CN102302772A - Duck hemorrhagic ovaritis (DHO) inactivated vaccine and preparation method thereof - Google Patents
Duck hemorrhagic ovaritis (DHO) inactivated vaccine and preparation method thereof Download PDFInfo
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Abstract
The invention relates to a duck hemorrhagic ovaritis (DHO) inactivated vaccine and a preparation method thereof. The preparation method comprises the following steps: reproducing virus by using 6-14-day-old chick embryos or duck embryos or Vero or CEF or BHK or PK15 cells based on a duck flavivirus strain WFG36 as a virus strain for producing the vaccine, wherein the microbial conservation number of the duck flavivirus strain is CGMCC No. 4718; and preparing antigens; inactivating; preparing the vaccine; and the like. By using the preparation method, the vaccine for safely and effectively preventing and controlling DHO can be prepared.
Description
Technical field
The present invention relates to a kind of duck hemorrhagic oophoritis inactivated vaccine and preparation method thereof, belong to the veterinary biologics technical field.
Background technology
Since 2010, a kind of novel epidemic disease has taken place in laying ducks that raise China some areas and kind duck; This disease is a main feature with sudden egg drop reduction; Mainly cause follicle distortion, degeneration, theca folliculi is congested, hemorrhage, the fallopian tube inflammatory lesion; Temporarily with this epidemic disease be called duck hemorrhagic oophoritis (Duck Hemorrhagic Ovaritis, DHO).We are separated to a strain virus on the morbidity kind duck of certain large-scale duck field, should virus be the RNA viruses that cyst membrane is arranged through Preliminary Identification, and RT-PCR identifies that banzi virus is positive, temporarily this virus is called duckling virus (Duck Flavivirus).
Because this novel pandemic is not had the specific treatment method, the development of vaccine is the first-selected measure of this disease of control.
Summary of the invention
The strain duckling virus WFG36 strain that the objective of the invention is to utilize the inventor to separate voluntarily, identify, preserve is used Strain as production of vaccine; The Embryo Gallus domesticus through using 6～14 ages in days or the duck embryo of 6～14 ages in days or Vero cell or CEF cell or bhk cell or PK15 cell carry out the breeding and the antigenic preparation of virus; Again through deactivation, join step such as Seedling, prepare a kind of safe and effective novel duck disease vaccine---duck hemorrhagic oophoritis inactivated vaccine.
1. production of vaccine is used Strain
(1) viral source
Vaccine is made and check to use seed culture of viruses be (Duck Flavivirus) the WFG36 strain of duckling virus, separates obtaining by the inventor in the kind duck body of certain plant of area, Shandong, and identifies, takes care of and supply.This Strain is delivered No. 3 China Committee for Culture Collection of Microorganisms of the Institute of Microorganism, Academia Sinica common micro-organisms center preservations in Yard 1, BeiChen xi Road, Chaoyang District, Beijing City on April 12nd, 2011, is numbered: CGMCC No.4718.
(2) Strain characteristic
1) viral level
Seed culture of viruses is made 10 times of serial dilutions with sterile saline, get 10
-2, 10
-3, 10
-43 dilution factors are respectively through 5 pieces of yolk sac inoculation 7 age in days SPF Embryo Gallus domesticus, and the 0.1ml/ embryo is put 37 ℃ and continued to hatch; Dead embryo discards and disregards within 24 hours, and dead Embryo Gallus domesticus takes out at any time after 24 hours, places 4 ℃ of preservations; Observation to 168 hour is calculated ELD according to the chicken embryo death number
50, every 0.1ml viral level answers>=10
3.0ELD
50
2) to the virulence of Embryo Gallus domesticus
Seed culture of viruses is done 100 times of dilutions with sterile saline, 10 pieces of yolk sac inoculation 7 age in days susceptible Embryo Gallus domesticus, the 0.1ml/ embryo is put 37 ℃ and is continued to hatch, Embryo Gallus domesticus should be in the inoculation back 24～168 hours dead more than 8 pieces, it is hemorrhage that general appears in dead fetus.
3) immunogenicity
Through yolk sac inoculation 7 age in days susceptible Embryo Gallus domesticus, results infect blastochyle and idiosome with seed culture of viruses, after idiosome adds normal saline and grinds, mix with blastochyle, and after the formalin deactivation, (water: ratio oil phase) was processed oil emulsion inactivated vaccine in 1: 2.
