CN106282193B - Human pancreatic polypeptide aptamer and its screening technique and application - Google Patents
Human pancreatic polypeptide aptamer and its screening technique and application Download PDFInfo
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Abstract
The invention discloses a kind of human pancreatic polypeptide aptamer and its screening technique and application, human pancreatic polypeptide aptamer is with any one in Q10,5 '-Q11-FAM-3 ', 5 '-Q12-FAM-3 ', 5 '-BHQ1-Q13-3 ', 5 '-Q14-BHQ1-3 ', 5 '-SH-Q16-3 ', 5 '-SH-Q17-3 '.Its screening technique include synthesis random library and primer, GO-SELEX screening, PCR amplification library, the single-stranded library preparation DNA, repeat screening, it is negative screen, multi-turns screen.Human pancreatic polypeptide aptamer of the invention can be applied to the kit of preparation detection human pancreatic polypeptide, have affinity more higher than protein antibodies and specificity, non-immunogenicity, can chemical synthesis, molecular weight it is small, it is stables, be easy to the advantages such as preservation and label.
Description
Technical field
The present invention relates to field of biotechnology, it is related to a kind of aptamer and its screening and application, more particularly to a kind of
It can be used for detecting aptamer and its screening technique and the application of human pancreatic polypeptide.
Background technique
Human pancreatic polypeptide by pancreatic secretion a kind of polypeptide hormone being made of 36 amino acid.Human pancreatic polypeptide has as follows
Physiological action: (1) inhibiting the discharge of cholecystokinin and pancreatin, makes smooth muscle of bile vesica relaxation, can reduce the pressure in gall-bladder, gallbladder
General pipeline sphincter anxiety is reinforced, and inhibits bile to duodenal discharge；(2) inhibit postprandial pancreatic juice and bile secretion, inhibit pancreas
The facilitation to pancreatic secretion such as secretin and cholecystokinin；(3) inhibit gastric acid caused by pentagastrin in the gastrointestinal tract
Secretion；(4) secretion for inhibiting Plasma Motilin Levels and Colonic Motility, increases the pressure of inferior esophageal sphincter, inhibits body of stomach myoelectrical activity.Nucleic acid is suitable
Ligand (i.e. aptamer) is aglucon phyletic evolution (the systematic evolution of ligands by index concentration
By exponential enrichment) one section of single stranded DNA (DNA) filtering out or RNA (ribonucleic acid) sequence
Column.Aptamer itself can form specific tertiary structure, with target high specific, combine to high-affinity.In addition, nucleic acid
That there are also molecular weight is small, nontoxic for aptamers, the characteristics of being easily-synthesized and modifying.Since aptamer easy can rapidly synthesize, at
This is lower, and size is smaller, and widely used, they have become the powerful for distinguishing intraor extracellular target molecule.Nucleic acid is suitable
The range of the identifiable object of ligand is very wide, it has developed into a kind of novel molecular probe in recent years, in medical application
Also there is certain progress in field.Therefore, the aptamer of human pancreatic polypeptide is screened for developing new detection method and studying its physiology
Effect has certain value.
Traditional aptamer screening technique generally requires fixed library or target, this includes covalently fixed and non-covalent
Fix two kinds of approach.Whether which kind of fixing means, the native conformation through fixed library or target all can to a certain extent
It is affected.In addition, carrier for fixing can also generate steric hindrance to the combination in library and target, these are all unfavorable for sieving
It selects and obtains the strong sequence of binding ability under free state, therefore develop revocable aptamer screening technique to seem especially heavy
It wants.It can be good at the characteristics of adsorbing single stranded DNA and can removing through centrifugation using graphene oxide herein, graphene oxide drawn
During entering to screening, non-specific adsorption is reduced, is effectively removed unbonded single stranded DNA, and do not influence aptamer
And the combination of target.This method greatly increases the efficiency of screening, has saved labour and has reduced costs.
Summary of the invention
The technical problem to be solved by the present invention is to overcome the deficiencies in the prior art, provide a kind of with higher than protein antibodies
Affinity and specificity, non-immunogenicity, can chemical synthesis, molecular weight it is small, it is stable, be easy to save and what is marked can be used for
Detect the aptamer of human pancreatic polypeptide；It additionally provides the screening technique of above-mentioned aptamer and detects human pancreatic polypeptide in preparation
Kit in application.
In order to solve the above technical problems, a kind of human pancreatic polypeptide aptamer is provided, the human pancreatic polypeptide nucleic acid adaptation
Body is any one in following sequence 1~7:
Sequence 1 is Q10, and the sequence of the Q10 is sequence shown in SEQ ID NO.1；
Sequence 2 is 5 '-Q11-FAM-3 ', and the sequence of the Q11 is sequence shown in SEQ ID NO.2；
Sequence 3 is 5 '-Q12-FAM-3 ', and the sequence of the Q12 is sequence shown in SEQ ID NO.3；
Sequence 4 is 5 '-BHQ1-Q13-3 ', and the sequence of the Q13 is sequence shown in SEQ ID NO.4；
Sequence 5 is 5 '-Q14-BHQ1-3 '；The sequence of the Q14 is sequence shown in SEQ ID NO.5；
Sequence 6 is 5 '-SH-Q16-3 ', and the sequence of the Q15 is sequence shown in SEQ ID NO.6；
Sequence 7 is 5 '-SH-Q17-3 ', and the sequence of the Q15 is sequence shown in SEQ ID NO.7.
Specifically:
Sequence 1:5 '-GGCACCGCTGTTTTAGCCTCGGCTGAGACAAGGGC-3 '.
Sequence 2:5 '-GGCACCGCTGTTTTAGCCTCGGCTGAGACAAGGGCT-FAM-3 '.
Sequence 3:5 '-CGTGCAATGTCGAATGCATGAGCAAACATGGCGAT-FAM-3 '.
