CN110257387A - The aptamer and its construction method of identification grass carp hemorrhagic disease virus and application - Google Patents
The aptamer and its construction method of identification grass carp hemorrhagic disease virus and application Download PDFInfo
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Abstract
A kind of aptamer, including following nucleotide sequence: TGAACCCACCTCAGGGCATCTTACATTTCTTCTAAGTTGTTACCATGTTT.The molecular weight of aptamer provided by the invention is smaller, short preparation period, favorable reproducibility, synthesizes convenient for iii vitro chemical, and convenient for label, sequence is stable and easy to transport and save.The aptamer exhales the lonely I type virus of intestines specificity with higher and affinity, and non-immunogenicity to grass carp.
Description
Technical field
The invention belongs to aquatic pathogenic bacterium detection technique fields, and in particular, to identify the nucleic acid of grass carp hemorrhagic disease virus
Aptamers and its screening technique and application.
Background technique
Guangxi is the big province of aquaculture in China, main breed variety include grass carp, channel catfish, grouper, grass carp,
Prawn, oyster, pteria martensii and various food plants.According to statistics, ended for the end of the year 2017, the fishery economic gross output value in Guangxi is
More than 62,100,000,000 yuan, aquaculture total amount is more than 320.76 ten thousand tons.However, the continuous expansion of aquaculture scale and cultivation density
Continue to increase breeding environment caused to deteriorate, all kinds of aquatic products epidemic disease cause of diseases are frequently broken out.Nanning, rivers and ponds, Liuzhou in 2018
Etc. the grass carp of ground pond and cage culture break out doubtful grass carp hemorrhage disease, by polymerizeing to pathogenic microorganism in illness grass carp
Enzyme chain reaction detection technique (polymerase chain reation, PCR) analyzes and identifies, it was demonstrated that grass carp exhales the lonely I type disease of intestines
Malicious (Grass carp reovirus, GCRV I) is principal causative cause of disease.Grass carp hemorrhage disease is the most common disease in grass carp cultivation
Viral disease, infective pathogen are grass carp reovirus (Grass carp reovirus, GCRV), also known as hemorrhagic disease of grass carp disease
Malicious (GCHV), is subordinate to Reoviridae, Aquareovirus category.GCRV virus is passed as a kind of highly pathogenic fish
It catches an illness poison, is usually expressed as explosion type infection, i.e., can lead to fish mortality in a very short period of time.At present caused by GCRV virus
Grass carp viral hemorrhagic disease is widely present in China, seriously affects the development of the culture fishery in China.Therefore, novel height is researched and developed
The antiviral drugs of effect, meanwhile, the detection skill that convenient, at low cost, time-consuming is short, accuracy is high is operated for GCRV viral progression
Art is most important for the harm of prevention and control grass carp GCRV virus.
Aptamer is aglucon phyletic evolution technology (the Systematic Evolution of of utilization index enrichment
Ligands by Exponential Enrichment technology, SELEX), being obtained in vitro by multi-turns screen,
It is capable of the single-stranded oligonucleotide of specific recognition target substance.Aptamer is high with specificity, compatibility is high, stability is strong,
Many advantages, such as chemically reactive synthesis and chemical modification.The novel inspection that aptamer is novel as one kind at present, is widely noticed
Survey and treatment tool, before wide application is shown in fields such as physianthropy research, medical diagnosis on disease, virus infection Mechanism Studies
Scape.
Summary of the invention
The object of the present invention is to provide a kind of aptamer for identifying grass carp hemorrhagic disease virus and its construction method and answer
With to realize that grass carp exhales highly sensitive, the specific detection of the lonely I type virus of intestines.
According to an aspect of the present invention, a kind of aptamer for identifying grass carp hemorrhagic disease virus is provided: including SEQ
The nucleotide sequence of ID NO.1.
Preferably, the nucleotide sequence including SEQ ID NO.2.
