CN103966222A - Decreasing DNA library concentration based aptamer screening method and aptamer - Google Patents
Decreasing DNA library concentration based aptamer screening method and aptamer Download PDFInfo
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Abstract
The invention relates to a decreasing DNA library concentration based aptamer screening method and an aptamer. The method includes: a protein-magnetic bead crosslinking step, i.e. subjecting control protein and target protein to crosslinking with magnetic beads respectively; a first-round screening step, i.e. adding a target protein-magnetic bead composite into an initial DNA library solution to conduct first-round screening; a repeated screening step, i.e. diluting the screening product of the previous round by 2-10 times to serve as the DNA library solution of the next round screening so as to carry out the second to the N-th round screening; and a DNA synthesis step, i.e. conducting cloning and sequencing on the final screening product, and synthesizing a numerically superior DNA sequence with high affinity, thus obtaining the aptamer. The method provided by the invention takes low concentration as a screening condition to screen out high affinity aptamer that can bind with the target protein under an extremely low concentration, thus greatly reducing the influence of PCR conditions on the screening process, significantly shortening the screening time, reducing the screening round number, and improving the success rate of screening.
Description
Technical field
The invention belongs to biology field, more particularly, relate to a kind of aptamer screening method and aptamer successively decreasing based on DNA library concentration.
Background technology
Aptamer refer to can with the oligonucleotide chain molecule of the strand of target molecule specific binding (DNA or RNA), be generally less than 100mer.Compared with antibody, between aptamer and target molecule, molecular recognition function is very similar to antibody, but the target molecule of effect is wider, comprise that toxin immunity originality is weak and do not there is immunogenic material and can identify the undistinguishable similar substance of monoclonal antibody, have higher specificity than antibody, avidity is higher.Because of the advantage of its structure and performance, aptamer is applied to biomedical fundamental research, diagnosis of disease and medicament research and development increasingly extensively.
Conventionally, aptamer is by exponential enrichment Fas lignand system evolution technology (Systematicevolution of ligands by exponential enrichment, SELEX), and screening obtains.The Basic Ways of exponential enrichment Fas lignand system evolution technology is that the DNA library solution and the target molecule that comprise jumbo random oligonucleotide sequence are hatched jointly, and separate with binding sequence not in connection with mixture by various separating pathways, wash-out, obtain the sequence of being combined with target molecule and these sequences are carried out to polymerase chain reaction (PCR) amplification, after being separated into strand, generate secondary library, be used further to lower whorl screening, after so number is taken turns repeatedly, pick out the aptamers of enrichment, and check order and obtain the oligonucleotide sequence of specific recognition target molecule, it is aptamer.Jumbo molecular library has almost been contained all possible three-dimensional conformation, can, for any target molecule screening, obtain the aptamer sequence of its specific recognition in theory.
At present not yet set up the standard screening flow process for any target, also can not ensure that all screenings must be effective.Technology screening process complexity, be filtered into power and be subject to the restriction of numerous influence factors.Especially PCR condition has obvious impact to SELEX screening process, and the PCR condition of a difference even causes falling flat of screening.
In order to obtain the aptamer of high-affinity combination, in the process of screening, the time of normally hatching by gradually reducing DNA library and target material, increase washing times and washing time and strengthen screening pressure with the amount and the incubation time that increase control substance of plant drug.But when DNA aptamers and target material are hatched, the concentration of DNA is to affect one of combination principal element between the two.But current SELEX technology, all adopts pcr amplification every after taking turns screening, hatches with DNA library and the target material of higher concentration, fail to utilize regulation and control concentration as the Main Means that improves screening pressure.
Summary of the invention
The technical problem to be solved in the present invention is, all adopt pcr amplification to cause technology screening process complexity, be filtered into the defect that power is low for existing aptamer screening method is every after taking turns screening, a kind of aptamer screening method and aptamer successively decreasing based on DNA library concentration is provided.
