CN105807049A - Hemagglutination inhibition test antigen for duck tembusu virus disease and preparation method thereof - Google Patents

Hemagglutination inhibition test antigen for duck tembusu virus disease and preparation method thereof Download PDF

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CN105807049A
CN105807049A CN201610096524.4A CN201610096524A CN105807049A CN 105807049 A CN105807049 A CN 105807049A CN 201610096524 A CN201610096524 A CN 201610096524A CN 105807049 A CN105807049 A CN 105807049A
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刘月焕
林健
王小蕾
杨志远
段会娟
赵际成
刘立新
潘洁
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Beijing Academy of Agriculture and Forestry Sciences
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Abstract

The invention relates to a hemagglutination inhibition test antigen for a duck tembusu virus disease and a preparation method thereof. A duck hemorrhagic ovarian inflammation virus HB strain is preserved at the typical culture preservation centre in China with the preservation number being CCTCC V201122; the strain comprises a specific gene sequene, and the GenBank accession number of the strain is JF523187. The strain is inoculated againt a neonatal rat proliferating virus; the hemagglutination inhibition test antigen for the duck tembusu virus disease is prepared through the technologies of hemagglutination inhibition nonspecific material (lipoprotein) removal, inactivation, addition of a protective agent and the like. The invention further discloses the preparation method of the hemagglutination inhibition test antigen by a duck tembusu virus.

Description

A kind of duck tembusu virus disease hemagglutination inhibition test antigen and preparation method thereof
Technical field
The present invention relates to a kind of duck tembusu virus disease hemagglutination inhibition test antigen and preparation method thereof, relate to biological engineering and field of immunology.
Background technology
Duck tembusu virus disease is a kind of new infectious disease of sending out or happen suddenly occurred for 2010, main clinical characteristics is that laying ducks egg production declines suddenly and Commercial meat-type duck death rate dramatically increases, the main substantially pathological changes of natural infection and artificial challenge's case is follicle deformation, congested, hemorrhage and spleen change in volume (infecting early stage spleen enlargement, later stage spleen reduces) and color blackening.Main histologic lesion is lymphocyte and skein cell activation hypertrophy, and the epidemic situation popular initial stage, according to main pathological change, by this disease called after " duck hemorrhagic ovaritis " temporarily.Then pass through Virus Isolation and DNA sequence homology analysis result shows, the virus causing above-mentioned pest of duck feelings is a kind of arbovirus, belong to flaviviridae Flavivirus, Ntaya virus groups, nearest with Tembusu virus sibship (Caoetal., 2011；Suetal.,2011；Yanetal.,2011).This name of disease is claimed unified for " duck tembusu virus is sick " by Chinese animal and veterinary association in 2011 first aquatic bird control and prevention of disease seminar, but up to the present, not yet has Uniform provisions or lifting manipulation that State-level names about this disease.It is noted that existing documents and materials show, Duckling flavivirus disease is likely to occur just on the ground such as Shijiazhuang City, Hebei Province, Handan early than nineteen ninety-five.Wen Libin etc. first from diarrhoea and lower limb paralysis sick duck liver is dirty, be separated to a strain virus spleen, this virus is spherical in shape, and size is about 30nm, has the single strand RNA virus of cyst membrane, expert's conclusion is that the virus separated seems to belong to flaviviridae, and is " duck viral encephalitis " by this disease preliminary designation.
Tembusu virus is separated in the mosquito body in Sarawak area in 1968-1970 first, is once once thinking that poultry was probably the natural reservoir (of bird flu viruses) (Plattetal., 1975) of virus.Tembusu virus be separated in Northern Thailand Japanese encephalitis Prevalent district mosquito body respectively at 1982 and 1992 (Leakeetal., 1986；Pandeyetal.,1999).2000, research worker can cause growth and development of chickens to be obstructed (Konoetal., 2000) after reporting the flaviviridae infections chicken of a kind of new Sitiawan by name, and the homology of Sitiawan virus and Tembusu viral nucleic acid is 92%.The Chinese nucleic acid very high homology that the Tembusu being separated in duck body is viral and Bagaza is viral (Tangetal., 2012；Yunetal.,2012).
In controlling the means taked of epidemic situation and measure, the work such as epidemic monitoring, serodiagnosis, vaccine immunity effect evaluation and laboratory animal screening is particularly important.Up to the present, the method of detection duck tembusu virus antibody has serum neutralization test (SerumNeutralizationTest, SNT), hemagglutination inhibition test (HemagglutinationInhibition, and elisa (Enzyme-LinkedImmunosorbentAssay HI), ELISA) etc., wherein HI test has sensitivity, special, easy, cost is low, detection morbidity duck group's positive rate is higher and can be used for the advantage of early diagnosis, is the prefered method of antibody test.HI test, for Epidemiological study, clinical diagnosis and vaccine immunity effect evaluation etc., plays a crucial role to controlling duck tembusu virus disease epidemic situation.
Summary of the invention
It is an object of the invention to overcome the deficiency not having hemagglutination inhibition test antigen and method in existing antibody test technology, there is provided a kind of duck tembusu virus disease hemagglutination inhibition test antigen and preparation method so that it is provide a kind of antibody test reagent accurate, sensitive, special, economic and simple to operate and method.
It is an object of the invention to be achieved through the following technical solutions:
A kind of hemagglutination inhibition test antigen prepared by duck tembusu virus.
Further, duck hemorrhagic ovaritis virus HB strain, preserving number is CCTCCV201122, is deposited in China typical culture collection on July 1st, 2011, and preservation address is: Wuhan, China university, Wuhan.
Use medicine, reagent and test kit prepared by described antigen.
The preparation method of described antigen, described preparation method includes following preparation process: 1) preparing duck blastema plinth seed culture of viruses after duck embryonic breeding kind duck tembusu virus, duck embryo went down to posterity less than 3 generations；2) duck blastema plinth seed culture of viruses inoculation neonatal rat being taken Mus brain and prepare HB strain seed culture of viruses, Mus brain went down to posterity less than 3 generations；3) HB strain virus is inoculated 2～4 age in days neonatal rats or C6/36 cell obtains virus liquid；4) inactivation；5) protective agent is added.
Further, step 1) concrete operation method be: the allantoic fluid of dead duck embryo and idiosome after taking inoculation duck tembusu virus, smash to pieces, dilution, freeze thawing, centrifugal, take supernatant, measure median lethal dose(LD 50) ELD50, and carry out steriling test, viral level is not less than 106.0ELD50/ 0.1ml and the qualified seed culture of viruses of steriling test are as duck blastema plinth seed culture of viruses.
