CN100572532C - A kind of duck plague vaccine and special strains thereof - Google Patents
A kind of duck plague vaccine and special strains thereof Download PDFInfo
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- CN100572532C CN100572532C CNB2007101783719A CN200710178371A CN100572532C CN 100572532 C CN100572532 C CN 100572532C CN B2007101783719 A CNB2007101783719 A CN B2007101783719A CN 200710178371 A CN200710178371 A CN 200710178371A CN 100572532 C CN100572532 C CN 100572532C
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Abstract
The invention discloses a kind of duck plague vaccine and special strains thereof.Duck plague vaccine of the present invention, its activeconstituents are that the duck plague simplexvirus AV1221 strain duck embryo of deactivation adapts to poison.Duck plague vaccine of the present invention uses inactivation of viruses to make, and security is good; Preservation, transportation (2-8 ℃) and easy to use, immune effect is stable.
Description
Technical field
The present invention relates to a kind of duck plague vaccine and special strains thereof.
Background technology
Duck plague (Duck Plague) has another name called duck viral enteritis, be a kind of acute, contact, the sepsis sexually transmitted disease of harm duck, goose and other Anseriformes birds, with blood vessel injury, digestive tract hemorrhage necrosis, lymphoid organ is impaired and parenchymatous organ's degeneration turns to principal character.The duck of any kind, age and sex can both infect, but the sickness rate of the duck that grows up is higher than young duck, wherein the mortality ratio with the duck of laying eggs the highest (Yin Zhen, Liu Jinghua. animal virology [M]. second edition. Beijing: Science Press, 1997:1073-1077).This disease by Baudet (BaudetA E R F.mortality in ducks in the Netherlands caused by a filterable virus.Fowl plague[J] .Tijdschr Diergeneeskd, 1923,50:455-459.) reported first is in Holland, (Huang Yongxian such as Huang Yinxian, Europe is kept and is expressed. intend the research [J] of duck plague. and south China agricultural college journal, 1959,1 (1): 67-78) find the existence of this disease first in China, become serious threat at present and supported one of main eqpidemic disease of duck industry in nineteen fifty-nine.This sick cause of disease be duck plague virus (Duck Plague Virus, DPV) or claim that (DuckEnteritis Virus, DEV), duck simplexvirus I type (Duck Herpesvirus Type 1) belongs to herpetoviridae to duck enteritis virus, does not adhere to separately.According to pertinent data (Yin Zhen, Liu Jinghua. animal virology [M]. second edition. Beijing: Science Press, 1997:1073-1077), isolating all over the world duck plague virus strain, be consistent on antigenicity, promptly have the common immunogenicity, all can produce mutual immunity.Since the sixties in 20th century, duck plague virus chicken embryo attenuated vaccine is used widely in the whole world.China also use duck plague chicken embryo or chick embryo fibroblast attenuated vaccine prevention duck plague (the Chinese veterinary drug allusion quotation council. People's Republic of China's veterinary drug allusion quotation [M]. the 2005 version. three ones. Beijing: Chinese agriculture press .2006.), and proves effective.
The inactivated vaccine security is good, does not need the cryopreservation transportation, thereby more can guarantee the result of use of vaccine.Existing duck plague attenuated live vaccines, in use because preservation transportation is improper, exist immuning failure possibility (poplar is yet to be built etc. the generation [J] of immunization failure and duck plague. Chinese poultry, 1998,20 (11): 20-21).
Summary of the invention
The purpose of this invention is to provide a kind of duck plague vaccine and special strains thereof.
Duck plague virus provided by the present invention is the duck embryo adaptation virus that duck plague simplexvirus AV1221 strain goes down to posterity and obtains through susceptible duck embryo.
Duck plague virus of the present invention specifically can be duck plague simplexvirus AV1221 strain and passes the duck embryo adaptation virus that two to seven generations obtained through susceptible duck embryo, is preferably duck plague simplexvirus AV1221 strain and passes the duck embryo adaptation virus that two to four generations obtained through susceptible duck embryo.
Wherein, the duck embryo that duck plague simplexvirus AV1221 strain obtains through susceptible duck embryo biography three generations adapts to poison-duck plague simplexvirus AV1221 DE3 strain and is preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center (being called for short CGMCC) (address is Da Tun road, Chaoyang District, BeiJing, China city) on November 27th, 2007, and preservation registration number is CGMCC No.2268.
Duck plague virus of the present invention can be used for preparing the duck plague inactivated vaccine.
Duck plague vaccine provided by the present invention, its activeconstituents are the duck embryo adaptation virus that the duck plague simplexvirus AV1221 strain of deactivation goes down to posterity and obtains through susceptible duck embryo.
Also contain the animal vaccine adjuvant in the described vaccine.Described adjuvant can be injection white oil, Si Ben-80, aluminum stearate and tween-80.
In the described duck plague vaccine, described activeconstituents is that the viral liquid deactivation with 94-96 capacity part obtains, and to be duck plague simplexvirus AV1221 strain adapt to the blastochyle that obtains behind the virus inoculation susceptible duck embryo through the susceptible duck embryo duck embryo that obtains that goes down to posterity to described viral liquid; Duck plague simplexvirus AV1221 strain is 10 through the susceptible duck embryo content that the duck embryo that obtains adapts to virus that goes down to posterity in the described viral liquid
4.5-10
5.5ELD
50/ 0.2ml (before the deactivation);
Contain injection white oil 138-288 capacity part in the described vaccine, Si Ben-80 5-24 capacity part, aluminum stearate 1.5-6 weight part, tween-80 4-8 capacity part;
Described capacity part: weight part=ml: g.
Described duck plague simplexvirus AV1221 strain can pass for two to seven generations through the duck embryo, was preferably through the duck embryo and passed for two to four generations.
Another object of the present invention provides a kind of method for preparing the duck plague vaccine.
