CN102533668A - Duck flavivirus, and vaccine and kit thereof - Google Patents

Duck flavivirus, and vaccine and kit thereof Download PDF

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CN102533668A
CN102533668A CN2010106010507A CN201010601050A CN102533668A CN 102533668 A CN102533668 A CN 102533668A CN 2010106010507 A CN2010106010507 A CN 2010106010507A CN 201010601050 A CN201010601050 A CN 201010601050A CN 102533668 A CN102533668 A CN 102533668A
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CN102533668B (en
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李泽君
颜丕熙
闫丽萍
滕巧泱
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Shanghai Veterinary Research Institute CAAS
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Abstract

The invention belongs to the field of bioengineering and discloses duck flavivirus. The amino acid sequence of an envelope protein E is shown as SEQ ID NO:4 or the homology of the amino acid sequence which is shown as the SEQ ID NO:4 is over 98 percent. The invention also discloses vaccine for preventing duck flavivirus, and a kit for detecting duck flavivirus; a new virus of duck flavivirus disease which causes output reduction and death of laying duck are separated and identified by various detection means, a method for diagnosing duck flavivirus disease is established, the vaccine for duck flavivirus disease is developed, important basis and means are provided for preventing duck flavivirus, and relative duck flavivirus diseases which cause the output reduction and death of laying duck can be prevented and diagnosed by using the vaccine and a diagnostic reagent which are developed by using the virus.

Description

A kind of duckling virus and vaccine thereof, test kit
Technical field:
The invention belongs to bioengineering field, relate in particular to a kind of virus, is a kind of duckling virus and vaccine thereof, test kit specifically.
Background technology:
China is a foster fowl big country, the aviculture fast development, and the year number of animals raised of poultry has reached 12,000,000,000 plumages, occupies first place in the world.The proportion of the economy of aviculture in national economy rises gradually, and its economy accounts for a big chunk of farmers' income.This shows that the healthy state of bird has very important effect to the development of Chinese national economy and society stable.In recent years, China aquatic bird aquaculture is continuing steady-state growth always.Add up according to Food and Argriculture OrganizationFAO (FAO); The year of China duck, goose raise total amount and reached nearly 5,000,000,000 plumages; Duck, goose YO reach 5,500,000 tons; All account for more than 75% of world's total amount, and the consumption proportion of aquatic bird meat such as duck, goose, egg is also in continuous rising, China has become the first in the world aquatic bird and has cultured big country and consumption big country.Though China aquatic bird number of animals raised was increasing year by year in recent years; Mass-producing, industrialization management level also obtain fast lifting; But the aquatic bird cultural technique is backward relatively, the especially generation of aquatic bird transmissible disease, and the mortality that causes aquatic bird to be cultured is high; Cause the annual loss of China's aquatic bird industry up to tens billion of yuans, become and perplexing the restraining factors that China's aquatic bird aquaculture develops in a healthy way.
The foster duck of China already faces the serious threat of multiple communicable disease.Since in April, 2010, at first in Shanghai, ground such as Zhejiang and the Jiangsu death of egg duck and the egg drop reduction that cause by unknown virus that take place, cause the egg duck field of nearly all (except that kind the duck) all to suffer the tremendous economic loss.It is useless exhausted that this sick cardinal symptom is that appetite appears in the egg duck, and egg drop reduction even stop to cause egg duck part dead, and mortality ratio can reach 5%～10%.Find that through cuing open the sick duck of inspection this disease is a manifest symptom with the spleen enlargement, morbidity liver in early stage has extravasated blood appearance to become, and the hemorrhage serious meeting of later stage liver is with tip-like white spotty necrosis.This disease constantly spreads in China, and all there is breaking out of this disease main egg duck production area, the whole nation at present.
Summary of the invention:
The object of the present invention is to provide a kind of duckling virus and vaccine thereof, test kit; Described this duckling virus and vaccine thereof, test kit will solve in the prior art that appetite is appearred in the egg duck is useless exhausted; Egg drop reduction even stop to cause the dead phenomenon treatment of egg duck part and the limited technical problem of means of prevention.
The invention provides a kind of duckling virus, the aminoacid sequence of its membranin M gene shown in SEQ ID NO:2 or with the homology of the aminoacid sequence shown in the SEQ ID NO:2 more than 98%.
Further, the nucleotide sequence of its membranin M gene shown in SEQ ID NO:1 or with the homology of nucleotide sequence shown in the SEQID NO:1 more than 98%.
Further, the aminoacid sequence of its envelope protein E protein gene shown in SEQ ID NO:4 or with the homology of the aminoacid sequence shown in the SEQ ID NO:4 more than 98%.
Further, the nucleotide sequence of its envelope protein E protein gene shown in SEQ ID NO:3 or with the homology of nucleotide sequence shown in the SEQ ID NO:3 more than 98%.
Further, the aminoacid sequence of its Nonstructural Protein NS5 protein gene shown in SEQ ID NO:6 or with the homology of the aminoacid sequence shown in the SEQ ID NO:6 more than 98%.
Further, the nucleotide sequence of its Nonstructural Protein NS5 protein gene shown in SEQ ID NO:5 or with the homology of nucleotide sequence shown in the SEQ ID NO:5 more than 98%.
The present invention also provides a kind of duckling virus, the aminoacid sequence of its envelope protein E protein gene shown in SEQ ID NO:4 or with the homology of the aminoacid sequence shown in the SEQ ID NO:4 more than 98%.
Further, the nucleotide sequence of its envelope protein E protein gene shown in SEQ ID NO:3 or with the homology of nucleotide sequence shown in the SEQ ID NO:3 more than 98%.
Further, the aminoacid sequence of its Nonstructural Protein NS5 protein gene shown in SEQ ID NO:6 or with the homology of the aminoacid sequence shown in the SEQ ID NO:6 more than 98%.
Further, the nucleotide sequence of its Nonstructural Protein NS5 protein gene shown in SEQ ID NO:5 or with the homology of nucleotide sequence shown in the SEQ ID NO:5 more than 98%.
The present invention also provides a kind of duckling virus, the aminoacid sequence of its Nonstructural Protein NS5 protein gene shown in SEQ ID NO:6 or with the homology of the aminoacid sequence shown in the SEQ ID NO:6 more than 98%.
Further, the nucleotide sequence of its Nonstructural Protein NS5 protein gene shown in SEQ ID NO:5 or with the homology of nucleotide sequence shown in the SEQ ID NO:5 more than 98%.
The present invention also provides a kind of duckling virus, and its preserving number is: CCTCC NO.V201032.
The present invention also provides a kind of antibody, and it is produced by above-mentioned a kind of duckling virus immunity.
The present invention also provides a kind of antibody, anti-above-mentioned duckling virus antigen.
The present invention also provides a kind of diagnostic reagent, contains above-mentioned antibody.
The present invention also provides a kind of dna molecular, its nucleotide sequence shown in SEQ ID NO:1 or with the homology of nucleotide sequence shown in the SEQ ID NO:1 more than 98%.
The present invention also provides a kind of dna molecular coded protein, its aminoacid sequence shown in SEQ IDNO:2 or with the homology of the aminoacid sequence shown in the SEQ ID NO:2 more than 98%.
The present invention also provides a kind of dna molecular, its nucleotide sequence shown in SEQ ID NO:3 or with the homology of nucleotide sequence shown in the SEQ ID NO:3 more than 98%.
The present invention also provides a kind of dna molecular coded protein, its aminoacid sequence shown in SEQ IDNO:4 or with the homology of the aminoacid sequence shown in the SEQ ID NO:4 more than 98%.
The present invention also provides a kind of dna molecular, its nucleotide sequence shown in SEQ ID NO:5 or with the homology of nucleotide sequence shown in the SEQ ID NO:5 more than 98%.
The present invention also provides a kind of dna molecular coded protein, its aminoacid sequence shown in SEQ IDNO:6 or with the homology of the aminoacid sequence shown in the SEQ ID NO:6 more than 98%.
The present invention also provides a kind of carrier, contain just like the nucleotide sequence shown in the SEQ ID NO:1 or with homology shown in the SEQ ID NO:1 at the nucleotide sequence more than 98%.
The present invention also provides a kind of carrier, contain just like the nucleotide sequence shown in the SEQ ID NO:3 or with homology shown in the SEQ ID NO:3 at the nucleotide sequence more than 98%.
The present invention also provides a kind of carrier, contain just like the nucleotide sequence shown in the SEQ ID NO:5 or with homology shown in the SEQ ID NO:5 at the nucleotide sequence more than 98%.
The present invention also provides a kind of carrier; Its expressed proteins contains just like the aminoacid sequence shown in SEQ ID NO:2 or SEQ IDNO:4 or the SEQ ID NO:6, perhaps with the homology of the aminoacid sequence shown in SEQ ID NO:2 or SEQ ID NO:4 or the SEQ ID NO:6 more than 98%.
The present invention also provides a kind of vaccine, contains above-mentioned carrier.
The present invention also provides a kind of vaccine, contains above-mentioned a kind of duckling virus of deactivation.
The present invention also provides a kind of vaccine, contains the above-mentioned a kind of duckling virus that causes weak or attenuation.
The present invention also provides a kind of test kit that detects above-mentioned duckling virus; Contain a pair of primer and probe; The sequence of one of them primer is shown in SEQ ID NO:7, and the sequence of another one primer is shown in SEQ ID NO:8, and the sequence of described probe is shown in SEQ ID NO:11.
The present invention also provides the test kit of the above-mentioned duckling virus of another kind of detection; Contain a pair of primer and probe; The sequence of one of them primer is shown in SEQ ID NO:9, and the sequence of another one primer is shown in SEQ IDNO:10, and the sequence of described probe is shown in SEQ ID NO:12.
