CN106337053A - Nucleic acid aptamer for detecting spring viremia of carp virus and application thereof - Google Patents
Nucleic acid aptamer for detecting spring viremia of carp virus and application thereof Download PDFInfo
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- CN106337053A CN106337053A CN201610903422.9A CN201610903422A CN106337053A CN 106337053 A CN106337053 A CN 106337053A CN 201610903422 A CN201610903422 A CN 201610903422A CN 106337053 A CN106337053 A CN 106337053A
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- 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
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Abstract
The invention discloses a nucleic acid aptamer for detecting spring viremia of carp virus and application thereof. The nucleic acid aptamer for detecting spring viremia of carp virus comprises one or two DNA fragments of a general formula as shown in a formula I; the formula I is 5'-P1-N-P2-3', wherein P1 and P2 are sequences as shown in Seq ID No. 1 and Seq ID No. 2 respectively, and N is a sequence as shown in Seq ID No. 3 or Seq ID No. 4. The nucleic acid aptamer for detecting spring viremia of carp virus can specifically and sensitively detect spring viremia of carp virus, and a novel approach and scheme are provided for detection of spring viremia of carp virus. The nucleic acid aptamer has higher practicality, and compared with enzyme-linked immunosorbent assay, the nucleic acid aptamer is low in cost and good in stability and simpler and more convenient to use.
Description
Technical field
The application is related to SVCV detection field, and more particularly to one kind is used for SVCV
The aptamer of detection and its application.
Background technology
Spring viremia (spring viremia of carp) is OIE (oie) register epidemic disease,
Fatality rate is high, and host is mainly cyprinid fish, causes very big threat to the bream aquaculture of China.The disease of spring viremia
Originally it was SVCV (spring viremia of carp virus, abridge svcv), belonged to Rhabdoviridae.
Svcv virion is in bullet shaped, long 90～180nm, wide 60～90nm, and genome length is 11019nt.
Detection for svcv mainly includes detection of nucleic acids and enzyme linked immunosorbent detection at present, for example, " the number that Wu Bin etc. delivers
Word rt-pcr detects that the method for spring viremia is set up and application " " Chinese animal quarantine ", 2015 (7): 72-76；An Wei etc.
" foundation of SVCV sybr green fluorescent quantitation rt-pcr method " " the Chinese Preventive Veterinary Medicine delivered
Report ", 2015,37 (9): 699-702；And, " the preparation of SVCV m monoclonal antibody that Li Zeming etc. delivers
And Cyprinus carpio natural immunity albumen trim32 characteristic preliminary study " " Hua Zhong Agriculture University ", 2015.
Aptamer (aptamer) is otherwise known as nucleic acid aptamer, is the single-stranded short dna or rna fragment of a class, due to it
There is certain space structure, to specific target molecule, there is affinity.Aptamer can be high with plurality of target material special
Property, combine with high selectivity, be therefore widely used in field of biosensors, and medical domain ahead of the curve, such as suppression people and animals
The aspect such as ill poison, antitumor drug research and development has application altogether.Traditional enzyme linked immunosorbent detection, because antigen-antibody reaction is sensitive
Degree and specificity are all preferable, and industrial technology is more ripe, and therefore, the many test kits on market are all based on the exploitation of this principle.
But, the protein that enzyme linked immunological the adopts easily degeneration by the such environmental effects such as ph, temperature, and synthesize expensive.Compare
For relatively, aptamer is made up of dna or rna, mainly dna, less than protein volume, and equally has and resist
Sensitivity and specificity that antigen-antibody reaction phase is equal to, simultaneously synthesizing be easier, stability more preferable.In the near future, nucleic acid
Aptamers are expected to replace traditional enzyme linked immunoassay.
Aptamer belongs to the technology comparing forward position, and the research being currently employed for Aquatic animals virus is also fewer, still
It is not related to research and the report of svcv aptamer or related reagent.
Content of the invention
The purpose of the application is to provide a kind of aptamer for SVCV detection and its application.
