CN104193804B - Duck tembusu virus cyst membrane E protein specific binding peptides and application thereof - Google Patents
Duck tembusu virus cyst membrane E protein specific binding peptides and application thereof Download PDFInfo
- Publication number
- CN104193804B CN104193804B CN201410361045.1A CN201410361045A CN104193804B CN 104193804 B CN104193804 B CN 104193804B CN 201410361045 A CN201410361045 A CN 201410361045A CN 104193804 B CN104193804 B CN 104193804B
- Authority
- CN
- China
- Prior art keywords
- duck tembusu
- tembusu virus
- specific binding
- binding peptides
- virus
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired - Fee Related
Links
Landscapes
- Peptides Or Proteins (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention discloses a kind of duck tembusu virus cyst membrane E protein specific binding peptides and application thereof, belong to animal virus technical field of molecular biology.The present invention obtains duck tembusu virus cyst membrane E protein specific binding peptides by phage random 12 peptide library selection, and its aminoacid sequence is: THRSWQGNSWYM.This specific binding peptides can suppress duck tembusu virus HN1 strain in the propagation of DEF, duck tembusu virus viral copy number in DEF all can be significantly lowered under variable concentrations, significantly reduce duck tembusu virus virus titer in DEF simultaneously, and the propagation of DEF is had no significant effect by itself.Duck tembusu virus cyst membrane E protein specific binding peptides has a good application prospect in preparing the sick medicine of anti-duck tembusu virus or feed additive, can be used for the preventing and treating that duck tembusu virus is sick.
Description
Technical field
The present invention relates to a kind of duck tembusu virus cyst membrane E protein specific binding peptides, also relate to this specificity knot
Close the application of peptide, belong to animal virus technical field of molecular biology.
Background technology
In recent years, southern china has broken out a kind of new duck viral infectious disease, mainly causes duck to lay eggs degradation, should
Sick propagation is rapid, coverage is wide, and morbidity early stage a certain proportion of nervous symptoms occurs for lasting high heat, morbidity later stage, shows
For paralysis, rolling, astasia and ataxia etc., some sick duck is even dead within a few days.Cuing open the dead duck of inspection, majority presents
Spleen and the obvious enlargement of kidney, laying ducks shows as hemorrhagic oophoritis.First this disease breaks out at Jiangsu and Zhejiang Provinces one band, spreads to good fortune subsequently
Build and the ground such as Anhui, afterwards in Jiangxi, Shandong, Hunan, Henan, Hebei breaks out in succession.Sick for this, abroad there is not been reported.
Studies in China personnel have carried out separation, morphological observation to its cause of disease, physicochemical property measures, RT-PCR detects and sequence analysis,
It has been determined that this disease pathogen is duck tembusu virus, therefore this disease is called duck tembusu virus disease.This disease sickness rate is high, it is fast to propagate
Speed, morbidity scope is extremely wide, causes heavy economic losses to duck culturing industry.
Duck tembusu virus (Duck tembusu virus, DTMUV) is a kind of novel banzi virus, belongs to flaviviridae not
Segmented single strand plus RNA virus, its virion is spherical in shape, diameter about 45～50nm, has cyst membrane and fibre to dash forward, virus about by
10990 nucleotide compositions.This virus is sensitive to heat, fatsolvent and sodium deoxycholate, just loses infection and live during pH<5 or pH>10
Property, more than 50 DEG C heating 60min are i.e. inactivated.DTMUV can be at duck embryo, Embryo Gallus domesticus, DEF and Vero, BHK, C6/
The part passage cells such as 36 fasten propagation.Its viral genome is read by 5 ' ends, the 3 ' noncoding regions held and a middle opening
Frame forms, 5 ' a length of 142nt in the noncoding region held, and containing I type m7GpppNp cap sequence, 3 ' hold non-coding head of district 618nt,
Not having poly (A) tail, open reading frame encodes the polyprotein precursor being made up of 3410 amino acid residues.Precursor protein exists
3 kinds of structural protein (C, prM and E) and 7 kinds of non-knots it are cut under the effect of host signal peptidase and virus serine protease
Structure albumen (NS1, NS2A, NS2B, NS3, NS4A, NS4B, NS5).
