CN105664150B

CN105664150B - A kind of newcastle disease virus, avian influenza virus and aviadenovirus triple inactivated vaccine

Info

Publication number: CN105664150B
Application number: CN201610099218.6A
Authority: CN
Inventors: 宫晓; 李陆梅; 程增青; 刘新文; 胡潇
Original assignee: Qingdao Yebio Bioengineering Co Ltd
Current assignee: Qingdao Yebio Bioengineering Co Ltd
Priority date: 2016-02-23
Filing date: 2016-02-23
Publication date: 2019-03-01
Anticipated expiration: 2036-02-23
Also published as: CN105664150A

Abstract

The present invention provides a kind of newcastle disease virus, avian influenza virus and aviadenovirus triple inactivated vaccine, the TCID of used YBAV-4 plants of 4 type aviadenovirus of I group new strains₅₀Potency is high, immunogenicity is good and can resist the attack of H9 hypotype and 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, triple vaccine no significant difference is stable effective；Efficacy test results prove that triple 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

A kind of newcastle disease virus, avian influenza virus and aviadenovirus triple inactivated vaccine

Technical field

The invention belongs to veterinary biologics technical fields, and in particular to a kind of newcastle disease virus, avian influenza virus and Aviadenovirus triple 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.

Bird flu be as caused by influenza A, Major Epidemic is in one of animals and humans infectiousness, the death rate High acute, highly contagious disease, betides all over the world, and often make a variation extensively, endangers very the mankind and aviculture Greatly.

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, newcastle disease, H9 subtype avian influenza are relatively conventional 2 kinds of important diseases for seriously threatening poultry； And chicken group is more and more to the infection of 4 type aviadenovirus of I group, this disease is easy to cause secondary infection epidemic disease, gives fowl industry raiser Many worries are brought, and vaccine especially inactivated vaccine is used for multiple times, not only increase chicken house man power and material burden, while more The secondary injection for grabbing chicken stress, also will affect production performance, chicken group caused to increase the neurological susceptibility of disease.In addition strain in recent years Constantly variation, although so that the vaccine for the multiple choices released, but still there is losing control of the situation of epidemic situation development, so needing It screens and obtains new popular strain to cope with new harm caused by variation.

Summary of the invention

The object of the present invention is to provide a kind of newcastle disease virus, avian influenza virus and aviadenovirus triple inactivated vaccine, To make up the deficiencies in the prior art.

Triple inactivated vaccine of the invention, wherein antigen is newcastle disease virus, avian influenza virus and the fowl adenopathy of inactivation Poison；

Wherein newcastle disease virus is preferably Sota plants of newcastle disease virus La；

Wherein avian influenza virus is preferably QDY plants of H9 subtype avian influenza virus (Avian influenza virus), in On April 29th, 2015 is deposited in Wuhan, China, the China typical culture collection center of Wuhan University, deposit number CCTCC NO:V201517。

Aviadenovirus is YBAV-4 plants of 4 type aviadenovirus of I group, is deposited in Wuhan, China, Wuhan on October 15th, 2015 The China typical culture collection center of university, deposit number are 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, avian flu venom, I group I fowl adenovirus venom are mixed；Take hybrid antigen liquid 95 Part, 5 parts of Tween 80s of sterilizing mix well；

Wherein the quantity of Newcastle Disease venom, avian flu venom, I group I fowl adenovirus is than being preferably 1:1:2；

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.

QDY plants of H9 subtype avian influenza virus used in vaccine of the invention and YBAV-4 plants of 4 type aviadenovirus of I group new poison The TCID of strain₅₀/EID₅₀Potency is high, immunogenicity is good and can resist the attack of H9 hypotype and 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.Storage life Analysis in test Jing Guo character, safety testing, potency test data, as a result compared with single seedling of similar product, three Seedling no significant difference, it is stable effective；Efficacy test results prove that triple vaccine and three kinds of single seedling antibody keep high level, than It is fast that similar product generates antibody, and control group antibody is feminine gender.

