CN104198736A - Application of efficiently and actively expressed protein in duck tembusu virus E protein core antigen domain - Google Patents
Application of efficiently and actively expressed protein in duck tembusu virus E protein core antigen domain Download PDFInfo
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- CN104198736A CN104198736A CN201410445731.7A CN201410445731A CN104198736A CN 104198736 A CN104198736 A CN 104198736A CN 201410445731 A CN201410445731 A CN 201410445731A CN 104198736 A CN104198736 A CN 104198736A
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
- G01N33/56983—Viruses
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
Abstract
The invention relates to a protein analyzing, preparing and diagnosing technology in the technical field of biotechnology and relates to application of efficiently and actively expressed protein in a duck tembusu virus E protein core antigen domain. A base sequence of the duck tembusu virus E protein core antigen domain is shown as a sequence 1 in a sequence table; an indirect ELISA detection method which is high sensitivity, accuracy and controllability is built on the basis of screening DTMUV antigen epitope core area; powerful tools and technical support are provided for effectively preventing and controlling transmission of the DTMUV.
Description
technical field
The present invention relates to protein analysis, preparation and diagnostic techniques in biological technical field, relate to the purposes of the duck tembusu virus E protein core antigenic domain albumen of high-efficiency activated expression.
background technology
2010, a kind of novel duck source flavivirus, in the outburst of scale duckery of China the extremely whole nation of rapid spread, was supported duck industry to China and has been brought huge economic loss.This cause of disease-duck tembusu virus (Duck Tembusu virus, DTMUV) is flaviviridae, Flavivirus member, for there being cyst membrane non-segmented negative single strand plus RNA virus.Duck mainly occurs that nervous symptoms, feed intake reduce, the degradation of laying eggs even has no harvest, and occurs mass mortality after infecting DTMUV.The other diseases such as the easy secondary disease virus hepatitis of duck, infectious serositis, Escherichia coli that infects DTMUV, owing to widely applying medicine, causes medicament residue serious after disease occurs, and has influence on duck quality and human health.In addition, aquatic bird is important " reservoir " of influenza virus at occurring in nature, once autoimmunity declines, will cause a large amount of propagation and the restructuring variation of influenza virus.
In view of DTMUV produces and potential significant damage, must set up rational prevention and control and monitoring measure, understand timely duck group's infection comprehensively and be with malicious situation.The Serology test of therefore, standardization, pin-point accuracy becomes inevitable.General Flavivirus Serology test has ELISA, AGP, hemagglutination-inhibition test (HI), complement fixation test (CFT) (CF) and neutralization experiment (NT) etc. in the world, but ELISA has high specificity, highly sensitive, detection speed is fast, the advantage such as easy and simple to handle.CN103869066A discloses a kind of tembusu virus double-antibody sandwich elisa detection method.Detection sensitivity and accuracy are the foundations that judges detection method quality.Disclosed ELISA detection method in prior art, sensitivity and accuracy, space all has greatly improved.
Summary of the invention
In order to solve the sensitivity and the not high problem of accuracy that exist in the ELISA detection method of tembusu virus in above prior art, the application of the duck tembusu virus E protein core antigenic domain albumen that the invention provides a kind of high-efficiency activated expression in the ELISA of tembusu virus detection method.
The present invention is achieved by the following measures:
Determining of duck tembusu virus E protein core antigenic domain of the present invention, to carry out homology modeling according to the expression product of duck tembusu virus E albumen, by analyzing its complete 3D structure, albumen composition and each compositing area fine structure, find that E albumen is folded into 3 different structure territory I-III (Domain I-III) at virus surface: the barrel-like structure that D I is made up of 8 β-pleated sheet sheets, being positioned at the center of albumen, is light gray region in figure; D II (figure Oxford gray region) is to form the dactylitic texture extending, and its sequence is interspersed among D I sequence, is and merges ring at D II end; Be D III (black region in figure) in the contrary direction of D II, for Ig sample (Immunoglobulin-like) domain, it is typical immunoglobulin structure, contain 7 β lamellas, find according to the research of other similar flaviviruss with to this region fine-structure distribution, this region is the main positions that virus antigen epitope exists simultaneously; In addition, by flavivirus being entered to the process study of host cell, finding that in DTMUV E albumen, His144, His285, His320 (are shown in that Fig. 1 marks) on the interface between D I and D III, participate in the conformational change that E albumen pH relies on, is also the main site of antibody effect.So from above protein structure, DTMUV antigen dominant area is positioned at the D III of E albumen.