The application employs technical scheme below:
The one side of the application discloses a kind of aptamer for SVCV detection, and this nucleic acid is fitted
Part includes one or two dna fragments of formula shown in formula one,
Formula one: 5 '-p1-n-p2-3’
Wherein, p1And p2It is respectively seq id no.1 and sequence shown in seq id no.2, n is seq id no.3 or seq
Sequence shown in id no.4；
Seq id no.1:5 '-ggacactcaaggttcatcag-3 '
Seq id no.2:5 '-gagtagttcaggataggacg-3 '
Seq id no.3:
5’-gctcccatgttttttccccctcctctccccctggttccta-3’
Seq id no.4:
5’-ttactgttttggtcttcattggtgcttggttcgctctcta-3’.
It should be noted that p in formula one1And p2For the application is specially designed can be special with SVCV
Property combine sequence, by p1And p2Sequence determine the detection specificity of the application aptamer, and n is then to be one section
The random sequences of insertion, length generally in 20～50nt, the application through researching and analysing final determine n be seq id no.3 or
Shown in seq id no.4 during sequence, the secondary structure of aptamer is proper, can preferably ensure spring viremia
The detection of virus.
Therefore, the aptamer of the application actually has two dna fragments, and that is, aptamer includes seq id
The dna fragment of sequence shown in no.5 and/or the dna fragment of sequence shown in seq id no.6；
Seq id no.5:
5’-ggacactcaaggttcatcaggctcccatgttttttccccctcctctccccctggttcctagagtag
ttcaggataggacg-3’
Seq id no.6:
5’-ggacactcaaggttcatcagttactgttttggtcttcattggtgcttggttcgctctctagagtag
ttcaggataggacg-3’.
It is appreciated that the aptamer of the two of the application dna fragments, because it has identical p1And p2Sequence, warp
Test confirms, both has very strong SVCV detection specificity, can be used in SVCV
Detection, therefore, this two dna fragment can be used alone it is also possible to use cooperatively, and here is not specifically limited.
Preferably, the dna fragment of aptamer has at least one functional group labelling, these functional group labellings
Including marking sulfhydryl, amino labeled, labelled with radioisotope, fluorescein labelling, biotin labeling, toxic label and enzyme mark
Note.
It is appreciated that the aptamer of the application has very strong specificity, can be used in SVCV
Detection, and according to different use environments or condition, the aptamer of the application can be carried out with various modifications, to coordinate tool
The signal display method of body or instrument, such as marking sulfhydryl, amino labeled, labelled with radioisotope, fluorescein labelling, biology
Plain labelling, toxic label and enzyme labelling.It should be noted that above-mentioned various functions group labelling, the acquisition of its labelling
Or preparation method is all known, these labellings are incorporated in the aptamer of the application the application so that nucleic acid is adapted to
Body possesses corresponding function, and then has expanded the application of aptamer.Certainly, the aptamer of the application can also
Do not carry out the above functional group labelling.
Preferably, the dna fragment of aptamer has during full sulfuration modification, end-block modification or peg modify extremely
Few one kind.Wherein peg modification refers to polyethyleneglycol modified with functional group.
Likewise, the aptamer to the application carries out full phosphorothioate, end-block modifies or peg modifies, these
The acquisition modified or preparation method are also known in itself, and the application is introduced in the aptamer of the application and is also
In order that it possesses corresponding function, these modifications are not equally the necessary requirements of the aptamer of the application, simply basis
Different use conditions or environment and selecting is carried out, and here is not specifically limited.
The another side of the application discloses the aptamer of the application in SVCV detection or in system
Application in standby SVCV detection kit or equipment.
The another side of the application discloses a kind of test kit for SVCV detection, contains in this test kit
There is the aptamer of the application.
It is appreciated that the aptamer of the application is inherently directed to what SVCV detected and studied,
It is of course possible to the aptamer of the application is made test kit, for example, its dna fragment is made dry powder or highly concentrated solution
Deposit in test kit, to facilitate SVCV to detect.Or dna fragment is fixed in ELISA Plate, with side
Just directly use.It is appreciated that in the test kit of application in addition to the aptamer containing the application, making for convenience
With, ELISA Plate and corresponding reagent can also be comprised, such as phosphate buffer pbs, phosphate Tween buffer pbst etc.,
This is not specifically limited.
