CN107875383A - Kind duck source goose parvovirus, the type adenovirus bivalent inactivated vaccine preparation method of duck 2 - Google Patents
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Abstract
The invention provides a kind duck source goose parvovirus, the type adenovirus bivalent inactivated vaccine preparation method of duck 2, the technical scheme devises targetedly process conditions according to the proliferative properties and immunogenicity of kind duck source goose parvovirus and the type adenovirus of duck 2.From the point of view of specific, the present invention, using LMH cell replicative adenovirus, is prepared into safely and effectively kind duck source goose parvovirus and adenovirus bivalent inactivated vaccine using chicken embryo propagation kind duck source goose parvovirus after being inactivated.Kind duck source goose parvovirus prepared using the present invention and adenovirus bivalent inactivated vaccine produce antibody morning, and protective rate is high.Vaccine prepared by the present invention be once inoculated with can prevent two disease, solve kind duck because vaccine is repeatedly inoculated with and caused by stress reaction.
Description
Technical field
The present invention relates to veterinary biologicses technical field, further to the technology of preparing of vaccine, and in particular to kind duck
Source goose parvovirus, the type adenovirus bivalent inactivated vaccine preparation method of duck 2.
Background technology
Kind duck gosling blast has been each duck culturing in the whole nation at present in kind duck raising area's eruption and prevalence in Fujian Province since 1997
Qu Junyou occurs.Clinically kind duck to suffer from diarrhoea, intestinal mucosa come off to form embolism as principal character, incidence of disease 50-70%, the death rate
40-60%.Clinically symptom management, provisions are unable to using Muscovy duck parvovirus live vaccine and gosling plague high immunity yolk antibody etc.
Duck industry causes larger economic loss.
Aviadenovirus is divided into I, II, III subgroup, and experts and scholars are mainly studied I and III duck group both at home and abroad at present.Duck
The type of adenovirus 2 belongs to I duck group's tentative species, is that Hungarian J.F.Bouquet in 1977 etc. is separated in kind duck earliest, it with
Known chicken and turkey it is viral not related, but can be grown in the cell of chicken and duck.Recent year researcher utilizes
Viral metagenomics are separated to the virus from Guangdong, Fujian etc. in succession, it is believed that the virus is a kind of duck source new virus.The disease is main
Occur in 20-30 age in days ducks, sick duck shows as One's spirits are drooping, depressed, row's ivory buff excrement, and serious even can be dead
Die.Pathological symptom is liver enlargement, bleeding, white spotty necrosis, and pancreas white point is downright bad, hydropericardium, and the bursa of farbricius reduces etc..Should
Sick spread speed is very fast, it is difficult to remove, larger economic loss is caused to duck culturing industry.
Kind duck source parvovirus and adenovirus are the Important Infectious Diseases of kind duck, and their often mixed infections, provisions
Duck industry causes larger economic loss, but there is no commercialized such bigeminy vaccine at present, and it is because of both to trace it to its cause
When animal is immunized as Combined vaccine in antigen, immunogenicity mutually forms influence, it tends to be difficult to while immune are reached to two kinds of viruses
Effect.
The content of the invention
It is contemplated that the technological deficiency for prior art, there is provided kind duck source goose parvovirus, the type adenovirus bigeminy of duck 2
Inactivated vaccine preparation method, to solve to lack the technical problem of such a vaccine in the prior art.
Another technical problem to be solved by the present invention is that in the prior art using kind duck source parvovirus and adenovirus as antigen
Bigeminy vaccine be difficult to produce effectively immune technical problem to two kinds of viruses simultaneously.