The technical solution adopted for the present invention to solve the technical problems is: construct a kind of aptamer screening method successively decreasing based on DNA library concentration, comprise the following steps:
Albumen magnetic bead cross-linking step, by crosslinked with magnetic bead respectively to reference protein and target protein, obtains reference protein magnetic bead mixture and target protein magnetic bead mixture;
First round screening step, in the solution of initial DNA library, add described target protein magnetic bead mixture just screening and wash-out after, carry out pcr amplification, be separated into strand, obtain first round screening product;
Repeat to screen step, previous round is screened to 2 ~ 10 times of DNA library solution as next round screening of product dilution, first add reference protein magnetic bead mixture to bear sieve, then add target protein magnetic bead mixture just to sieve, and after wash-out, obtain screening product and supernatant; Every DNA taking turns in screening product and supernatant is carried out to pcr amplification, and whether more every DNA amount of taking turns in screening product and supernatant meets preset requirement, finally screened product to carry out synthetic DNA step, carried out next round screening otherwise execution repeats to screen step;
Synthetic DNA step, carries out Cloning and sequencing to described final screening product, synthetic quantitatively dominant DNA sequence dna, and can and there is the DNA sequence dna of high-affinity as the aptamer of this albumen with described target protein specific binding after testing.
According in the aptamer screening method successively decreasing based on DNA library concentration of the present invention, described target protein is endothelium glycoprotein, and described reference protein is bovine serum albumin.
According in the aptamer screening method successively decreasing based on DNA library concentration of the present invention, described first round screening step specifically comprises:
Just sieving step: get initial DNA library solution cooling rapidly in 95 DEG C of heating juxtapositions on ice, add target protein magnetic bead mixture, be placed in 37 DEG C of shaking tables and hatch 1.5 ~ 2.5h, remove supernatant, with damping fluid washing three times;
Library elution step: heat 15 ~ 20 minutes at 95 degrees Celsius after adding sterilized water, cooled on ice, puts magnetic frame 3～5 minutes, gets supernatant;
Pcr amplification step: taking this supernatant as template DNA library solution, and by after 16 ~ 20 circulations of this template DNA library solution amplification, separate strand desalting treatment, obtain first round screening product.
According in the aptamer screening method successively decreasing based on DNA library concentration of the present invention, describedly repeat to screen step and specifically comprise:
The pre-treatment step in library: previous round is screened to product and get the DNA library solution as next round screening after 2 ~ 10 times of dilutions, cooling rapidly on ice 95 DEG C of heating juxtapositions;
Anti-sieve step: get reference protein magnetic bead mixture 3 ~ 10ug and add in the DNA library solution that pre-treatment obtains, 37 DEG C of shaking tables are hatched 30 minutes ~ 1.5 hours, puts and on magnetic frame, collects supernatant;
Just sieving step: by the supernatant dilution that anti-sieve obtains, add target protein magnetic bead mixture 3 ~ 10ug, 37 DEG C of shaking tables are hatched 30 minutes ~ 1.5 hours, remove supernatant, use buffer solution for cleaning three times, obtain DNA target protein magnetic bead mixture;
Library elution step: add sterilized water in DNA target protein magnetic bead mixture, 95 degrees Celsius are heated 15 ~ 20 minutes, and cooled on ice, puts magnetic frame 3～5 minutes, collects supernatant; Repeat with sterilized water washing, collect each supernatant and be mixed to get this and take turns screening product.
The present invention is also corresponding provides a kind of aptamer, adopts the aptamer screening method successively decreasing based on DNA library concentration as above to prepare.
Implement aptamer screening method and the aptamer successively decreasing based on DNA library concentration of the present invention, there is following beneficial effect: the present invention utilize lower concentration as screening pressure to filter out the aptamer of the high-affinity that can be combined with target protein under extremely low concentration, greatly reduce the impact of PCR condition on screening process, simplify whole screening process, significantly shorten the time of screening, reduce screening wheel number, improved the success ratio of screening.
