CN1458168A - Recombined pig influenza virus NP antigen, its preparing method and use in diagnosis - Google Patents
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Abstract
The present invention discloses recombinant pig influenza virus NP antigen and its preparation process and application in diagnosis. Type-A influenza virus genome consists of 8 segments of RNA, and its nucleoprotein (NP) has type and group specificity and is the basis of typing diagnosis. The recombinant pig influenza virus NP antigen has colibacillus prokaryotic expression system to express complete NP gene of separated pig influenza virus strain in China, and the expressed NP is used as antigen in detecting type-A influenza virus infection produced anti-NP protein antibody. The NP gene is the basis for classifying and diagnosing pig influenza virus with many subtypes. Therefore, the diagnosis technology established based on recombinant SIV NP antigen may be used in detecting type-A influenza virus infection of pig, fowl, horse and human.
Description
Technical field: the present invention relates to the Vet Biotechnology field, utilize escherichia expression system to express the NP albumen of swine influenza virus (SIV) H3N2, behind the purifying as diagnosis antigen.
Background technology:
Porcine influenza (Swine Influenza) is a kind of acute respiratory transmissible disease of the pig that causes of the swine influenza virus of the A of orthomyxoviridae family type Influenza Virus, shows as unexpected generation, bamboo telegraph, and the difficulty of being short of breath has the paroxysmal cough concurrently.After this get final product rehabilitation and produce strong persistent immunizing power, but also become secular carrier and toxin expelling person.The U.S. at first reported the generation porcine influenza in 1918, thought that now current influenza and human flu outbreak in 1918 are interrelated.People such as nineteen thirty Shope are separated to the first strain H1N1 hypotype porcine influenza from the Iowa.Successively from the pig body, be separated to the influenza virus of various hypotypes after this again.Porcine influenza is popular, the global distribution of region, and particularly H3N2 subtype distribution of porcine influenza is also all arranged in China each province and city.In addition, when analyzing by nineteen sixty-eight Mao flu virus H3N2 hypotype is originated from, people infer and the pig that progressively is confirmed plays a part the intermediate host in the generation of human influenza, and pig is that fowl source and Mammals influenza virus are responsive unique animal.It can make avian influenza virus pass through reorganization and infect give the people, also can make the early stage influenza virus of people be able to store in the pig body, and can be over a period to come infected person once more.This makes that the public health meaning of porcine influenza is more outstanding, promises to be the early warning signal of human next flu outbreak.
Influenza virus is a minus-stranded rna virus, and viral genome is made up of 8 segmented RNA, and the virus protein of main coding has 5 peptide species, be hemagglutinin (HA), neuraminidase (NA), stromatin (MA), nucleoprotein (NP) and saccharan (PB1, PB2, PA).HA and NA are the main immunogenic albumen of virus, can induce body to produce the protective capability of anti-virus infection.NP has type and group specificity, is the basis of influenza virus somatotype and diagnosis, but is not the main immunogenic albumen of virus.Therefore, utilize NP to detect influenza infection, do not influence the differential diagnosis of HA/NA subunit vaccine or live vector vaccine immune swine.
Summary of the invention:
The nucleoprotein that the objective of the invention is to utilize prokaryotic expression system to efficiently express swine influenza virus is set up the antibody diagnosis technology of swine influenza virus and other A type influenza infections as antigen.
The object of the present invention is achieved like this:
Reorganization swine influenza virus NP antigen, described antigen is the reorganization nucleoprotein antigen of diagnosis A type influenza infection, its the has adopted escherichia coli prokaryotic expression system expression complete NP gene of China's swine influenza virus (SIV) strain isolated (H3N2 hypotype), the anti-NP protein antibodies that utilizes the NP albumen of expressing to produce as Detection of antigen A type influenza infection.
Described reorganization swine influenza virus NP antigen adopts escherichia expression system to express the NP albumen of swine influenza virus H3N2 hypotype, behind the purifying as A type influenza infection diagnosis antigen.