To realize above technical purpose, the present invention uses following technical scheme：
Kind duck source goose parvovirus, the type adenovirus bivalent inactivated vaccine preparation method of duck 2, comprise the following steps：
1) infection kind duck source goose parvovirus, 9 age in days kind ducks are taken, its liver, spleen or intestinal tissue is taken, is passed through after grinding
8000rpm rotating speeds centrifuge 10min, take supernatant, as parvovirus liquid；
2) the type adenovirus of infected duck 2,16 age in days kind ducks are taken, takes its liver or spleen tissue, after grinding through 8000rpm rotating speeds from
Heart 10min, take supernatant, as the type adenopathy venom of duck 2；
3) utilize the filter filtration step 1 in 0.22 μm of aperture) obtained by parvovirus liquid, 10-29~11 are inoculated in after dilution
Age in days is non-to exempt from chicken embryo, 37 DEG C of cultures, discards the non-specific dead germs of 24h, and 24h-120h dead germs are placed in into 4 DEG C of refrigeration 4-24h, harvest urine
Cyst fluid, by it in chicken embryo the generation of blind passage 3~5, until chicken embryo occur it is lethal, and the death time control in 96h, harvest dead germ
Allantoic fluid；According to same method numerous malicious 5~8 generation, while viral level is determined, take virus titer highest allantoic fluid stand-by；
4) utilize the filter filtration step 2 in 0.22 μm of aperture) obtained by the type adenopathy venom of duck 2, it is then pressed into cell liquid
The 0.1-2% of volume is inoculated into long into the LMH cells of individual layer state, 37 DEG C of culture 72-96h, harvest virus, multigelation 3
Secondary, in the generation of blind passage 3~5, until lesion occurs in 75% cell, and lesion time control harvests virus liquid in 72h；According to same
Quadrat method numerous malicious 5~8 generation, while viral level is determined, take virus titer highest virus liquid stand-by；
5) step 3) products therefrom is subjected to 2.5 times of volume concentrations using the method that is concentrated by ultrafiltration, obtains parvovirus antigen
Liquid；Step 4) products therefrom is subjected to 2 times of volume concentrations using the method that is concentrated by ultrafiltration, obtains the type Adenovirus Antigen liquid of duck 2；
6) respectively inactivation step 5) obtained by parvovirus antigen liquid and the type Adenovirus Antigen liquid of duck 2；
7) by the parvovirus antigen liquid after inactivation and the type Adenovirus Antigen liquid of duck 2 after inactivation respectively with 3500r/min
Rotating speed centrifuges 10min, is then mixed in equal volume after filtering respectively through 8 layers of filtered through gauze again, adds mixeding liquid volume 4%
Tween 80, mix, obtain vaccine aqueous phase；
8) Marcol52 white oils are taken, 1.2% (w/v) aluminum stearate is added thereto, is heated after mixing, to aluminum stearate
The Span 80 of white oil dosage 6/94 (w/w) is added after being completely dissolved thereto, heats while stirring to being completely dissolved, produces vaccine
Oil phase；
9) the vaccine aqueous phase obtained by the vaccine oil phase obtained by step 8) and step 7) is pressed 3:2 (w/w) ratio mixing, breast
Change, that is, obtain kind duck source goose parvovirus, the type adenovirus bivalent inactivated vaccine of duck 2.
Preferably, its kind of duck source of step 3) virus titer highest allantoic fluid goose parvovirus potency >=10- 5.5EID50/0.1ml。
Preferably, its type adenovirus titers >=10 of duck 2 of step 4) the virus titer highest virus liquid-6.0TCID50/
0.1ml。
Preferably, step 5) the ultrafiltration concentration method is implemented using 100KD films bag；More excellent, the 100KD
Film bag is produced by Millipore Corp..
Preferably, inactivation includes following operation described in step 6)：Added into antigen liquid formaldehyde to final concentration of 1~
2 ‰ (v/v), shake up, to inactivate 16-20 hours under the conditions of 180r/min rotating speeds, 37 DEG C.
Implement preferably, the emulsification described in step 9) is the mulser produced using Fu Luke companies, specifically include
Operate below：Take the vaccine oil phase of formula ratio to be added in container, start mulser to A shelves, add the vaccine aqueous phase of formula ratio,
Continue to shear 2min, mulser is placed in B shelves, shears 3min.
The invention provides a kind duck source goose parvovirus, the type adenovirus bivalent inactivated vaccine preparation method of duck 2, the technical side
Case devises targetedly technique bar according to the proliferative properties and immunogenicity of kind duck source goose parvovirus and the type adenovirus of duck 2
Part.From the point of view of specific, the present invention, using LMH cell replicative adenovirus, is inactivated using chicken embryo propagation kind duck source goose parvovirus
After be prepared into safely and effectively kind duck source goose parvovirus and adenovirus bivalent inactivated vaccine.Kind duck source prepared using the present invention
Goose parvovirus and adenovirus bivalent inactivated vaccine produce antibody morning, and protective rate is high.Vaccine prepared by the present invention is once inoculated with can
With prevention two disease, solve kind duck because vaccine is repeatedly inoculated with and caused by stress reaction.