Duck tembusu virus E protein core antigenic domain of the present invention (be shown in sequence table shown in sequence 1 by base sequence, hereinafter to be referred as E D III) expression, to determine on the basis of core antigenic domain, first design the pcr amplification of duck tembusu virus E protein D III and express primer E D III exp-1:5 '-CATGCCATGGAAAGGCATGACCTACCCGATGTG-3 ' (comprising NcoI restriction enzyme site), E D III exp-2:5 '-CCGCTCGAGACTTCTATGCCACTGGTACCT-3 ' (comprising XhoI restriction enzyme site), for the clone of duck tembusu virus E D III.Adopt after NcoI and XhoI double digestion PCR product, be connected in the pET32a of same double digestion, build recombinant expression plasmid pET32a/E D III, then transform Bacillus coli expression bacterial strain Rosetta gami B (DE3) and carry out the high-efficiency activated expression of E D III.The demonstration of SDS-PAGE result, when temperature is 18 DEG C, IPTG concentration is that 0.2mM spends the night while inducing cultivation, has realized the solution expression with high efficiency of E D III albumen, expressing quantity can account for 42% of bacterial protein, and albumen has natural activity.
The expression of duck tembusu virus E protein core antigenic domain of the present invention also comprises the high-purity preparation of expressing protein.Thalline carries out ultrasonication, and albumin in collection selects nickel post to carry out purifying.First with containing 50mM Tris and 300mM NaCl(pH value 8.0) damping fluid balance cylinder, then make upper albumin through cylinder with suitable speed, finally carry out gradient elution with the imidazoles of variable concentrations, eluent is 50mM Tris, the Imidazole(pH value 8.0 of 300mM NaCl and 0 mM, 20mM, 40 mM, 50 mM, 250mM variable concentrations), collect the albumen of wash-out and dialyse under the condition of 20mM Tris (pH7.4).The high purity protein that final acquisition purity is 90%.
Duck tembusu virus antibody indirect ELISA diagnostic method of the present invention is characterized in that: use the high-purity activity E D III albumen of prokaryotic expression as ELISA coating protein, every hole adds the coating buffer of 100 L containing the E D III albumen of 0.5ug/ml, after room temperature effect 30min, 4 DEG C are spent the night, PBST washes three times, 37 DEG C of sealing 2h, then add the duck tembusu virus serum of 10000 times of dilutions to be checked, every hole 100 L, room temperature effect 0.5h, PBST washes the anti-duck ELIAS secondary antibody of rabbit that adds 100 L 1:5000 dilutions after plate 3 times, room temperature 0.5h, PBST washes plate 3 times, finally add 100 L1mg/mLTMB substrates, after room temperature effect 15min, add stop buffer, in microplate reader, measure OD450 value.The positive and negative control are established in reaction simultaneously, and negative positive criterion is for when OD450 detected value-negative control average/positive control average-negative control average, and it is 0.2 positive that gained ratio is more than or equal to, and are less than or equal to 0.2 negative.
ELISA detection kit provided by the invention, is characterized in that:
Elisa plate used be with 100mL containing 4 DEG C of coated spending the night after the coating buffer room temperature effect 30min of 0.5mg/mL duck tembusu virus E D III albumen, PBST washes plate 3 times, adds confining liquid, the ELISA Plate of 37 DEG C of sealing 2h.
Coating buffer used is for containing 15mM Na
2cO
3, 35mM NaHCO
3, the solution of pH 9.6.
