CN105567698B - The aptamer K19 and its screening technique of ray Angiostatin 1 and application - Google Patents

The aptamer K19 and its screening technique of ray Angiostatin 1 and application Download PDF

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CN105567698B
CN105567698B CN201610101377.5A CN201610101377A CN105567698B CN 105567698 B CN105567698 B CN 105567698B CN 201610101377 A CN201610101377 A CN 201610101377A CN 105567698 B CN105567698 B CN 105567698B
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aptamer
functional areas
ray angiostatin
albumen
ray
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沈巍
张垒
张勇
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Qingdao English bioscience Biotechnology Co., Ltd.
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Abstract

One group of aptamer and preparation method thereof that can identify 1 functional areas albumen of ray Angiostatin.Oligonucleotide sequence includes No.2~11 SEQ ID, is respectively provided with higher affine specificity, can be used for the detection of 1 functional areas albumen of ray Angiostatin.

Description

The aptamer K19 and its screening technique of ray Angiostatin 1 and application
Technical field
The invention belongs to biotechnologies, specifically, the present invention relates to a kind of ray Angiostatin 1 adaptations Son and its screening technique and application.
Background technology
In recent years, promising replacement molecule of the oligonucleotide aptamer as antibody molecule, research more induces one to note Mesh.Oligonucleotide aptamer is by SELEX technologies (Systematic evolution of ligands by exponential Enrichment) biological libraries technology screening obtains, and the principle of the technology is exactly to utilize Protocols in Molecular Biology, and structure is artificial The single-stranded random oligonucleotide library of synthesis, random sequence length is in 20-100 base or so.Utilize single stranded oligonucleotide The flexible and changeable characteristic of molecular configurations interacts random oligonucleotide library and target molecule, retains with spatial conformation and target The oligonucleotides that molecule combines, through repeated amplification, the several cycles of screening, you can make the oligonucleotides sequence with the target specific bond Row are enriched with, and the specific oligonucleotide acid aptamer of a variety of target molecules, i.e. aptamer are finally obtained.Utilize SELEX technology screenings The pattern of the aptamer identification molecules of acquisition is similar with protein antibodies, but compared with protide antibody, nucleic acid aglucon has more More superiority can be external artificial synthesized if do not limited by immune condition and immunogenicity, and denaturation is reversible with renaturation, can modify And be conducive to long-term preservation and room temperature transport etc..Importantly, aptamer has higher specificity or even energy than antibody Identify the undistinguishable protein molecule of monoclonal antibody.And the target molecule of aptamer is very extensive, as low as dye molecule, and it is big to complete Whole virion and bacterial pathogens, or even complete cell can also go out high-affinity by cutting down SELEX technology screenings Oligonucleotide aptamer.
1 functional areas of ray Angiostatin hereinafter referred to as ray functional areas, ray Angiostatin 1 are that we are first The protein (molecular weight 42KD) separated in a kind of secondary ray tissue from South China Sea.Result of study is shown：Ray angiogenesis Inhibiting factor 1 significantly inhibits chick chorioallantoic membrane itself and KB cell's induction chick chorioallantoic membrane blood vessel life At；Either intraperitoneal injection or gavage all significantly inhibit the growth and transfer of nude mouse Lewis lung cancer, and it is micro- to reduce tumor tissues Vessel density lowers the expression of angiogenic factors VEGF；Also there is high inhibition to the transfer of nude mouse Melanoma B16 cell Effect；Lower the expression for promoting transfer factor CD44v6 and ErBb2；It is more aobvious with effect when 5 FU 5 fluorouracil (5-FU) use in conjunction It writes.The anti-cancer drugs Avastin that the action target spot of ray Angiostatin 1 is developed with gene technology research institute of the U.S. is not Together.Avastin is gene engineering product, is the antibody of VEGF, and action target spot is VEGF；Ray Angiostatin 1 is Natural products, it acts not only on VEGF, also acts on bFGF and PDGF.Therefore, for 1 function of ray Angiostatin It is the consistent pursuit in this field that area, which carries out the discriminating of specificity and screening,.
