Example 1
1. Preparation of sea purse angiogenesis inhibiting factor 1 functional zone protein
The method for obtaining the sea purse angiogenesis inhibiting factor 1 functional domain protein with the same biological activity as the sea purse angiogenesis inhibiting factor 1 functional domain protein by adopting a yeast recombinant expression mode well known by a person skilled in the art, wherein the protein sequence is shown as SEQ ID NO: 1 is shown in the specification; the concentration of the protein solution was 15 mg/ml.
2. Synthesis of libraries and primers
2.1 Synthesis of ssDNA oligonucleotide library for screening (5' -TCA GTC GCT TCG CCG TCT CCTTC-)
N35- - -GCA CAA GAG GGA GAC CCC AGA GGG-3'), wherein N35 is 35 random oligonucleotides;
primer P1: TCAGTCGCTTCGCCGTCTCCTTC, respectively;
primer P2: CCCTCTGGGGTCTCCCTCTTGTGC are provided.
2.2, SELEX screening of the aptamers, wherein the specific method comprises the following steps:
2.2.1 binding and separating of ssDNA and sea purse angiogenesis inhibitor 1 functional domain protein, the specific method is as follows:
taking 4 mu L of a 100 mu M ssDNA oligonucleotide library, diluting to 100 mu L with 2 multiplied by binding buffer solution, denaturing at 95 ℃ for 5min, adding 100 mu L of ray angiogenesis inhibiting factor 1 functional domain protein after ice bath for 10min, binding by a shaking table for 30min, centrifuging at 6000rpm for 5min, discarding supernatant, washing precipitate with 1 multiplied by binding buffer solution, and discarding supernatant; adding 100 mu L of 1 Xbinding buffer solution into the sediment, heating at 96 ℃ for 5min, then centrifuging at 15000rpm for 10min, taking supernatant, heating and centrifuging the sediment again, and combining the supernatant to obtain a ssDNA secondary library with affinity to the protein of the functional region of the sea purse angiogenesis inhibitor 1; the 2 × binding buffer solution is a solution obtained by diluting the 20 × binding buffer solution by 10 times with double distilled water, and the 1 × binding buffer solution is a solution obtained by diluting the 20 × binding buffer solution by 20 times with double distilled water; the 20 × binding buffer formulation was 1M NaCl, 50mM KCl, 500mM Tris-HCl, 10mM MgCl2, pH 7.4.
2.2.2 binding and separating of ssDNA and sea purse angiogenesis inhibitor 1 functional domain protein, the specific method is as follows:
the ssDNA which is obtained by separation in the step 2.2.1 and can be combined with the protein of the sea purse angiogenesis inhibiting factor 1 functional area is combined with 100 mu l of the protein of the sea purse angiogenesis inhibiting factor 1 functional area for 30min in a shaking table, and the subsequent step is synchronous with the step 2.2.1, so that a ssDNA secondary library which has affinity with the protein of the sea purse angiogenesis inhibiting factor 1 functional area can be separated.
2.2.3 asymmetric PCR amplification of ssDNA, the specific procedure is as follows:
carrying out asymmetric PCR amplification on the ssDNA secondary library obtained by separation in the step 2.2.2, wherein the asymmetric PCR amplification system with the total volume of 25 mu l: 10 × PCR buffer: 2 mu l of the solution; p1(10 μ M): 1 mul; p2(0.2 μ M): 1 mul; dNTPs (2.5 mM each): 0.4 μ l; MgCl2(25 mM): 1.2 μ l; ssDNA template (0.2. mu.g/. mu.l): 2 mu l of the solution; taq DNA polymerase (5 u/. mu.l): 0.2 μ l; ddH 2O: 17.2 μ l; PCR reaction parameters: pre-denaturation at 94 ℃ for 4min, 40 cycles of denaturation at 94 ℃ for 30s, annealing at 58 ℃ for 30s, extension at 72 ℃ for 20s, and final extension at 72 ℃ for 7 min;
2.2.4 determination of affinity, the specific method is as follows:
2.2.4.1 amplification: using primer P1 with digoxin label to do asymmetric PCR to amplify the screened ssDNA secondary library, wherein the amplification conditions and parameters are the same as those of the asymmetric PCR amplification system and parameters in step 2.2.3;
2.2.4.2 binding to proteins: taking 100 mu L of PCR product obtained by amplification in the step 2.2.4.1, carrying out denaturation at 95 ℃ for 5min, carrying out ice bath for 10min, then adding the PCR product into 100 mu L of protein, fully mixing, combining the protein at room temperature for 30min, then centrifuging at 6000rpm, separating protein and supernatant, wherein the protein contains ssDNA (single-stranded deoxyribonucleic acid) which is combined with the protein and is provided with a digoxin label, the supernatant is unbound ssDNA, and meanwhile, a blank without ssDNA is made, namely 2 × binding buffer solution is used for replacing the PCR product, and the operation is carried out in the same way;
2.2.4.3 washing: washing the protein with 1 × 500 μ L of binding buffer solution for 1 time, centrifuging at 6000rpm, removing supernatant, and collecting protein;
2.2.4.4 binding to enzyme-labeled rabbit anti-digoxin antibody: adding 100 μ L of enzyme-labeled rabbit anti-digoxin antibody diluted by 1: 900TBS into protein, mixing thoroughly, reacting for 10min to combine with digoxin-labeled ssDNA in protein;
the TBS is 0.5M Tris-NaCl solution, and the preparation method comprises the following steps: dissolving 8.5-9 g NaCl in water, adding 100ml of Tris-HCl (0.5M, pH7.6) solution, and finally adding water to a constant volume of 1L; preparation method of 0.5M Tris-HCl (pH7.6, 100ml) solution: weighing 6.06g of Tris, adding 40ml of double distilled water for dissolution, dropwise adding concentrated HCl to adjust the pH value to 7.6, and fixing the volume to 100 ml.