The application simultaneously discloses a kind of preparation side of the aptamer for SVCV detection again
Method, including the aptamers storehouse of formula shown in synthesis type two, then passes through index concentration Fas lignand system evolution technology to aptamers storehouse
Screened, obtained the aptamer for SVCV detection；
Formula two: 5 '-p1-n’-p2-3’
Wherein, p1And p2It is respectively seq id no.1 and sequence shown in seq id no.2, n ' is the stochastic ordering of 20～50nt
Row.
It should be noted that wherein, index concentration Fas lignand system evolution technology refers to selective expansion of
Ligands by exponential enrichment, abridge selex, is the routine techniquess of aptamer screening, here is not
It is specifically limited.It is critical only that of the application establishes p1And p2It is respectively seq id no.1 and sequence shown in seq id no.2
, there is the aptamers storehouse of formula shown in formula two.As previously described, p1And p2Sequence be just to determine the application nucleic acid fit
The specific key of detection of part.
Preferably, the aptamer that the preparation method of the application is also included using enriching primer obtains to screening is carried out
Pcr amplification enrichment, the forward primer of enriching primer is sequence shown in seq id no.1, and the downstream primer of enriching primer is seq
Sequence shown in id no.7, and 5 ' ends of downstream primer have phosphorylation modification, after the completion of pcr amplification, using Exonucleolytic
Enzymic digestion carries the antisense strand of phosphorylation modification, that is, obtain single-stranded aptamer；
Seq id no.7:5 '-cgtcctatcctgaactactc-3 '.
Preferably, in described formula two, n ' is the random sequences of 40nt.
The application has the beneficial effects that:
The aptamer for SVCV detection of the application, can specifically and sensitively detect Cyprinus carpio
Spring viremia virus, is that the detection of SVCV provides a kind of new approach and scheme.The nucleic acid of the application
Aptamers have higher practicality, and compared with enzyme linked immunosorbent detection, low cost, good stability, using simpler convenience.
Brief description
Fig. 1 is the response rate that the embodiment of the present application amplifying nucleic acid aptamers often take turns screening；
Fig. 2 is the secondary structure figure of two aptamers screening acquisition in the embodiment of the present application, and wherein a fits for nucleic acid
The secondary structure of part a2, b is the secondary structure of aptamer a15；
Fig. 3 is the combination response rate result figure that in the embodiment of the present application, fluorescence pcr measures the aptamer that screening obtains；
Fig. 4 is that in the embodiment of the present application, two aptamers combine the gel retardation assasy result figure after svcv, wherein,
A is the result figure of aptamer a2, and b is the result figure of aptamer a15；
Fig. 5 is two aptamer virus neutralization experimental result pictures in the embodiment of the present application, and wherein, a is adapted to for nucleic acid
Body a2 and svcv, b are that only aptamer a2, c is right for the positive for only aptamers a15, e for aptamer a15 and svcv, d
It is negative control according to, f.
Specific embodiment
Below by specific embodiment, the application is described in further detail.Following examples only are entered to advance to the application
One step explanation, should not be construed as the restriction to the application.
Embodiment
First, material and method
1.1 strains and cell line
The svcv a-1 strain that this example adopts is separated by Animal ＆. Plant Inspection and Quarantine Techn Center, Shenzhen Bureau of Impor
And preserve.Cyprinus carpio epithelium oncocyte (abbreviation epc) cell line is by Shenzhen Entry-Exit Inspection and Quarantine Bureau animals and plants inspection and quarantine technology
Center preserves.
The design in 1.2 aptamers storehouses and synthesis
The aptamers storehouse of this example has formula shown in formula two,
Formula two: 5 '-p1-n’-p2-3’
Wherein, p1And p2It is respectively seq id no.1 and sequence shown in seq id no.2, n ' is the stochastic ordering of 20～50nt
Row.p1And p2It is the sequence of specific bond SVCV, n ' is generated by Synesis Company at random for random sequences, this example
Specifically synthesize the aptamers storehouse that n ' is 40nt.