Based on considerations above, the present invention is used using 1 functional areas albumen of ray Angiostatin as purpose target protein SELEX technologies obtain 10 special aptamer of 1 functional areas albumen of ray Angiostatin, and combination application can be fast Fast, sensitive, special detects 1 functional areas albumen of ray Angiostatin.Due to single strand dna oligonucleotide aptamer It can stablize, synthesize convenient and inexpensively, after modification can be directly used for fluorescence or chemiluminescence, chromophoric method detection target bandage, because This is easy to operate, direct.
Invention content
The purpose of the present invention is to provide not only having the characteristics that quick, the easy to operate, stability of detection is higher than antibody, and And one group of few nucleosides that can identify 1 functional areas albumen of ray Angiostatin that preparation method is easy, manufacturing cycle is shorter Acid sequence and preparation method thereof.
The oligonucleotide sequence that can identify 1 functional areas albumen of ray Angiostatin, including SEQ ID No.2-11, and can be completed to 1 functional areas albumen of ray Angiostatin using an oligonucleotide sequence therein Recognition detection；
The preparation method packet of the described one group oligonucleotide sequence that can identify 1 functional areas albumen of ray Angiostatin Include following steps：
1, ssDNA oligonucleotide library (5 '-TCA GTC GCT TCG CCG TCT CCTs of the synthesis for screening TC----
N35----GCA CAA GAG GGA GAC CCC AGA GGG-3 '), wherein N35 is 35 random oligonucleotides；
2, SELEX sieves are carried out after mixing oligonucleotide library with 1 functional areas albumen of ray Angiostatin respectively Choosing obtains aptamer enriched library；
3, after the completion of SELEX screenings, cloning and sequencing is carried out to the aptamer enriched library of acquisition；
4, the high copy ssDNA occurred in sequencing result is selected, carries out the verification of affine specificity, screening is obtained and can be identified The oligonucleotide sequence of 1 functional areas albumen of ray Angiostatin.
Specific implementation mode
Embodiment 1
1, the preparation of 1 functional areas albumen of ray Angiostatin
Using yeast well known to those skilled in the art recombinant expression by the way of obtain with ray Agiogenesis inhibition because The 1 functional areas albumen of ray Angiostatin of the identical bioactivity of 1 functional areas albumen of son, the protein sequence such as SEQ ID NO：Shown in 1；A concentration of 15mg/ml of protein solution.
2, the synthesis in library and primer
2.1, ssDNA oligonucleotide library (5 '-TCA GTC GCT TCG CCG TCT CCTs of the synthesis for screening TC----
N35----GCA CAA GAG GGA GAC CCC AGA GGG-3 '), wherein N35 is 35 random oligonucleotides；
Primer P1：TCAGTCGCTTCGCCGTCTCCTTC；
Primer P2：CCCTCTGGGGTCTCCCTCTTGTGC.
2.2, the SELEX screenings of aptamer, the specific method is as follows：
2.2.1 the combination of 1 functional areas albumen of ssDNA and ray Angiostatin, detach, the specific method is as follows：
The 4 μ L of ssDNA oligonucleotide libraries for taking 100 μM are diluted to 100 μ l, 95 DEG C of denaturation with 2 × combination buffer It is added 100 μ l rays Angiostatin, 1 functional areas albumen after 5min, ice bath 10min, shaking table combination 30min, then 6000rpm centrifuges 5min, abandons supernatant, then washes precipitation with 1 × combination buffer, abandon supernatant；1 × combination is added in precipitation 100 μ L of buffer solution, 96 DEG C of heating 5min, then 15000rpm centrifugations 10min, takes supernatant, and precipitation is heated and centrifuged again, Merge supernatant, then separates the ssDNA grade text for obtaining having affinity with 1 functional areas albumen of ray Angiostatin Library；2 × the combination buffer is that 20 × combination buffer distilled water dilutes the solution after 10 times, described 1 × combination buffering Liquid is that 20 × combination buffer distilled water dilutes the solution after 20 times；20 × combination buffer formula be 1M NaCl, 50mM KCl、500mM Tris-HCl、10mM MgCl2、pH 7.4。
2.2.2ssDNA it and the combination of 1 functional areas albumen of ray Angiostatin, detaches, the specific method is as follows：