Preferably, at least one nucleotide on nucleotide sequence be phosphorylated, sulfhydrylation, methylation, amination or
Isotopologue.
Preferably, marker is combined on nucleotide sequence, marker is selected from fluorescent material, luminescent material, biotin
One of enzyme or more than one.
Preferably, fluorescent material in hydroxyl fluorescein, fluorescein isothiocynate or carboxyl tetramethylrhodamine one
Kind or more than one.
According to another aspect of the present invention, a kind of aptamer constructing above-mentioned identification grass carp hemorrhagic disease virus is provided
Method, using SELEX technology, comprising the following steps: (1) establish single stranded DNA random library Library50, single stranded DNA is random
The nucleotide sequence of library Library50 are as follows:
5'-GTCTGAAGTAGACGCAGGAG(50N)AGTCACACCTGAGTAAGCGT；
(2) the lonely I type virus infected cell screening single stranded DNA random library Library50 of intestines is exhaled using grass carp, obtains grass carp
Exhale the specific DNA libraries of the lonely I type virus of intestines；(3) using specific DNA libraries as template, SEQ ID is classified as with nucleotides sequence
The primer that primer, the nucleotides sequence of NO.3 is classified as SEQ ID NO.4 carries out PCR amplification.
Preferably, the amplification program of PCR amplification is: 94 DEG C 5 minutes, 94 DEG C 1 minute, 56 DEG C 30 seconds, 72 DEG C 1 minute, 25
Wheel circulation；72 DEG C 5 minutes.
According to another aspect of the present invention, the aptamer of above-mentioned identification grass carp hemorrhagic disease virus is provided in detection grass
Fish exhales the application in the lonely I type virus of intestines.
According to another aspect of the present invention, the quantitative fluorescent PCR for providing a kind of grass carp hemorrhagic disease virus quickly detects
Kit: including fluorescent molecule detection probe, using the aptamer of above-mentioned identification grass carp hemorrhagic disease virus as fluorescent molecule
Detection probe, marker are hydroxyl fluorescein.
The molecular weight of the aptamer of identification grass carp hemorrhagic disease virus provided by the invention is smaller, short preparation period, weight
Existing property is good, synthesizes convenient for iii vitro chemical, and convenient for label, sequence is stable and easy to transport and save.The aptamer exhales grass carp
Intestines orphan's I type virus specificity with higher and affinity, and non-immunogenicity.In addition, forming the core of the aptamer
Nucleotide sequence is easy to be substituted or modify.Partially replaced or the above-mentioned aptamer after modification is able to maintain and original
The essentially identical or similar molecular structure of aptamer, physicochemical property and function equally can effectively realize that grass carp exhales intestines
The detection of lonely I type virus.Aptamer provided by the invention is marked using hydroxyl fluorescein, as a result, by the aptamer
It is fabricated to fluorescence probe, it is combined with real-time fluorescence quantitative PCR detection technique, can be realized and the lonely I type disease of intestines is exhaled to grass carp
The high specific of poison, highly sensitive real-time qualitative, quantitative detection.
Detailed description of the invention
Fig. 1 is the second level knot of the aptamer for the identification grass carp hemorrhagic disease virus that nucleotides sequence is classified as SEQ ID NO.2
Structure prognostic chart；
Fig. 2 is the sample F AM fluorescent value test result of flow cytomery in embodiment 2；
Fig. 3 is the observed result of laser scanning co-focusing microscope in embodiment 2: (a) the light microscopic figure of control sample,
(b) fluorogram of control sample, (c) the light microscopic figure of test group sample, (d) fluorogram of test group sample.
Specific embodiment
It in order to enable those skilled in the art to better understand the solution of the present invention, below will be to the skill in the embodiment of the present invention
Art scheme is clearly and completely described, it is clear that and the described embodiment is only a part of the embodiment of the present invention, without
It is whole embodiments.