The antigenic cultural method of a kind of reorganization swine influenza virus NP, utilize the RT-PCR method to clone to be located away from the NP gene of H3N2 hypotype swine influenza virus, then complete NP gene being got off with digestion with restriction enzyme is connected with the prokaryotic expression carrier pET30 of same treatment again, obtains to contain SIV NP expression of gene plasmid pETNP;
With pETNP transformation receptor bacterium BL21, the single bacterium colony of picking carries out inducing and acclimating with inductor with different concns, and collects sample at different time.Confirm that through the SDS-PAGE electrophoretic analysis the best induced concentration of the expression of NP and definite IPTG and induction time are respectively 1mM and 6 hours;
Protein band behind the SDS-PAGE is changeed film, and Western-blot test has confirmed that expressed reorganization nucleoprotein size conforms to and has reactive behavior.By the selectivity experiment of specificity, susceptibility and working conditions, set up the reorganization nucleoprotein fine jade expansion diagnostic techniques that swine influenza virus infects.
A kind of application of swine influenza virus NP antigen in diagnosis of recombinating, this reorganization nucleoprotein antigen is used for fine jade and expands diagnostic techniques (AGIP), enzyme linked immunosorbent assay (ELISA), dot-ELISA (Dot-ELISA), immunofluorescence antibody test (IF) and immunity seal stain test (Western Blot).
A kind of recombinate swine influenza virus NP antigen and the application in diagnosis thereof, this reorganization nucleoprotein antigen can be used for detecting A type influenza infection, comprises swine influenza virus, avian influenza virus, equine influenza virus and human influenza virus infect.The present invention has following positively effect:
1. provide important techniques basis to the epidemiology generaI investigation of porcine influenza and terrain raiser to the diagnosis of this disease based on being established as of the diagnostic techniques of reorganization SIV nucleoprotein antigen in China.
2. the reorganization nucleoprotein of being produced is not only produced easily, and is simple to operate and purity is higher, has good specificity and susceptibility.The fine jade of setting up expands diagnostic techniques and also is easy to be accepted by the user.
3. the bag of will recombinating behind the nucleoprotein purifying also can be set up the higher ELISA diagnostic method of susceptibility by the polystyrene micro-reaction plate.Because nucleoprotein is A type influenza virus type specific antigen, the diagnostic techniques of setting up based on reorganization SIV nucleoprotein antigen also can be used for A type avian influenza virus, equine influenza virus and human influenza virus's INFECTION IN DETECTION.
4. set up and be easy to the porcine influenza diagnostic method promoted simply, easily and fast, solved China and be badly in need of the subject matter that solves.Making processes is simple, and cost is low, is convenient to promote the use of.
5. because the HA/HI technology also is easy to generate nonspecific result except that the certain technical qualification of needs.And the nucleoprotein of in prokaryotic expression system, expressing not only expression amount is high but also have a biological respinse activity, it is a kind of novel method that is used to produce diagnostic antigen recently, utilize the biological activity of expression product can directly detect viral infection, and expressed recombinant protein merged histidine-taggedly, and this also makes simultaneously the protein purification easy handling.Provide technical support for setting up more responsive diagnostic methods such as ELISA.
6. porcine influenza is a kind of universal mass-sending sexually transmitted disease, not only distributes wide but also takes place frequently in the Chinese Pigs influenza, its meaning of anti-system of porcine influenza not only is the meaning of animal doctor's loimology, and also has far-reaching public health meaning.Porcine influenza is the main immunosuppressive disease of pig, also is the main inducing that influences the growth of growing and fattening pigs in the large-scale pig farm and cause the sow miscarriage except that causing respiratory tract disease.Pig is propagated in the chain between the kind of influenza virus " fowl-pig and human ", " mixing tank " that has served as viral reorganization and duplicated.With respect to bird flu, the research of the porcine influenza of China is started late, and the task of pendulum in face of researcher also shouldered heavy responsibilities.And to a kind of research of transmissible disease, primarily be to set up suitable diagnostic method, finish the generaI investigation of this disease and formulate rational program from epidemiology for anti-system, control its popular purpose that reaches prevention.This product is that new approach has been started in preventing and controlling.