The method for preparing the duck plague vaccine provided by the present invention, be with following oil phase and water according to (1-3): 1 volume ratio is mixed and made into emulsion;
Described water can be prepared as follows: after the viral liquid deactivation with 94-98 capacity part, add aseptic tween-80 2-6 capacity part, mixing obtains water; Described viral liquid is the blastochyle that duck plague simplexvirus AV1221 strain obtains after susceptible duck embryo goes down to posterity the duck embryo adaptation virus inoculation susceptible duck embryo that obtains; Duck plague simplexvirus AV1221 strain is 10 through the susceptible duck embryo content that the duck embryo that obtains adapts to virus that goes down to posterity in the described viral liquid
4.5-10
5.5ELD
50/ 0.2ml (before the deactivation);
Described oil phase can be prepared as follows: in the ratio preparation oil phase of injection white oil 92-96 capacity part, Si Ben-80 4-8 capacity part and aluminum stearate 1-2 weight part;
Described capacity part: weight part=ml: g.
In this method, described duck plague simplexvirus AV1221 strain passed for two to seven generations through the duck embryo, was preferably through the duck embryo and passed for two to four generations.
Experiment showed, with duck plague simplexvirus AV1221 strain duck embryo and adapt to systemic adverse reactions and the tangible local reaction that the malicious duck plague vaccine for preparing does not cause laying ducks.Vaccine uses different immunization routes on different varieties, the duck at age (4 ages in days～12 monthly ages), all can induce to produce protection more than 80%.Behind immune duck of vaccine, different time induces the NAT (GMT) of generation to be respectively, 7 days is 3.2,10 days to be 4.6,14 days to be to be 7 in 8,21 days, 30 days and 60 days; Exempt to produce in back 10 days 80% and attack the poison protection, can produce protection fully in 14 days and 21 days.Behind the vaccine immunity duck,, can resist the attack of strong poison effectively although induce the NAT of generation lower.
Duck plague vaccine of the present invention uses inactivation of viruses to make, and security is good; Preservation, transportation (2-8 ℃) and easy to use, immune effect is stable.
Embodiment
Embodiment 1, utilize the duck embryo of duck plague simplexvirus AV1221 strain to adapt to poison preparation duck plague vaccine
One, the production of the duck plague simplexvirus AV1221 strain foundation of kind of malicious seed lot
Go down to posterity by the duck embryo, the duck embryo of having set up duck plague simplexvirus AV1221 strain adapts to poison.By comparison to characteristics such as the immunogenicity of 3 strain DPV, virulence, select duck plague simplexvirus AV1221 strain DE2 for duck embryo poison as seedling with kind of a poison, duck plague virus NJ strain D
5Use strong poison for duck hepatic tissue poison as checking.Use the duck embryo to AV1221 strain DE
2Poison has carried out continuous 6 times and has gone down to posterity.DE2 generation～DE7 is shown for the qualification result of freeze-drying kind poison: do not find that bacterium, mould, mycoplasma and exogenous virus pollute; Viral level is that every 0.2mL contains 10
4.38～10
5.25ELD
50Can be neutralized by the DPV antiserum(antisera); Make inactivated vaccine, immunity all can produce protection fully after becoming duck.According to test-results, set up and produced with kind of the seed lot of poison.
The used experiment material of following experiment is as follows:
A, viral duck plague simplexvirus AV1221 strain, duck hepatic tissue poison is available from China Veterinery Drug Inspection Office; Duck plague virus NJ strain (DPV NJ strain) (available from China Veterinery Drug Inspection Office) duck hepatic tissue poison.
B, antiserum(antisera) DPV antiserum(antisera) (available from China Veterinery Drug Inspection Office).According to existing " People's Republic of China's veterinary drug allusion quotation " (the Chinese veterinary drug allusion quotation council. People's Republic of China's veterinary drug allusion quotation [M]. the 2005 version. three ones. Beijing: Chinese agriculture press, 2006.) regulation the neutralizing antibody measuring method carry out titration, the result shows that NAT is 1: 16.
C, chicken embryo 9-10 age in days SPF chicken embryo, logical laboratory animal company limited provides by Beijing Cimmeria dimension.Be used for exogenous virus and pollute check.
D, duck embryo 11-12 age in days susceptible duck embryo, popular from no bird flu and duck plague, the kind duck group who did not inject the duck plague vaccine.
E, 2～12 monthly age of tame duck susceptible sheldrake or cherry valley duck from no bird flu and the popular duck group of duck plague, were not injected the duck plague vaccine.
Concrete experimental technique and result are as follows:
1, the duck embryo adapts to the preparation and the viral level mensuration of poison
1) virus goes down to posterity, and obtains the duck embryo and adapts to poison
Duck plague simplexvirus AV1221 strain goes down to posterity with susceptible duck embryo.After using sterile saline that virus is diluted, through chorioallantoic membrane inoculation duck embryo, every embryo 0.2mL.Cultivate for 37 ℃ and observed 6-8 days.Collect the blastochyle and the chorioallantoic membrane of dead embryo after 48 hours, add penicillin and Streptomycin sulphate after the homogenate, make the final concentration of penicillin and Streptomycin sulphate be 100-400 unit/ml, make the embryo poison, obtain first-generation duck embryo and adapt to poison, be that duck plague simplexvirus AV1221 strain duck embryo passes monobasic duck embryo poison (name is called duck plague simplexvirus AV1221 DE1 strain), be used to prepare freeze-drying kind poison or go down to posterity.Duck plague simplexvirus AV1221 strain duck embryo biography monobasic duck embryo poison is inoculated susceptible duck embryo more according to the method described above to go down to posterity, obtain duck plague simplexvirus AV1221 strain duck embryo and pass bibasic duck embryo poison (s-generation duck embryo adaptation poison, name is called duck plague simplexvirus AV1221 DE2 strain), go down to posterity successively, obtain duck plague simplexvirus AV1221 strain duck embryo and pass triple-substituted duck embryo poison (third generation duck embryo adaptation poison, name is called duck plague simplexvirus AV1221 DE3 strain), duck plague simplexvirus AV1221 strain duck embryo passes duck embryo poison (the 4th generation duck embryo adaptation poison in four generations, name is called duck plague simplexvirus AV1221 DE4 strain), duck plague simplexvirus AV1221 strain duck embryo passes duck embryo poison (the 5th generation duck embryo adaptation poison in five generations, name is called duck plague simplexvirus AV1221 DE5 strain), duck plague simplexvirus AV1221 strain duck embryo passes hexabasic duck embryo poison (the 6th generation duck embryo adaptation poison, name is called duck plague simplexvirus AV1221 DE6 strain), duck plague simplexvirus AV1221 strain duck embryo passes the duck embryo poison (the 7th generation duck embryo adaptation poison, name is called duck plague simplexvirus AV1221 DE7 strain) in seven generations.Wherein, duck plague simplexvirus AV1221DE3 strain is preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center on November 27th, 2007, and preservation registration number is CGMCC No.2268.