Get 10 7～10 healthy susceptible ducklings of age in days, be equally divided into two groups, 5 as test group; 5 as the blank group; Every nape subcutaneous injection of test group vaccine 0.5ml inoculates back 21 days, carries out counteracting toxic substances with duckling virus WFG36 strain as the counteracting toxic substances strain; Eye dripping, collunarium 0.2ml/ only, intramuscular injection 0.3ml/ is only.Behind the counteracting toxic substances the 7th day, gather the anticoagulation of every duck, separated plasma, respectively through 5 pieces of yolk sac inoculation 7 age in days susceptible Embryo Gallus domesticus, the 0.2ml/ embryo, the inoculation back was observed 7 days, immune group should at least 4 virus separate negatively, matched group should at least 4 viruses separate positive.
4) pure
Undertaken by existing " Chinese veterinary drug allusion quotation " appendix, should not have antibacterial, mycete, mycoplasma and exogenous virus and pollute.
5) specificity
With seed culture of viruses with 100 times of sterile saline dilutions, after the anti-duckling virus-specific of equivalent serum mixes, in 37 ℃ and 1 hour; 10 pieces of yolk sac inoculation 7 age in days SPF Embryo Gallus domesticus, the 0.2ml/ embryo was observed 168 hours; Should not cause the dead or infection of specificity, non-specific dead Embryo Gallus domesticus should be no more than 1 piece.
2. the method for preparing of a duck hemorrhagic oophoritis inactivated vaccine; Mainly be: use duckling virus WFG36 strain malicious with planting as production of vaccine; The Embryo Gallus domesticus of use 6～14 ages in days or the duck embryo of 6～14 ages in days or Vero cell or CEF cell or bhk cell or PK15 cell carry out the breeding and the antigenic preparation of virus, and every 0.1ml antigenic virus content answers>=10
3.0ELD
50Antigen is produced water by conventional method after formalin or beta-propiolactone or the deactivation of divinyl imines, the oil phase of processing with mineral oil mixes the inactivated oil Adjuvanted vaccines.
Following preparation process is specifically arranged:
(1) select duckling virus WFG36 strain malicious with planting as production of vaccine;
That (2) selects 6～14 ages in days uses material to the duck embryo of duckling virus susceptible or Vero cell or CEF cell or bhk cell or PK15 cell as virus breeding and antigen preparation to the Embryo Gallus domesticus of duckling virus susceptible or 6～14 ages in days;
(3) antigenic preparation can realize through following 6 kinds of approach:
1) uses material with the susceptible Embryo Gallus domesticus of 6～14 ages in days as virus breeding and antigen preparation
Planting poison inoculation susceptible Embryo Gallus domesticus will produce with planting malicious by every embryo>=10
1.0ELD
50Dose inoculation to susceptible embryo yolk sac or allantoic cavity, pin hole is sealed with wax candle in inoculation back, puts 37 ℃ and hatches;
Shine egg 1 every day after hatching and observe susceptible egg inoculation, discards the dead embryo in 24 hours, and per afterwards 12 hours photograph eggs once take out dead embryo at any time, place 2～8 ℃ of preservations.After hatching completion in 168 hours, take out whole Embryo Gallus domesticus, place 2～8 ℃ of coolings;
Gather in the crops viral liquid refrigerative Embryo Gallus domesticus is taken out, aseptic results idiosome, blastochyle and CAM, ground and mixed.
2) use material with the susceptible duck embryo of 6～14 ages in days as virus breeding and antigen preparation
Planting poison inoculation susceptible duck embryo will produce with planting malicious by every embryo>=10
1.0ELD
50Dose inoculation to susceptible embryo yolk sac or allantoic cavity, pin hole is sealed with wax candle in inoculation back, puts 37 ℃ and hatches;
Shine egg 1 every day after hatching and observe susceptible duck embryonic breeding kind, discards the dead embryo in 24 hours, and per afterwards 12 hours photograph eggs once take out dead embryo at any time, place 2～8 ℃ of preservations.After hatching completion in 168 hours, take out whole duck embryos, place 2～8 ℃ of coolings;
Gather in the crops viral liquid refrigerative duck embryo is taken out, aseptic results idiosome, blastochyle and CAM, ground and mixed.
