CN102304494B - Duck hemorrhagic ovaritis virus strain, inactivated vaccine and preparation method thereof - Google Patents
Duck hemorrhagic ovaritis virus strain, inactivated vaccine and preparation method thereof Download PDFInfo
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- CN102304494B CN102304494B CN 201110255115 CN201110255115A CN102304494B CN 102304494 B CN102304494 B CN 102304494B CN 201110255115 CN201110255115 CN 201110255115 CN 201110255115 A CN201110255115 A CN 201110255115A CN 102304494 B CN102304494 B CN 102304494B
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Abstract
The invention discloses a duck hemorrhagic ovaritis virus strain, an inactivated vaccine and a preparation method thereof. The duck hemorrhagic ovaritis virus is named duck hemorrhagic ovaritis flavivirus HB strain, and the collection number of the duck hemorrhagic ovaritis flavivirus HB strain is CCTCC V201122. The virus is ssRNA virus with cyst membrane; virions are basically spherical, and the diameter is 40 to 60 nanometers; the virus is sensitive to ether and chloroform; most detergents can quickly inactivate the virus; the virus can also be inactivated when fumigated in 0.1-percent formalin for 6 hours at 37 DEG C; and the duck hemorrhagic ovaritis virus cannot agglutinate red blood cells of chickens, ducks and turkeys, but can agglutinate 1-percent red blood cells of doves. The inactivated vaccine is prepared by inoculating the duck hemorrhagic ovaritis virus, harvesting, inactivating, concentrating and emulsifying. The vaccine has the advantages of high safety, quick immune response, long immune duration and the like.
Description
Technical field
The present invention relates to a kind of virus stain and inactivated vaccine, relate in particular to a kind of duck hemorrhagic ovaritis virus strain, inactivated vaccine and preparation method thereof, belong to technical field of bioengineering.
Background technology
Duck hemorrhagic ovaritis (Duck Hemorrhagic, DH) be recently popular, acute, the height contact a kind of duck transmissible disease, its cause of disease is duck hemorrhagic ovaritis virus (Duck Hemorrhagic Ovaritis Virus, DHOV), belong to the member of flaviviridae at virus taxis, infect the different days duck, cause the duck ovarian hemorrhage, egg drop reduction, ovary, testis, uterine tube and atrophy of vas deferens, the Commercial meat-type duck death rate increases.
From in June, 2010, since the area such as this interior syndrome state Guangdong, Guangxi, Fujian, Jiangxi, Zhejiang, Anhui, Jiangsu, Shandong, Hebei and Beijing, duck hemorrhagic ovaritis has become one of main epidemic disease of the foster duck industry of recent harm China.This virus main harm egg duck (Shaoxing duck, Jinyun Domestic Duck, mountain sheldrake, gold are decided duck, Kang Beier duck, Taiwan White and changed duck etc.), meat kind duck (cherry valley duck, Beijing duck etc.), wild duck kind duck.Almost 100% morbidity of duck group that is contaminted, mortality ratio is 5%～30% not wait.Disease latent period is short, and morbidity is anxious, can cause mine massively appetite and egg productivity of duck sharply to descend duck group even total crop failure behind the virus infection duck group in 3～5 days.About 200 age in days duck groups' egg productivity returns to premorbid level needs 30 days or longer time, and the large age in days duck mass-sending of part is eliminated owing to production performance descends after being ill.Planting the rear fertility rate and hatchability of planting egg of duck morbidity reduces.Death rate obviously increased after Commercial meat-type duck infected.Therefore, this disease provisions duck already brings huge financial loss, and this is so that support the duck industry and once be driven to the last ditch.
In the research of virus taxis status, the people such as Cao Zhenzhen, Zhang Cun is by viral and Flavivirus ntaya virus group (the Ntay a virus group to duck hemorrhagic ovaritis, NTAV group) and encephalitis b virus group (Japanese encephalitis virus group, JEV group) selects the known virus of NS1 sequence to carry out sequence homology analysis and evolutionary analysis in, find that the genetic distance of the bagaza virus of this virus and flaviviridae Flavivirus is positioned at the level of viral taxonomy kind.The people's such as Su Jingliang, Wan Chun and, Shi Shaohua research also shows and separates causing of obtaining the lay eggs homology of the nucleotide sequence 9-777 position of rapid drawdown new virus and tembusu virus (TMUV) of kind of (egg) duck is the highest, is 88.7%; Phylogenetic analysis shows that the WR strain virus has similar ancestors to tembusu virus, Saint Louis' encephalitis virus (SLEV), Israel's turkey meningitis virus (ITV), ntaya virus (NTAV).