7. the reorganization nucleoprotein fine jade that infects of swine influenza virus expands being established as in China of diagnostic techniques and this disease popular is generally investigated and promptly and accurately diagnosis and prevent that this disease from providing technical foundation.With swine influenza virus reorganization nucleoprotein is that many-sided Application and Development can be carried out in the basis, and for example: a. expands can the achieve a butt joint differential diagnosis of pig farm porcine influenza infection of kind of HA/NA subunit vaccine or live vector vaccine of diagnostic techniques based on the fine jade of reorganization nucleoprotein; B. be that the proteic purifying of NP can be carried out in the basis with reorganization nucleoprotein, and then can be used for studying the proteic functional structure of NP etc.; C. be that the proteic purifying of NP can be carried out in the basis with reorganization nucleoprotein, and then set up than fine jade and expand more responsive ELISA, Dot-ELISA, immunofluorescence and immunity seal stain wait diagnostic method.
Description of drawings:
Fig. 1, swine influenza virus reorganization nucleoprotein expression vector PETNP plasmid structural representation.
Fig. 2, expression plasmid pET-NP be abduction delivering product S DD-PAGE electrophoresis result in recipient bacterium BL21.
1. molecular weight of albumen Marker.2. empty carrier induced product contrast; 3-6.IPTG induce the expression product behind the different time.
Fig. 3, the structure schema of swine influenza virus NP Prokaryotic Expression and serodiagnosis technology.
The specific embodiment of the present invention:
Embodiment 1:
Reorganization swine influenza virus NP antigen, described antigen is the reorganization nucleoprotein antigen of diagnosis A type influenza infection, its the has adopted escherichia coli prokaryotic expression system expression complete NP gene of China's swine influenza virus (SIV) strain isolated (H3N2 hypotype), the anti-NP protein antibodies that utilizes the NP albumen of expressing to produce as Detection of antigen A type influenza infection.
Embodiment 2:
Described reorganization swine influenza virus NP antigen adopts escherichia expression system to express the NP albumen of swine influenza virus H3N2 hypotype, behind the purifying as A type influenza infection diagnosis antigen.
Embodiment 3:
The antigenic cultural method of a kind of reorganization swine influenza virus NP, utilize the RT-PCR method to clone to be located away from the NP gene of H3N2 hypotype swine influenza virus, then complete NP gene being got off with digestion with restriction enzyme is connected with the prokaryotic expression carrier pET30 of same treatment again, obtains to contain SIV NP expression of gene plasmid pETNP;
With pETNP transformation receptor bacterium BL21, the single bacterium colony of picking carries out inducing and acclimating with inductor with different concns, and collects sample at different time.Confirm that through the SDS-PAGE electrophoretic analysis the best induced concentration of the expression of NP and definite IPTG and induction time are respectively 1mM and 6 hours;
Protein band behind the SDS-PAGE is changeed film, and Western-blot test has confirmed that expressed reorganization nucleoprotein size conforms to and has reactive behavior.By the selectivity experiment of specificity, susceptibility and working conditions, set up the reorganization nucleoprotein fine jade expansion diagnostic techniques that swine influenza virus infects.
Embodiment 4:
A kind of application of swine influenza virus NP antigen in diagnosis of recombinating, this reorganization nucleoprotein antigen is used for fine jade and expands diagnostic techniques (AGIP), enzyme linked immunosorbent assay (ELISA), dot-ELISA (Dot-ELISA), immunofluorescence antibody test (IF) and immunity seal stain test (Western Blot).
Embodiment 5:
A kind of recombinate swine influenza virus NP antigen and the application in diagnosis thereof, this reorganization nucleoprotein antigen can be used for detecting A type influenza infection, comprises swine influenza virus, avian influenza virus, equine influenza virus and human influenza virus infect.
Embodiment 6:
The expression and the preparation method of reorganization SIV nucleoprotein antigen:
One, the cultivation of SIV and the extraction of RNA
Get H1N1 kind poison allantoic cavity inoculation 9-11 age in days SPF chicken embryo, gather in the crops allantoic fluid after 4 days, ultracentrifugation behind the low-speed centrifugal, the TE of no Rnase A (PH=8.0) damping fluid dissolution precipitation, the extraction of RNA is undertaken by Trizol test kit specification sheets; The RNA that is extracted is used for RT-PCR.
