CN103820397B - A kind of kind Duck parvovirus and application thereof - Google Patents
A kind of kind Duck parvovirus and application thereof Download PDFInfo
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Abstract
The object of this invention is to provide a kind of kind Duck parvovirus, for YBMDV strain, the China Committee for Culture Collection of Microorganisms's common micro-organisms center that has been preserved in No. 3 Institute of Microorganism, Academia Sinica in Yard 1, BeiChen xi Road, Chaoyang District, Beijing City on November 08th, 2013, deposit number is CGMCCNo.8504. It is little that a kind Duck parvovirus for the present invention's screening has toxicity, and the feature that immunogenicity is high, with a kind Duck parvovirus YBMDP strain inoculation kind duck embryo, gather in the crops respectively dead embryo idiosome and blastochyle, after grinding, freeze thawing, collect virus liquid, after ultrafiltration concentration, formalin deactivation, the adjuvant mixing and emulsifying that refuels is made vaccine. Can improve the antibody of kind of kind duck, ensure its filial generation maternal antibody level, the duckling parvovirus infections that prevention kind Duck parvovirus causes, this vaccine has advantages of efficiently, security is good.
Description
Technical field
The invention belongs to poultry sex pheromone triage techniques field, be specifically related to a kind of kind duck thinSmall virus and in the application of preparing in vaccine, antibody.
Background technology
Kind Duck parvovirus disease is the acute or subacute infectious disease of the one of the young kind duck of infringement, be withBreathe, the downright bad and hemorrhage infectious disease for principal character of diarrhoea and pancreas; There is hyperinfection,And M & M is all very high, often causes serious economic loss. This disease mainly betidesThe young bird in 1～3 week age kind duck, especially the young bird kind duck of 10 about ages in days, and when reach 3 week age withAbove just substantially do not fall ill. This disease comes across Chinese good fortune early than the middle and later periods eighties 20th centuryBuild the area with French western Brittany, early 1990s just recognizes that it is to be different fromThe independent disease of gosling plague. If kind duck to death is dealt with improperly, often cause the climing of virusProlong, produce larger harm. Therefore, just need to filter out the height of tiring, immunogenicity good kindDuck parvovirus is prepared vaccine. And, because causing the vaccine valence using, virus variation fallsLow, also need the Strain that filters out variation to prepare the vaccine that curative effect is higher.
Summary of the invention
The object of this invention is to provide a kind of kind Duck parvovirus filtering out, and in preparation epidemic diseaseApplication in seedling; With vaccine prepared by this virus have efficiently, security is good, protective rate is highAdvantage, thus make up the deficiencies in the prior art.
First the present invention provides a kind of kind Duck parvovirus, is YBMDV strain, in November, 2013Within 08th, be preserved in Yard 1, BeiChen xi Road, Chaoyang District, Beijing City No. 3 Institute of Microorganism, Academia SinicaChina Committee for Culture Collection of Microorganisms's common micro-organisms center, deposit number is CGMCCNo.8504。
Of the present invention kind of Duck parvovirus is for the preparation of inactivated vaccine;
Of the present invention kind of Duck parvovirus is also for the preparation of immune antiboidy.
It is little that a kind Duck parvovirus for the present invention's screening has toxicity, and the high feature of immunogenicity, usesKind Duck parvovirus YBMDP strain inoculation kind duck embryo, gathers in the crops respectively dead embryo idiosome and blastochyle, through grindingAfter mill, freeze thawing, collect virus liquid, after ultrafiltration concentration, formalin deactivation, the adjuvant that refuels is mixedClose emulsification and make vaccine. Can improve the antibody of kind of kind duck, ensure its filial generation maternal antibody level,The duckling parvovirus infections that prevention kind Duck parvovirus causes, this vaccine has efficiently, safetyThe advantage that property is good.
Detailed description of the invention
Below in conjunction with specific embodiment, the present invention is described in detail.