Brief description of the drawings
Below in conjunction with drawings and Examples, the invention will be further described, in accompanying drawing:
Fig. 1 is the schema of the aptamer screening method successively decreasing based on DNA library concentration according to a preferred embodiment of the invention;
Fig. 2 is the process schematic diagram of the aptamer screening method successively decreasing based on DNA library concentration according to a preferred embodiment of the invention;
Fig. 3 A-3G is that in the aptamer screening method successively decreasing based on DNA library concentration according to a preferred embodiment of the invention, each wheel screens the electrophorogram of DNA after PCR in product and supernatant;
Fig. 4 A-4B is laser co-focusing experiment light field and the details in a play not acted out on stage, but told through dialogues figure of the aptamer screening method empty control group successively decreasing based on DNA library concentration according to a preferred embodiment of the invention;
Fig. 5 A-5F is laser co-focusing experiment light field and the details in a play not acted out on stage, but told through dialogues figure of bovine serum albumin control group in the aptamer screening method successively decreasing based on DNA library concentration according to a preferred embodiment of the invention;
Fig. 6 A-6F is laser co-focusing experiment light field and the details in a play not acted out on stage, but told through dialogues figure of endothelium glycoprotein experimental group in the aptamer screening method successively decreasing based on DNA library concentration according to a preferred embodiment of the invention;
Fig. 7 A-7G is dominant DNA sequence dna in library and the simulation secondary structure figure thereof selecting in the aptamer screening method successively decreasing based on DNA library concentration according to a preferred embodiment of the invention;
Fig. 8 A-8B is in the aptamer screening method successively decreasing based on DNA library concentration according to a preferred embodiment of the invention, the flow cytometer detection result figure of E-A1 and protein binding situation.
Embodiment
In order to make object of the present invention, technical scheme and advantage clearer, below in conjunction with drawings and Examples, the present invention is further elaborated.
The present invention is mainly by jointly hatching the DNA library solution and the target protein that comprise jumbo random oligonucleotide sequence, and the sequence of being combined with target protein with not binding sequence separation, wash-out, acquisition in connection with mixture by various separating pathways these sequences are carried out to polymerase chain reaction (PCR) amplification, after being separated into strand, generate secondary library, be used further to lower whorl screening.Take turns since second, previous round library is diluted, made its concentration successively decrease and screen by wheel, and no longer carry out pcr amplification, after number is taken turns repeatedly, screening product is cloned, checked order, synthesize the DNA sequence dna that quantity is dominant in library, after surveying the investigation of Kd value, obtain the oligonucleotide sequence of specific recognition target molecule, i.e. aptamer.
Referring to Fig. 1, is the schema of the aptamer screening method successively decreasing based on DNA library concentration according to a preferred embodiment of the invention.As shown in Figure 1, the aptamer screening method successively decreasing based on DNA library concentration that this embodiment provides mainly comprises the following steps:
First, in step S101, carry out albumen magnetic bead cross-linking step, by crosslinked with magnetic bead respectively to reference protein and target protein, obtain reference protein magnetic bead mixture and target protein magnetic bead mixture.
Subsequently, in step S102, carry out first round screening step, in the solution of initial DNA library, add described target protein magnetic bead mixture just screening and wash-out after, carry out pcr amplification, be separated into strand, obtain first round screening product.
Subsequently, in step S103, execution repeats to screen step, previous round is screened to 2 ~ 10 times of DNA library solution as next round screening of product dilution, first add reference protein magnetic bead mixture to bear sieve, add again target protein magnetic bead mixture just to sieve, and after wash-out, obtain screening product and supernatant; Every DNA taking turns in screening product and supernatant is carried out to pcr amplification, and whether more every DNA amount of taking turns in screening product and supernatant meets preset requirement, finally screened product to carry out synthetic DNA step, carried out next round screening otherwise execution repeats to screen step.
Finally, in step S104, carry out synthetic DNA step, described final screening product is carried out to Cloning and sequencing, synthetic quantitatively dominant DNA sequence dna, and can and there is the DNA sequence dna of high-affinity as the aptamer of this albumen with described target protein specific binding after testing.
The present invention also corresponding providing adopts the worth aptamer of the method.