3) use material with the Vero cell as virus breeding and antigen preparation
Kind of malicious inoculating cell will be inoculated on the Vero cell of monolayer after will producing and diluting with normal saline with kind of poison, and the cell bottle placed on the Rotary Machine cultivate;
Observe with results and connect the every 12h observation in poison back once, be cultured to 72-96h, the results venom will connect poison cell and place-20 ℃.
4) use material with the CEF cell as virus breeding and antigen preparation
After kind of malicious inoculating cell will be produced and dilute with normal saline with kind of poison, be inoculated into monolayer CEF cell after, be positioned on the Rotary Machine and cultivate;
Observe to meet the every 12h in poison back with results and observe once, when the cell typical cytopathic, continue to be cultured to 72-96h, reach about 80% to cytopathy, the results venom places-20 ℃.
5) use material with bhk cell as virus breeding and antigen preparation
Kind of malicious inoculating cell will be inoculated on the bhk cell of monolayer after will producing and diluting with normal saline with kind of poison, and the cell bottle placed on the Rotary Machine cultivate;
Observe with results and connect the every 12h observation in poison back once, be cultured to 72-96h, the results venom will connect poison cell and place-20 ℃.
6) use material with the PK15 cell as virus breeding and antigen preparation
Kind of malicious inoculating cell will be inoculated on the PK15 cell of monolayer after will producing and diluting with normal saline with kind of poison, and the cell bottle placed on the Rotary Machine cultivate;
Observe with results and connect the every 12h observation in poison back once, be cultured to 72-96h, the results venom will connect poison cell and place-20 ℃.
(4) antigen (the viral liquid of results) of preparation; Should by " Chinese veterinary drug allusion quotation " (Chinese veterinary drug allusion quotation committee. three ones of in 2005 versions of People's Republic of China's veterinary drug allusion quotation. Chinese agriculture publishing house; 2006; The present invention is called for short " Chinese veterinary drug allusion quotation ") appendix makes steriling test by bottle, and samples 1 part and measures viral level (ELD
50), answer asepsis growth, every 0.1ml viral level answers>=10
3.0ELD
50
(5) viral liquid deactivation adds formalin (or beta-propiolactone or divinyl imines) in the viral liquid that is up to the standards, and shakes up to be placed on 37 ℃ and to carry out deactivation;
Steriling test carries out steriling test by " Chinese veterinary drug allusion quotation " appendix to inactivation of viruses liquid, answers asepsis growth.
Viral liquid after the deactivation, 10 pieces of yolk sac inoculation 7 age in days SPF Embryo Gallus domesticus, 0.2ml/ embryo are got in deactivation check; Putting 36～37 ℃ hatches; Dead embryo discards in 24 hours, observes to 168 hours, and the non-specific death of Embryo Gallus domesticus should be no more than 1 piece; Check fetus one by one, lesions such as liver, kidney be hemorrhage all should not occur.
(6) after oil emulsion inactivated vaccine preparation will be joined the oil phase that Seedling uses (94 parts of injection white oils, Jia Siben-806 part, aluminium stearate 2 parts) and water (96 parts of inactivation antigens, 4 parts of tween 80s) preparation completion; Proportioning ratio in 2: 1 parts of oil phase and waters places emulsion tank to carry out emulsifying; After the emulsifying; Get 8～10ml centrifugal 15 minutes, layering should not occur with 3000r/min.Carry out aseptic subpackagedly after emulsifying is accomplished, can obtain the finished product vaccine.
Advantage of the present invention
The present invention relates to a kind of duck hemorrhagic oophoritis inactivated vaccine and preparation method thereof.It number is that the strain duckling virus WFG36 strain of CGMCC No.4718 is as the Strain of producing vaccine that the present invention utilizes microbial preservation; The Embryo Gallus domesticus through using 6～14 ages in days or the duck embryo of 6～14 ages in days or Vero cell or CEF cell or bhk cell or PK15 cell carry out the breeding and the antigenic preparation of virus; Again through deactivation, join step such as Seedling, prepare the vaccine of a kind of safe and effective prevention and control duck hemorrhagic oophoritis.
The practical implementation method
Embodiment
Following examples further specify the present invention, but not as limitation of the present invention.
