CN105770885B - A kind of newcastle disease virus, aviadenovirus bivalent inactivated vaccine - Google Patents
A kind of newcastle disease virus, aviadenovirus bivalent inactivated vaccine Download PDFInfo
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/12—Viral antigens
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
- A61K2039/525—Virus
- A61K2039/5252—Virus inactivated (killed)
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/55—Medicinal preparations containing antigens or antibodies characterised by the host/recipient, e.g. newborn with maternal antibodies
- A61K2039/552—Veterinary vaccine
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/70—Multivalent vaccine
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
- C12N2710/00011—Details
- C12N2710/10011—Adenoviridae
- C12N2710/10211—Aviadenovirus, e.g. fowl adenovirus A
- C12N2710/10234—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2760/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses negative-sense
- C12N2760/00011—Details
- C12N2760/18011—Paramyxoviridae
- C12N2760/18111—Avulavirus, e.g. Newcastle disease virus
- C12N2760/18134—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
Abstract
The present invention provides a kind of newcastle disease virus, aviadenovirus bivalent inactivated vaccine, the TCID of the new strain of YBAV-4 plants of 4 type aviadenovirus of I group used in vaccine of the invention50Potency is high, immunogenicity is good and can resist the attack of aviadenovirus disease each place separation poison.The good security of vaccine prepared by the present invention does not occur any locally and systemically adverse reaction as caused by vaccine.Analysis in storage life test Jing Guo character, safety testing, potency test data, as a result compared with single seedling of similar product, Combined vaccine no significant difference is stable effective；Efficacy test results prove that Combined vaccine and three kinds of single seedling antibody keep high level, and it is fast to generate antibody than similar product, and control group antibody is feminine gender.
Description
Technical field
The invention belongs to veterinary biologics technical fields, and in particular to a kind of newcastle disease virus, aviadenovirus bigeminy
Inactivated vaccine.
Background technique
Newcastle disease has very high disease incidence and case fatality rate, all over the world to have generation more, and once outburst will be supported to fowl
It grows industry and brings crushing blow, be a kind of Infectious Diseases for endangering aviculture.OIE is classified as A class epidemic disease.
By the epidemiological survey to I group I fowl adenovirus, disease disease incidence in China chicken group is higher, can pass through level
It is propagated with vertical two kinds of approach, and is in rise year by year trend.The host range of morbidity is also increasingly wider, white meat-type chickens, Breeder hens,
Morbidity increase trend can be presented after infection morbidity, especially 2010 year in laying hen, yellow plumage chicken, there is stream in China
Row.Many I group I fowl adenovirus can replicate in healthy carcass, and symptom is very slight or does not show infection symptoms, but 4 type of I group
Aviadenovirus exception can directly cause chicken mass-sending disease, and major lesions show as hydropericardium and liver, kidney enlargement, this disease is in 1963
Year occurs in the U.S. for the first time, then occurs in succession all over the world, is whole world poultry and the common zymad of wild fowl.1976
Year, this disease occurred for the first time for Taiwan Province, China, in all parts of the country later to have the report that this disease occurs, and in trend is risen year by year, gave chicken
Aquaculture brings serious harm.
In recent years, H9 subtype avian influenza is the relatively conventional important diseases for seriously threatening poultry；And chicken group is to I group 4
The infection of type aviadenovirus is more and more, this disease is easy to cause secondary infection epidemic disease, and to fowl, industry raiser brings many worries,
And vaccine especially inactivated vaccine is used for multiple times, not only increase chicken house man power and material burden, while repeatedly grabbing the injection of chicken
Stress, it also will affect production performance, chicken group caused to increase the neurological susceptibility of disease.In addition the continuous variation of strain in recent years, makes
Although obtaining the vaccine for the multiple choices released, but still occur losing control of the situation of epidemic situation development, so needing to screen acquisition
New popular strain come cope with variation caused by new harm.
Summary of the invention
The object of the present invention is to provide a kind of newcastle disease virus, aviadenovirus bivalent inactivated vaccine, to make up existing
The deficiency of technology.
Bivalent inactivated vaccine of the invention, wherein antigen is the newcastle disease virus and aviadenovirus of inactivation；
Wherein newcastle disease virus is preferably Sota plants of newcastle disease virus La；
Aviadenovirus is YBAV-4 plants of 4 type aviadenovirus of I group, and deposit number is CCTCC No.V201541.
Above-mentioned inactivated vaccine, wherein the inactivation of virus uses formalin-inactivated；
Inactivated vaccine of the invention the preparation method is as follows:
1) oil is mutually prepared:
Take 95 parts of mineral oil, 1 part of aluminum stearate, after being uniformly mixed in oily phase preparation tank and being heated to 80 DEG C, then plus 5 parts
This 80 (Span-80) of department complete oil after cooling and mutually prepare until temperature maintains 40 minutes when reaching 115 DEG C；
2) prepared by water phase:
The Newcastle Disease venom of inactivation, I group I fowl adenovirus venom are mixed；95 parts of hybrid antigen liquid are taken, 5 parts of sterilizing are spat
Temperature 80, mixes well；
Wherein the quantity of Newcastle Disease venom, I group I fowl adenovirus is than being preferably 1:2；
3) it emulsifies
2 parts of oily phase is taken to be put into emulsion tank, after being added 1 part of water phase, then with the 3500r/min stirring cream of completion in 30~40 minutes
Change preparation.