Seq id no.1:5 '-ggacactcaaggttcatcag-3 '
Seq id no.2:5 '-gagtagttcaggataggacg-3 '
Formula two:
5’-ggacactcaaggttcatcag-n’-gagtagttcaggataggacg-3’
Meanwhile, this example have also been devised the enriching primer pair for aptamer pcr amplification enrichment, enriching primer
Forward primer is sequence shown in seq id no.1, and the downstream primer of enriching primer is sequence shown in seq id no.7, and under
5 ' ends of trip primer have phosphorylation modification.
Seq id no.7:5 '-cgtcctatcctgaactactc-3 '.
The aptamers storehouse of this example and enriching primer pair, by Sangon Biotech's synthesis.
The purification of 1.3 svcv
Cultivate epc cell line in 25 DEG C, after passing on 24h, inoculate svcv.After cell produces 90% pathological changes, freeze thawing 3 times,
4 DEG C of centrifugation 30min of 12000g, collect viral supernatant suspension；The viral supernatant suspension collected is carried out with 4 DEG C of hypervelocities of 140000g
Centrifugation 4h, removes supernatant, and precipitation is resuspended with a small amount of pbs, i.e. pbs virus re-suspension liquid.Prepare 30%, 45%, 60% saccharose gradient,
Pbs virus re-suspension liquid is added on 30% sucrose interface, 4 DEG C of ultracentrifugation 3h of 100000g, collects 30% and divide with 45% concentration
Precipitation at boundary, with appropriate pbs dissolving, 4 DEG C of ultracentrifugation 4h of 140000g, removes supernatant, precipitation is resuspended with appropriate pbs, that is, obtain
Obtain the svcv of purification.
The mensure of 1.4 purification svcv amounts
The protein content determination of purification svcv uses modified form bca determination of protein concentration test kit, biological purchased from raw work
Engineering (Shanghai) limited company, the mensure of virus titer tcid50 uses reed-muench method.
The selex screening of 1.5 aptamers
By 500ng purification svcv and epc cell debriss coated elisa plate corning respectively, 4 DEG C overnight after, pbs and pbst
Respectively wash 2 times.The aptamers storehouse of 5od about 5.5nmole is dissolved in pbs, 95 DEG C of heating 10min, it is immediately placed on 10min on ice.To fit
Part storehouse adds to the coated hole of epc cell debriss, and room temperature carries out negative screening with reference to 1h, removes the nucleic acid being combined with epc fragment and fits
Part.The supernatant of negative screening is added to the coated hole of svcv, room temperature combines 1h.Washed with pbs after 5 times, add protease k to disappear
Change, discharge the aptamer of combination.Digestive system supernatant uses qiaquick nucleotide removal kit
(qiagen) aptamer is reclaimed.
10 are carried out to the aptamer reclaiming-2、10-3、10-4、10-5、10-6Dilute again, fitted with the nucleic acid that each dilutes
Part is template, using enriching primer pair, carries out fluorescence pcr detection to the aptamer of screening, measures it and combine the response rate.
Wherein, the downstream primer of enriching primer pair is sequence shown in seq id no.7, and its 5 ' end does not have phosphorylation modification.Fluorescence pcr adopts
Sybr premix ex taq with takara company.
Fluorescence pcr reaction total system is 25 μ l, comprising: water 8 μ l, sybr premix ex taq 12.5 μ l, forward primer
1 μ l, downstream primer 1 μ l, rox 0.5 μ l, template 2 μ l.
Reaction condition is: 95 DEG C of 30s, subsequently into 40 circulations 95 DEG C of 5s, 60 DEG C of 34s.And 60 DEG C be collect fluorescence.
In this example, the response rate is calculated according to the ct value of fluorescence pcr, specifically, the storehouse of concentration known before screening is entered
10 times of gradient dilutions of row, carry out fluorescence pcr, obtain the ct value of sample before screening and concentration or dilution matched curve；So
Sample after afterwards screening being reclaimed carries out fluorescence pcr, obtains its concentration or dilution angle value according to its ct value, and this value characterizes this example
The response rate.
The enrichment of 1.6 aptamers
To the aptamer reclaiming, using enriching primer pair, carry out conventional pcr amplification, the downstream primer of enriching primer
Sequence shown in seq id no.7, and 5 ' ends of downstream primer have phosphorylation modification.The conventional pcr of this example adopts
The ex taq of takara and buffer.
