CN1769294A - Recombinant akabane virus capsid protein, its preparation method and uses - Google Patents
Recombinant akabane virus capsid protein, its preparation method and uses Download PDFInfo
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Abstract
The invention discloses a novel recombinant Akabane virus nucleotide capsid protein, gene encoding the recombinant protein and the process for preparing the recombinant protein. The recombinant protein has the amino acid sequence represented by SEQ ID No:1, the gene encoding the recombinant Akabane virus nucleotide capsid protein has the amino acid sequence represented by SEQ ID No:2. The preparing process of the recombinant protain comprises the following steps: constructing recombinant Akabane virus nucleotide capsid protein prokaryotic expression vectors, transforming bacillus coli with the recombinant Akabane virus nucleotide capsid protein prokaryotic expression vectors, inducing recombinant nucleotide capsid protein expression with IPTG, reclaiming and purifying the expressed recombinant nucleotide capsid protein.
Description
Technical field
The present invention relates to a kind of recombinant protein, relate in particular to a kind of recombinant akabane virus capsid protein, this recombinant akabane virus capsid protein gene of encoding, the preparation method of this recombinant akabane virus capsid protein and the application in detection or diagnosis Akabane Disease poison antigen thereof belong to the genetically engineered field.
Background technology
The Akabane Disease poison, claim Ah card's pinta poison (Akabane virus again, AKAV) be the arboviruses that belongs to the hot ripple virus groups of Bunyaviridae Bunyavirus, can cause ox, sheep, goat production congenital abnormality fetus, with stream, early, stillbirth and congenital archrogryposis and hydranencephaly disease (Arthrogryposis-Hydraencephaly, AH syndromes) be feature.Separate at first obtaining AKAV virus in the mosquito Cules Tritaeniorhynchis that in the red plumage in Gunma Prefecture, Japan village cowshed, gathers in nineteen fifty-nine and the AedesVexans body, so called after Akabane Disease poison.
AKAV is the sub-thread minus-stranded rna virus, its nucleic acid by big (L, about 7000nt), in (M, 4309nt), little (S, 858nt) three sections compositions.L sections coding L albumen, it has transcribes and replication activity; M sections coding membrane glycoprotein G1, G2 and Nonstructural Protein NSm; S sections coding nucleocapsid protein N (702bp) and Nonstructural Protein Ns (276bp).To 23 strain isolateds (comprising the OBE-1 strain), phylogenetic analysis is the result show, N Argine Monohydrochloride 97%-100% homology has high conservative, does the ELISA demonstration with the N protein monoclonal antibody that the OBE-1 strain is made, and all strains all have very high reactive behavior.
Akabane Disease once was very popular to the west of the Japanese Northeast at 1972-1975, caused enormous economic loss.At present, in East Asia, South East Asia, the many countries in the Middle East and East Africa is separated to virus or detects positive antibody.According to epidemiology survey, the many areas in China Taiwan and continent also have this disease popular, and this disease was once broken out in 1996-1997 Shanghai, and miscarriage in the cattle farm, premature labor, stillbirth and odd-shaped fetus account for 20%-30%, cause enormous economic loss to cattle-raising.This disease is listed in two class zoonosis, the Quarantine Objects of attaching most importance in China.
But the researchist in Taiwan finds pig natural infection AKAV recently, it can propagate evidence through mouth and nose, they have carried out serosurvey to 1088 pigs, confirmation has 816 pigs to be the serology positive reaction, positive rate reaches 75%, and also can examine neutralizing antibody in the piglet body, show that pig may become the AKAV ring of the life history.Thought the cattle infected AKAV non-evident sympton of growing up, but the report cattle infected of growing up can cause encephalomyelitis recently in the past.Factor to sum up, this sick difficulty of following prevention and control will strengthen, in case its loss of outbreak of epidemic also can be bigger., strengthen its quarantine is best prevention and control measure also not when China is very popular in present this disease.