The TCID of the new strain of YBAV-4 plants of 4 type aviadenovirus of the group of I used in vaccine of the invention50Potency height, immunogene
Property it is good and the malicious attack of aviadenovirus disease each place separation can be resisted.The good security of vaccine prepared by the present invention, does not occur
Any locally and systemically adverse reaction as caused by vaccine.Pass through character, safety testing, potency test number in storage life test
According to analysis, as a result compared with single seedling of similar product, Combined vaccine no significant difference, it is stable effectively；Efficacy test results
It proves, Combined vaccine and three kinds of single seedling antibody keep high level, and it is fast to generate antibody than similar product, and control group antibody is yin
Property.
Specific embodiment
Applicant screens the 4 type aviadenovirus of I group for obtaining one plant of novel variant, by the virus and newcastle disease virus one
Get up to prepare combined vaccine, to facilitate the present invention.
The present invention is described in detail below with reference to embodiment.
The screening of 1:YBAV-4 plants of strains of embodiment
1, since two thousand and ten, part Breeder hens, laying hen and the numb chicken in the area such as Shandong, Jiangsu go out for epidemiological survey
One kind is showed with death rate height, dissection is mainly shown as the disease with the characteristics of liver enlargement, hydropericardium, through clinical investigation and reality
Room detection is tested, tentative diagnosis is hydropericardium hepatitis syndrome caused by I group of C-4 type aviadenovirus.2010, inventor was from mountain
Eastern Zibo farm, which has in the chicken liver of dying of illness of inclusion body hepatitis and hydropericardium classical symptom, to be successfully separated to 1 strain virus.
2, it after virus purification takes the liver for the chicken that dies of illness to grind, is made of the ratio addition sterile saline in 1:5 outstanding
Liquid；After multigelation 3 times, 3000r/min is centrifuged 30min, takes supernatant；It is added penicillin and streptomysin each 10000IU/ml, 4 DEG C
Overnight, it filters through millipore filter, is saved backup after steriling test is qualified.By the virus liquid of above-mentioned preparation with the agent of 0.2ml/ embryo
Amount is inoculated with 6.5 age in days SPF chicken embryos through yolk bag approach, abandons dead germ in for 24 hours, takes the allantois of dead chicken embryo in inoculation 48h~168h
The hepatic tissue of the 3rd generation dead germ is observed in liquid and fetus, continuous passage after handling in aforementioned manners, and it is short that chicken embryo shows as dead germ, idiosome
Small, hypoevolutism, fetus curling, liver enlargement and matter are crisp, and embryo is congested.Collect dead germ allantoic fluid and fetus, -20 DEG C of preservations.
3, viral identification
3.1 blood clotting CHARACTERISTICS IDENTIFICATION aseptic collection SPF chickens and duck blood 5~10ml of liquid, are washed 3~5 times, last physiology repeatedly
Haemocyte mud is diluted to 0.8%, 1% and 2% concentration by salt water, and 4 DEG C save backup.Whether detection isolated strain according to a conventional method
With the characteristic for being aggregated these red blood cells.III group I fowl adenovirus EDSV-76 is set simultaneously as agglutinating reaction positive control.As a result: point
It cannot be aggregated SPF chicken and duck red blood cell from poison, even if changing the concentration of red blood cell, can not be allowed to be aggregated.III group I fowl adenovirus
EDSV-76 can be aggregated the red blood cell of chicken, duck.
3.2 physicochemical properties examine the method introduced referring to " animal virology ", and virus liquid is respectively with 5-bromouracil-
After 2 '-deoxyribonucleosides (BUDR), chloroform, ether, hydrochloric acid (pH3), sodium hydroxide (pH10), temperature (60 DEG C, 1h) processing, inoculation
Chicken embryo (0.2ml/ embryo) separately sets physiological saline processing group as control.Chicken embryo lesion is observed after being inoculated with 5d.As a result: separation poison point
Not after BUDR, sodium hydroxide (pH10) and 60 DEG C, 1h processing, inoculated into chick embryo, chicken embryo is acted normally, and PCR detection is negative.Show
BUDR can inhibit duplication of the virus in chicken embryo, and the nucleic acid type of isolated strain is DNA, and virus is not alkaline-resisting, to thermo-responsive, 60
DEG C, 1h can be inactivated.And the strain handled through ether, chloroform and hydrochloric acid (pH3), proliferation of the virus in chicken embryo is not influenced, out
Now apparent chicken embryo lesion, PCR testing result are positive.Show that virus does not have lipid cyst membrane, has resistance to ether and chloroform, it is resistance to
Acid.
3.3 serological Identification
3.3.1 group specificity identification and utilization agar gel diffusion test (AGP) prepares agar gel plate to separation poison
Strain carries out group specificity identification.After agar solidification, being punched with punch, perforation pattern is central 1 hole surrounding, 6 hole, aperture 4mm,
Pitch-row is 4mm, bottom hole closing.Virus to be checked is placed in interstitial hole, and holes around adds I group I fowl adenovirus type strain, the capital EDSV-76 911
Strain standard positive serum and negative serum.Fine jade expansion plate is placed in cover in wet box and is acted on for 37 DEG C, 24~48h sees whether occur
Coagulation sedimentation line.As a result: separate malicious antigen and be only capable of occurring obvious sediment line with I group I fowl adenovirus, 4 type positive serum, and with III group
911 plants of the capital aviadenovirus EDSV-76 does not occur precipitation line between standard positive serum and negative serum.