Reaction system is 50 μ l, comprising: water 18 μ l, ex taq mix 25 μ l, forward primer 1 μ l, downstream primer 1 μ l, mould
Plate 5 μ l.
Reaction condition is: 95 DEG C of 2min；Subsequently into 15 circulations 95 DEG C of 30s, 52 DEG C of 30s, 72 DEG C of 20s；Loop ends
Afterwards, 72 DEG C of 3min.
After pcr reaction terminates, using lambda exonuclease, purchased from new england biolabs, pcr is expanded
Product is digested.
Digestions system includes for 50 μ l: excision enzyme 0.5 μ l, buffer 5 μ l, pcr product 25 μ l, water 19.5 μ l.
Remove the reverse strand of phosphorylation modification in 37 DEG C of 30min enzyme action, that is, obtain single-stranded aptamer.
Aptamer after single stranded is re-used for " the selex screenings of 1.5 aptamers ".Repeat screening process
For several times, the number of times being washed using pbs before being stepped up Proteinase K digestion, until the response rate no longer improves.This example specifically repeat into
9 selex that gone screen.
The monoclonal of 1.7 aptamers and sequencing
Last wheel is combined with the aptamer of rear recovery, using enriching primer to carrying out conventional pcr amplification, wherein rich
The downstream primer integrating primer is as sequence shown in seq id no.7, and 5 ' ends of downstream primer do not have phosphorylation modification.
Reaction system includes for 50 μ l: water 18 μ l, ex taq mix 25 μ l, forward primer 1 μ l, downstream primer 1 μ l, mould
Plate 5 μ l.Reaction condition is: 95 DEG C of 2min；Subsequently into 15 circulations 95 DEG C of 30s, 52 DEG C of 30s, 72 DEG C of 20s；After loop ends,
72℃3min.
Conventional pcr product is connected on pmd18-t carrier, is transformed into competence escherichia coli dh5 α, coats ammonia benzyl
Penicillin resistance flat board.Picking monoclonal, is combined after power confirmation by method 1.5, send Hua Da gene to be sequenced.
The method reclaiming is qiaquick nucleotide removal kit (qiagen).Carrier be
Takara's, 10 μ l systems, wherein water 3 μ l, buffer 5 μ l, carrier 1 μ l, pcr product 1 μ l, 16 DEG C of connection 3h.Conversion is exactly
After mixing with carrier, 42 DEG C of 45s, it is immediately placed on ice.
In this example, the confirmation of adhesion is exactly the method by measuring the response rate, because being through pbs washing before reclaiming
It is possible to think that recovery is returned it is simply that combine on svcv.Because during screening, to about the 8th wheel, adhesion
Just do not increase, maintain a maintenance level it is believed that sequence in storehouse, average adhesion is exactly substantially 12 × 10-4.And
The sequence confirming later, clone gets.Through the calculating of the response rate, find to be also probably 12 × 10-4, this just illustrates its knot
Make a concerted effort also suitable with original storehouse.
1.8 secondary structure analysis
According to the sequencing result of aptamer, using its secondary structure of mfold software analysis, choose free energy δ g
Low, i.e. the most stable of conformation of structure.
1.9 adhesion analyses
According to sequencing result, the aptamer of acquisition is synthesized by Sangon Biotech (Shanghai) Co., Ltd..For
Verify its adhesion, using the aptamer of 10ng synthesis, according to described in " the selex screenings of 1.5 aptamers "
Method is combined with svcv, washs, enzymic digestion and recovery, and measures it using the fluorescence pcr described in it and combine the response rate.
During being combined with svcv, setting epc cell debriss are coated hole as negative control, arrange bsa and are coated hole
As blank.
1.10 gel retardation assasy
By 95 DEG C of heating 10min of aptamer of 1 μ g synthesis, it is immediately placed on 10min on ice.Respectively with 12 μ g, 2.4 μ
G, the svcv room temperature of 0.48 μ g mass gradient combine 1h.Setting negative control 12 μ g, the epc cell debriss of 2.4 μ g simultaneously, and 12
μ g bsa blank, is combined 1h with aptamer in room temperature.
