CN108866063A - A kind of aptamer and its preparation method and application of PEG modification - Google Patents
A kind of aptamer and its preparation method and application of PEG modification Download PDFInfo
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- CN108866063A CN108866063A CN201810687339.1A CN201810687339A CN108866063A CN 108866063 A CN108866063 A CN 108866063A CN 201810687339 A CN201810687339 A CN 201810687339A CN 108866063 A CN108866063 A CN 108866063A
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- aptamer
- peg
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- preparation
- peg modification
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
- C12N15/115—Aptamers, i.e. nucleic acids binding a target molecule specifically and with high affinity without hybridising therewith ; Nucleic acids binding to non-nucleic acids, e.g. aptamers
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/70—Carbohydrates; Sugars; Derivatives thereof
- A61K31/7088—Compounds having three or more nucleosides or nucleotides
- A61K31/711—Natural deoxyribonucleic acids, i.e. containing only 2'-deoxyriboses attached to adenine, guanine, cytosine or thymine and having 3'-5' phosphodiester links
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/12—Antivirals
- A61P31/14—Antivirals for RNA viruses
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2310/00—Structure or type of the nucleic acid
- C12N2310/10—Type of nucleic acid
- C12N2310/16—Aptamers
Abstract
The invention discloses a kind of aptamers and its preparation method and application of PEG modification.The present invention carries out PEG modification for GCRV aptamer, with the PEG in different reaction time, reaction ratio different molecular weight is modified and filters out optimal reaction condition, and does not influence cell viability and to GCRV infected with inhibiting effect.The aptamer for the GCRV that the present invention modifies has the application prospect of preparation viral disease therapeutic agent, it may have prepares the application prospect of GCRV molecular probe, detection reagent.
Description
Technical field
The invention belongs to field of biological detection, and in particular to a kind of nucleic acid adaptation of the grass carp reovirus of PEG modification
Preparation and effect.
Background technique
Hemorrhagic disease of grass carp caused by grass carp reovirus (GCRV), causes fish body mortality, the death rate be up to 85% with
On cause the loss of grass carp aquaculture serious.Hemorrhagic disease of grass carp is once broken out, currently without effectively antiviral treatment method.Cause
This, hemorrhagic disease of grass carp becomes a bottleneck of grass carp cultivation, seriously constrains the cultivation of grass carp.
Aptamers (Aptamer) are a kind of oligonucleotide sequences (RNA or DNA) obtained through in-vitro screening technology, with phase
The ligand answered has the affinity of stringent recognition capability and height.Aptamers have that high specific, target molecule are wide, are easy to close in vitro
At the advantages that, wide application prospect is shown in basic research, clinical diagnosis and treatment.But the adaptation being not decorated
Body is degraded quickly in vivo, plays a role and has a significant impact in an experiment to it.
Summary of the invention
It is an object of the present invention to provide a kind of aptamers of PEG modification.
The object of the invention is another to be to provide a kind of aptamer preparation method of PEG modification.
The object of the invention is further to provide a kind of application of the aptamer of PEG modification.
The technical solution adopted by the present invention is that：
A kind of aptamer preparation method of PEG modification, it is shown that steps are as follows：
1) it is vibrated after mixing PEG, EDCHCl and NHS；
2) aptamer is first added in the MES buffer that pH is 4~7, is then added to the mixed liquor of step 1) preparation,
Vibrate the aptamer modified after 2~5h to get PEG.
Further, PEG described in step 1)：EDC·HCl：The molar ratio of NHS is 2~6：2~6：1.
Further, the 10~25min of concussion reaction under the speed of 60~100rpm of oscillation described in step 1).
Further, the 2~5h of concussion reaction under the speed of 60~100rpm of oscillation described in step 2).
Further, the volume ratio of PEG, aptamer described in step 2) and MES buffer is 1:10~100：1.
Further, the aptamer is the aptamer of grass carp reovirus.
The aptamer of the PEG modification of method preparation described in any of the above embodiments.
Further, the aptamer of PEG modification is the grass carp reovirus nucleic acid adaptation of PEG modification
Body.
The aptamer of PEG modification described above is in preparation prevention and/or treatment grass carp reovirus kit
Application.
Further, the aptamer of PEG modification is the nucleic acid adaptation of the PEG modification of grassland reovirus
Body.