The present invention utilize regulation and control concentration this with the closely-related factor of DNA avidity, as raising screening pressure means, filtered out the target protein aptamer with high-affinity; The present invention need to use pcr amplification except first round screening, all the other pcr amplifications of taking turns screening are all for screening the preparation before checking and the cloning and sequencing of product, and not as the amplification in next round screening library, therefore significantly reduced the impact of PCR condition on screening process, simplify whole screening process, greatly shorten the time of screening, improved the success ratio of screening.
Referring to Fig. 2, is the process schematic diagram of the aptamer screening method successively decreasing based on DNA library concentration according to a preferred embodiment of the invention.In the present embodiment, getting target protein is endothelium glycoprotein (Endoglin), and reference protein is bovine serum albumin (BSA).Embodiment is as follows:
(1) library and primer pre-treatment step
Use sterilized water that upstream and downstream primer is configured to 50uM concentration, DNA library is dissolved as the solution of 50uM with PBS, be all stored in 20 DEG C.And configuring the PCR solution system that library final concentration is 5nM, set temperature grads PCR is optimized the amplification temperature in this DNA library, selects specific amplification the most obviously and the pcr amplification optimum temperuture as subsequent step without the assorted temperature of being with.
(2) albumen magnetic bead cross-linking step
This step is as shown in step S201 in Fig. 2, specific as follows:
1. get the magnetic bead of 150 ~ 300 μ l, with the MES (MES) of 25mM, its pH value is 5.0 washed twice and abandons supernatant.
2. the EDC(ethylene dichloride that is 50mg/ml with MES configuration final concentration) and NHS(N-N-Hydroxysuccinimide).
3. get each 75 ~ 150 μ l of EDC and NHS to the magnetic bead cleaning, fully mix in room temperature shaking table and hatch 30 ~ 45 minutes, put magnetic frame upper 4 minute, remove supernatant.Clean twice with the MES of 150 ~ 300 μ l.Magnetic bead activates.
4. get respectively bovine serum albumin and endothelium glycoprotein 25ug adds in the magnetic bead of activation, room temperature underlying shaking table is hatched 30 ~ 45 minutes, abandons supernatant.
5. use the Tris(Tutofusin tris of 50mM), its pH value is 7.4, the magnetic bead of cleaning coating protein totally 4 times.Finally use PBS (containing 0.02%NaN
3) magnetic bead to the 0.1 ~ 0.5ug/ul of the resuspended coating protein of 125ul ~ 250ul.
(3) first round screening step
This step is as shown in step S202-S204 in Fig. 2, specific as follows:
1. just sieving step: as shown in step S202 in Fig. 2, get appropriate initial DNA library solution cooling rapidly in 95 DEG C of heating juxtapositions on ice, add with in the washed endothelium glycoprotein-magnetic bead of PBS mixture.37 DEG C of shaking tables are hatched 1.5 ~ 2.5h, remove supernatant, wash three times obtain DNA-endothelium glycoprotein-magnetic bead mixture with cleaning buffer solution.
2. library elution step: as shown in step S203 in Fig. 2, add 500 microlitre sterilized waters just sieving in DNA-endothelium glycoprotein-magnetic bead mixture that step obtains.95 degrees Celsius are heated 15 ~ 20 minutes, and cooled on ice, puts magnetic frame 3～5 minutes, collect supernatant.
3. pcr amplification step: as shown in step S204 in Fig. 2, taking gained supernatant in the elution step of library as template DNA library solution, and by after 16 ~ 20 circulations of this template DNA library solution amplification, then separate strand desalting treatment, obtain first round screening product.
4. measuring process: this step can further be surveyed A value, the amount of substance of calculating first round screening product.
(4) repeat to screen step and second take turns the screening of screening ~ the N wheel
This step is as shown in step S205-S206 in Fig. 2, specific as follows:
1. the pre-treatment step in library: previous round is screened to product and get 1/2 as the follow-up detection of mentioning, the other 1/2 DNA library solution as next round screening, with after 2 ~ 10 times of binding buffer liquid (Binding Buffer) dilutions, 95 DEG C of heating juxtapositions are cooling rapidly, stand-by on ice.