3.3.2 type specificity identification I group I fowl adenovirus, 1~12 type standard positive serum makees 1:10 dilution first, then presses
Version " Chinese veterinary pharmacopoeia " annex fixed virus diluted blood heat-clearing method in 2010, by I group I fowl adenovirus, 1~12 type standard strain, separation
Poison carries out cross neutralization test to I group I fowl adenovirus, 1~12 type standard positive serum, records neutralization titer result.As a result: separation
Poison surveys the neutralization titer (1:501) of 4 type standard positive serums and the neutralization titer of 4 type standard strains, 4 type standard positive serums of survey
(1:537) is closer to；Isolated strain surveys the neutralization titer of other type standard positive serums in 1:10 or less.Showing separation strains is
4 type of serum.
3.4 PCR detection and the sterile grinding of gene sequencing diseased chicken liver, multigelation 3 times, are mentioned using pillar animal DNA
It takes kit to extract viral DNA, carries out PCR detection.1% agarose gel electrophoresis observes result.Positive sample is subjected to Hexon
Gene sequencing, and carry out phylogenetic analysis.It is compared according to Hexon gene order and phylogenetic analysis result can be seen that
Separation poison belongs to same branch with I group I fowl adenovirus, closest with 4 type homology of serum, but there is also the differences in sequence；With
6 type of serum, 7 types, 8a and 8b type homology are lower.
The Strain is preserved in the China typical culture collection center of Wuhan Wuhan University on October 15th, 2015, protects
Hiding number is CCTCC No.V201541.
The preparation of 2:YBAV-4 plants of seeds culture of viruses of embodiment
(1) chicken liver cell optimal culture condition is studied
1, the influence that cell density grows cell by 5 kinds of different densities (1~50,000/ml, 5~100,000/ml, 10
~15 ten thousand/ml, 15~200,000/ml, 20~250,000/ml) chicken liver cell be inoculated with respectively with a batch of 25cm2Cell
Bottle, 225cm2Cell bottle, 3000ml rolling bottle in 10 layer cell factories, are cultivated under the conditions of same with the DMEM nutrient solution of same batch,
Each density is inoculated with 5 bottles/2, the time required to culture, observation cell grow up to fine and close single layer under the conditions of and the form of cell.
2, the newborn bovine serum that newborn bovine serum content produces the influence that cell is grown with same producer, is pressed respectively
6%, 8%, 10%, 12% ratio is added in DMEM culture solution, cultivates with a collection of chicken liver cell, the whole density of cell is 15
~20 ten thousand/ml, the nutrient solution of every kind of serum-concentration is inoculated with same batch 25cm2Cell bottle, 225cm2Cell bottle, 3000ml turn
Bottle, 10 layer cell factories each 5 bottles/2, observation cell grow up to fine and close single layer required time and cellular morphology.
3, cell dissociation buffer used in the influence that pancreatin grows cell is 0.02%EDTA-0.25% trypsin solution, to thin
After born of the same parents digest well, a part of cell culture container goes digestive juice that nutrient solution is added, and another part cell culture container does not discard
Digestive juice is directly added into nutrient solution.Nutrient solution be pH value be 7.0~7.2, the DMEM nutrient solution containing 10% newborn bovine serum, cell
Density is 15~200,000/ml.It is inoculated with same batch 25cm2Cell bottle, 225cm2Cell bottle, 3000ml rolling bottle, 10 confluent monolayer cells
Factory each 5 bottles/2, under the conditions of culture, observation cell grow up to fine and close single layer the time required to and cellular morphology.
4, the other conditions of influence that nutrient solution pH value grows cell are all the same, only the pH value of nutrient solution adjust separately for
6.8,7.0,7.2,7.4, the nutrient solution of different pH value is inoculated with 25cm respectively2Cell bottle, 225cm2Cell bottle, 3000ml rolling bottle, 10
Layer cell factory each 5 bottles/2, observation nutrient solution color change, cell grow up to fine and close single layer required time and cellular morphology.
(2) YBAV-4 plants of optimal culture condition researchs of 4 type aviadenovirus of I group
1, the determination of toxic dose most preferably is connect respectively in different culture vessels with 0.1%, 0.5%, 1%, 2%, 5% 5
The YBAV-4 strain virus liquid of a various dose is inoculated with chicken liver cell, observes and records the time for cytopathy occur and lesion journey
Degree measures respectively after 80% or more cells showed cytopathic (hereinafter referred to as CPE) harvest virus liquid, multigelation 2 times
Its viral level (TCID50), with TCID50The toxic dose that connects of soprano is most preferably to connect toxic dose.
2, it most preferably connects the determination of malicious time and YBAV-4 strain virus liquid is bred under three kinds of growth conditions with cell, work as chicken gizzard
Cell grows up to 60~70% single layers, grows up to 70~80% single layers and grows up to 90% or more single layer virus inoculation, connects by 1% amount
Poison, 37 DEG C, 5%CO2Incubator culture, harvests virus liquid when CPE occurs in 80% or more cell, after multigelation 2 times, point
Its TCID is not measured50, with TCID50The malicious time that connects of soprano is most preferably to connect the malicious time.