By step 2.2.1 it is isolated can with the protein bound ssDNA in 1 functional areas of ray Angiostatin, then With 100 μ l rays Angiostatin, 1 functional areas albumen shaking table combination 30min, subsequent step can then divide with step 2.2.1 From to the ssDNA secondary libraries for having affinity with 1 functional areas albumen of ray Angiostatin.
2.2.3 asymmetric PCR expands ssDNA, and the specific method is as follows：
Asymmetric PCR amplification carried out to the ssDNA secondary libraries that step 2.2.2 separation obtains, total volume be 25 μ l not Non-symmetric PCR amplification system is：10 × PCR buffer solutions：2μl；P1(10μM)：1μl；P2(0.2μM)：1μl；DNTP (each 2.5mM)： 0.4μl；MgCl2(25mM)：1.2μl；SsDNA templates (0.2 μ g/ μ l)：2μl；Taq archaeal dna polymerases (5u/ μ l)：0.2μl； ddH2O：17.2μl；PCR response parameters：Then 94 DEG C of pre-degeneration 4min carry out 40 cycles, 94 DEG C of denaturation 30s, 58 DEG C of annealing 30s, 72 DEG C of extensions 20s, last 72 DEG C of extensions 7min；
2.2.4 the measurement of affinity, the specific method is as follows：
2.2.4.1 amplification：The ssDNA grade screened is expanded with the primer P1 asymmetric PCRs with digoxigenin labeled Library, amplification condition and parameter are identical as the asymmetric PCR amplification system and parameter of step 2.2.3；
2.2.4.2 with protein binding：The 100 μ L of PCR product of step 2.2.4.1 amplification gained are taken, 95 DEG C are denaturalized 5min, ice It is added in 100 μ L albumen, is sufficiently mixed after bath 10min, combine 30min at room temperature, then 6000rpm is centrifuged, protein isolate With supernatant, include in albumen with the ssDNA with digoxigenin labeled that is combined in albumen, be unbonded in supernatant SsDNA, while doing a blank for being not added with ssDNA, i.e., PCR product is replaced with 2 × combination buffer, equally carries out aforesaid operations；
2.2.4.3 washing：Albumen is washed 1 time with 1 × combination buffer, 500 μ L, 6000rpm centrifugations abandon supernatant, take egg In vain；
2.2.4.4 it is combined with enzyme mark rabbit-anti DigiTAb：100 μ L, 1: 900TBS diluted excess is added in albumen Enzyme mark rabbit-anti DigiTAb, after being sufficiently mixed, react 10min, be allowed to and the ssDNA of the digoxigenin labeled in albumen tie It closes；
The TBS is 0.5M Tris-NaCl solution, and preparation method is：First water-soluble 8.5~9g NaCl, then add Tris- HCl (0.5M, pH7.6) solution 100ml, finally plus water is settled to 1L；0.5M Tris-HCl (pH7.6,100ml) solution is prepared Method：Tris 6.06g are weighed, distilled water 40ml dissolvings are added, dense HCl tune pH to 7.6 is added dropwise, is settled to 100ml.
2.2.4.5 washing：6000rpm is centrifuged, and is removed supernatant, then washed 3 times with 1 × combination buffer, 500 μ L, is obtained albumen；
2.2.4.6TMB (tetramethyl benzidine) develops the color：400 μ L distilled waters are added, albumen is resuspended, adds 200 μ L TMB Developing solution after being protected from light colour developing 10min, terminates reaction with 2mol/L H2SO4200 μ L, measures the light absorption value OD450 at 450nm, The value reflects that the affinity of the ssDNA combined with bacterium, i.e. OD combine, and blank equally carries out above-mentioned steps 2.2.4.3, 2.2.4.4,2.2.4.5 and 2.2.4.6 obtains the corresponding absorbance OD blank of blank；
The TMB developing solutions are prepared using conventional preparation method.
2.2.4.7 the molar concentration of DNA in PCR product is measured：The PCR product for taking step 2.2.4.1 amplification gained, with Know that the initial libraries ssDNA of concentration gradient are standard items, uses Bandscan softwares as image analysis software, using ethidium bromide Agarose gel electrophoresis method quantitative determines the DNA content in PCR product, obtains the molar concentration of corresponding DNA, and then can calculate Go out the DNA molal quantitys in 100 μ L PCR products.
2.2.4.8 calculating the affinity in corresponding library：
2.3 repeat to screen, and specific method is：Screening library using the product of each round asymmetric PCR as next round, weight Multiple above-mentioned SELEX screening steps 2.2, until affinity no longer rises, the screening for finally passing through 16 wheels obtains the suitable of ssDNA Gamete enriched library.After asymmetric PCR expands, the step of condition is with front, clones and be sequenced, obtain copy number highest 20 20 aptamers are carried out affine specificity verification, obtain 10 to ray Angiostatin by a effective ssDNA respectively 1 functional areas albumen has the oligonucleotide sequence (aptamer) of preferable affine specificity, and particular sequence is as follows：