DPV NJ strain is gone down to posterity with 12 monthly age susceptible sheldrakes.After virus diluted, intramuscular injection became duck.The liver of the dead duck of aseptic collection typical case morbidity is made the hepatic tissue poison, is used to prepare freeze-drying kind poison or goes down to posterity.DPV NJ strain passed for 5 generations altogether, obtained duck plague virus NJ strain and became duck to pass 5 generation viruses (name is called NJ D5 or NJ strain D5 poison)
2) viral level is measured
Duck plague simplexvirus AV1221 strain duck embryo adapts to poison (duck plague simplexvirus AV1221 DE2-DE7 strain) and carries out viral level mensuration with susceptible duck embryo respectively.Use sterile saline that virus is made 10 times of serial dilutions, get diluent through chorioallantoic membrane 8 inoculation duck embryos, 5 pieces of each extent of dilution inoculations, every embryo 0.2mL.Cultivate for 37 ℃ and observed 6-8 days.The death condition of record embryo.Calculate ELD according to the Reed-Muench method
50(medium lethal dose).
The result shows that every 0.2mL duck plague simplexvirus AV1221 DE2-DE7 strain contains 10
4.38-10
6.18ELD
50Virus.
2, specificity is identified
Use sterile saline respectively the virus and the duck plague virus NJ D5 of duck plague simplexvirus AV1221 DE2-DE7 strain to be diluted to 200MLD/mL, mix with equivalent duck plague virus antiserum(antisera) respectively; Establish virus control (100ELD simultaneously
50/ 0.2mL or 100MLD/mL) and the physiological saline contrast.Room temperature effect 1 hour.10 pieces of susceptible duck embryos are respectively inoculated in AV1221DE2-DE7 strain of duck plague simplexvirus and anti-serum mixture and contrast, and route of inoculation is a chorioallantoic membrane, every embryo 0.2mL.Cultivate for 37 ℃ and observed 6-8 days, the death condition of record embryo.5 of 12 monthly age susceptible sheldrakes are respectively inoculated in duck plague virus NJD5 and anti-serum mixture and contrast, every intramuscular injection 1mL, and isolated rearing was observed 10-14 days, the death condition of record duck.
With the duck plague virus antiserum(antisera) result's (table 1) that 6 strain virus carry out the specificity evaluation is shown, 10 pieces of duck embryos of inoculation duck plague simplexvirus AV1221 DE2 strain and anti-serum mixture are all survived, and as virus control, 10 pieces of duck embryos of inoculation duck plague simplexvirus AV1221 DE2 strain are all dead; 10 pieces of duck embryos of inoculation duck plague simplexvirus AV1221DE3 strain CGMCC No.2268 and anti-serum mixture are all survived, and as virus control, 10 pieces of duck embryos of inoculation duck plague simplexvirus AV1221 DE3 strain CGMCC No.2268 are all dead; 10 pieces of duck embryos of inoculation duck plague simplexvirus AV1221 DE4 strain and anti-serum mixture are all survived, and as virus control, 10 pieces of duck embryos of inoculation duck plague simplexvirus AV1221 DE4 strain are all dead; 10 pieces of duck embryos of inoculation duck plague simplexvirus AV1221DE5 strain and anti-serum mixture are all survived, and as virus control, 10 pieces of duck embryos of inoculation duck plague simplexvirus AV1221 DE5 strain are all dead; 10 pieces of duck embryos of inoculation duck plague simplexvirus AV1221 DE6 strain and anti-serum mixture are all survived, and as virus control, 10 pieces of duck embryos of inoculation duck plague simplexvirus AV1221 DE6 strain are all dead; 10 pieces of duck embryos of inoculation duck plague simplexvirus AV1221 DE7 strain and anti-serum mixture are all survived, and as virus control, 10 pieces of duck embryos of inoculation duck plague simplexvirus AV1221 DE7 strain are all dead; 5 of inoculation duck plague virus NJ D5 and anti-serum mixture become duck all strong alive; And as virus control, 5 of inoculation duck plague virus NJ D5 become duck all dead.Illustrate that duck plague simplexvirus AV1221 DE2-DE7 strain and NJ D5 can be neutralized by the DPV antiserum(antisera).
Table 17 strain DPV specificity qualification results
3, pure property check
According to existing " People's Republic of China's veterinary drug allusion quotation " (the Chinese veterinary drug allusion quotation council. People's Republic of China's veterinary drug allusion quotation [M]. the 2005 version. three ones. Beijing: Chinese agriculture press, .2006.) regulation is polluted check (wherein exogenous virus pollutes check employing chick embryo method) to carry out bacterium, mould, mycoplasma and exogenous virus as the poison of sowing: duck plague simplexvirus AV1221 DE2-DE7 strain and duck plague virus NJ D5.Assay shows, does not find in duck plague simplexvirus AV1221 DE2-DE7 strain and the duck plague virus NJ D5 kind poison that bacterium, mould, mycoplasma and exogenous virus pollute.