Further, 5 ' flag F AM fluorescence report group of described probe, 3 ' end TAMRA fluorescent quenching group.
A kind of duckling virus of the present invention; Its preserving number is CCTCC NO.V201032; Classification called after Flavivirus Tembusu virus (Fengxian 101 strains of duckling virus); This virus is preserved in the Chinese typical culture collection center (CCTCC), and the address at Chinese typical culture collection center is: Luojiashan, Wuchang, Wuhan City, Hubei Province (Wuhan University), preservation date is on December 8th, 2010.
The present invention adopts various analyzing and testing means; Separate, identified the virus of the initiation egg duck underproduction that a strain is new and dead duckling virus disease; And its biological characteristics made studied in great detail; Obtain the main biological characteristics of this cause of disease and main gene order, set up, develop to this sick vaccine to this sick diagnostic method; For the control of duckling virus disease provides important evidence and means, utilize the vaccine of these virus research and development can prevent and diagnose the relevant duckling virus disease that causes the underproduction of egg duck and death with diagnostic reagent.
Description of drawings:
After Fig. 1 has shown duckling virus infection chicken embryo, cause that idiosome is hemorrhage, the phenomenon of oedema.
Fig. 2 has shown the electrophoresis picture of virogene and RNase and DNase digestion product, and a virogene is handled through RNase, and the b virogene is handled through DNase, the virogene that c handles without enzyme.
Fig. 3 is that PCR identifies electrophorogram.
Fig. 4 is the Electronic Speculum picture of virus particle.
Fig. 5 shown and attacked the pathological change of malicious duck spleen, wherein A group and the obvious enlargement of B group duck spleen, and C organizes the obvious no change of duck.
Fig. 6 has shown and has attacked the pathological change only of malicious duck that A shows that spleen lymphocyte is downright bad; B shows that liver is congested, hemorrhage, hepatic necrosis; C shows that kidney is hemorrhage, and renal cells is downright bad; D shows hemorrhage, the lymphocytic infiltration of lungs.
Fig. 7 is that fluorescence quantitative RT-RCR detects, and A is the kinetic curve of virus, and B is the typical curve of virus.
Fig. 8 is a virus (PRRSV) TaqMan PCR method susceptibility test amplification curve diagram.
Fig. 9 has shown the typical curve of ELISA reagent.
Figure 10 is duckling virus isolated strain of the present invention and Tembusu virus N S5 gene nucleotide series comparison diagram.
Figure 11 is duckling virus isolated strain of the present invention and Tembusu virus N S5 Argine Monohydrochloride sequence comparison diagram.
Figure 12 is duckling virus of the present invention Fengxian 101 strains heredity derivation analytical results figure.
Embodiment:
Embodiment 1 pathogen separation
Get the spleen tissue of morbidity duck, add 1ml PBS (Streptomycin sulphate that contains 100 unit penicillium mould and 50ug) and grind.After centrifugal, get supernatant, after the degerming of 0.2um filter, inoculation SPF chicken embryo.Discard chicken embryo dead in the 24h; Dead chicken embryo behind the collection 24h, visible chorioallantoic membrane thickens the hemorrhage (see figure 1) of idiosome general; Collect allantoic fluid, chicken embryo idiosome and chorioallantoic membrane thereof respectively.
The evaluation of embodiment 2 viruses main location in the chicken embryo
Get idiosome leach liquor and allantoic fluid respectively by ten doubling dilutions to 10 -1, 10 -2, 10 -3, 10 -4, each titre 100ul is put in 37 ℃ of incubators and hatches through 3 piece of 9 age in days SPF chicken embryo of allantoic cavity inoculation, observes and record chicken embryo death situation.The result finds and should virus mainly be present in chicken embryo idiosome and the chorioallantoic membrane thereof that the viral level in the allantoic fluid has only 1/10th of above-mentioned position.
Embodiment 3 virus amplification
After the idiosome of collecting and chorioallantoic membrane thereof ground, centrifuging and taking supernatant behind the multigelation three times, was put in 37 ℃ of incubators and hatches through 40 piece of 9 age in days SPF chicken embryo of allantoic cavity inoculation with ten times of sterilization PBS dilutions.Discard chicken embryo dead in the 24h; Inoculation back 48-120h, the chicken embryo is all dead; Collect chicken embryo idiosome, chorioallantoic membrane and allantoic fluid thereof respectively.
The purifying of embodiment 4 viruses
The idiosome of collecting and chorioallantoic membrane fully ground process suspension, multigelation three times.Adopt the sucrose density gradient centrifugation purified virus, operation steps is following: 1) with 7500rpm high speed centrifugation 30min, obtain supernatant, with 10000rpm high speed centrifugation 30min, obtain supernatant again.2) with the gained supernatant with 40000rpm ultracentrifugation 2h, will precipitate with 10ml STE and dissolve.3) in the ultracentrifugation pipe of two 38ml, add above-mentioned that 5ml obtained earlier respectively through STE dissolved virus liquid; Add 30%, 45%, 60% sucrose solution then successively from centrifuge tube bottom with the minute hand head; With 40000rpm ultracentrifugation 2.5h; The result can find a band clearly between 45% and 60%, the liquid of drawing this band is to the ultracentrifugation pipe of 38ml.4) remove sucrose: with the virus of STE damping fluid dilution purifying,, then with 40000rpm ultracentrifugation 3h, the gained deposition is purified viral liquid with the dissolving of 2mlSTE damping fluid.
Embodiment 5 viruses are identified
5.1 the evaluation of cause of disease nucleic acid type
The spissated virus of purifying is extracted viral RNA with QIAGEN RNeasy plus mini Kit, and the 5ulRNA extract adds 37 ℃ of incubation 30min of 1ulRNase (20mg/ul); DNase digestion: 10 * buffer 1ul, RNase Inhibitor 0.5ul, DNase I 1ul, H20 (RNase-free) 2.5ul, RNA extract 5ul, 37C incubation 30min.Electrophoresis result is seen shown in Figure 2, through DNase digestion after product and all visible electrophoretic band of viral genome, and does not have electrophoretic band through the product of RNase digestion.Analysis can know virus to be RNA viruses.
5.2 the PCR of cause of disease identifies
Utilize M-MLV ThermoScript II (Takara company) and random primer with the viral RNA reverse transcription; With the reverse transcription product is that template utilizes the Auele Specific Primer of bird flu virus, NDV, the sick virus of RE hyperplasia, I type DHV, reovirus, parvovirus, birnavirus to carry out pcr amplification respectively, PCR result all negative (figure slightly).With the reverse transcription product is template, increases with the special PCR primer of duckling virus, and product can obtain special 250bp and the band of 300bp through agarose gel electrophoresis, sees Fig. 3.
5.3 the electron microscopic observation of virus particle
Certain density viral liquid is observed with the JEM2100 transmission electron microscope through phosphoric acid tungsten negative staining.The virus particle size is about 50nm under the visible mirror in this experimental result, is shaped as icosahedron ball-like structure (Fig. 4).
5.4 the evaluation of virus envelope
Get the idiosome leach liquor of 1ml gained, add the analytically pure chloroform of 50ul, place 4 ℃ of vibration mixing 10min, 500rpm subsequently, centrifugal 5min draws supernatant, and ten doubling dilutions are 10 -1, 10 -2, 10 -3, 10 -4, each titre is got 100ul through 3 pieces of instar chicken embryos on the 9th of allantoic cavity inoculation, hatches observed and recorded chicken embryo death situation in 37 ℃ of incubators; Control group adds the saline water of equal volume, handles equally and titration.Experimental result shows that chloroform is handled virus and lost infection ability, and therefore, this virus has cyst membrane.
5.5 the genome sequence of virus is measured
With purified virus, extract viral RNA with QIAGEN RNeasy plus mini Kit, utilize the cDNA Larbrary Construction Kit test kit construction cDNA library of Takara company; From the library, take out part bacterium liquid, coat on the LB/Amp Agar Plating and cultivate, the random choose bacterial clone; Check order after extracting plasmid; Utilize Seqman software (DNASTAR) to splice, after removal host's (chicken embryo) gene order, obtain this viral gene order.The nucleotide sequence of duckling virus membrane antigen M gene is shown in SEQID NO:1; The aminoacid sequence of duckling virus membrane antigen M gene is shown in SEQ ID NO:2; The nucleotide sequence of duckling virus envelope protein gene E is shown in SEQ ID NO:3; The amino acid acid sequence of duckling virus envelope protein gene E is shown in SEQ ID NO:4; The nucleotide sequence of duckling virus nonstructural protein gene NS5 is shown in SEQ ID NO:5, and the amino acid acid sequence of duckling virus nonstructural protein gene NS5 is shown in SEQ ID NO:6.
Through studying the genetic evolution of relative conservative gene NS5, find that the homology of Tembusu virus in duckling virus Fengxian 101 strains and the flaviviridae Flavivirus is the highest, the nucleotide sequence homology of the two NS5 gene is 88.3%, sees Figure 10.The proteic amino acid sequence homology of the two NS5 is 95.7%, sees Figure 11.Flavivirus different virus strain isolated is carried out evolutionary analysis, find that two strain virus sibships are nearest,, see Figure 12 dropping on the systematic evolution tree in the same branch.Therefore, duckling virus of the present invention Fengxian 101 strains are variation strains of Tembusu virus.
