, prepared the aptamer of Protein-tyrosine-phosphatase SHP2.Patent CN201110148431.9 and patent CN201110148603.2, announced fit and derivative, screening method and the application with HCV NS5A protein nucleic acid of HCV E1E2 albumen, wherein the fit derivative of Nucleotide comprises the improved derivative of RNA aptamer, skeleton after replacement, transform the derivative after peptide nucleic acid(PNA) as and connects the derivative after upper Geigers.Patent CN201210214996.7, the aptamer, micro-fluidic chip and screening method and the application for screening that can be used for detecting myohaemoglobin have been announced, the screening method that the aforementioned micro-fluidic chip of a kind of use screens described aptamer is disclosed, comprise and optimize nucleic acid library, primary dcreening operation, purifying, the several key steps of circulation, the detection method of this invention has advantages of high-affinity and high specific.

As from the foregoing, aptamer is because the chemical antibody characteristic of its uniqueness becomes a new generation for molecular medicine or the targeting vector of specific protein, and for detection of the target molecule material of its specific recognition.But the efficient specific recognition research that is directed to glycolated hemoglobin GHb at present based on aptamer also lacks very much, and be directed to the aptamer of glycolated hemoglobin GHb and screen preparation method and there is not yet report.

Summary of the invention

The technical problem to be solved in the present invention is the deficiency that overcomes existing glycolated hemoglobin detection technique, fill up the aptamer and the screening preparation method blank thereof that yet there are no glycolated hemoglobin GHb, a kind of glycolated hemoglobin GHb aptamer and preparation method thereof is provided, aptamer provided by the present invention is shown in SEQ ID NO:1 ~ SEQ ID NO:4, names respectively GHb1, GHb2, GHb3, GHb4.

The solution of the present invention is by such realization:

A glycolated hemoglobin aptamer, is characterized in that, the nucleotides sequence of described aptamer has been classified sequence dna fragment shown in SEQ ID NO:2 as, called after GHb2, and its sequence is:

SEQ?ID?NO:2：gggaggcatg?tgatttaagg?cgcggcagat?gttggaaccc。。

Prepare an above arbitrary described glycolated hemoglobin aptamer method, it is characterized in that comprising the following steps:

(1) build random sequence oligonucleotides storehouse: synthetic single stranded DNA sequence, building storage capacity is 10 ⁵~ 10 ⁶single stranded DNA random sequence oligonucleotides storehouse, the DNA sequence dna that single stranded DNA random oligonucleotide storehouse comprises is:

5’-GACCATACCAGCTTATTCAATT-N _25~60-AGATAGTAAGTGCAATCTGGC-3’

Stochastic sequence N in the middle of the DNA sequence dna in described single stranded DNA random oligonucleotide storehouse comprises _{25 ~ 60}with two ends fixed sequence program, described middle stochastic sequence N _{25 ~ 60}be the stochastic sequence of 25 ~ 60 bases, described two ends fixed sequence program is: 5 '-GACCATACCAGCTTATTCAATT, and 3 '-CGGTCTAACGTGAATGATAGA, described two ends fixed sequence program is pcr amplification primer land;

(2) prepare glycolated hemoglobin-micro-magnetic bead mixture: will have the amino micro-magnetic bead of activation in coupling buffer, to mix according to the ratio of 0.05 ~ 0.2ml:1mg with glycolated hemoglobin, add again 100 ~ 200ul coupling agent solution, light mixed 20 ~ 30 hours under 25oC reaction conditions, make glycolated hemoglobin C-terminal covalently bound to micro-magnetic bead, in tripping device, with scavenging solution washing, obtain glycolated hemoglobin-micro-magnetic bead mixture;

(3) screen for the first time target material-nucleic acid complexes: 2 ~ 5nmol single stranded DNA random sequence oligonucleotides storehouse is dissolved in to binding buffer liquid, carry out heat treated, single-stranded DNA banks with in conjunction with the magnetic bead of glycolated hemoglobin, hatch after, collect not the liquid in conjunction with the DNA random oligonucleotide storehouse of magnetic bead; By step 2) glycolated hemoglobin-micro-magnetic bead mixture collects and is not combined DNA random oligonucleotide storehouse 37oC incubation 15 ~ 40min in binding buffer liquid of magnetic bead after thermal treatment, with elution buffer, wash glycolated hemoglobin-oligonucleotide-micro-magnetic bead mixture, remove that be not combined with glycolated hemoglobin and oligonucleotide sequence non-specific binding, glycolated hemoglobin-oligonucleotide-micro-magnetic bead mixture 92oC in elution buffer is hatched after 5min, reclaims the elution buffer with single stranded oligonucleotide sequence.