Sequence 4:5 '-BHQ1-TGGCACCGCTGTTTTAGCCTCGGCTGAGACAAGGGC-3 '.
Sequence 5:5 '-GGCACCGCTGTTTTAGCCTCGGCTGAGACAAGGGCT-BHQ1-3 '.
Sequence 6:5 '-SH-TTTTTTTTTTGGCACCGCTGTTTTAGCCTCGGCTGAGACAAGGGC-3 '.
Sequence 7:5 '-SH-TTTTTTTTTTCGTGCAATGTCGAATGCATGAGCAAACATGGCGAT-3 '.
Above-mentioned human pancreatic polypeptide aptamer, it is preferred that a certain position on the nucleotide sequence of the sequence 1~7
It is phosphorylated, methylates, amination, sulfhydrylation or isotopologue.Above-mentioned nucleotide sequence is phosphorylated, methylates, amino
Performance will not change after change, sulfhydrylation or isotopologue, can also identify human pancreatic polypeptide.
Above-mentioned human pancreatic polypeptide aptamer, it is preferred that be combined with biology on the nucleotide sequence of the sequence 1~7
Element, digoxin, fluorescent material, nano material or enzyme label.Above-mentioned biotin, digoxin, fluorescent material, enzyme label are combinable
In any position of nucleotide sequence.
Above-mentioned human pancreatic polypeptide aptamer, it is preferred that the fluorescent material includes fluorescein, rhodamine, anthocyanidin.
Above-mentioned human pancreatic polypeptide aptamer, it is preferred that the nano material is gold nano grain.
Above-mentioned human pancreatic polypeptide aptamer, it is preferred that the enzyme is labeled as horseradish peroxidase.
Above-mentioned human pancreatic polypeptide aptamer, it is preferred that the human pancreatic polypeptide aptamer further includes following three kinds
Any one in sequence:
(1) with the homology of the nucleotide sequence of the human pancreatic polypeptide aptamer 80% or more；
(2) sequence hybridized with the nucleotide sequence of the human pancreatic polypeptide aptamer；
(3) RNA sequence of the nucleotide sequence transcription of the human pancreatic polypeptide aptamer.
Above-mentioned human pancreatic polypeptide aptamer, it is preferred that the human pancreatic polypeptide aptamer further includes people's pancreas
The phosphorothioate backbone or the human pancreatic polypeptide nucleic acid that the skeleton of the nucleotide sequence of polypeptide-nucleic acid aptamers derives are suitable
The corresponding peptide nucleic acid that ligand is transformed into.
As a general technical idea, the present invention also provides a kind of screening sides of above-mentioned human pancreatic polypeptide aptamer
Method, comprising the following steps:
S1, synthesis random library and primer:
Random library uses RS35 random library: 5 '-AGCGTCGGATACCACTACTA-NNNNNNNNNNNNNNNNNNN
NNNNNNNNNNNNNNNN-ATCATGGAGTTCGTGGTCAG-3 ',
5 ' primers: 5 '-FAM-AGCGTCGGATACCACTACTA-3 ',
3 ' primers: 5 '-Biotin-CTGACCACGAACTCCATGAT-3 '；
S2, GO-SELEX screening: human pancreatic polypeptide dry powder is dissolved in buffer solution, is incubated for random sequence；So
The buffer containing graphene oxide is added afterwards to be incubated for, supernatant is collected by centrifugation, as human pancreatic polypeptide specific nucleic acid is adapted to
Body library；
S3, PCR amplification library: carrying out PCR amplification for the human pancreatic polypeptide specific nucleic acid aptamers library, obtains amplification and produces
Object；
S4, it prepares the single-stranded library DNA: supernatant is removed into the agarose microbeads centrifugation of Streptavidin modification, then by the expansion
Volume increase object is incubated at normal temperature with agarose microbeads；Then lye is added into agarose microbeads, reacts, be centrifuged, receive under room temperature
Collect supernatant；The supernatant is crossed except collecting the solution to drip, the single-stranded library as DNA after salt plug；
S5, it repeats to screen: the single-stranded library the DNA being substituted into the random library in the step S2, and repeats above-mentioned step
The process of rapid S2~S4 is at least once；
S6, negative screening: bovine serum albumin(BSA), human serum albumins, hemoglobin dry powder are dissolved in buffer solution,
Then it is incubated for the single-stranded library the DNA；Then the buffer containing graphene oxide is added to be incubated for, is discarded through centrifugation
Clearly, it adds buffer solution and graphene oxide is uniformly dispersed again, collect graphene oxide solution at this time；
S7, multi-turns screen: the single-stranded library DNA of the substitution of graphene oxide solution collected by S6 S5 is continued in repetition
State the operating process of step S5~S6；Until filtering out the aptamer met the requirements to the recognition capability of human pancreatic polypeptide.
Above-mentioned screening technique, it is preferred that in the step S2 specifically: human pancreatic polypeptide dry powder is dissolved in buffer solution
In, 2h is incubated at 37 DEG C with random sequence；Then the buffer containing graphene oxide is added and is incubated for 25min at 25 DEG C,
Supernatant, as human pancreatic polypeptide specific nucleic acid aptamers library is collected by centrifugation；The random sequence and the graphene oxide
Ratio is 1.67nmol/mg.
Above-mentioned screening technique, it is preferred that in the S3 step, the process conditions of the PCR amplification are as follows: 94 DEG C of initial denaturations
10min is denaturalized 30sec, 56.9 DEG C of annealing 30sec, 72 DEG C of extension 30sec, 10~20 wheel of circulation, finally in 72 DEG C at 94 DEG C
Lower last extension 7min；In the step S4, the amplified production is 0.5h with the time that agarose microbeads are incubated at normal temperature,
Lye is added into agarose microbeads, reacts 15min under room temperature.
As same technical concept of the invention, the present invention also provides a kind of human pancreatic polypeptide aptamers to make
Application in the kit of standby detection human pancreatic polypeptide.