E albumen of the present invention is the main immunizing antigen of duckling virus, so the foundation of vaccine development, diagnosis and antibody detection method is main relevant with the albumen of E genetic expression.We not only can contain the consistent duckling virus isolated strain of sequence SEQ ID NO:4 that utilizes with our report; Also capable of using containing reports with us that sequence SEQ ID NO:4 homology is higher than 98% strain and at 4 of E protein 14s, 218 and 381 and the consistent flavivirus strain isolated of the corresponding amino acid of sequence SEQ ID NO:4; Develop inactivated vaccine, cause weak or artificial constructed infections clone through going down to posterity and cause weak less toxic vaccine, and set up PCR, Real-time PCR, ELISA and fine jade and expand diagnostic method.Utilize expression vector, express the consistent E albumen of sequence SEQ ID NO:4 with our report; Or express with us and report that sequence SEQ ID NO:4 homology is higher than 98% E albumen, this albumen while 144,218 with 381 with annex in the consistent albumen of the corresponding amino acid of sequence SEQ ID NO:4; And express 10 of partial continuous with the virus sequence of upper amino acid and our report 4 consistent or homology more than 98% and contain with annex sequence SEQ ID NO:4 in the peptide sequence corresponding nucleotide sequences of one of 144,218 and 381 corresponding amino acid develop dna vaccination, vector-viral vaccine (comprising paramyxovirus vector, poxvirus vector, adenovirus carrier, herpesvirus vector, baculovirus etc.) and prokaryotic expression and develop polypeptide vaccine.
Gene M is another immunizing antigen of duckling virus; Not only can utilize the consistent duckling virus isolated strain of virus sequence SEQ ID NO:2 that contains with our report; Also capable of using containing with us reports that sequence SEQ ID NO:2 homology is higher than 98% strain and the consistent flavivirus strain isolated of the corresponding amino acid of sequence SEQ ID NO:2 in 38 in M albumen and 72 and annex; Develop inactivated vaccine, cause weak or artificial constructed infections clone through going down to posterity and cause weak less toxic vaccine, and set up PCR, Real-time PCR, ELISA and fine jade and expand diagnostic method.Utilize expression vector, express the consistent M albumen of sequence SEQ ID NO:2 with our report; Or express and we report that sequence SEQ ID NO:2 homology is higher than 98% M albumen, 38 in this albumen with 72 with annex in the consistent albumen of sequence 2 corresponding amino acid; And express 10 of partial continuous with the virus sequence of upper amino acid and our report 2 consistent or homology more than 98% and contain with annex sequence SEQ ID NO:2 in the peptide sequence corresponding nucleotide sequences of one of 38 and 72 corresponding amino acid develop dna vaccination, vector-viral vaccine (comprising paramyxovirus vector, poxvirus vector, adenovirus carrier, herpesvirus vector, baculovirus etc.) and prokaryotic expression and develop polypeptide vaccine.
NS5 is conservative relatively for duckling virus nonstructural protein gene, in virus replication, plays an important role.We not only can utilize contains the duckling virus isolated strain consistent with the virus sequence 6 of report; Also capable of using containing with us reports that sequence SEQ ID NO:6 homology is higher than 98% strain and the consistent flavivirus strain isolated of the corresponding amino acid of sequence SEQ ID NO:6 in NS5 protein 18 position, 131 and 247 and annex; Develop inactivated vaccine, cause weak or artificial constructed infections clone through going down to posterity and cause weak less toxic vaccine, and set up PCR, Real-time PCR, ELISA and fine jade and expand diagnostic method.Utilize expression vector, express the consistent NS5 albumen of sequence SEQ ID NO:6 with our report; Or express with us and report that sequence 6 homologys are higher than 98% NS5 albumen, this albumen while 18,131 with 247 with annex in the consistent albumen of the corresponding amino acid of sequence SEQ ID NO:6; And express 10 of partial continuous with the virus sequence of upper amino acid and our report 6 consistent or homology more than 98% and contain with annex sequence SEQ IDNO:6 in the peptide sequence corresponding nucleotide sequences of one of 18,131 and 247 corresponding amino acid develop dna vaccination, vector-viral vaccine (comprising paramyxovirus vector, poxvirus vector, adenovirus carrier, herpesvirus vector, baculovirus etc.) and prokaryotic expression and develop polypeptide vaccine.
Embodiment 6 animal models are set up
Get healthy sheldrake in nine ages in week, be divided into three groups of A, B and C, 10 every group.A group: 10 4ELD 50Virus 100ul trans-oral infects, the normal raising; B group: 10 4ELD 50Virus 100ul infects through intramuscular injection, the normal raising; The C group is as contrast.Visible two days later A and B group duck spirit are depressed, and food consumption reduces; C group duck is asymptomatic.Attack poison back the 4th day and cut open three of inspections respectively for every group, the A group is organized the duck liver surface with B has tangible point-like ecchymosis, or white needle point sampling point shape necrosis region; The obvious enlargement of spleen, the surface is piebald shape.C group duck does not have this type of pathological change.The spleen picture of A and B group duck is seen Fig. 5, and Fig. 6 is seen in pathological change.Titration of virus is found and should virus in tissues such as duck liver, spleen, lungs, tracheae, kidney and brain, can be duplicated, produces higher virus titer.
A little less than the causing of embodiment 7 virus
Idiosome of collecting and the aseptic grinding of chorioallantoic membrane are processed suspension, and the centrifugal 45min of 10000rpm gets its supernatant and is used for virus weakening.
A little less than 7.1 cell causes: 1) with this supernatant to 10 of sterilization PBS dilution -2, get 100ul and be added on well-grown CEF cell, be put in 37 ℃ of cell culture incubator internal adsorption 1～2h, wash once with PBS subsequently, to wash the not virus of absorption off, add fresh medium again and place 37 ℃ to contain 5%CO 2Cell culture incubator in cultivate.2) draw cell conditioned medium behind the 48h and make virus be adsorbed in another well-grown CEF cell, be put in 37 ℃ and contain 5%CO with quadrat method 2Cell culture incubator in cultivate.3) continuous passage to the is gathered in the crops the cell conditioned medium liquid that contains virus after 20 generations, is diluted to 10 -1, 10 -2, 10 -3, 10 -4, three piece of 9 age in days SPF chicken embryo of each titre inoculation is put in 37 ℃ of incubators and hatches, and discards chicken embryo dead in the 24h; Remove the chicken embryo of non-specific death, collect dead chicken embryo, calculate ELD 50, the result shows through going down to posterity on the CEF cell a little less than virus is significantly caused after 20 times.
7.2 the chicken embryo causes weak method: 1) with 100 times of viral suspension supernatant dilutions,, be put in 37 ℃ of incubators and hatch, discard chicken embryo dead in the 24h, remove the chicken embryo of non-specific death, collect dead chicken embryo through 10 piece of 9 age in days SPF chicken embryo of allantoic cavity inoculation.2) get dead chick embryo allantoic liquid,, be put in 37 ℃ of incubators and hatch by inoculating 10 piece of 9 age in days SPF chicken embryo behind above-mentioned same method and the serum mixing once more; Discard chicken embryo dead in the 24h; Remove the chicken embryo of non-specific death, collect dead chicken embryo, more than 30 generations of continuous passage.
7.3 idiosome and chorioallantoic membrane supernatant after viral virulence experiment collecting cell supernatant or the grinding, infection laying ducks in 26 age in week, check virus weakening situation.Be divided into three groups of A, B and C, 10 every group.A group: 10 4ELD 50Cell attenuated virus 100ul infects through intramuscular injection, the normal raising; B group: 10 4ELD 50Chicken embryo attenuated virus 100ul infects through intramuscular injection, the normal raising; The C group is as contrast.All duck food consumptions are normal two days later, do not see that spirit is depressed, and A group and B group egg productivity slightly descend; C group duck is asymptomatic.Attack poison back the 4th day and cut open three of inspections respectively for every group, the A group is organized the duck liver with B and is not seen tangible pathology; Not enlargement of spleen.Titration of virus discovery virus has in tissues such as duck spleen and digestive tube necessarily duplicates, and virus titer is lower.
7.4 less toxic vaccine development: cause weak virus after propagation on corresponding SPF chicken embryo or the cell with above-mentioned, centrifugal removal impurity mixes the back packing with protective material, and freeze-drying on Freeze Drying Equipment is used to develop less toxic vaccine.Carry out the less toxic vaccine development by " veterinary biologics standard ".
The preparation of embodiment 8 inactivated vaccines
8.1 virus multiplication
After the idiosome of collecting and chorioallantoic membrane thereof ground, centrifuging and taking supernatant behind the multigelation three times, was put in 37 ℃ of incubators and hatches through 40 piece of 9 age in days SPF chicken embryo of allantoic cavity inoculation with ten times of sterilization PBS dilutions.Discard chicken embryo dead in the 24h; Inoculation back 72h, the chicken embryo is all dead; Collect chicken embryo idiosome, chorioallantoic membrane and allantoic fluid thereof respectively.
8.2 chicken embryo MDT (MDT) is measured
Method by 49 pages of " veterinary biologics standard " appendix is carried out.Calculation result MDT is 67.75 hours.
8.3 toxicity test to the chicken embryo
To plant poison and do 100 times of dilutions with sterile saline, inoculation 10 age in days SPF chicken embryos are 100 in the allantoic cavity, and every embryo 0.1mL puts 37 ℃ and continues to hatch, and 6～8 hours photograph embryos once write down dead embryo number and idiosome pathology in 24～120 hours.The result sees table 1.
Table 1: to the mensuration of embryo toxicity power
Figure BSA00000395589400101
8.4 viral level is measured
Make 10 times of serial dilutions by containing viral liquid with sterile saline, get 10 -1, 10 -2, 10 -3, 10 -4, 10 -5Five extent of dilution are inoculated 10 age in days SPF chicken embryos respectively in allantoic cavity, 3 pieces of embryos of each titre inoculation, every embryo 0.1mL; Putting 37 ℃ continues to hatch; Dead embryo discards and disregards before 24 hours, and 48～120 hours dead chicken embryos are in time taken out, and checks chorioallantoic membrane and idiosome one by one; Remove non-specific dead embryo, calculate ELD 50, the result sees table 2.