(4) prepare secondary single strand dna oligonucleotide storehouse: will in step 3), reclaim and obtain carrying out pcr amplification with the elution buffer of single stranded oligonucleotide sequence, pcr amplification product be take the micro-magnetic bead of Streptavidin and is carried out separation as matrix, through alkaline denaturation, unwind, filter, purifying, obtain secondary single strand dna oligonucleotide storehouse, for next round, screen.

(5) screen and identify glycolated hemoglobin aptamer: it is matrix separation screening that the micro-magnetic bead of Streptavidin is taken turns to through 12 ~ 20 in the secondary single strand dna oligonucleotide storehouse of step 4) gained, obtain the few nucleotide sequence of target, clone and the few nucleotide sequence of the described target that checks order, by enzyme, join specificity and the avidity that aptamer adsorption measurement identifies that it is combined with glycolated hemoglobin.

A kind of above-described glycolated hemoglobin aptamer method of preparing, the PCR primer sequence that described pcr amplification is used is:

Primer 1:5 '-ATACCAGCTTATTCAATT-3 '

Primer 2: 5 '-Biotin-AGATTGCACTTACTATCT-3 '

5 ' end of described pcr amplification primer 2 is containing biotin labeling;

Described pcr amplification processing condition are: 94oC denaturation 5 min, and 94oC 30s, 47oC 1min, 72oC 1min, 10 ~ 25 circulations of increasing, final extension is 72oC 10min.

A kind of above-described glycolated hemoglobin aptamer method of preparing, described coupling buffer is containing 20mM potassium phosphate salt damping fluid, 0.15M NaCl, 1mM DTT, pH 5.5; Described coupling agent solution is the dimethylaminopropyl ethyl carboxamide containing 57%; Described binding buffer liquid is for containing 100 mM NaCl, 20 mM Tris-HCl pH 7.6,2 mM MgCl ₂, 5mM KCl, 1 mM CaCl ₂, 0.02% Tween 20,1 mM DTT; Described thermal treatment is 90 oC heating 10min, is placed in 10min on ice, and then temperature is placed 5min; Described elution buffer is for containing 20 mM Tris-HCl pH7.6,200 mM NaCl, 10 mM EDTA.

Above-described glycolated hemoglobin aptamer or the described glycolated hemoglobin aptamer preparing, it is characterized in that, described aptamer is used for identification, detects glycolated hemoglobin, or for the preparation of detecting in the test kit of glycolated hemoglobin.

As a total technical conceive, the present invention also provides the derivative of more than one arbitrary described glycolated hemoglobin aptamers, and described derivative comprises any one in following four kinds:

(1) base A, T, C or G on the optional position of aptamer described in claim 1 are replaced as to rare base and methylate after purine, dihydrouracil or xanthoglobulin, the nucleic acid aptamer derivative obtaining;

(2) phosphorothioate backbone that the skeleton of the nucleotide sequence of aptamer described in claim 1 derives;

(3) peptide nucleic acid(PNA) that described in claim 1, aptamer is transformed into;

(4) the lock nucleic acid that described in claim 1, aptamer is transformed into.

Above-described a kind of glycolated hemoglobin nucleic acid aptamer derivative, is characterized in that, described aptamer or its derivative are used for identification, detect glycolated hemoglobin, or for the preparation of detecting in the test kit of glycolated hemoglobin.

Substantive distinguishing features of the present invention and marked improvement are: the aptamer molecular weight that (1) screens is little, and nontoxicity is conducive to the design of molecular probe, are easy to synthetic and mark; (2) aptamer special identification glycolated hemoglobin only, not in conjunction with non-glycolated hemoglobin and other glycated proteins molecule, in conjunction with the ability of glycolated hemoglobin, be 20 ~ 80 times in conjunction with the ability of non-glycolated hemoglobin Hb, can become the reagent of differentiating glycolated hemoglobin, improve specificity and the sensitivity of detection method, simplification detection method, reduces costs.

Accompanying drawing explanation

Fig. 1. enzyme connection aptamer adsorption measurement detects the binding ability curve of aptamer GHb1 and glycolated hemoglobin.In Fig. 1, X-coordinate is aptamer dna concentration, and ordinate zou is OD relative value, represent the binding curve of aptamer GHb1 and glycolated hemoglobin, represent the binding curve of aptamer GHb1 and non-glycolated hemoglobin Hb.

Fig. 2. enzyme connection aptamer adsorption measurement detects the binding ability curve of aptamer GHb2 and glycolated hemoglobin.In Fig. 2, X-coordinate is aptamer dna concentration, and ordinate zou is OD relative value, represent the binding curve of aptamer GHb2 and glycolated hemoglobin, represent the binding curve of aptamer GHb2 and non-glycolated hemoglobin Hb.

Fig. 3. enzyme connection aptamer adsorption measurement detects the binding ability curve of aptamer GHb3 and glycolated hemoglobin.In Fig. 3, X-coordinate is aptamer dna concentration, and ordinate zou is OD relative value, represent the binding curve of aptamer GHb3 and glycolated hemoglobin, represent the binding curve of aptamer GHb3 and non-glycolated hemoglobin Hb.