PBST used is that every 1000mL solution is containing 8g NaCl, 0.2g KCl, 2.9g Na
2hPO412H
2o, 0.2g KH
2pO
4, 0.5mL tween-20.
Substrate buffer solution used is that every 500mL solution is containing 14.2g citric acid, 10.5g Na
2hPO
412H
2o.
Substrate solution used is TMB mother liquor (0.1g/100mL absolute ethyl alcohol) 10mL, substrate solution 90mL, H
2o
2750uL is formulated before use.
Confining liquid used is the PBST containing 1%BSA.
ELIAS secondary antibody used is to dilute with PBST 1:5000.
Positive serum reference standards used, for using duck tembusu virus purifying E D III albumen to be prepared from through four immune new zealand white rabbits, saves backup as a standard positive serum-20 DEG C packing.
Yin and yang attribute result used is judged: when OD450 detected value-negative control average/positive control average-negative control average, it is 0.2 positive that gained ratio is more than or equal to, and is less than or equal to 0.2 negative.
Beneficial effect of the present invention:
On the basis of screening DTMUV epitope nucleus, hypersensitivity, accuracy and handling strong indirect ELISA detection method are set up, for the propagation of effective prevention and control DTMUV provides strong tools and techniques to support.
Brief description of the drawings
Fig. 1. E albumen 3D structural representation,
Wherein D I is left end light gray region, and D II is stage casing dark grey area, and D III is right-hand member black region;
Fig. 2. duck tembusu virus abduction delivering figure,
Wherein: M:Markers, 1: do not induce supernatant, 2: not induced precipitation, 18 DEG C of 3:0.2mM IPTG spend the night (supernatant); 18 DEG C of 4:0.2mM IPTG spend the night (precipitation); 25 DEG C of 5:0.2mM IPTG spend the night (supernatant); 25 DEG C of 6:0.2mM IPTG spend the night (precipitation); 30 DEG C of 7:0.2mM IPTG spend the night (supernatant); 30 DEG C of 37 DEG C of (supernatant) 5h of (precipitation) 9:0.2mM IPTG that spend the night of 8:0.2mM IPTG; 37 DEG C of 5h(precipitations of 10:0.2mM IPTG);
Fig. 3. the qualification of duck tembusu virus E D III purity of protein,
Wherein: M:Markers, 1,2 is E D III protein purification product.
Embodiment
Below in conjunction with specific embodiment, further illustrate related content of the present invention.Not marked concrete test method in following example, conventionally according to the condition described in molecular cloning laboratory manual, or the condition providing according to manufacturer.
the acquisition of embodiment 1 antigen protein
The expression of duck tembusu virus E protein core antigenic domain (hereinafter to be referred as E D III), to determine on the basis of core antigenic domain, first design the pcr amplification of duck tembusu virus E protein D III and express primer E D III exp-1:5 '-CATGCCATGGAAAGGCATGACCTACCCGATGTG-3 ' (comprising NcoI restriction enzyme site), E D III exp-2:5 '-CCGCTCGAGACTTCTATGCCACTGGTACCT-3 ' (comprising XhoI restriction enzyme site), for the clone of duck tembusu virus E D III.Adopt after NcoI and XhoI double digestion PCR product, be connected in the pET32a of same double digestion, build recombinant expression plasmid pET32a/E D III, then transform Bacillus coli expression bacterial strain Rosetta gami B (DE3) and carry out the high-efficiency activated expression of E D III.The demonstration of SDS-PAGE result, when temperature is 18 DEG C, IPTG concentration is that 0.2mM spends the night while inducing cultivation, has realized the solution expression with high efficiency of E D III albumen, expressing quantity can account for 42% of bacterial protein, and albumen has natural activity.