Table 2: the mensuration of viral level
Figure BSA00000395589400111
8.5 inactivation of virus
After 8.5.1 idiosome that inactivation test is collected the first step and chorioallantoic membrane grind, multigelation three times, 4 ℃ of centrifugal 45min of 10000rpm get supernatant; Add formalin solution, mixing, the final concentration that makes formaldehyde is 3 ‰; In another bottle of impouring, fail to contact inactivator behind the adding formaldehyde, be put in 37 ℃ of deactivation 24h (reaching 37 ℃ with temperature in the bottle picks up counting) then with near adherent virus avoiding bottleneck; Jolting is 3～4 times during deactivation, then takes out, and places 4 ℃ of preservations subsequent use.
8.5.2 inactivating efficacy check:
8.5.2.1 10 pieces of 10 age in days SPF chicken embryos are got in chick embryo method check, every piece through allantoic cavity inoculation inactivation of viruses liquid 0.2ml, and continuously observe five day according to egg 2 times every day, and the chicken embryo does not have specificity death, and 120 as a child checked all do not have obvious pathology to all chicken embryos.
8.5.2.2 sheldrake method check: with 9 age in week 40 of sheldrakes, be divided into 4 groups, first group 10, the inactivation of viruses liquid 0.5mL of 5 times of amount of vaccine of every intramuscular inoculation; Second group 10, the inactivation of viruses liquid 0.5mL of 5 times of amount of vaccine of every collunarium inoculation; The 3rd group 10, every collunarium inoculation 0.5mL saline water is as contrast; The 4th group 10, every intramuscular inoculation 0.5mL saline water is as contrast; Under equal conditions in shield retaining, raise respectively for four groups, observed 10, the record reaction.Four groups of ducks all do not have death, cut open after 4 days and kill three, and the result shows that liver, the splenomegaly of inactivation of viruses liquid inoculation group and control group duck is little consistent, do not have swelling and pathology such as hemorrhage.The virus separating resulting shows that each internal organs of two groups of ducks all are not separated to the virus of the chicken embryo that can cause death.
8.6 vaccine method of manufacture
Get 92 parts of No. 10 white oils, add 6 parts of departments this 80, after the mixing, add 2 parts in Triple Pressed Stearic Acid aluminium, with add be stirred to transparent till, 115 ℃ of autoclaving 40min, the cooling back is subsequent use.Get 8 parts of Tween-80 sterilization in the bottle of band granulated glass sphere of packing into, add respectively 46 parts of deactivation blastochyle and deactivation Frustrate blood and mycoplasma bacterium liquid after the cooling respectively, till shake well dissolves tween 80 fully.Get oil phase and be put in colloidal mill or the emulsion tank for 5 parts, start the motor slow rotation and stir, add 3 parts of waters simultaneously slowly; Stirred 2 minutes with 10000rpm again after adding, then, add 1 part of water fast; With same speed emulsification 1.5min; Before stopping emulsification, add 1% Thiomersalate solution, making its ultimate density is 0.01%, milky oil emulsion inactivated vaccine.Carry out quality test by associated viscera.
8.7 vaccine safety test
To 2 different duck kind groups of every batch of vaccine test, 6 the week age SPF egg duck, 4 the week age duckling each 10, random packet.For 6 the week age SPF egg duck, 4 the week age duckling, adopt this oil-emulsion inactivated vaccine of subcutaneous multi-point injection 5mL/ plumage; In addition, control group is set, neck subcutaneous or intramuscular injection single-point or multiple spot saline water 0.5mL/ plumage or 1mL/ plumage were observed 30 days, had or not fervescence, lassitude, and appetite stimulator and other abnormal responses, the result sees table 3.
Table 3: vaccine safety test
Figure BSA00000395589400121
8.8 immuning effect test
1) 6 the week age egg duck immuning effect test, every plumage neck subcutaneous injection 0.3mL.Test situation is seen table 4.
2) 4 the week age duckling immuning effect test, every plumage neck subcutaneous injection 0.3mL.Test situation is seen table 4.
Table 4: vaccine immunity efficacy determinations
Figure BSA00000395589400131
8.9 minimum immune dosage is measured
With 6 age in week SPF egg duck and 4 age in week duckling with 0.1mL, 0.2mL, 0.3mL, 0.5mL, the subcutaneous vaccination of 1mL different dosages neck, 10 every group of SPF ducks, 4 age in week 40 every group of ducklings.Control group is 10 for every group.Attack poison after the blood sampling in 21 days, attack poison and separated virus in back 5 days.
8.10 immune kinetic determination
With 10 age in days SPF ducklings, every plumage neck subcutaneous injection 0.2mL is respectively at taking a blood sample in 3,7,10,14 and 21 days after the immunity and attacking poison.
8.11 the immunoprotection phase is measured
Get 120 of SPF ducks, 80 of test group, 40 of control groups; Test group is pressed the homemade oil-emulsion vaccine of dosage subcutaneous vaccination of 0.5ml/ plumage; Control group inoculation saline water 0.5ml/ plumage, every respectively at the inoculation back at a distance from 60 duck vein separation of 3 days random acquisitions serum, every after 30 days at a distance from blood sampling in 7 days 1 time; Until back 180 days of inoculation, the serum of collection detected serum antibody.
8.12 attack malicious protection test
Get the SPF duck of duration of immunity determination test group, respectively at after the immunity 21,30,45,60,75,90,115,130,145,160 days, stochastic sampling, each 5, with 10 4ELD 50Strong malicious intramuscular inoculation is attacked; Simultaneously, randomly draw 2 chickens of control group and attack, slaughtered the test duck in back 10 days, write down this virus infection situation respectively, measure protection ratio, and record attacks the variation of body weight and egg productivity in the poison 10 days, protect effect to see table 5 in attacking poison with identical method.
Table 5: deactivation vaccine is attacked poison protection effect measuring
Figure BSA00000395589400132
Figure BSA00000395589400141
Embodiment 9DNA vaccine development
9.1M pcr amplification with the E gene
With RNA extraction agent box extracting virus total RNA, become cDNA article one chain through the random primer reverse transcription.With cDNA article one chain, with Auele Specific Primer difference pcr amplification M gene and E gene.The pcr amplification condition: 94 ℃ of preparatory sex change 3min, 94 ℃ of sex change 30s, 53 ℃ of annealing 30s, 72 ℃ are extended 30s, totally 30 circulations, last 72 ℃ of insulation 10min.The PCR product is visible respectively 250bp and the big or small band of 1500bp in 1.0% agarose gel electrophoresis, and size is consistent with expected results.Behind the PCR product rubber tapping purifying with M and E gene, be connected in the pMD19-T carrier, and send order-checking.Confirm that through the sequencing result analysis sequence is correct, do not have sudden change.
9.2M structure and evaluation with E gene eucaryon expression plasmid pCAGGS-M and pCAGGS-E
With the PCR product of above-mentioned rubber tapping purifying with Hind III and Xho I double digestion after, be connected in the pCAGGS carrier that same double digestion is handled.Linked system is following: 10 * buffer 1ul, T4DNA ligase enzyme 1ul, enzyme cut handle carrier pCAGGS and PCR product each 1 and 7ul, room temperature connection 2h.The product that is connected of this M and E gene is transformed DH5 α competent cell respectively, and containing 37 ℃ of cultivations on the LB flat board of penbritin.Behind the 16-20h, the single bacterium colony of picking, extracting DNA carry out enzyme and cut evaluation (Hind III and Xho I).Enzyme is cut product and is observed in 1.0% agarose electrophoresis.The result is visible, can cut out the band of size for 250bp and 1500bp behind pCAGGS-M and the pCAGGS-E recombinant plasmid double digestion respectively.Send order-checking with pCAGGS-M and pCAGGS-E recombinant plasmid then.Sequencing result confirms that the M and the E gene order of the pCAGGS carrier that inserts are errorless.These results prove that pCAGGS-M and pCAGGS-E recombinant plasmid have made up success.