Fig. 4. enzyme connection aptamer adsorption measurement detects the binding ability curve of aptamer GHb4 and glycolated hemoglobin.In Fig. 4, X-coordinate is aptamer dna concentration, and ordinate zou is OD relative value, represent the binding curve of aptamer GHb4 and glycolated hemoglobin, represent the binding curve of aptamer GHb4 and non-glycolated hemoglobin Hb.

Embodiment

Below in conjunction with Figure of description and specific embodiment, the invention will be further described.The experimental technique using in following embodiment if no special instructions, is ordinary method.The material using in following embodiment, reagent etc., if no special instructions, obtain in all can be from commercial channels.

embodiment 1the preparation of the aptamer GHb1 of glycolated hemoglobin specific combination

(1) build random sequence oligonucleotides storehouse: synthetic single stranded DNA sequence, building storage capacity is 1 * 10 ⁵single stranded DNA random sequence oligonucleotides storehouse, the DNA sequence dna that single stranded DNA random oligonucleotide storehouse comprises is:

5’-GACCATACCAGCTTATTCAATT-N _25~40-AGATAGTAAGTGCAATCTGGC-3’

Stochastic sequence N in the middle of the DNA sequence dna in described single stranded DNA random oligonucleotide storehouse comprises _{25 ~ 60}with two ends fixed sequence program, described middle stochastic sequence N _{25 ~ 40}be the stochastic sequence of 25 ~ 40 bases, described two ends fixed sequence program is: 5 '-GACCATACCAGCTTATTCAATT, and 3 '-CGGTCTAACGTGAATGATAGA, described two ends fixed sequence program is pcr amplification primer land;

(2) prepare glycolated hemoglobin-micro-magnetic bead mixture: by 0.25ml with the amino micro-magnetic bead of activation and 5mg glycolated hemoglobin at coupling buffer (20mM potassium phosphate salt damping fluid, 0.15M NaCl, 1mM DTT, pH 5.5) middle mixing, add 100ul coupling agent solution (the dimethylaminopropyl ethyl carboxamide containing 57%), light mixed 20 hours under 25oC reaction conditions, make glycolated hemoglobin C-terminal covalently bound to micro-magnetic bead, in tripping device, with scavenging solution washing, obtain glycolated hemoglobin-micro-magnetic bead mixture.

(3) screen for the first time target material-nucleic acid complexes: 2nmol single-stranded DNA banks is dissolved in to binding buffer liquid (100 mM NaCl, 20 mM Tris-HCl pH 7.6,2 mM MgCl2,5mM KCl, 1 mM CaCl2,0.02% Tween 20,1 mM DTT), carry out heat treated, 90 oC heating 10min, are placed in 10min on ice, and then temperature is placed 5min, single-stranded DNA banks, after hatching in conjunction with the magnetic bead of glycolated hemoglobin, is collected not the liquid in conjunction with the DNA random oligonucleotide storehouse of magnetic bead, by step 2) glycolated hemoglobin-micro-magnetic bead mixture collects and is not combined DNA random oligonucleotide storehouse 37oC incubation 30min in binding buffer liquid of magnetic bead after thermal treatment, with elution buffer (20 mM Tris-HCl pH7.6, 200 mM NaCl, 10 mM EDTA) washing glycolated hemoglobin-oligonucleotide-micro-magnetic bead mixture, remove that be not combined with glycolated hemoglobin and oligonucleotide sequence non-specific binding, glycolated hemoglobin-oligonucleotide-micro-magnetic bead mixture 92oC in elution buffer is hatched after 5min, recovery is with the elution buffer of single stranded oligonucleotide sequence.

(4) prepare secondary single strand dna oligonucleotide storehouse: will in step 3), reclaim and obtain carrying out pcr amplification with the elution buffer of single stranded oligonucleotide sequence, pcr amplification product be take the micro-magnetic bead of Streptavidin and is carried out separation as matrix, through alkaline denaturation, unwind, filter, purifying, obtain secondary single strand dna oligonucleotide storehouse, for next round, screen; Pcr amplification processing condition are: 94oC denaturation 5 min, 94oC 30s, 47oC 1min, 72oC 1min, 16 circulations of increasing, final extension is 72oC 10min, primer 1:5 '-ATACCAGCTTATTCAATT-3 ', primer 2: 5 '-Biotin-AGATTGCACTTACTATCT-3 '

, 5 ' end biotin labeling of primer 2.

(5) screen and identify glycolated hemoglobin aptamer: it is matrix separation screening that the micro-magnetic bead of Streptavidin is taken turns to through 15 in the secondary single strand dna oligonucleotide storehouse of step 4) gained, obtain the few nucleotide sequence of target, clone and the few nucleotide sequence of the described target that checks order, the few nucleotide sequence of target is SEQ ID NO:1, called after GHb1.