Further, step 2) concrete operation method be: utilize duck blastema plinth seed culture of viruses, with 100ELD50The dosage of/0.1ml inoculates 2～4 age in days neonatal rats through brain, gathers in the crops spiritual depressed and the obvious Mus brain of clinical symptoms that trembles, grinds, dilution, and freeze thawing is centrifuged, and takes supernatant, mensuration median lethal dose(LD 50) ELD50, and carry out steriling test, median lethal dose(LD 50) is not less than 106.0ELD50/0.1ml, and HB strain seed culture of viruses based on the qualified seed culture of viruses of steriling test.
Further, step 3) HB strain virus inoculation 2～4 age in days neonatal rats, take out the obvious murine brains of clinical symptoms such as spirit is depressed, tremble, remove non-specific blood clotting mortifier and obtain virus liquid, described virus liquid viral level is not less than 106.0ELD50/0.1ml。
Further, the method removing non-specific blood clotting inhibitor thing is: cerebral tissue adds the aseptic sucrose of final concentration of 8.5% after melting, homogenate, under agitated conditions, homogenate is added dropwise in acetone, centrifugal segregation supernatant, collection pellet frozen dries, add the precipitation after physiological saline solution dissolves lyophilization, centrifugal 30～60 minutes of 5000～10000g, collect supernatant.
Further, step 3) in HB strain virus inoculation C6/36 cell line, collect supernatant concentration when 75%CPE occurs in cell and obtain virus liquid, described virus liquid viral level is not less than 106.0TCID50/0.1ml。
Further, step 4) concrete operations be: adding formalin in above-mentioned virus liquid, inactivate 2～3 days, described formalin concentration is 37～40%, and addition concentration of formaldehyde is 0.02%～0.2%；Step 5) concrete operations be: add gelatin freeze drying protectant or glycerol, prepare gelation dry solid-state antigen or liquid stable antigen.
The invention has the beneficial effects as follows:
The present invention is Isolation of duck tembusu virus (HB strain) from the sick duck of infected duck tembusu virus; by in isolated viral brain or intraperitoneal inoculation neonatal rat (or C6/36 cell) cultivate; results infect cerebral tissue (or Cell sap); the virus wherein cultivated through Mus brain processes through sucrose-acetone, adds suitable protective agent and make after formalin-inactivated.Detection for duck tembusu virus disease antibody.This antigen has the following characteristics that
Stability: depositing 24 months for 2～8 DEG C, HA titer is still not less than 1:128.
Specificity: duck tembusu virus positive serum be should be positive reaction (HI titer >=1:10) by antigen, all should be negative reaction (HI titer 1:10) to duck tembusu virus negative serum, duck plague virus positive serum, DHV positive serum, newcastle, bird flu virus H5, H9 hypotype positive serum.
Sensitivity: can detect that vaccine immunity 2～3 weeks and the viral infection HI antibody in duck and goose serum after 4～5 days.
The innovative point of the present invention: there is not yet both at home and abroad the research of duck tembusu virus disease hemagglutination inhibition test antigen report, by us for many years with systematic study it is shown that develop antigen stable, special and sensitive, compensate for the blank of this external diagnosis reagent.
The key problem in technology point of the present invention: (1) clear and definite basic seed culture of viruses and produce the generation of seed culture of viruses；(2) remove viral hemoagglutination inhibiting substances and enable the erythrocytic technique of virus coagulation；(3) technique that inactivation has infection activity virus；(4) the preservation state (liquid or lyophilizing) of antigen.
Detailed description of the invention
Embodiment 1
Prepare antigen raw material:
1) sensitive (selecting 2～4 age in days closed colony or inbred line) neonatal rat, in brain approach, according to 25 μ l/ only (containing about 100ELD50) dose inoculation duck tembusu virus, observe to 144 hours, neonatal rat occurs that spirit is depressed and the clinical symptoms such as tremble, and neonatal rat M & M should be greater than 90%.
2) duck tembusu virus, belongs to flaviviridae member, comprises specific gene sequence, and it is JF523187 in GenBank accession number, and isolated strain gene sequencing result shows, strain is the highest with tembusu virus homology.Virion size 30～50nm, spherical, there are cyst membrane and fibre to dash forward, it is possible to coagulation 0.25～0.6% goose and Columba livia erythrocyte.Virus can growth on closed colony or inbred line Mus brain, Embryo Gallus domesticus, duck embryo, DEF, C6/36 (aedes albopictus cell), BHK21 (breast hamster kidney cell) and Vero (African green monkey kidney cell) cell.
This virus main infection duck, goose and chicken etc., different days duck, goose and chicken all can infect, but duck and goose infects sequela, and clinical symptoms becomes apparent from.Virus mainly encroaches on the organ or tissues such as reproductive system, nervous system, immune system, respiratory system, digestive system and urinary system, and major lesions is hemorrhagic oophoritis, gangrenosum acne orchitis, gangrenosum acne splenitis, acute nonsuppurative hepatitis, interstitial pneumonia, interstitial nephritis and apyetous encephalitis.Owing to virus causes duck splenic injury, lymphocyte such as CD4 and CD8 etc. is downright bad and runs off, and quantity declines, and causes immunosuppressant, is easily caused other pathogenic microorganism secondary infection, and death rate increases.Meanwhile, the reproductive system of viral damage sexual maturity duck, morbidity laying ducks main manifestations feed intake and egg production sharply decline, follicle deformation and hemorrhage, fallopian tube atrophy, drake testis and atrophy of vas deferens.The duck and goose death rate of forth infection increases, and laying rate, fertility rate and hatchability are low.
This virus has characteristics that
Duck tembusu virus is ssRNA virus, has cyst membrane, surface to have fine prominent.Virion is substantially spherical, diameter 30～50nm.The resistance of duck tembusu virus environment to external world is more weak.Virus is sensitive to ether, chloroform.Most of detergents can by its rapid inactivation.Under 37 DEG C of conditions, just can inactivate with the stifling 6h of 0.1% formalin.Virus is deposited a few week in 4 DEG C, deposits some months in-20 DEG C, and its appeal is unaffected.Duck going out property oophoritis virus can not coagulation Adult Chicken, duck and turkey erythrocyte, it is possible to the goose of coagulation 0.25～0.5% and Columba livia erythrocyte.This virus can in 2～4 age in days Mus brains, 9～13 age in days duck embryo allantoic cavitys, embryo allantocherion, 9～10 age in days chick embryo allantoic cavities and embryo allantocherion, 6 age in days chick embryo yolk sacs and 7～8 age in days duck embryo yolk sac are cultivated, it is possible to cultivate on DEF, C6/36, BHK21 cell and Vero cell.