4, to the pathogenic test of this animal
Use sterile saline respectively 10 times of serial dilutions to be made in duck plague simplexvirus AV1221 DE2 strain and duck plague virus NJ D5, get diluent and inoculate 3 monthly age susceptible sheldrakes, 5 of each extent of dilution inoculations, every intramuscular injection 1mL.Isolated rearing was observed 10-14 days, record typical case morbidity and death condition.With the high dilution of the virus that causes the death of whole inoculation duck as MLD (minimum lethal dose).
The result is as shown in table 2, shows NJ D
5Stronger pathogenic to becoming duck to have, its minimum lethal dose is 10
-8/ 1g organizes poison.And duck plague simplexvirus AV1221 DE2 to become duck pathogenic a little less than.
Table 23 strain DPV is to becoming the pathogenicity test results of duck
Annotate: ND, expression undetermined.In the mark of " morbidity and dead " row, denominator is represented the total number of elements of one-tenth duck inoculated, divides that subrepresentation is strong lives or dead number of elements.
5, immunogenicity
With the duck plague simplexvirus AV1221 DE2-DE7 of duck embryo culture, the method according to the preparation in the following step 2 0601 prepares vaccine respectively, 5 of each immunization 65 age in days susceptible sheldrakes, every subcutaneous injection 0.5mL.After 21-28 days,, use duck plague virus NJ D5 virus to attack poison, every duck intramuscular injection 1000MLD/1mL together with 5 of the contrast ducks (not immunity) of condition.Isolated rearing was observed 10-14 days, and the morbidity death condition of duck respectively organized in record.Finish strong the living of duck with the observation period and be judged to be protection.
The result is as shown in table 3, behind the vaccine immunity duck of duck plague simplexvirus AV1221 DE2-DE7 preparation, all can resist the DPV strong virus attack for 4, and immunogenicity is good.
Table 3
Strain virus immunogenicity determining result
Annotate: ND, expression is immunity not.In the mark of " protection result " row, denominator is represented the total number of elements of one-tenth duck inoculated, divides subrepresentation strong duck number of elements of living.
6, produce with the foundation and the evaluation of planting malicious seed lot
According to above-mentioned test-results, select duck plague simplexvirus AV1221 strain as the seedling strain, duck plague virus NJ strain is as the check virulent strain.In view of the above, as primordial seed, the s-generation duck plague simplexvirus AV1221 DE2 behind adaptation duck embryo begins to set up basic seed and seeding with duck plague simplexvirus AV1221 strain duck hepatic tissue poison.Use susceptible duck embryo that duck plague simplexvirus AV1221 DE2 has been carried out continuous passage, obtain duck plague simplexvirus AV1221 DE3 strain CGMCC No.2268, duck plague simplexvirus AV1221 DE4, duck plague simplexvirus AV1221 DE5, duck plague simplexvirus AV1221 DE6 and duck plague simplexvirus AV1221 DE7, and make freeze-drying kind poison.According to veterinary biologics experimental study technical director principle [M] (Ministry of Agriculture's veterinary drug is evaluated the center. the 2006 version) regulation, each has been done comprehensive evaluation for kind of a poison.The result is as follows:
1) viral level uses the duck embryo that prepared each generation kind poison of duck plague simplexvirus AV1221 strain has been carried out viral level mensuration according to the method for step 1.Result such as table 4, the viral level that shows each generation kind poison are that every 0.2mL contains 10
4.38～10
5.25ELD
50, differ less than 1 titre.Show that virus keeps stable in the process of going down to posterity.
Table 4 AV1221 kind viral disease poison assay result
2) virulence of duck embryo is done 10 with the kind poison of each generation with physiological saline
2Doubly after the dilution, inoculate 10 pieces of 11 age in days susceptible duck embryos through chorioallantoic membrane.Every embryo 0.2mL.Cultivate for 37 ℃ and observed 6-8 days.As a result, the virus of 5 generations duck embryo more than 9 pieces that all can cause death.
3) pure property is carried out steriling test, mycoplasma check and exogenous virus check according to the method for step 3 to each generation kind poison, and result such as table 5 show from the kind poison of 5 generations not detect bacterium, mould and mycoplasma contamination, from DE
2, DE
5And DE
7In generation, plants and do not detect the exogenous virus pollution in the poison.
The pure property assay of table 5 AV1221 kind poison
Annotate: "-", expression is negative.ND is not for doing this check.
4) specificity according to the method for step 2 to DE
2, DE
4, and DE
7Plant poison and carried out the specificity evaluation.The result shows that the virus of 3 generations all can be neutralized by the DPV antiserum(antisera).
5) immunogenicity prepares vaccine, immune 3 monthly age susceptible sheldrakes with duck plague simplexvirus AV1221 DE2-DE7 according to the method for the preparation in the following step 2 0601 respectively.After 21-28 days, use the DPV strong virus attack.Protection the results are shown in Table 6, shows 6 generation virus vaccines immune ducks, all can produce protection fully after attacking poison.Show that virus immunogenicity in the duck embryo goes down to posterity process keeps stable.
Table 6 AV1221 kind poison immunogenicity determining result
Annotate: ND, expression is immunity not.In the mark of " protection result " row, denominator represents to attack the total number of elements of one-tenth duck of poison, divides subrepresentation strong duck number of elements of living.
6) plant poison and use determining of generation
According to qualification result to 6 generation kind poison, determine that kind of the highest use generation of poison was the 7th generation, 2-4 is on behalf of basic seed, and the use generation of seeding was no more than for 3 generations.
