CN102304494A - 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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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 stain, 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), on virus taxis, belong to the member of flaviviridae, infect the different days duck; Cause the duck ovarian hemorrhage; Egg drop reduction, ovary, testis, uterine tube and atrophy of vas deferens, meat sold on the market 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 eqpidemic disease of the foster duck industry of recent harm China.This virus main harm egg duck (Shaoxing duck, Jinyun sheldrake, mountain sheldrake, gold are decided duck, Kang Beier duck, Taiwan and changed duck etc. in vain), meat kind duck (cherry valley duck, Beijing duck etc.), wild duck kind duck.Almost 100% morbidity of duck crowd 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 crowd even total crop failure behind the virus infection duck crowd in 3～5 days.About 200 age in days duck crowds' egg productivity returns to premorbid level to be needed 30 days or the longer time, and the big age in days duck mass-sending of part is eliminated owing to production performance descends after being ill.Plant duck morbidity back and plant the rate of fertilization and the hatching rate reduction of egg.The meat sold on the market duck infects the back death rate obviously to be increased.Therefore, this disease provisions duck already brings enormous economic loss, and this makes that supporting the duck industry once was driven to the last ditch.
In the research of virus taxis status; People such as Cao Zhenzhen, Zhang Cun is through viral and Flavivirus ntaya virus crowd (the Ntay a virus group to duck hemorrhagic ovaritis; NTAV crowd) and encephalitis b virus crowd (Japanese encephalitis virus group; JEV crowd) 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.People's such as Su Jingliang, Wan Chun and, Shi Shaohua research also shows and separates causing of obtaining the lay eggs homology of nucleotide sequence 9-777 position and tembusu virus (TMUV) of rapid drawdown new virus of kind of (egg) duck is the highest, is 88.7%; Genetic evolution analysis revealed WR strain virus has similar ancestors with tembusu virus, Saint Louis' encephalitis virus (SLEV), Israel's turkey meningitis virus (ITV), ntaya virus (NTAV).
Aspect diagnosis, people's report can adopt clinical symptom such as Cao Zhenzhen, Wan Chun and, Su Jingliang, Teng Qiao are vast, reach histopathology variation, pcr amplification and virus substantially and method such as separate and make a definite diagnosis this disease.But owing to do not find better vaccine strain, extensive and high laboratory host, the vaccine evaluation model of cultivating virus of tiring not to set up as yet, restricting developing vaccines at present at present.
Therefore, support the duck hemorrhagic ovaritis vaccine that duck is already needed a kind of good immune effect badly at present.
Summary of the invention
One of the object of the invention provides a kind of duck hemorrhagic ovaritis virus stain, so that 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 said duck hemorrhagic ovaritis inactivated vaccine.
Virus stain called after Flavivirus duck hemorrhagic ovaritis virus HB according to the invention strain is adopted ordinary method to inoculate responsive duck embryo and is separated acquisition, and preserving number is CCTCC V201122; Said duck hemorrhagic ovaritis virus is ssRNA virus, and cyst membrane is arranged; It is spherical that virion is substantially, diameter 40～60nm; The said virus resistibility of environment to external world is stronger, in 4 ℃, deposits several weeks, in-20 ℃, deposits some months, and its infectivity is all unaffected; Said 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; Said virus can not the aggegation chicken, the red corpuscle of duck, turkey, can aggegation 1% dove red blood corpuscle; Said 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 on DEF, BHK21 cell and Vero cell, cultivate.
A kind of duck hemorrhagic ovaritis inactivated vaccine; Said inactivated vaccine by duck hemorrhagic ovaritis virus through inoculation, results, deactivation, concentrate and emulsification and getting; Said duck hemorrhagic ovaritis virus stain 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, through the formaldehyde solution deactivation, concentrate and emulsification makes; Concrete steps are following:
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: the death in 48～120 hours of results inoculation back and the tangible duck embryo of lesion idiosome, chorioallantoic membrane and allantoic fluid, 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: in duck embryo tissue juice, add mass concentration 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 handling 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, add Si Ben-80, be oil phase after the sterilization by (V/V) 4% by (W/V) 0.8%～2.0%;
Water and oil phase are pressed 1:1.67～1:3 mixed emulsification, 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, through the formaldehyde solution deactivation, concentrate and emulsification makes; 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: the inoculation back produced cytopathy (CPE) in 72～96 hours and reaches 80%～90% o'clock harvested cell; And will fill the sedimentary aseptic beaker of harvested cell and be placed in 0 ℃ of ice bath with cracking 30s/ time under 85% power condition of lysis appearance; 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: in above-mentioned virocyte lysate, add mass concentration 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: in the import white oil, add aluminum stearate by (W/V) 0.8% ~ 2.0%, add Si Ben-80 by (V/V) 4%, the sterilization back is an oil phase;
Water and oil phase are pressed 1:1.67～1:3 mixed emulsification, duck hemorrhagic ovaritis deactivation vaccine, detect its viral level and be not less than 10
6.0TCID
50/ plumage part.
Vaccine according to the invention has following characteristics:
Said 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 not all seen 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%;
Unusual clinical manifestation such as significantly relevant with this disease food consumption and egg drop reduction does not take place in the laying ducks field of using this vaccine, and meat sold on the market duck death rate obviously descends, and butchers the superfine rate in back 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
Do further explanation below in conjunction with embodiment, these instances 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 according to the invention strain was deposited in Chinese typical culture collection center (being called for short CCTCC) on 07 01st, 2011, preserving number is CCTCC V201122; Said virus is ssRNA virus, and cyst membrane is arranged; It is spherical that virion is substantially, diameter 40～60nm; The said virus resistibility of environment to external world is stronger, in 4 ℃, deposits several weeks, in-20 ℃, deposits some months, and its infectivity is all unaffected; Said 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; Said virus can not the aggegation chicken, the red corpuscle of duck, turkey, can aggegation 1% dove red blood corpuscle; Said 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 on DEF, BHK21 cell and Vero cell, cultivate.