The above-mentioned aptamer of the present embodiment can derive the following nucleic acid aptamer derivative that can be used for detecting glycolated hemoglobin: base A, T, C or G on the aptamer optional position of the present embodiment are replaced as to rare base and methylate after purine, dihydrouracil or xanthoglobulin, the nucleic acid aptamer derivative obtaining; The phosphorothioate backbone that the skeleton of the nucleotide sequence of the aptamer of the present embodiment derives; The peptide nucleic acid(PNA) that the aptamer of the present embodiment is transformed into; The lock nucleic acid that the aptamer of the present embodiment is transformed into.

embodiment 2the preparation of the aptamer GHb2 of glycolated hemoglobin specific combination

(1) build random sequence oligonucleotides storehouse: synthetic single stranded DNA sequence, building storage capacity is 3 * 10 ⁵single stranded DNA random sequence oligonucleotides storehouse, the DNA sequence dna that single stranded DNA random oligonucleotide storehouse comprises is:

5’-GACCATACCAGCTTATTCAATT-N _30~45-AGATAGTAAGTGCAATCTGGC-3’

Stochastic sequence N in the middle of the DNA sequence dna in described single stranded DNA random oligonucleotide storehouse comprises _{25 ~ 60}with two ends fixed sequence program, described middle stochastic sequence N _{30 ~ 45}be the stochastic sequence of 30 ~ 45 bases, described two ends fixed sequence program is: 5 '-GACCATACCAGCTTATTCAATT, and 3 '-CGGTCTAACGTGAATGATAGA, described two ends fixed sequence program is pcr amplification primer land;

(2) prepare glycolated hemoglobin-micro-magnetic bead mixture: by 1.0ml with the amino micro-magnetic bead of activation and 5mg glycolated hemoglobin at coupling buffer (20mM potassium phosphate salt damping fluid, 0.15M NaCl, 1mM DTT, pH 5.5) middle mixing, add 120ul coupling agent solution (the dimethylaminopropyl ethyl carboxamide containing 57%), light mixed 25 hours under 25oC reaction conditions, make glycolated hemoglobin C-terminal covalently bound to micro-magnetic bead, in tripping device, with scavenging solution washing, obtain glycolated hemoglobin-micro-magnetic bead mixture.

(3) screen for the first time target material-nucleic acid complexes: 5nmol single-stranded DNA banks is dissolved in to binding buffer liquid (100 mM NaCl, 20 mM Tris-HCl pH 7.6,2 mM MgCl2,5mM KCl, 1 mM CaCl2,0.02% Tween 20,1 mM DTT), carry out heat treated, 90 oC heating 10min, are placed in 10min on ice, and then temperature is placed 5min, single-stranded DNA banks, after hatching in conjunction with the magnetic bead of glycolated hemoglobin, is collected not the liquid in conjunction with the DNA random oligonucleotide storehouse of magnetic bead, by step 2) glycolated hemoglobin-micro-magnetic bead mixture collects and is not combined DNA random oligonucleotide storehouse 37oC incubation 40min in binding buffer liquid of magnetic bead after thermal treatment, with elution buffer (20 mM Tris-HCl pH7.6, 200 mM NaCl, 10 mM EDTA) washing glycolated hemoglobin-oligonucleotide-micro-magnetic bead mixture, remove that be not combined with glycolated hemoglobin and oligonucleotide sequence non-specific binding, glycolated hemoglobin-oligonucleotide-micro-magnetic bead mixture 92oC in elution buffer is hatched after 5min, recovery is with the elution buffer of single stranded oligonucleotide sequence.

(4) prepare secondary single strand dna oligonucleotide storehouse: will in step 3), reclaim and obtain carrying out pcr amplification with the elution buffer of single stranded oligonucleotide sequence, pcr amplification product be take the micro-magnetic bead of Streptavidin and is carried out separation as matrix, through alkaline denaturation, unwind, filter, purifying, obtain secondary single strand dna oligonucleotide storehouse, for next round, screen; Pcr amplification processing condition are: 94oC denaturation 5 min, 94oC 30s, 47oC 1min, 72oC 1min, 12 circulations of increasing, final extension is 72oC 10min, primer 1:5 '-ATACCAGCTTATTCAATT-3 ', primer 2: 5 '-Biotin-AGATTGCACTTACTATCT-3 '

, 5 ' end biotin labeling of primer 2.

(5) screen and identify glycolated hemoglobin aptamer: it is matrix separation screening that the micro-magnetic bead of Streptavidin is taken turns to through 10 in the secondary single strand dna oligonucleotide storehouse of step 4) gained, obtain the few nucleotide sequence of target, clone and the few nucleotide sequence of the described target that checks order, the few nucleotide sequence of target is SEQ ID NO:2, called after GHb2.