One, the screening of YBMDV strain
Acute kind Duck parvovirus disease has been broken out duck field in Zhejiang Province in 2011, gets the pancreas of the duck that dies of illnessDirty and enteron aisle, uses pancreas and intestinal tissue without physiological saline homogenate and makes 20% suspension, 3000r/minCentrifugal 15min, inoculates respectively 13 ages in days kind duck embryo through allantoic cavity after getting supernatant degerming, and hatching 120 is littleTime, collect allantoic fluid and idiosome tissue, after homogenate, multigelation 3 times, gets supernatant frozen.The virus liquid of results has carried out the sides such as viral level, immunogenicity, specificity and pure property after purifiedThe analyzing and testing of the virus characteristic of face, result shows that this strain viral level is 105.7ELD50/ 0.2ml,Little immunizing dose is 102.0ELD50/ 0.2ml, this virus only with a kind Duck parvovirus generation specific reaction,Pollute without bacterium, mycoplasma and exogenous virus, be suitable as seedling strain. To screen YBMDV strainBe preserved in No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City Chinese science on November 08th, 2013China Committee for Culture Collection of Microorganisms's common micro-organisms center of institute of microbiology of institute, preservationBe numbered CGMCCNo.8504.
Proterties for the strain of the present invention screening detects, and result shows, this strain virus belongs to tinyVirus, circular without cyst membrane, diameter in 20 μ m left and right, insensitive to ether, trypsase, acid,To red blood cell without agglutination phenomenon. This virus can cause that the urgency taking hemorrhagic enteritis as feature occurs young kind duckProperty septic infectious disease. Vaccine immunity prepared by this strain is grown up after kind duck, can protect immunity 4 monthsThe attack of epidemic isolates is avoided in interior kind filial generation that duck produces. This strain is done after 100 times of dilutions, with equivalent young birdThe sick antiserum neutralization of kind Duck parvovirus, inoculation 13 ages in days kind duck embryo, result shows, neutralization group kindDuck embryo is strong living all, and virus control group kind duck embryo is all dead.
The structural gene VP of the YBMDV strain to screening checks order, and result VP total length is 2199bp,732 amino acid of encoding; The blast sequence analysis of NCBI shows, the structural gene VP of YBMDV strainThere are differences site with the VP of published kind of Duck parvovirus, homology is 97.7%～98.3%(existing 9-16 amino acid whose difference).
In order to detect above-mentioned amino acid whose difference to the antigenic impact of YBMDV strain, next with YBMDV strainPrepare antigen and detect its immunogenicity.
Two, the preparation of antigen for vaccine
1. seedling will be produced the strain with seed culture of viruses YBMDV with the preparation of virus liquid, allantoic cavity inoculation 12～14 age in days susceptibles kind duck embryo, every embryo 0.2ml, hatches for 36～37 DEG C, 2 photograph embryos inspection every day.Select after inoculation deadly in 48～120 hours, and idiosome has extensively hemorrhagely, and hair follicle is hemorrhage, liverDirty sex change and necrosis, fine hair allantois has the embryo of Mild edema, gets its allantoic fluid and idiosome and puts into groupKnit in bruisher and pulverize, the tissue of getting after pulverizing adds physiological saline to mix by 1:2; Put 2～8 DEG C 4～12 hours, collect allantoic fluid, amniotic fluid and idiosome (remove head and four limbs). Even by blastochyleSlurry, freeze thawing 3 times, the centrifugal 30min of 4000r/min, gets supernatant and is mixed in sterile chamber, puts2～8 DEG C of preservations.
2. deactivation is placed in YBMDV strain virus liquid in deactivation bottle, is metered into 10% formaldehyde moltenLiquid, with adding with shaking, fully mixes it, and the ultimate density of formalin is 0.2%. Add formaldehydeAfter solution, pour in another deactivation bottle, fail to contact deactivation with near the virus of avoiding adhering to bottleneckAgent. 37 DEG C of deactivations were taken out after 16 hours, put 2～8 DEG C of preservations.
3. the inspection of semifinished product
(1) steriling test is got the blastochyle of deactivation, is undertaken by existing " Chinese veterinary pharmacopoeia " annexSteriling test.
(2) viral level is measured virus liquid sterile saline is made to 10 times of serial dilutions,Get 10-2、10-3、10-4、10-54 dilution factors, allantoic cavity inoculation 12～14 ages in days are easy respectivelySense kind duck embryo, every embryo 0.2ml establishes 5 pieces of inoculation physiological saline contrasts, every embryo 0.2ml simultaneously.Put 36～37 DEG C and continue to hatch, per sunshine, embryo 2 times, observed 168 hours. With chicken embryo death alsoOccur that chorioallantoic membrane oedema thickens, the systemic bleeding venereal disease changes such as head, neck, back are judged to infection, meterCalculate ELD50。
(3) deactivation inspection was by the virus liquid allantoic cavity inoculation after YBMDV strain deactivation 12～14 daysAge 10 pieces of susceptible kind duck embryos, every embryo 0.2ml, puts 36～37 DEG C and continues to hatch, Continuous Observation 168Hour.