Two, design is synthetic be used to the to increase primer of SIV NP gene
According to a pair of Auele Specific Primer of A/Swine/Hong Kong/126/82 (H3N2) influenzae strain virus NP gene sequences Design among the GeneBank at the NP gene.Upstream primer is: 5 ' ACTCACTGGGTACCATCAAAGTCATGG 3 '; Downstream primer is: 5 ' ACAAGCTTATTTTTCTTCAATTGTCGT 3 '.
Three, the NP Gene RT-PCR
1, the reverse transcription of SIV RNA
Get above-mentioned viral RNA solution, add the NP upstream primer 1 μ l of dNTPs 4 μ l, the 20pmol/L of 5 * Buffer, 5 μ l, 10mmol/L in the reverse transcription test kit, in instantaneous centrifugal rearmounted 95 ℃ of water-baths 1 minute, took out rapidly in the rearmounted ice bath 5 minutes; Add 40U/ μ l RNasin 1 μ l again, 10U/ μ lAMV-RNase 1 μ l, behind the mixing in 42 ℃ of water-baths 1.5 hours.5 minutes deactivation AMV-RNase of the most rearmounted 95 ℃ of water-baths took out rapidly in the rearmounted ice bath 5 minutes.
2, the PCR of NP gene
Get 5 above-mentioned μ l cDNA products as pcr template, (50 μ l reaction system) formed in the PCR reaction:
TaKaRa?LA?Taq(5u/μl)0.5μl
10 * LA pCR
TMBuffer II (contains Mg
2+) 5 μ l
DNTP Mixture (each 2.5mM) 4 μ l
Primer P1 (10pmol/ μ l) 2 μ l
Primer P2 (10pmol/ μ l) 2 μ l
Sterilization deionized water 31.5 μ l
The PCR reaction conditions: 95 ℃ of pre-sex change 10 minutes, 94 ℃ 1 minute, 55 ℃ 1 minute, 72 ℃ 1.5 minutes, carry out 35 circulations, reaction finishes preceding 72 ℃ and extended 10 minutes.
Four, the structure of expression vector pET-NP
1, the PCR product is reclaimed test kit (Shanghai China Shun bio-engineering corporation product) glue and reclaims with glue after, (the precious biotech firm in Dalian) is connected with the PMD18-T carrier, its system is: PCR product 4 μ l, T carrier 1 μ l, Solution I 5 μ l.In 16 ℃ of connections will connect product conversion DH5 α competence bacterium after 6 hours after, some white colonies of random extraction, extract plasmid in a small amount with alkaline lysis, carrying out enzyme with KpnI then cuts, 0.8% agarose gel electrophoresis therefrom filters out the segmental recombinant plasmid of the external source that contains the 1.5kb size (called after TNP).
2, above-mentioned recombinant plasmid TNP and prokaryotic expression carrier PET30 (a) are digested with the KpnI restriction enzyme, reclaiming test kit with glue then reclaims, handle with the SalI digestion with restriction enzyme, glue reclaims test kit and reclaims fragment and the same prokaryotic expression carrier of handling that contains the NP gene again.With the fragment after reclaiming and vector plasmid PET30 (a) with 3: 1 mixings of mol ratio, with 16 ℃ of connection of spending the night of T4 dna ligase (Shanghai bio-engineering corporation), the competence of connection product conversion BL21 strain preparation.Some bacterium colonies of picking extract plasmid and keep bacterial classification in a small amount after 37 ℃ of overnight incubation in the LB that contains kantlex (available from Shanghai China Shun biotech firm) at random.Plasmid screens positive recombinant plasmid with the KpnI/SalI double digestion, and called after pET-NP.PET-NP is served the order-checking of Hai Boya biotech firm.