Compared with the prior art, the advantages of the present invention are as follows:
(1) the present invention provides a kind of human pancreatic polypeptide aptamers, have higher affinity compared to protein antibodies
With specificity；The identification human pancreatic polypeptide of energy specificity and high-affinity, dissociation constant is within nanomolar range.
(2) the present invention provides a kind of human pancreatic polypeptide aptamer, non-immunogenicities；Can iii vitro chemical synthesis, point
Son amount is small, different parts can be modified and be replaced, and sequence is stablized, and is easy to save；(label is not needed convenient for label
Secondary antibody).
(3) the present invention provides a kind of screening techniques of human pancreatic polypeptide aptamer, using graphene oxide auxiliary
The screening of human pancreatic polypeptide aptamer, can greatly improve the efficiency of screening, to reduce cost, save labour.
(4) application the present invention provides a kind of human pancreatic polypeptide aptamer in detection human pancreatic polypeptide, operation is more
Simply, rapidly, and the period is short, favorable reproducibility.Since the synthesis cost of aptamer is at low cost compared with Antibody preparation, so detection
Cost is also corresponding low.The target molecule that it is combined is studied using aptamer of the invention, can also be best understood from
Its physiological function has good application prospect in terms of the diagnosis of pancreatic polypeptide-producing tumor.
Detailed description of the invention
In order to make the object, technical scheme and advantages of the embodiment of the invention clearer, below in conjunction with the embodiment of the present invention
In attached drawing, the technical scheme in the embodiment of the invention is clearly and completely described.
Fig. 1 is the principle and key step schematic diagram of graphene oxide assisting sifting in the embodiment of the present invention, and mode one is
The process of positive-selecting, mode two are the process that the combination of negative and positive-selecting carries out.
Fig. 2 is the secondary structure schematic diagram of the Q10 aptamer in the embodiment of the present invention using the simulation of IDTDNA software.
Fig. 3 is that the secondary structure of the FAM-Q11 aptamer in the embodiment of the present invention using the simulation of IDTDNA software is shown
It is intended to.
Fig. 4 is that the secondary structure of the FAM-Q12 aptamer in the embodiment of the present invention using the simulation of IDTDNA software is shown
It is intended to.
Fig. 5 is that the secondary structure of the BHQ1-Q13 aptamer in the embodiment of the present invention using the simulation of IDTDNA software is shown
It is intended to.
Fig. 6 is that the secondary structure of the BHQ1-Q14 aptamer in the embodiment of the present invention using the simulation of IDTDNA software is shown
It is intended to.
Fig. 7 is that the secondary structure of the SH-Q16 aptamer in the embodiment of the present invention using the simulation of IDTDNA software is illustrated
Figure.
Fig. 8 is that the secondary structure of the SH-Q17 aptamer in the embodiment of the present invention using the simulation of IDTDNA software is illustrated
Figure.
Fig. 9 is that native polyacrylamide gel electrophoresis investigates sequence Q10 to human pancreatic polypeptide combination in the embodiment of the present invention
The testing result of situation.
Figure 10 is the testing result that multi-function microplate reader investigates Q11 situation in conjunction with human pancreatic polypeptide in the embodiment of the present invention.
Figure 11 is the testing result that multi-function microplate reader investigates Q11 and human pancreatic polypeptide binding ability in the embodiment of the present invention.
Figure 12 is that Fluorescence Spectrometer investigates influence of the incubation time to Q11 in conjunction with human pancreatic polypeptide in the embodiment of the present invention
Testing result.
Figure 13 is the detection that Fluorescence Spectrometer investigates influence of the temperature to Q11 in conjunction with human pancreatic polypeptide in the embodiment of the present invention
As a result.
Figure 14 is the difference that multi-function microplate reader detects Q11 and human pancreatic polypeptide antibody combining site.
Figure 15 is that multi-function microplate reader detects selectivity of the Q11 in conjunction with human pancreatic polypeptide.
Figure 16 is that Fluorescence Spectrometer detects mode of the Q12 and Q13 in conjunction with human pancreatic polypeptide.
Figure 17 is the binding site that Fluorescence Spectrometer detects Q14 and Q15 and human pancreatic polypeptide.
Figure 18 dynamic light scattering method detects Q16 and Q17 to the detection effect of human pancreatic polypeptide.
Figure 19 dynamic light scattering method detects the selectivity of Q16 and Q17 detection human pancreatic polypeptide.
Specific embodiment
Below in conjunction with Figure of description and specific preferred embodiment, the invention will be further described, but not therefore and
It limits the scope of the invention.
Embodiment
Material employed in following embodiment and instrument are commercially available.
Embodiment 1:
A kind of aptamer can be used for detecting human pancreatic polypeptide, comprising:
Sequence 1 (Q10):
5’-GGCACCGCTGTTTTAGCCTCGGCTGAGACAAGGGC-3’(SEQ ID NO.1)。
Sequence 2 (FAM-Q11):
5’-GGCACCGCTGTTTTAGCCTCGGCTGAGACAAGGGCT-FAM-3’(SEQ ID NO.2)。
Sequence 3 (FAM-Q12):
5’-CGTGCAATGTCGAATGCATGAGCAAACATGGCGAT-FAM-3’(SEQ ID NO.3)。
Sequence 4 (BHQ1-Q13):
5’-BHQ1-TGGCACCGCTGTTTTAGCCTCGGCTGAGACAAGGGC-3’(SEQ ID NO.4)。
Sequence 5 (BHQ1-Q14):
5’-GGCACCGCTGTTTTAGCCTCGGCTGAGACAAGGGCT-BHQ1-3’(SEQ ID NO.5)。
Sequence 6 (SH-Q16):
5’-SH-TTTTTTTTTTGGCACCGCTGTTTTAGCCTCGGCTGAGACAAGGGC-3’(SEQ ID NO.6)。
Sequence 7 (SH-Q17):
5’-SH-TTTTTTTTTTCGTGCAATGTCGAATGCATGAGCAAACATGGCGAT-3’(SEQ ID NO.7)。
Negative control (FAM-Random):
5’-NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN-FAM-3’。
It closes probe (FAM-Q15):
5’-FAM-AGCCTTTGTTTCATCCGAGG-3’。
Assist probes (SH-Q18):
5’-SH-TTTTTTTTTT-3’。
Note: N represents any base in A, T, G, C.In above-mentioned sequence of the present embodiment, seven to can be used for detecting people's pancreas more
Peptide, respectively Q10, FAM-Q11, FAM-Q12, BHQ1-Q13, BHQ1-Q14, SH-Q16, SH-Q17；FAM-Random is as yin
Property control；FAM-Q15 is as closing probe；SH-Q18 is as assist probes.