The expression of duck tembusu virus E protein core antigenic domain of the present invention also comprises the high-purity preparation of expressing protein.Thalline carries out ultrasonication, and albumin in collection selects nickel post to carry out purifying.First with containing 50mM Tris and 300mM NaCl(pH value 8.0) damping fluid balance cylinder, then make upper albumin through cylinder with suitable speed, finally carry out gradient elution with the imidazoles of variable concentrations, eluent is 50mM Tris, the Imidazole(pH value 8.0 of 300mM NaCl and 0 mM, 20mM, 40 mM, 50 mM, 250mM variable concentrations), collect the albumen of wash-out and dialyse under the condition of 20mM Tris (pH7.4).The high purity protein that final acquisition purity is 90%.
embodiment 2
Working concentration and the time of the best coated concentration of duck tembusu virus E D III indirect ELISA method antigen, best serum dilution, the best coated condition of antigen, ELIAS secondary antibody obtain in the following manner:
(1) determining of the best coated concentration of antigen and best serum dilution
The concentration of antigen and serum is determined employing square formation titrimetry.E D III antigen protein is carried out to doubling dilution with 20mM Tris (pH7.4), final concentration is respectively 1 μ g/ml, 0.5 μ g/ml, and 0.25 μ g/ml, 0.125 μ g/ml, the horizontal coated elisa plate of every hole 100 μ L, 4 DEG C are spent the night.Then use PBST liquid to wash three times, add confining liquid, 37 DEG C of sealing 2h.Yin and yang attribute serum starts to carry out the longitudinal doubling dilution of ELISA Plate from 1:2000, and dilutability is respectively 1:2000,1:4000, and 1:8000,1:16000, every hole adds 100 L, room temperature effect 0.5h, PBST washes plate 3 times.Then add 2000 times of dilution goat-anti duck ELIAS secondary antibody ELIAS secondary antibody 100 L, room temperature 0.5h, PBST washes plate 3 times, finally adds 100 L1mg/mL tmb substrates, after room temperature effect 15min, adds stop buffer, measures OD450 value in microplate reader.Finally determine the optimum dilution degree of the coated concentration harmonizing yinyang serum of the best of antigen according to P/N value.When antigen concentration is 0.5 μ g/ml, when serum dilution 1:10000, the OD450 value of positive serum is 1.0303(≈ 1), feminine gender is that 0.1597, P/N value is 6.4493(to the maximum and sees the following form).Therefore 0.05 μ g antigen is coated optium concentration, and serum 1:10000 dilution is best dilute concentration.
Titration square formation OD value
(2) determining of the best coated condition of antigen
On the antigen coated concentration of the best and the definite basis of serum dilution, the coated of E D III antigen acts on 20min, 30min, 45min, 60min respectively under room temperature (25 DEG C) and 37 DEG C of conditions, and then 4 DEG C are spent the night, other conditions constant (1) together.In the time of room temperature effect 30min, positive serum OD450 value is 1.0994, and negative serum OD450 value is that 0.3156, P/N value is maximum, is 3.4835.
The best coated condition of antigen
(3) determining of the working concentration of ELIAS secondary antibody and time
Select 96 hole elisa plates, every hole adds the E D III albumen of 100 L 0.5 g/ml, and for coated elisa plate, after coating buffer room temperature effect 30min, 4 DEG C are spent the night, and then use PBST liquid to wash three times, add confining liquid, 37 DEG C of sealing 2h.Add the yin and yang attribute serum of 1:10000 dilution, every hole 100 L, room temperature effect 0.5h, PBST washes plate 3 times.The working concentration of anti-rabbit of HRP mark duck ELIAS secondary antibody is carried out to 1:1000,1:2000,1:3000,1:4000,1:5000,1:6000 dilution, room temperature is placed as respectively 15min, 30 min, 45 min, 60 min, and PBST washes plate 3 times, finally adds 100 L1mg/mL tmb substrates, after room temperature effect 15min, add stop buffer, in microplate reader, measure OD450 value.ELIAS secondary antibody is carried out 1:5000 dilution, room temperature effect 30min, and it is maximum that positive and negative OD450 value differs, and P/N value is maximum.