After room temperature combines 1h, using 2% agarose gel electrophoresiies, and with sybr gold nucleic acid gel
Stain, life technology dyeing, gel imaging instrument carries out observation and takes pictures.
1.11 virus neutralization tests
By 95 DEG C of heating 10min of 15 μ g aptamer, it is immediately placed on 10min on ice.With 6.3 × 103tcid50/ml
Svcv room temperature combines 1h, is then seeded into passing on the epc cell line of 24h.Arrange simultaneously and inoculate only after aptamer room temperature 1h
Pass on the hole of the epc cell line of 24h, inoculation after setting svcv room temperature 1h passes on the hole of the epc cell line of 24h as positive right
According to inoculation after setting pbs room temperature 1h passes on the hole of the epc cell line of 24h as negative control.After inoculation epc cell line, 20 DEG C
Culture observed pathological changes situation after 5 days.
2nd, result
The mensure of 2.1 svcv amounts
Using bca determination of protein concentration test kit, svcv after purification is measured, result shows, records protein and contain
Measure as 1.28mg/ml, titre is 6.3 × 109tcid50/ml.
The enrichment of 2.2 aptamers
The combination response rate that aptamer measures for the first time according to the selex of 1.5 aptamers " screening ", that is, first
The wheel response rate, is re-used for through " enrichments of 1.6 aptamers " after that the selex of 1.5 aptamers " screen " measure
It is the second wheel response rate in conjunction with the response rate, by that analogy, this example has specifically carried out nine wheel determination of recovery rates.
Result, as shown in Figure 1 it is seen that the increase of the front four-wheel response rate is inconspicuous, starts to dramatically increase from the 5th wheel, to the 8th
Wheel tends towards stability, and the screening product therefore choosing the 9th wheel carries out follow-up cloning and sequencing.
2.3 cloning and sequencings and secondary structure analysis
30 monoclonals of this example picking, are combined after power confirms by method 1.5, choose optimal 2 grams of adhesion
Grand be sequenced, numbering be respectively a2 and a15.Sequencing result is shown in Table 1.Secondary structure to two aptamers of a2 and a15
Analysis result is shown in Fig. 2, is respectively provided with multiple loop-stem structures.
Table 1 nucleic acid aptamer sequence
| Title | Sequence (5 ' -3 ') | seq id no. |
| a2 | ggacactcaaggttcatcaggctcccatgttttttccccctcctctccccctggttcctagagtagttcaggataggacg | 5 |
| a15 | ggacactcaaggttcatcagttactgttttggtcttcattggtgcttggttcgctctctagagtagttcaggataggacg | 6 |
" gctcccatgttttttccccctcctctccccctggttccta " i.e. seq id no.3 in table 1, and
" ttactgttttggtcttcattggtgcttggttcgctctcta " i.e. seq id no.4 is insertion in nucleic acid aptamer sequence
Random sequences.
2.4 adhesion checkings
This example has synthesized two aptamers of a2 and a15, for svcv detection.A2 and a15 nucleic acid adaptation to synthesis
Body is combined power checking.
Result as shown in figure 3, in figure curve 1-10 is sequentially, Concentraton gradient 10-2, a2, a15, Concentraton gradient 10-3, concentration
Gradient 10-4, a15 negative control, a2 negative control, Concentraton gradient 10-5, Concentraton gradient 10-6, blank.Wherein Concentraton gradient
10-2, Concentraton gradient 10-3, Concentraton gradient 10-4, Concentraton gradient 10-5, Concentraton gradient 10-6, refer to screen back through last wheel
The gradient dilution sample of the aptamer harvesting；A15 negative control, a2 negative control refer to directly by a15 and a2 nucleic acid
Aptamers are added to the sample that epc cell debriss are coated hole；A2, a15 refer to the aptamer sample synthesizing.
It can be seen that, a2 and a15 has a close combination response rate, respectively 1.43 × 10-3With 1.11 × 10-3.And it is negative right
According to the response rate be respectively 2.28 × 10-5With 2.96 × 10-5.