Embodiment 1 identifies the screening and preparation of the aptamer of grass carp hemorrhagic disease virus
S1. the synthesis of the building in the library random single chain DNA (ssDNA) and primer
SsDNA pool Library50 is constructed, both ends are fixed sequence program, and intermediate 50 nucleotide are random sequence,
Nucleotide sequence is as follows:
5’-GTCTGAAGTAGACGCAGGAG(50N)AGTCACACCTGAGTAAGCGT。
5 ' primers (SEQ ID NO.3): 5 '-FAM-GTCTGAAGTAGACGCAGGAG-3 '.
3 ' primers (SEQ ID NO.4): 5 '-Biotin-ACGCTTACTCAGGTGTGACT-3 '.
SsDNA pool and primer are synthesized by the raw work biology Co., Ltd in Shanghai.
S2.SELEX screens (being equivalent to positive-selecting)
The above-mentioned ssDNA pool of 10nmol is dissolved in 500 μ L PBS, 92 DEG C of water bath with thermostatic control 5min, then rapidly
It is inserted into ice, ice bath 10min, by treated, ssDNA pool is incubated on ice with I type virus infected cell of grass carp GCRV
1h。
After the completion of hatching combination, centrifugation removes supernatant, and it is thin to wash I type virus infection of grass carp GCRV with the process PBS of 10mL
Supernatant, as I type virus infected cell of specific recognition grass carp GCRV is collected by centrifugation in born of the same parents, 92 DEG C of waters bath with thermostatic control 10min, 12000g
SsDNA nucleic acid library.
S3.PCR amplification
The library ssDNA for the I type virus infected cell of identification grass carp GCRV for taking 100 μ L to screen and 5 ' primers, 3 ' are drawn
Object carries out PCR amplification.Following (1000 μ L): 10 × Buffer 100 μ L, dNTP Mix (2.5mM) the 80 μ L of PCR reaction system,
The primer of 40 μ L, the SEQ ID NO.4 of primer of SEQ ID NO.3,100 μ L, rTaq enzyme of the library ssDNA 12.5 μ L, ddH2O
627.5μL.The amplification program of PCR are as follows: 94 DEG C of 5min, 94 DEG C of 1min, 56 DEG C of 30sec, 72 DEG C of 1min are recycled by 25 wheels；72
℃5min.The supernatant obtained after the screening of first round SELEX will be completely used for carrying out PCR amplification, obtain double-strandednucleic acid after amplification
(dsDNA) library.
The preparation in the library S4.ssDNA
The library dsDNA made from the magnetic bead and S3 of 100 μ L streptavidins label is incubated for 20min at normal temperature, is utilized
The affinity interaction of streptavidin, is integrated to magnetic bead surfaces for dsDNA on biotin and magnetic bead on dsDNA, utilizes magnetism point
From supernatant is removed on device, magnetic bead is washed with 2mL PBS, 200 μ L NaOH solutions (200mM) are then added in EP pipe, room temperature is anti-
10min is answered, dsDNA is made to be denaturalized unwinding, a chain with biotin stays on magnetic bead in conjunction with streptavidin, after completion of the reaction
It recycles to obtain supernatant using magnetic separation rack；Supernatant is added and removes salt plug after sterile water washing, in the work of gravity
It is dripped off naturally with lower.500 μ L PBS are added into filtrate, are collected into the solution containing the library ssDNA, the sieve for next round
Choosing.
The repeated screening in the library S5.ssDNA
The library ssDNA obtained in S4 is replaced into the ssDNA pool in S2, repeats the sieve of SELEX shown in S2-S4
Choosing, producing process 9 times of PCR amplification and the library ssDNA.
S6. negative screening
The second wheel of S5 and the second wheel are screened the obtained library ssDNA dissolution, 92 DEG C of waters bath with thermostatic control, ice baths later after,
It is incubated for 1h on ice with grass carp normal cell, supernatant solution is collected by centrifugation after the completion of being incubated for；Then by this supernatant solution and grass carp
Normal cell carries out ice bath combination；After the completion of hatching combination, supernatant is collected, at this point, the supernatant being collected into is by negative sieve
The library ssDNA of choosing.