Five, the abduction delivering of NP gene
The above-mentioned BL21 bacterial classification that contains recombinant plasmid pET-NP is rule containing on the LB flat board of kantlex, 37 ℃ of overnight incubation, the single bacterium colony of picking at random contains among the antibiotic LB of Kna in 10ml and to be cultured to OD in 37 ℃ of shaking tables
600Adding inductor IPTG (sec.-propyl-β-D thiogalactoside, the precious biotech firm in Dalian product) during for 0.6-0.7 to final concentration 1mM, continues jolting and cultivated 3-6 hour, the results bacterium.After centrifugal thalline is washed twice with the damping fluid of 0.5M Nacl, 20mM Triscl (pH=7.6), use 1mlPBS (PH=7.4) suspension cell then, behind ultrasonic treatment, add 2 * sample-loading buffer (100m mol/LTrisHClPH=6.8,200mmol/L dithiothreitol (DTT), 4%SDS, 0.2% tetrabromophenol sulfonphthalein, 20% glycerine), carry out 10% polyacrylamide gel electrophoresis (SDS-PAGE) analysis after boiling sex change, Coomassie brilliant blue dyeing, observations.The result has expressed the fusion rotein of about 60ku after inducing on the bacterium that has the PETNP plasmid.And the contrast bacterium does not have corresponding band.Thin layer scanning is analyzed, and this albumen can account for about 30% of tropina total amount.
Six, the mensuration of protein-active
1, Western-blot analyzes: expressing protein is through being transferred to behind the SDS-PAGE electrophoresis on the nitrocellulose filter (NC film), with 37 ℃ of incubation 1-2 of PBS (PH=7.4) hours sealing NC films that contain 5% (w/v) skim-milk; Add the 37 ℃ of incubations of PBS 1 hour contain 5% skim-milk, 1% porcine influenza standard positive serum then; Wash once with 150mmol/L NaCl, 50mmol/L TrisCl (PH=7.5) behind the PBS washing film three times, add 37 ℃ of incubations of the anti-pig IgG of rabbit 1 hour of the horseradish peroxidase-labeled of diluting then with TrisCl damping fluid 1/5000; With 150mmol/L NaCl, 50mmol/L TrisCl (PH=7.5) washing three times, add and contain 6mg diaminobenzidine, 10 μ l 30%H
2O
2The colour developing liquid of TrisCl (0.01mol/L PH=7.6) react, observe when the protein band color depth of formation reaches requirement and use water rinse, put into PBS and preserve.The result can see and the corresponding reaction band of SDS-PAGE specific band.
2, agar diffusion test:prepare the flat board that contains 1% agar powder with physiological saline, punch tool punching back spirit lamp heated base back cover, centre hole adds expression product then, and peripheral hole adds various hypotype porcine influenza positive serums.Do the positive and negative control with totivirus antigen and BL21 bacterium split product simultaneously, 37 ℃ of of of of of of of wet of in boxes, place the back observations of spending the night.The result has formed specific reaction precipitation line between expression product and totivirus antigen and positive serum, and does not form