2. the anti-step of sieving: get bovine serum albumin-magnetic bead mixture 3 ~ 10ug, by 250 ~ 500uL binding buffer liquid washed twice, add above-mentioned library, 37 DEG C of shaking tables are hatched 30 minutes ~ 1.5 hours (this time extends along with the increase of screening wheel number).Put and on magnetic frame, collect supernatant.
3. just sieving step: by counter sieve that step obtains on reset and add binding buffer liquid and be diluted to 1mL, add with in the washed endothelium glycoprotein-magnetic bead of PBS mixture 3 ~ 10ug.37 DEG C of shaking tables are hatched 30 minutes ~ 1.5 hours (this time reduces along with the increase of screening wheel number), pipette supernatant, are labeled as " N wheel is just sieving supernatant ", with cleaning buffer solution washing three times, obtain DNA-endothelium glycoprotein-magnetic bead mixture.
4. library elution step: as shown in step S206 in Fig. 2, add 200 microlitre sterilized waters in DNA-endothelium glycoprotein-magnetic bead mixture.95 degrees Celsius are heated 15 ~ 20 minutes, and cooled on ice, puts magnetic frame 3～5 minutes, collect supernatant.Repeat to wash magnetic bead with sterilized water, collect each supernatant and mix, collect altogether epicycle screening product, i.e. the DNA library of 500ul ~ 1ml.
(5) detect library and protein binding situation
The aforementioned screening wheel number that repeats to screen step is determined with the result of electrophoresis or laser co-focusing.Generally, do 3 ~ 4 and take turns screening.
1. pcr amplification and electrophoresis.Using each product of taking turns screening and supernatant as template, make pcr amplification with same loop number, contrast the DNA amount that each takes turns screening product and supernatant, judge this takes turns in screening whether to occupy advantage with the amount of the DNA of endothelium Glycoprotein binding, be to have met preset requirement, otherwise continue next round screening.
2. laser co-focusing.Each product of taking turns screening is carried out to pcr amplification (and using fluorescent dye primer), be separated into strand, hatch with target protein and reference protein, adopt laser co-focusing evaluation in conjunction with effect, judge whether to carry out next round screening according to preset requirement.
(6) clone, order-checking, synthetic DNA step.
This step is as shown in step S207-S208 in Fig. 2, specific as follows:
First final screening product is carried out to the clone as shown in step S207, then enter the order-checking as shown in step S208, analyze sequencing result, be chosen in dominant DNA sequence dna in library and synthesize, obtain required aptamer.
(7) avidity of investigation aptamer and target protein.
By synthetic with fluorescently-labeled DNA and will be with the each 100nM of fluorescently-labeled irrelevant sequence and endothelium glycoprotein to hatch in aforementioned (5), utilize flow cytometer to survey fluorescence intensity change, and extrapolate kd value, determine the specific recognition capability of this sequence for target, can and there is the DNA sequence dna of high-affinity as the aptamer of this albumen with target protein specific binding.The high-affinity that aptamer has target molecule.This area basic technology personnel know compared with antibody, aptamer and micromolecular equilibrium dissociation constant micromole between nmole; And the equilibrium dissociation constant between biomacromolecule is generally between Na Mo and picomole.Therefore, the target molecule endothelium glycoprotein adopting for the present invention is small molecules, the equilibrium dissociation constant that therefore high-affinity refers to DNA sequence dna and this endothelium glycoprotein micromole between nmole.