3, the determination of optimum culturing temperature is grown up to 4 type aviadenovirus YBAV-4 strain virus liquid of I group by 1% amount inoculation
The chicken liver cell of good single layer is respectively placed in 34 DEG C, 36 DEG C, 37 DEG C, 38 DEG C of cultures, the time and disease that observation cytopathy occurs
Change degree harvests cell venom when CPE occurs in 80% cell, measures its TCID respectively after multigelation 2 times50, with TCID50
The cultivation temperature of soprano is optimum culturing temperature.
4, the best determination for receiving the malicious time is grown up to 4 type aviadenovirus YBAV-4 strain virus liquid of I group by 1% amount inoculation
The chicken liver cell of good single layer, respectively at 37 DEG C, 5%CO2Incubator culture, when cell occurs 70%, 80%, 90% or so
It harvests virus liquid when CPE, after multigelation 2 times, measures its TCID respectively50, with TCID50The receipts of the soprano malicious time is best
Receive the malicious time.
5, best maintained liquid serum content, which is determined, is inoculated with 4 type aviadenovirus YBAV-4 strain virus liquid of I group by 1% amount
Grow up to the chicken liver cell of good single layer, newborn bovine serum content is respectively 1%, 2%, 3% in maintaining liquid, respectively at 37 DEG C, 5%
CO2Incubator culture, cell venom, multigelation is harvested when CPE occurs in 80% cell at the time that observation cytopathy occurs
Its TCID is measured after 2 times respectively50, with TCID50The serum content of soprano is best maintained liquid serum content.
6, according to above-mentioned test result, we select most preferably to connect malicious mode, most preferably connect toxic dose, is best verification test
Connect the malicious time, optimum culturing temperature, it is best receive the malicious time and be prepared for 3 batches of virus liquids, after virus liquid multigelation 2 times, measurement
The viral level of virus liquid.
Embodiment 3: the preparation of newcastle disease, aviadenovirus (I group, 4 types) antigen
1. Millipore is concentrated by ultrafiltration machine and uses, maintain condition and application method, the operation instruction provided by producer into
Row.
2. concentrated effect detects
Antigen valence in provirus liquid (HA and/EID is concentrated in the measurement that keeps sample before the effect inspection concentration of 2.1 concentrated antigens50/
TCID50), concentrate antigen valence (HA) is measured by sampling at any time in concentration process, determines cycles of concentration, the concentrate after concentration stays
Sample measures antigen valence in concentrate (HA and/EID50/TCID50)。
(antigen concentration is maximum in concentrate at this time, in filter liquor when the detection of 2.2 filter liquor antigens takes concentration to close to an end
A possibility that leaking out antigen is maximum) filter liquor, measurement HA-HI test (HA) is respectively adopted, (whether there is or not diseases living for observation for inoculation SPF chicken embryo
Poison leaks out), have nonantigenic composition in inoculation SPF chicken (having detected whether antigenic substance leakage) etc. approach detection omission timber.
Of different sizes, the retention (filtering) of different ultrafiltration membranes of 2.3 concentration membranous type number selection different virus antigen compositions
Aperture is different, therefore the ultrafiltration membrane of different pore size model (1#, 2#, 3#, 4#, 5#) is selected to carry out rejection tests to different virus,
And result and concentration required time are examined according to antigen testing result, concentration effect in filter liquor, determine that two kinds of viruses, which are concentrated, to be resisted respectively
Membranous type number is most preferably concentrated used in original.
The best model of 2.4 concentrated effect stability tests is concentrated film and is concentrated NDV, FADV blastochyle each 3 batches, detection concentration
The stability of effect.
2.5 cycles of concentration test with best model concentration film, by NDV, FADV blastochyle be respectively concentrated into original volume 1/2,
1/3,1/4,1/5, the EID of the different cycles of concentration of measurement50/TCID50, determine suitable cycles of concentration.
The analysis of 2.6 concentrated costs calculates concentrated cost according to material consumption when concentration.
(1) malicious (bacterium) kind should reach required standard:
Use Sota plants of newcastle disease virus La, YBAV-4 plants of 4 type aviadenovirus of I group as antigen seed culture of viruses.
(2) antigen preparation and the inspection of semifinished product:
The 1 production preparation of seed culture of viruses
The preparation of 1.1 Sota plants of newcastle disease virus La seeds culture of viruses:
Seed culture of viruses sterile saline or PBS are made into appropriate dilution (such as 10-4Or 10-5), allantoic cavity is interior to be inoculated with 10 ages in days
SPF chicken embryo, every embryo 0.1ml.72~120 hours dead and apparent chicken embryos of lesion, harvest chicken embryo liquid (allantois respectively after choosing inoculation
Liquid and amniotic fluid), loaded in sterilization container.To examine it is sterile, to 1% chicken red blood cell agglutination titer not less than 1:512 (micromethod)
The mixing of chicken embryo liquid, quantitative separating indicate harvest date, Virus passages and loading amount, freezen protective in aseptic bottle.
The YBAV-4 plants of seed culture of viruses preparations of 1.2 I groups of 4 type aviadenovirus
1.2.1 the chicken liver cell to grow fine is selected in seed culture of viruses breeding, discards original fluid, and addition contains 1% seed culture of viruses
Maintaining liquid is set 37 DEG C and is cultivated 36~48 hours, harvests when cytopathy is up to 80% or more, freeze thawing 2 times, be sub-packed in sterilization container
Interior, sampling is identified.Indicate harvest date, Virus passages etc..