Three, the preparation of inactivated vaccine: carry out vaccine system through semi-finished product antigen after the assay was approvedStandby (in following preparation, each liquid component is counted by volume).
(1) 95 parts of white oils for animals are got in oil phase preparation, and 1 part of aluminum stearate, is placed in oil phase systemIn standby tank, be heated to after 80 DEG C, then Jia Siben-805 part, while reaching 115 DEG C to temperature, dimensionHold 30min, cooling rear for subsequent use.
(2) water is prepared the YBMDV strain virus liquid proper proportion mixing that deactivation is up to the standards,Make to be not less than 10 containing YBMDV strain virus content in every 0.2ml water4.0ELD50. Get telling after sterilizing-805 parts of temperature, add in Agitation Tank, add 95 parts of hybrid antigen liquid simultaneously, start stirring motorStir 20～30min, Tween-80 is dissolved completely.
(3) emulsification is got 2 parts of oil phases and is put in high-speed shearing machine, starts motor slow rotation and stirsMix, slowly add 1 part of water simultaneously, with 10000r/min, emulsification 5 minutes. After emulsification,Get 10ml, with 3000r/min centrifugal 15 minutes, the water that separate out at the pipe end should be no more than 0.5ml.
Four, vaccine product inspection
(1) proterties
Outward appearance vaccine should be milky emulsion, and free from admixture and external packing should be qualified.
Formulation is water-in-oil type. Get a clean suction pipe, draw a small amount of vaccine and splash in cold water,Except the 1st, all should indiffusion.
Stability is drawn vaccine 10ml and is added in centrifuge tube, centrifugal 15 points with 3000r/minClock, the water that separate out at the pipe end should be no more than 0.5ml.
Viscosity is undertaken by existing " Chinese veterinary pharmacopoeia " annex, should conform with the regulations.
(2) loading quantity inspection is undertaken by existing " Chinese veterinary pharmacopoeia " annex, should conform with the regulations.
(3) steriling test is undertaken by existing " Chinese veterinary pharmacopoeia " annex, should conform with the regulations.
(4) 10 of 21 ages in days kind ducks, every neck hypodermic injection vaccine for safety verification2.0ml establishes 5 of contrasts simultaneously, raises Continuous Observation 14 days, note under identical conditionRecord test chicken is searched for food, drinking-water and clinical setting. Should not occur any part of being caused by vaccine andSystemic adverse reactions.
(5) efficacy test
1. 10 of 21～35 age in days SPF chickens, every neck hypodermic injection for serological methodVaccine, 0.5ml/ only, separately gets 5 with not immune comparing of age in days chicken, exempts from blood sampling latter 21 daysSeparation of serum, measures respectively kind Duck parvovirus fine jade and expands antibody. Immune group AGP antibody titer,Should be not less than 1:4, control group should be all negative.
2. filial generation maternal antibody with attack poison protection be related to 20 of 90 age in days kind kind ducks, every neckPortion's hypodermic injection vaccine, 2ml/ only, separately gets 10 with not immune comparing of age in days kind kind duck. InLatter 15 days identical approach same doses of immunity again immunity once, are collected the kind of producing while opening April in postpartumEgg incubation hatching, in the time of young kind of duck 7 age in days, immune group filial generation is got 10, and control group filial generation is got10, attack malicious YBMDP strain, every intramuscular injection 0.5ml. Immune group filial generation protective rate should be notLower than 8, the control group incidence of disease should be not less than 9.
3. protect (screening of strain) by popular each place poison to the poison of attacking of local epidemic isolatesInactivated vaccine is prepared respectively in strain, gets 20 of 90 age in days kind kind ducks, every neck hypodermic injection epidemic diseaseSeedling, 2ml/ only, separately gets 10 with not immune comparing of age in days kind kind duck. In immunity latter 15 daysIdentical approach same dose again immunity once, is collected to open and is produced the hatching of breeding eggs hatching of producing in 1 month,In the time of young kind of duck 7 age in days, immune group filial generation is got 10, and control group filial generation is got 10, attacks poison 6Strain each place separated strain, every intramuscular injection 0.5ml. Malicious protection situation is attacked in observation.