Q10, FAM-Q11, FAM-Q12, BHQ1-Q13, BHQ1-Q14, SH-Q16, SH-Q17 mainly use following screening side
Method screens to obtain, as shown in Figure 1, specifically includes the following steps:
A kind of screening technique of the human pancreatic polypeptide aptamer of the present embodiment, comprising the following steps:
1, random single-stranded DNA banks and primer shown in following sequence are synthesized:
Random library RS35:
5 '-AGCGTCGGATACCACTACTA-NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN (N represents A, T,
One of tetra- bases of G, C)-ATCATGGAGTTCGTGGTCAG-3 '；
5 ' primers: 5 '-FAM-AGCGTCGGATACCACTACTA-3 '；
3 ' primers: 5 '-Biotin-CTGACCACGAACTCCATGAT-3 '.
2, the preparation of buffer solution:
The preparation of 2.1 1 × Binding Buffer (pH=7.35): 2.383g HEPES, 3.510g are weighed respectively
NaCl, 0.186g KCl and 0.203g MgCl2·6H2O is dissolved in 200mL aqua sterilisa, is adjusted pH value to 7.35, is added water
It is settled to 500mL and obtains 1 × Binding Buffer, wherein the concentration of HEPES is 20mM, and the concentration of NaCl is 120mM, KCl
Concentration be 5mM, the concentration of MgCl2 is 2mM, then saves 1 × Binding Buffer in 4 DEG C of refrigerators.
2.2 concentration are the preparation of the PBS solution (pH=7.5) of 20mM: weighing 0.2940g NaH2PO4·2H2O,
2.8960g Na2HPO4·12H2O, 4.390g NaCl add aqua sterilisa to 200mL, adjust pH to 7.5, constant volume in 500mL volumetric flask,
4 DEG C of preservations.
2.3 concentration are the preparation of the NaOH solution of 200mM: weighing 0.800g NaOH, add a small amount of aqua sterilisa to dissolve, constant volume
In 100mL volumetric flask, 4 DEG C of preservations.
The preparation of 2.4 antibody incubation liquid (20mM PBS, 0.05%Tween-20, pH=7.5): 0.2940g is weighed
NaH2PO4·2H2O, 2.8960g Na2HPO4·12H2O, 4.390g NaCl add aqua sterilisa to 200mL, and 50 μ L Tween- are added
20, pH to 7.5 is adjusted, constant volume is in 500mL volumetric flask, 4 DEG C of preservations.
2.5 concentration are the preparation of the PBS solution (pH=7.4) of 30mM: weighing 0.0889g NaH2PO4·2H2O,
0.8699g Na2HPO4·12H2O, 1.7532g NaCl add aqua sterilisa to 60mL, and after completely dissolution, constant volume is in 100mL capacity
Bottle, 4 DEG C of preservations.
3, GO-SELEX screening obtains human pancreatic polypeptide specific nucleic acid aptamer library, and principle is as shown in Figure 1:
The pretreatment of 3.1 random libraries: the centrifuge tube equipped with 5nmol random library RS35 is placed in a centrifuge, in 9000g
Lower centrifugation 10min, carefully takes out centrifuge tube, avoids shaking, and 1 × Binding Buffer of 400 μ L is rapidly added, by centrifuge tube
It is placed at 95 DEG C after heating 5min, takes out immediately and ice bath 5min, further take out to be placed in and maintained after 5min obtains pretreatment at room temperature
Random library.
3.2 are incubated for: it is molten to obtain human pancreatic polypeptide with 1 × Binding Buffer of 50 μ L, 200 μ g human pancreatic polypeptide dry powder of dissolution
Human pancreatic polypeptide solution is added in step 3.1 in pretreated random library, is incubated for 2h at 37 DEG C by liquid.
3.3 dissociation: graphene oxide dispersion (final concentration of 30 μ of graphene oxide dispersion is added after the completion of being incubated for
G/mL), 25min is adsorbed at 25 DEG C, 10min is then centrifuged at 20800g, collects supernatant, as screening gained human pancreatic polypeptide
Specific nucleic acid aptamers library.
4, it carries out PCR amplification library: taking 100 μ L to screen resulting human pancreatic polypeptide specific nucleic acid aptamers library and carry out routine
PCR amplification, amplification condition are as follows: initial denaturation 10min at 94 DEG C is denaturalized 30sec at 94 DEG C, and anneal 30sec at 56.9 DEG C, at 72 DEG C
Extend 30sec, 10 wheel of circulation finally finally extends 7min at 72 DEG C.The first round screening after need by all human pancreatic polypeptide
Specific nucleic acid aptamer library expands 10 circulations in advance, then carries out the amplification of this step, obtains amplified production.