The beneficial effects of the invention are as follows：
The GCRV aptamer of PEG modification prepared by the present invention, with different reaction time, reaction ratio different molecular
The PEG of amount is modified and is filtered out optimal reaction condition, and stability is good and does not influence cell viability, and to GCRV's
Infected with inhibiting effect, there is the application prospect of preparation viral disease therapeutic agent, it may have preparation GCRV molecular probe, inspection
The application prospect of test agent.
Detailed description of the invention
Fig. 1 is the gel electrophoresis figure of PEG- aptamer of the present invention, 1DNA marker；2PEG modification of nucleic acids aptamers
4h is reacted in 80rpm；3PEG modification of nucleic acids aptamers react 30min in 80rpm；4 aptamers.
Fig. 2 is the gel electrophoresis figure of the PEG- aptamer of differential responses ratio of the present invention, 1DNA marker；2 nucleic acid
Aptamers：PEG is 1:1；3 aptamers：PEG is 1:30；4 aptamers：PEG is 1:50；5 aptamers：PEG is
1:80；6 aptamers：PEG is 1:100.
Fig. 3 is the gel electrophoresis figure for the aptamer that the PEG of different molecular weight is modified, 1.marker；2.PEG 2000
Modification of nucleic acids is adapted to precursor reactant 4h；3.PEG 5000 modification of nucleic acids are adapted to precursor reactant 4h；7500 modification of nucleic acids aptamers of 4.PEG
React 4h；2000 modification of nucleic acids of 5.PEG is adapted to precursor reactant 2h；5000 modification of nucleic acids of 6.PEG is adapted to precursor reactant 2h；
7.PEG7500 modification of nucleic acids is adapted to precursor reactant 2h；8. aptamer.
Fig. 4 is that MTT measures cell viability.
After Fig. 5 is modification of nucleic acids aptamers and GCRV while being added to cell, the inhibitory effect of aptamer.
Fig. 6 is that GCRV, the inhibitory effect of aptamer is added in the addition of modification of nucleic acids aptamers afterwards for 24 hours.
Fig. 7 is that GCRV infects for 24 hours addition modification of nucleic acids aptamers afterwards, the inhibitory effect of aptamer.
Specific embodiment
The present invention is further illustrated with reference to the accompanying drawing, but is not limited to embodiment.
1 virus and cell
Fat head Minnow muscle cell cell (FHM cell), GCRV are saved by this laboratory；GCRV be stored in -80 DEG C it is spare.
28 DEG C of FHM cell are incubated at the fetal calf serum M199 culture medium containing 10%.
The characteristic of 2DNA synthesis and aptamer
It screens to obtain suitable aptamer by SELEX method.Aptamer library is made in 95 DEG C of effect 5min
It is denaturalized, and is then quickly placed at and places 10min on ice.The plank of coated GCRV is added in the aptamer library of denaturation, in 4 DEG C
It is incubated for 60min.Then plank is eluted 3 times with PBS, removes unbound sequence.By plank in 95 DEG C of effect 10min, make to bind
Sequence dissociation, recycles the sequence of binding.The sequence of recycling carries out PCR amplification as template, separates the ssDNA of acquisition for next
Wheel screening.Reversed screening is added in every 5 wheel, i.e., is coated with plank with cell liquid, unbound sequence is recycled after incubation.It is gradually increased and incubates
Temperature is educated, is gradually increased to 28 DEG C from 4 DEG C.12 wheel of screening is repeated, screening is terminated.
FT6:CATTGTGTTATACGAGAATGACTCCGTTAAAATT(SEQ ID NO.1)
FT7:GACAGTATACTATATTTTTTCTAAATGTTTCTTTT(SEQ ID NO.2)
FT8:TACCGAGTGGTAGAATTTGCTTGGATTATTTTTTA(SEQ ID NO.3)
FT9:GATTGTGCAACGTTCCTGTCGTGTTGTCCATAGAT(SEQ ID NO.4)
Embodiment 1
1) by 1- (3- dimethyl aminopropyl) -3- ethyl-carbodiimide hydrochloride of PEG5000,0.400M (also known as EDC
HCl) and the n-hydroxysuccinimide of 0.100M (NHS) molar ratio is 4：4：After 1 (each 50 μ L) mixing, in 80rpm item
Concussion reaction 15min under part.
It 2) will be 1 with PEG volume ratio:30 FT6 aptamer is added to the 2- (N- morpholine) that the pH of 50 μ L is 6
Ethanesulfonic acid buffer (also known as MES buffer) is then added to the mixed liquor of step 1) preparation, 4h is shaken under the conditions of 80rpm, reacts
After carry out detected through gel electrophoresis.