E protein is a kind of envelope protein, encodes 501 aminoacid, there is high conservative region and change in its aminoacid sequence
Different district.E protein is positioned at ripe surfaces of viral particles, merges and virus assembly mistake at viruses adsorption receptor and host cell membrane
Journey has important effect, and containing multiple epitope, these epi-positions and virus recognition of host cell-membrane receptor, absorption and
Entering cell, viral is pathogenic closely related with induction immunne response etc., plays an important role in virus infects.It addition, E egg
In vain also there is preferable immunogenicity and reactionogenicity, be the main protection antigen of host's anti-infectious immunity, be also that detection should
The preferable target antigen of special viral antibody.
Display technique of bacteriophage is the effective means of a research protein molecule interphase interaction of rising in recent years, tool
There are quick, efficient, phenotype and the feature such as genotype is consistent, are widely used in peptide medicament, peptide for inhibiting, antigen mimicking in recent years
The research fields such as the screening of epi-position.Utilize display technique of bacteriophage screening duck tembusu virus cyst membrane E protein specific binding
Polypeptide, and study this polypeptide to DTMUV impact in terms of DEF propagation, can be that the preventing and treating of DTMUV provides one
Individual new approach, has preferable application prospect.
Summary of the invention
It is an object of the invention to provide a kind of duck tembusu virus cyst membrane E protein specific binding peptides.
Meanwhile, the present invention also provides for the application of a kind of duck tembusu virus cyst membrane E protein specific binding peptides.
In order to realize object above, the technical solution adopted in the present invention is:
Duck tembusu virus cyst membrane E protein specific binding peptides, its aminoacid sequence is as shown in SEQ ID NO:1.
Described duck tembusu virus is duck tembusu virus HN1 strain.
The application of duck tembusu virus cyst membrane E protein specific binding peptides, specially specific binding peptides is preparing anti-duck
The medicine of tembusu virus sick (suppression duck tembusu virus propagation, as suppression duck tembusu virus is bred at DEF)
Application in thing or feed additive.
Beneficial effects of the present invention:
The present invention obtains DTMUV HN1 strain E protein by gene engineering method, and using DTMUV HN1 strain E protein as target
Mark, utilizes phage random 12 peptide library selection to obtain duck tembusu virus cyst membrane E protein specific binding peptides, its aminoacid sequence
For: THRSWQGNSWYM.This specific binding peptides can suppress duck tembusu virus HN1 strain in the propagation of DEF.
The impact that DEF is bred by standard mtt assay detection E protein specific binding peptides, result shows, polypeptide itself is right
The propagation of DEF has no significant effect.Quantitative fluorescent PCR and TCID50 detection E protein specific binding peptides are smooth to duck
The virus impact in terms of DEF propagation of cloth Soviet Union, result shows, this polypeptide variable concentrations (0.02 μ g/mL,
0.2 μ g/mL, 2 μ g/mL, 20 μ g/mL) under all can significantly lower duck tembusu virus virus in DEF and copy
Shellfish number, significantly reduces duck tembusu virus virus titer in DEF simultaneously.Result shows, E protein is special
Property combine Toplink suppression duck tembusu virus in the propagation of DEF, have in the preventing and treating that duck tembusu virus is sick
Good application prospect.
Accompanying drawing explanation
Fig. 1 is that in the embodiment of the present invention 1, recombinant expression plasmid pET32a-E builds schematic diagram；
Fig. 2 is the ELISA detection Phage Display Peptide combination activity to target molecule in the embodiment of the present invention 1；
Fig. 3 is embodiment 1 pnagus medius positive colony Competitive assays result of the test；
Fig. 4 is the impact that in test example, DEF is bred by DRMUV HN1 strain E protein specific binding peptides.