The above-mentioned aptamer of the present embodiment can derive the following nucleic acid aptamer derivative that can be used for detecting glycolated hemoglobin: base A, T, C or G on the aptamer optional position of the present embodiment are replaced as to rare base and methylate after purine, dihydrouracil or xanthoglobulin, the nucleic acid aptamer derivative obtaining; The phosphorothioate backbone that the skeleton of the nucleotide sequence of the aptamer of the present embodiment derives; The peptide nucleic acid(PNA) that the aptamer of the present embodiment is transformed into; The lock nucleic acid that the aptamer of the present embodiment is transformed into.

embodiment 3the preparation of the aptamer GHb3 of glycolated hemoglobin specific combination

(1) build random sequence oligonucleotides storehouse: synthetic single stranded DNA sequence, building storage capacity is 1 * 10 ⁶single stranded DNA random sequence oligonucleotides storehouse, the DNA sequence dna that single stranded DNA random oligonucleotide storehouse comprises is:

5’-GACCATACCAGCTTATTCAATT-N _35~60-AGATAGTAAGTGCAATCTGGC-3’

Stochastic sequence N in the middle of the DNA sequence dna in described single stranded DNA random oligonucleotide storehouse comprises _{25 ~ 60}with two ends fixed sequence program, described middle stochastic sequence N _{35 ~ 60}be the stochastic sequence of 35 ~ 60 bases, described two ends fixed sequence program is: 5 '-GACCATACCAGCTTATTCAATT, and 3 '-CGGTCTAACGTGAATGATAGA, described two ends fixed sequence program is pcr amplification primer land;

(2) prepare glycolated hemoglobin-micro-magnetic bead mixture: by 0.5ml with the amino micro-magnetic bead of activation and 5mg glycolated hemoglobin at coupling buffer (20mM potassium phosphate salt damping fluid, 0.15M NaCl, 1mM DTT, pH 5.5) middle mixing, add 160ul coupling agent solution (the dimethylaminopropyl ethyl carboxamide containing 57%), light mixed 28 hours under 25oC reaction conditions, make glycolated hemoglobin C-terminal covalently bound to micro-magnetic bead, in tripping device, with scavenging solution washing, obtain glycolated hemoglobin-micro-magnetic bead mixture.

(3) screen for the first time target material-nucleic acid complexes: 3nmol single-stranded DNA banks is dissolved in to binding buffer liquid (100 mM NaCl, 20 mM Tris-HCl pH 7.6,2 mM MgCl2,5mM KCl, 1 mM CaCl2,0.02% Tween 20,1 mM DTT), carry out heat treated, 90 oC heating 10min, are placed in 10min on ice, and then temperature is placed 5min, single-stranded DNA banks, after hatching in conjunction with the magnetic bead of glycolated hemoglobin, is collected not the liquid in conjunction with the DNA random oligonucleotide storehouse of magnetic bead, by step 2) glycolated hemoglobin-micro-magnetic bead mixture collects and is not combined DNA random oligonucleotide storehouse 37oC incubation 20min in binding buffer liquid of magnetic bead after thermal treatment, with elution buffer (20 mM Tris-HCl pH7.6, 200 mM NaCl, 10 mM EDTA) washing glycolated hemoglobin-oligonucleotide-micro-magnetic bead mixture, remove that be not combined with glycolated hemoglobin and oligonucleotide sequence non-specific binding, glycolated hemoglobin-oligonucleotide-micro-magnetic bead mixture 92oC in elution buffer is hatched after 5min, recovery is with the elution buffer of single stranded oligonucleotide sequence.

(4) prepare secondary single strand dna oligonucleotide storehouse: will in step 3), reclaim and obtain carrying out pcr amplification with the elution buffer of single stranded oligonucleotide sequence, pcr amplification product be take the micro-magnetic bead of Streptavidin and is carried out separation as matrix, through alkaline denaturation, unwind, filter, purifying, obtain secondary single strand dna oligonucleotide storehouse, for next round, screen; Pcr amplification processing condition are: 94oC denaturation 5 min, 94oC 30s, 47oC 1min, 72oC 1min, 18 circulations of increasing, final extension is 72oC 10min, primer 1:5 '-ATACCAGCTTATTCAATT-3 ', primer 2: 5 '-Biotin-AGATTGCACTTACTATCT-3 '

, 5 ' end biotin labeling of primer 2.

(5) screen and identify glycolated hemoglobin aptamer: it is matrix separation screening that the micro-magnetic bead of Streptavidin is taken turns to through 20 in the secondary single strand dna oligonucleotide storehouse of step 4) gained, obtain the few nucleotide sequence of target, clone and the few nucleotide sequence of the described target that checks order, the few nucleotide sequence of target is SEQ ID NO:3, called after GHb3.