In view of this disease causes enormous economic loss to cattle-raising, strengthen the diagnosis of this disease is prevented and treated show particularly urgent.So far, the diagnostic method of domestic report has microneutralization test, agar diffusion test and PCR and ELISA diagnostic method, and these methods all have its weak point, or operates loaded down with trivial detailsly, or is subject to maternal antibody and disturbs, or technical equipment is had relatively high expectations.In conjunction with producing actual needs, using indirect fluorescent antibody technique, directly to detect AKAV antigen in the tissue be a kind of easy, quick, accurate, diagnostic method that susceptibility is high.But the prerequisite that this method can be used is the AKAV antigen protein that need prepare high specific, and this antigen protein also need possess easy purifying preparation, cost is low, does not have the advantages such as danger of the poison that looses.
Summary of the invention
Technical problem to be solved by this invention is to overcome the deficiencies in the prior art, one specific specificity height is provided, easily purifying preparation, the reorganization AKAV nucleocapsid protein that cost is low, this reorganization AKAV nucleocapsid protein can be prepared into antibody serum, and easy, accurate detection goes out the AKAV antigen in the tissue.
Technical problem to be solved by this invention is that following technological approaches is realized:
A kind of reorganization AKAV nucleocapsid protein has the aminoacid sequence shown in the SEQ ID NO:1.
Another technical problem to be solved by this invention provides the above-mentioned reorganization AKAV of a kind of coding nucleocapsid protein gene.
Another technical problem to be solved by this invention realizes by following technological approaches:
The above-mentioned reorganization AKAV of a kind of coding nucleocapsid protein gene has the nucleotide sequence shown in the SEQ ID NO:2.
Another technical problem to be solved by this invention provides the recombinate method of AKAV nucleocapsid protein of a kind of the present invention of preparation.
Another technical problem to be solved by this invention realizes by following technological approaches:
The recombinate method of AKAV nucleocapsid protein of a kind of the present invention of preparation may further comprise the steps:
Make up reorganization AKAV nucleocapsid protein gene prokaryotic carrier; With this reorganization AKAV nucleocapsid protein gene prokaryotic carrier transformed into escherichia coli; Induce the reorganization nucleocapsid protein to express, reclaim and the expressed reorganization nucleocapsid protein of purifying.
Coding AKAV nucleocapsid protein gene is inserted between the suitable restriction enzyme site of prokaryotic expression carrier, makes that coding AKAV nucleocapsid protein gene is exercisable to be connected with the prokaryotic expression regulating and controlling sequence.As the most preferred embodiment of the present invention, be preferably coding AKAV nucleocapsid protein gene is inserted between the BamHI and XhoI restriction enzyme site on the prokaryotic expression carrier pET-28a (+), nucleocapsid protein prokaryotic expression carrier PET-N obtains recombinating; Resulting reorganization nucleocapsid protein prokaryotic expression carrier by this area ordinary method transformed into escherichia coli competent cell, as the most preferred embodiment of the present invention, is preferably transformed into escherichia coli (E.coli) BL21 (DE3); Induce the expression of reorganization AKAV nucleoprotein with IPTG, preferred, the condition of inducing reorganization AKAV nucleoprotein to express with IPTG is as follows: bacterium liquid OD
600nmIt is that the IPTG of 0.6-1.5mM induces that value begins during for 0.5-0.6 with concentration, and induction time is 4-5 hour.Expressed reorganization nucleocapsid protein can be by the ordinary method purifying of this area.The recombinant protein of about 27ku (801bp) that recombinant bacterial strain of the present invention is expressed for contain N aminopeptidase gene acid sequence (699bp, 19-26ku) and the fusion rotein of pET-28a (+) carrier part sequence (102bp, about 3ku).In pET-28a (+) part one His tag sequence (containing 6 successive Histidines) is arranged, therefore available ProBond
TMPurification System (contains Ni
2+) the method purification of recombinant proteins of affinity chromatography.