The preparation method of a kind of hemagglutination inhibition test antigen prepared by duck tembusu virus, comprises the following steps:
1) duck blastema plinth seed culture of viruses will be prepared after duck embryonic breeding kind duck tembusu virus, duck embryo went down to posterity less than 3 generations: take allantoic fluid and the idiosome of dead duck embryo after inoculating duck tembusu virus, smash to pieces, dilution proportion according to W/V=1:10, freeze thawing 1～2 time (discharging virus), centrifugal 30 minutes of 5000g, take supernatant, subpackage, 1.0ml/ props up.Measure Embryo Gallus domesticus median lethal dose(LD 50) (ELD50), and carry out steriling test according to Chinese veterinary pharmacopoeia.Viral level is not less than 106.0ELD50/ 0.1ml, and seed culture of viruses based on the qualified seed culture of viruses of steriling test, duck embryo went down to posterity less than 3 generations (F1～F3 generation).
2) duck blastema plinth seed culture of viruses inoculation neonatal rat being taken Mus brain and prepare HB strain seed culture of viruses, Mus brain went down to posterity less than 3 generations: utilize duck blastema plinth seed culture of viruses, with 100ELD50The obvious Mus brains of clinical symptoms such as the dosage of/0.1ml inoculates 2～4 age in days neonatal rats through brain, and results spirit is depressed and trembles.According to the dilution proportion of W/V=1:10 after grinding, freeze thawing 1 time, centrifugal 30 minutes of 5000g, take supernatant, subpackage, 1.0ml/ props up.Measure Embryo Gallus domesticus median lethal dose(LD 50) (ELD50), and carry out steriling test according to Chinese veterinary pharmacopoeia.Embryo Gallus domesticus median lethal dose(LD 50) is not less than 106.0ELD50/ 0.1ml, and seed culture of viruses based on the qualified seed culture of viruses of steriling test, Mus brain went down to posterity less than 3 generations (F1～F3 generation).
3) HB strain virus is inoculated 2～4 age in days neonatal rats and obtain virus liquid: by virus inoculation 2～4 age in days neonatal rat, the viral level of propagation should be not less than 106.0ELD50/0.1ml.The removal of non-specific blood clotting mortifier (lipid material): after cerebral tissue melts, adds the final concentration of sucrose of aseptic 8.5%, homogenate according to certain W/V ratio.Under agitated conditions, homogenate is added dropwise over acetone, 2 (centrifugal segregation supernatant of extracting, collection pellet frozen dries, add the precipitation after physiological saline solution dissolves lyophilization), centrifugal 30～60 minutes of 5000～10000g, supernatant is the virus liquid without non-specific blood clotting mortifier.
4) inactivation: add formalin in above-mentioned virus liquid, inactivate 2～3 days.Described formalin concentration is 37～40%, and adding concentration of formaldehyde is 0.02%～0.2%.
5) protective agent is added: add gelatin freeze drying protectant or glycerol according to a certain percentage, prepare gelation dry solid-state antigen or liquid stable antigen.
The quality standard of antigen:
Steriling test: undertaken by existing " Chinese veterinary pharmacopoeia ", answer asepsis growth.
HA titer: undertaken by note 1, should be not less than 1:128 to the HA titer of 0.33% goose hematid (note 3).
Specific assay: duck tembusu virus positive serum be should be positive reaction (HI titer >=1:10) by antigen, all should be negative reaction (HI titer 1:10) to duck tembusu virus negative serum, duck plague virus positive serum, DHV positive serum, newcastle, bird flu virus H5, H9 hypotype positive serum.
Effect and purposes: for the detection of duck tembusu virus antibody.
Storage: 2～8 DEG C of preservations, effect duration is 24 months.
Specification: 1ml/ bottle.
Embodiment 2
Prepare antigen raw material:
1) C6/36 cell (100TCID50The dose inoculation of/0.1ml grows up to the cell of monolayer, observes to 120 hours, and typical CPE pathological changes occurs in cell).
2) duck tembusu virus, belongs to flaviviridae member, comprises specific gene sequence, and it is JF523187 in GenBank accession number, and isolated strain gene sequencing result shows, strain is the highest with tembusu virus homology.Virion size 30～50nm, spherical, there are cyst membrane and fibre to dash forward, it is possible to coagulation 0.25～0.5% goose and Columba livia erythrocyte.Virus can growth on closed colony or inbred line Mus brain, Embryo Gallus domesticus, duck embryo, DEF, C6/36 (aedes albopictus cell), BHK21 (breast hamster kidney cell) and Vero (African green monkey kidney cell) cell.
This virus main infection duck, goose and chicken etc., different days duck, goose and chicken all can infect, but duck and goose infects sequela, and clinical symptoms becomes apparent from.Virus mainly encroaches on the organ or tissues such as reproductive system, nervous system, immune system, respiratory system, digestive system and urinary system, and major lesions is hemorrhagic oophoritis, gangrenosum acne orchitis, gangrenosum acne splenitis, acute nonsuppurative hepatitis, interstitial pneumonia, interstitial nephritis and apyetous encephalitis.Owing to virus causes duck splenic injury, lymphocyte such as CD4 and CD8 etc. is downright bad and runs off, and quantity declines, and causes immunosuppressant, is easily caused other pathogenic microorganism secondary infection, and death rate increases.Meanwhile, the reproductive system of viral damage sexual maturity duck, morbidity laying ducks main manifestations feed intake and egg production sharply decline, follicle deformation and hemorrhage, fallopian tube atrophy, drake testis and atrophy of vas deferens.The duck and goose death rate of forth infection increases, and laying rate, fertility rate and hatchability are low.
This virus has characteristics that
Duck tembusu virus is ssRNA virus, has cyst membrane, surface to have fine prominent.Virion is substantially spherical, diameter 30～50nm.The resistance of duck tembusu virus environment to external world is more weak.Virus is sensitive to ether, chloroform.Most of detergents can by its rapid inactivation.Under 37 DEG C of conditions, just can inactivate with the stifling 6h of 0.1% formalin.Virus is deposited a few week in 4 DEG C, deposits some months in-20 DEG C, and its appeal is unaffected.Duck going out property oophoritis virus can not coagulation Adult Chicken, duck and turkey erythrocyte, it is possible to the goose of coagulation 0.25～0.5% and Columba livia erythrocyte.This virus can in 2～4 age in days Mus brains, 9～13 age in days duck embryo allantoic cavitys, embryo allantocherion, 9～10 age in days chick embryo allantoic cavities and embryo allantocherion, 6 age in days chick embryo yolk sacs and 7～8 age in days duck embryo yolk sac are cultivated, it is possible to cultivate on DEF, C6/36, BHK21 cell and Vero cell.
The preparation method of a kind of hemagglutination inhibition test antigen prepared by duck tembusu virus, comprises the following steps:
1) duck blastema plinth seed culture of viruses will be prepared after duck embryonic breeding kind duck tembusu virus, duck embryo went down to posterity less than 3 generations: take allantoic fluid and the idiosome of dead duck embryo after inoculating duck tembusu virus, smash to pieces, dilution proportion according to W/V=1:10, freeze thawing 1～2 time (discharging virus), centrifugal 30 minutes of 5000g, take supernatant, subpackage, 1.0ml/ props up.Measure Embryo Gallus domesticus median lethal dose(LD 50) (ELD50), and carry out steriling test according to Chinese veterinary pharmacopoeia.Viral level is not less than 106.0ELD50/ 0.1ml, and seed culture of viruses based on the qualified seed culture of viruses of steriling test, duck embryo went down to posterity less than 3 generations (F1～F3 generation).