Detailed description of the invention
The present invention is only described in further detail by following embodiment, but does not constitute any limitation of the invention.
Embodiment 1
The screening of duck tembusu virus cyst membrane E protein specific binding peptides in the present embodiment, comprises the following steps:
1) conventional method prepares DTMUV HN1 strain cyst membrane E protein
(1) structure of recombinant expression plasmid pET32a-E
According to the gene order (serial number: JQ669731) of the DTMUV HN1 strain E protein that GenBank delivers, utilize
DNAStar software analysis, designs a pair specific primer P1 and P2, primer two ends add respectively restriction enzyme site EcoR I and
Xba I and protectiveness base (synthesis of Takara company), underscore part is restriction enzyme site.
Forward primer P1:5 '-CCGGAATTCTTCAGCTGTCTGGGGATGCA-3 ' is (such as SEQ ID NO:2, underscore portion
It is divided into EcoR I restriction enzyme site)；
Downstream primer P2:5 '-CGATCTAGAATTGACATTTACTGCCAGG-3 ' is (such as SEQ ID NO:3, underscore portion
It is divided into Xba I restriction enzyme site).
Conventional method extracts DTMUV HN1 strain RNA (viral RNA extracts test kit, Nanjing Tiangen company limited), logical
Cross conventional reverse transcriptional PCR and obtain the full length gene sequence (Invitrogen One step RT-PCR test kit) of E protein, by obtain
E protein gene is cloned in pMD18-T carrier, and checks order.By the E protein gene purpose fragment warp on pMD18-T carrier
After EcoR I and Xba I enzyme action, it is cloned into pET32a carrier, builds recombinant expression plasmid pET32a-E, and with EcoR I and Xba
I double digestion and PCR identify, identify that positive plasmid is recombinant expression plasmid pET32a-E, serve sea Invitrogen
Company's order-checking (see Fig. 1).
(2) abduction delivering of recombinant expression plasmid pET32a-E and purification
Use CaCl2Conversion method, is transformed into e. coli bl21 (DE3) competent cell by recombiant plasmid pET32a-E
In, screening positive clone, it is the positive gene engineered strain of gene containing DTMUV HN1 strain E protein.By positive gene engineering
Bacterial strain, in 37 DEG C of cultivations, treats A600Value reaches when 0.5 (0.4～0.6), adds IPTG to final concentration of 0.8mM, induces
Express.Collect the thalline that different induction time is expressed, ultrasonic disruption, take cleer and peaceful precipitation respectively after being centrifuged and carry out SDS-PAGE
Electrophoresis, and be one to resist with duck anti-DTMUV-1 serum, the rabbit anti-duck IgG of HRP labelling is two anti-expression product to be carried out Western
Blot analyzes, and finally develops the color with DAB colour reagent box.The bacterium solution of abduction delivering is centrifuged and gathers in the crops precipitation, with cleaning mixture (5mM
Imidazoles, 0.5M NaCl, 20mM Tris-HCl, pH 7.9) after resuspended thalline, centrifugal, by solubility table after ultrasonic treatment
Reaching supernatant protein N i post affinity chromatograph to be purified, specific operation process is with reference to Novagen company His bind
Purification kit description, E protein, after ni-sepharose purification, i.e. obtains DTMUV HN1 strain E protein.
2) the affine screening in phage random 12 peptide storehouse
(1) the DTMUV HN1 strain E protein of purification it is coated
The E protein that said gene engineering is expressed is diluted to 10 μ g/mL with TBS, takes the E protein after dilution and be coated enzyme mark
Plate, 100 μ L/ holes, place in wet box 4 DEG C overnight；Tipping next day is coated liquid, adds the TBST washing 3 containing 0.05%Tween 20
Secondary (standing 3min), kowtows dry every time, then adds each holes with the TBS containing 5% defatted milk powder by 100 μ L/ holes, puts in wet box 37 DEG C
Close 2h；Tipping confining liquid, washs 3 times with the TBST solution containing 0.05%Tween 20, kowtows dry, the ELISA Plate envelope that will be coated
Mouthful, stand-by in 4 DEG C of preservations.