Embodiment 2
1) by 1- (3- dimethyl aminopropyl) -3- ethyl-carbodiimide hydrochloride of PEG5000,0.400M (also known as EDC
HCl) and the n-hydroxysuccinimide of 0.100M (NHS) molar ratio is 5：5：After 1 (each 50 μ L) mixing, in revolving speed
Concussion reaction 10min under the conditions of 80rpm.
It 2) will be 1 with PEG volume ratio:10 FT7 aptamer is added to 50 μ L, the pH that concentration is 20ml/L is 4
2- (N- morpholine) ethanesulfonic acid buffer (also known as MES buffer) is then added to the mixed liquor of step 1) preparation, is in revolving speed
2h is shaken under the conditions of 60rpm, carries out detected through gel electrophoresis after reaction.
Embodiment 3
A kind of aptamer preparation method of PEG modification, it is shown that steps are as follows：
1) by 1- (3- dimethyl aminopropyl) -3- ethyl-carbodiimide hydrochloride of PEG5000,0.400M (also known as EDC
HCl) and the n-hydroxysuccinimide of 0.100M (NHS) molar ratio is 2：2：After 1 (each 50 μ L) mixing, in revolving speed
20min is shaken under the conditions of 100rpm.
It 2) will be 1 with PEG volume ratio:50 FT8 aptamer is added to the 2- (N- morpholine) that the pH of 50 μ L is 7
Ethanesulfonic acid buffer (also known as MES buffer) is then added to the mixed liquor of step 1) preparation, shakes under the conditions of revolving speed is 80rpm
5h carries out detected through gel electrophoresis after reaction.
Embodiment 4
A kind of aptamer preparation method of PEG modification, it is shown that steps are as follows：
1) by 1- (3- dimethyl aminopropyl) -3- ethyl-carbodiimide hydrochloride of PEG5000,0.400M (also known as EDC
HCl) and the n-hydroxysuccinimide of 0.100M (NHS) molar ratio is 6：6：After 1 (each 50 μ L) mixing, in revolving speed
25min is shaken under the conditions of 80rpm.
It 2) will be 1 with PEG volume ratio:100 FT9 aptamer is added to the 2- (N- morpholine) that the pH of 50 μ L is 6
Ethanesulfonic acid buffer (also known as MES buffer) is then added to the mixed liquor of step 1) preparation, shakes under the conditions of revolving speed is 80rpm
5h carries out detected through gel electrophoresis after reaction.
1. the aptamer effect that the PEG of differential responses time is modified
As the result is shown：The amount of the amount 4h of PEG modification of nucleic acids aptamers is significantly more than 30min reacting dose (Fig. 1).
2. modifying the aptamer effect of the PEG of different proportion
As the result is shown：Choose aptamer：PEG is that molar ratio is 1:1；1:30；1:50；1:80；1:100 ratios into
Row reaction, gel electrophoresis result, which is shown, works as aptamer：PEG molar ratio is 1:When 30, aptamer modified effect
Preferably (Fig. 2).
The aptamer preparation method of the PEG of 3 modification different molecular sizes
As the result is shown：It is reacted, is coagulated with aptamer with PEG2000, PEG5000, PEG7500 of different molecular weight respectively
Gel electrophoresis detection, the band that discovery reacts 4 hours is brighter, therefore it is best (the 2nd~4 swimming lane of Fig. 3) to react 4 hours effects.
Further effect detection is made to the aptamer of the PEG of above-mentioned preparation below.
The measurement of 1 cell viability
Method：By FHM cell inoculation in 96 orifice plates, it is isometric to 80% when, be separately added into PEG2000- aptamer,
PEG5000- aptamer, PEG7500- aptamer, 28 DEG C of culture 48h add the MTT solution of every 20 μ l of hole, and 28
DEG C culture 4h, using elisa plate reader wavelength be 490nm read absorbance.
As a result：As shown in figure 4, PEG2000- aptamer, PEG5000- aptamer, PEG7500- nucleic acid adaptation
Body is added to FHM cell, and MTT solution is added in 28 DEG C of culture 48h, is read using elisa plate reader in 490nm and shows do not have
Significantly affect cell viability.