5, it prepares the single-stranded library DNA: supernatant is removed into the agarose microbeads 2200g centrifugation of 200 μ L Streptavidins modification, then
It is washed with 500 μ L PBS, supernatant is removed in centrifugation；Repeated washing is twice.By the resulting amplified production of PCR amplification in step 3 and agar
Sugared microballoon is incubated for half an hour at normal temperature, passes through the parent of the Streptavidin on the biotin and agarose microbeads on amplified production
Double-stranded DNA is sufficiently captured to agarose microbeads surface with effect.Then supernatant is removed in 2200g centrifugation, with PBS centrifuge washing three
It is secondary；Add the denaturation treatment that 500 μ L of 200mM NaOH solution is used for double-stranded DNA into agarose microbeads, normal-temperature reaction
15min, 2200g are centrifuged 5min, collect supernatant；After the salt plug sterile water washing of 10mL, collection is obtained after addition alkaline denaturation
Supernatant drips off naturally.1mL sterile water is added, collects the solution to drip, this is the single-stranded library DNA.
6, repeat to screen: the random library in the single-stranded library alternative steps 3 of the DNA that step 5 is obtained repeats the above steps
Positive-selecting, PCR amplification shown in 3~5 and single-stranded DNA banks process is produced, is repeated 4 times.
7, negative screening: the 5th wheel screening and after, be with human serum albumins, bovine serum albumin(BSA), hemoglobin
The single-stranded library the DNA screened after step 5 is carried out negative screening, the concrete operations of feminine gender screening are as follows: by human serum by control
Albumin, bovine serum albumin(BSA), hemoglobin dry powder are dissolved in buffer solution, then mono- with the DNA that screens after step 5
Chain library is incubated for；Then the buffer containing graphene oxide is added to be incubated for, is discarded supernatant through centrifugation, it is molten to add buffering
Graphene oxide is uniformly dispersed by liquid again, collects graphene oxide solution at this time.
8, multi-turns screen: the single-stranded library DNA in graphene oxide solution alternative steps 6 collected by step 7 is continued
The operating process for repeating above-mentioned steps 6~7, after first round screening, the library amount for being incubated for screening is about
500pmol, since third round screening, the ratio of target and random library is begun to decline, and ratio drops to 1 from 10；It repeats to sieve
The gained single-stranded library DNA is monitored to the enhancing situation of human pancreatic polypeptide recognition capability, until 18 wheels with Fluorescence Spectrometer during choosing
The single-stranded library DNA meets the requirements the recognition capability of human pancreatic polypeptide after screening, and products therefrom is analyzed through high-throughput sequence.To sequencing
As a result after finishing analysis, seven high sequences of degree of enrichment finally can be obtained: Q10, Q11, Q12, Q13, Q14, Q16, Q17, this seven
Sequence is the aptamer that can be used for detecting human pancreatic polypeptide of above-mentioned the present embodiment.
It investigates 1: Sketch of secondary structure being carried out to seven sequences of the present embodiment with IDTDNA software.With IDTDNA software pair
For structural schematic diagram after seven sequences progress Sketch of secondary structure of the present embodiment as shown in Fig. 2 to Fig. 8, analog result shows seven
The secondary structure of sequence all contains stem ring part, shows that it can further be curled into tertiary structure in conjunction with human pancreatic polypeptide.
Investigation 2: the combination effect of non denatured polyacrylamide gel electrophoresis detection sequence 1 (Q10) and human pancreatic polypeptide.
Specific investigation method is the following steps are included: carry out denaturation treatment for Q10: after heating 5min at 95 DEG C, taking out immediately
And ice bath 5min, then 5min is maintained at room temperature.Experimental group: taking 5 μ L concentration is that 5 μM of DNA sequence dnas being denaturalized are with 5 μ L concentration
100 μM of human pancreatic polypeptide mixes in one 200 μ L PCR pipes；Control group: taking 5 μ L concentration is 5 μM of DNA sequence dnas being denaturalized and 5 μ
1 × Binding Buffer of L is mixed in another 200 μ L PCR pipe, and two PCR pipes are all incubated for 2h at 25 DEG C respectively.It incubates
After educating, to 3 μ L loading buffers are added in 10 μ L samples of experimental group and control group, (sample-loading buffer contains 0.25%
Bromophenol blue, 0.25% dimethylbenzene blueness FF, 30% glycerol), 2 μ LSYBR GOLD dyestuffs obtain mixed liquor, mixed liquor is mixed
Afterwards, 10 μ L are taken to click and enter in the loading wells of 18% non-denaturing polyacrylamide gel, with constant current mode 10mA in 1 × tbe buffer liquid
Middle carry out electrophoresis, electrophoresis tank should be protected from light.Stop electrophoresis when purple bromophenol blue band is run to from gel bottom about 1cm, takes out and clap
According to.
Non-denaturing polyacrylamide gel preparation method (10mL system): 30% acrylamide/methylene diacrylamide
6mL, 5 × tbe buffer liquid 2mL, ultrapure water 1.93mL, 10% ammonium persulfate, 70 7 μ L of μ L, TEMED.
The preparation method of electrophoretic buffer (TBE, 5 ×/500mL): Tris-base 27g, Na2EDTA·2H2O 1.86g,
Boric acid 13.75g is added 400mL ultrapure water, is adjusted to pH=8.3, and by solution constant volume in 500mL volumetric flask.
Testing result is as shown in figure 9, swimming lane 1 is control group, and swimming lane 2 is experimental group, and there are one relative to swimming lane 1 for swimming lane 2
Positioning moves, be human pancreatic polypeptide with Q10 ining conjunction with after influence Q10 secondary structure to influence its electrophoretic velocity, illustrate the present embodiment
Q10 aptamer have recognition capability to human pancreatic polypeptide.
It investigates 3: being imitated with the combination of multi-function microplate reader, graphene oxide detection sequence 2 (FAM-Q11) and human pancreatic polypeptide
Fruit.