The above-mentioned aptamer of the present embodiment can derive the following nucleic acid aptamer derivative that can be used for detecting glycolated hemoglobin: base A, T, C or G on the aptamer optional position of the present embodiment are replaced as to rare base and methylate after purine, dihydrouracil or xanthoglobulin, the nucleic acid aptamer derivative obtaining; The phosphorothioate backbone that the skeleton of the nucleotide sequence of the aptamer of the present embodiment derives; The peptide nucleic acid(PNA) that the aptamer of the present embodiment is transformed into; The lock nucleic acid that the aptamer of the present embodiment is transformed into.

embodiment 4the preparation of the aptamer GHb4 of glycolated hemoglobin specific combination

(1) build random sequence oligonucleotides storehouse: synthetic single stranded DNA sequence, building storage capacity is 7 * 10 ⁵single stranded DNA random sequence oligonucleotides storehouse, the DNA sequence dna that single stranded DNA random oligonucleotide storehouse comprises is:

5’-GACCATACCAGCTTATTCAATT-N _30~50-AGATAGTAAGTGCAATCTGGC-3’

Stochastic sequence N in the middle of the DNA sequence dna in described single stranded DNA random oligonucleotide storehouse comprises _{25 ~ 60}with two ends fixed sequence program, described middle stochastic sequence N _{30 ~ 50}be the stochastic sequence of 30 ~ 50 bases, described two ends fixed sequence program is: 5 '-GACCATACCAGCTTATTCAATT, and 3 '-CGGTCTAACGTGAATGATAGA, described two ends fixed sequence program is pcr amplification primer land;

(2) prepare glycolated hemoglobin-micro-magnetic bead mixture: by 0.75ml with the amino micro-magnetic bead of activation and 5mg glycolated hemoglobin at coupling buffer (20mM potassium phosphate salt damping fluid, 0.15M NaCl, 1mM DTT, pH 5.5) middle mixing, add 200ul coupling agent solution (the dimethylaminopropyl ethyl carboxamide containing 57%), light mixed 30 hours under 25oC reaction conditions, make glycolated hemoglobin C-terminal covalently bound to micro-magnetic bead, in tripping device, with scavenging solution washing, obtain glycolated hemoglobin-micro-magnetic bead mixture.

(3) screen for the first time target material-nucleic acid complexes: 4nmol single-stranded DNA banks is dissolved in to binding buffer liquid (100 mM NaCl, 20 mM Tris-HCl pH 7.6,2 mM MgCl2,5mM KCl, 1 mM CaCl2,0.02% Tween 20,1 mM DTT), carry out heat treated, 90 oC heating 10min, are placed in 10min on ice, and then temperature is placed 5min, single-stranded DNA banks, after hatching in conjunction with the magnetic bead of glycolated hemoglobin, is collected not the liquid in conjunction with the DNA random oligonucleotide storehouse of magnetic bead, by step 2) glycolated hemoglobin-micro-magnetic bead mixture collects and is not combined DNA random oligonucleotide storehouse 37oC incubation 15min in binding buffer liquid of magnetic bead after thermal treatment, with elution buffer (20 mM Tris-HCl pH7.6, 200 mM NaCl, 10 mM EDTA) washing glycolated hemoglobin-oligonucleotide-micro-magnetic bead mixture, remove that be not combined with glycolated hemoglobin and oligonucleotide sequence non-specific binding, glycolated hemoglobin-oligonucleotide-micro-magnetic bead mixture 92oC in elution buffer is hatched after 5min, recovery is with the elution buffer of single stranded oligonucleotide sequence.

(4) prepare secondary single strand dna oligonucleotide storehouse: will in step 3), reclaim and obtain carrying out pcr amplification with the elution buffer of single stranded oligonucleotide sequence, pcr amplification product be take the micro-magnetic bead of Streptavidin and is carried out separation as matrix, through alkaline denaturation, unwind, filter, purifying, obtain secondary single strand dna oligonucleotide storehouse, for next round, screen; Pcr amplification processing condition are: 94oC denaturation 5 min, 94oC 30s, 47oC 1min, 72oC 1min, 20 circulations of increasing, final extension is 72oC 10min, primer 1:5 '-ATACCAGCTTATTCAATT-3 ', primer 2: 5 '-Biotin-AGATTGCACTTACTATCT-3 '

, 5 ' end biotin labeling of primer 2.

(5) screen and identify glycolated hemoglobin aptamer: it is matrix separation screening that the micro-magnetic bead of Streptavidin is taken turns to through 25 in the secondary single strand dna oligonucleotide storehouse of step 4) gained, obtain the few nucleotide sequence of target, clone and the few nucleotide sequence of the described target that checks order, the few nucleotide sequence of target is SEQ ID NO:4, called after GHb4.