(2) affine screening
With TBST dilution primary libraries (being purchased from U.S. New England Biolabs, NEB company) to 1 × 1011pfu/
ML, takes the library liquid 100 μ L after dilution and joins in the ELISA Plate hole being coated E protein in advance, places in wet box 4 DEG C overnight.Secondary
Abandon liquid in hole day, and clean 10 times with TBST, in micropore, add 100 μ L 0.2M Glycine-HCl (pH 2.2), in room temperature
Gentle shake 10min, eluent sucks in another clean microcentrifugal tube, adds 15 μ L 1M Tris-HCl (pH 9.1) and neutralizes
Above-mentioned eluent.Measuring the titre of a small amount of (～1 μ L) eluate, residue eluate joins (bacterium in 20mL ER2738 culture
Body is in logarithm early stage), 37 DEG C are aggressively shaken cultivation 4.5h.Being proceeded to by culture in centrifuge tube, 4 DEG C 12,000rpm is centrifuged
10min.Supernatant proceeds in another centrifuge tube, then is centrifuged.Take 80% supernatant to proceed to, in new pipe, add the PEG/ of 1/6 volume
NaCl, 4 DEG C of precipitates overnight.Next day, 4 DEG C 12,000rpm is centrifuged PEG and precipitates 15min.Abandon supernatant, ofer short duration centrifugal, discard residual
Supernatant.Precipitate is resuspended in 1mL TBS, and suspension proceeds in microcentrifugal tube, and 4 DEG C of centrifugal 5min make residual cells precipitate.On
Proceed to clearly another fresh microcentrifugal tube, by the PEG/NaCl reprecipitation of 1/6 volume.(15～60min is equal to hatch 30min on ice
Can).4 DEG C of centrifugal 10min, abandon supernatant, ofer short duration centrifugal, inhale with micropipettor and abandon remaining supernatant.Precipitate is resuspended in 200 μ L
TBS, 0.02%NaN3In.Centrifugal 1min, precipitates the insoluble matter of any remnants.Supernatant proceeds to, in new centrifuge tube, be first
Bacteriophage elution thing after wheel amplification.After measuring amplification according to conventional M13 method with LB/IPTG/Xgal flat board, eluate drips
Degree.Phagocytosis body fluid after being expanded the first round is diluted to 1 × 10 with TBS11Pfu/mL, takes 100 μ L and joins and be coated E protein in advance
ELISA Plate hole in carry out second and take turns screening, 37 DEG C of incubation 2h, abandon liquid in hole, and with the TBST solution containing 0.05%Tween 20
Clean 10 times, the amplification of phage and the mensuration of titre after ibid screening, repeat above step and carry out third round screening.
In screening process, the phagocytosis scale of construction phagocytosis scale of construction often taking turns input screening is designated as Input, affording is designated as
Output, com-parison and analysis is often taken turns output/input ratio (Input/Output) situation, is reflected the enrichment of specific binding phage
Degree.Taking turns screening through 3 to find: the phage that elutriation obtains gets more and more, specificity is increasingly stronger, wherein third round phage
Titre is 2.3 × 106pfu/mL.Phage ratio for input and output improves (see table 1) by wheel, shows have specific binding effect
Phage show preferable concentration effect.