2 external inhibition virus infection experiments
Method：By FHM cell inoculation in 96 orifice plates, it is long to 80% when, carry out following three groups of experiments：(1) GCRV (MOI=
0.5) PEG2000- aptamer, PEG5000- aptamer, PEG7500- aptamer (300nM) are added simultaneously
FHM cell；(2) after FHM cell is infected 1 hour by GCRV (MOI=0.5), by PEG2000- aptamer, PEG5000- core
Sour aptamers, PEG7500- aptamer (300nM) are added in culture medium；(3) FHM cell is adapted to PEG2000- nucleic acid
Body, PEG5000- aptamer after PEG7500- aptamer (300nM) is incubated for 1 hour, are felt with GCRV (MOI=0.5)
Contaminate FHM cell.Finally, by FHM cell in 28 DEG C of culture 48h, and virus titer is measured using Reed-Muench method.
As a result：As a result as shown in Fig. 5~Fig. 7, PEG2000- aptamer, PEG5000- aptamer,
PEG7500- aptamer, which is added separately to FHM cell, can inhibit virus multiplication, and the aptamer of PEG modification can be with
Play the role of inhibiting virus infection.
The aptamer and GCRV of modification are added to cell (Fig. 5) simultaneously, the aptamer cell of modification shifts to an earlier date for 24 hours
Obvious (Fig. 7) is added for 24 hours than virus infection in the inhibitory effect of (Fig. 6) being added, the virus of both afterwards.Prove that PEG can prolong
The degradation of slow aptamer, the aptamer of PEG modification, which can extend, inhibits anti-grass carp reovirus infection
Effect.
The above embodiment is a preferred embodiment of the present invention, but embodiments of the present invention are not by above-described embodiment
Limitation, other any changes, modifications, substitutions, combinations, simplifications made without departing from the spirit and principles of the present invention,
It should be equivalent substitute mode, be included within the scope of the present invention.
SEQUENCE LISTING
<110>China's Pearl River Fishery Research Institute of Aquatic Science Research Institute
<120>A kind of aptamer and its preparation method and application of PEG modification
<130>
<160> 4
<170> PatentIn version 3.5
<210> 1
<211> 34
<212> DNA
<213>Artificial primer
<400> 1
cattgtgtta tacgagaatg actccgttaa aatt 34
<210> 2
<211> 35
<212> DNA
<213>Artificial primer
<400> 2
gacagtatac tatatttttt ctaaatgttt ctttt 35
<210> 3
<211> 35
<212> DNA
<213>Artificial primer
<400> 3
taccgagtgg tagaatttgc ttggattatt tttta 35
<210> 4
<211> 35
<212> DNA
<213>Artificial primer
<400> 4
gattgtgcaa cgttcctgtc gtgttgtcca tagat 35
Claims (10)
1. a kind of aptamer preparation method of PEG modification, which is characterized in that shown in steps are as follows：
1) it is vibrated after mixing PEG, EDCHCl and NHS；
2) aptamer is first added in the MES buffer that pH is 4~7, is then added to the mixed liquor of step 1) preparation, oscillation 2
To get the aptamer of PEG modification after~5h.
2. the method according to claim 1, wherein PEG described in step 1)：EDC·HCl：The molar ratio of NHS
It is 2~6：2~6：1.
3. the method according to claim 1, wherein oscillation described in step 1) is under the speed of 60~100rpm
10~25min of concussion reaction.
4. the method according to claim 1, wherein oscillation described in step 2) is under the speed of 60~100rpm
2~5h of concussion reaction.
5. the method according to claim 1, wherein PEG described in step 2), aptamer and MES buffering
The volume ratio of liquid is 1:10~100：1.
6. method according to claims 1 to 5, which is characterized in that the aptamer of the PEG modification is exhaled for grass carp
The aptamer of the PEG modification of the lonely virus of intestines.
7. the aptamer of the PEG modification of the described in any item method preparations of claim 1~6.
8. the aptamer of PEG modification according to claim 7, which is characterized in that the nucleic acid of the PEG modification is suitable
Ligand is the aptamer that the PEG of grass carp reovirus is modified.
9. the aptamer of the modification of PEG described in claim 7 is in preparation prevention and/or treatment grass carp reovirus kit
In application.
10. application according to claim 9, which is characterized in that the aptamer of the PEG modification is that intestines are exhaled on grassland
The aptamer of the PEG modification of lonely virus.
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Cited By (1)
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| CN110257386A (en) * | 2019-07-04 | 2019-09-20 | 广西科学院 | The aptamer and its construction method of anti-I virus of grass carp GCRV and application |
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Application publication date: 20181123 |