The present invention's AKAV nucleocapsid protein of recombinating can efficiently express in intestinal bacteria, expressed recombinant protein only contains the carrier part amino acid of about 3kD, sequence and native protein are approaching, this is to utilize the 26S Proteasome Structure and Function of recombinant protein research AKAV nucleocapsid protein to provide convenience, also for easy, detect AKAV antigen fast and accurately and other applied researcies are laid a good foundation.Show through Western blot, fine jade expansion detection and immunofluorescence, ELISA application test, the present invention recombinate the AKAV nucleocapsid protein have the height immunologic competence, the antiserum(antisera) that utilizes this recombinant protein to be prepared into adopts indirect fluorescent antibody technique can accurate detection to go out AKAV antigen in the tissue.The antiserum(antisera) that is prepared into by this reorganization AKAV nucleocapsid protein is compared with the hyper-immune serum of AKAV totivirus immunity preparation and is had the following advantages: the easy purifying preparation of antigen, cost is low, and (engineering bacteria is behind the ultrasonic disruption high speed centrifugation, the recombinant protein of high density is all at supernatant, exist with water-soluble form, this makes it can directly carry out purifying and obtains a large amount of highly purified fusion roteins, avoided sex change, complicated processes such as renaturation), and there is not the danger of the poison that looses, the most important thing is that the present invention's AKAV nucleocapsid protein of recombinating has the specificity of height, has guaranteed to utilize this recombinant protein to detect the specificity of AKAV antigen method.
Description of drawings
Fig. 1 cuts for recombinant plasmid PMD-S enzyme and PCR identifies;
1: λ-EcoT14I digest Marker; 2:EcoRI and SalI double digestion; The S fragment that 3:PCR amplifies; The water contrast of 4:PCR amplification; 5:DL2000Marker.
Fig. 2 cuts for recombinant plasmid PET-N enzyme and PCR identifies;
1: λ-EcoT14I digest Marker; 2:BamHI and XhoI double digestion; The N gene fragment that 3:PCR amplifies; The water contrast of 4:PCR amplification; 5:DL2000Marker.
Fig. 3 is the SDS-PAGE electrophoretogram of recombinant protein;
1: albumen Marker; The contrast of 2:BL21 bacterium; Transformed bacteria contrast before 3:IPTG induces; Transformed bacteria after 4:IPTG induces; 5: the fusion rotein behind the purifying.
Fig. 4 is that the Western blot of recombinant protein analyzes;
1: albumen Marker; The contrast of 2:BL21 bacterium; Transformed bacteria contrast before 3:IPTG induces; Transformed bacteria after 4:IPTG induces; 5: the fusion rotein behind the purifying.
Fig. 5 is recombinant bacterial strain SDS-PAGE thin layer scanning result (maximum peak is a fusion rotein);
Fig. 6 is inductive condition optimization Test result;
Series 1:1～7 are induction time 1～individual hour (D
600nm=0.60, IPTG=1mM);
Series 2: D when beginning to induce
600nmValue, 1～6 is respectively 0.10,0.35, and 0.51,0.73,0.91,1.03 (induced 3.5 hours, IPTG=1mM); Series 3:IPTG (mM) amount, 1～8 is respectively 0.05,0.1, and 0.3,0.6,0.8,1.0,1.5,2.0 (induced D 3.5 hours
600nm=0.61).
Fig. 7 infects BHK21 cell behind the AKAV and suckling mouse brain tissue and the two negative control to be observed under fluorescent microscope, and as seen infecting in the cell cytosol of AKAV has positive fluorescent signal, and negative control cell then can not detect.
A: the BHK21 cell that connects poison; B: negative control BHK21 cell; C: attack malicious mouse brain section; D: negative control mouse brain section (1,2 magnification 200 *; 3,4 magnifications 400 *).
Further describe beneficial effect of the present invention by the following examples, it should be understood that these embodiment only are used for the purpose of illustration, never limit protection scope of the present invention.