The screening enrichment to phage that table 1 three-wheel is affine
3) amplification of monoclonal phage
ER2738 is inoculated in 20mL LB culture medium, cultivates to slightly muddy for 37 DEG C.Wash in a pan from third round with the suction nozzle of sterilizing
Select random picking 10 clone respectively in thing.Each phage clone joins often in pipe ER2738 culture fluid, 37 DEG C of cultivations
4.5h.By above-mentioned culture in 4 DEG C 12,000rpm is centrifuged 10min, and supernatant moves into new centrifuge tube, then is centrifuged.Take 80% supernatant
In new centrifuge tube, add the PEG/NaCl of 1/6 volume.4 DEG C of precipitates overnight (at least 1h).4 DEG C of 12,000rpm 15min are centrifugal heavy
Form sediment, abandon supernatant, then carry out of short duration centrifugal, discard remaining supernatant.Precipitation is resuspended in 1mL TBS, and suspension is proceeded to microcentrifugation
Pipe, 4 DEG C of centrifugal 5min remove the residual cell in precipitation.Supernatant proceeds to new microcentrifugal tube, adds the PEG/NaCl of 1/6 volume
Reprecipitation.Act on 30min (15～60min) on ice.4 DEG C of centrifugal 10min, abandon supernatant, then carry out of short duration centrifugal, discard residual
Remaining supernatant.Precipitation is resuspended in 50 μ L TBS, and M13 method carries out phage titre mensuration routinely.
4) ELISA detection phage display small peptide to target molecule combination activity
The most specific binding with E protein in order to detect the phage clone that obtains of screening, survey after third round is screened and drip
20 single phage clones of random choose in the agar plate of degree, are coated elisa plate with E protein and do indirect ELISA test.With 100
The target molecule E protein of μ L 10 μ g/mL (is dissolved in 0.1M pH 8.6NaHCO3In) it is coated elisa plate, and set PBS control.Close
In the wet box of envelope, 4 DEG C are coated overnight.After confining liquid 4 DEG C closes 1h, PBST washs, and adds biting of 100 μ L single clones to be measured
Thalline supernatant (is diluted to: 1011Pfu/mL) it is small peptide to be measured, room temperature reaction 1h.After PBST washing, add and be diluted to working concentration
Anti-M13-HRP conjugate, room temperature reaction 1h.PBST fully washs, add tmb substrate chromophoric solution, room temperature 30min, after add
The H of 50 μ L 2M2SO4Terminate reaction, survey A450 value.
Result shows the positive findings (see Fig. 2) that wherein 10 clone's displays are stronger.Be respectively designated as P1, P2, P3, P4,
P5、P6、P7、P8、P9、P10。
5) Competitive assays test
By above-mentioned 10 clones, do competitive ELISA experiment, with identify further the Phage Display Peptide that screened whether with
E protein is specific binding.In wet box, it is coated target molecule E protein and closes (method is ibid), adding 100 μ L variable concentrations
(2.5 μ g/mL, 5 μ g/mL, 10 μ g/mL, 15 μ g/mL, 20 μ g/mL) E protein and above-mentioned monoclonal phage (it is diluted to:
1011Pfu/mL) equal-volume mixing, room temperature effect 10min, every hole 100 μ L, room temperature jog hatches 1h.PBST washes 6 times, adds anti-
M13-HRP antibody, room temperature jog is hatched 1h, PBST and is washed 6 times, adds tmb substrate colour developing, surveys A450 value.
The computing formula of suppression ratio sees below formula (1):
Suppression ratio (%)=(A450 value after not suppressing A450 value-suppression)/do not suppress A450 value × 100% (1)
Result shows, with E protein be combined in advance can block phage clone P3, P8 be coated E protein combination (see
Fig. 3).The specific binding peptides that 12 peptides are E protein inserted in phage P3, P8 is described.
6) preparation of single stranded phage DNA
Dilute ER2738 overnight culture by 1:100 and be inoculated in LB culture medium, being dispensed in culture tube, often pipe 1mL.With
Sterilizing suction nozzle is chosen a blue plaque and (is selected, to ensure that each plaque only contains from the total amount flat board less than 100 plaques
One DNA sequence) add in above-mentioned 1mL culture tube, each clone one pipe to be identified.5h cultivated by 37 DEG C of shaking tables, and (4.5～5h is equal
Can).Culture proceeds in microcentrifugal tube, centrifugal 30s.Supernatant proceeds in new pipe, then is centrifuged, then by 500 μ L containing phage
Supernatant proceed in new centrifuge tube.Adding 200 μ L PEG/NaCl, reverse mixing, room temperature places 10min.Centrifugal 10min, abandons
Clearly, ofer short duration centrifugal, careful suction abandons remaining supernatant.By 70% washing with alcohol precipitation, the most after drying, precipitation is resuspended in 30 μ L
In TE (10mM Tris-HCl, pH 8.0,1mM EDTA).