Two, the preparation of duck plague vaccine and potency test and proof test
This step uses basis kind poison and the duck embryo set up to prepare 3 batches of seedings, and breeds, after the collection blastochyle virus, the deactivation of 0.1-0.2% formaldehyde solution, prepared 3 batches of duck plague inactivated vaccines with the mineral oil adjuvant emulsion through the duck embryo with these seeds.Use into duck and measure, the vaccine minimum immune dosage is 0.12mL, determines that using dosage is every plumage part 0.5mL.Single dose, single dose repeat and overdose proof test result is, 3 batches of vaccines do not cause the systemic adverse reactions and the tangible local reaction of laying ducks.The immune challenge test result of 3 batches of vaccines shows that vaccine uses different immunization routes on different varieties, the duck at age (4 ages in days～12 monthly ages), all can induce to produce protection more than 80%.Behind immune duck of vaccine, different time induces the NAT (GMT) of generation to be respectively, 7 days is 3.2,10 days to be 4.6,14 days to be to be 7 in 8,21 days, 30 days and 60 days; Exempt to produce in back 10 days 80% and attack the poison protection, can produce protection fully in 14 days and 21 days.Behind the vaccine immunity duck,, can resist the attack of strong poison effectively although induce the NAT of generation lower.
The used experiment material of following experiment except that A-G, the same step 1 of other material source.
A, injection white oil MARCOL 52 are available from French Esso company, lot number VG3288982P.
B, tween-80 CRILLET 4 are available from Singapore CRODA company, lot number 15488.
C, Si Ben-80 CRILL 4 are available from Singapore CRODA company, lot number 15438.
D, aluminum stearate are available from the abundant bright Industrial Co., Ltd. in Shanghai, lot number 070706.
E, duck embryo 11-12 age in days susceptible duck embryo, popular from no bird flu and duck plague, the kind duck group who did not inject the duck plague vaccine.Be used for seeding, the viral preparation of seedling, viral level is measured, the production specificity evaluation of seed etc.
F, tame duck 4 ages in days～12 monthly age susceptible sheldrakes, cherry valley duck or gold are decided duck, from no bird flu and the popular duck group of duck plague, do not inject the duck plague vaccine.The safety testing and the potency test that are used for vaccine.
The duck plague simplexvirus AV1221 strain seedling of G, the preparation of viral step 1 is planted poison (DE with the basis
2～DE
4In generation, is malicious), ELD
50〉=10
-4.38/ 0.2mL, DPV NJ strain D
5In generation,, tissue was malicious, and MLD is 10
-8/ g is used for the check of vaccine.
1, vaccine production
1) seeding preparation and check
Respectively with after duck plague simplexvirus AV1221 DE2, duck plague simplexvirus AV1221 DE3 strain CGMCC No.2268 and the malicious rehydration of duck plague simplexvirus AV1221 DE4 freeze-drying basis kind, use sterile saline to dilute, through chorioallantoic membrane inoculation susceptible duck embryo, every embryo 0.2mL.Cultivate for 37 ℃ and observed 4-6 days.Collect embryo dead after 48 hours, aseptic collection blastochyle and chorioallantoic membrane add penicillin and Streptomycin sulphate after the homogenate, make the final concentration of penicillin and Streptomycin sulphate be 100-400 unit/ml, make duck embryo poison.As seeding.Carry out viral level mensuration, specificity evaluation and the check of pure property according to the method for step 1.
Use duck plague simplexvirus AV1221 DE2, duck plague simplexvirus AV1221 DE3 strain CGMCCNo.2268 and duck plague simplexvirus AV1221 DE4 basis to plant poison respectively and prepared 3 batches of seedings, every crowd of 40mL.Assay is 3 batches does not all have bacterium, mould, mycoplasma and exogenous virus pollution, can be neutralized by the DPV antiserum(antisera).The viral level difference 10 of duck plague simplexvirus AV1221 DE2, duck plague simplexvirus AV1221 DE3 strain CGMCC No.2268 and duck plague simplexvirus AV1221 DE4 seed
5.38, 10
5.25With 10
4.83ELD
50/ 0.2mL.
2) seedling prepares with viral liquid
Use sterile saline that the seeding of step 1) is diluted, through allantoic cavity inoculation duck embryo, every embryo 0.2mL.Cultivate for 37 ℃ and observed 4-6 days.Collect the dead embryo and the embryo of living after 48 hours, the aseptic collection blastochyle.As the viral liquid of seedling.
The result uses 3 batches of seedings to prepare 3 batches of viral liquid respectively, every crowd of 200mL: duck plague simplexvirus AV1221 DE2 virus liquid, duck plague simplexvirus AV1221 DE3 strain CGMCC No.2268 virus liquid and duck plague simplexvirus AV1221 DE4 virus liquid, its viral level is respectively 10
5.0, 10
4.5With 10
4.83ELD
50/ 0.2mL.
3) inactivation of virus and check
To step 2) in three batches of viral liquid of preparation, add formaldehyde solution, the final concentration that makes formaldehyde is 0.1-0.2% (volumn concentration), fully falls bottle behind the mixing; 37 ℃ deactivation 24-36 hour, every therebetween interval 4-8 hour jolting 1 time.After deactivation was finished, the deactivation check was done in sampling.Use 10 pieces of 11 age in days susceptible duck embryos, every embryo is cultivated for 37 ℃ and was observed 7 days through chorioallantoic membrane inoculation 0.2mL stoste.And method like this, get a blastochyle blind passage generation.The result shows that the viral liquid after 3 batches of deactivations connects and passed for 2 generations, all do not cause embryo death on the duck embryo.Show that virus has been inactivated thoroughly.
4) vaccine preparation
With three batches of vaccines 0601,0602 and 0603 of viral liquid preparation through the step 3) inactivation treatment.
I) 0601 preparation
Following capacity part: weight part=ml: g.
A) oil phase preparation
Ratio preparation oil phase in injection white oil 94 capacity parts, Si Ben-80 6 capacity part and aluminum stearate 1.5 weight parts.
The injection that takes a morsel white oil mixes with aluminum stearate, and heating is dissolved to translucent and mixed with full dose Si Ben-80 and injection white oil, and through 116 ℃ of sterilizations 30 minutes, it was standby to be chilled to room temperature.Obtain oil phase.