Embodiment
Main test materials and source
1.1 bacterial strain kind poison
The BHK21 cell is preserved by the Harbin veterinary institute; AKAV OBE-1 strain kind poison and positive serum are so kind as to give by Japanese Agriculture, Forestry and Fisheries Ministry animal health institute.
1.2 main agents
Various e. coli strains restriction enzymes, ligase enzyme, ThermoScript II, Taq enzyme, pMD18-TVector, pET-28a (+) expression vector, DNA Marker are precious biotechnology (Dalian) company limited product; Plasmid extraction agent box (production code member: W5002 is as follows), glue in a small amount reclaims small volume of reagent box (W5212) available from Shanghai Hua Shun; RNeasy
Mini Kit (74106), QAquickPCR Purification Kit (28104) are available from QIAGEN company; Albumen Marker (SM0431) is available from Fermentas company; The anti-ox IgG of horseradish peroxidase-labeled rabbit (A5295) is available from SIGMA company; ProBond
TMPurification System (K850-01) is available from Invitrogen company.
1.3 numerous poison and TCID50 measure
To plant poison and insert the BHK21 cell, and treat to receive poison when CPE appears in 70% cell, with cell freeze thawing three times, centrifugal 15 minutes of 1500g, getting supernatant, to measure malicious valency with 96 orifice plates be 10
6TCID
50/ ml, packing is also frozen in-80 ℃.
The structure of [embodiment 1] reorganization AKAV nucleocapsid protein gene prokaryotic carrier
Use RNeasy
Mini Kit RNA extraction agent box extracts the total RNA of AKAV, by match Parkson company synthetic primer, and amplification S sections gene, expection amplification length 840bp, primer sequence: p1:5-TAA CTA CGC ATT GCA ATG GC-3 (SEQ ID NO:3); P2:5-AGT AGT GTGCTC CAC-3 (SEQ ID NO:4).Amplification condition: 42 ℃ of 1h, enter circulation after 94 ℃ of 5min sex change, loop parameter is 94 ℃ of 60s, 55 ℃ of 30s, 72 ℃ of 60s, after 30 circulations, 72 ℃ are extended 10min, and the 10g/L agarose gel electrophoresis is identified the PCR product.
Be connected with pMD18-T Vector with the PCR purified product, Transformed E .coli JM109, coating contains the LB solid medium of penbritin (100ug/ml), X-gale and IPTG, the single bacterium colony of picking white after 37 ℃ of incubated overnight, be seeded to the liquid LB that contains penbritin (100ug/ml), after 37 ℃ of shaking tables are cultivated 8 hours,, serve the order-checking of Hai Boya company after PCR, double digestion are identified correctly and confirm, identify correct recombinant plasmid called after PMD-S with test kit extracting plasmid.With primer p1 and p2, enzyme EcoRI with recombinant plasmid PMD-S is carried out PCR to SalI and enzyme is cut evaluation (Fig. 1); The result is all consistent with expected results.
Having the primer of restriction enzyme site with design, is that the template subclone goes out the N gene fragment, primer sequence: p1:5-AGA with the PMD-S plasmid
GGA TCCATG GCA AAT CAA TTC-3 (BamHI) (SEQ ID NO:5); P2:5-TAG
CTC GAGTTA GAT CTG GAT AC-3 (XhoI) (SEQ ID NO:6).With BamHI and XhoI purifying N gene fragment and the pET-28a (+) that amplifies carried out double digestion respectively, enzyme is cut the purified back of product and is connected with connecting solution I (precious company), Transformed E .coli JM109 then, coating contains the LB substratum of kantlex (50ug/ml), the smooth single bacterium colony of picking, shake bacterium and cultivate the back, serve the order-checking of Hai Boya company after PCR, double digestion are identified correctly and confirm, identify correct recombinant plasmid called after PET-N with test kit extracting plasmid.With primer p1 and p2, enzyme BamHI with recombinant plasmid PET-N is carried out PCR to XhoI and enzyme is cut evaluation (Fig. 2), the result is all consistent with expected results, and this recombinant plasmid PET-N is through sequencing, and measurement result is shown in SEQ IDNO:2.。
[embodiment 2] inductive condition optimization Test
Recombinant plasmid PET-N is transformed into E.coli BL21 (DE3) expressive host bacterium, picking list bacterium colony inserts 3mlLB substratum (all substratum all contain kantlex) from the LB flat board, 37 ℃ of incubated overnight, insert 37 ℃ of cultivations of 100ml 2 * YT substratum with 150 then, do the inductive condition optimization Test, compare with inductive bacterium not, do different induction times respectively, different D
600nmValue is initial induces, and best inductive condition is determined in different IPTG amount induction experiments at last.