2.5 gel retardation assasy
This example has carried out gel retardation assasy to aptamer a2 and a15 respectively.As shown in figure 4, in figure, a is result
The result figure of aptamer a2, b is the result figure of aptamer a15, two in figures of a and b, and swimming lane 1,2,3 is respectively each
From aptamer and 12 μ g, 2.4 μ g, the svcv of 0.48 μ g mass gradient result, swimming lane 4 and 5 be negative control 12 μ g,
The result of the epc cell debriss of 2.4 μ g, swimming lane 6 is the result of bsa blank；M is dl500 marker.
Result shows, the gel in the hole that aptamer is combined with svcv, i.e. the visible bright band of swimming lane 1 and 2, and with svcv
Amount minimizing and weaken, and svcv amount reduce again, there is no bright band, i.e. swimming lane 3；Negative control, i.e. swimming lane 4 and 5, and and empty
White comparison is swimming lane 6, is showed no and blocks.Aptamer a2 and a15 of this explanation this example can be with svcv specific bond.
2.6 virus neutralization experiments
Aptamer a2 and a15 of this example is carried out virus neutralization tests respectively.Result is as shown in figure 5, in figure a is core
Sour aptamers a2 are that only the observed result figure of inoculation aptamer a2 to epc cell line, c are with the observed result figure of svcv, b
Aptamer a15 is with svcv is inoculated into the observed result figure of epc cell line, d is inoculated into epc cell line for only aptamers a15
Observed result figure, e is inoculated into the positive control observed result figure of epc cell line for svcv, f is negative control.
Result shows, only connects the Positive control wells of malicious svcv, i.e. e figure, (the abbreviation of obvious CPE
cpe)；Inoculate after svcv and aptamer a2 incubation, obvious cpe does not occur, be i.e. a figure；The hole of svcv and a15 incubation occurs
Faint cpe, i.e. c figure.And only add the hole of aptamer a2 and a15 to be showed no to epc cell generation toxicity, i.e. b figure and d
Figure；Illustrate that a2 and a15 has neutralization and inhibitory action to svcv, and, two aptamers of a2 and a15 of this example are to thin
The equal avirulence of born of the same parents.
Aptamer has certain space conformation due to it, has the ability combining with target substance.This example is used
Selex screening technique, filters out two aptamer a2 and a15 for svcv first, and, all possesses preferable combination
Ability.The important step of screening of aptamer is the immobilization of target substance, and labeling magnetic mainly taken by many documents at present
The method that pearls knot closes expressing protein；And this example takes the coated method of ELISA Plate, target substance is totiviruss after purification, totiviruss
Compare the albumen of expression, Antigen conformation is more accurate, improve the success rate of screening；And ELISA Plate has to nucleic acid absorption
Low, can adsorb no label protein and be easy to washing the advantages of.Certainly, this is likely to lead to the absorption of foreign protein, therefore this example
Each wheel screening all adds negative screening, to ensure to remove nonspecific aptamer in time.
This example employs the test method of three kinds of different principle, and the aptamer filtering out is verified.Fluorescence pcr
Method by measure the response rate verify its adhesion, this is consistent with selex screening process method, and mensure the response rate
3 wheels last with selex are close, illustrate that the binding ability of aptamer that this example screening obtains is consistent with expection.Gel blocking
It is to test the method for nucleic acid protein interaction it can also be used to verify aptamers adhesion, the gel blocking result of this example is said
Bright, two kinds of aptamers of a2 and a15 that screening obtains all can be combined with svcv, causes a certain amount of nucleic acid to hinder in gel pore
Stagnant, and the gradient with svcv amount reduces and weakens, and there is certain proportion relation.The virus neutralization experimental result of this example is said
Bright, two aptamers that this example screening obtains can produce suppression to svcv, and does not produce toxicity to normal cell, proves
Aptamer can combine with svcv.
Above content is further description the application made with reference to specific embodiment it is impossible to assert this Shen
Being embodied as please is confined to these explanations.For the application person of an ordinary skill in the technical field, do not taking off
On the premise of the application design, some simple deduction or replace can also be made, all should be considered as belonging to the protection of the application
Scope.