The selection of 2 seedling materials
The well-developed susceptible chicken embryo of 10~11 age in days of 2.1 newcastle disease virus seedling materials (ND HI antibody≤1:
4)。
2.2 I group I fowl adenovirus seedling material chicken liver cells.
The preparation of 3 antigen for vaccine liquid
3.1 newcastle disease virus antigens
3.1.1 inoculation takes production seed culture of viruses, makees appropriate dilution (such as 10 with sterilizing PBS- 4Or 10- 5), it presses and " automatically connects
Kind machine operation instruction " it requires, it is inoculated with instar chicken embryo on the 10th~11,0.2ml is inoculated in every embryo allantoic cavity, sets 36~37 DEG C and continue to incubate
It educates, it is not necessary to turn over embryo.
3.1.2 after being incubated for and observing egg inoculation, every sunshine embryo 1 time is incubated for 96 hours, all takes out, and according to embryo, abandons
Dead germ is removed, embryo gas chamber living is upright upwards, set 2~8 DEG C of coolings 12~24 hours.
3.1.3 it harvests and takes out cooling chicken embryo, by " full-automatic cropper operation instruction " requirement, harvest chicken embryo liquid.
It draws blastochyle to be placed in sterilization container, sampling measurement erythrocyte agglutination valence, agglutination titer should be discarded lower than 1:256 person.The embryo of harvest
It saves, should be no more than 5 at 2~8 DEG C before liquid inactivation.
3.1.4 it is concentrated by the blastochyle of harvest under the conditions of 2~8 DEG C, the erythrocyte agglutination valence of sampling measurement at any time is hot thin
Born of the same parents' agglutination titer stops concentration when being not less than 1:1024.Keep sample, carry out the inspection of semifinished product, remaining blastochyle is inactivated immediately.
3.1.5 it inactivates and imports virus liquid in inactivation tank, metered 10% formalin is sufficiently mixed, formaldehyde is molten
The ultimate density of liquid is 0.1%.37 DEG C of inactivations are taken out 16 hours (reaching 37 DEG C of beginning timing with temperature in tank) afterwards, set 2~8 DEG C
It saves, should be no more than 1 month.
3.2 I group I fowl adenovirus antigens
3.2.1 cell preparation is set in 37 DEG C of water-baths from taking-up cryopreservation tube in liquid nitrogen container and is melted, and cell immigration is equipped with
In the centrifuge tube of 10ml culture solution, 1000r/min is centrifuged 5 minutes.With the culture solution suspension cell for containing 20% newborn bovine serum, set
37 DEG C, 5%CO2Incubator culture, when growing up to good single layer with pancreas enzyme -EDTA vitellophag.
3.2.2 prepared by antigen
3.2.2.1 the seed cell that cell monolayer culture will be enlarged by culture is inoculated into cell factory, 37 DEG C of cultures.
3.2.2.2 it connects poison and selects the chicken liver cell to grow fine, discard original fluid, the dimension containing 1% seed culture of viruses is added
Liquid is held, 37 DEG C is set and continues to cultivate.
3.2.2.3 after observation connects poison with harvest, daily observation 2 times records cytopathy situation.When cytopathy reaches
It is harvested when 80% or more, freeze thawing 2 times, sampling carries out the inspection of semifinished product.- 15 DEG C of preservations should be no more than 30.
3.2.2.4 it is concentrated by the venom of harvest under the conditions of 2~8 DEG C, is concentrated 2~3 times with the machine of ultrafiltration concentration, keeps sample,
The inspection of semifinished product is carried out, remaining blastochyle is inactivated immediately.
3.2.2.5 it inactivates and imports virus liquid in inactivation tank, metered 10% formalin is sufficiently mixed, formaldehyde
The ultimate density of solution is 0.2%.37 DEG C of inactivations are taken out 16 hours (reaching 37 DEG C of beginning timing with temperature in tank) afterwards, set 2~8
DEG C save, should be no more than 1 month.
4 inspection of semifinished product
4.1 newcastle disease parts
4.1.1 the measurement of erythrocyte agglutination valence takes the virus liquid before inactivation, is surveyed by existing " Chinese veterinary pharmacopoeia " annex
Fixed, erythrocyte agglutination valence should be not less than 1:1024.
4.1.2 10 times of the virus liquid work taken out before inactivation is serially diluted by viral level measurement, takes 10- 7、10- 8、10? 93 dilutions, each allantoic cavity is interior to be inoculated with 10~11 5 pieces of age in days SPF chicken embryos, and every embryo 0.1m1 sets 36~37 DEG C and continues to be incubated for, often
It sunshine embryo 2 times, observes 5.Erythrocyte agglutination valence is measured by embryo, is not less than 1:128 person, is judged to infect, calculates EID50.Often
0.1ml viral level answers >=109 . 0EID50。
4.1.3 steriling test takes the virus liquid after inactivation, tests, answers sterile by existing " Chinese veterinary pharmacopoeia " annex
Growth.