Different as shown in Figure 6 induction times and different D when beginning to induce
600nmValue has a significant effect to the Expression of Fusion Protein rate, and different IP TG amount is induced then not clearly.Determine D
600nmValue is initially to induce the reorganization nucleocapsid protein to express with IPTG at 0.5～0.6 o'clock, and that IPTG is 0.6～1.5mM, and induction time is 4～5 hours.
Evaluation, purifying and the expression amount of [embodiment 3] reorganization AKAV nucleocapsid protein are measured
Engineering bacteria after inducing, through ultrasonic degradation, centrifugal 15 minutes of 12000 * g after testing expressing protein all at supernatant, the sample band with compare, many bright bands at 27ku place, big or small consistent with expection, ProBond
TMThen only surplus this band behind the Purification System purifying, this illustrates that it really is the fusion rotein of abduction delivering.Do an anti-effect with the anti-AKAV positive serum of ox, the anti-ox IgG of horseradish peroxidase-labeled rabbit does two anti-effects, and DAB colour developing back result shows that correspondence position all has obvious band, and contrast does not have, and this shows that expressed fusion protein has immunologic competence (Fig. 3, Fig. 4).
Recombination fusion protein of the present invention adopts the software of GENETYX-WIN Version5.1 to infer that its result has the aminoacid sequence shown in the SEQ ID NO:1.
Results after inducing bacterium liquid and with 4 ℃ of centrifugal 10min of 1500 * g, abandon supernatant, use then with the long-pending PBS solution of bacteria liquid suspend-centrifugal-abandon supernatant to give a baby a bath on the third day after its birth time, suspend with the long-pending PBS of 1/10 bacteria liquid at last, ultrasonic degradation is to transparent, with 4 ℃ of centrifugal 15min of 12000 * g, get cleer and peaceful precipitation and be equipped with inspection.After 12% SDS-PAGE detects the positive, with Western blot detection of active, do one anti-ly with the anti-AKAV positive serum of ox, the anti-ox IgG of horseradish peroxidase-labeled rabbit is two anti-, the DAB colour developing detects the expressing fusion protein rate by thin layer scanning.Detection shows that fusion rotein accounts for solubility bacterial protein per-cent and is up to 58.5% (Fig. 5) to engineering bacteria SDS-PAGE with thin layer chromatography scanner.
[test example 1] the present invention AKAV nucleocapsid protein of recombinating prepares antiserum(antisera) and detects the AKAV antigen test
1 material and method
1.1 material
1.1.1 planting malicious AKAV OBE-1 strain kind poison is so kind as to give by Japanese Agriculture, Forestry and Fisheries Ministry animal health institute.
1.1.2 a preparation anti-and the anti-AKAV reorganization of two anti-rabbits nucleocapsid protein serum sees for details following; FITC mark goat anti-rabbit igg (H+L) (article No. ZF-0311, lot number 64419; Antibody diluent: glycerine=1: 1,0.75mg/ml) available from Bioisystech Co., Ltd of China fir Golden Bridge in Beijing.