Aspect diagnosis, Cao Zhenzhen, Wan Chun with, people's report can the adopt clinical symptom such as Su Jingliang, Teng Qiao be vast, substantially reach histopathology variation, pcr amplification and virus and the method such as separate and make a definite diagnosis this disease.But owing to do not find better vaccine strain, laboratory host, vaccine evaluation model extensive and high-titer cultivation virus not yet to set up, restricting the at present exploitation of vaccine at present.
Therefore, support at present the duck hemorrhagic ovaritis vaccine that duck is already needed a kind of good immune effect badly.
Summary of the invention
One of the object of the invention provides a kind of duck hemorrhagic ovaritis virus strain, in order to be that the duck hemorrhagic ovaritis vaccine of developing good immune effect lays the foundation;
Two of the object of the invention provides a kind of duck hemorrhagic ovaritis inactivated vaccine;
Three of the object of the invention provides the preparation method of described duck hemorrhagic ovaritis inactivated vaccine.
Virus stain called after Flavivirus duck hemorrhagic ovaritis virus HB of the present invention strain is adopted ordinary method to inoculate responsive duck embryo and is separated acquisition, and preserving number is CCTCC V201122; Described duck hemorrhagic ovaritis virus is ssRNA virus, and cyst membrane is arranged; It is spherical that virion is substantially, diameter 40～60nm; The described virus to external world resistibility of environment is stronger, deposits several weeks in 4 ℃, deposits some months in-20 ℃, and its infectivity is all unaffected; Described virus is responsive to ether, chloroform; Most of stain removers can be with its rapid deactivation; Under 37 ℃ of conditions, stifling 6h just can be its deactivation with 0.1% formalin; Described virus can not the aggegation chicken, the red corpuscle of duck, turkey, can aggegation 1% dove red blood corpuscle; Described virus can be at 9～13 age in days duck embryo allantoic cavities, embryo chorioallantoic membrane, 9～10 age in days chick embryo allantoic cavities, embryo chorioallantoic membrane, 6 age in days chick embryo yolk sacs are cultivated in 7～8 age in days duck embryo yolk sac, also can cultivate at DEF, BHK21 cell and Vero cell.
A kind of duck hemorrhagic ovaritis inactivated vaccine, described inactivated vaccine is got through inoculation, results, deactivation, concentrated and emulsification by duck hemorrhagic ovaritis virus, described duck hemorrhagic ovaritis virus strain is Flavivirus duck hemorrhagic ovaritis virus HB strain, and preserving number is CCTCC V201122.
A kind of method for preparing above-mentioned duck hemorrhagic ovaritis inactivated vaccine, it in responsive duck embryo, gathers in the crops toxic duck embryo tissue juice with duck hemorrhagic ovaritis virus inoculation, makes through formaldehyde solution deactivation, concentrated and emulsification; Concrete steps are as follows:
A. the inoculation: with duck hemorrhagic ovaritis virus through the allantoic cavity approach according to 10
2～10
4ELD
50/ piece dose inoculation in the responsive duck embryo of 10～13 ages in days, hatch for 37 ℃;
B. gather in the crops: death in 48～120 hours and the obvious duck embryo of lesion idiosome, chorioallantoic membrane and allantoic fluid after the results inoculation, 20000r/min smashs 2min to pieces, and multigelation 2 times is stored in-20 ℃ and is duck embryo tissue juice; According to medium lethal dose, the median infective dose measuring method of " People's Republic of China's veterinary drug allusion quotation " (three ones of two 〇, one 〇 versions) appendix 7 regulations, carry out viral level and measure, virus of proliferation is tired and is answered 〉=10
6.0ELD
50/ 0.1ml;
C. deactivation: add mass concentration in the duck embryo tissue juice and be 37% formalin and make its final content reach 0.08%～0.2%, shake up, 37 ℃ of deactivations 16～36 hours or 4 ℃ of deactivations 5～7 days;
D. emulsification:
Water preparation: above-mentioned inactivation of viruses tissue juice is adopted the centrifugal 15min of 3500r/min, abandons sediment, supernatant liquor dilutes 1～3 times with the PBS of aseptic 0.1M pH7.2, be concentrated into original volume with 50KD film bag again, 2～4 times so repeatedly, the duck embryo tissue juice residual formaldehyde of processing deactivation is not more than 0.1%, and total protein content is not more than 400 μ g/ml, adds aseptic tween-80 by (V/V) 4%～6%, stir, be water;
Oil phase preparation: in the import white oil, add aluminum stearate by (W/V) 0.8%～2.0%, add Si Ben-80 by (V/V) 4%, be oil phase after the sterilization;
With water and oil phase in 1:1.67～1:3 ratio mixing and emulsifying, duck hemorrhagic ovaritis inactivated vaccine, viral level is not less than 10
6.0ELD
50/ plumage part.