2) duck blastema plinth seed culture of viruses inoculation neonatal rat being taken Mus brain and prepare HB strain seed culture of viruses, Mus brain went down to posterity less than 3 generations: utilize duck blastema plinth seed culture of viruses, with 100ELD50The obvious Mus brains of clinical symptoms such as the dosage of/0.1ml inoculates 2～4 age in days neonatal rats through brain, and results spirit is depressed and trembles.According to the dilution proportion of W/V=1:10 after grinding, freeze thawing 1 time, centrifugal 30 minutes of 5000g, take supernatant, subpackage, 1.0ml/ props up.Measure Embryo Gallus domesticus median lethal dose(LD 50) (ELD50), and carry out steriling test according to Chinese veterinary pharmacopoeia.Embryo Gallus domesticus median lethal dose(LD 50) is not less than 106.0ELD50/ 0.1ml, and seed culture of viruses based on the qualified seed culture of viruses of steriling test, Mus brain went down to posterity less than 3 generations (F1～F3 generation).
3) HB strain virus inoculation C6/36 cell line, collects supernatant concentration and obtains virus liquid when 75%CPE occurs in cell, and described virus liquid viral level is not less than 106.0TCID50/0.1ml。。
4) inactivation: add formalin in above-mentioned virus liquid, inactivate 2～3 days.Described formalin concentration is 37～40%, and adding concentration of formaldehyde is 0.02%～0.2%.
5) protective agent is added: add gelatin freeze drying protectant or glycerol according to a certain percentage, prepare gelation dry solid-state antigen or liquid stable antigen.
The quality standard of antigen:
Steriling test: undertaken by existing " Chinese veterinary pharmacopoeia ", answer asepsis growth.
HA titer: undertaken by note 1, should be not less than 1:128 to the HA titer of 0.33% goose hematid (note 3).
Specific assay: duck tembusu virus positive serum be should be positive reaction (HI titer >=1:10) by antigen, all should be negative reaction (HI titer 1:10) to duck tembusu virus negative serum, duck plague virus positive serum, DHV positive serum, newcastle, bird flu virus H5, H9 hypotype positive serum.
Effect and purposes: for the detection of duck tembusu virus antibody.
Storage: 2～8 DEG C of preservations, effect duration is 24 months.
Specification: 1ml/ bottle.
Embodiment 3
Having optimization on the basis of embodiment 1 further, material therefor reagent is identical with embodiment 1.
Duck tembusu virus HB strain (F1 generation) is as seed culture of viruses, with 25 μ l/ only (containing 100ELD50) dosage intracranial inoculation 2～4 age in days KM kind neonatal rat.Take out the obvious Mus of clinical symptoms such as spirit is depressed, tremble in time, put less than-20 DEG C preservations.Neonatal rat is taken out, uses iodine tincture disinfection brain, aseptically remove brain skin and skull, take out cerebral tissue, put in sterilization container.Final concentration of 8.5% aseptic sucrose solution, homogenate is added according to the ratio of W/V=1:4.Under agitated conditions, homogenate is added dropwise in the cold acetone of 10～30 times of volumes of homogenate amount.500g is centrifuged 5min, abandoning supernatant, the extruding of precipitate Glass rod is ground, suspends with the cold acetone of amount same as described above, ice bath 1 hour, and precipitate extruding is ground by period with sterile glass rod.500g is centrifuged 5min, abandoning supernatant.Collecting in sterile test tube by the precipitate containing a small amount of acetone, 500g is centrifuged 5min, abandoning supernatant, is divided in drying tube by precipitate ,-70 DEG C of freezings 1 hour, vacuum lyophilization 2～3 hours.According to the ratio of former homogenate amount V/V=1/1, add physiological saline solution, dissolve dry precipitate 12～24 hours.Centrifugal 1 hour of 8000g, the formaldehyde taking supernatant addition final concentration of 0.05% inactivates.Take 6 age in days SPF Embryo Gallus domesticus 10 pieces, through yolk sac inoculation supernatant, every embryo 0.2ml, put 36～37 DEG C and continue to hatch.24 hours photograph embryos, Embryo Gallus domesticus dead in 24 hours is judged to nonspecific death, and Embryo Gallus domesticus nonspecific death should less than 2 pieces.After 24 hours, per sunshine, embryo 2 times, observed to 168 hours, and Embryo Gallus domesticus is all survived, and was judged to inactivation completely.Within 24～168 hours, dead embryo takes out in time, gathers in the crops idiosome, and blind passage 1 generation, as without chicken embryo death, being judged to inactivation completely.It is ratio addition sterile glycerol in checking qualified supernatant of 3:1 by volume, after mixing, is liquid stable antigen.
Stability: antigen 2～8 DEG C is deposited 24 months, HA titer is still not less than the operation art formula of 1:128, HA test and sees note 1.
Specificity: duck tembusu virus positive serum be should be positive reaction (HI titer >=1:10) by antigen, all should be negative reaction (HI titer 1:10) to duck tembusu virus negative serum, duck plague virus positive serum, DHV positive serum, newcastle, bird flu virus H5, H9 hypotype positive serum.The operation art formula of HI test is shown in note 2.
Sensitivity: can detect that vaccine immunity 2～3 weeks and the wild virus infection HI antibody in duck, goose serum after 4～5 days.
Embodiment 4
Having optimization on the basis of embodiment 1 further, material therefor reagent is identical with embodiment 1.