precipitation line between BL21 bacterium split product and the positive serum; Do not form precipitation line between expression product and totivirus antigen and the negative serum. ( H3N2 ) NP：ATGGCGCTTCAAGGCACCAAACGATCTTATGAGCAGATGGAAACTGGTGGGGAACGCCAGAATGCTACTGAGATCAGAGCATCTGTTGGGAGAATGGTTGGTGGAATCGGAAGATTCTACATACAGATGTGCACTGAACTCAAACTCAGCGACTACGAAGGCAGGCTGATCCAAAACAGCATAACAATAGAGAGAATGGTCCTCTCTGCATTTGATGAGAGGAGAAACAGATACCTGGAAGAAAATCCCAGTGCGGGGAAGGACCCGAAGAAAACTGGAGGCCCTATCTACAAAAGGAGGGAAGGAAAGTGGGTGAGAGAGCTGATTTTGTATGATAAGGAGGAGATCAGGAGAATTTGGCGTCAAGCGAACAATGGAGAAGACGCAACTGCTGGTCTCACCCATCTGATGATCTGGCATTCCAATCTGAATGATGCCACATATCAAAGAACAAGAGCTCTCGTGCGTACTGGAATGGACCCCAGAATGTGCTCTCTGATGCAAGGATCAACTCTCCCGAGGAGATCTGGAGCTGCGGGTGCAGCGGTAAAGGGAGTTGGAACGATGGTAATGGAACTAATTCGGATGGTAAAGCGAGGGATCAATGATCGGAATTTCTGGAGAGGTGAAAATGGACGAAGAACAAGAATTGCATATGAGAGGATGTGCAACATCCTCAAAGGGAAATTCCAAACAGCAGCACAGCGAGCAATGATGGACCAGGTGAGAGAAAGCAGGAATCCTGGGAATGCTGAGATTGAAGACCTTATCTTTCTGGCTCGGTCTGCACTCATTCTGAGAGGATCAGTGGCTCATAAGTCCTGCTTGCCTGCTTGTGTATATGGACTTGCTGTGGCCAGTGGATACGACTTTGAGAGAGAGGGGTACTCTCTGGTCGGAATAGATCCTTTCCGTCTGCTTCAAAACAGCCAGGTGTTAAGCCTCATTAGACCAAATGAGAATCCGGCACATAAGAGTCAACTGGTATGGATGGCATGCCATTCTGCAGCATTTGAAGACCTGAGAGTATCAAGCTTCATCAGAGGGACAAGAGTGATCCCAAGAGGACAACTGTCCACCAGAGGAGTTCAAATAGCTTCTAATGAGAACATGGAAACAATGGACTCCAGTACTCTTGAACTGAGAAGCAGATACTGGGCTATAAGGACTAGGAGTGGAGGAAACACCAACCAACAGAGAGCATCTGCAGGGCAAATCAGTGTGCAACCCACTTTCTCGGTACAGAGAAATCTTCCTTTCGAGAGAGCGACCATCATGGCGGCATTTACAGGGAACACTGAAGGCAGAACATCTGACATGAGGACTGAAATCATAAGAATGATGGAAAGTGCCAGACCAGAAGATGTGTCTTTCCAGGGGCGGGGAGTCTTCGAGCTCTCGGACGAAAAGGCAACGAACCCGATCGTGCCTTCCTTTGACATGAGTAATGAGGGATCTTATTTCTTCGGAGACAATGCAGAGGAGTATGACAATTAA ( H3N2 ) NP：GTIKVMALQGTKRSYEQMETGGERQNATEIRASVGRMVGGIGRFYIQMCTELKLSDYEGRLIQNSITIERMVLSAFDERRNRYLEENPSAGKDPKKTGGPIYKRREGKWVRELILYDKEEIRRIWRQANNGEDATAGLTHLMIWHSNLNDATYQRTRALVRTGMDPRMCSLMQGSTLPRRSGAAGAAVKGVGTMVMELIRMVKRGINDRNFWRGENGRRTRIAYERMCNILKGKFQTAAQRAMMDQVRESRNPGNAEIEDLIFLARSALILRGSVAHKSCLPACVYGLAVASGYDFEREGYSLVGIDPFRLLQNSQVLSLIRPNENPAHKSQLVWMACHSAAFEDLRVSSFIRGTRVIPRGQLSTRGVQIASNENMETMDSSTLELRSRYWAIRTRSGGNTNQQRASAGQISVQPTFSVQRNLPFERATIMAAFTGNTEGRTSDMRTEIIRMMESARPEDVS
FQGRGVFELSDEKATNPIVPSFDMSNEGSYFFGDNAEEYDNZ
Claims (5)
1, reorganization swine influenza virus NP antigen, it is characterized in that: described antigen is the reorganization nucleoprotein antigen of diagnosis A type influenza infection, its the has adopted escherichia coli prokaryotic expression system expression complete NP gene of China's swine influenza virus (SIV) strain isolated (H3N2 hypotype), the anti-NP protein antibodies that utilizes the NP albumen of expressing to produce as Detection of antigen A type influenza infection.
2, according to the described reorganization swine influenza virus of claim 1 NP antigen, it is characterized in that: adopt escherichia expression system to express the NP albumen of swine influenza virus H3N2 hypotype, behind the purifying as A type influenza infection diagnosis antigen.