1.1.3 numerous malicious material B HK21 culturing cell grinds preservation by breathing out beast; A brood of Kunming white mouse is 93 age in days suckling mouses and a female mouse wherein, and grinding Experimental Animal Center by the Kazakhstan beast provides.
1.2 method
1.2.1 numerous poison and TCID50 measure and will plant poison and insert the BHK21 cell, treat to receive poison when CPE appears in 70% cell, and with cell freeze thawing three times, centrifugal 15 minutes of 1500g, getting supernatant, to measure malicious valency with 96 orifice plates be 10
6TCID50/ml, packing is also frozen in-80 ℃.
1.2.2 the preparation of the anti-AKAV of rabbit reorganization nucleocapsid protein serum: adopt the purified reorganization nucleoprotein of the embodiment of the invention 3, it is 460ug/ml that the nucleoprotein of will recombinating is mixed with concentration, the rabbit at immune then 2 monthly ages.Head exempts from, the subcutaneous multi-point injection of the complete freund adjuvant of 1ml recombinant protein solution and equivalent (Sigma company) emulsification back part; Two exempt from after two weeks, get the incomplete subcutaneous multiple spot of freund adjuvant emulsification back part and the leg muscle injection of 1ml reorganization nucleoprotein solution and equivalent; Two Zhou Housan exempt from again, get the injection of the direct ear vein of 1.3ml recombinant protein solution, week back heart blood sampling, and separation of serum-20 is ℃ frozen.Estimate this serum with breathing out the beastly agp antigen that grinds preservation, with still visible clear precipitation line after 64 times of its dilutions.
1.2.3 cell connects poison BHK21 cell dissociation adherent on the culturing bottle got off after, dispel with the DMEM that contains 8% foetal calf serum and to suspend and counting, final concentration of cells is 1 * 10
5Individual/ml, with every hole 1ml it is inserted 24 orifice plates then, after 20 hours (this moment cell area account for the culture plate area 70～80%) the sucking-off substratum, wash twice with PBS, sample aperture adds the viral liquid of 200ulDMEM dilution, and (DMEM liquid: viral liquid=199: 1, malicious valency are 2 * 10
4TCID50/ml), control wells adds 200ulDMEM solution, and 37 ℃ of senses are made to add after 1 hour the DMEM that contains 2% foetal calf serum and kept liquid, at 37 ℃ of 5%CO
2Cultivated 14 hours in the incubator.
1.2.4 suckling mouse attack 6 suckling mouses of poison respectively intracranial inoculation 1.2.1 poison valency be 10
6TCID50/ml virus liquid 10ul, its excess-three is only inoculated equivalent DMEM in contrast, and tail end is cut off a little to show difference.
1.2.5 antigen sheet treatment process:
1.2.5.1 culturing cell discards the substratum in the hole, dries up behind the cell to fix 30 minutes with 4 ℃ of 70% cold ethanol and develop a film each 5 minutes 3 times with the PBS of pH7.4 then; Anti-do every hole, 1: 20 dilution back with PBS and add 200ul one, 37 ℃ of effects 30 minutes are taken out the back and are washed 3 times with PBS; Two is anti-with every hole adding 200ul after 1: the 100 times of dilution of sample diluting liquid do that contains 0.01 ‰ ivens orchid, and 37 ℃ act on 30 minutes, take out the back and wash 3 times with PBS, use fluorescence microscope after drying up immediately.
1.2.5.2 will connecing the taking-up with the contrast suckling mouse brain that occurred nervous symptoms in malicious back 5～6 days, the suckling mouse brain frozen section freezes on-24 ℃ of freezing microtome load sample plates, cut out the 5um slab, after launching gently, immediately it is adhered on the slide glass that poly-lysine handled; Treat to put into after slice, thin piece dries 4 ℃ of acetone and fix 5 minutes, take out the back room temperature and dry, in washing tank, develop a film 3 times each 5 minutes then with PBS; I is resisted with dripping on the sample after the dilution in 1: 20 of PBS do, slice, thin piece is put into 37 ℃ of effects of wet box 30 minutes, wash 3 times with PBS after the taking-up; Anti-do 1: 100 times of dilution with the sample diluting liquid that contains 0.01 ‰ ivens orchid and be added drop-wise on the sample two, 37 ℃ of effects 30 minutes are taken out the back and are washed 3 times with PBS, dry up the back mounting and use fluorescence microscope immediately.