9.3M development with E gene DNA vaccine
From the LB solid culture, chosen single bacterium colony, be inoculated in the test tube that fills 3ml LB liquid nutrient medium, 37 ℃ of incubated overnight.The centrifugal supernatant of abandoning of bacterium liquid, bacterial sediment carries out ultrapure plasmid extraction with the intracellular toxin test kit that goes of OMEGA company.1) dips in to go bail for transfering loop and deposit glycerol stock (containing plasmid 3048-HA or 2842 or 2843 or 1238), containing the line of Amp LB planar surface, 37 ℃ of hold over night; 2) picking list bacterium colony is inoculated in the 5mlAmp LB substratum, and 37 ℃ of shaking culture are to OD 600Value is 1.0-1.5; 3) balance pillar: add 2ml solution E 4, make under its spontaneous current; 4) collect 3ml overnight culture, centrifugal thorough removal substratum; 5) suspension thalline: contain the solution S olutionI suspension thalline of RNase A with 0.25ml, until becoming the homogenate shape; 6) a cracking bacterium: add 0.25ml solution S olutionII again, put upside down 5 times with mixing (not concussion gently! ); 7) neutralization: add 0.125ml solution B ufferN3, put upside down 5 times at once with mixing gently, until forming white flocks, room temperature 12,000 * g, centrifugal 10min; 8) carefully supernatant is poured in the clean 1.5ml centrifuge tube, the ETR Solution (blueness) that adds 0.1 times of volume puts upside down 7-10 time, then ice bath 10min to supernatant; 9) add 42 ℃ of water-bath 5min, muddy once more, room temperature 12,000 * g, centrifugal 3min, ETR solution will form a blue layering in the centrifuge tube bottom; 10) supernatant is moved in the new 1.5ml centrifuge tube, adds the absolute ethyl alcohol of 0.5 times of volume, puts upside down 6-7 time, and room temperature is placed 1-2min; 11) above-mentioned mixed solution is poured a new HiBand DNAMini post into, be placed on the collection tube of a 2ml, and room temperature 10,000 * g, centrifugal 1min makes lysate flow through pillar; 12) discard liquid in the collection tube, remaining mixed solution is joined in the pillar, room temperature 10,000 * g, centrifugal 1min makes lysate pass through pillar fully; Add 500ul BufferHB to pillar, 10,000 centrifugal 1min under the room temperature, the washing pillar is guaranteed to remove remaining protein, to obtain high-quality DNA; 13) abandon liquid, with 700ulDNA Wash buffer washing pillar, 10,000 * g under the room temperature, centrifugal 1min discards liquid; 14) repeat, add DNAWash buffer again; 15) abandon liquid, idle running, the centrifugal 2min of room temperature 12,000 * g removes liquid; 16) place a clean 1.5ml centrifuge tube to pillar, the Endotoxin-Free Elution buffer that adds 30-50ul is to pillar, and room temperature leaves standstill 2min, and the centrifugal 1min of room temperature 12,000 * g can wash twice with eluted dna; 17) electrophoresis detection, and with Nanodrop2000c ultra-violet and visible spectrophotometer measurement OD260, OD280, estimation dna content and purity.The pCAGGS-M and the pCAGGS-E plasmid that obtain by aforesaid operations are diluted to 1mg/ml with 0.85% saline water, are dna vaccination M and E.
9.4 duck immunoprotection experiment
9.4.1 40 ducks of immunization method are divided into 4 groups at random, 10 every group: 1) recombinant plasmid pCAGGS-M group; 2) recombinant plasmid pCAGGS-E group; 3) vector plasmid pCAGGS group; 4) blank group.Every duck is injected the pre-treatment of appropriate hydrochloric acid bupivacaine in tibialis anterior in immunity the last week.Duck tibialis anterior intramuscular injection 0.1ml (1mg/ml) plasmid, 1 time/week, continuous 3 weeks.Get duck serum respectively at two, three weeks behind the last booster immunization, after all around and splenocyte is measured multinomial amynologic index.
9.4.2 the mensuration duck of antiviral protection ratio immunity dna vaccination is after 28 days, each experimental group intramuscular inoculation 10 4ELD 50Virus liquid.Observe and write down death, the incidence of duck every day.The result sees table 6; Recombinant plasmid pCAGGS-M; Recombinant plasmid pCAGGS-E immune group is attacked malicious sequela rate and is respectively 80% and 20%, shows that dna vaccination recombinant plasmid pCAGGS-M or recombinant plasmid pCAGGS-E have certain immunoprotection to duck, can be used as dna vaccination and uses.
The antiviral protection ratio of table 6:DNA vaccine is measured
Figure BSA00000395589400161
Embodiment 10 viral diagnosis methods
10.1 quantitative fluorescent PCR (Real-time PCR) detection method
10.1.1 primer and probe: utilize bioinformatic analysis software MegAlign software that the full genome of 5 strain ducklings virus that this research department records is carried out the conservative property analysis; Adopt Primer Express 2.0 softwares; Assist and carry out the primer probe design, carry out broad spectrum and specificity in its kind of Blast analysis verification then.Primer, probe are given birth to the synthetic and mark of worker's biotechnology ltd by Shanghai, and probe 5 ', 3 ' end be flag F AM fluorescence report group and TAMRA fluorescent quenching group respectively.In addition, designed the conventional PCR primer of two covers to this gene order.Primer and probe are synthetic by Shanghai Invitrogen company.
Primer sequence is following:
PCR1016-1038CTGGCTTGTTAACAGAGACTGGT
PCR1126-1105GCGTGGGCCTCCTCAAATTCTA
PCR1366-1086TGTCTTATGCAGGTACCGATG
PCR1470-1450CGTATGGGTTGACTGTTATCA
The Taqman probe sequence is following:
FXV?probe?1041-1056CATGACCTCAATTTAC
FXV?probe?1397-1414AGTTCCCATATCCATGTC
10.1.2 the extraction of viral RNA and cDNA are synthetic: extract test kit with RNA and extract total RNA of viral cultures and the homogenate of duck viscera tissue respectively, be dissolved in 30ul DEPC treating water, be used for synthetic cDNA by its process specifications.Getting total RNA 10.5ul adds in the 20ul reverse transcription system; Wherein comprise 4ul 5 * reverse transcription Buffer, 2uldNTPmix (10mmol/L), 1ul 9-mer random primer (50mmol/L), 2ul AMV (5u/ul) and 0.5ul RNA enzyme inhibitors (40u/ul); Gently behind the mixing in 42 ℃ of water-bath 1h; Last ice bath 2min is used for quantitative fluorescent PCR or puts 20 ℃ of preservations subsequent use then.
10.1.3 the preparation of positive criteria article: the E gene fragment of conventional pcr amplification duckling virus; The PCR product reclaims and behind the test kit purifying, connects into the pMD18-T carrier; Be converted into DH5 α competent escherichia coli cell, extract the positive colony plasmid of identifying and pass through the sequencing analysis verification through PCR, after the mensuration DNA concentration; The copy number of base of calculation article by formula, copy number (copies)=(quality/molecular weight) * 6.0 * 10 23 Reach 10 with 10 times of serial dilution plasmid standard to lower limits 0Copies/ul.
10.1.4 the optimization of reaction system and condition: in same concentrations template and reaction system, adopt matrix method to optimize the optimum concn of primer and probe.In the fluorescence quantitative RT-RCR reaction conditions,, select best Tm value that the positive nucleus acid template of same concentrations is detected simultaneously in conjunction with adjust different Tm values (50 ℃～65 ℃) with the grads PCR appearance.This research is optimized primer and concentration and probe concentration, to improve the amplification efficiency and the susceptibility of reaction.The result shows that working as primer concentration is 10pmol/ul; Concentration and probe concentration is 50pmol/ul; And the Tm temperature is when being 54 ℃, can obtain minimum Ct value and high fluorescent increased value, and optimizing the afterreaction system is to comprise in every 25ul reaction solution: 10 * Ex Taq Buffer (Mg2+Plus) 2.5ul; DNTPmix (each 2.5mmol/L) 2.5ul; Each 1ul of upstream and downstream primer (10pmol/ul); Probe (l0pmol/ul) 0.5ul; Ex Taq SH (5u/ul) 0.2ul, template (plasmid or cDNA) 1ul mends DEPC water to 25ul.Detect by following reaction parameter with the Mx3005p quantitative real time PCR Instrument then: 95 ℃ of sex change 5min, with 95 ℃ of 20s, 40 circulations of 54 ℃ of 1min amplifications are carried out the single-point fluoroscopic examination at 54 ℃.
10.1.5 the foundation of typical curve: with 10 times of serial dilution plasmid standards 10 1Copies/ul～10 7Copies/ul is a template, after quantitative real time PCR Instrument detects, calculates Ct value and drawing standard curve.Getting concentration is 2.96 * 10 1Copies/ul～2.96 * 10 7The standard substance template of copies/ul is carried out fluorescent quantitative PCR and is set up typical curve.Obtaining the typical curve equation is y=-3.447 * LOG (X)+40.74, coefficient R 2=0.999; Amplification efficiency E=0.95 (seeing Fig. 7 A and B).
10.1.6 specificity, susceptibility, replica test: the experimental analysis of detection architecture, all utilize the analysis of virus infected cell supernatant, carry out RNA extraction, rt earlier, be Real-time PCR then and detect.Detect the duckling virus strain of 3 strain different areas respectively, examine the conservative property of this detection architecture in the virus kind; Choose the relevant flavivirus epidemic encephalitis type B of this virus respectively and carry out check and analysis, to examine the specificity of this system; The sensitivity analysis experiment is at first carried out the virus plaque titration experiments to viral sample, uses serum-free cell culture medium with 10 times of dilutions then, obtains 1～10 5PFU/ml serial dilution virus sample suspension carries out check and analysis respectively, studies it and detects lower limit; Different sample a, b, c, the d of 4 template content of this experimental selection carries out 5 times respectively and detects.Divide and carry out for 2 times, be respectively 2 holes, the parallel detection in 3 holes; Experimental result Ct value to obtaining is carried out statistical analysis, and the MV and the variation coefficient of calculating each test sample Ct value are stable or repeatable to estimate it.
All between 18～31 circulations, the result all is judged to be the positive to the Ct value that warp detection 3 strain duckling viruses obtain.Further through experimental verification the conservative property of primer probe, in duckling virus is planted, have the wider spectrum that contains.Be the specificity of confirmatory experiment method, the method for setting up with us detects japanese encephalitis virus (JEV) virus of same Tobamovirus, and detected result all is negative, and shows that we have specificity by the designed primer probe.Viral sample with serial dilution is carried out RNA extraction, rt and Real-time pcr analysis respectively, and it is as shown in Figure 8 that experiment obtains amplification curve.If the Ct value is with 35 cycle criterion results' positive and negative, then the detection lower limit of this method can be judged to be 10PFU/ml, if the Ct value is limited to 1PFU/ml under with 38 cycle criterion results, then detecting.Sample concentration is when 10PFU/ml is above, and the Ct value can be judged to be the positive all less than 35, and the result sees Fig. 8.