7) positive bacteriophage determined dna sequence
After the single stranded DNA of the phage positive colony extracting above-mentioned numbered P8, with-28gIII (such as SEQ ID NO:4 institute
Show) and-96gIII sequencing primer (as shown in SEQ ID NO:5) transfer to Shanghai Invitrongen company to check order, to speculate its phase
The aminoacid sequence answered, result shows, the aminoacid sequence of the DTMUV HN1 strain E protein specific binding peptides obtained is P8-
12:THRSWQGNSWYM (as shown in SEQ ID NO.1).
8) chemically synthesized polypeptide
Use solid-phase synthesis synthesis aforementioned polypeptides P8-12, by peptide biology company limited of Shanghai section synthesize, purity 95% with
On.
Embodiment 2
In the present embodiment, duck tembusu virus cyst membrane E protein specific binding peptides is preparing the medicine that anti-duck tembusu virus is sick
Application in thing, comprises the following steps: take the powder after duck tembusu virus cyst membrane E protein specific binding peptide purification lyophilizing
(duck tembusu virus cyst membrane E protein specific binding peptides is synthesized by Solid phase peptide synthssis mode, and RP-HPLC method is pure
Changing, after purification lyophilizing, purity reaches more than 95%), commonly use supplementary product starch with field of medicaments, sucrose, Pulvis Talci mix, be inhibited
The tablet medicine of duck tembusu virus propagation, duck tembusu virus cyst membrane E protein specific binding peptides is as in antiviral drugs
Major antiviral active component, principal agent every content is 0.1g, and the percentage composition of principal agent is 50%.Or polypeptide powder is used
PBS dissolves, and is configured to the polypeptide solution of variable concentrations, is used for suppressing duck tembusu virus proliferation test.
Embodiment 3
In the present embodiment, duck tembusu virus cyst membrane E protein specific binding peptides is in preparation suppression duck tembusu virus propagation
Feed additive in application, comprise the following steps: duck tembusu virus cyst membrane E protein specific binding peptides is frozen after purification
Dry, obtain purity and reach the polypeptide powder (purification uses high performance liquid chromatography, and lyophilizing uses freeze-drying) of more than 95%.Can
Add the duck tembusu virus cyst membrane E protein specific binding peptides of 0.2～0.5 ‰ by feed relative, feedstuff the most per ton adds many
Peptide powder 200～500 grams.
Test example
(1) impact that DEF is bred by DTMUV HN1 strain E protein specific binding peptides P8-12
1mm is cut into after rinsing 5 times (4～5 times) except the duck embryonic PBS of brain, eye, extremity, internal organs3(0.5～
1.5mm3) piece of tissue；Tissue is moved into culture bottle, is inverted culture bottle, is placed in 37 DEG C, 5%CO2Cultivate in incubator
4.5h (4～5h)；Complete adherent rear addition culture fluid 9ml (8～10mL) of piece of tissue, culture fluid composition: DMEM+
10% superfine hyclone (FBS)；Culture fluid in reject culture bottle, after residue in PBS washing culture bottle 2 times, uses pancreas
Add culture fluid 9mL (8～10mL) after protease digestion and terminate digestion；Postdigestive cell average mark loads 2 cultivations
In Ping, put into 37 DEG C, 5%CO2Incubator continues cultivate to 2 × 105Cells/mL, is divided into 5 groups, and the 1st to the 4th group respectively
Add the P8-12 peptide of variable concentrations (0.02 μ g/mL, 0.2 μ g/mL, 2 μ g/mL, 20 μ g/mL).5th group for adding PBS blank
Group, after continuing to cultivate 48h, uses mtt assay detection DEF proliferative conditions.