Embodiment 1
Production prepares with seed culture of viruses
(1) seed culture of viruses breeding
Seed culture of viruses is diluted 100 times with sterile saline, yolk sac inoculation 6～14 age in days SPF Embryo Gallus domesticus), the 0.1ml/ embryo; Discard dead embryo in 24 hours, per afterwards 12 hours photograph embryos once take out dead embryo at any time; Place 2～8 ℃ of preservations, inoculate back 168 hours, all embryos of living are put 2～8 ℃ of cool overnight; Gather in the crops the blastochyle of all Embryo Gallus domesticus, be loaded in the sterilization container.The quantitative packing of blastochyle that check is aseptic, freezing preservation is indicated the harvest date, seed culture of viruses generation etc.
(2) seed culture of viruses is identified
Viral level is made 10 times of serial dilutions with seed culture of viruses with sterile saline, gets 10
-2, 10
-3, 10
-43 dilution factors are respectively through 5 pieces of yolk sac inoculation 6～14 age in days age in days SPF Embryo Gallus domesticus, and the 0.1ml/ embryo is put 37 ℃ and continued to hatch; Dead embryo discards and disregards within 24 hours, and dead Embryo Gallus domesticus takes out at any time after 24 hours, places 4 ℃ of preservations; Observation to 168 hour is calculated ELD according to the chicken embryo death number
50, every 0.1ml viral level answers>=10
3.0ELD
50
Purely undertaken, should not have antibacterial, mycete, mycoplasma and exogenous virus and pollute by existing " Chinese veterinary drug allusion quotation " appendix.
Carry out according to above explanation, should be up to specification.The seed culture of viruses that meets above standard is used seed culture of viruses as production.
(3) seed culture of viruses is preserved
Noxious dampness is below-15 ℃, and tentative is 12 months; The lyophilizing poison is below-70 ℃, and tentative is 24 months.
(4) seed culture of viruses subculture
Should be no more than for 3 generations.
Embodiment 2
The preparation of duck hemorrhagic oophoritis inactivated vaccine
(1) seedling is with the breeding of venom
1) inoculation is got and is produced with kind of a poison, with 100 times of sterile saline dilutions, and yolk sac inoculation 6～14 age in days age in days susceptible Embryo Gallus domesticus (or healthy duck embryo), the 0.1ml/ embryo, pin hole is sealed with wax candle in the inoculation back, puts 37 ℃ and continues to hatch, needn't egg-turning.
2) hatch and observe after the egg inoculation every day according to egg 1 time, discard dead embryo in 24 hours, per afterwards 12 hours photograph eggs once take out dead embryo at any time, place 2～8 ℃ of preservations, to 168 hours, take out whole Embryo Gallus domesticus, place 2～8 ℃ of cool overnight.
3) results are taken out refrigerative Embryo Gallus domesticus, and aseptic results idiosome and blastochyle should be noted inspection to every piece of Embryo Gallus domesticus before drawing blastochyle, and all fetuses are corrupt, blastochyle is muddy and the suspicious person of any pollution is arranged, and discarding need not.Idiosome, blastochyle, the CAM of results mixes with blastochyle after adding the sterile saline grinding, and sampling is tested.Preserve subsequent use below-15 ℃.
The viral liquid of 4) check results should be made steriling test by bottle by existing " Chinese veterinary drug allusion quotation " appendix, and a viral level (ELD that measures of sampling
50), answer asepsis growth, every 0.1ml viral level answers>=10
3.0ELD
50
(2) in the viral liquid that is up to the standards, to add formalin to final concentration be 0.1% in deactivation, fully shakes up immediately, picks up counting when being warming up to 37 ℃; Deactivation 48 hours, after deactivation finishes, sampling respectively; Carry out the deactivation check, the viral liquid of deactivation is put 2～8 ℃ of preservations, should be no more than 30.
(3) inspection of semifinished product
1) steriling test carries out steriling test by existing " Chinese veterinary drug allusion quotation " appendix to inactivation of viruses liquid, answers asepsis growth.
2) viral liquid after the deactivation, 10 pieces of yolk sac inoculation 7 age in days SPF Embryo Gallus domesticus, 0.2ml/ embryo are got in deactivation check; Putting 36～37 ℃ hatches; Dead embryo discards in 24 hours, observes to 168 hours, and the non-specific death of Embryo Gallus domesticus should be no more than 1 piece; Check fetus one by one, lesions such as liver, kidney be hemorrhage all should not occur.