The method of the above-mentioned duck hemorrhagic ovaritis inactivated vaccine of another kind of preparation provided by the invention is:
In DEF, results contain poison cell with duck hemorrhagic ovaritis virus inoculation for it, make through formaldehyde solution deactivation, concentrated and emulsification; Specifically carry out as follows:
A. inoculation: duck hemorrhagic ovaritis virocyte is adapted to malicious ratio in 0.02%～1% be inoculated in the DEF individual layer;
B. gather in the crops: inoculation generation in rear 72～96 hours cytopathy (CPE) harvested cell when reaching 80%～90%, and the aseptic beaker that will fill harvested cell precipitation is placed in 0 ℃ of ice bath with cracking 30s/ time under 85% power condition of lysis instrument, cracking 3 times ,-20 ℃ of frozen virocyte lysates that are; Carry out viral level according to " People's Republic of China's veterinary drug allusion quotation " (three ones of two 〇, one 〇 versions) appendix 7 medium lethal doses, median infective dose measuring method and measure, virus of proliferation is tired and is answered 〉=10
6.0TCID
50/ 0.1ml;
C. deactivation: add mass concentration in the above-mentioned virocyte lysate and be 37% formalin and make its final content reach 0.05%～0.12%, shake up, 37 ℃ of deactivations 16～36 hours or 4 ℃ of deactivations 5～7 days;
D emulsification:
Water preparation: above-mentioned inactivation of viruses tissue juice is adopted the centrifugal 15min of 3500r/min, abandon sediment, supernatant liquor dilutes 1 times with the PBS of aseptic 0.1M pH7.2, be concentrated into original volume with 50KD film bag again, 2～4 times so repeatedly, the cell culture fluid residual formaldehyde of deactivation is not more than 0.1%, and total protein content is not more than 400 μ g/ml; And press V/V adding 4%～6% aseptic tween-80, and stir, be water;
The oil phase preparation: adding aluminum stearate by (W/V) 0.8% ~ 2.0% in the import white oil, add Si Ben-80 by (V/V) 4%, is oil phase after the sterilization;
With water and oil phase in 1:1.67～1:3 ratio mixing and emulsifying, duck hemorrhagic ovaritis deactivation vaccine, detect its viral level and be not less than 10
6.0TCID
50/ plumage part.
Vaccine of the present invention has following characteristics:
Described vaccine adopts the import white oil to prepare as oily adjuvant, and little, the good stability of viscosity was preserved 3 months at 37 ℃, deposited 12 months for 4 ℃, and it is unusual layering, variable color etc. not to occur;
Safe good perfection adopts 2 times of using dosage immune ducks, observes for 2 weeks, and the mental status, food consumption and egg duck egg productivity etc. are showed no any unusual;
Immunne response produces rapidly, and immune duration is long, with its immune duck, produces immunne response in 10 days, and 2 can produce stronger immunizing power in all, and duration of immunity at least 5 months is attacked malicious protection ratio 〉=80%;
The unusual clinical manifestation such as significantly relevant with this disease food consumption and egg drop reduction does not occur in the laying ducks field of using this vaccine, and the Commercial meat-type duck death rate obviously descends, and butchers rear superfine rate and improves;
Result of study shows that vaccine of the present invention is stable, safety, effective.For the control of this disease provides a kind of practicable inactivated vaccine.
Embodiment
Be described further below in conjunction with embodiment, these examples only are used for explanation, and are not used in the protection domain of restriction this patent.