This step uses grass carp normal cell for control, by the library ssDNA screened after S5 carry out negative screening with
Improve the screening efficiency in the library ssDNA.
S7.9 wheel screening
The supernatant that will be collected into S6, by the library ssDNA of the PCR amplification of S3 and S4 preparation after, be repeated in into
The process of row S6, S2, S3 and S4, using the library ssDNA obtained by flow cytomery to I type virus infected cell of grass carp GCRV
The situation of change of recognition capability repeats the screening of 9 wheels, and the obtained library ssDNA is to I type virus infected cell of grass carp GCRV
Recognition capability reaches most strong.By gained amplified production after cloning and sequencing is analyzed, finally obtains and can be used for detecting I type of grass carp GCRV
The ssDNA aptamer of virus infected cell, nucleotide sequence are as follows:
TGAACCCACCTCAGGGCATCTTACATTTCTTCTAAGTTGTTACCATGTTT
(SEQ ID NO.1),
Or,
GTCTGAAGTAGACGCAGGAGTGAACCCACCTCAGGGCATCTTACATTTCTTCTAAGTTGTTACCATGT
TTAGTCACACCTGAGTAAGCGT(SEQ ID NO.2)。
Do you utilize MFOLD software (http://mfold.rna.albany.edu/? q=mfold/DNA-Folding-Form)
On-line prediction nucleotides sequence is classified as the secondary structure of the aptamer of SEQ ID NO.2, and prediction result is as shown in Figure 1, nucleic acid
Aptamers form special loop-stem structure and hairpin structure.
Embodiment 2
2.1 key instrument
Attune NxT flow cytometer (the silent winged generation that science and technology of match), FV3000 laser scanning co-focusing microscope (Olympic
Bath).
2.2 experimental implementation
The SEQ ID NO.2 aptamer that marks embodiment 1 to prepare respectively using hydroxyl fluorescein (FAM) and random
The library ssDNA Library50.
Test group: the SEQ ID NO.2 aptamer that 0.1nmol FAM is marked is dissolved in 500 μ L PBS, 92 DEG C
Water bath with thermostatic control 5min, is then rapidly inserted into ice, ice bath 10min, will treated SEQ ID NO.2 aptamer and grass carp
I type virus infected cell of GCRV is incubated for 1h on ice.After the completion of hatching combination, centrifugation removes supernatant.
Control group: the ssDNA pool Library50 that 0.1nmol FAM is marked is dissolved in 500 μ LPBS, 92 DEG C
Water bath with thermostatic control 5min, is then rapidly inserted into ice, ice bath 10min, will treated ssDNA pool Library50 and grass
I type virus infected cell of fish GCRV is incubated for 1h on ice.After the completion of hatching combination, centrifugation removes supernatant.
Flow cytometer and laser scanning co-focusing microscope detection test group and control group are utilized respectively by being incubated for institute
Obtained cell precipitate.
2.3 experimental result
The testing result of flow cytometer is apparently higher than control group as shown in Fig. 2, measuring fluorescent value corresponding with test group.
The testing result of laser scanning co-focusing microscope is as shown in figure 3, control group is barely perceivable apparent fluorescent effect, relatively
For, fluorescent effect corresponding to experimental group is obvious, and cell sample issues strong green light.
The test result of flow cytometer and laser scanning co-focusing microscope explanation, with ssDNA pool
Library50 is compared, and the SEQ ID NO.2 aptamer of FAM label has I type virus infected cell of grass carp GCRV higher
Affinity and specificity.
The above embodiments are merely illustrative of the technical solutions of the present invention rather than limiting the scope of the invention, although ginseng
The present invention is explained in detail according to preferred embodiment, those skilled in the art should understand that, it can be to of the invention
Technical solution is modified or replaced equivalently, without departing from the spirit and scope of technical solution of the present invention.