(4) oil emulsion inactivated vaccine preparation
1) 94 parts of injection white oils are got in oil phase preparation, Jia Siben-80 6 part, after the mixing, add 2 parts in stearic acid aluminum, with add be stirred to transparent till, autoclaving is subsequent use.
2) 96 parts of inactivation antigens are got in the water preparation, and 4 parts of the tween 80s of adding sterilization fully stir, and tween 80 is dissolved fully.
3) emulsifying is got oil phase and is placed the oil phase jar for 2 parts, starts motor and stirs, and slowly adds 1 part of water simultaneously, changes in the emulsion tank after adding, and gets final product in 60 minutes with 3000r/min emulsifying.Before emulsifying is ended, add 1% thimerosal solution, making its final concentration is 0.01%.After the emulsifying, get 8～10ml centrifugal 15 minutes, layering should not occur with 3000r/min.
(5) packing
Under aseptic condition, quantitatively packing seals.
Embodiment 3
The preparation of duck hemorrhagic oophoritis inactivated vaccine
(1) seedling is with the breeding of venom---with Vero cell preparation antigen
1) plant malicious inoculating cell will produce dilute with normal saline with kind of poison after, be inoculated on the Vero cell of monolayer, and the cell bottle placed on the Rotary Machine cultivate;
2) observation connects the every 12h observation in poison back once with results, is cultured to 72h, gathers in the crops venom, will connect poison cell and place-20 ℃.
The viral liquid of 3) check results should be made steriling test by bottle by existing " Chinese veterinary drug allusion quotation " appendix, and a viral level (ELD that measures of sampling
50), answer asepsis growth, every 0.1ml viral level answers>=10
3.0ELD
50
(2) in the viral liquid that is up to the standards, to add formalin to final concentration be 0.1% in deactivation, fully shakes up immediately, picks up counting when being warming up to 37 ℃; Deactivation 48 hours, after deactivation finishes, sampling respectively; Carry out the deactivation check, the viral liquid of deactivation is put 2～8 ℃ of preservations, should be no more than 30.
(3) inspection of semifinished product
1) steriling test carries out steriling test by existing " Chinese veterinary drug allusion quotation " appendix to inactivation of viruses liquid, answers asepsis growth.
2) viral liquid after the deactivation, 10 pieces of yolk sac inoculation 7 age in days SPF Embryo Gallus domesticus, 0.2ml/ embryo are got in deactivation check; Putting 36～37 ℃ hatches; Dead embryo discards in 24 hours, observes to 168 hours, and the non-specific death of Embryo Gallus domesticus should be no more than 1 piece; Check fetus one by one, lesions such as liver, kidney be hemorrhage all should not occur.
(4) oil emulsion inactivated vaccine preparation
1) 94 parts of injection white oils are got in oil phase preparation, Jia Siben-80 6 part, after the mixing, add 2 parts in stearic acid aluminum, with add be stirred to transparent till, autoclaving is subsequent use.
2) 96 parts of inactivation antigens are got in the water preparation, and 4 parts of the tween 80s of adding sterilization fully stir, and tween 80 is dissolved fully.
3) emulsifying is got oil phase and is placed the oil phase jar for 2 parts, starts motor and stirs, and slowly adds 1 part of water simultaneously, changes in the emulsion tank after adding, and gets final product in 60 minutes with 3000r/min emulsifying.Before emulsifying is ended, add 1% thimerosal solution, making its final concentration is 0.01%.After the emulsifying, get 8～10ml centrifugal 15 minutes, layering should not occur with 3000r/min.
(5) packing
Under aseptic condition, quantitatively packing seals.
Embodiment 4
(1) preparation of duck hemorrhagic oophoritis inactivated vaccine---with CEF cell preparation antigen
1) plant malicious inoculating cell will produce dilute with normal saline with kind of poison after, be inoculated into monolayer CEF cell after, be positioned on the Rotary Machine and cultivate;
2) observe and to meet the every 12h in poison back with results and observe once, when typical cytopathic appears in cell, continue to be cultured to 72h, reach about 80% to cytopathy, the results venom places-20 ℃.
The viral liquid of 3) check results should be made steriling test by bottle by existing " Chinese veterinary drug allusion quotation " appendix, and a viral level (ELD that measures of sampling
50), answer asepsis growth, every 0.1ml viral level answers>=10
3.0ELD
50
(2) in the viral liquid that is up to the standards, to add formalin to final concentration be 0.1% in deactivation, fully shakes up immediately, picks up counting when being warming up to 37 ℃; Deactivation 48 hours, after deactivation finishes, sampling respectively; Carry out the deactivation check, the viral liquid of deactivation is put 2～8 ℃ of preservations, should be no more than 30.