Claims (9)
1. a kind of aptamer for SVCV detection it is characterised in that: described aptamer includes
One or two dna fragments of formula shown in formula one,
Formula one: 5 '-p1-n-p2-3’
Wherein, p1And p2It is respectively seq id no.1 and sequence shown in seq id no.2, n is seq id no.3 or seq id
Sequence shown in no.4；
Seq id no.1:5 '-ggacactcaaggttcatcag-3 '
Seq id no.2:5 '-gagtagttcaggataggacg-3 '
Seq id no.3:
5’-gctcccatgttttttccccctcctctccccctggttccta-3’
Seq id no.4:
5’-ttactgttttggtcttcattggtgcttggttcgctctcta-3’.
2. aptamer according to claim 1 it is characterised in that: described aptamer includes seq id no.5
The dna fragment of shown sequence and/or the dna fragment of sequence shown in seq id no.6；
Seq id no.5:
5’-ggacactcaaggttcatcaggctcccatgttttttccccctcctctccccctggttcctagagtagttca
ggataggacg-3’
Seq id no.6:
5’-ggacactcaaggttcatcagttactgttttggtcttcattggtgcttggttcgctctctagagtagttca
ggataggacg-3’.
3. aptamer according to claim 1 and 2 it is characterised in that: in the dna fragment of described aptamer have
There is at least one functional group labelling, described functional group labelling includes marking sulfhydryl, amino labeled, radiosiotope mark
Note, fluorescein labelling, biotin labeling, toxic label and enzyme labelling.
4. aptamer according to claim 1 and 2 it is characterised in that: the dna fragment of described aptamer has
At least one in full phosphorothioate, end-block modification or peg modification.
5. the aptamer according to any one of claim 1-4 is in SVCV detection or in preparation Cyprinus carpio
Application in spring viremia virus detection kit or equipment.
6. a kind of test kit for SVCV detection it is characterised in that: in described test kit contain have the right will
Seek the aptamer described in any one of 1-4.
7. a kind of preparation method of the aptamer for SVCV detection it is characterised in that: include synthesis
The aptamers storehouse of formula shown in formula two, is then sieved to described aptamers storehouse by index concentration Fas lignand system evolution technology
Choosing, obtains the aptamer for SVCV detection；
Formula two: 5 '-p1-n’-p2-3’
Wherein, p1And p2It is respectively seq id no.1 and sequence shown in seq id no.2, n ' is the random sequences of 20～50nt.
8. preparation method according to claim 7 it is characterised in that: also include using enriching primer to screening obtain core
Sour aptamers carry out pcr amplification enrichment, and the forward primer of described enriching primer is sequence, described enrichment shown in seq id no.1
The downstream primer of primer is sequence shown in seq id no.7, and 5 ' ends of described downstream primer have phosphorylation modification,
After the completion of pcr amplification, carry the antisense strand of phosphorylation modification using exonuclease digestion, that is, obtain single-stranded nucleic acid adaptation
Body；
Seq id no.7:5 '-cgtcctatcctgaactactc-3 '.
9. the preparation method according to claim 7 or 8 it is characterised in that: in described formula two, n ' be 40nt stochastic ordering
Row.
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| CN109182455A (en) * | 2018-08-08 | 2019-01-11 | 淮海工学院 | A kind of screening technique of protein aptamer |
| CN114814213A (en) * | 2022-06-10 | 2022-07-29 | 尹伟力 | Method for establishing rapid detection of spring blood disease virus of carp by using aptamer gold probe technology |
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| CN105002298A (en) * | 2015-05-21 | 2015-10-28 | 上海市水产研究所 | Fluorescent quantitative PCR detection method of spring viraemia of carp virus |
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| CN105002298A (en) * | 2015-05-21 | 2015-10-28 | 上海市水产研究所 | Fluorescent quantitative PCR detection method of spring viraemia of carp virus |
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| LI PENGFEI 等: "Isolation and characterization of a new class of DNA aptamers specific binding to Singapore grouper iridovirus (SGIV) with antiviral activities", 《VIRUS REASEARCH》 * |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109182455A (en) * | 2018-08-08 | 2019-01-11 | 淮海工学院 | A kind of screening technique of protein aptamer |
| CN114814213A (en) * | 2022-06-10 | 2022-07-29 | 尹伟力 | Method for establishing rapid detection of spring blood disease virus of carp by using aptamer gold probe technology |
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