Embodiment
The embodiment of the present invention will be described in detail below.In order to avoid excessive unnecessary details,
It is will not be described in detail in following examples to belonging to known structure or function.
Approximating language used in following examples can be used for quantitative expression, show do not changing the feelings of basic function
Under condition quantity can be allowed to have certain variation.Therefore, it is accurate that the numerical value corrected with the language such as " about ", " left and right " is not limited to this
Numerical value is in itself.In certain embodiments, the numerical value for " about " representing to allow its amendment is in the scope of positive and negative 10 (10%)
Interior change, such as, " about 100 " represent can be any numerical value between 90 to 110.In addition, " the about first numerical value arrives
In the statement of second value ", the first and second numerical value two values are at about corrected.In some cases, approximating language
May be relevant with the precision of measuring instrument.
In addition to being defined, technology used and scientific terminology have and art technology people of the present invention in following examples
The identical meanings that member is commonly understood by.
Test reagent consumptive material used, is routine biochemistry reagent unless otherwise specified in following examples；The experiment
Method, it is conventional method unless otherwise specified；Quantitative test in following examples, it is respectively provided with and repeats to test three times, as a result
Average；% in following examples, it is weight/mass percentage composition unless otherwise instructed.
1. the separation and identification of virus
The separation and identification of 1.1 kinds of duck source goose parvovirus
Pathological material of disease is collected in falling ill kind duck from the age in days of ZhangZhou kind, Fujian Province duck field 9, takes its liver, spleen and enteron aisle, by tissue grinder,
8000rpm centrifuges 10min, takes supernatant to enter performing PCR amplification to its VP3 gene, and further carry out sequencing identification, it was demonstrated that the virus
For a kind duck source Goose Parvovirus, MGPV-FJ-2016 is named as.
The separation and identification of 1.2 kinds of Ana 1 aviadenoviruses
From the age in days of Guangdong Province kind duck field 16 morbidity kind duck collection pathological material of disease, take its liver and spleen to be ground, 8000rpm from
Heart 10min, take supernatant to enter performing PCR amplification to its Hexon GFP, and further carry out sequencing identification, it was demonstrated that the virus is
The type of Ana 1 aviadenovirus 2, is named as DAdV-2-GD-2016.
2. the propagation of virus
(1) propagation of MGPV-FJ-2016 kinds poison
The MGPV-FJ-2016 viral supernatants after being centrifuged in step 1) are filtered using 0.22um filters, 10-2Dilution
After be inoculated in that 9~11 ages in days are non-to exempt from chicken embryo (no gosling plague antibody), 37 DEG C of cultures.The non-specific dead germs of 24h are discarded, by 24h-120h
Dead germ is placed in 4 DEG C of cold egg 4-24h.Harvest allantoic fluid, by it in chicken embryo the generation of blind passage 3~5, until chicken embryo occur it is lethal, and
Death time is controlled in 96h, and harvest dead germ allantoic fluid is named as P1.
According to same method numerous malicious 5~8 generation, while viral level is determined, 1 is the results are shown in Table, by the poison of potency highest generation
Preparation for vaccine.By viral level measurement result regulation vaccine >=10 are answered with toxic effect valency-5.5EID50/0.1ml。
The viral level measurement result (10 of table 1n EID50/0.1ml)
| P1 | P2 | P3 | P4 | P5 | P6 | P7 | P8 |
| 4.0 | 4.83 | 5.5 | 5.83 | 5.83 | 5.5 | 5.83 | 5.5 |
2.1.1 the propagation of DAdV-2-GD-2016 kinds poison
The DAdV-2-GD-2016 viral supernatants after being centrifuged in step 1) are filtered using 0.22um filters, treat LMH
During cell length to individual layer, by the 0.1-2% virus inoculations of cell liquid volume, 37 DEG C of culture 72-96h, harvest virus, multigelation
3 times.In the generation of blind passage 3~5, until lesion occurs in 75% cell, and lesion time control, in 72h, harvest viral nomenclature is P1.