Refer to Fig. 3 A-3G, be respectively each wheel in the aptamer screening method successively decreasing based on DNA library concentration according to a preferred embodiment of the invention and screen the electrophorogram of DNA after PCR in product and supernatant.Wherein, Fig. 3 A-3C is respectively second and takes turns and just sieve product, and second takes turns and just sieve the cleer and peaceful second electrophorogram of taking turns anti-sieve product.Fig. 3 D-3E is respectively third round and is just sieving product and third round and just sieving the electrophorogram of supernatant.Fig. 3 F-3G is respectively fourth round and is just sieving product and fourth round and just sieving the electrophorogram of supernatant.The cycle number of the digitized representation PCR that in each figure, below indicates.As can be seen, second takes turns after screening, and the amount calibration sieve product that is just sieving DNA in supernatant is many, instead sieves product and just sieves product amount similar (seeing Fig. 3 A, 3B and 3C).Along with the increase of screening wheel number, the DNA just sieving in supernatant reduces gradually, to fourth round, is just sieving the DNA many (seeing Fig. 3 F-3G) in product calibration sieve supernatant, and the concentration of DNA library solution while hatching with endothelium glycoprotein in taking turns due to this is much smaller than 1 ~ 30nM.Therefore advise second taking turns, the product P CR amplification of third round and fourth round, characterize to analyze with laser co-focusing.
Refer to laser co-focusing experiment light field and details in a play not acted out on stage, but told through dialogues figure that Fig. 4 A-4B is respectively the aptamer screening method empty control group successively decreasing based on DNA library concentration according to a preferred embodiment of the invention.This blank group is with fluorescently-labeled endothelium glycoprotein-M270 magnetic bead mixture.
Fig. 5 A-5F is respectively laser co-focusing experiment light field and the details in a play not acted out on stage, but told through dialogues figure of bovine serum albumin control group in the aptamer screening method successively decreasing based on DNA library concentration according to a preferred embodiment of the invention.This bovine serum albumin control group is that each wheel screened the result of doing laser co-focusing after hatching with bovine serum albumin-M270 magnetic bead mixture respectively after fluorescent mark for product.Wherein, Fig. 5 A and 5B are respectively second and take turns the light field and the details in a play not acted out on stage, but told through dialogues figure that after screening product and bovine serum albumin-M270 magnetic bead mixture are hatched, do laser co-focusing; Fig. 5 C and 5D are respectively the light field and the details in a play not acted out on stage, but told through dialogues figure that after third round screening product and bovine serum albumin-M270 magnetic bead mixture are hatched, do laser co-focusing; Fig. 5 E and 5F are respectively the light field and the details in a play not acted out on stage, but told through dialogues figure that after fourth round screening product and bovine serum albumin-M270 magnetic bead mixture are hatched, do laser co-focusing.
Fig. 6 A-6F is respectively laser co-focusing experiment light field and the details in a play not acted out on stage, but told through dialogues figure of endothelium glycoprotein experimental group in the aptamer screening method successively decreasing based on DNA library concentration according to a preferred embodiment of the invention.This endothelium glycoprotein experimental group is that each wheel screened the result of doing laser co-focusing after hatching with endothelium glycoprotein-M270 magnetic bead mixture respectively after fluorescent mark for product.Wherein, Fig. 6 A and 6B are respectively second and take turns the light field and the details in a play not acted out on stage, but told through dialogues figure that after screening product and endothelium glycoprotein-M270 magnetic bead mixture are hatched, do laser co-focusing; Fig. 6 C and 6D are respectively the light field and the details in a play not acted out on stage, but told through dialogues figure that after third round screening product and endothelium glycoprotein-M270 magnetic bead mixture are hatched, do laser co-focusing; Fig. 6 E and 6F are respectively the light field and the details in a play not acted out on stage, but told through dialogues figure that after fourth round screening product and endothelium glycoprotein-M270 magnetic bead mixture are hatched, do laser co-focusing.
The condition of aforementioned all laser co-focusing experiments is identical.From figure, show, endothelium glycoprotein experimental group is taken turns visible fluorescence at third and fourth.And bovine serum albumin control group has no fluorescence, show that this two-wheeled screening product and endothelium Glycoprotein binding are obviously strong compared with reference protein, therefore can advise carrying out cloning and sequencing after third round screening or fourth round screening.
Refer to Fig. 7 A-7G, in the aptamer screening method successively decreasing based on DNA library concentration according to a preferred embodiment of the invention, from clone, sequencing result, select dominant DNA sequence dna and simulation secondary structure figure thereof in library.Cloning and sequencing result shows that second takes turns the order-checking of screening product, wherein has 3% for same sequence.In third round screening product, this sequence reaches 25%.In fourth round screening product, this sequence that checks order reaches 68%, claims that this sequence is E-A1.Wherein, the cloning and sequencing result figure that Fig. 7 A is E-A1; Fig. 7 B-7G is the multiple analog secondary structure figure of E-A1.