The stability analysis experimental result: the Ct value that 4 different concns sample duplicate detection are obtained for 5 times gathers, and through calculating the MV and the standard deviation of Ct value, further can obtain the variation coefficient of each test item.4 test sample all can be judged to be the positive; And the Ct value variation coefficient (is 3.32% to the maximum in the reasonable scope; All less than 5%), can reach a conclusion: the Real-time PGR detection method of structure has satisfactory stability property, for quantitative analysis is laid a good foundation.
Separates 10.1.7 clinical sample detects with virus: the method detection of Preliminary Applications foundation should virus artificial challenge duck clinical sample part: picked up from 500 parts of geographic duck pathological tissues such as Zhejiang Jiaxing, Fengxian, Shanghai City in April, 2010 to October and grind separately, utilize Real-time PCR check and analysis to carry this viral situation.
21 portions of pathological material of disease lapping liquids are inoculated the SPF embryo respectively carry out the virus separation, the result is presented at amounting in 11 parts of pathological material of diseases of the peripheral epidemic season in 2010 in Shanghai, obtains virus totally 3 strains through separation and evaluation; TaqManPCR detects with Ct value＜35 and the S-shaped positive decision principle of amplification curve: the Real-time PCR detected result of 11 parts of pathological material of diseases of Shanghai periphery shows 6 parts and can be judged to be virus-positive, is respectively SH103, SH109, SH110, GS3, GS4 and GS6; And viral separating resulting only shows that isolation identification obtains duckling virus in SH103, SH109 and GS3.
Can find out that from The above results the TaqManPCR detection method and the viral separation method of virus relatively have higher susceptibility, viral isolating positive findings detects all positive at Real-time PCR.Through this experiment tentative confirmation this method have feasibility in the context of detection of virus, highly sensitive in viral separation method.
10.2 two-way agarose diffusion test: dissolve agarose, the agarose that on the level table, will dissolve is poured in the plate, processes the thick sepharose of the about 3～4mm of thickness, punches according to desired shape after cooling.Antigen and antibody are added respectively in the aperture, make antigen and antibody mutual diffusion mutually on agar plate.When two diffusion circles meet, be specific combination and ratio when suitable like antigen and antibody, will form the deposition of immune complex, this deposition can present an opaque and white precipitation line in agar.If antigen and irrelevant antibody just precipitation line can not occur, therefore can pass through this test, identify the duckling virus antigen with specific antibody, otherwise or with known duckling virus antigen evaluation antibody.
10.3ELISA method is identified
10.3.1 be used to detect the double antibody sandwich method of duckling virus antigen
10.3.1.1ELISA the preparation of double antibody sandwich method detection reagent
Encapsulate enzyme-linked reaction plate with anti-duckling viral monoclonal antibodies 2M1 strain, and carry out system optimization, select best preparation technology encapsulating concentration, temperature, time and sealing condition etc.With the anti-4F3 strain of anti-duckling viral monoclonal of mark horseradish peroxidase (HRP), adopt the square formation volumetry to select the best effort concentration of enzymic-labelled antibody.After the matched proportion density of other auxiliary reagent is optimized, be assembled into test kit.
It is 10 μ g/ml that optimum antibody encapsulates concentration, and the working concentration of enzyme labelling thing is 1: 4,000.Optimum test condition: application of sample 100 μ l, put 37 ℃ of water-bath 30min, the enzyme-added affinity tag 100 μ l in washing back put 37 ℃ of water-bath 20min, add developer after the washing, put 37 ℃ of water-bath 10min, after the termination, measure the OD of each reacting hole in the 450nm wavelength 450Value.
10.3.1.2 antigen quantitative criterion curve plotting
Virus-positive is made 1.5 times of serial dilutions with reference to article (tire and be 10EU/ml), measure the OD of each concentration 450Value is calculated through linear regression equation, confirms best quantitative concentration range, and the mensuration of calculating reagent is limited the quantity of.The absorption OD of sample in the establishing criteria curve 450Value is carried out Linear correlative analysis to measured value.The drawing standard curve is seen Fig. 9.Final definite antigenic quantitative scope is 0.131～0.666EU/ml.Sample concentration and mensuration OD in this is interval 450Value is good linear relation (r=0.996), and it is quantitative accurate to guarantee, minimum quantitative limit value is 0.131EU/ml.
10.3.1.3 reagent specificity checking
Bovine serum albumin, duck plague vaccine, duck parvovirus vaccine and hepatitis B vaccine in the cell culture fluid that this virus culture is used, SPF chick embryo allantoic liquid, the sample diluting liquid are measured, the specificity of examination reagent.
Test-results shows that this virus is positive reaction with reference to the detected result of article and SPF chick embryo allantoic liquid to be checked and virus-culturing fluid, measures OD 450Value and antigen concentration are good dependency.And other vaccines (duck plague vaccine, duck parvovirus vaccine, Vaccinum Encephalitidis Epidemicae) and the cell tissue liquid, the BSA solution that are used to produce are negative reaction (A＜0101).Proof ELISA reagent is special to this antigenic mensuration result, and non-this antigenic component can not produce cross reaction in detection, see table 7.
The specificity test of table 7ELISA reagent
10.3.1.4 reagent accurate degree and accuracy validation
Be formulated in the high, medium and low 3 parts of quantitative samples of this antigen in the measurement range with these viral positive criteria article, carry out replication 10 times, calculate the precision and the accuracy of reagent.
High, medium and low 3 parts of quantitative samples in the quantitative scope of ELISA reagent are carried out 10 times measure, through statistical procedures, the variation coefficient (CV) is respectively 8.3%, 11.0% and 10.0%.The measured value of sample and the recovery of theoretical value are respectively 99%, 98% and 102%, meet " Chinese biological goods rules " (2000 editions) join diagnostic reagent precision and accuracy to enzyme requirement.
10.3.1.5 reagent stability test
Carry out the stability test of reagent by " Chinese biological goods rules " (2000 editions) requirement, test kit (the whole series) is deposited different time at 37 ℃, observe the stability of each component in the reagent.
Detect identical quantitative reference article and sample to be checked in 37 ℃ of reagent of preserving 3d and 6d with the reagent of 2～8 ℃ of preservations, 3 groups of results are through statistics F check (variance analysis) degree of freedom (4,12), F=0.035＜F (0.05)=3.26, P＞0.05.Analyze according to the P value, the reagent detected result difference nonsignificance of the reagent after accelerating the failure and 2～8 ℃ of preservations is seen table 8.
The test of table 8ELISA reagent stability
Figure BSA00000395589400212
10.3.1.6ELISA the comparison of detected result and RT-PCR test detected result
Is standard with virus-positive with reference to article, measures the virus antigen content that 10 batches of SPF chick embryo allantoic liquids increase with ELISA, and the RT-PCR test detected result with these 10 batches of chick embryo allantoic liquids compares simultaneously, observes the dependency that two kinds of methods are measured results.The result that the RT-PCR test of 10 batches of chick embryo allantoic liquids and ELISA detect antigenic content sees table 9.RT-PCR contrast ELISA detected result in the table.RT-PCR identifies that the male sample also is positive in the ELISA reaction.
The comparison of table 9ELISA detected result and RT-PCR test detected result
Figure BSA00000395589400221
10.3.1.7 repeatability
With positive reference article is quantitative criterion; Operation rules and regulations according to test kit; Virus of A that the Fengxian is separated to and B replication 9 times are done 3 dilution mensuration to every batch of sample to be checked in each test, calculate each dilution ELISA unit (EU/ml) respectively; Get its MV, checking quantitative accuracy of reagent and test bay error.Through statistical procedures, the actual measurement antigenic content MV of A strain virus is (5.92 ± 0.16) EU/ml, and the variation coefficient is 2.7%; The actual measurement antigenic content MV of B strain is (1.72 ± 0.08) EU/ml, and the variation coefficient is 4.4%, explains that the test bay error of reagent is less, and the accuracy of mensuration is high.
Concrete ELISA reactions step is following: (following test no longer repeats)
Encapsulate: using 0.05M PH9.6 carbonate to encapsulate damping fluid is 1～10 μ g/ml with antibody dilution to protein contnt.In the reacting hole of each XPS, add 0.1ml, 4 ℃ are spent the night.Discard solution in the hole next day, washes 3 times each 3 minutes with lavation buffer solution.(being called for short washing, down together).
Application of sample: the sample 0.1ml to be checked that adds certain dilution puts 37 ℃ and hatched 1 hour in the above-mentioned reacting hole that has encapsulated.Washing then.(setting up blank well simultaneously, negative control hole and positive control hole).
Add enzyme labelled antibody: in each reacting hole, add enzyme labelled antibody (extent of dilution after the titration) 0.1ml of fresh dilution.Hatched 0.5～1 hour washing for 37 ℃.
Add substrate solution colour developing: in each reacting hole, add the tmb substrate solution 0.1ml of interim preparation, 37 ℃ 10～30 minutes.
Termination reaction: in each reacting hole, add 2M sulfuric acid 0.05ml.
The result judges: can be on white background, and the result directly detects by an unaided eye: color is dark more in the reacting hole, and positive degree is strong more, and negative reaction is colourless or extremely shallow, according to the depth of be color, with "+", "-" number expression.Also can survey the OD value: on the ELISA detector, locate, survey each hole OD value with zeroing back, blank hole in 450nm (if with ABTS colour developing, then 410nm), if it is greater than 2.1 times of the negative control OD value of stipulating, promptly positive.