Duck tembusu virus HB strain (F1 generation) is as seed culture of viruses, with 25 μ l/ only (containing about 100ELD50) dosage intracranial inoculation 2～4 age in days ICR kind neonatal rat.Take out the obvious Mus of clinical symptoms such as spirit is depressed, tremble in time, put less than-20 DEG C preservations.Neonatal rat is taken out, uses iodine tincture disinfection brain, aseptically remove brain skin and skull, take out cerebral tissue, put in sterilization container.Final concentration of 8.5% aseptic sucrose solution, homogenate is added according to the ratio of W/V=1:4.Under agitated conditions, homogenate is added dropwise in the cold acetone of 10～30 times of volumes of homogenate amount.500g is centrifuged 5min, abandoning supernatant, is shredded with aseptic scissors by precipitate, suspends with the cold acetone of amount same as described above, ice bath 1 hour, and precipitate extruding is ground by period with sterile glass rod.500g is centrifuged 5min, abandoning supernatant.Collecting in sterile test tube by the precipitate containing a small amount of acetone, 500g is centrifuged 5min, abandoning supernatant, is divided in drying tube by precipitate ,-70 DEG C of freezings 1 hour, vacuum lyophilization 2～3 hours.According to the ratio of former homogenate amount V/V=1/1, add physiological saline solution, dissolve dry precipitate 12～24 hours.Centrifugal 1 hour of 8000g, the formaldehyde taking supernatant addition final concentration of 0.05% inactivates.Take 6 age in days SPF Embryo Gallus domesticus 10 pieces, through yolk sac inoculation supernatant, every embryo 0.2ml, put 36～37 DEG C and continue to hatch.24 hours photograph embryos, Embryo Gallus domesticus dead in 24 hours is judged to nonspecific death, and Embryo Gallus domesticus nonspecific death should less than 2 pieces.After 24 hours, per sunshine, embryo 2 times, observed to 168 hours, and Embryo Gallus domesticus is all survived, and was judged to inactivation completely.Within 24～168 hours, dead embryo takes out in time, gathers in the crops idiosome, and blind passage 1 generation, as without chicken embryo death, being judged to inactivation completely.It is ratio addition gelatin protective agent, lyophilizing in checking qualified supernatant of 4:1 by volume, is freeze-dried antigen.
Stability: antigen 2～8 DEG C is deposited 24 months, HA titer is still not less than the operation art formula of 1:128, HA test and sees note 1.
Specificity: duck tembusu virus positive serum be should be positive reaction (HI titer >=1:10) by antigen, all should be negative reaction (HI titer 1:10) to duck tembusu virus negative serum, duck plague virus positive serum, DHV positive serum, newcastle, bird flu virus H5, H9 hypotype positive serum.The operation art formula of HI test is shown in note 2.
Sensitivity: can detect that vaccine immunity 2～3 weeks and the wild virus infection HI antibody in duck, goose serum after 4～5 days.
Embodiment 5
Having optimization on the basis of embodiment 1 further, material therefor reagent is identical with embodiment 1.
Duck tembusu virus HB strain (F2 generation) is as seed culture of viruses, with 25 μ l/ only (containing about 100ELD50) dosage intracranial inoculation 2～4 age in days NIH kind neonatal rat.Take out the obvious Mus of clinical symptoms such as spirit is depressed, tremble in time, put less than-20 DEG C preservations.Neonatal rat is taken out, uses iodine tincture disinfection brain, aseptically remove brain skin and skull, take out cerebral tissue, put in sterilization container.The final concentration of sucrose of aseptic 8.5%, homogenate is added according to the ratio of W/V=1:4.Under agitated conditions, homogenate is added dropwise in the cold acetone of 10～30 times of volumes of homogenate amount.500g is centrifuged 5min, abandoning supernatant, is shredded with aseptic scissors by precipitate, suspends with the cold acetone of amount same as described above, ice bath 1 hour, and precipitate extruding is ground by period with sterile glass rod.500g is centrifuged 5min, abandoning supernatant.Collecting in sterile test tube by the precipitate containing a small amount of acetone, 500g is centrifuged 5min, abandoning supernatant, is divided in drying tube by precipitate ,-70 DEG C of freezings 1 hour, vacuum lyophilization 2～3 hours.According to the ratio of former homogenate amount V/V=1/1, add physiological saline solution, dissolve dry precipitate 12～24 hours.Centrifugal 1 hour of 8000g, the formaldehyde taking supernatant addition final concentration of 0.02% inactivates.Take 6 age in days SPF Embryo Gallus domesticus 10 pieces, through yolk sac inoculation supernatant, every embryo 0.2ml, put 36～37 DEG C and continue to hatch.24 hours photograph embryos, Embryo Gallus domesticus dead in 24 hours is judged to nonspecific death, and Embryo Gallus domesticus nonspecific death should less than 2 pieces.After 24 hours, per sunshine, embryo 2 times, observed to 168 hours, and Embryo Gallus domesticus is all survived, and was judged to inactivation completely.Within 24～168 hours, having dead germ to take out in time, gather in the crops idiosome, in blind passage 1 generation, as without chicken embryo death, being judged to inactivation completely.It is ratio addition sterile glycerol in checking qualified supernatant of 3:1 by volume, after mixing, is liquid stable antigen.
Stability: antigen 2～8 DEG C is deposited 24 months, HA titer is still not less than the operation art formula of 1:128, HA test and sees note 1.
Specificity: duck tembusu virus positive serum be should be positive reaction (HI titer >=1:10) by antigen, all should be negative reaction (HI titer 1:10) to duck tembusu virus negative serum, duck plague virus positive serum, DHV positive serum, newcastle, bird flu virus H5, H9 hypotype positive serum.The operation art formula of HI test is shown in note 2.
Sensitivity: can detect that vaccine immunity 2 weeks and the wild virus infection HI antibody in duck, goose serum after 4～5 days.
Embodiment 6
Having optimization on the basis of embodiment 1 further, material therefor reagent is identical with embodiment 1.
Duck tembusu virus HB strain (F2 generation) is as seed culture of viruses, with every 25 μ l/ only (containing 100ELD50) dosage intracranial inoculation 2～4 age in days CFW kind neonatal rat.Take out the obvious Mus of clinical symptoms such as spirit is depressed, tremble in time, put less than-20 DEG C preservations.Neonatal rat is taken out, uses iodine tincture disinfection brain, aseptically remove brain skin and skull, take out cerebral tissue, put in sterilization container.Final concentration of 8.5% aseptic sucrose solution, homogenate is added according to the ratio of W/V=1:4.Under agitated conditions, homogenate is added dropwise in the cold acetone of 10～30 times of volumes of homogenate amount.500g is centrifuged 5min, abandoning supernatant, is shredded with aseptic scissors by precipitate, suspends with the cold acetone of amount same as described above, ice bath 1 hour, and precipitate extruding is ground by period with sterile glass rod.500g is centrifuged 5min, abandoning supernatant.Collecting in sterile test tube by the precipitate containing a small amount of acetone, 500g is centrifuged 5min, abandoning supernatant, is divided in drying tube by precipitate ,-70 DEG C of freezings 1 hour, vacuum lyophilization 2～3 hours.According to the ratio of former homogenate amount V/V=1/1, add physiological saline solution, dissolve dry precipitate 12～24 hours.Centrifugal 1 hour of 8000g, the formaldehyde taking supernatant addition final concentration of 0.02% inactivates.Take 6 age in days SPF Embryo Gallus domesticus 10 pieces, through yolk sac inoculation supernatant, every embryo 0.2ml, put 36～37 DEG C and continue to hatch.24 hours photograph embryos, Embryo Gallus domesticus dead in 24 hours is judged to nonspecific death, and Embryo Gallus domesticus nonspecific death should less than 2 pieces.After 24 hours, per sunshine, embryo 2 times, observed to 168 hours, and Embryo Gallus domesticus is all survived, and was judged to inactivation completely.Within 24～168 hours, having dead germ to take out in time, gather in the crops idiosome, in blind passage 1 generation, as without chicken embryo death, being judged to inactivation completely.It is ratio addition gelatin protective agent, lyophilizing in checking qualified supernatant of 4:1 by volume, is freeze-dried antigen.