2.2.4.5 washing: centrifuging at 6000rpm, removing supernatant, and washing with 1 × 500 μ L buffer solution for 3 times to obtain protein;
2.2.4.6TMB (tetramethylbenzidine) color development: adding 400 mu L of double distilled water resuspended protein, then adding 200 mu L of TMB color development solution, after color development for 10min in a dark place, terminating the reaction by 2mol/L H2SO4200 mu L, measuring the absorbance OD450 at 450nm, wherein the absorbance OD450 reflects the affinity of ssDNA bound with bacteria, namely OD binding, and carrying out the steps 2.2.4.3, 2.2.4.4, 2.2.4.5 and 2.2.4.6 in the blank to obtain the blank corresponding to the absorbance OD blank;
the TMB color developing solution is prepared by using a conventional preparation method.
2.2.4.7 determination of the molar concentration of DNA in the PCR product: taking the PCR product obtained by the amplification in the step 2.2.4.1, taking an initial ssDNA library with a known concentration gradient as a standard, taking Bandscan software as image analysis software, quantitatively measuring the DNA content in the PCR product by ethidium bromide agarose gel electrophoresis to obtain the molar concentration of the corresponding DNA, and further calculating the DNA mole number in 100 mu L of the PCR product.
2.2.4.8 calculation of the affinity of the corresponding library:
2.3 repeated screening, the specific method is as follows: and (3) taking the product of each round of asymmetric PCR as a screening library of the next round, repeating the SELEX screening step 2.2 until the affinity is not increased any more, and finally obtaining the aptamer enrichment library of the ssDNA through 16 rounds of screening. After asymmetric PCR amplification, under the same conditions as the previous steps, cloning and sequencing to obtain 20 effective ssDNAs with the highest copy number, and respectively carrying out affinity specificity verification on 20 aptamers to obtain 10 oligonucleotide sequences (aptamers) with better affinity specificity to the functional region protein of the sea purse angiogenesis inhibitor 1, wherein the specific sequences are as follows:
the affinity data are as follows:
aptamer name
|
Affinity of
|
Aptamer name
|
Affinity of
|
K2
|
0.62
|
K15
|
0.43
|
K6
|
0.53
|
K16
|
0.51
|
K9
|
0.50
|
K17
|
0.59
|
K10
|
0.47
|
K19
|
0.62
|
K14
|
0.60
|
K20
|
0.49 |
2.4 analysis of specificity and affinity of 20 aptamers
Incubating the fluorescence-labeled aptamer sequences with the functional domain protein of the sea purse angiogenesis inhibiting factor 1, performing flow cytometry detection, wherein 10 sequences show high fluorescence intensity, performing a nonlinear regression curve on a saturation curve by using GraphPad prism5.0 software, and performing the same experimental operation on 10 high-affinity aptamer sequences respectively to obtain Kd values each of which is configured:
aptamer name
|
Kd value (nM)
|
Aptamer name
|
Kd value (nM)
|
K2
|
35.27
|
K15
|
59.23
|
K6
|
51.73
|
K16
|
38.97
|
K9
|
43.81
|
K17
|
44.83
|
K10
|
50.46
|
K19
|
34.57
|
K14
|
35.89
|
K20
|
47.81 |
The Kd value of K20 is the smallest, which indicates that the protein can be combined with the target protein rapidly and has stable structure and is not easy to separate.
The DNAMAN software is adopted to construct the secondary structures of 10 aptamers and calculate the minimum free energy of the aptamers, the minimum free energy of the structures is small, and the structures are relatively stable.