10.3.2 detect the indirect method of corresponding antibodies with duckling virus-virus antigen:
10.3.2.1 antigenic preparation and purifying:
Normal antigen: duckling virus is inoculated 9 age in days SPF chicken embryos through allantoic cavity, collects dead chicken embryo idiosome, chorioallantoic membrane and allantoic fluid thereof after 24 hours respectively.After the freeze thawing 3 times, grind, 10, the centrifugal 30min of 000r/min gets supernatant, and surveys protein content 1.9789mg/mL.
Specific antigen: with above-mentioned duckling virus supernatant in 4 ℃, 40, the centrifugal 3h of 000r/min, collecting precipitation is in an amount of PBS.After 20% sucrose is centrifugal, use determined by ultraviolet spectrophotometry antigen protein content to be 6.3125mg/mL again.
10.3.2.2 confirming of judgement criteria
Select OD according to GB 450Value reads absorbancy.Under the situation that the yin, yang contrast is set up, blood serum special antigen to be checked hole 0D value >=0.2, blood serum special antigen hole OD value to be checked/negative control specific antigen hole OD value >=2.1 are judged to the positive.
10.3.2.3 confirming of normal antigen and specific antigen, enzyme conjugates best effort concentration:
With the yin, yang control serum with examine serum 1: 40 dilution fully.Normal antigen encapsulates with 0.05,0.1,0.2,0.4 μ g/mL respectively; Specific antigen encapsulates with 5,10,20 μ g/mL respectively; Goat-anti pig IgG-HRP is with 1: 5000～1: 64000 doubling dilution, through square formation confirm normally, specific antigen and enzyme conjugates best effort concentration are respectively 0.2,10 μ g/mL and 1: 20000.
10.3.2.4 specificity test:
Under the condition that this virus yin, yang contrast is all set up, all there is not specific reaction with duck plague, duck hepatitis, duck influenza, duck newcastle disease and duck parvovirus yin, yang serum.
10.3.2.5 stability test:
3 different batches antigen coated microplates with 4 ℃ of placements 0,30 and 90d detect known 4 parts of feminine genders and 4 parts of positive serums simultaneously, calculate OD 450Be worth velocity of variation (relative deviation), confirm the time stability of ELISA method.The OD value velocity of variation (relative deviation) of 8 parts of serum is all less than 25%.
10.3.2.6 accuracy test:
Use with a collection of plate with the same extent of dilution of a positive serum and to do 20 holes simultaneously, calculate OD 450The variation coefficient of value.With 4 different batches antigen coated microplates of 4 ℃ of placements 0,30,60 and 90d, detect same 8 parts of positive serums and same 8 parts of negative serums of different antibodies level simultaneously, replica test between criticizing calculates interassay coefficient of variation.
The variation coefficient (CV) in normally, specific antigen is criticized is respectively 8.3% and 6.4%; Normally, specific antigen is criticized an average coefficient of variation (CV) and is respectively 9.7% and 11.5%.
10.3.2.7 susceptibility test:
This virus antibody positive serum was made serial doubling dilution since 1: 40, detect the positive hole (OD of maximum dilution ratio with the ELISA method of setting up 450Value>=0.2) be its detection sensitivity, the result sees table 10.Table 10 indirect ELISA assay sensitivity is measured result (OD 450Value)
Figure BSA00000395589400241
10.3.2.8 confidence level:
With the ELISA method of setting up, the known yin, yang serum that application commercialization ELISA antibody assay kit was detected detects the confidence level of checking this method for 60 parts.
The ELISA method detects known yin, yang serum result and sees table 11.46 parts of negative serums detect 3 parts of positives, 43 parts of feminine genders, and it is 93.5% that negative serum detects coincidence rate; 6 parts of positive serum detected results are all positive, and positive coincidence rate is 100%; In 8 parts of suspicious serum, detect 7 parts of positives, 1 part of feminine gender.
Table 11ELISA method detects oneself and knows positive and negative serum result
Figure BSA00000395589400242
10.3.3 detect the indirect method of duckling antiviral antibody as antigen with the albumen of prokaryotic expression:
10.3.3.1 antigen prepd antigen utilizes the pET-32a prokaryotic expression system to express and purification of Recombinant duckling virus E protein.
10.3.3.2 the righttest definite square formation volumetry of using that encapsulates concentration and serum optimum dilution degree of antigen; With the reorganization duckling virus E protein (1.722mg/mL) of 0.05mol/L pH 9.6 carbonate buffer solutions after with purifying successively by 1.0,0.8,0.4,0.2,0.1, the 0.05g/ hole dilutes; Every hole 100uL encapsulates elisa plate, and 4 ℃ are spent the night.Positive serum and negative serum are done dilution in 1: 20,1: 40,1: 80,1: 160,1: 320,1: 640 respectively, and ELIAS secondary antibody is done dilution in 1: 5000, according to the indirect ELISA program determination.Measure OD 450Value value and P/N value are selected positive OD 450Value is greater than 1, negative OD 450The value value is less than 0.2, and antigen coated concentration and serum diluting multiple when the P/N value is maximum are best effort concentration.
The square formation titration results is with OD 450The value value is that the best encapsulates concentration near 1.0 o'clock the extent of dilution that encapsulates, and at this moment, error is minimum, and reaction is the sensitiveest.When detecting antigen is 2ug/mL, and serum dilution is 1: 160 o'clock, OD 450The value value approaches 1.0 most, therefore, confirms that the righttest antigenic concentration that encapsulates is 2ug/mL, and serum dilution is 1: 160 (table 12).
The righttest the confirming of concentration and serum optimum dilution degree that encapsulate of table 12 antigen
Figure BSA00000395589400252
Annotate: numerical value is OD in the table 450Value.
10.3.3.3 antigen the best encapsulates confirming of condition
With the righttest antigen concentration coated elisa plate, be divided into 6 groups, the 1st group 37 ℃ encapsulate 1h; The 2nd group 37 ℃ encapsulate 2h; The 3rd group 37 ℃ encapsulate 4h; The 4th group 4 ℃ are spent the night and encapsulate; The 5th group 37 ℃ encapsulate 1h and add 4 ℃ and spend the night and encapsulate; The 6th group 37 ℃ encapsulate 2h and add 4 ℃ and spend the night, and other conditions are constant, respectively organize negative hole, positive hole, control wells OD 450Value and P/N value confirm that the best encapsulates condition.
With detecting antigen 2ug/mL coated elisa plate, the result sees table 13, and is the highest with the 5th group P/N value, reaches 7.51, and the result shows, 37 ℃ encapsulate 1h and add 4 ℃ and spend the night to encapsulate to the best and encapsulate condition.
The righttest condition that encapsulates of table 13 antigen is confirmed
Figure BSA00000395589400261
10.3.3.4 confirming of confining liquid
Encapsulate the condition coated elisa plate with the righttest antigen concentration and the best, after the washing, use the PBST that contains 5% skimming milk, 1% gelatin, 1%BSA, 10% calf serum respectively as confining liquid, the 200uL/ hole, other conditions are constant, select suitable confining liquid.Carry out ELISA behind 37 ℃ of sealing 2h and detect, measure OD 450Value and P/N value select P/N value the maximum as confining liquid.
When with the PBST that contains 1%BSA the P/N value during as confining liquid maximum, be 7.676, in view of the above, the PBST of selection 1%BSA is as confining liquid.
10.3.3.5 confirming of best off-period
With best antigen concentration with encapsulate the condition coated elisa plate, after the washing, add confining liquid, the 200uL/ hole is divided into 5 groups.The 1st group 37 ℃ sealing 1h; The 2nd group 37 ℃ sealing 2h; The 3rd group 37 ℃ sealing 3h; The 4th group 37 ℃ sealing 4h; The 5th group 37 ℃ the sealing 30min, other conditions are constant, with P/N value the highest be the best off-period.With best antigen concentration with encapsulate the condition coated elisa plate, after the washing, add confining liquid, the 200uL/ hole, test result analysis, with 37 ℃ of sealing 2h, P/N value is 7.738 to the maximum, therefore selects 37 ℃ of sealing 2h to be the best off-period.
10.3.3.6 the selection of two anti-working concentrations
ELIAS secondary antibody is done dilution in 1: 2000,1: 4000,1: 8000,1: 16000, and other condition is constant, and antigen and serum are pressed the optimum dilution degree dilution, measure OD 450Value and P/N value.
With best antigen concentration with encapsulate the condition coated elisa plate, after the washing, add confining liquid, the 200uL/ hole, test result analysis, with 37 ℃ of sealing 2h, P/N value is 7.738 to the maximum, therefore selects 37 ℃ of sealing 2h to be the best off-period.
10.3.3.7 the selection of substrate-function time
With best antigen diluent degree coated elisa plate, the anti-duck IgG of rabbit HRP labeling antibody all adopts optimum dilution degree with negative, positive serum, and the substrate reactions time is respectively room temperature 5,10,15,30min, and other condition is constant, makes an experiment by ordinary method, measures OD 450Value and P/N value.
10.3.3.8ELISA confirming of yin and yang attribute threshold value
ELISA method by above-mentioned foundation detects 30 parts of duckling virus negative serums to be checked, measures its OD 450Value is also carried out statistical analysis, confirms the threshold value OD of positive serum 450Value>=0.2.
10.3.3.9 replica test in batch
Use the recombinant antigen with a collection of preparation to encapsulate different elisa plates, get the serum of 6 parts of different antibodies levels, in identical conditions, press the indirect ELISA program determination, every part of blood sample is done 5 hole parallel tests, and the result is carried out statistical analysis.
With 5 parts of positive serums and 1 part of negative serum being carried out the repeatability detection with batch recombinant antigen; With its result of statistical analysis, its variation coefficient is between 1.57%-7.43%, less than 10%; Explain that same sample degree of variation in a collection of test is very little, have better repeatability.