Stability: antigen 2～8 DEG C is deposited 24 months, HA titer is still not less than the operation art formula of 1:128, HA test and sees note 1.
Specificity: duck tembusu virus positive serum be should be positive reaction (HI titer >=1:10) by antigen, all should be negative reaction (HI titer 1:10) to duck tembusu virus negative serum, duck plague virus positive serum, DHV positive serum, newcastle, bird flu virus H5, H9 hypotype positive serum.The operation art formula of HI test is shown in note 2.
Sensitivity: can detect that vaccine immunity 2～3 weeks and the wild virus infection HI antibody in duck, goose serum after 4～5 days.
Embodiment 7
Having optimization on the basis of embodiment 1 further, material therefor reagent is identical with embodiment 1.
Duck tembusu virus HB strain (F3 generation) is as seed culture of viruses, with every 25 μ l/ only (containing 100ELD50) dosage intracranial inoculation 2～4 age in days C3H/He kind neonatal rat.Take out the obvious Mus of clinical symptoms such as spirit is depressed, tremble in time, put less than-20 DEG C preservations.Neonatal rat is taken out, uses iodine tincture disinfection brain, aseptically remove brain skin and skull, take out cerebral tissue, put in sterilization container.Final concentration of 8.5% aseptic sucrose solution, homogenate is added according to the ratio of W/V=1:4.Under agitated conditions, homogenate is added dropwise in the cold acetone of 10～30 times of volumes of homogenate amount.500g is centrifuged 5min, abandoning supernatant, is shredded with aseptic scissors by precipitate, suspends with the cold acetone of amount same as described above, ice bath 1 hour, and precipitate extruding is ground by period with sterile glass rod.500g is centrifuged 5min, abandoning supernatant.Collecting in sterile test tube by the precipitate containing a small amount of acetone, 500g is centrifuged 5min, abandoning supernatant, is divided in drying tube by precipitate ,-70 DEG C of freezings 1 hour, vacuum lyophilization 2～3 hours.According to the ratio of former homogenate amount V/V=1/1, add physiological saline solution, dissolve dry precipitate 12～24 hours.Centrifugal 1 hour of 8000g, the formaldehyde taking supernatant addition final concentration of 0.03% inactivates.Take 6 age in days SPF Embryo Gallus domesticus 10 pieces, through yolk sac inoculation supernatant, every embryo 0.2ml, put 36～37 DEG C and continue to hatch.24 hours photograph embryos, Embryo Gallus domesticus dead in 24 hours is judged to nonspecific death, and Embryo Gallus domesticus nonspecific death should less than 2 pieces.After 24 hours, per sunshine, embryo 2 times, observed to 168 hours, and Embryo Gallus domesticus is all survived, and was judged to inactivation completely.Within 24～168 hours, having dead germ to take out in time, gather in the crops idiosome, in blind passage 1 generation, as without chicken embryo death, being judged to inactivation completely.It is ratio addition sterile glycerol in checking qualified supernatant of 3:1 by volume, after mixing, is liquid stable antigen.
Stability: antigen 2～8 DEG C is deposited 24 months, HA titer is still not less than the operation art formula of 1:128, HA test and sees note 1.
Specificity: duck tembusu virus positive serum be should be positive reaction (HI titer >=1:10) by antigen, all should be negative reaction (HI titer 1:10) to duck tembusu virus negative serum, duck plague virus positive serum, DHV positive serum, newcastle, bird flu virus H5, H9 hypotype positive serum.The operation art formula of HI test is shown in note 2.
Sensitivity: can detect that vaccine immunity 2 weeks and the wild virus infection HI antibody in duck, goose serum after 4～5 days.
Embodiment 8
Having optimization on the basis of embodiment 1 further, material therefor reagent is identical with embodiment 1.
Duck tembusu virus HB strain (F3 generation) is as seed culture of viruses, with every 25 μ l/ only (containing 100ELD50) dosage intracranial inoculation 2～4 age in days BALB/c kind neonatal rat.Take out the obvious Mus of clinical symptoms such as spirit is depressed, tremble in time, put less than-20 DEG C preservations.Neonatal rat is taken out, uses iodine tincture disinfection brain, aseptically remove brain skin and skull, take out cerebral tissue, put in sterilization container.Final concentration of 8.5% aseptic sucrose solution, homogenate is added according to the ratio of W/V=1:4.Under agitated conditions, homogenate is added dropwise in the cold acetone of 10～30 times of volumes of homogenate amount.500g is centrifuged 5min, abandoning supernatant, is shredded with aseptic scissors by precipitate, suspends with the cold acetone of amount same as described above, ice bath 1 hour, and precipitate extruding is ground by period with sterile glass rod.500g is centrifuged 5min, abandoning supernatant.Collecting in sterile test tube by the precipitate containing a small amount of acetone, 500g is centrifuged 5min, abandoning supernatant, is divided in drying tube by precipitate ,-70 DEG C of freezings 1 hour, vacuum lyophilization 2～3 hours.According to the ratio of former homogenate amount V/V=1/1, add physiological saline solution, dissolve dry precipitate 12～24 hours.Centrifugal 1 hour of 8000g, the formaldehyde taking supernatant addition final concentration of 0.03% inactivates.Take 6 age in days SPF Embryo Gallus domesticus 10 pieces, through yolk sac inoculation supernatant, every embryo 0.2ml, put 36～37 DEG C and continue to hatch.24 hours photograph embryos, Embryo Gallus domesticus dead in 24 hours is judged to nonspecific death, and Embryo Gallus domesticus nonspecific death should less than 2 pieces.After 24 hours, per sunshine, embryo 2 times, observed to 168 hours, and Embryo Gallus domesticus is all survived, and was judged to inactivation completely.Within 24～168 hours, having dead germ to take out in time, gather in the crops idiosome, in blind passage 1 generation, as without chicken embryo death, being judged to inactivation completely.It is ratio addition sterilizing gelatin protective agent, lyophilizing in checking qualified supernatant of 3:1 by volume, is freeze-dried antigen.
Stability: antigen 2～8 DEG C is deposited 24 months, HA titer is still not less than the operation art formula of 1:128, HA test and sees note 1.
Specificity: duck tembusu virus positive serum be should be positive reaction (HI titer >=1:10) by antigen, all should be negative reaction (HI titer 1:10) to duck tembusu virus negative serum, duck plague virus positive serum, DHV positive serum, newcastle, bird flu virus H5, H9 hypotype positive serum.The operation art formula of HI test is shown in note 2.