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: the death in 48～120 hours of results inoculation back and the tangible duck embryo of lesion idiosome, chorioallantoic membrane and allantoic fluid, 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: in duck embryo tissue juice, adding mass concentration and be 37% formalin and making its final content is 0.2%, shakes up back 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, add tween-80 by (V/V) 4% again, be oil phase after the sterilization by (W/V) 0.8%;
Water and oil phase are pressed the emulsification of 1:3 mixed, duck hemorrhagic ovaritis deactivation vaccine, viral level is 10
6.1ELD
50/ plumage part.
Said 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, do not see any unusual.
Efficacy test: produced immunizing power in 10～14 days behind this vaccine of test duck inoculation, 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: the inoculation back produced cytopathy (CPE) in 72～96 hours and reaches 80%～90% o'clock harvested cell; And will fill the sedimentary aseptic beaker of harvested cell and be placed in 0 ℃ of ice bath with cracking 30s/ time under 85% power condition of lysis appearance; 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: in the virocyte lysate, add mass concentration and be 37% formalin and make its final content reach 0.1%, shake up back 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, add Si Ben-80 by (V/V) 4% again, be oil phase after the sterilization by (W/V) 2.0%;
Water and oil phase are pressed the emulsification of 1:1.67 mixed, 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, do not see any unusual.
Efficacy test: produced immunizing power in 10～14 days behind this vaccine of test duck inoculation, duration of immunity is 5 months, and the malicious protection ratio of attacking in the duration of immunity is 80%.
Claims (4)
1. a duck hemorrhagic ovaritis virus stain is characterized in that, said virus stain called after Flavivirus duck hemorrhagic ovaritis virus HB strain, and preserving number is CCTCC V201122; Said virus is ssRNA virus, and cyst membrane is arranged; It is spherical that virion is substantially, diameter 40～60nm; The said virus resistibility of environment to external world is stronger, in 4 ℃, deposits several weeks, in-20 ℃, deposits some months, and its infectivity is all unaffected; Said 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; Said virus can not the aggegation chicken, the red corpuscle of duck, turkey, can aggegation 1% dove red blood corpuscle.
2. duck hemorrhagic ovaritis inactivated vaccine; It is characterized in that; Said inactivated vaccine by duck hemorrhagic ovaritis virus through inoculation, results, deactivation, concentrate and emulsification and getting; Said duck hemorrhagic ovaritis virus stain is Flavivirus duck hemorrhagic ovaritis virus HB strain, and preserving number is CCTCC NO:V201122.
One kind prepare as as described in the claim 2 the method for duck hemorrhagic ovaritis inactivated vaccine, it is characterized in that it in responsive duck embryo, gathers in the crops toxic duck embryo tissue juice with duck hemorrhagic ovaritis virus inoculation, through the formaldehyde solution deactivation, concentrate and emulsification makes; Concrete steps are following:
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: the death in 48～120 hours of results inoculation back and the tangible duck embryo of lesion idiosome, chorioallantoic membrane and allantoic fluid, 20000r/min smashs 2min to pieces, and multigelation 2 times is stored in-20 ℃ and is duck embryo tissue juice; Medium lethal dose, median infective dose measuring method according to People's Republic of China's veterinary drug allusion quotation, three appendix of two 〇, one 〇 version, 7 regulations carry out viral level and measure, and virus of proliferation is tired and answered>=10
6.0ELD
50/ 0.1ml;
C. deactivation: in duck embryo tissue juice, add mass concentration 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 deactivation duck embryo tissue juice is adopted the centrifugal 15min of 3500r/min, abandons sediment; Supernatant liquor is with 1～3 times of aseptic 0.1M pH7.2 PBS dilution; Be concentrated into original volume with 50KD film bag again; 2～4 times so repeatedly; The duck embryo tissue juice residual formaldehyde of handling 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, add Si Ben-80, be oil phase after the sterilization by (V/V) 4% by (W/V) 0.8%～2.0%;
Water and oil phase are pressed 1:1.67～1:3 mixed emulsification, duck hemorrhagic ovaritis inactivated vaccine, its viral level is not less than 10
6.0ELD
50/ plumage part.
4. a method for preparing like inactivated vaccine as described in the claim 2 is characterized in that, in DEF, results contain poison cell with duck hemorrhagic ovaritis virus inoculation for it, through the formaldehyde solution deactivation, concentrate and emulsification makes; It carries 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: the inoculation back produced cytopathy (CPE) in 72～96 hours and reaches 80%～90% o'clock harvested cell; And will fill the sedimentary aseptic beaker of harvested cell and be placed in 0 ℃ of ice bath with cracking 30s/ time under 85% power condition of lysis appearance; 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 virus of proliferation is tired and answered>=10
6.0TCID
50/ 0.1ml;
C. deactivation: in above-mentioned virocyte lysate, add mass concentration 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, 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 by the aseptic tween-80 of (V/V) 4%～6% adding, stir, be water;
The 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%, the sterilization back is an oil phase;
Water and oil phase are pressed 1:1.67～1:3 mixed emulsification, 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 |
| CN102586193A (en) * | 2012-02-10 | 2012-07-18 | 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 |
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| 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 |
| CN105807049A (en) * | 2016-02-22 | 2016-07-27 | 北京市农林科学院 | Hemagglutination inhibition test antigen for duck tembusu virus disease and preparation method thereof |
| CN110898218A (en) * | 2019-12-30 | 2020-03-24 | 瑞普（保定）生物药业有限公司 | Duck tembusu virus disease inactivated vaccine and preparation method thereof |
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