Embodiment 1
One, the preparation of antigen for vaccine
1. seedling will be produced the strain with seed culture of viruses YBMDV with the preparation of virus liquid, allantoic cavity inoculation 12～14 age in days susceptibles kind duck embryo, every embryo 0.2ml, hatches for 36～37 DEG C, 2 photograph embryos inspection every day.Select after inoculation deadly in 48～120 hours, and idiosome has extensively hemorrhagely, and hair follicle is hemorrhage, liverDirty sex change and necrosis, CAM has kind duck embryo of Mild edema, gets its allantoic fluid and kind duckIdiosome is put into tissue mashing machine and is pulverized, and the tissue of getting after pulverizing adds physiological saline mixed by 1:2Close; Put 2～8 DEG C 4～12 hours, collect allantoic fluid, amniotic fluid and idiosome (remove head and four limbs).With blastochyle homogenate, freeze thawing 3 times, the centrifugal 30min of 4000r/min, gets supernatant and is mixed in aseptic appearanceIn device, put 2～8 DEG C of preservations. (referring to table 1).
The preparation of table 1 venom
| Strain title | Seedling material | Inoculation embryo number (piece) | Embryo age in days | Results venom (ml) |
| YBMDP strain | Kind duck embryo | 100 | 13 | 1200 |
2. deactivation is poured virus liquid in deactivation bottle into, is metered into 10% formalin, makes itFully mix, the ultimate density of formalin is 0.2%. After adding formalin, pour another deactivation intoIn tank, fail to contact inactivator with near the virus of avoiding adhering to tank mouth. 37 DEG C of deactivations 16 hoursRear taking-up, puts 2～8 DEG C of preservations.
3. the vaccine inspection of semifinished product
(1) steriling test is got the blastochyle of deactivation, undertaken by existing " Chinese veterinary pharmacopoeia " annex,Asepsis growth.
(2) viral level is measured virus liquid sterile saline is made to 10 times of serial dilutions,Get 10-2、10-3、10-4、10-54 dilution factors, allantoic cavity inoculation 12～14 ages in days are easy respectivelySense kind duck embryo, every embryo 0.2ml establishes 5 pieces of inoculation physiological saline contrasts, every embryo 0.2ml simultaneously.Put 36～37 DEG C and continue to hatch, per sunshine, embryo 2 times, observed 168 hours. With chicken embryo death alsoOccur that chorioallantoic membrane oedema thickens, the systemic bleeding venereal disease changes such as head, neck, back are judged to infection, meterCalculate ELD50Be 105.7ELD50/0.2ml。
(3) deactivation inspection is by virus liquid allantoic cavity inoculation 12～14 age in days susceptibles kind duck embryo 10Piece, every embryo 0.2ml, puts 36～37 DEG C and continues to hatch, Continuous Observation 168 hours, and duck embryo is equalWithout dead.
Two, the preparation of inactivated vaccine: carry out vaccine system through semi-finished product antigen after the assay was approvedStandby (in following preparation, each liquid component is counted by volume, referring to table 2).
(1) 95 parts of white oils for animals are got in oil phase preparation, and 1 part of aluminum stearate, is placed in oil phase systemIn standby tank, be heated to after 80 DEG C, then Jia Siben-805 part, while reaching 115 DEG C to temperature, dimensionHold 30min, cooling rear for subsequent use.
(2) water is prepared the virus liquid mixing that deactivation is up to the standards, and detects every 0.2ml waterMutually, be not less than 10 containing YBMDP strain virus content5.7ELD50. Get 5 parts of Tween-80s after sterilizing,Add in Agitation Tank, add 95 parts of hybrid antigen liquid simultaneously, start stirring motor and stir 20～30min,Tween-80 is dissolved completely.
(3) emulsification is got 2 parts of oil phases and is put in high-speed shearing machine, starts motor slow rotation and stirsMix, slowly add 1 part of water simultaneously, with 10000r/min, emulsification 5 minutes. After emulsification,Get 10ml, with 3000r/min centrifugal 15 minutes, separated out without water at the pipe end.
(4) packing quantitative separating, seals.
The emulsification of table 2 vaccine and packing
Three, vaccine product inspection
(1) proterties
Appearance milky white emulsion.
Formulation water-in-oil type, gets a clean suction pipe, draws a small amount of vaccine and drips in cold water, is oilDrip shape indiffusion.
Stability is drawn vaccine 10ml and is added in centrifuge tube, centrifugal 15 points with 3000r/minClock, separate out without water at the pipe end.
Viscosity is undertaken by current edition " Chinese veterinary pharmacopoeia " annex, is 58.6cp.
(2) loading quantity inspection is undertaken by current edition " Chinese veterinary pharmacopoeia " annex, meets standard.
(3) steriling test is undertaken by current edition " Chinese veterinary pharmacopoeia " annex, asepsis growth.