Fig. 8 A-8B is in the aptamer screening method successively decreasing based on DNA library concentration according to a preferred embodiment of the invention, the flow cytometer detection result figure of E-A1 and protein binding situation.The fluorescently-labeled E-A1 of anamorphic zone tests fluorescence intensity with flow cytometer after library and endothelium glycoprotein are hatched.Wherein Fig. 8 A is that flow cytometer detects, the irrelevant sequence of contrast and E-A1 respectively with the rear fluorescence intensity change figure of hatching of endothelium glycoprotein.Curve K1 is blank, and curve K2 is irrelevant sequence, and curve K3 is E-A1.Fig. 8 B is for surveying its fluorescence intensity, the matching of calculated equilibrium constant and row E-A1 and endothelium glycoprotein equilibrium dissociation constant by streaming; The kd that calculates E-A1 is about 6.9nM ± 0.56, experimental results show that the present invention screens the E-A1 and the target protein that obtain and has high affinity, may serve as the molecular probe that endothelium glycoprotein detects.
The present invention is described according to specific embodiment, but it will be understood by those skilled in the art that in the time not departing from the scope of the invention, can carry out various variations and be equal to replacement.In addition,, for adapting to specific occasion or the material of the technology of the present invention, can carry out many amendments and not depart from its protection domain the present invention.Therefore, the present invention is not limited to specific embodiment disclosed herein, and comprises all embodiment that drop into claim protection domain.
Claims (5)
1. the aptamer screening method successively decreasing based on DNA library concentration, is characterized in that, comprises the following steps:
Albumen magnetic bead cross-linking step, by crosslinked with magnetic bead respectively to reference protein and target protein, obtains reference protein magnetic bead mixture and target protein magnetic bead mixture;
First round screening step, in the solution of initial DNA library, add described target protein magnetic bead mixture just screening and wash-out after, carry out pcr amplification, be separated into strand, obtain first round screening product;
Repeat to screen step, previous round is screened to 2 ~ 10 times of DNA library solution as next round screening of product dilution, first add reference protein magnetic bead mixture to bear sieve, then add target protein magnetic bead mixture just to sieve, and after wash-out, obtain screening product and supernatant; Every DNA taking turns in screening product and supernatant is carried out to pcr amplification, and whether more every DNA amount of taking turns in screening product and supernatant meets preset requirement, finally screened product to carry out synthetic DNA step, carried out next round screening otherwise execution repeats to screen step;
Synthetic DNA step, carries out Cloning and sequencing to described final screening product, synthetic quantitatively dominant DNA sequence dna, and can and there is the DNA sequence dna of high-affinity as the aptamer of this albumen with described target protein specific binding after testing.
2. the aptamer screening method successively decreasing based on DNA library concentration according to claim 1, is characterized in that, described target protein is endothelium glycoprotein, and described reference protein is bovine serum albumin.
3. the aptamer screening method successively decreasing based on DNA library concentration according to claim 1, is characterized in that, described first round screening step specifically comprises:
Just sieving step: get initial DNA library solution cooling rapidly in 95 DEG C of heating juxtapositions on ice, add target protein magnetic bead mixture, be placed in 37 DEG C of shaking tables and hatch 1.5 ~ 2.5h, remove supernatant, with damping fluid washing three times;
Library elution step: heat 15 ~ 20 minutes at 95 DEG C after adding sterilized water, cooled on ice, puts magnetic frame 3～5 minutes, gets supernatant;
Pcr amplification step: taking this supernatant as template DNA library solution, and by after 16 ~ 20 circulations of this template DNA library solution amplification, separate strand desalting treatment, obtain first round screening product.