B) water preparation
With 96 weight part steps 2) viral level be 10
5.0ELD
50The duck plague simplexvirus AV1221 DE2 virus liquid of/0.2mL (before the deactivation) through the step 3) deactivation after the assay was approved, adds sterilization tween-80 4 capacity parts, behind the shake well, puts 2-8 ℃ of placement and spends the night, and dissolves fully up to tween-80.Obtain water.
According to oil phase and 1.5: 1 volume ratio of water, oil phase is added in the homogenate cup, when slowly running, slowly add water.Added behind the water homogenate 2-3 minute, and stopped to add before the emulsification 0.01% Thiomersalate of emulsion gross weight, obtain lot number and be 0601 500mL vaccine.
Ii) 0602 preparation
Following capacity part: weight part=ml: g.
A) oil phase preparation
Ratio preparation oil phase in injection white oil 92 capacity parts, Si Ben-80 8 capacity part and aluminum stearate 1 weight part.
The injection that takes a morsel white oil mixes with aluminum stearate, and heating is dissolved to translucent and mixed with full dose Si Ben-80 and injection white oil, and through 116 ℃ of sterilizations 30 minutes, it was standby to be chilled to room temperature.Obtain oil phase.
B) water preparation
With 94 weight part steps 2) viral level be 10
4.5ELD
50The duck plague simplexvirus AV1221 DE3 strain CGMCC No.2268 virus liquid of/0.2mL (before the deactivation) through the step 3) deactivation after the assay was approved, adds sterilization tween-80 4 capacity parts, behind the shake well, puts 2-8 ℃ of placement and spends the night, and dissolves fully up to tween-80.Obtain water.
According to oil phase and 2: 1 volume ratio of water, oil phase is added in the homogenate cup, when slowly running, slowly add water.Added behind the water homogenate 2-3 minute, and stopped to add before the emulsification 0.01% Thiomersalate of emulsion gross weight, obtain lot number and be 0602 500mL vaccine.
Iii) 0603 preparation
Following capacity part: weight part=ml: g.
A) oil phase preparation
Ratio preparation oil phase in injection white oil 95 capacity parts, Si Ben-80 5 capacity part and aluminum stearate 2 weight parts.
The injection that takes a morsel white oil mixes with aluminum stearate, and heating is dissolved to translucent and mixed with full dose Si Ben-80 and injection white oil, and through 116 ℃ of sterilizations 30 minutes, it was standby to be chilled to room temperature.Obtain oil phase.
B) water preparation
With 96 weight part steps 2) viral level be 10
4.83ELD
50The duck plague simplexvirus AV1221 DE4 virus liquid of/0.2mL (before the deactivation) through the step 3) deactivation after the assay was approved, adds sterilization tween-80 4 weight parts, behind the shake well, puts 2-8 ℃ of placement and spends the night, and dissolves fully up to tween-80.Obtain water.
According to oil phase and 3: 1 weight ratio of water, oil phase is added in the homogenate cup, when slowly running, slowly add water.Added behind the water homogenate 2-3 minute, and stopped to add before the emulsification 0.01% Thiomersalate of emulsion gross weight, obtain lot number and be 0603 500mL vaccine.
Lot number is respectively 0601,0602 and 0603 500mL vaccine, quantitatively packing, after the labeling, 2～8 ℃ of preservations.Sampling simultaneously according to the rules (the Chinese veterinary drug allusion quotation council. People's Republic of China's veterinary drug allusion quotation [M]. the 2005 version. three ones. Beijing: Chinese agriculture press, 2006.) do steriling test, physical behavior check, formaldehyde and Thiomersalate assay.As a result, 3 batches of equal asepsis growths of vaccine; Physical behavior check, formaldehyde and Thiomersalate assay are all up to specification.
2, minimum immune dosage is measured
Use 0601 batch of vaccine, dosage be respectively 0.5mL/ only, 0.25mL/ only and 0.12mL/ only.5 94 age in days susceptible sheldrakes of each dosage injection, 5 together with condition after 24 days contrast ducks (not carrying out immunity), with duck plague virus NJ D5 (the NJ strain D of step 1 preparation
5Poison) attacks every duck intramuscular injection 10
3MLD/1mL.Isolated rearing was observed 10-14 days, and the morbidity death condition of duck respectively organized in record.Finish strong the living of duck with the observation period and be judged to be protection.The result is as shown in table 7.
Table 7 duck plague inactivated vaccine minimum immune dosage measurement result
Annotate: *, molecule is the strong duck quantity of living, denominator is for attacking malicious duck quantity.*, 0.25mL group duck dies unexpectedly between duration of immunity 1.ND, expression is immunity not.
The result shows that the 0.25mL vaccine immunity can produce the protection of 4/4 duck, and the 0.12mL vaccine produces 4/5 protection.Minimum immune dosage is 0.12mL.In order to leave surplus capacity, adopt the using dosage of 0.5mL as vaccine.
3, immuning effect test uses 3 batches of vaccines, adopt subcutaneous (deciding duck) or muscle (with 12 monthly age sheldrakes) injecting immune respectively with 4 age in days cherry valley ducks and 4 monthly ages gold, 5 of the every batch of vaccine immunities, every 0.5mL (4 age in days cherry valley ducks after 14 days with dosage booster immunization 1 time).5 together with condition after 21 days contrast ducks (not carrying out immunity), with duck plague virus NJ D5 (the NJ strain D of step 1 preparation
5Poison) attacks every duck intramuscular injection 10
3MLD/1mL.Isolated rearing was observed 10-14 days, and the morbidity death condition of duck respectively organized in record.Finish strong the living of duck with the observation period and be judged to be protection.The result is shown in table 8-10.
The immunity gold of 3 batches of duck plague inactivated vaccines of table 8 subcutaneous route is decided duck challenge test result
Annotate: ND, expression is immunity not.In the mark of " protection " row, denominator represents to attack the total number of elements of one-tenth duck of poison, divides subrepresentation strong duck number of elements of living.