(3) inspection of semifinished product
1) steriling test carries out steriling test by existing " Chinese veterinary drug allusion quotation " appendix to inactivation of viruses liquid, answers asepsis growth.
2) viral liquid after the deactivation, 10 pieces of yolk sac inoculation 7 age in days SPF Embryo Gallus domesticus, 0.2ml/ embryo are got in deactivation check; Putting 36～37 ℃ hatches; Dead embryo discards in 24 hours, observes to 168 hours, and the non-specific death of Embryo Gallus domesticus should be no more than 1 piece; Check fetus one by one, lesions such as liver, kidney be hemorrhage all should not occur.
(4) oil emulsion inactivated vaccine preparation
1) 94 parts of injection white oils are got in oil phase preparation, Jia Siben-80 6 part, after the mixing, add 2 parts in stearic acid aluminum, with add be stirred to transparent till, autoclaving is subsequent use.
2) 96 parts of inactivation antigens are got in the water preparation, and 4 parts of the tween 80s of adding sterilization fully stir, and tween 80 is dissolved fully.
3) emulsifying is got oil phase and is placed the oil phase jar for 2 parts, starts motor and stirs, and slowly adds 1 part of water simultaneously, changes in the emulsion tank after adding, and gets final product in 60 minutes with 3000r/min emulsifying.Before emulsifying is ended, add 1% thimerosal solution, making its final concentration is 0.01%.After the emulsifying, get 8～10ml centrifugal 15 minutes, layering should not occur with 3000r/min.
(5) packing
Under aseptic condition, quantitatively packing seals.
Embodiment 5
(1) preparation of duck hemorrhagic oophoritis inactivated vaccine---prepare antigen with bhk cell
1) plant malicious inoculating cell will produce dilute with normal saline with kind of poison after, be inoculated on the bhk cell of monolayer, and the cell bottle placed on the Rotary Machine cultivate;
2) observation connects the every 12h observation in poison back once with results, is cultured to 96h, gathers in the crops venom, will connect poison cell and place-20 ℃.
The viral liquid of 3) check results should be made steriling test by bottle by existing " Chinese veterinary drug allusion quotation " appendix, and a viral level (ELD that measures of sampling
50), answer asepsis growth, every 0.1ml viral level answers>=10
3.0ELD
50
(2) in the viral liquid that is up to the standards, to add formalin to final concentration be 0.1% in deactivation, fully shakes up immediately, picks up counting when being warming up to 37 ℃; Deactivation 48 hours, after deactivation finishes, sampling respectively; Carry out the deactivation check, the viral liquid of deactivation is put 2～8 ℃ of preservations, should be no more than 30.
(3) inspection of semifinished product
1) steriling test carries out steriling test by existing " Chinese veterinary drug allusion quotation " appendix to inactivation of viruses liquid, answers asepsis growth.
2) viral liquid after the deactivation, 10 pieces of yolk sac inoculation 7 age in days SPF Embryo Gallus domesticus, 0.2ml/ embryo are got in deactivation check; Putting 36～37 ℃ hatches; Dead embryo discards in 24 hours, observes to 168 hours, and the non-specific death of Embryo Gallus domesticus should be no more than 1 piece; Check fetus one by one, lesions such as liver, kidney be hemorrhage all should not occur.
(4) oil emulsion inactivated vaccine preparation
1) 94 parts of injection white oils are got in oil phase preparation, Jia Siben-80 6 part, after the mixing, add 2 parts in stearic acid aluminum, with add be stirred to transparent till, autoclaving is subsequent use.
2) 96 parts of inactivation antigens are got in the water preparation, and 4 parts of the tween 80s of adding sterilization fully stir, and tween 80 is dissolved fully.
3) emulsifying is got oil phase and is placed the oil phase jar for 2 parts, starts motor and stirs, and slowly adds 1 part of water simultaneously, changes in the emulsion tank after adding, and gets final product in 60 minutes with 3000r/min emulsifying.Before emulsifying is ended, add 1% thimerosal solution, making its final concentration is 0.01%.After the emulsifying, get 8～10ml centrifugal 15 minutes, layering should not occur with 3000r/min.