(4) 10 of 21 ages in days kind ducks, every neck hypodermic injection vaccine for safety verification2.0ml establishes 5 of contrasts simultaneously, raises Continuous Observation 14 days, examination under identical conditionTest all strong living of kind duck, and all do not occur any part and general reaction causing because of vaccine.
(5) efficacy test
1. use 10 of 28 age in days SPF chickens, every neck hypodermic injection vaccine 0.5ml, separately gets 5Only, with not immune comparing of age in days chicken, exempt from latter 21 days, blood sampling separation of serum, measures kind duck tinyVirus AGP antibody titer. 10 chicken antibodies of immune group are tired and are all not less than as 1:4, control group5 chickens are all negative. (referring to table 3).
Table 3 vaccine potency assay
2. filial generation maternal antibody with attack poison protection be related to 20 of 90 age in days kind kind ducks, every neckPortion's hypodermic injection vaccine, 2ml/ only, separately gets 10 with not immune comparing of age in days kind duck. In exempting fromAfter epidemic disease identical approach same dose on the 15th again immunity once, collect kind of the egg that produces while opening April in postpartumHatching and nestling, in the time of young kind of duck 7 age in days, immune group filial generation is got 10, and control group filial generation gets 10Only, attack malicious YBMDP strain, every intramuscular injection 0.5ml. 9 of immune group filial generation protections, contrastGroup is morbidity all.
3. the poison of attacking of local epidemic isolates is protected to (seed selection of strain) 90 age in days kind kind ducks 20Only, every neck hypodermic injection vaccine, 2ml/ only, separately get 10 not immune with age in days kind kind duckCompare. In latter 15 days identical approach same doses of immunity again immunity once, collect to open and produce 1The hatching of breeding eggs hatching of producing in individual month, in the time of young kind of duck 7 age in days, immune group filial generation is got 10,Control group filial generation is got 10, attacks malicious 6 strain each place separated strains, every intramuscular injection 0.5ml.Result shows, a kind Duck parvovirus inactivated vaccine of preparing with YBMDP strain other poison of comparingInactivated vaccine prepared by strain, can resist each place and separate malicious attack. Its seed culture of viruses immunogenicity is good,It is the comparatively ideal seedling strain of a strain. And, kind Duck parvovirus of preparing with YBMDP strainInactivated vaccine shows for the poison experiment of attacking of YBMDP strain, the exempting from of vaccine prepared by YBMDP strainEpidemic disease effect is best, and supposition causes (referring to table 4) because YBMDP pnca gene morphs.
Table 4 is to the malicious protection of attacking of local epidemic isolates
Therefore, the YBMD strain of the present invention screening can be used as seedling strain prepare inactivated vaccine andYolk antibody.
Claims (1)
1. a kind Duck parvovirus, is characterized in that, described kind Duck parvovirus is YBMDP strain, inBe preserved in the micro-life of the Chinese Academy of Sciences of No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City on November 08th, 2013China Committee for Culture Collection of Microorganisms's common micro-organisms center of thing research institute, deposit number is CGMCCNo.8504。
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN104611389A (en) * | 2014-12-10 | 2015-05-13 | 沈阳药科大学 | Fermentation optimizing technology for producing secalonic acid D (SAD) by using P.oxalicum |
| CN105153303B (en) * | 2015-09-21 | 2018-09-25 | 山东农业大学 | A kind of Yolk antibody and preparation method thereof of prevention Duck parvovirus |
| CN105219735B (en) * | 2015-09-21 | 2018-04-03 | 山东农业大学 | A kind of Duck parvovirus strain and its inactivated vaccine and preparation method |
| CN105154410B (en) * | 2015-09-21 | 2018-04-03 | 山东农业大学 | A kind of Duck parvovirus strain and its live vaccine |
| CN105585632A (en) * | 2016-03-22 | 2016-05-18 | 重庆三杰众鑫生物工程有限公司 | Meat duck parvovirus refined yolk antibody |
| CN105949307B (en) * | 2016-06-18 | 2019-11-22 | 青岛易邦生物工程有限公司 | It is a kind of for preventing and treating a kind Yolk antibody for duck source gosling plague |
| CN106139140B (en) * | 2016-06-30 | 2019-05-31 | 青岛真源生物技术有限公司 | A kind of Muscovy duck parvovirus subunit vaccine |
| CN108707588B (en) * | 2018-06-05 | 2021-11-16 | 山东信得科技股份有限公司 | Muscovy duck parvovirus strain and application thereof |
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