Sensitivity: can detect that vaccine immunity 2～3 weeks and the wild virus infection HI antibody in duck, goose serum after 4～5 days.
Embodiment 9
Having optimization on the basis of embodiment 2 further, material therefor reagent is identical with embodiment 2.
Duck tembusu virus HB strain is as seed culture of viruses, and inoculation C6/36 cell monolayer after 1000 times of dilutions, in 50ml Tissue Culture Flask, inoculation 0.3ml, adds maintenance medium 7ml, continue at 28 DEG C of quiescent culture after inoculation.Every day 2 observation of cell pathological changes (CytopathicEffect, CPE), when there is the CPE of 50% in cell, harvesting and supernatant.Supernatant is centrifuged 30 minutes with the rotating speed of 3000 revs/min, draws supernatant in bag filter, concentrate 5～10 times with the Polyethylene Glycol (PEG) of molecular weight 10000.Concentrated solution adds the formaldehyde of final concentration of 0.05% inactivate.Take 6 age in days SPF Embryo Gallus domesticus 10 pieces, through yolk sac inoculation supernatant, every embryo 0.2ml, put 36～37 DEG C and continue to hatch.24 hours photograph embryos, Embryo Gallus domesticus dead in 24 hours is judged to nonspecific death, and Embryo Gallus domesticus nonspecific death should less than 2 pieces.After 24 hours, per sunshine, embryo 2 times, observed to 168 hours, and Embryo Gallus domesticus is all survived, and was judged to inactivation completely.Within 24～168 hours, having dead germ to take out in time, gather in the crops idiosome, in blind passage 1 generation, as without chicken embryo death, being judged to inactivation completely.It is ratio addition sterile glycerol in checking qualified supernatant of 3:1 by volume, after mixing, is liquid stable antigen.
Stability: antigen 2～8 DEG C is deposited 24 months, HA titer is still not less than the operation art formula of 1:128, HA test and sees note 1.
Specificity: duck tembusu virus positive serum be should be positive reaction (HI titer >=1:10) by antigen, all should be negative reaction (HI titer 1:10) to duck tembusu virus negative serum, duck plague virus positive serum, DHV positive serum, newcastle, bird flu virus H5, H9 hypotype positive serum.The operation art formula of HI test is shown in note 2.
Sensitivity: can detect that vaccine immunity 2～3 weeks and the wild virus infection HI antibody in duck, goose serum after 5～6 days.
Embodiment 10
Having optimization on the basis of embodiment 2 further, material therefor reagent is identical with embodiment 2.
Duck tembusu virus HB strain is as seed culture of viruses, and inoculation C6/36 cell monolayer after 1000 times of dilutions, in 100ml Tissue Culture Flask, inoculation 0.3ml, adds maintenance medium 10ml, continue at 33 DEG C of quiescent culture after inoculation.Every day 2 observation of cell pathological changes (CytopathicEffect, CPE), when there is the CPE of 50% in cell, harvesting and supernatant.Supernatant is centrifuged 30 minutes with the rotating speed of 3000 revs/min, draws supernatant in bag filter, concentrate 5～10 times with the Polyethylene Glycol (PEG) of molecular weight 10000.Concentrated solution adds the formaldehyde of final concentration of 0.05% inactivate.Take 6 age in days SPF Embryo Gallus domesticus 10 pieces, through yolk sac inoculation supernatant, every embryo 0.2ml, put 36～37 DEG C and continue to hatch.24 hours photograph embryos, Embryo Gallus domesticus dead in 24 hours is judged to nonspecific death, and Embryo Gallus domesticus nonspecific death should less than 2 pieces.After 24 hours, per sunshine, embryo 2 times, observed to 168 hours, and Embryo Gallus domesticus is all survived, and was judged to inactivation completely.Within 24～168 hours, having dead germ to take out in time, gather in the crops idiosome, in blind passage 1 generation, as without chicken embryo death, being judged to inactivation completely.It is ratio addition sterilizing gelatin protective agent, lyophilizing in checking qualified supernatant of 3:1 by volume, is freeze-dried antigen.
Stability: antigen 2～8 DEG C is deposited 24 months, HA titer is still not less than the operation art formula of 1:128, HA test and sees note 1.
Specificity: duck tembusu virus positive serum be should be positive reaction (HI titer >=1:10) by antigen, all should be negative reaction (HI titer 1:10) to duck tembusu virus negative serum, duck plague virus positive serum, DHV positive serum, newcastle, bird flu virus H5, H9 hypotype positive serum.The operation art formula of HI test is shown in note 2.
Sensitivity: can detect that vaccine immunity 2～3 weeks and the wild virus infection HI antibody in duck, goose serum after 5～6 days.
Note
1.HA titration operation art formula adopts the 96 U-shaped micro plates in hole to test, and reaction cumulative volume is μ l.By table 1 with antigen specific diluent by 2 times of serial dilutions of antigen, be subsequently adding the goose erythrocyte (preparation method is shown in note 3) of 0.33%, gently mixing after put into wet box mid-37 DEG C 60 minutes, observe agglutination.
Table 1HA titration operation art formula
Result decision method: blood-coagulation-board is tilted 45° angle 20～30 seconds.The erythrocyte of 100% is judged to " ++++" by virus institute's coagulation (not trickling), and the most high dilution of " +++～++++" is as judging terminal occurring.
2.HI titration operation art formula
The preparation of 2.14 HA working unit antigen liquids: the measurement result according to agglutination test, with the antigen of 4 work units of antigen specific diluent preparing of sterilizing.Hemagglutinative titer is divided by the pre-dilution multiple that 8 is antigen.Being exemplified below: the HA titer of antigen is 128, extension rate is 128/8=16,16 times of dilutions, and namely the antigen liquid of 1ml joins and is diluted in the antigen specific diluent of 15ml.Antigen has been prepared and should have been re-started demarcation.
The operational approach of 2.2HI test: apply the 96 U-shaped micro plates in hole and test, the reaction cumulative volume of hemagglutination test is 100 μ l, after utilizing antigen specific diluent that testing sample (serum pre-treating method to be checked is shown in note 4) is done continuous doubling dilution, add 4 HA unit antigens, put in wet box, 2～8 DEG C overnight, add the goose erythrocyte of 0.33%, gently mixing after put into wet box mid-37 DEG C 60 minutes, observe agglutination, test sets positive and negative serum control and erythrocyte comparison, and concrete operation method is in Table 2.
Table 2HI test operation art formula
Result decision method: occur that obvious hemagglutination inhibition starts to judge with positive control serum, when the HI titer of comparison positive and negative serum differs less than 1 titre with known titer, 1 unit antigen occurs +++～++++cohesion, result of the test is set up.Blood-coagulation-board is tilted 45° angle, and the most high dilution of hemagglutination activity completely or largely repressed serum to there are 4 working unit antigens is as judging terminal.HI antibody titer >=the 1:20 of serum to be checked is judged to the positive.