ELIAS secondary antibody the best use of condition
the susceptibility of embodiment 3 duck tembusu virus E D III indirect ELISAs
Select 96 hole elisa plates, every hole adds the E D III albumen of 100 L 0.5 g/ml, and for coated elisa plate, after coating buffer room temperature effect 30min, 4 DEG C are spent the night, and then use PBST liquid to wash three times, add confining liquid, 37 DEG C of sealing 2h.Add 1:2000-1:32000 doubling dilution DTMUV positive serum, every hole 100 L, room temperature effect 0.5h, PBST washes the anti-duck ELIAS secondary antibody of rabbit that adds again 100 L 1:5000 dilutions after plate 3 times, room temperature 0.5h, PBST washes plate 3 times, finally add 100 L1mg/mL tmb substrates, after room temperature effect 15min, add stop buffer, in microplate reader, measure OD450 value, result is judged the computing method that use OD450 detected value-negative control average/positive control average-negative control average, positive in the time that gained ratio is more than or equal to 0.2, be less than or equal to 0.2 o'clock negative.Measurement result, in the time that serum dilutes according to 1:16000, testing result is positive higher than 0.2, and 1:32000 measured value is less than 0.2, negative, and the susceptibility of system can reach 1:16000.
Up to the present, also indirect ELISA method (the animal medicine progress that has report to use duck tembusu virus E albumen to set up as envelope antigen, 2012,33(12): 17-22), its definite serum optimum dilution degree is 1:320, though do not carry out sensitivity experiments, two orders of magnitude (10 of optimum dilution degree that the dilutability using uses lower than this patent
2doubly), almost there is not comparability, reflect the height of the detection sensitivity of this programme.
the specificity of embodiment 4 duck tembusu virus E D III indirect ELISAs
Taking duck virus hepatitis virus, duck reovirus, duck Avian pneumo-encephalitis virus, avian influenza virus, avian leukosis virus, duck plague virus positive serum as contrast, carry out the specific detection of indirect ELISA method.Select 96 hole elisa plates, every hole adds the E D III albumen of 100 L 0.5 g/ml, and for coated elisa plate, after coating buffer room temperature effect 30min, 4 DEG C are spent the night, and then use PBST liquid to wash three times, add confining liquid, 37 DEG C of sealing 2h.The serum that adds 1:1000 doubly to dilute, every hole 100 L, room temperature effect 0.5h, PBST washes the anti-duck ELIAS secondary antibody of rabbit that adds again 100 L 1:5000 dilutions after plate 3 times, room temperature 0.5h, PBST washes plate 3 times, finally adds 100 L1mg/mL tmb substrates, after room temperature effect 15min, add stop buffer, in microplate reader, measure OD450 value.The positive and negative control hole are established in reaction simultaneously, and result is judged the computing method that use OD450 detected value-negative control average/positive control average-negative control average, positive in the time that gained ratio is more than or equal to 0.2, be less than or equal to 0.2 o'clock negative.The positive serum of result DHV, ARV, NDV, AIV, ALV, DPV and negative control detected value are all less than 0.2, and testing result is negative, and duck tembusu virus measurement result is greater than 0.2, presents the positive, show that the method specificity is good.