Virus stain called after Flavivirus duck hemorrhagic ovary illness in eye poison HB of the present invention strain was deposited in Chinese Typical Representative culture collection center (being called for short CCTCC) on 07 01st, 2011, preserving number is CCTCC V201122; Described virus is ssRNA virus, and cyst membrane is arranged; It is spherical that virion is substantially, diameter 40～60nm; The described virus to external world resistibility of environment is stronger, deposits several weeks in 4 ℃, deposits some months in-20 ℃, and its infectivity is all unaffected; Described virus is responsive to ether, chloroform; Most of stain removers can be with its rapid deactivation; Under 37 ℃ of conditions, stifling 6h just can be its deactivation with 0.1% formalin; Described virus can not the aggegation chicken, the red corpuscle of duck, turkey, can aggegation 1% dove red blood corpuscle; Described virus can be at 9～13 age in days duck embryo allantoic cavities, embryo chorioallantoic membrane, 9～10 age in days chick embryo allantoic cavities, embryo chorioallantoic membrane, 6 age in days chick embryo yolk sacs are cultivated in 7～8 age in days duck embryo yolk sac, also can cultivate at DEF, BHK21 cell and Vero cell.
Embodiment 1
A. the inoculation: with duck hemorrhagic ovaritis virus through the allantoic cavity approach according to 10
3ELD
50/ piece dose inoculation in the responsive duck embryo of 10 ages in days, hatch for 37 ℃;
B. gather in the crops: death in 48～120 hours and the obvious duck embryo of lesion idiosome, chorioallantoic membrane and allantoic fluid after the results inoculation, 20000r/min smashs 2min to pieces, and multigelation 2 times is stored in-20 ℃ and is duck embryo tissue juice; According to medium lethal dose, the median infective dose measuring method of " People's Republic of China's veterinary drug allusion quotation " (three ones of two 〇, one 〇 versions) appendix 7 regulations, carry out viral level and measure, it is 10 that virus of proliferation is tired
7.1ELD
50/ 0.1ml;
C. deactivation: adding mass concentration in the duck embryo tissue juice and be 37% formalin and making its final content is 0.2%, shakes up rear 37 ℃ of deactivations 16 hours;
D. emulsification:
Water preparation: adopt the centrifugal 15min of 3500r/min to abandon sediment in above-mentioned deactivation duck embryo tissue juice, supernatant liquor dilutes 2 times with the PBS of aseptic 0.1M pH7.2, be concentrated into original volume with 50KD film bag again, 4 times so repeatedly, the duck embryo tissue juice residual formaldehyde of deactivation is 0.08%, and total protein content is 380 μ g/ml, and adds aseptic tween-80 by (V/V) 6%, stir, be water;
Oil phase preparation: in the import white oil, add aluminum stearate by (W/V) 0.8%, add tween-80 by (V/V) 4% again, be oil phase after the sterilization;
With water and oil phase in 1:3 ratio mixing and emulsifying, duck hemorrhagic ovaritis deactivation vaccine, viral level is 10
6.1ELD
50/ plumage part.
Described vaccine, has good stability for the water-in-oil type through property determination, and viscosity is little, and is safe and reliable.This vaccine is preserved at 4 ℃ and was not occurred layering in 12 months.
Safety verification: adopt 2 times of using dosage immune ducks, observed for 2 weeks, have no any unusual.
Efficacy test: the test duck inoculates behind this vaccine 10～14 days and produces immunizing power, and duration of immunity is 5 months, and the malicious protection ratio of attacking in the duration of immunity is 80%.
Embodiment 2
A. inoculation: duck hemorrhagic ovaritis virocyte is adapted to malicious ratio in 0.1% be inoculated in the DEF individual layer;
B. gather in the crops: inoculation generation in rear 72～96 hours cytopathy (CPE) harvested cell when reaching 80%～90%, and the aseptic beaker that will fill harvested cell precipitation is placed in 0 ℃ of ice bath with cracking 30s/ time under 85% power condition of lysis instrument, cracking 3 times ,-20 ℃ of frozen virocyte lysates that are; Medium lethal dose, median infective dose measuring method according to " People's Republic of China's veterinary drug allusion quotation " (three ones of two 〇, one 〇 versions) appendix 7 regulations carry out viral level mensuration, and it is 10 that virus of proliferation is tired
6.5TCID
50/ 0.1ml;
C. deactivation: add mass concentration in the virocyte lysate and be 37% formalin and make its final content reach 0.1%, shake up rear 4 ℃ of deactivations 7 days;
D emulsification:
Water preparation: adopt the centrifugal 15min of 3500r/min to abandon sediment above-mentioned inactivation of viruses cell pyrolysis liquid, supernatant liquor dilutes 1 times with the PBS of aseptic 0.1M pH7.2, be concentrated into original volume with 50KD film bag again, 2 times so repeatedly, the cell pyrolysis liquid residual formaldehyde of deactivation is 0.06%, and total protein content is 200 μ g/ml; And by the aseptic tween-80 of (V/V) 6% adding, stir, be water;
In the import white oil, add aluminum stearate by (W/V) 2.0%, add Si Ben-80 by (V/V) 4% again, be oil phase after the sterilization;
With water and oil phase in 1:1.67 ratio mixing and emulsifying, duck hemorrhagic ovaritis deactivation vaccine, viral level is not less than 10
6.0TCID
50/ plumage part.