According to same method numerous malicious 5~8 generation, while viral level is determined, 2 are the results are shown in Table, by the poison of potency highest generation
Preparation for vaccine.By viral level measurement result regulation vaccine >=10 are answered with toxic effect valency-6.0TCID50/0.1ml。
The viral level measurement result (10 of table 2n TCID50/0.1ml)
| P1 | P2 | P3 | P4 | P5 | P6 | P7 | P8 |
| 5.0 | 6.0 | 6.38 | 6.5 | 6.38 | 6.38 | 6.5 | 6.0 |
3. the preparation of vaccine
The concentration of 3.1 viruses
Using the method that is concentrated by ultrafiltration (Millipore Corp. 100KD films bag) respectively by MGPV-FJ-2016 and DAdV-2-GD-
2016 carry out 2.5 times and 2 times concentrations, the preparation for bigeminy vaccine.
The inactivation of 3.2 viruses
Two kinds of cell venom are placed in sterilization container respectively, formalin is added according to virus liquid volume and adjusts formaldehyde
Final concentration fully shakes up venom, with 180r/min rotating speeds, 16-20 hours is inactivated under the conditions of 37 DEG C to 1-2 ‰ (v/v)；
The preparation of 3.3 vaccines
3.3.1 the preparation of aqueous phase
Two virus liquids after inactivation are centrifuged into 10min with 3500r/min respectively, then through 8 layers of filtered through gauze.After processing
Two kinds of virus liquids are with 1：1 volume ratio mixing, then Tween-80 is added by the 4% of admixture poison liquid product, fully mix.
3.3.2 the preparation of oil phase
94 parts of Marcol52 white oils, 1.2% (w/v) aluminum stearate is added, is heated after mixing, it is completely molten to aluminum stearate
6 parts of Span-80 is added after solution, heats while stirring to being completely dissolved, produces oil phase.
3.3.3 emulsification
Take 3 parts of oil phases to be added in container, start cutter (FLUKO) to A shelves, be slowly added to 2 parts of aqueous phases, continue to shear
2min, cutter is placed in B shelves, shears 3min.
4. the safety examination of vaccine
Vaccine is carried out to the safety test of 1 overdose (1mL) and single dose repeated inoculation (0.2mL × 2) respectively.Observation
14 days, test chicken without any locally and systemically adverse reaction, showed that above-mentioned vaccine safety is good
5. vaccine potency is examined
5.1 serological methods are examined
1 age in days kind duck 20 is taken, is divided into 2 groups, every group of 10 chickens, the 1st group is injected 0.2ml vaccines through leg muscle, the 2nd group
Phosphate buffer 0.2mL is injected as compareing, raises under similarity condition, is taken a blood sample weekly after immune, separate serum, until immune
The 8th week afterwards, specific antibody is detected using agarose diffusion test (AGP), the results are shown in Table 3.
Antibody level measurement result (nlog2) after the vaccine immunity of table 3
5.2 Immunization methods are examined
1 Day-old Broiler Chickens 40 are taken, are divided into 4 groups, every group of 10 chickens, the 1st group and the 2nd group is injected through leg muscle respectively
0.2ml vaccines, the 3rd group and the 4th group not immune as malicious control is attacked, and is raised under similarity condition.14d after immune, take the 1st group and
3rd group to attack GPV respectively malicious by force, 0.2ml/ plumages, attacks 14d cut open inspections after poison, tissues observed lesion, determines protective rate, be shown in Table 2.Exempt from
21d after epidemic disease, the 2nd group and the 4th group malicious by force, 0.2ml/ plumages of attacking DAdV respectively attack 7d cut open inspections after poison, tissues observed lesion, it is determined that
Protective rate, it is shown in Table 4.
Table 4 attacks malicious protective rate result
Embodiments of the invention are described in detail above, but the content is only presently preferred embodiments of the present invention,
It is not intended to limit the invention.All all any modification, equivalent and improvement done in the application range of the present invention etc., all should
Within protection scope of the present invention.