The computing formula of Relative cell proliferation rate sees below formula (2):
Relative cell proliferation rate=experimental group OD value/matched group OD value × 100% (2)
The computing formula of cell inhibitory rate sees below formula (3):
Cell inhibitory rate=(matched group OD value-experimental group OD value)/matched group OD value × 100% (3)
Result shows, compared with PBS blank group, the P8-12 peptide group cell under experimental concentration is relative to the equal nothing of the rate of increase
Notable change (P > 0.05) (see Fig. 4), shows that DTMUV HN1 strain E protein specific binding peptides P8-12 itself is to duck embryo fibroblast
The propagation of cell has no significant effect.
(2) shadow that DTMUV HN1 strain is bred by DTMUV E protein specific binding peptides P8-12 at DEF
Ring
DEF is cultivated to 2 × 105Cells/mL, rinses by the DMEM culture medium containing 1%FBS, adds
Plasma-free DMEM medium, then subpackage is in 96 porocyte culture plates, and experiment is divided into 6 groups, and the 1st～5 group adds virus and infect multiple
Number is the DTMUV virus liquid of 0.01MOI.For adding PBS blank group, respectively organize cell and cultivate 4h, discard infection liquid, PBS for 6th group
Rinse cell 2 times, add the DMEM culture medium containing 10%FBS, then in the 1st group to the 4th group, be separately added into variable concentrations (0.02
μ g/mL, 0.2 μ g/mL, 2 μ g/mL, 20 μ g/mL) P8-12 peptide.After continuing to cultivate 48h, observed and recorded pathological changes, and press Reed
And Munch formula calculates the TCID50 of each group, uses fluorescence quantitative PCR method detection DTMUV viral copies simultaneously.
Fluorescent quantitative PCR result shows, compared with the 5th group, and the 1st group to the 4th group of polypeptide P8-12 adding variable concentrations
(0.02 μ g/mL, 0.2 μ g/mL, 2 μ g/mL, 20 μ g/mL) all can significantly lower DTMUV HN1 strain in DEF
Viral copy number, measure different experiments group DTMUV HN1 strain TCID50, result displays that, the polypeptide P8-12 of variable concentrations
All can significantly reduce DTMUV HN1 strain virus titer in DEF.Result above shows, DTMUV HN1 strain E
Protein-specific binding peptide P8-12 can suppress DTMUV HN1 strain in the propagation of DEF.
Claims (4)
1. duck tembusu virus cyst membrane E protein specific binding peptides, it is characterised in that: its aminoacid sequence such as SEQ ID NO:1
Shown in.
Duck tembusu virus cyst membrane E protein specific binding peptides the most according to claim 1, it is characterised in that: described
Duck tembusu virus is duck tembusu virus HN1 strain.
3. an application for duck tembusu virus cyst membrane E protein specific binding peptides as claimed in claim 1 or 2, its feature
It is: described duck tembusu virus cyst membrane E protein specific binding peptides is preparing the sick medicine of anti-duck tembusu virus or feedstuff
Application in additive.