FAM-Q11 is subjected to denaturation treatment according to the method for investigating 2.By the FAM-Q11 being denaturalized and final concentration of 1 μM
Human pancreatic polypeptide mixes in EP pipe, and the final concentration of FAM-Q11 is respectively 10,25,50,75,100,150,200,300nM.Mixing
It being placed in well in 25 DEG C of insulating boxs and is incubated for 2h, the rear ratio that graphene oxide is added and controls itself and FAM-Q11 is 2nmol/mg, then
It is incubated for 25min.Supernatant is collected after being centrifuged 15min with 20800g, utilizes the fluorescence of multi-function microplate reader detection supernatant.With glimmering
Light changing value is ordinate, and the concentration of DNA is abscissa, by Y=Bmax × X/ (Kd+ X) equation simulation curve, obtain this implementation
The dissociation constant of example FAM-Q11 draws curve such as Figure 10, and this makes it possible to obtain dissociation constant KdAs shown in table 1 below.
Table 1: dissociation constant result
| Sequence names | Dissociation constant (nanomole) |
| Q11 | 24.72±13.40 |
As shown in Figure 10, the combination of FAM-Q11 and human pancreatic polypeptide meets Michaelis-Menten equation, is typical albumen and ligand binding
Mode；Dissociation constant KdIn nanomole rank, show that the combination of FAM-Q11 and human pancreatic polypeptide has stronger affinity.
Investigate 4: the combination of multi-function microplate reader, graphene oxide detection aptamer FAM-Q11 and human pancreatic polypeptide is imitated
Fruit and specificity.
FAM-Q11 and negative control (FAM-Random) are subjected to denaturation treatment respectively according to the method for investigating 2.It will denaturation
Good FAM-Q11, FAM-Random mixes FAM-Q11, FAM- in black ELISA Plate with the human pancreatic polypeptide of various concentration
Random final concentration is 50nM, and the final concentration of human pancreatic polypeptide is respectively 0,0,100,250,500,1000,1250,1500nM.
ELISA Plate is placed in 25 DEG C of insulating boxs after mixing and is incubated for 2h, it is rear that graphene oxide is added and makes its final concentration of 18 μ g/mL,
25min is incubated in 25 DEG C of insulating boxs, and (graphite oxide is not added after incubation as fluorescence initial value in first group of 0nM human pancreatic polypeptide
Alkene).The fluorescence of multi-function microplate reader detection FAM is utilized after incubation.
As a result as shown in figure 11: relative to random sequence FAM-Random, signal of the FAM-Q11 in conjunction with human pancreatic polypeptide is more
By force, illustrate that the FAM-Q11 of the present embodiment has strong specific recognition capability to human pancreatic polypeptide.
Investigate 5: Fluorescence Spectrometer, graphene oxide detection incubation time are to aptamer FAM-Q11 and human pancreatic polypeptide
Combination effect influence.
FAM-Q11 is subjected to denaturation treatment according to the method for investigating 2.By the FAM-Q11 being denaturalized with it is final concentration of
The human pancreatic polypeptide of 1500nM mixes, and addition is dense eventually after being incubated for 15min, 30min, 45min, 60min, 90min respectively at 25 DEG C
Degree is the graphene oxide of 18 μ g/mL, then is incubated for 25min, and the final concentration of FAM-Q11 is 50nM at this time.Finally with fluorescence light
Spectrometer detects the fluorescence of FAM, and calculates the fluorescence recovery rate under different incubation times.
As a result as shown in figure 12, fluorescence recovery rate basically reaches platform after 45min, illustrates the FAM- of the present embodiment
The combination of Q11 and human pancreatic polypeptide is very fast.
Investigate 6: Fluorescence Spectrometer, graphene oxide detect temperature to the knot of aptamer FAM-Q11 and human pancreatic polypeptide
Close the influence of effect.
FAM-Q11 is subjected to denaturation treatment according to the method for investigating 2.It is respectively with final concentration by the FAM-Q11 being denaturalized
The human pancreatic polypeptide of 0nM and 1500nM mixes, and is incubated under 4 DEG C, 15 DEG C, 25 DEG C, 37 DEG C of four different temperatures, the end of DNA sequence dna
Concentration is 50nM.After being incubated for 2h, graphene oxide is added and makes its final concentration of 18 μ g/mL, then is incubated for 25min.Finally use
Fluorescence Spectrometer detects the fluorescence of FAM, and calculates the fluorescence recovery rate under different temperatures.
As a result as shown in figure 13, the fluorescence recovery rate difference at four temperature is little, illustrates temperature to the present embodiment
The combination of FAM-Q11 and human pancreatic polypeptide influences unobvious.
Investigate 7: multi-function microplate reader detects the difference of aptamer FAM-Q11 and human pancreatic polypeptide antibody combining site.
Fixation of the human pancreatic polypeptide antibody on magnetic bead: taking the 50 coated magnetic beads of μ L albumin A, discards supernatant through magnetic field separation
Liquid.200 μ L antibody incubation liquid (2.4 parts that antibody incubation liquid configuration method is shown in embodiment 1) are added to be washed, through magnetic field point
Supernatant is abandoned, washs magnetic bead three times repeatedly；5 μ g human pancreatic polypeptide antibody are added after washing, are incubated for 30min at 37 DEG C；It incubates
It educates and discards supernatant liquid through magnetic field separation after terminating, and wash magnetic bead three times with 200 μ L antibody incubation liquid, be finally dispersed in magnetic bead
In 500 μ L antibody incubation liquid.
FAM-Q11 is subjected to denaturation treatment according to the method for investigating 2.The FAM-Q11 that has been denaturalized and final concentration be respectively 0,
100, the human pancreatic polypeptide mixing of 200nM, the final concentration of FAM-Q11 is 20nM, is incubated for 1.5h at 25 DEG C.It is solid that 20 μ L are then added
The magnetic bead of human pancreatic polypeptide antibody is set, then is incubated for 30min.Incubation separates through magnetic field after terminating and takes supernatant, with multifunctional enzyme mark
Instrument detects the fluorescence of FAM in supernatant.