4. the aptamer screening method successively decreasing based on DNA library concentration according to claim 3, is characterized in that, describedly repeats to screen step and specifically comprises:
The pre-treatment step in library: previous round is screened to product and get the DNA library solution as next round screening after 2 ~ 10 times of dilutions, cooling rapidly on ice 95 DEG C of heating juxtapositions;
Anti-sieve step: get reference protein magnetic bead mixture 3 ~ 10ug and add in the DNA library solution that pre-treatment obtains, 37 DEG C of shaking tables are hatched 30 minutes ~ 1.5 hours, puts and on magnetic frame, collects supernatant;
Just sieving step: by the supernatant dilution that anti-sieve obtains, add target protein magnetic bead mixture 3 ~ 10ug, 37 DEG C of shaking tables are hatched 30 minutes ~ 1.5 hours, remove supernatant, use buffer solution for cleaning three times, obtain DNA target protein magnetic bead mixture;
Library elution step: add sterilized water in DNA target protein magnetic bead mixture, 95 degrees Celsius are heated 15 ~ 20 minutes, and cooled on ice, puts magnetic frame 3～5 minutes, collects supernatant; Repeat with sterilized water washing, collect each supernatant and be mixed to get this and take turns screening product.
5. an aptamer, is characterized in that, adopts and prepares according to the aptamer screening method successively decreasing based on DNA library concentration described in any one in claim 1-4.
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| CN109182455A (en) * | 2018-08-08 | 2019-01-11 | 淮海工学院 | A kind of screening technique of protein aptamer |
| CN109576272A (en) * | 2018-07-02 | 2019-04-05 | 广西医科大学 | A kind of DKK-1 aptamer and its application |
| CN110577979A (en) * | 2018-06-08 | 2019-12-17 | 首都师范大学 | rapid screening method of aptamer based on crosslinking reaction and structure switch type aptamer obtained through screening |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN104789696A (en) * | 2015-03-20 | 2015-07-22 | 中国科学院南海海洋研究所 | DNA (Deoxyribose Nucleic Acid) aptamer for detecting grouper iridovirus infection, as well as screening method and application of DNA aptamer |
| CN104789570A (en) * | 2015-03-20 | 2015-07-22 | 中国科学院南海海洋研究所 | DNA (Deoxyribose Nucleic Acid) aptamer for detecting grouper iridovirus infection, as well as screening method and application of DNA aptamer |
| CN104789696B (en) * | 2015-03-20 | 2017-07-11 | 中国科学院南海海洋研究所 | A kind of DNA aptamers and its screening technique and application for being used to detect grouper irido virus infection |
| CN104789570B (en) * | 2015-03-20 | 2018-07-06 | 中国科学院南海海洋研究所 | A kind of DNA aptamers for being used to detect grouper irido virus infection and its screening technique and application |
| CN105106973A (en) * | 2015-09-21 | 2015-12-02 | 广西医科大学 | Tumor vessel specificity chemotaxis CTL (cytotoxic T lymphocyte) nano system as well as preparation and application thereof |
| CN105106973B (en) * | 2015-09-21 | 2018-08-07 | 广西医科大学 | Tumor vessel specificity chemotactic CTL nanosystems and its preparation and application |
| CN107129988A (en) * | 2016-02-29 | 2017-09-05 | 广西医科大学 | The aptamer of specific binding CD3 a kind of and its screening technique and application |
| CN106872680A (en) * | 2017-01-06 | 2017-06-20 | 秦皇岛拜恩发生物技术有限公司 | Serum composition target aptamer screening technique |
| CN110577979A (en) * | 2018-06-08 | 2019-12-17 | 首都师范大学 | rapid screening method of aptamer based on crosslinking reaction and structure switch type aptamer obtained through screening |
| CN110577979B (en) * | 2018-06-08 | 2022-09-27 | 首都师范大学 | Rapid screening method of aptamer based on crosslinking reaction and structure switch type aptamer obtained through screening |
| CN109576272A (en) * | 2018-07-02 | 2019-04-05 | 广西医科大学 | A kind of DKK-1 aptamer and its application |
| CN109182455A (en) * | 2018-08-08 | 2019-01-11 | 淮海工学院 | A kind of screening technique of protein aptamer |
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