The application of duck tembusu virus cyst membrane E protein specific binding peptides the most according to claim 3, it is characterised in that:
Described duck tembusu virus cyst membrane E protein specific binding peptides increases at DEF at preparation suppression duck tembusu virus
Application in the medicine grown or feed additive.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410361045.1A CN104193804B (en) | 2014-07-25 | 2014-07-25 | Duck tembusu virus cyst membrane E protein specific binding peptides and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410361045.1A CN104193804B (en) | 2014-07-25 | 2014-07-25 | Duck tembusu virus cyst membrane E protein specific binding peptides and application thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN104193804A CN104193804A (en) | 2014-12-10 |
| CN104193804B true CN104193804B (en) | 2016-08-24 |
Family
ID=52079200
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201410361045.1A Expired - Fee Related CN104193804B (en) | 2014-07-25 | 2014-07-25 | Duck tembusu virus cyst membrane E protein specific binding peptides and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN104193804B (en) |
Families Citing this family (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109865138B (en) * | 2017-12-05 | 2022-03-22 | 江苏省农业科学院 | Application of tembusu virus E protein receptor binding domain |
| CN114315991A (en) * | 2020-10-30 | 2022-04-12 | 广西壮族自治区动物疫病预防控制中心 | Competitive ELISA method based on duck flavivirus E protein and monoclonal antibody thereof |
Family Cites Families (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101412750B (en) * | 2008-10-08 | 2011-06-15 | 南京农业大学 | Bursin specific binding peptide and screening method thereof |
| CN102586193B (en) * | 2012-02-10 | 2013-06-19 | 中国农业科学院上海兽医研究所 | Monoclonal antibody for resisting duck tembusu virus, hybridoma strain and application thereof |
-
2014
- 2014-07-25 CN CN201410361045.1A patent/CN104193804B/en not_active Expired - Fee Related
Also Published As
| Publication number | Publication date |
|---|---|
| CN104193804A (en) | 2014-12-10 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Liu et al. | Genetic diversity of avian infectious bronchitis coronavirus strains isolated in China between 1995 and 2004 | |
| Li et al. | Isolation and genetic analysis revealed no predominant new strains of avian infectious bronchitis virus circulating in South China during 2004–2008 | |
| CN104193805B (en) | Duck tembusu virus envelop E protein inhibitory peptide and application thereof | |
| CN107619822B (en) | Porcine epidemic diarrhea virus low virulent strain, and culture method and application thereof | |
| Liao et al. | Genetic characterization of a novel astrovirus in Pekin ducks | |
| CN101724605B (en) | Foot-and-mouth disease virus (FMDV) resistant monoclonal antibody and identified epitope and application thereof | |
| CN107987136A (en) | ZIKV-NS1 albumen and its application in zika virus transmission_blocking vaccine is prepared | |
| Chen et al. | Recognition of linear B-cell epitope of betanodavirus coat protein by RG-M18 neutralizing mAB inhibits giant grouper nervous necrosis virus (GGNNV) infection | |
| CN104193804B (en) | Duck tembusu virus cyst membrane E protein specific binding peptides and application thereof | |
| CN114874995B (en) | Swine fever virus 2E rns Monoclonal antibody hybridoma cell strain of protein and application | |
| CN102690327A (en) | Enterovirus 71 neutralized epitope polypeptide and application thereof | |
| Wang et al. | The dynamics of Chinese variant porcine epidemic diarrhea virus production in Vero cells and intestines of 2-day old piglets | |
| KR101885573B1 (en) | Affibody specific to dengue virus | |
| CN104193803B (en) | DHV I type VP1 albumen peptide for inhibiting and application thereof | |
| CN109402065A (en) | One kind kind duck C hypotype fowl metapneumovirus low virulent strain and its preparation and application | |
| CN107698665A (en) | A kind of antiviral polypeptide, encoding gene, carrier, Host Strains and application | |
| CN103013929B (en) | Avian influenza virus, and detection kit and vaccine thereof | |
| CN105037499B (en) | It is directed to people's histamine receptor 4 using phage antibody library（HR4）Epitope mimic peptide screening and its vaccine construction method | |
| CN107602672A (en) | The adenovirus cilia protein peptide of recombination expression, adenovirus subunit vaccine and preparation method thereof | |
| US20210040153A1 (en) | Oligopeptide having dengue virus replication inhibition function and application thereof | |
| CN110845584A (en) | Hog cholera virus envelope protein oligomeric protein body and preparation method and application thereof | |
| CN112618542B (en) | Use of HSP70 inhibitors for broad spectrum anti-flavivirus activity | |
| Zhou et al. | An affinity peptide exerts antiviral activity by strongly binding nervous necrosis virus to block viral entry | |
| Xu et al. | Recombinant scFv antibodies against infectious pancreatic necrosis virus isolated by flow cytometry | |
| CN104163854B (en) | DHV I type VP1 protein-specific binding peptide and application thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20160824 Termination date: 20170725 |
|
| CF01 | Termination of patent right due to non-payment of annual fee |