As a result as shown in figure 14: FAM-Q11 is added after being incubated for various concentration human pancreatic polypeptide and is fixed with human pancreatic polypeptide antibody
Magnetic bead, after incubation in supernatant fluorescence decline, can also be shown by antibody capture after illustrating human pancreatic polypeptide in conjunction with FAM-Q11
Site of the site that FAM-Q11 and human pancreatic polypeptide combine in conjunction with antibody and human pancreatic polypeptide is different.
Investigate 8: multi-function microplate reader detects selectivity of the aptamer FAM-Q11 in conjunction with human pancreatic polypeptide.
Human pancreatic polypeptide antibody is fixed on magnetic bead according to the method for investigating 7.
FAM-Q11 is subjected to denaturation treatment according to the method for investigating 2.The FAM-Q11 being denaturalized respectively with human pancreatic polypeptide, pancreas
Island element, glucagon, protein kinase A peptide substrate, glutathione mixing, the final concentration of FAM-Q11 is 20nM, human pancreatic polypeptide
Final concentration be respectively 0,200nM, insulin final concentration be respectively 0,200nM, glucagon final concentration be respectively 0,
200nM, protein kinase A peptide substrate final concentration be respectively 0,200nM, glutathione final concentration be respectively 0,200nM, 25 DEG C
Lower incubation 1.5h.20 μ L are then added and fix the magnetic bead of human pancreatic polypeptide antibody, then are incubated for 30min.Incubation terminate after through magnetic field
Separation takes supernatant, and the fluorescence of FAM in supernatant is detected with multi-function microplate reader, and calculates change in fluorescence rate.
As a result as shown in figure 15, the magnetic bead for being fixed with human pancreatic polypeptide antibody is added in FAM-Q11 and different material after being incubated for, only
Have human pancreatic polypeptide group supernatant fluorescence decline, illustrate only human pancreatic polypeptide after in conjunction with FAM-Q11 can also antibody capture, have
There is specificity.
Investigate 9: Fluorescence Spectrometer detects the present embodiment two aptamer FAM-Q12 (sequence 3), BHQ1-Q13 (sequences
Column 4) with the binding pattern of human pancreatic polypeptide.
According to the method for investigating 2, denaturation treatment is carried out after FAM-Q12 and BHQ1-Q13 is mixed.The FAM- that will be denaturalized
Q12 and BHQ1-Q13 respectively with final concentration of 0,10,20,50, the human pancreatic polypeptide of 100nM mix, 4h, FAM- are incubated at 25 DEG C
The final concentration of Q12 and BHQ1-Q13 is 100nM.The fluorescence of FAM in Fluorescence Spectrometer detection solution is utilized after incubation.
As a result as shown in figure 16: with the increase of human pancreatic polypeptide concentration, the fluorescence intensity of solution declines, and illustrates fluorophor
Distance with quenching group is furthered, and fluorescence is quenched, this show FAM-Q12 and BHQ1-Q13 can simultaneously with human pancreatic polypeptide knot
It closes, forms sandwich structure.
Also, SH-Q16 and SH-Q17 can also form sandwich knot simultaneously in conjunction with pancreatic polypeptide in the present embodiment
Structure.
Investigate 10: Fluorescence Spectrometer detects aptamer BHQ1-Q14 (sequence 5), FAM-Q15 (closing probe) and people
The binding site of pancreatic polypeptide.
BHQ1-Q14 and FAM-Q15 are made annealing treatment: BHQ1-Q14 and FAM-Q15 are mixed be placed in 95 DEG C plus
Hot 5min, being placed on room temperature makes its natural cooling 2h.It is respectively 0nM by the BHQ1-Q14 to have annealed and FAM-Q15 and final concentration
It is mixed with the human pancreatic polypeptide of 50nM, 2h is incubated at 25 DEG C, wherein the final concentration of 20nM of BHQ1-Q14, the end of FAM-Q15 are dense
Degree is 25nM.A control group separately is set as the BHQ1-Q14 of final concentration 20nM, is also incubated for 2h at 25 DEG C.Fluorescence is used after incubation
The fluorescence of spectrometer detection FAM.
As a result as shown in figure 17: the Q14 of quenching group is marked as identification sequence, the Q15 of fluorophor is marked as envelope
Close sequence, the fluorescence of the fluorescence Q14 more individual than control group after Q14 and Q15 annealing reduces many, illustrates annealed processing
Q11 and Q15 can be very good together with hybridization, and the distance to have furthered between fluorophor and quenching group, fluorescence is quenched.And it passes through
The Q14 and Q15 of annealing compare Q14 and Q15 compound after human pancreatic polypeptide is added, and fluorescence rises, and illustrate human pancreatic polypeptide energy
In conjunction with Q14, the hybrid structure of Q14 and Q15 are destroyed, so that the distance of quenching group and fluorophor becomes remote.And due to Q15
Closed is the 3 ' ends of Q14, illustrates that human pancreatic polypeptide may be combined with the 3 ' ends of Q14.
Investigate 11: it is (auxiliary that dynamic light scattering method detects the present embodiment SH-Q16 (sequence 6), SH-Q17 (sequence 7), SH-Q18
Help probe) to the detection effect of human pancreatic polypeptide.
The synthesis of gold nano grain: after the gold chloride of 100mL 0.01% is boiled, it is molten that 1% trisodium citrate of 3mL is added
Liquid continues heating and maintains fluidized state, and solution becomes claret, then boils 15 to 20min, can stop heating.It is cold to solution
But it is settled to 100mL afterwards.