Claims (6)
1. kind duck source goose parvovirus, the type adenovirus bivalent inactivated vaccine preparation method of duck 2, it is characterised in that including following step
Suddenly：
1) infection kind duck source goose parvovirus, 9 age in days kind ducks are taken, its liver, spleen or intestinal tissue are taken, through 8000rpm after grinding
Rotating speed centrifuges 10min, takes supernatant, as parvovirus liquid；
2) the type adenovirus of infected duck 2,16 age in days kind ducks are taken, takes its liver or spleen tissue, is centrifuged after grinding through 8000rpm rotating speeds
10min, take supernatant, as the type adenopathy venom of duck 2；
3) utilize the filter filtration step 1 in 0.22 μm of aperture) obtained by parvovirus liquid, 10-29~11 ages in days are inoculated in after dilution
It is non-to exempt from chicken embryo, 37 DEG C of cultures, the non-specific dead germs of 24h are discarded, 24h-120h dead germs are placed in 4 DEG C of refrigeration 4-24h, harvest allantois
Liquid, by it in chicken embryo the generation of blind passage 3~5, until chicken embryo occur it is lethal, and the death time control in 96h, harvest dead germ urine
Cyst fluid；According to same method numerous malicious 5~8 generation, while viral level is determined, take virus titer highest allantoic fluid stand-by；
4) utilize the filter filtration step 2 in 0.22 μm of aperture) obtained by the type adenopathy venom of duck 2, it is then pressed into cell liquid volume
0.1-2% be inoculated into long into the LMH cells of individual layer state, 37 DEG C of culture 72-96h, harvest virus, multigelation 3 times is blind
Passed for 3~5 generations, until lesion occurs in 75% cell, and lesion time control harvests virus liquid in 72h；According to same method
Numerous malicious 5~8 generation, while viral level is determined, take virus titer highest virus liquid stand-by；
5) step 3) products therefrom is subjected to 2.5 times of volume concentrations using the method that is concentrated by ultrafiltration, obtains parvovirus antigen liquid；Profit
Step 4) products therefrom is subjected to 2 times of volume concentrations with the method for ultrafiltration concentration, obtains the type Adenovirus Antigen liquid of duck 2；
6) respectively inactivation step 5) obtained by parvovirus antigen liquid and the type Adenovirus Antigen liquid of duck 2；
7) by the parvovirus antigen liquid after inactivation and the type Adenovirus Antigen liquid of duck 2 after inactivation respectively with 3500r/min rotating speeds
10min is centrifuged, is then mixed in equal volume after filtering respectively through 8 layers of filtered through gauze again, adds the tween of mixeding liquid volume 4%
80, mix, obtain vaccine aqueous phase；
8) Marcol52 white oils are taken, 1.2% (w/v) aluminum stearate is added thereto, is heated after mixing, it is complete to aluminum stearate
The Span 80 of white oil dosage 6/94 (w/w) is added after dissolving thereto, is heated while stirring to being completely dissolved, produces vaccine oil
Phase；
9) the vaccine aqueous phase obtained by the vaccine oil phase obtained by step 8) and step 7) is pressed 3:2 (w/w) ratio mixing, emulsification,
Obtain kind duck source goose parvovirus, the type adenovirus bivalent inactivated vaccine of duck 2.
2. according to claim a kind of duck source goose parvovirus, the type adenovirus bivalent inactivated vaccine preparation method of duck 2, its
It is characterised by its kind of duck source of step 3) virus titer highest allantoic fluid goose parvovirus potency >=10-5.5EID50/
0.1ml。
3. according to claim a kind of duck source goose parvovirus, the type adenovirus bivalent inactivated vaccine preparation method of duck 2, its
It is characterised by its type adenovirus titers >=10 of duck 2 of step 4) the virus titer highest virus liquid-6.0TCID50/0.1ml。
4. according to claim a kind of duck source goose parvovirus, the type adenovirus bivalent inactivated vaccine preparation method of duck 2, its
It is characterised by that step 5) the ultrafiltration concentration method is implemented using 100KD films bag.