4.1.4 inactivation, which is examined, takes 10 6 pieces of age in days SPF chicken embryos, inactivation of viruses liquid is inoculated in allantoic cavity, every embryo 0.2ml is set
36~37 DEG C are continued to be incubated for, every sunshine embryo 2 times, are observed 5, and chicken embryo nonspecific death should be no more than 1 piece.All blastochyles are distinguished
Measure erythrocyte agglutination valence, should all not be aggregated, by a blastochyle harvest blind passage generation again, still without agglutination titer when, be judged to inactivate
Entirely.
4.2 I group I fowl adenovirus parts
4.2.1 viral level measurement takes the virus liquid before inactivation to be measured, and every 0.1ml viral level answers >=
107 . 3TCID50。
4.2.2 steriling test takes the virus liquid after inactivation, tests, answers sterile by existing " Chinese veterinary pharmacopoeia " annex
Growth.
4.2.3 inactivation, which is examined, makees 10 times of dilutions for the virus liquid after inactivation, is inoculated with chicken liver cell (24 holes grown fine
Cell plates) 4 holes, every hole 0.2ml, supplement maintaining liquid to 2.0ml；It sets nonvaccinated chicken liver cell simultaneously and makees blank control, 37 DEG C,
5%CO2Incubator culture is observed 120 hours.Cytopathy should all not occur in cell control well and sample well.Culture is received
A blind passage generation after multigelation is obtained, culture 120 hours is continued, when sample well does not occur cytopathy still, is judged to inactivating completely.
Embodiment 4: the preparation of vaccine
Mutually preparation takes 95 parts of mineral oil to 1 oil, 1 part of aluminum stearate, is uniformly mixed in oily phase preparation tank and is heated to 80
After DEG C, then Jia Siben -805 parts, until temperature maintains 40 minutes when reaching 115 DEG C, it is spare after cooling.
The preparation of 2 water phases mixes the Newcastle Disease venom of inactivation, I group I fowl adenovirus venom with 1:2 ratio.Take mixing anti-
95 parts of stoste, 5 parts of the Tween-80 of sterilizing mixes well, is completely dissolved Tween-80.
3 emulsifications take 2 parts of oily phase to be put into emulsion tank, start motor, slow rotation stirring, while water phase 1 being added slowly
After part, then with 3500r/min stirring 30~40 minutes.After emulsification, take vaccine 10ml be added centrifuge tube in, with 3000r/min from
The heart 15 minutes, the water phase that tube bottom is precipitated should be no more than 0.5ml.
4 packing quantitative separatings, seal, and adhesive label, set 2~8 DEG C of preservations.
(4) safety test of vaccine
1. the safety test that a single dose of the various route of inoculation of pair target animals is inoculated with takes 1 age in days SPF chicken to be divided into 3 groups,
Every group 10,1401 batches of inactivated vaccines are subcutaneously injected in the 1st group of neck, and 0.3ml/ is only；2nd group of intramuscular injection, 1401 batches of inactivated vaccines,
0.3ml/ is only；3rd group of intramuscular injection physiological saline 0.3ml/ is only compared.22 age in days SPF chickens are taken to be divided into 3 groups, every group 10,
1401 batches of inactivated vaccines are subcutaneously injected in 1 group of neck, and 0.5ml/ is only；2nd group of intramuscular injection, 1401 batches of inactivated vaccines, 0.5ml/ is only；3rd group
Intramuscular injection physiological saline 0.5ml/ is only compared, and is raised in isolator respectively, is observed continuously 14.As a result neck is subcutaneously infused
It penetrates and obvious adverse reaction is not caused to injection site and whole body with two kinds of approach of intramuscular injection, the test chicken within the entire observation period
Feeding drinking-water is normal, dissects within 15 days after exempting from, injection site absorbs good, it was demonstrated that the vaccine is through two kinds of injecting pathways to SPF chicken
It is safe.
Safety test of the different injecting pathways of table 1 to SPF chicken
Note: "-" indicates that chicken feeding, drinking-water, excrement, spirit are normal.
2. the safety test that pair target animals single dose inoculation, single dose repeated inoculation, an overdose are inoculated with
Single dose is inoculated with safety test
1 age in days SPF chicken 20 is taken, is divided into 2 groups, every group 10.1401 batches of Combined vaccines are subcutaneously injected in 1st group of neck,
0.3ml/ is only；Physiological saline is subcutaneously injected in 2nd group of neck, and 0.3ml/ only, observe 14, record feeding, drink by raising in isolator
Situations such as water, excrement, exempts from the absorbing state of latter 15 days anatomic observation injection site lesions and vaccine.22 age in days SPF chickens 20 are taken,
It is divided into 2 groups, every group 10.1401 batches of Combined vaccines are subcutaneously injected in 3rd group of neck, and 0.5ml/ is only；Physiology is subcutaneously injected in 4th group of neck
Situations such as salt water, 0.5ml/ only, are raised in isolator, observed 14, record feeding, drinking-water, excrement, exempts to be dissected and observed for latter 15 days
The absorbing state of injection site lesion and vaccine.It the results are shown in Table 2.