This vaccine, has good stability for the water-in-oil type through property determination, and viscosity is little, and is safe and reliable.This vaccine is preserved at 4 ℃ and was not occurred layering in 12 months.
Safety verification: adopt 2 times of using dosage immune ducks, observed for 2 weeks, have no any unusual.
Efficacy test: the test duck inoculates behind this vaccine 10～14 days and produces immunizing power, and duration of immunity is 5 months, and the malicious protection ratio of attacking in the duration of immunity is 80%.
Claims (3)
1. duck hemorrhagic ovaritis virus strain, it is characterized in that, (Flaviviridae Flavivirus Duck Hemorrhagic Ovaritis Virus) the HB strain of described virus stain called after Flavivirus duck hemorrhagic ovaritis virus, preserving number is CCTCC V201122; Described virus is ssRNA virus, and cyst membrane is arranged; It is spherical that virion is substantially, diameter 40～60nm; The described virus to external world resistibility of environment is stronger, deposits several weeks in 4 ℃, deposits some months in-20 ℃, and its infectivity is all unaffected; Described virus is responsive to ether, chloroform; Most of stain removers can be with its rapid deactivation; Under 37 ℃ of conditions, stifling 6h just can be its deactivation with 0.1% formalin; Described virus can not the aggegation chicken, the red corpuscle of duck, turkey, can aggegation 1% dove red blood corpuscle; Described virus can be at 9～13 age in days duck embryo allantoic cavities, embryo chorioallantoic membrane, 9～10 age in days chick embryo allantoic cavities, embryo chorioallantoic membrane, and 6 age in days chick embryo yolk sacs are cultivated in 7～8 age in days duck embryo yolk sac; Perhaps cultivate at DEF, BHK21 cell and Vero cell.
2. duck hemorrhagic ovaritis inactivated vaccine, it is characterized in that, described inactivated vaccine is got through inoculation, results, deactivation, concentrated and emulsification by duck hemorrhagic ovaritis virus, described duck hemorrhagic ovaritis virus strain is Flavivirus duck hemorrhagic ovaritis virus HB strain, and preserving number is CCTCC V201122;
It is prepared by following steps:
A. the inoculation: with duck hemorrhagic ovaritis virus through the allantoic cavity approach according to 10
2～10
4ELD
50/ piece dose inoculation in the responsive duck embryo of 10～13 ages in days, hatch for 37 ℃;
B. gather in the crops: death in 48～120 hours and the obvious duck embryo of lesion idiosome, chorioallantoic membrane and allantoic fluid after the results inoculation, 20000r/min smashs 2min to pieces, and multigelation 2 times is stored in-20 ℃ and is duck embryo tissue juice; According to medium lethal dose, the median infective dose measuring method of three appendix of " People's Republic of China's veterinary drug allusion quotation " two 〇, one 〇 version 7 regulation, carry out viral level and measure, virus of proliferation tires 〉=and 10
6.0ELD
50/ 0.1ml;
C. deactivation: add mass concentration in the duck embryo tissue juice and be 37% formalin and make its final content reach 0.08%～0.2%, shake up, 37 ℃ of deactivations 16～36 hours or 4 ℃ of deactivations 5～7 days;
D. concentrated and emulsification:
Water preparation: above-mentioned deactivation duck embryo tissue juice is adopted the centrifugal 15min of 3500r/min, abandons sediment, supernatant liquor dilutes 1～3 times with aseptic 0.1M pH7.2 PBS, be concentrated into original volume with 50KD film bag again, 2～4 times so repeatedly, the duck embryo tissue juice residual formaldehyde of processing deactivation is not more than 0.1%, and total protein content is not more than 400 μ g/ml, presses V/V 4%～6% and adds aseptic tween-80, stir, be water;
Oil phase preparation: in the import white oil, press W/V 0.8%～2.0% and add aluminum stearate, press V/V 4% and add Si Ben-80, be oil phase after the sterilization;
With water and oil phase in 1:1.67～1:3 ratio mixing and emulsifying, duck hemorrhagic ovaritis inactivated vaccine, its viral level is not less than 10
6.0ELD
50/ plumage part.