(5) packing
Under aseptic condition, quantitatively packing seals.
Embodiment 6
(1) preparation of duck hemorrhagic oophoritis inactivated vaccine---with PK15 cell preparation antigen
1) plant malicious inoculating cell will produce dilute with normal saline with kind of poison after, be inoculated on the PK15 cell of monolayer, and the cell bottle placed on the Rotary Machine cultivate;
2) observation connects the every 12h observation in poison back once with results, is cultured to 96h, gathers in the crops venom, will connect poison cell and place-20 ℃.
The viral liquid of 3) check results should be made steriling test by bottle by existing " Chinese veterinary drug allusion quotation " appendix, and a viral level (ELD that measures of sampling
50), answer asepsis growth, every 0.1ml viral level answers>=10
3.0ELD
50
(2) in the viral liquid that is up to the standards, to add formalin to final concentration be 0.1% in deactivation, fully shakes up immediately, picks up counting when being warming up to 37 ℃; Deactivation 48 hours, after deactivation finishes, sampling respectively; Carry out the deactivation check, the viral liquid of deactivation is put 2～8 ℃ of preservations, should be no more than 30.
(3) inspection of semifinished product
1) steriling test carries out steriling test by existing " Chinese veterinary drug allusion quotation " appendix to inactivation of viruses liquid, answers asepsis growth.
2) viral liquid after the deactivation, 10 pieces of yolk sac inoculation 7 age in days SPF Embryo Gallus domesticus, 0.2ml/ embryo are got in deactivation check; Putting 36～37 ℃ hatches; Dead embryo discards in 24 hours, observes to 168 hours, and the non-specific death of Embryo Gallus domesticus should be no more than 1 piece; Check fetus one by one, lesions such as liver, kidney be hemorrhage all should not occur.
(4) oil emulsion inactivated vaccine preparation
1) 94 parts of injection white oils are got in oil phase preparation, Jia Siben-80 6 part, after the mixing, add 2 parts in stearic acid aluminum, with add be stirred to transparent till, autoclaving is subsequent use.
2) 96 parts of inactivation antigens are got in the water preparation, and 4 parts of the tween 80s of adding sterilization fully stir, and tween 80 is dissolved fully.
3) emulsifying is got oil phase and is placed the oil phase jar for 2 parts, starts motor and stirs, and slowly adds 1 part of water simultaneously, changes in the emulsion tank after adding, and gets final product in 60 minutes with 3000r/min emulsifying.Before emulsifying is ended, add 1% thimerosal solution, making its final concentration is 0.01%.After the emulsifying, get 8～10ml centrifugal 15 minutes, layering should not occur with 3000r/min.
(5) packing
Under aseptic condition, quantitatively packing seals.
Embodiment 7
The check of duck hemorrhagic oophoritis inactivated vaccine
[character] appearance milky white Emulsion.
The dosage form water-in-oil type.Get a cleaning suction pipe, draw a small amount of vaccine and drip in cold water, except that the 1st, all should indiffusion.
Stability is drawn vaccine 10ml and is added in the centrifuge tube, and with 3000r/min centrifugal 15 minutes, the water that separate out at the pipe end should no more than 0.5ml.
Viscosity is drawn about 25 ℃ vaccine 1.0ml with 1ml suction pipe (the end opening internal diameter is 1.2mm, and internal diameter suitable for reading is 2.7mm), makes its vertical outflow naturally, and record flows out the 0.4ml required time, should be in 8 seconds.
[steriling test] undertaken by the method for " Chinese veterinary drug allusion quotation " regulation, answers asepsis growth.
[safety verification] get 1～2 age in week 10 of SPF ducks, the subcutaneous or intramuscular injection vaccine 1ml of nape observed 14 days respectively, any part and the systemic adverse reactions that are caused by vaccine injection should not occur.
[efficacy test] got 10 7～10 age in days SPF ducklings, is equally divided into two groups, and 5 as test group; 5 as the blank group, and every nape subcutaneous injection of test group vaccine 0.3ml inoculates back 21 days; With the strong malicious counteracting toxic substances of duckling virus, eye dripping, collunarium 0.2ml/ only, intramuscular injection 0.3ml/ is only.Behind the counteracting toxic substances the 7th day, gather the anticoagulation of every duck, separated plasma, respectively through 5 pieces of yolk sac inoculation 7 age in days susceptible Embryo Gallus domesticus, the 0.2ml/ embryo, the inoculation back was observed 7 days, test group should at least 4 virus separate negatively, matched group should at least 4 viruses separate positive.