Single dose repeated inoculation safety testing
1 age in days SPF chicken 20 is taken, is divided into 2 groups, every group 10.1401 batches of Combined vaccines are subcutaneously injected in 1st group of neck,
Only, in immune latter 14 days, same dosage inoculated 1 time 0.3ml/ again；Physiological saline, 0.3ml/ is subcutaneously injected in 2nd group of neck
Only, after injection 14 days again same dosage inject again 1 time, raising in isolator, two exempt from after observe again 14, record feeding,
Situations such as drinking-water, excrement, two exempt from the absorbing state of latter 15 days anatomic observation injection site lesions and vaccine.Take 22 age in days SPF chickens
20, it is divided into 2 groups, every group 10.3rd group of neck 1402 batches of Combined vaccines of subcutaneous injection, 0.5ml/, again in immune latter 14 days
Same dosage inoculates 1 time；4th group of neck is subcutaneously injected physiological saline, 0.5ml/ only, the same dosage again on the 14th after injection
Inject again 1 time, raising in isolator, two exempt from after observe again 14, situations such as record feeding, drinking-water, excrement, two exempt from after solve within 15th
Cut open the absorbing state of observation injection site lesion and vaccine.It the results are shown in Table 3.
Table 2 the result shows that, after the inoculation of vaccine single dose, observe 14, injection site and whole body do not cause it is obvious not
Good reaction is dissected for 15 days after exempting from, and injection site absorbs well no swelling, and inflammation etc., test chicken is searched for food within the entire observation period
It drinks water normal.Table 3 the result shows that, after vaccine secondary inoculation, observe 14, injection site and whole body do not cause it is obvious not
Good reaction, injection site absorb well no swelling, inflammation etc..Test chicken feeding drinking-water is normal within the entire observation period.
The safety test of 2 SPF chicken single dose of table inoculation
Note: 1, "-" indicates that chicken feeding, drinking-water, excrement, spirit are normal；Similarly hereinafter.2, immunization route is subcutaneous using neck
Injection.
The safety test of 3 SPF chicken single dose repeated inoculation of table
One time overdose is inoculated with safety testing
With 1 age in days SPF chicken 40, it is divided into 4 groups, every group 10,1401 batches of Combined vaccines are subcutaneously injected in the 1st group of neck,
Only, 1402 batches of Combined vaccines are subcutaneously injected in the 2nd group of neck to 0.6ml/, and only, 1403 batches of bigeminy are subcutaneously injected in the 3rd group of neck to 0.6ml/
Seedling, only, physiological saline is subcutaneously injected in the 4th group of neck to 0.6ml/, and 0.6ml/ only, observe to 14 days, record is adopted by raising in isolator
Before eating, drink water, being immune and exempt from rear SPF chicken weight on the 15th etc., the absorbing state of anatomic observation injection site lesion and vaccine.With 7
Age in days SPF chicken 40, is divided into 4 groups, and every group 10, the 1st group of neck is subcutaneously injected 1401 batches of Combined vaccines, 1.0ml/ only, the 2nd group of chest
1402 batches of Combined vaccines of portion's intramuscular injection, 1.0ml/, the 3rd group of neck 1403 batches of Combined vaccines of subcutaneous injection, 1.0ml/, the 4th group
Physiological saline is subcutaneously injected in neck, and 0.6ml/ only, observed to 14 days by raising in isolator, record feeding, drinking-water, it is immune before and
SPF chicken weight on the 15th etc. after exempting from, is dissected and observed the absorbing state of injection site lesion and vaccine.With 22 age in days SPF chickens 40, divide
It is 4 groups, every group 10, the 1st group of chest muscle injects 1401 batches of Combined vaccines, and only, the 2nd group of neck is subcutaneously injected 1402 batches to 1.0ml/
Combined vaccine, only, 1403 batches of Combined vaccines are subcutaneously injected in the 3rd group of neck to 1.0ml/, and only, physiology is subcutaneously injected in the 4th group of neck to 1.0ml/
Salt water, 0.6ml/, the interior raising of isolator was observed to 14 days, recorded before searching for food, drink water, being immune and exempt from rear SPF chicken weight on the 15th
Deng the absorbing state of anatomic observation injection site lesion and vaccine.With 270 age in days SPF chickens 40, it is divided into 4 groups, every group 10,
1401 batches of Combined vaccines are subcutaneously injected in 1st group of neck, and only, the 2nd group of chest muscle injects 1402 batches of Combined vaccines, 1.0ml/ to 1.0ml/
Only, the 3rd group of neck 1403 batches of Combined vaccines of subcutaneous injection, 1.0ml/, the 4th group of neck subcutaneous injection physiological saline, 1.0ml/,
Raising in isolator, is observed 60, record clinical symptoms, drinking-water, feeding and laying rate situation.
The safety test of a 41 age in days SPF overdose of chicken of table inoculation
The safety test of a 57 age in days SPF overdose of chicken of table inoculation
The safety test of a 6 22 age in days SPF overdose of chicken of table inoculation
The safety test of a 7 270 age in days SPF overdose of laying hen of table inoculation
Note: every group of experimental animal number is 10, and injection dosage is 1.0ml/.
Table 4~7 the result shows that, after vaccine overdose inoculation, observe 14, do not cause in injection site and whole body
Obvious adverse reaction, vaccine injection group and the weight gain of control group SPF chicken are absorbed without too big variation, anatomic observation, injection site vaccine
Well, no swelling, inflammation etc., test chicken feeding drinking-water is normal within the entire observation period.Laying hen test result shows bigeminy
Seedling within laying period be immunized be to laying period laying hen it is safe, laying rate, weight are substantially unaffected.