The modification of SH-Q16, SH-Q17 and SH-Q18 on gold nano grain surface: by 10 μ L concentration be 100 μM of SH-Q16,
10 μ L concentration are 100 μM of SH-Q18 and 10 μ L concentration are 100 μM of SH-Q17,10 μ L concentration are that 100 μM of SH-Q18 are separately added into
All it is (to do two pipe Duplicate Samples) in 1mL gold nano grain to volume, is incubated for 16h at 4 DEG C.Then 500 μ L 30mM are added
PBS, then 40h is incubated at 4 DEG C.30min is centrifuged at 4 DEG C with the revolving speed of 20800g after incubation and removes supernatant, will be precipitated
It is uniformly dispersed in 1 × Binding Buffer of 1mL, then 30min is centrifuged at 4 DEG C with the revolving speed of 20800g and removes supernatant,
Repeat dispersion, centrifugation goes supernatant step twice.It obtains the co-modified gold particle of SH-Q16, SH-Q18 and SH-Q17, SH-Q18 is total
(SH-16 and SH-17 have recognition reaction as detection probe to the gold particle of modification, and SH-Q18 does not have recognition reaction, is only intended to
Detection probe is adjusted in the modification density on gold particle surface, SH-Q18 and SH-Q16 are modified jointly in Yijin pellet surface, SH-
Q18 and SH-Q17 is again common to be modified on another gold particle surface).
The SH-Q17 gold particle mixing that the gold particle for the SH-Q16 for taking 20 μ L to modify and 20 μ L have been modified, it is more to be added people's pancreas
Peptide, the final concentration of human pancreatic polypeptide are respectively 0,1,2,5,10,15nM, are incubated for 2h at 25 DEG C after mixing.It is utilized after incubation
The partial size of the gold particle of dynamic light scattering method test sample.
As a result as shown in figure 18: with the raising of human pancreatic polypeptide concentration, the partial size of gold particle increases, and illustrates human pancreatic polypeptide energy
Simultaneously in conjunction with SH-Q16 and SH-Q17 on gold particle, the distance between gold particle is drawn in, partial size is made to become larger.And the grain of gold particle
Diameter variation, linear equation y=0.138+1.229x in a linear relationship with the concentration of human pancreatic polypeptide, related coefficient 0.9936,
Detection is limited to 37pM.Illustrate that there is preferable effect, inspection using SH-Q16, SH-Q17 and dynamic light scattering method detection human pancreatic polypeptide
It is lower to survey limit.
Investigate 12: dynamic light scattering method detects the selectivity of two aptamer detection human pancreatic polypeptides of the present embodiment.
The modification of the synthesis of gold nano grain and SH-Q16, SH-Q17 and SH-Q18 on the gold particle is with reference to investigation 11.
It takes that 20 μ L have modified the gold particle of SH-Q16 and 20 μ L have modified the mixing of SH-Q17 gold particle, it is more to be separately added into people's pancreas
Peptide, bovine serum albumin(BSA), human serum albumins, immunoglobulin G, insulin, glucagon, the final concentration of six kinds of substances is all
For 15nM, 2h is incubated at 25 DEG C after mixing.The partial size of the gold particle of dynamic light scattering method test sample is utilized after incubation.
As a result as shown in figure 19: modified the gold particle of SH-Q16 and modified SH-Q17 gold particle only and human pancreatic polypeptide
Change of size is more apparent after incubation, and change of size is little after being incubated for other materials, illustrates that SH-Q16 and SH-Q17 and people's pancreas are more
The combination of peptide has certain specificity.
The above described is only a preferred embodiment of the present invention, being not intended to limit the present invention in any form.Though
So the present invention is disclosed as above with preferred embodiment, and however, it is not intended to limit the invention.It is any to be familiar with those skilled in the art
Member, in the case where not departing from Spirit Essence of the invention and technical solution, all using in the methods and techniques of the disclosure above
Appearance makes many possible changes and modifications or equivalent example modified to equivalent change to technical solution of the present invention.Therefore,
Anything that does not depart from the technical scheme of the invention, according to the technical essence of the invention any letter made to the above embodiment
Single modification, equivalent replacement, equivalence changes and modification, all of which are still within the scope of protection of the technical scheme of the invention.
Claims (5)
1. a kind of human pancreatic polypeptide aptamer, which is characterized in that the human pancreatic polypeptide aptamer be following sequence 1,2,
4, any one in 5,6:
Sequence 1 is Q10, and the sequence of the Q10 is sequence shown in SEQ ID NO.1；
Sequence 2 is 5 '-Q11-FAM-3 ', and the sequence of the Q11 is sequence shown in SEQ ID NO.2；
Sequence 4 is 5 '-BHQ1-Q13-3 ', and the sequence of the Q13 is sequence shown in SEQ ID NO.4；
Sequence 5 is 5 '-Q14-BHQ1-3 '；The sequence of the Q14 is sequence shown in SEQ ID NO.5；
Sequence 6 is 5 '-SH-Q16-3 ', and the sequence of the Q16 is sequence shown in SEQ ID NO.6；
Or, the human pancreatic polypeptide aptamer are as follows: the mixture of sequence 4 and sequence 3, sequence 3 are 5 '-Q12-FAM-3 ', institute
The sequence for stating Q12 is sequence shown in SEQ ID NO.3；
Or, the human pancreatic polypeptide aptamer are as follows: the mixture of sequence 6 and sequence 7, sequence 7 is 5 '-SH-Q17-3 ', described
The sequence of Q17 is sequence shown in SEQ ID NO.7.
2. human pancreatic polypeptide aptamer according to claim 1, which is characterized in that the nucleotides sequence of the sequence 1~7
Biotin, digoxin, fluorescent material, nano material or enzyme label are combined on column.
3. human pancreatic polypeptide aptamer according to claim 2, which is characterized in that the fluorescent material includes fluorescence
Element；The nano material is gold nano grain；The enzyme is labeled as horseradish peroxidase.
4. human pancreatic polypeptide aptamer according to claim 3, which is characterized in that fluorescein is rhodamine or cyanine
Element.
5. human pancreatic polypeptide aptamer described in a kind of any one of claims 1 to 4 is in the reagent of preparation detection human pancreatic polypeptide
Application in box.
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