The specific data of affinity are as follows：
Aptamer title Affinity Aptamer title Affinity
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, the specificity and compatibility of 20 aptamers are analyzed
The adaptor sequence of fluorescent marker is incubated with 1 functional areas albumen of ray Angiostatin, carries out fluidic cell Analysis detection, wherein 10 sequences show high fluorescent, is done using GraphPad Prism5.0 softwares for saturation curve Nonlinear regression curve uses identical experimental implementation to 10 high-affinity adaptor sequences respectively, and it is to match to have obtained every The Kd values set：
Aptamer title Kd values (nM) Aptamer title Kd values (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 3589 K20 4781
The Kd values of wherein K20 are minimum, and explanation can quickly be combined with target protein and stable structure is not readily separated.
Using 10 aptamers of DNAMAN software buildings secondary structure and calculate their minimum free energy, tie Structure minimum free energy is also all smaller, and structure is also stablized relatively.
2.5 aptamer specificity are analyzed
BSA, human hemoglobin, 1 functional areas albumen of ray Angiostatin and 10 aptamers are respectively adopted to carry out Specific detection, by combining experiment to find that this 10 sequences are not combined with BSA or human hemoglobin, and only with ray blood Pipe generates 1 functional areas protein binding of inhibiting factor and keeps higher specificity.
Sequence table
＜ 110 ＞ is brave
The aptamer K19 and its screening technique of 120 ＞ rays Angiostatins 1 of ＜ and application
〈160〉13
〈210〉1
〈211〉225
〈212〉PRT
213 ＞ rays of ＜
〈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
213 ＞ artificial sequences of ＜
〈400〉K2
1 TCAGTCGCTT CGCCGTCTCC TTCATGATCG CGCTGACAAA TTAGGCCATT CAATCAGAGC
61 ACAAGAGGGA GACCCCAGAG GG
〈210〉3
<211〉 84
〈212〉DNA
213 ＞ artificial sequences of ＜
〈400〉K6
1 TCAGTCGCTT CGCCGTCTCC TTCCCGTGAT GAATTGCTGA TGAGCGCAGC ATGGAGCTGC
61 ACAAGAGGGA GACCCCAGAG GG
〈210〉4
<211〉 84
〈212〉DNA
213 ＞ artificial sequences of ＜
〈400〉K9
1 TCAGTCGCTT CGCCGTCTCC TTCTGACGCA TTCGGATCCA AGTTAATTAA ATAACTGCGC
61 ACAAGAGGGA GACCCCAGAG GG
〈210〉5
<211〉 84
〈212〉DNA
213 ＞ artificial sequences of ＜
〈400〉K10
1 TCAGTCGCTT CGCCGTCTCC TTCATTGCAA CCTGAGGCCA TGGGACAGAC CATGATAGGC
61 ACAAGAGGGA GACCCCAGAG GG
〈210〉6
<211〉 84
〈212〉DNA
213 ＞ artificial sequences of ＜
〈400〉K14
1 TCAGTCGCTT CGCCGTCTCC TTCAACTTGG ACCCTTGAGC GATGAAGTAA CGGTTTACGC
61 ACAAGAGGGA GACCCCAGAG GG
〈210〉7
<211〉 84
〈212〉DNA
213 ＞ artificial sequences of ＜
〈400〉K15
1 TCAGTCGCTT CGCCGTCTCC TTCGCACTGC TACCGATATT ACATATATGG AGATACAGGC
61 ACAAGAGGGA GACCCCAGAG GG
〈210〉8
<211〉 84
〈212〉DNA
213 ＞ artificial sequences of ＜
〈400〉K16
1 TCAGTCGCTT CGCCGTCTCC TTCGGCCGAG TAACAGATTG GAACCCAACT GAGTGAGAGC
61 ACAAGAGGGA GACCCCAGAG GG
〈210〉9
<211〉 84
〈212〉DNA
213 ＞ artificial sequences of ＜
〈400〉K17
1 TCAGTCGCTT CGCCGTCTCC TTCTTATGGA CGAGTAGAGG TACGATGACC CAATGATTGC
61 ACAAGAGGGA GACCCCAGAG GG
〈210〉10
<211〉 84
〈212〉DNA
213 ＞ artificial sequences of ＜
〈400〉K19
1 TCAGTCGCTT CGCCGTCTCC TTCCGATTGA GGGAGATTAC GCATATGAGT ACAACTGAGC
61 ACAAGAGGGA GACCCCAGAG GG
〈210〉11
<211〉 84
〈212〉DNA
213 ＞ artificial sequences of ＜
〈400〉K20
1 TCAGTCGCTT CGCCGTCTCC TTCTTTAGAC CCGATAATGT TGTTTTGGTG ACCGAATTGC
61 ACAAGAGGGA GACCCCAGAG GG
〈210〉12
<211〉23
〈212〉DNA
213 ＞ artificial sequences of ＜
〈400〉P1
TCAGTCGCTT CGCCGTCTCC TTC
〈210〉13
<211〉 24
〈212〉DNA
213 ＞ artificial sequences of ＜
〈400〉P2
CCCTCTGGGG TCTCCCTCTT GTGC

Claims (2)

1. a kind of oligonucleotide sequence of 1 functional areas albumen of identification ray Angiostatin, it is characterised in that its sequence is Shown in SEQ ID No.10.
2. oligonucleotide sequence shown in claim 1 is used to screen the application of 1 functional areas albumen of ray Angiostatin.
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CN105693849A (en) * 2014-07-13 2016-06-22 马海龙 Aptamer NKXA5 specifically for Neutrokine-alpha protein and application of aptamer NKXA5
CN105693848A (en) * 2014-07-13 2016-06-22 马海龙 Aptamer NKXA7 specifically for Neutrokine-alpha protein and application of aptamer NKXA7

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