SEQUENCE LISTING
<110>Guangxi Academy Of Sciences
<120>aptamer and its construction method of identification grass carp hemorrhagic disease virus and application
<130>
<160> 4
<170> PatentIn version 3.5
<210> 1
<211> 50
<212> DNA
<213>artificial sequence
<400> 1
tgaacccacc tcagggcatc ttacatttct tctaagttgt taccatgttt 50
<210> 2
<211> 90
<212> DNA
<213>artificial sequence
<400> 2
gtctgaagta gacgcaggag tgaacccacc tcagggcatc ttacatttct tctaagttgt 60
taccatgttt agtcacacct gagtaagcgt 90
<210> 3
<211> 20
<212> DNA
<213>artificial sequence
<400> 3
gtctgaagta gacgcaggag 20
<210> 4
<211> 20
<212> DNA
<213> GTCTGAAGTAGACGCAGGAG
<400> 4
acgcttactc aggtgtgact 20
Claims (10)
1. a kind of aptamer for identifying grass carp hemorrhagic disease virus, it is characterised in that: the nucleotides sequence including SEQ ID NO.1
Column.
2. the aptamer of identification grass carp hemorrhagic disease virus as described in claim 1, it is characterised in that: including SEQ ID
The nucleotide sequence of NO.2.
3. the aptamer of identification grass carp hemorrhagic disease virus as described in claim 1, it is characterised in that: on its nucleotide sequence
At least one nucleotide be phosphorylated, sulfhydrylation, methylation, amination or isotopologue.
4. identifying the aptamer of grass carp hemorrhagic disease virus as described in any one of claim 1-3, it is characterised in that: its nucleosides
Marker is combined on acid sequence, the marker is selected from one of fluorescent material, luminescent material, biotin or enzyme or a kind of
More than.
5. the aptamer of identification grass carp hemorrhagic disease virus as claimed in claim 4, it is characterised in that: the fluorescent material choosing
From one of hydroxyl fluorescein, fluorescein isothiocynate or carboxyl tetramethylrhodamine or more than one.
6. a kind of method that building identifies the aptamer of grass carp hemorrhagic disease virus as described in any one of claims 1 or 2,
It is characterized in that, using SELEX technology, comprising the following steps:
(1) single stranded DNA random library Library50, the nucleotide sequence of the single stranded DNA random library Library50 are established
Are as follows:
5'-GTCTGAAGTAGACGCAGGAG(50N)AGTCACACCTGAGTAAGCGT；
(2) it exhales the lonely I type virus infected cell of intestines to screen the single stranded DNA random library Library50 using grass carp, obtains grass carp
Exhale the specific DNA libraries of the lonely I type virus of intestines；
(3) using the specific DNA libraries as template, primer, the nucleotide sequence of SEQ ID NO.3 are classified as with nucleotides sequence
PCR amplification is carried out for the primer of SEQ ID NO.4.
7. the method for the aptamer of building identification grass carp hemorrhagic disease virus as claimed in claim 6, which is characterized in that described
The amplification program of PCR amplification is:
94 DEG C 5 minutes, 94 DEG C 1 minute, 56 DEG C 30 seconds, 72 DEG C 1 minute, 25 wheel circulation；
72 DEG C 5 minutes.
8. identifying that the aptamer of grass carp hemorrhagic disease virus exhales intestines orphan I in detection grass carp as described in any one of claim 1-3
Application in type virus.
9. the aptamer of identification grass carp hemorrhagic disease virus is exhaled in the lonely I type virus of intestines in detection grass carp as claimed in claim 4
Application.
10. a kind of quantitative fluorescent PCR quick detection kit of grass carp hemorrhagic disease virus, it is characterised in that: including fluorescence point
Sub- detection probe, to identify that the aptamer of grass carp hemorrhagic disease virus is examined as the fluorescent molecule as claimed in claim 4
Probing needle, the marker are hydroxyl fluorescein.
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