The immune challenge test result of 3 batches of duck plague inactivated vaccines of table 9 intramuscular routes
Annotate: ND, expression is immunity not.In the mark of " protection " row, denominator represents to attack the total number of elements of one-tenth duck of poison, divides subrepresentation strong duck number of elements of living.
The duckling immunity challenge test result of 3 batches of duck plague inactivated vaccines of table 10
Annotate: ND, expression is immunity not.In the mark of " protection " row, denominator represents to attack the total number of elements of one-tenth duck of poison, divides subrepresentation strong duck number of elements of living.*, respectively there is 1 wing number to come off between duration of immunity.*, unexpected death is 1 when attacking the poison injection.
The result shows, the 3 batches of duck plague inactivated vaccines adopt the different approaches immunity to the duck of different varieties, different ages (4 monthly ages and 12 monthly ages), all can produce fully and protect.3 batches of vaccines adopt second immunisation to 4 age in days cherry valley ducks, and 1 batch produces 4/5 protection, and 2 batches produce 4/4 protection.
Determine according to The above results, adopt the method for testing efficacy of immune challenge test as vaccine.Use 2～12 the monthly age 5 of ducks, every subcutaneous injection vaccine 0.5mL after 21～28 days, together with 5 of the contrast ducks (not carrying out immunity) of condition, uses 10
-3The MLD virulent virus is attacked.Observed 14 days, with 5/5 death of contrast duck, immune duck 4/5 protection is as rendeing a service criterion of acceptability.
4, immune generation phase test
Use 0602 batch of vaccine, 10 of subcutaneous route immunity 12 monthly age sheldrakes, every 0.5mL.Other gets 10 and (does not carry out immunity) in contrast.Respectively at exempting from respectively to get 5 immune ducks and contrast duck in back 10 days and 14 days, with duck plague virus NJ D5 (the NJ strain D of step 1 preparation
5Poison) attacks every duck intramuscular injection 10
3MLD/1mL.Isolated rearing was observed 14 days, and the morbidity death condition of duck respectively organized in record.Finish strong the living of duck with the observation period and be judged to be protection.The result is as shown in table 11.
Table 11 duck plague inactivated vaccine immunity generation phase test-results
Annotate: in the mark of " protection " row, denominator represents to attack the total number of elements of one-tenth duck of poison, divides subrepresentation strong duck number of elements of living.
Test-results shows, the vaccine of 1 using dosage can produce the protection of 4/5 duck in once immune back 10 days, and once immune back 14 days and 21 days (table 9) can produce fully to be protected.
5, neutralizing antibody produces
Use 0602 batch of vaccine, 15 of subcutaneous route immunity 12 monthly age sheldrakes, every 0.5mL.Respectively at the blood of 5 ducks of 7,10,14,21,30 and 60 days random acquisitions before the immunity, after the immunity, separation of serum; Simultaneously carry out challenge test respectively at respectively randomly drawing 5 ducks in 10 and 14 days after the immunity.Use DPV AV1222 (available from China Veterinery Drug Inspection Office) strain E
62Generation virus (breeding of chicken embryo the 62nd generation poison) and SPF chicken embryo are measured the NAT of serum, and calculate geometric mean titer (GMT) that each is organized.
Result such as table 12.
Table 12 duck plague inactivated vaccine neutralizing antibody produces test-results
The result shows that immunity began to produce neutralizing antibody in back 7 days, obviously rose, and maintain on certain level in 14 days.The situation that antibody produces with attack poison protection basically identical as a result.
6,3 batches of vaccines are used in proof test, and 21 ages in days gold decides duck or 12 monthly age sheldrakes have carried out single dose, single dose repeats and the overdose safety testing.The result is shown in table 13-15.
3 batches of duck plague inactivated vaccines of table 13 single dose proof test result
| The vaccine lot number | Injecting pathway and dosage | Duck varieties and age | Observe result after 14 days | Reaction |
| 0601 | The subcutaneous 0.5mL/ of nape portion only | 21 ages in days gold is decided duck | 5/5 strong living | Injecting had the food of subtracting in back 24 hours slightly, and injection site nodosity slightly in 7 days disappeared after 7 days. |
| 0601 | The subcutaneous 0.5mL/ of nape portion only | 12 monthly age sheldrakes | 5/5 strong living | Injecting had the food of subtracting in back 24 hours slightly, and injection site nodosity slightly in 7 days disappeared after 7 days. |
| 0602 | The subcutaneous 0.5mL/ of nape portion only | 21 ages in days gold is decided duck | 5/5 strong living | Injecting had the food of subtracting in back 24 hours slightly, and injection site nodosity slightly in 7 days disappeared after 7 days. |
| 0602 | The subcutaneous 0.5mL/ of nape portion only | 12 monthly age sheldrakes | 5/5 strong living | Injecting had the food of subtracting in back 24 hours slightly, and injection site nodosity slightly in 7 days disappeared after 7 days. |
| 0603 | The subcutaneous 0.5mL/ of nape portion only | 21 ages in days gold is decided duck | 5/5 strong living | Injecting had the food of subtracting in back 24 hours slightly, and injection site nodosity slightly in 7 days disappeared after 7 days. |
| 0603 | The subcutaneous 0.5mL/ of nape portion only | 12 monthly age sheldrakes | 5/5 strong living | Injecting had the food of subtracting in back 24 hours slightly, and injection site nodosity slightly in 7 days disappeared after 7 days. |
Annotate: in the mark of " observing result after 14 days " row, denominator is represented the total number of elements of one-tenth duck of immunity, divides subrepresentation strong duck number of elements of living.