The above-mentioned aptamer of the present embodiment can derive the following nucleic acid aptamer derivative that can be used for detecting glycolated hemoglobin: base A, T, C or G on the aptamer optional position of the present embodiment are replaced as to rare base and methylate after purine, dihydrouracil or xanthoglobulin, the nucleic acid aptamer derivative obtaining; The phosphorothioate backbone that the skeleton of the nucleotide sequence of the aptamer of the present embodiment derives; The peptide nucleic acid(PNA) that the aptamer of the present embodiment is transformed into; The lock nucleic acid that the aptamer of the present embodiment is transformed into.

embodiment 5enzyme connection aptamer adsorption measurement detects the binding ability of aptamer GHb1, GHb2, GHb3, GHb4 and glycolated hemoglobin

1) external synthesizing held biotin labeled glycolated hemoglobin aptamer with 5 '.

2) on 96 hole elisa plates, add the coating buffer (Na2CO3 15mM, NaHCO3 35mM, NaCl 0.2M) with 0.1mg/ml glycolated hemoglobin and oxyphorase contrast, every hole 100 μ L, set PBS hole for zeroing hole, 4 ℃ of overnight incubation.

3) with phosphoric acid elutriant washing elisa plate, then confining liquid is coated with 96 hole elisa plates, every hole 100 μ L, and elutriant (PBS pH 7.4,0.05% Tween) washing is used in 37 ℃ of sealings after 1 hour.

4) biotin labeled aptamer is carried out to gradient dilution with phosphoric acid buffer, concentration is respectively 0 nml/L, 5 nml/L, 10 nml/L, 20 nml/L, 50 nml/L, 100nml/L, 200 nml/L, by the non-96 hole elisa plates that Jie Ru not be coated with glycolated hemoglobin and oxyphorase of the aptamer of 100 μ L dilutions, hatch 30min for 25 ℃.

5) with after elutriant wash-out elisa plate, the Streptavidin of horseradish peroxidase-labeled is added to 100 μ L with PBS by the rear every hole of 1:1000 dilution, hatch 30min for 37 ℃.

6) with elutriant, wash elisa plate, then every hole add horseradish peroxidase chromogenic substrate TMB (3,3', 5,5'-tetramethybenzidine) 80 μ L colour developing 2min, are to detect absorbancy under 450 nm wavelength in OD value after termination reaction.

7) use the mapping of Sigma plot software, calculate the interactional dissociation constant of screened aptamer and glycolated hemoglobin (kd).

The binding ability of aptamer GHb1, GHb2, GHb3, GHb4 and the glycolated hemoglobin detecting is as shown in Fig. 1 ~ 4.

Claims (8)

1. a glycolated hemoglobin aptamer, is characterized in that, the nucleotides sequence of described aptamer has been classified sequence dna fragment shown in SEQ ID NO:2 as, and its sequence is:

SEQ?ID?NO:2：gggaggcatg?tgatttaagg?cgcggcagat?gttggaaccc。

2. a method of preparing the arbitrary described glycolated hemoglobin aptamer of claim 1, is characterized in that, comprises the following steps:

(1) build random sequence oligonucleotides storehouse: synthetic single stranded DNA sequence, building storage capacity is 10 ⁵~ 10 ⁶single stranded DNA random sequence oligonucleotides storehouse, the DNA sequence dna that single stranded DNA random oligonucleotide storehouse comprises is:

5’-GACCATACCAGCTTATTCAATT-N _25~60-AGATAGTAAGTGCAATCTGGC-3’

Stochastic sequence N in the middle of the DNA sequence dna in described single stranded DNA random oligonucleotide storehouse comprises _{25 ~ 60}with two ends fixed sequence program, described middle stochastic sequence N _{25 ~ 60}be the stochastic sequence of 25 ~ 60 bases, described two ends fixed sequence program is: 5 '-GACCATACCAGCTTATTCAATT, and 3 '-CGGTCTAACGTGAATGATAGA, described two ends fixed sequence program is pcr amplification primer land;

(2) prepare glycolated hemoglobin-micro-magnetic bead mixture: will have the amino micro-magnetic bead of activation in coupling buffer, to mix according to the ratio of 0.05 ~ 0.2ml:1mg with glycolated hemoglobin, add again 100 ~ 200ul coupling agent solution, light mixed 20 ~ 30 hours under 25oC reaction conditions, make glycolated hemoglobin C-terminal covalently bound to micro-magnetic bead, in tripping device, with scavenging solution washing, obtain glycolated hemoglobin-micro-magnetic bead mixture;