embodiment 5 replica tests
In being divided into batch revision test and batch between revision test.While test in carrying out batch, select 96 hole elisa plates, with the antigen coated different elisa plate of same batch of preparation, every hole adds the E D III albumen of 100 L 0.5 g/ml, and after coating buffer room temperature effect 30min, 4 DEG C are spent the night, and then uses PBST liquid to wash three times, add confining liquid, 37 DEG C of sealing 2h.50 parts of serum to be checked that add 1:1000 doubly to dilute, every part of serum repeats 3 times, every hole 100 L, room temperature effect 0.5h, PBST washes the anti-duck ELIAS secondary antibody of rabbit that adds again 100 L 1:5000 dilutions after plate 3 times, room temperature 0.5h, PBST washes plate 3 times, finally add 100 L1mg/mL tmb substrates, after room temperature effect 15min, add stop buffer, in microplate reader, measure OD450 value.The positive and negative control hole are established in reaction simultaneously.In batch in replica test coefficient of variation maximum be 2.63%, and minimum be only 1.05%.While test between batch, select 96 hole elisa plates, the antigen of preparing with 3 batches of different batches is coated elisa plate respectively, every hole adds the E D III albumen of 100 L 0.5 g/ml, and after coating buffer room temperature effect 30min, 4 DEG C are spent the night, and then uses PBST liquid to wash three times, add confining liquid, 37 DEG C of sealing 2h.50 parts of serum to be checked that add 1:1000 doubly to dilute, every hole 100 L, room temperature effect 0.5h, PBST washes the goat-anti duck ELIAS secondary antibody that adds again 100 L 1:5000 dilutions after plate 3 times, room temperature 0.5h, PBST washes plate 3 times, finally adds 100 L1mg/mL tmb substrates, after room temperature effect 15min, add stop buffer, in microplate reader, measure OD450 value.The positive and negative control hole are established in reaction simultaneously.Calculate the coefficient of variation according to OD value equally.The coefficient of variation of preparing antigen-reactive result with a serum and different batches is all below 3.7%, and repeatability is good.
embodiment 6 contrast property tests
Opening antenatal duck (47 week age) serum to 78 of clinical collection parts uses respectively the indirect ELISA method of agar gel diffusion test (AGP), antibody neutralization test and foundation to contrast detection.Indirect ELISA method detects 18 of positive quantity, and negative quantity is 60, and AGP and neutralizing antibody test the positive quantity detecting and be respectively 2 and 5, all far below the Positive rate of indirect ELISA method.
Indirect ELISA detection method and agar method of diffusion, antibody neutralization method detect contrast test
| Detection method | Positive quantity | Negative quantity |
| Indirect ELISA | 18 | 60 |
| AGP | 2 | 76 |
| Antibody neutralization | 5 | 73 |
Above-described embodiment is preferably embodiment of the present invention; but embodiments of the present invention are not subject to the restriction of embodiment; other is any does not deviate from change, modification, the combination made under Spirit Essence of the present invention and principle, substitute, simplify and all should be equivalent substitute mode, within being included in protection scope of the present invention.
<110> Institute of Animal Science and Veterinary Medicine, Shandong Academy of Agricul
The purposes of the duck tembusu virus E protein core antigenic domain albumen of the high-efficiency activated expression of <120>
<160>3
<210>1
<211>309
<212>DNA
<213> duck tembusu virus (Duck Tembusu virus)
<220>
<223>
<400>1
aaaggaatga?cctacccgat?gtgtagcaat?acattttccc?tagtgaagaa?tcctaccgac?60
actgggcatg?gcactgtcgt?ggtggaattg?tcttatgcag?gtaccgatgg?gccctgtaga?120
gttcccatat?ccatgtcggc?agatctgaat?gacatgacac?cagttggacg?cttgataaca?180
gtcaatccat?acgtgtcgac?ctcctccacg?ggtgccaaga?taatggtgga?agtggaacct?240
ccattcgggg?attcattcat?cttagtagga?agtggaaaag?gacagatcag?gtaccagtgg?300
catagaagt 309
<210>2
<211>33
<212>DNA
<213> is manually synthetic
<220>
<223>
<400>2
CATGCCATGG?AAAGGCATGA?CCTACCCGAT?GTG?33
<210>2
<211>30
<212>DNA
<213> is manually synthetic
<220>
<223>
<400>3
CCGCTCGAGA?CTTCTATGCC?ACTGGTACCT?30
Claims (9)
1. the application of the duck tembusu virus E protein core antigenic domain expressing protein of high-efficiency activated expression in the ELISA of duck tembusu virus detection method, duck tembusu virus E protein core antigenic domain base sequence is as shown in sequence in sequence table 1.