10.3.3.10 replica test between batch
Encapsulate different elisa plates with 3 different batches preparations and purified recombinant albumen, get the serum of 6 parts of different antibodies levels, press the indirect ELISA program determination, the result is carried out statistical analysis in identical conditions.
The result is through statistical analysis, and its variation coefficient less than 10%, explains that same sample degree of variation in the different batches antigen test is very little between 1.80%-8.82%, have better repeatability.
Figure ISA00000395589600021
Figure ISA00000395589600031
Figure ISA00000395589600041
Figure ISA00000395589600081
Figure ISA00000395589600091
Figure ISA00000395589600111

Claims (30)

1. duckling virus is characterized in that: the aminoacid sequence of its membranin M gene shown in SEQ ID NO:2, perhaps with the homology of the aminoacid sequence shown in the SEQ ID NO:2 more than 98%.
2. a kind of duckling virus as claimed in claim 1 is characterized in that: the aminoacid sequence of its envelope protein E protein gene shown in SEQ ID NO:4, perhaps with the homology of the aminoacid sequence shown in the SEQ ID NO:4 more than 98%.
3. according to claim 1 or claim 2 a kind of duckling virus is characterized in that: the aminoacid sequence of its Nonstructural Protein NS5 protein gene shown in SEQ ID NO:6, perhaps with the homology of the aminoacid sequence shown in the SEQ ID NO:6 more than 98%.
4. duckling virus is characterized in that: the aminoacid sequence of its envelope protein E protein gene shown in SEQ ID NO:4, perhaps with the homology of the aminoacid sequence shown in the SEQ ID NO:4 more than 98%.
5. a kind of duckling virus as claimed in claim 4 is characterized in that: the aminoacid sequence of its Nonstructural Protein NS5 protein gene shown in SEQ ID NO:6, perhaps with the homology of the aminoacid sequence shown in the SEQ ID NO:6 more than 98%.
6. duckling virus is characterized in that: the aminoacid sequence of its Nonstructural Protein NS5 protein gene shown in SEQ IDNO:6, perhaps with the homology of the aminoacid sequence shown in the SEQ ID NO:6 more than 98%.
7. duckling virus, its preserving number is: CCTCC NO.V201032.
8. a kind of duckling virus as claimed in claim 1 is characterized in that: the proteic nucleotide sequence of its membranin M shown in SEQ ID NO:1, perhaps with the homology of nucleotide sequence shown in the SEQ ID NO:1 more than 98%.
9. a kind of duckling virus as claimed in claim 4 is characterized in that: the proteic nucleotide sequence of its envelope protein E shown in SEQ ID NO:3, perhaps with the homology of nucleotide sequence shown in the SEQ ID NO:3 more than 98%.
10. a kind of duckling virus as claimed in claim 6 is characterized in that: the proteic nucleotide sequence of its Nonstructural Protein NS5 shown in SEQ ID NO:5, perhaps with the homology of nucleotide sequence shown in the SEQ ID NO:5 more than 98%.
11. an antibody is characterized in that: by claim 1 or claim 2 or claim 3 or claim 4 or claim 5 or claim 6 or the described a kind of duckling virus immunity generation of claim 7.
12. an antibody is characterized in that: anti-claim 1 or claim 2 or claim 3 or claim 4 or claim 5 or claim 6 or the described a kind of duckling virus antigen of claim 7.
13. a diagnostic reagent is characterized in that: contain claim 11 or the described antibody of claim 12.
14. a dna molecular is characterized in that: its nucleotide sequence shown in SEQ ID NO:1, perhaps with the homology of nucleotide sequence shown in the SEQID NO:1 more than 98%.
15. a dna molecular is characterized in that: coded protein, its aminoacid sequence shown in SEQ ID NO:2, perhaps with the homology of the aminoacid sequence shown in the SEQ ID NO:2 more than 98%.
16. a dna molecular is characterized in that: its nucleotide sequence shown in SEQ ID NO:3, perhaps with the homology of nucleotide sequence shown in the SEQID NO:3 more than 98%.
17. a dna molecular is characterized in that: coded protein, its aminoacid sequence shown in SEQ ID NO:4, perhaps with the homology of the aminoacid sequence shown in the SEQ ID NO:4 more than 98%.
18. a dna molecular is characterized in that: its nucleotide sequence shown in SEQ ID NO:5, perhaps with the homology of nucleotide sequence shown in the SEQID NO:5 more than 98%.
19. a dna molecular is characterized in that: coded protein, its aminoacid sequence shown in SEQ ID NO:6, perhaps with the homology of the aminoacid sequence shown in the SEQ ID NO:6 more than 98%.
20. a carrier is characterized in that, contain just like the nucleotide sequence shown in the SEQ ID NO:1, perhaps with homology shown in the SEQID NO:1 at the nucleotide sequence more than 98%.
21. a carrier is characterized in that, contain just like the nucleotide sequence shown in the SEQ IDNO:3, perhaps with homology shown in the SEQID NO:3 at the nucleotide sequence more than 98%.
22. a carrier is characterized in that, contain just like the nucleotide sequence shown in the SEQ ID NO:5, perhaps with homology shown in the SEQID NO:5 at the nucleotide sequence more than 98%.
23. carrier; It is characterized in that; Its expressed proteins contains just like the aminoacid sequence shown in SEQ ID NO:2 or SEQ ID NO:4 or the SEQ ID NO:6, perhaps with the homology of the aminoacid sequence shown in SEQ ID NO:2 or SEQ IDNO:4 or the SEQ ID NO:6 more than 98%.
24. a vaccine is characterized in that: contain the described carrier of claim 20 or claim 21 or claim 22.
25. a vaccine is characterized in that: the claim 1 or claim 2 or claim 3 or claim 4 or claim 5 or claim 6 or the described duckling virus of claim 7 that contain deactivation.
26. a vaccine is characterized in that: contain the claim 1 or claim 2 or claim 3 or claim 4 or claim 5 or claim 6 or the described duckling virus of claim 7 that cause weak or attenuation.
27. a test right requires 1 or the test kit of claim 2 or claim 3 or claim 4 or claim 5 or claim 6 or the described duckling virus of claim 7; It is characterized in that: contain a pair of primer and probe; The sequence of one of them primer is shown in SEQ ID NO:7; The sequence of another one primer is shown in SEQ ID NO:8, and the sequence of described probe is shown in SEQ ID NO:11.
28. a kind of test kit that detects duckling virus as claimed in claim 27 is characterized in that: 5 ' flag F AM fluorescence report group of described probe, 3 ' end TAMRA fluorescent quenching group.
29. a test right requires 1 or the test kit of claim 2 or claim 3 or claim 4 or claim 5 or claim 6 or the described a kind of duckling virus of claim 7; It is characterized in that: contain a pair of primer and probe; The sequence of one of them primer is shown in SEQ ID NO:9; The sequence of another one primer is shown in SEQ ID NO:10, and the sequence of described probe is shown in SEQ ID NO:12.
30. a kind of test kit that detects duckling virus as claimed in claim 29 is characterized in that: 5 ' flag F AM fluorescence report group of described probe, 3 ' end TAMRA fluorescent quenching group.
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CN102793918A (en) * 2012-08-01 2012-11-28 中国农业科学院哈尔滨兽医研究所 Duck flavivirus subunit vaccine, as well as a preparation method and application thereof
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CN103571798A (en) * 2012-08-07 2014-02-12 中国农业科学院上海兽医研究所 Duck tembusu virus low virulent strain and application thereof
CN103571798B (en) * 2012-08-07 2015-11-04 中国农业科学院上海兽医研究所 Duck tembusu virus low virulent strain and application thereof
CN103123351B (en) * 2012-11-12 2015-02-25 福州大北农生物技术有限公司 Method for testing exogenous virus of SPF (specific pathogen free) egg
CN103123351A (en) * 2012-11-12 2013-05-29 福州大北农生物技术有限公司 Method for testing exogenous virus of SPF (specific pathogen free) egg
CN102925592A (en) * 2012-11-28 2013-02-13 湖北省农业科学院畜牧兽医研究所 Fluorescence quantitative RT-PCR (reverse transcription-polymerase chain reaction) rapid diagnostic kit for specific detection of duck flavivirus infection and application method thereof
CN102925592B (en) * 2012-11-28 2013-12-25 湖北省农业科学院畜牧兽医研究所 Fluorescence quantitative RT-PCR (reverse transcription-polymerase chain reaction) rapid diagnostic kit for specific detection of duck flavivirus infection
CN103675275A (en) * 2013-12-30 2014-03-26 山东滨州博莱威生物技术有限公司 Duck flavivirus detection reagent kit
CN104046595A (en) * 2014-06-26 2014-09-17 中国科学院武汉病毒研究所 Chick embryo separating method for improving avian influenza virus separation rate
CN104046596A (en) * 2014-06-26 2014-09-17 中国科学院武汉病毒研究所 Chick embryo allantois membrane separation method capable of enhancing separation rate of avian influenza virus
CN106729694A (en) * 2016-12-20 2017-05-31 天津瑞普生物技术股份有限公司 A kind of I group of 4 type aviadenovirus DNA vaccination and its application
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
CN108676078A (en) * 2018-05-23 2018-10-19 江苏省农业科学院 Cause the application of the antigen of tembusu virus antibody-dependant humidification
CN111321122A (en) * 2018-12-13 2020-06-23 中国农业科学院上海兽医研究所(中国动物卫生与流行病学中心上海分中心) Duck egg laying-reduction syndrome virus and vaccine thereof
CN111321122B (en) * 2018-12-13 2022-11-29 中国农业科学院上海兽医研究所(中国动物卫生与流行病学中心上海分中心) Duck egg laying-reduction syndrome virus and vaccine thereof

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