3. duck hemorrhagic ovaritis inactivated vaccine, it is characterized in that, described inactivated vaccine is got through inoculation, results, deactivation, concentrated and emulsification by duck hemorrhagic ovaritis virus, described duck hemorrhagic ovaritis virus strain is Flavivirus duck hemorrhagic ovaritis virus HB strain, and preserving number is CCTCC V201122;
It is prepared by following steps:
A. inoculation: duck hemorrhagic ovaritis virocyte is adapted to malicious ratio in 0.02%～1% be inoculated in the DEF individual layer,
B. gather in the crops: harvested cell when the generation cytopathy reached 80%～90% in 72～96 hours after the inoculation, and the aseptic beaker that will fill harvested cell precipitation is placed in 0 ℃ of ice bath with cracking 30s/ time under 85% power condition of lysis instrument, cracking 3 times ,-20 ℃ of frozen virocyte lysates that are; Carrying out viral level according to medium lethal dose, the median infective dose measuring method of three appendix of " People's Republic of China's veterinary drug allusion quotation " two 〇, one 〇 version 7 regulation and measure, virus of proliferation tires 〉=and 10
6.0TCID
50/ 0.1ml;
C. deactivation: add mass concentration in the above-mentioned virocyte lysate and be 37% formalin and make its final content reach 0.05%～0.12%, shake up, 37 ℃ of deactivations 16～36 hours or 4 ℃ of deactivations 5～7 days; Concentrated and the emulsification of d: water preparation: above-mentioned inactivation of viruses tissue juice is adopted the centrifugal 15min of 3500r/min, abandon sediment, supernatant liquor dilutes 1 times with the PBS of aseptic 0.1M, pH 7.2, be concentrated into original volume with 50KD film bag again, 2～4 times so repeatedly, the cell culture fluid residual formaldehyde of deactivation is not more than 0.1%, and total protein content is not more than 400 μ g/ml; And press the aseptic tween-80 of V/V 4%～6% adding, and stir, be water; The oil phase preparation: pressing W/V 0.8% ~ 2.0% and add aluminum stearate in the import white oil, press V/V 4% and add Si Ben-80, is oil phase after the sterilization; With water and oil phase in 1:1.67～1:3 ratio mixing and emulsifying, duck hemorrhagic ovaritis deactivation vaccine, detect its viral level and be not less than 10
6.0TCID
50/ plumage part.
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| CN102488893B (en) * | 2011-12-28 | 2013-07-17 | 瑞普（保定）生物药业有限公司 | Duck hemorrhagic oophoritis inactivated vaccine production method by using cell line and product thereof |
| CN102586193B (en) * | 2012-02-10 | 2013-06-19 | 中国农业科学院上海兽医研究所 | Monoclonal antibody for resisting duck tembusu virus, hybridoma strain and application thereof |
| CN102749427B (en) * | 2012-06-15 | 2015-12-09 | 北京市农林科学院畜牧兽医研究所 | A kind of method for testing efficacy of duck hemorrhagic oaritis inactivated vaccine |
| CN102735808B (en) * | 2012-06-15 | 2015-07-08 | 北京市农林科学院畜牧兽医研究所 | Effect testing method for duck hemorrhagic ovaritis inactivated vaccine |
| CN104306966A (en) * | 2014-10-14 | 2015-01-28 | 龙明珠 | Duck plague tissue inactivated vaccine, preparation method and application |
| CN105807049B (en) * | 2016-02-22 | 2017-03-29 | 北京市农林科学院 | A kind of duck tembusu virus disease hemagglutination inhibition test antigen and preparation method thereof |
| CN110898218B (en) * | 2019-12-30 | 2023-10-10 | 瑞普（保定）生物药业有限公司 | Duck tembusu virus inactivated vaccine and preparation method thereof |
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