2. application according to claim 1, is characterized in that duck tembusu virus E protein core antigenic domain expressing protein obtains by following steps:
The pcr amplification of design duck tembusu virus E protein D III is expressed primer:
E?DⅢ?exp-1:?5’-CATGCCATGGAAAGGCATGACCTACCCGATGTG-3’，
E?DⅢ?exp-2:?5’-CCGCTCGAGACTTCTATGCCACTGGTACCT-3’，
For the clone of duck tembusu virus E protein core antigen gene, adopt after NcoI and XhoI double digestion PCR product, be connected in the plasmid of same double digestion, build recombinant expression plasmid, transform Bacillus coli expression bacterial strain and carry out activity expression.
3. application according to claim 2, is characterized in that Bacillus coli expression bacterial strain is at 18 DEG C, IPTG concentration be 0.2mM spend the night induction cultivate.
4. according to the application described in claim 2 or 3, it is characterized in that coli somatic to carry out ultrasonication, albumin in collection, selects nickel post to carry out purifying to obtain the high-purity expressing protein of duck tembusu virus E protein core antigenic domain.
5. application according to claim 4, it is characterized in that first with pH value be 8.0, containing the damping fluid balance cylinder of 50mM Tris and 300mM NaCl, then make albumin through cylinder, finally carry out gradient elution with imidazoles, eluent is 50mM Tris, and 300mM NaCl and concentration are respectively the imidazoles of 0 mM, 20mM, 40 mM, 50 mM, 250mM, and pH value is 8.0, collect the albumen of wash-out and dialyse under the condition of pH7.4,20mM Tris, the high purity protein that acquisition purity is 90%.
6. according to the application described in any one in claim 1-5, it is characterized in that duck tembusu virus E protein core antigenic domain expressing protein is as ELISA coating protein, detect for the ELISA of duck tembusu virus.
7. application according to claim 6, the coated concentration that it is characterized in that ELISA coating protein is 0.5mg/mL.
8. application according to claim 6, it is characterized in that elisa plate used is containing 4 DEG C of coated spending the night after the coating buffer room temperature effect 30min of 0.5mg/mL duck flavivirus E D III albumen with 100uL, PBST washes plate 3 times, adds confining liquid, the ELISA Plate of 37 DEG C of sealing 2h.
9. application according to claim 8, is characterized in that coating buffer used is for containing 15mM NaCO
3, 35mM NaHCO
3, the solution of pH 9.6; PBST used is that every 1000mL solution is containing 8gNaCl, 0.2gKCl, 2.9g Na
2hPO4.12H
2o, 0.2g KH
2pO
4, 0.5mL tween-20; Substrate buffer solution used is that every 500mL solution is containing 14.2g citric acid, 10.5g Na
2hPO
4.12H
2o; Substrate solution used is 0.1g/100mL absolute ethyl alcohol 10mL, substrate solution 90mL, H
2o
2750uL is formulated before use; Confining liquid used is the PBST containing 1%BSA; ELIAS secondary antibody used is to dilute with PBST 1:5000.
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105087607A (en) * | 2015-09-28 | 2015-11-25 | 山东出入境检验检疫局检验检疫技术中心 | Method for prokaryotic expression of third structural domain of DTMUV (duck Tembusu virus) E protein as well as application of method |
| CN107656066A (en) * | 2017-09-07 | 2018-02-02 | 华中农业大学 | A kind of duck tembusu virus E protein truncated protein and application |
| CN113501866A (en) * | 2021-08-02 | 2021-10-15 | 山东省农业科学院畜牧兽医研究所 | Duck tembusu virus inhibitor |
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| CN113501866A (en) * | 2021-08-02 | 2021-10-15 | 山东省农业科学院畜牧兽医研究所 | Duck tembusu virus inhibitor |
| CN115353564A (en) * | 2022-08-08 | 2022-11-18 | 华中农业大学 | Duck tembusu virus monoclonal antibody EDIII-Mab and detection kit and application thereof |
| CN115353564B (en) * | 2022-08-08 | 2024-03-26 | 华中农业大学 | Duck tembusu virus monoclonal antibody EDIII-Mab, detection kit and application thereof |
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