3, the antigenic cultural method of a kind of reorganization swine influenza virus NP, it is characterized in that: the NP gene that utilizes the RT-PCR method to clone to be located away from H3N2 hypotype swine influenza virus, then complete NP gene being got off with digestion with restriction enzyme is connected with the prokaryotic expression carrier pET30 of same treatment again, obtains to contain SIV NP expression of gene plasmid pETNP;
With pETNP transformation receptor bacterium BL21, the single bacterium colony of picking carries out inducing and acclimating with inductor with different concns, and collects sample at different time.Confirm that through the SDS-PAGE electrophoretic analysis the best induced concentration of the expression of NP and definite IPTG and induction time are respectively 1mM and 6 hours;
Protein band behind the SDS-PAGE is changeed film, and Western-blot test has confirmed that expressed reorganization nucleoprotein size conforms to and has reactive behavior.By the selectivity experiment of specificity, susceptibility and working conditions, set up the reorganization nucleoprotein fine jade expansion diagnostic techniques that swine influenza virus infects.
4, a kind of application of swine influenza virus NP antigen in diagnosis of recombinating, this reorganization nucleoprotein antigen is used for fine jade and expands diagnostic techniques (AGIP), enzyme linked immunosorbent assay (ELISA), dot-ELISA (Dot-ELISA), immunofluorescence antibody test (IF) and immunity seal stain test (Western Blot).
5, a kind of recombinate swine influenza virus NP antigen and the application in diagnosis thereof, this reorganization nucleoprotein antigen can be used for detecting A type influenza infection, comprises swine influenza virus, avian influenza virus, equine influenza virus and human influenza virus infect.
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| CN101871944A (en) * | 2010-06-02 | 2010-10-27 | 中国农业科学院茶叶研究所 | Qualitative detection method of tea geometrid nuclear polyhedrosis virus |
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| CN107099512A (en) * | 2005-04-21 | 2017-08-29 | 佛罗里达大学研究基金公司 | The material and method controlled for respiratory disease in canid |
| CN111171146A (en) * | 2020-02-20 | 2020-05-19 | 西北农林科技大学 | Nano antibody for resisting H9N2 subtype avian influenza virus, preparation method and application |
| CN112326963A (en) * | 2020-11-09 | 2021-02-05 | 扬州大学 | Application of eukaryotic expression influenza A virus PB2cap protein |
| CN113150128A (en) * | 2020-11-24 | 2021-07-23 | 中国农业科学院哈尔滨兽医研究所（中国动物卫生与流行病学中心哈尔滨分中心） | Monoclonal antibody of influenza virus NP protein and application thereof |
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2003
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| CN107099512A (en) * | 2005-04-21 | 2017-08-29 | 佛罗里达大学研究基金公司 | The material and method controlled for respiratory disease in canid |
| CN103451158B (en) * | 2005-04-21 | 2018-11-23 | 佛罗里达大学研究基金公司 | The substance and method controlled for respiratory disease in canid |
| CN101871944A (en) * | 2010-06-02 | 2010-10-27 | 中国农业科学院茶叶研究所 | Qualitative detection method of tea geometrid nuclear polyhedrosis virus |
| CN101871944B (en) * | 2010-06-02 | 2013-07-10 | 中国农业科学院茶叶研究所 | Qualitative detection method of tea geometrid nuclear polyhedrosis virus |
| CN103336121A (en) * | 2013-06-13 | 2013-10-02 | 华中农业大学 | Swine influenza virus H3 subtype colloidal gold detection kit as well as preparation method and application thereof |
| CN103336121B (en) * | 2013-06-13 | 2015-03-04 | 华中农业大学 | Swine influenza virus H3 subtype colloidal gold detection kit as well as preparation method thereof |
| CN111171146A (en) * | 2020-02-20 | 2020-05-19 | 西北农林科技大学 | Nano antibody for resisting H9N2 subtype avian influenza virus, preparation method and application |
| CN111171146B (en) * | 2020-02-20 | 2022-03-04 | 西北农林科技大学 | Nano antibody for resisting H9N2 subtype avian influenza virus, preparation method and application |
| CN112326963A (en) * | 2020-11-09 | 2021-02-05 | 扬州大学 | Application of eukaryotic expression influenza A virus PB2cap protein |
| CN112326963B (en) * | 2020-11-09 | 2024-02-06 | 扬州大学 | Application of eukaryotic expression type A influenza virus PB2cap protein |
| CN113150128A (en) * | 2020-11-24 | 2021-07-23 | 中国农业科学院哈尔滨兽医研究所（中国动物卫生与流行病学中心哈尔滨分中心） | Monoclonal antibody of influenza virus NP protein and application thereof |
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