5. according to claim a kind of duck source goose parvovirus, the type adenovirus bivalent inactivated vaccine preparation method of duck 2, its
It is characterised by that inactivation includes following operation described in step 6)：Formaldehyde is added into antigen liquid to final concentration of 1~2 ‰ (v/v),
Shake up, to inactivate 16-20 hours under the conditions of 180r/min rotating speeds, 37 DEG C.
6. according to claim a kind of duck source goose parvovirus, the type adenovirus bivalent inactivated vaccine preparation method of duck 2, its
It is that the mulser produced using Fu Luke companies is implemented to be characterised by the emulsification described in step 9), specifically includes following operation：
Take the vaccine oil phase of formula ratio to be added in container, start mulser to A shelves, add the vaccine aqueous phase of formula ratio, continue to shear
2min, mulser is placed in B shelves, shears 3min.
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| Application Number | Priority Date | Filing Date | Title |
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| CN201711339929.7A CN107875383A (en) | 2017-12-14 | 2017-12-14 | Kind duck source goose parvovirus, the type adenovirus bivalent inactivated vaccine preparation method of duck 2 |
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|---|---|---|---|
| CN201711339929.7A CN107875383A (en) | 2017-12-14 | 2017-12-14 | Kind duck source goose parvovirus, the type adenovirus bivalent inactivated vaccine preparation method of duck 2 |
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
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| CN108607095A (en) * | 2018-06-05 | 2018-10-02 | 山东信得科技股份有限公司 | A kind of strain of goose parvovirus and its application |
| CN108714211A (en) * | 2018-06-05 | 2018-10-30 | 山东信得科技股份有限公司 | A kind of goose parvovirus and 2 type adenovirus bivalent inactivated vaccine of duck |
| CN108939063A (en) * | 2018-06-05 | 2018-12-07 | 山东信得科技股份有限公司 | A kind of kind duck triple inactivated vaccine |
| CN111000990A (en) * | 2019-12-19 | 2020-04-14 | 广东渔跃生物技术有限公司 | Duck tembusu virus and duck adenovirus bivalent inactivated vaccine and preparation method thereof |
| CN113185607A (en) * | 2021-03-22 | 2021-07-30 | 华南农业大学 | Preparation method of multivalent avian adenovirus egg yolk antibody |
| CN114891094A (en) * | 2022-05-05 | 2022-08-12 | 安徽省农业科学院畜牧兽医研究所 | Preparation method of avian adenovirus and Muscovy duck parvovirus bigeminal egg yolk antibody |
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Cited By (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108465107A (en) * | 2018-06-05 | 2018-08-31 | 山东信得科技股份有限公司 | A kind of 2 type adenovirus of duck and Muscovy duck parvovirus disease bivalent inactivated vaccine |
| CN108607095A (en) * | 2018-06-05 | 2018-10-02 | 山东信得科技股份有限公司 | A kind of strain of goose parvovirus and its application |
| CN108714211A (en) * | 2018-06-05 | 2018-10-30 | 山东信得科技股份有限公司 | A kind of goose parvovirus and 2 type adenovirus bivalent inactivated vaccine of duck |
| CN108939063A (en) * | 2018-06-05 | 2018-12-07 | 山东信得科技股份有限公司 | A kind of kind duck triple inactivated vaccine |
| CN108714211B (en) * | 2018-06-05 | 2021-12-10 | 山东信得科技股份有限公司 | Goose parvovirus and duck type 2 adenovirus bivalent inactivated vaccine |
| CN108939063B (en) * | 2018-06-05 | 2022-06-03 | 山东信得科技股份有限公司 | Muscovy duck triple inactivated vaccine |
| CN111000990A (en) * | 2019-12-19 | 2020-04-14 | 广东渔跃生物技术有限公司 | Duck tembusu virus and duck adenovirus bivalent inactivated vaccine and preparation method thereof |
| CN113185607A (en) * | 2021-03-22 | 2021-07-30 | 华南农业大学 | Preparation method of multivalent avian adenovirus egg yolk antibody |
| CN114891094A (en) * | 2022-05-05 | 2022-08-12 | 安徽省农业科学院畜牧兽医研究所 | Preparation method of avian adenovirus and Muscovy duck parvovirus bigeminal egg yolk antibody |
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