3 batches of duck plague inactivated vaccines of table 14 single dose repeats the proof test result
| The vaccine lot number | Injecting pathway and dosage | Duck varieties and age | Observe result after 14 days | Reaction |
| 0601 | The subcutaneous 0.5mL/ of nape portion only, after 21 days with this dosage of approach duplicate injection | 12 monthly age sheldrakes | 5/5 strong living | Injecting had the food of subtracting in back 24 hours slightly, and injection site nodosity slightly in 7 days disappeared after 7 days. |
| 0602 | The subcutaneous 0.5mL/ of nape portion only, after 21 days with this dosage of approach duplicate injection | 12 monthly age sheldrakes | 5/5 strong living | Injecting had the food of subtracting in back 24 hours slightly, and injection site nodosity slightly in 7 days disappeared after 7 days. |
| 0603 | The subcutaneous 0.5mL/ of nape portion only, after 21 days with this dosage of approach duplicate injection | 12 monthly age sheldrakes | 5/5 strong living | Injecting had the food of subtracting in back 24 hours slightly, and injection site nodosity slightly in 7 days disappeared after 7 days. |
Annotate: in the mark of " observing result after 14 days " row, denominator is represented the total number of elements of one-tenth duck of immunity, divides subrepresentation strong duck number of elements of living.
3 batches of duck plague inactivated vaccines of table 15 overdose repeats the proof test result
| The vaccine lot number | Injecting pathway and dosage | Duck varieties and age | Observe result after 14 days | Reaction |
| 0601 | The subcutaneous 1mL/ of nape portion only | 12 monthly age sheldrakes | 5/5 strong living | Injecting had the food of subtracting in back 24 hours slightly, and injection site nodosity slightly in 7 days disappeared after 7 days. |
| 0602 | The subcutaneous 1mL/ of nape portion only | 12 monthly age sheldrakes | 5/5 strong living | Injecting had the food of subtracting in back 24 hours slightly, and injection site nodosity slightly in 7 days disappeared after 7 days. |
| 0603 | The subcutaneous 1mL/ of nape portion only | 12 monthly age sheldrakes | 5/5 strong living | Injecting had the food of subtracting in back 24 hours slightly, and injection site nodosity slightly in 7 days disappeared after 7 days. |
Annotate: in the mark of " observing result after 14 days " row, denominator is represented the total number of elements of one-tenth duck of immunity, divides subrepresentation strong duck number of elements of living.
3 batches of vaccines are 3 kinds of safety testing results show, removing the injection back had the food of subtracting the same day slightly, and systemic adverse reactions outside the nodosity, is not seen slightly in the part in 7 days.Determine thus, use 2～12 the monthly age 5 of ducks, every subcutaneous injection 1mL vaccine.Observed 10～14 days, 5 all strong work of duck are up to the standards for vaccine safety.
Claims (7)
1, a kind of duck plague virus is that duck plague simplexvirus AV1221 strain passes the duck embryo adaptation strain duck plague simplexvirus AV1221 DE3 strain that the three generations obtains through susceptible duck embryo, and its preservation registration number is CGMCC No.2268.
2, the application of the described duck plague virus of claim 1 in preparation duck plague vaccine.
3, a kind of duck plague vaccine, its activeconstituents are the described duck plague viruses of the claim 1 of deactivation.
4, vaccine according to claim 3 is characterized in that: also contain adjuvant in the described vaccine.
5, vaccine according to claim 4 is characterized in that: described adjuvant is injection white oil, Si Ben-80, aluminum stearate and tween-80.
6, vaccine according to claim 5, it is characterized in that: described activeconstituents is that the viral liquid deactivation with the described duck plague virus of claim 1 of 94-96 capacity part obtains, described viral liquid is the blastochyle that obtains behind the described duck plague virus inoculation of the claim 1 susceptible duck embryo, and the content of the described duck plague virus of claim 1 is 10 in the described viral liquid
4.5-10
5.5ELD
50/ 0.2ml;
Contain injection white oil 138-288 capacity part in the described vaccine, Si Ben-80 5-24 capacity part, aluminum stearate 1.5-6 weight part, tween-80 4-8 capacity part; Described capacity part: weight part=ml: g.
7, a kind of method for preparing the duck plague vaccine, be with following oil phase and water according to (1-3): 1 volume ratio is mixed and made into emulsion;
Described water is prepared as follows: after the viral liquid deactivation with the described duck plague virus of claim 1 of 94-98 capacity part, add aseptic tween-80 2-6 capacity part, mixing obtains water; Described viral liquid is the blastochyle that obtains behind the described duck plague virus inoculation of the claim 1 susceptible duck embryo; The content of the described duck plague virus of claim 1 is 10 in the described viral liquid
4.5-10
5.5ELD
50/ 0.2ml;
Described oil phase is prepared as follows: in the ratio preparation oil phase of injection white oil 92-96 capacity part, Si Ben-80 4-8 capacity part and aluminum stearate 1-2 weight part;
Described capacity part: weight part=ml: g.
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| CNB2007101783719A CN100572532C (en) | 2007-11-29 | 2007-11-29 | A kind of duck plague vaccine and special strains thereof |
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| CN100572532C true CN100572532C (en) | 2009-12-23 |
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| CN102174091B (en) * | 2011-01-25 | 2013-04-24 | 四川农业大学 | Truncated and expressed duck plague virus (DPV) recombinant envelope gI protein and preparation method and application thereof |
| CN102643932A (en) * | 2012-04-20 | 2012-08-22 | 四川农业大学 | Polymerase chain reaction (PCR) detection method for distinguishing virulent strains and vaccine strains of duck plague virus (DPV) |
| CN102716483A (en) * | 2012-05-31 | 2012-10-10 | 郑州后羿制药有限公司 | Duck plague yolk antibody freeze-dried powder and preparation method thereof |
| CN103224913B (en) * | 2013-05-08 | 2014-05-14 | 中国兽医药品监察所 | Duck plague live vaccine and preparation method thereof |
| CN104436186A (en) * | 2013-09-18 | 2015-03-25 | 普莱柯生物工程股份有限公司 | Vaccine composition and preparation method and application thereof |
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