(5) immune period test
The antibody dynamic regularity and immune duration of 1 age in days and 22 age in days SPF chickens are carried out with 3 batches of seedlings that laboratory is manufactured experimently
Research.1 age in days SPF chicken is immunized in Combined vaccine, and test result is shown, the part NDV: after immune in 21 days, 5 months ND antibody >=
6.0log2 attacks 10/10 protection after poison；6 months after immune, antibody minority is down to 4log2 hereinafter, attacking 5/10~6/10 guarantor after poison
Shield；Rather than immunized controls chicken attacks equal 5/5 death after poison.Aviadenovirus part: it is anti-to detect that fine jade expands for a small number of chickens on the 7th after immune
Body, 21 days~6 months fine jades expand antibody equal 7/10 and with positive, attack malicious immune group within 21 days, 5,6 months after immune and reach after exempting from
8/10 or more protection；Control group attacks equal 8/10 or more morbidity after poison.From the point of view of result above, Combined vaccine is inoculated with 1 age in days SPF
Chicken (0.3ml/ is only) still was able to reach ideal protecting effect afterwards in 5 months.22 age in days SPF chickens, test result is immunized in Combined vaccine
It shows, 7 months individual chicken antibodies are down to threshold levels after vaccine immunity.Immune group laying rate is produced with Normal group after attacking poison
Egg rate difference is little, and attacks malicious control group laying rate of chicken and be decreased obviously.It can be seen that the Combined vaccine ND part protection period is up to 7
Month.From the point of view of result above, Combined vaccine is inoculated with 22 age in days SPF chickens (0.5ml/ is only) and still is able to reach ideal guarantor afterwards in 7 months
Protect effect.In view of feeding environment and practical condition, to prevent newcastle disease, H9 subtype avian influenza, I group of 4 type fowl gland
Virus generation, immunizing dose and duration of immunity are set to by we: 3 week old and within chicken, 0.3ml/ only, duration of immunity be 4 months.3
The above chicken of week old, only, duration of immunity is 6 months to 0.5ml/.
The 81 age in days SPF chicken immune phase of table tests
Moreover, inactivated vaccine prepared by the present invention is significantly better than commercially available I for YBAV-4 plants of the immune effect for attacking poison
4 type aviadenovirus vaccines of group, thus it is speculated that be caused by being morphed due to YBAV-4 pnca gene.
Claims (3)
1. a kind of newcastle disease virus, aviadenovirus bivalent inactivated vaccine, which is characterized in that the bivalent inactivated vaccine,
The antigen used is the newcastle disease virus and aviadenovirus of inactivation；The newcastle disease virus is newcastle disease virus La
Sota plants；The aviadenovirus is YBAV-4 plants of 4 type aviadenovirus of I group, and deposit number is CCTCC No.V201541.
2. bivalent inactivated vaccine as described in claim 1, which is characterized in that the inactivation of the virus uses formalin-inactivated.
3. the preparation method of bivalent inactivated vaccine as described in claim 1, which is characterized in that the method includes following
Step:
1) oil is mutually prepared:
Take 95 parts of mineral oil, 1 part of aluminum stearate, after being uniformly mixed in oily phase preparation tank and being heated to 80 DEG C, then plus 5 parts of departments
80, until temperature maintains 40 minutes when reaching 115 DEG C, oil is completed after cooling and is mutually prepared；
2) prepared by water phase:
The Newcastle disease venom of inactivation, aviadenovirus venom are mixed；Take 95 parts of hybrid antigen liquid, 5 parts of Tween 80s of sterilizing,
It mixes well；
3) it emulsifies
It takes 2 parts of oily phase to be put into emulsion tank, after being added 1 part of water phase, then the emulsification of completion in 30~40 minutes is stirred with 3500r/min and is made
It is standby.
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| WO2007072916A1 (en) * | 2005-12-22 | 2007-06-28 | Juridical Foundation The Chemo-Sero-Therapeutic Research Institute | Vaccine for in ovo inoculation |
| CN102068694A (en) * | 2010-12-29 | 2011-05-25 | 青岛易邦生物工程有限公司 | Method for producing triple inactivated vaccine for newcastle disease, infectious bronchitis and infectious bursal disease |
| CN104922663A (en) * | 2015-07-07 | 2015-09-23 | 青岛易邦生物工程有限公司 | New castle disease and H9 subtype bird flu bivalent vaccine |
| CN105031638A (en) * | 2015-07-07 | 2015-11-11 | 青岛易邦生物工程有限公司 | Trivalent inactivated vaccine against Newcastle disease, avian influenza and infectious bursal disease |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2007072916A1 (en) * | 2005-12-22 | 2007-06-28 | Juridical Foundation The Chemo-Sero-Therapeutic Research Institute | Vaccine for in ovo inoculation |
| CN102068694A (en) * | 2010-12-29 | 2011-05-25 | 青岛易邦生物工程有限公司 | Method for producing triple inactivated vaccine for newcastle disease, infectious bronchitis and infectious bursal disease |
| CN104922663A (en) * | 2015-07-07 | 2015-09-23 | 青岛易邦生物工程有限公司 | New castle disease and H9 subtype bird flu bivalent vaccine |
| CN105031638A (en) * | 2015-07-07 | 2015-11-11 | 青岛易邦生物工程有限公司 | Trivalent inactivated vaccine against Newcastle disease, avian influenza and infectious bursal disease |
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