2 test-results
2.1BHK21 cell
Observe under fluorescent microscope by the culturing cell sample of virus infection, as seen have in the cytoplasm of single or agglomerating cell very strong green fluorescence signal is arranged, control cells does not then have, and sees Fig. 7.
2.1 suckling mouse
The The above results explanation, adopt the recombinate antiserum(antisera) of the prepared one-tenth of AKAV nucleocapsid protein of the present invention, adopt indirect fluorescent antibody technique can accurate detection to go out AKAV antigen in the tissue, this method specificity height and action required time are short, the needs of suitable clinical diagnosis.
SEQUENCE?LISTING
＜110〉Scientia Agricultura Sinica research institute Harbin veterinary institute
＜120〉recombinant akabane virus capsid protein, its preparation method and application
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Claims (9)
1. a recombinant akabane virus capsid protein is characterized in that having the aminoacid sequence shown in the SEQ ID NO:1.
2. the recombinant akabane virus capsid protein gene of the claim 1 of encoding is characterized in that having the nucleotide sequence shown in the SEQ ID NO:2.
3. method for preparing the recombinant akabane virus capsid protein of claim 1 may further comprise the steps:
Make up recombinant akabane virus capsid protein gene prokaryotic carrier; With this prokaryotic expression carrier transformed into escherichia coli; Induce the reorganization nucleocapsid protein to express, reclaim and the expressed reorganization nucleocapsid protein of purifying.
4. according to the method for claim 3, it is characterized in that described recombinant akabane virus capsid protein gene prokaryotic carrier is PET-N.
5. according to the method for claim 3, it is characterized in that described intestinal bacteria are BL21 (DE3).
6. according to the method for claim 3, it is characterized in that described inducing is as bacterium liquid OD
600nmValue is that the IPTG of 0.6-1.5mM induces with concentration when the 0.5-0.6, and induction time is 4-5 hour.
7. according to the method for claim 3, it is characterized in that adopting the method purification of recombinant proteins of affinity chromatography.
8. the application of the recombinant akabane virus capsid protein of claim 1 in preparation diagnosis or detection Akabane Disease poison antigen-drug.
9. the application of the coding recombinant akabane virus capsid protein gene of claim 2 in preparation diagnosis or detection Akabane Disease poison antigen-drug.
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| CN101949936A (en) * | 2010-08-02 | 2011-01-19 | 宁波检验检疫科学技术研究院 | Colloidal gold test strip for detecting antibodies against akabane virus and preparation method thereof |
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| EP2713161A1 (en) * | 2012-09-27 | 2014-04-02 | Idexx Laboratories, Inc. | Method of detection of Schmallenberg virus (SBV) and kit |
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| JP2015535841A (en) * | 2012-09-27 | 2015-12-17 | アイデックス ラボラトリーズ インコーポレイテッドＩＤＥＸＸ Ｌａｂｏｒａｔｏｒｉｅｓ， Ｉｎｃ． | Schmalenberg virus (SBV) detection method and kit |
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| CN107119058B (en) * | 2017-05-10 | 2020-12-08 | 斯澳生物科技（苏州）有限公司 | MS2 capsid protein fusion expression method and application thereof |
| CN115057925A (en) * | 2022-06-22 | 2022-09-16 | 中国检验检疫科学研究院 | Anti-akabane virus monoclonal antibody and application thereof |
| CN115057925B (en) * | 2022-06-22 | 2024-03-26 | 中国检验检疫科学研究院 | Anti-akabane virus monoclonal antibody and application thereof |
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