[formaldehyde, the antiseptic mercurials determination of residual amount] undertaken by the method for " Chinese veterinary drug allusion quotation " regulation respectively, should meet the regulation of veterinary biologics general rule.
Claims (3)
1. duck hemorrhagic oophoritis inactivated vaccine; It is characterized in that vaccine involved in the present invention contains (Duck Flavivirus) the WFG36 strain of strain duckling virus; This Strain is delivered No. 3 China Committee for Culture Collection of Microorganisms of the Institute of Microorganism, Academia Sinica common micro-organisms center preservations in Yard 1, BeiChen xi Road, Chaoyang District, Beijing City on April 12nd, 2011, is numbered: CGMCC No.4718.
2. a method for preparing of making the said duck hemorrhagic of claim 1 oophoritis inactivated vaccine is characterized in that using duckling virus WFG36 strain malicious with planting as production of vaccine; Use the Embryo Gallus domesticus of 6～14 ages in days or the duck embryo of 6～14 ages in days to carry out the breeding and the antigenic preparation of virus; Every 0.1ml antigenic virus content answers>=10
3.0ELD
50Produce water by conventional method after antigen process formalin or beta-propiolactone or the deactivation of divinyl imines, the oil phase of processing with mineral oil mixes the inactivated oil Adjuvanted vaccines.
3. the method for preparing of duck hemorrhagic oophoritis inactivated vaccine as claimed in claim 2 is characterized in that the Embryo Gallus domesticus of available Vero cell or CEF cell or bhk cell or PK15 cell replacement 6～14 ages in days or the duck embryo of 6～14 ages in days carry out the breeding and the antigenic preparation of virus.
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Cited By (11)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102397539A (en) * | 2011-11-25 | 2012-04-04 | 广东省农业科学院兽医研究所 | Duck flavivirus inactivated vaccine and preparation method thereof |
| CN102488893A (en) * | 2011-12-28 | 2012-06-13 | 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 |
| CN102628030A (en) * | 2012-01-20 | 2012-08-08 | 宁波大学 | Chicken embryo culture method of Portunus tritubereulatus reovirus |
| CN102735807A (en) * | 2012-06-15 | 2012-10-17 | 北京市农林科学院畜牧兽医研究所 | Effect testing method for duck hemorrhagic ovaritis inactivated vaccine |
| CN102749427A (en) * | 2012-06-15 | 2012-10-24 | 北京市农林科学院畜牧兽医研究所 | Method for testing efficacy of duck hemorrhagic ovaritis inactivated vaccine |
| CN102793918A (en) * | 2012-08-01 | 2012-11-28 | 中国农业科学院哈尔滨兽医研究所 | Duck flavivirus subunit vaccine, as well as a preparation method and application thereof |
| CN103143009A (en) * | 2013-03-07 | 2013-06-12 | 齐鲁动物保健品有限公司 | Method for producing duck tembusu virus inactivated vaccines in large scale |
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| CN103157102A (en) * | 2012-12-27 | 2013-06-19 | 瑞普（保定）生物药业有限公司 | Method for preparing duck hemorrhagic ovaritis inactivated vaccines |
| CN103263386A (en) * | 2013-06-07 | 2013-08-28 | 成都乾坤动物药业有限公司 | Preparation process of saligenin emulsion injection |
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-
2011
- 2011-08-31 CN CN201110254517.XA patent/CN102302772B/en active Active
Non-Patent Citations (3)
| Title |
|---|
| ROEHRIG JT: "Antigenic structure of Flavivirus proteins", 《ADV VIRUS RES.》 * |
| 曹贞贞等: "鸭出血性卵巢炎的初步研究", 《中国兽医杂志》 * |
| 滕巧泱等: "一种新的黄病毒导致蛋鸭产蛋下降及死亡", 《中国动物传染病学报》 * |
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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 |
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| CN102793918B (en) * | 2012-08-01 | 2014-09-10 | 中国农业科学院哈尔滨兽医研究所 | Duck flavivirus subunit vaccine, as well as a preparation method and application thereof |
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