2.5 aptamer specificity assay
The specificity detection is respectively carried out by adopting BSA, human hemoglobin, ray angiogenesis inhibiting factor 1 functional zone protein and 10 aptamers, and the combination test shows that the 10 sequences are not combined with the BSA or the human hemoglobin, but are only combined with the ray angiogenesis inhibiting factor 1 functional zone protein to keep higher specificity.
Sequence listing
110 Zhang Yong
Aptamer K20 of < 120 > ray angiogenesis inhibitor 1, and screening method and application thereof
〈160〉13
〈210〉1
〈211〉225
〈212〉PRT
Ray (213)
〈400〉1
TLDIYKQLRD KETPSGFTLD DVIQTGVDNP GHPFIMTVGC VAGDEESYEV FKALFDPVIQ 60
DRHGGYKPTD KHKTDLNHEN LKGGDDLDPN YVLSSRVRTG RSIKGIALPP HCSRGERRLV 120
EKLCLEGLAT LTGEFQGKYY PLTTMSDAEQ QQLIDDHFLF DKPVSPLLLA SGMARDWPDA 180
RGIWHNNDKT FLVWVNEEDH LRVISMQKGG NMKEVFRRFC VGLKK 225
〈210〉2
<211〉 84
〈212〉DNA
Artificial sequence of < 213 >
〈400〉K2
1 TCAGTCGCTT CGCCGTCTCC TTCATGATCG CGCTGACAAA TTAGGCCATT CAATCAGAGC
61 ACAAGAGGGA GACCCCAGAG GG
〈210〉3
<211〉 84
〈212〉DNA
Artificial sequence of < 213 >
〈400〉K6
1 TCAGTCGCTT CGCCGTCTCC TTCCCGTGAT GAATTGCTGA TGAGCGCAGC ATGGAGCTGC
61 ACAAGAGGGA GACCCCAGAG GG
〈210〉4
<211〉 84
〈212〉DNA
Artificial sequence of < 213 >
〈400〉K9
1 TCAGTCGCTT CGCCGTCTCC TTCTGACGCA TTCGGATCCA AGTTAATTAA ATAACTGCGC
61 ACAAGAGGGA GACCCCAGAG GG
〈210〉5
<211〉 84
〈212〉DNA
Artificial sequence of < 213 >
〈400〉K10
1 TCAGTCGCTT CGCCGTCTCC TTCATTGCAA CCTGAGGCCA TGGGACAGAC CATGATAGGC
61 ACAAGAGGGA GACCCCAGAG GG
〈210〉6
<211〉 84
〈212〉DNA
Artificial sequence of < 213 >
〈400〉K14
1 TCAGTCGCTT CGCCGTCTCC TTCAACTTGG ACCCTTGAGC GATGAAGTAA CGGTTTACGC
61 ACAAGAGGGA GACCCCAGAG GG
〈210〉7
<211〉 84
〈212〉DNA
Artificial sequence of < 213 >
〈400〉K15
1 TCAGTCGCTT CGCCGTCTCC TTCGCACTGC TACCGATATT ACATATATGG AGATACAGGC
61 ACAAGAGGGA GACCCCAGAG GG
〈210〉8
<211〉 84
〈212〉DNA
Artificial sequence of < 213 >
〈400〉K16
1 TCAGTCGCTT CGCCGTCTCC TTCGGCCGAG TAACAGATTG GAACCCAACT GAGTGAGAGC
61 ACAAGAGGGA GACCCCAGAG GG
〈210〉9
<211〉 84
〈212〉DNA
Artificial sequence of < 213 >
〈400〉K17
1 TCAGTCGCTT CGCCGTCTCC TTCTTATGGA CGAGTAGAGG TACGATGACC CAATGATTGC
61 ACAAGAGGGA GACCCCAGAG GG
〈210〉10
<211〉 84
〈212〉DNA
Artificial sequence of < 213 >
〈400〉K19
1 TCAGTCGCTT CGCCGTCTCC TTCCGATTGA GGGAGATTAC GCATATGAGT ACAACTGAGC
61 ACAAGAGGGA GACCCCAGAG GG
〈210〉11
<211〉 84
〈212〉DNA
Artificial sequence of < 213 >
〈400〉K20
1 TCAGTCGCTT CGCCGTCTCC TTCTTTAGAC CCGATAATGT TGTTTTGGTG ACCGAATTGC
61 ACAAGAGGGA GACCCCAGAG GG
〈210〉12
<211〉23
〈212〉DNA
Artificial sequence of < 213 >
〈400〉P1
TCAGTCGCTT CGCCGTCTCC TTC
〈210〉13
<211〉 24
〈212〉DNA
Artificial sequence of < 213 >
〈400〉P2
CCCTCTGGGG TCTCCCTCTT GTGC