(3) screen for the first time target material-nucleic acid complexes: 2 ~ 5nmol single stranded DNA random sequence oligonucleotides storehouse is dissolved in to binding buffer liquid, carry out heat treated, single-stranded DNA banks with in conjunction with the magnetic bead of glycolated hemoglobin, hatch after, collect not the liquid in conjunction with the DNA random oligonucleotide storehouse of magnetic bead; By step 2) glycolated hemoglobin-micro-magnetic bead mixture collects and is not combined DNA random oligonucleotide storehouse 37oC incubation 15 ~ 40min in binding buffer liquid of magnetic bead after thermal treatment, with elution buffer, wash glycolated hemoglobin-oligonucleotide-micro-magnetic bead mixture, remove that be not combined with glycolated hemoglobin and oligonucleotide sequence non-specific binding, glycolated hemoglobin-oligonucleotide-micro-magnetic bead mixture 92oC in elution buffer is hatched after 5min, reclaims the elution buffer with single stranded oligonucleotide sequence;

(4) prepare secondary single strand dna oligonucleotide storehouse: will in step 3), reclaim and obtain carrying out pcr amplification with the elution buffer of single stranded oligonucleotide sequence, pcr amplification product be take the micro-magnetic bead of Streptavidin and is carried out separation as matrix, through alkaline denaturation, unwind, filter, purifying, obtain secondary single strand dna oligonucleotide storehouse, for next round, screen;

(5) screen and identify glycolated hemoglobin aptamer: it is matrix separation screening that the micro-magnetic bead of Streptavidin is taken turns to through 12 ~ 20 in the secondary single strand dna oligonucleotide storehouse of step 4) gained, obtain the few nucleotide sequence of target, clone and the few nucleotide sequence of the described target that checks order, by enzyme, join specificity and the avidity that aptamer adsorption measurement identifies that it is combined with glycolated hemoglobin.

3. a kind of method of preparing glycolated hemoglobin aptamer according to claim 2, is characterized in that: the PCR primer sequence that described pcr amplification is used is:

Primer 1:5 '-ATACCAGCTTATTCAATT-3 '

Primer 2: 5 '-Biotin-AGATTGCACTTACTATCT-3 '

5 ' end of described pcr amplification primer 2 is containing biotin labeling;

Described pcr amplification processing condition are: 94oC denaturation 5 min, and 94oC 30s, 47oC 1min, 72oC 1min, 10 ~ 25 circulations of increasing, final extension is 72oC 10min.

4. a kind of method of preparing glycolated hemoglobin aptamer according to claim 2, described coupling buffer is for containing 20mM potassium phosphate salt damping fluid, 0.15M NaCl, 1mM DTT, pH 5.5; Described coupling agent solution is the dimethylaminopropyl ethyl carboxamide containing 57%; Described binding buffer liquid is for containing 100 mM NaCl, 20 mM Tris-HCl pH 7.6,2 mM MgCl ₂, 5mM KCl, 1 mM CaCl ₂, 0.02% Tween 20,1 mM DTT; Described thermal treatment is 90 oC heating 10min, is placed in 10min on ice, and then temperature is placed 5min; Described elution buffer is for containing 20 mM Tris-HCl pH7.6,200 mM NaCl, 10 mM EDTA.

5. the glycolated hemoglobin aptamer preparing according to method described in arbitrary described glycolated hemoglobin aptamer or claim 2 ~ 4 in claim 1, it is characterized in that, described aptamer is used for identification, detects glycolated hemoglobin, or for the preparation of the test kit that detects glycolated hemoglobin.

6. a derivative for arbitrary described glycolated hemoglobin aptamer in claim 1, described derivative comprises any one in following four kinds:

(1) base A, T, C or G on the optional position of aptamer described in claim 1 are replaced as to rare base and methylate after purine, dihydrouracil or xanthoglobulin, the nucleic acid aptamer derivative obtaining;

(2) phosphorothioate backbone that the skeleton of the nucleotide sequence of aptamer described in claim 1 derives;

(3) peptide nucleic acid(PNA) that described in claim 1, aptamer is transformed into;

(4) the lock nucleic acid that described in claim 1, aptamer is transformed into.

7. a derivative for the glycolated hemoglobin aptamer preparing described in arbitrary in claim 2 ~ 4, described derivative comprises any one in following four kinds:

(1) base A, T, C or G on the optional position of aptamer described in claim 1 are replaced as to rare base and methylate after purine, dihydrouracil or xanthoglobulin, the nucleic acid aptamer derivative obtaining;

(2) phosphorothioate backbone that the skeleton of the nucleotide sequence of aptamer described in claim 1 derives;

(3) peptide nucleic acid(PNA) that described in claim 1, aptamer is transformed into;

(4) the lock nucleic acid that described in claim 1, aptamer is transformed into.

8. according to a kind of glycolated hemoglobin nucleic acid aptamer derivative described in claim 6 or 7, it is characterized in that, described aptamer or its derivative are used for identification, detect glycolated hemoglobin, or for the preparation of the test kit that detects glycolated hemoglobin.