3.0.33% the preparation of goose hematid suspension
Gather the blood of 2～4 12～24 monthly age duck tembusu virus disease negative antibody geese, mix with equivalent A Shi liquid, then with 0.01M, pH7.2～7.6PBS washs 3 times, it is centrifuged 15 minutes with 1500rpm, the erythrocyte VAD of deposition is configured to 0.33% erythrocyte (V/V) suspension, put 4～8 DEG C standby.
4. serum processes operational approach
The serum to be checked 56～60 DEG C of above-mentioned process is inactivated 30 minutes by 4.1.
4.2 serum 100 μ l+400 μ l25% kaolin.
4.3 acutely shake serum/kaolin mixture, and every 5min shakes once, totally 20 ± 5min.
4.41000g is centrifuged, 20min.
4.5 add 100 μ l10% goose hematids.
4.6 are shaken gently for the supernatant every 5min, make erythrocyte keep suspended state.
4.71500rpm is centrifuged 20 ± 5min.Erythrocyte can be deposited on kaolin upper strata.
Supernatant is moved in new pipe by 4.8, and this liquid is considered as the serum to be checked of 1:10 dilution.
The preparation of 5.25% Poldeman Suspension (KaolinSuspension)
Kaolin 25g
PBS100ml
6. antigen specific diluent
1.5M sodium chloride solution: NaCl87.7g, adds distilled water to dissolving, total amount 1000ml；
0.5M boric acid solution: H3BO330.92g, adds distilled water and heats to dissolving, adjusts total amount to 700ml after cooling；
1N sodium hydroxide solution: NaOH40.0g, distilled water extremely dissolves, total amount 1000ml；
PH9.0 boric acid sodium chloride solution: 1.5M sodium chloride solution 80ml, 0.5M boric acid solution 100ml, 1N sodium hydroxide solution 24ml, adds distilled water to 1000ml；
4% bovine serum albumin solution: bovine serum albumin V4.0g, pH9.0 boric acid sodium chloride solution 90ml, corrects pH to 9.0 with 1N sodium hydroxide solution and pH9.0 boric acid sodium chloride solution, and termination capacity is 100ml；
Antigenic dilution: 0.1% bovine serum albumin boric acid sodium chloride solution (BABS): 4% bovine serum albumin solution 2.5ml, pH9.0 boric acid sodium chloride solution 97.5ml.
7.VAD
1.5M sodium chloride solution: NaCl87.7g, adds distilled water to dissolving, total amount 1000ml；
0.5M disodium phosphate soln: Na2HPO4 12H2O17.66g, adds distilled water to dissolving, total amount 100ml；
1.0M sodium dihydrogen phosphate: NaH2PO4 2H2O156g, adds distilled water to dissolving, total amount 1000ml；
Take 1.5M sodium chloride solution 100ml, 0.5M disodium phosphate soln 62ml, 1.0M sodium dihydrogen phosphate 160ml, add distilled water to 1000ml, be configured to VAD solution.After this solution mixes with equivalent pH9.0BABS, pH value is 6.2.

Claims (10)

1. the hemagglutination inhibition test antigen that one kind is prepared by duck tembusu virus.
2. antigen according to claim 1, it is characterised in that: duck hemorrhagic ovaritis virus HB strain, preserving number is CCTCCV201122, is deposited in China typical culture collection on July 1st, 2011.
3. use medicine, reagent and test kit prepared by antigen described in claim 1 or 2.
4. the preparation method of antigen described in claim 1, it is characterised in that: described preparation method includes following preparation process: 1) preparing duck blastema plinth seed culture of viruses after duck embryonic breeding kind duck tembusu virus, duck embryo went down to posterity less than 3 generations；2) duck blastema plinth seed culture of viruses inoculation neonatal rat being taken Mus brain and prepare HB strain seed culture of viruses, Mus brain went down to posterity less than 3 generations；3) HB strain virus is inoculated 2～4 age in days neonatal rats or C6/36 cell obtains virus liquid；4) inactivation；5) protective agent is added.
5. the preparation method of antigen according to claim 4, it is characterised in that: step 1) concrete operation method be: the allantoic fluid of dead duck embryo and idiosome after taking inoculation duck tembusu virus, smash to pieces, dilution, freeze thawing, centrifugal, take supernatant, measure median lethal dose(LD 50) ELD50, and carry out steriling test, viral level is not less than 106.0ELD50/ 0.1ml and the qualified seed culture of viruses of steriling test are as duck blastema plinth seed culture of viruses.
6. the preparation method of antigen according to claim 4, it is characterised in that: step 2) concrete operation method be: utilize duck blastema plinth seed culture of viruses, with 100ELD50The dosage of/0.1ml inoculates 2～4 age in days neonatal rats through brain, gathers in the crops spiritual depressed and the obvious Mus brain of clinical symptoms that trembles, grinds, dilution, and freeze thawing is centrifuged, and takes supernatant, mensuration median lethal dose(LD 50) ELD50, and carry out steriling test, median lethal dose(LD 50) is not less than 106.0ELD50HB strain seed culture of viruses based on/0.1ml and the qualified seed culture of viruses of steriling test.
7. the preparation method of antigen according to claim 4, it is characterized in that: step 3) HB strain seed culture of viruses inoculation 2～4 age in days neonatal rats, take out the obvious murine brains of clinical symptoms such as spirit is depressed, tremble, removing non-specific blood clotting mortifier and obtain virus liquid, described virus liquid viral level is not less than 106.0ELD50/0.1ml。
8. the preparation method of antigen according to claim 7, it is characterized in that: the method removing non-specific blood clotting inhibitor thing is: cerebral tissue adds the aseptic sucrose of final concentration of 8.5% after melting, homogenate, under agitated conditions, homogenate is added dropwise in acetone, centrifugal segregation supernatant, collection pellet frozen dries, add the precipitation after physiological saline solution dissolves lyophilization, centrifugal 30～60 minutes of 5000～10000g, collect supernatant.
9. the preparation method of antigen according to claim 4, it is characterised in that: step 3) in HB strain seed culture of viruses inoculation C6/36 cell line, collect supernatant concentration when 75%CPE occurs in cell and obtain virus liquid, described virus liquid viral level is not less than 106.0TCID50/0.1ml.Centrifugal 20 minutes of 2000g, collects supernatant, 2～8 DEG C of preservations.
10. the preparation method of antigen according to claim 4, it is characterized in that: step 4) concrete operations be: in above-mentioned virus liquid add formalin, inactivateing 2～3 days, described formalin concentration is 37～40%, and adding concentration of formaldehyde is 0.02%～0.2%；Step 5) concrete operations be: add gelatin freeze drying protectant or glycerol, prepare gelation dry solid-state antigen or liquid stable antigen.
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