Specific embodiment

Applicant, which screens, obtains the 4 type aviadenovirus of I group of one plant of novel variant, by the virus and newcastle disease virus, Avian influenza virus comes together 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 occur for epidemiological survey It is a kind of with death rate height, dissection is mainly shown as the disease with the characteristics of liver enlargement, hydropericardium, through clinical investigation and experiment Room detection, tentative diagnosis are hydropericardium hepatitis syndrome caused by I group of C-4 type aviadenovirus.2010, inventor was from Shandong 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, after virus purification takes the liver for the chicken that dies of illness to grind, suspension is made with sterile saline is added in the ratio of 1:5； After multigelation 3 times, 3000r/min is centrifuged 30min, takes supernatant；Penicillin and each 10000IU/ml of streptomysin, 4 DEG C of mistakes are added Night filters through millipore filter, saves backup after steriling test is qualified.By the virus liquid of above-mentioned preparation with the dosage of 0.2ml/ embryo, 6.5 age in days SPF chicken embryos are inoculated with through yolk bag approach, abandon dead germ in for 24 hours, take the allantoic fluid of dead chicken embryo in inoculation 48h~168h And the hepatic tissue of the 3rd generation dead germ is observed in fetus, continuous passage after handling in aforementioned manners, 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 salt repeatedly Haemocyte mud is diluted to 0.8%, 1% and 2% concentration by water, and 4 DEG C save backup.Whether detection isolated strain has according to a conventional method There is 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: separation Poison cannot be aggregated SPF chicken and duck red blood cell, 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 -2 ' - After deoxyribonucleoside (BUDR), chloroform, ether, hydrochloric acid (pH3), sodium hydroxide (pH10), temperature (60 DEG C, 1h) processing, it is inoculated with 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 is respectively 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 isolated strain Carry out group specificity identification.It after agar solidification, is punched with punch, perforation pattern is 6 hole of central 1 hole surrounding, aperture 4mm, hole Away from for 4mm, bottom hole closing.Virus to be checked is placed in interstitial hole, and holes around adds 911 plants of I group I fowl adenovirus type strain, the capital EDSV-76 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 to coagulate Collect precipitation 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 of fowl 911 plants of the capital adenovirus 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.

3.4PCR detection and the sterile grinding of gene sequencing diseased chicken liver multigelation 3 times, are extracted using pillar animal DNA Kit extracts viral DNA, carries out PCR detection.1% agarose gel electrophoresis observes result.Positive sample is subjected to Hexon base Because of sequencing, and carry out phylogenetic analysis.It is compared according to Hexon gene order and phylogenetic analysis result can be seen that point Belong to same branch with I group I fowl adenovirus from poison, it is closest with 4 type homology of serum, but there is also the differences in sequence；With blood Clear 6 type, 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~ 150000/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, respectively by 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 rolling bottle, 10 Layer cell factory each 5 bottles/2, observation cell grows 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 various dose is inoculated with chicken liver cell, observes and records the time for cytopathy occur and lesion degree, After 80% or more cells showed cytopathic (hereinafter referred to as CPE) harvest virus liquid, multigelation 2 times, it is measured respectively 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, when chicken gizzard is thin Born of the same parents grow up to 60~70% single layers, grow up to 70~80% single layers and grow up to 90% or more single layer virus inoculation, connect poison by 1% amount, 37 DEG C, 5%CO₂Incubator culture, harvests virus liquid when CPE occurs in 80% or more cell, after multigelation 2 times, respectively Measure its TCID₅₀, 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 good 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 lesion that observation cytopathy occurs Degree harvests cell venom when CPE occurs in 80% cell, measures its TCID respectively after multigelation 2 times₅₀, with TCID₅₀Most The cultivation temperature of high person 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 good The chicken liver cell of good single layer, respectively at 37 DEG C, 5%CO₂Incubator culture, when 70%, 80%, 90% or so CPE occurs in cell When harvest virus liquid, after multigelation 2 times, measure its TCID respectively₅₀, with TCID₅₀The receipts of the soprano malicious time is best receipts poison Time.

5, best maintained liquid serum content, which is determined, is inoculated with length by 1% amount for 4 type aviadenovirus YBAV-4 strain virus liquid of I group At 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, most preferably connect verification test Malicious time, optimum culturing temperature, best receipts malicious time are prepared for 3 batches of virus liquids, after virus liquid multigelation 2 times, measurement disease The viral level of venom.

Embodiment 3: the preparation of newcastle disease, H9 subtype avian influenza, aviadenovirus (I group, 4 types) antigen

1.Millipore is concentrated by ultrafiltration machine and uses, and maintains 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, leaks in filter liquor when the detection of 2.2 filter liquor antigens takes concentration to close to an end It is maximum a possibility that antigen out) filter liquor, be respectively adopted that measurement HA-HI test (HA), (whether there is or not live virus for observation for inoculation SPF chicken embryo Leak out), have nonantigenic composition in inoculation SPF chicken (having detected whether antigenic substance leakage) etc. approach detection omission timber.

Of different sizes, retention (filtering) hole of different ultrafiltration membranes of 2.3 concentration membranous type number selection different virus antigen compositions Diameter 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 The time required to according to antigen testing result, concentration effect inspection result and concentration in filter liquor, two kinds of viral antigens of concentration are determined respectively Best concentration membranous type number used.

The best model of 2.4 concentrated effect stability tests is concentrated film and is concentrated NDV, AIV, FADV blastochyle each 3 batches, detects dense The stability of contracting effect.

Film is concentrated with best model in the test of 2.5 cycles of concentration, and NDV, AIV, FADV blastochyle are respectively concentrated into the 1/ of original volume 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, QDY plants of avian influenza virus, 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 QDY plants of seed culture of viruses preparations of 1.2H9 subtype avian influenza virus:

1.2.1 seed culture of viruses sterilizing PBS is made appropriate dilution (such as 10 by seed culture of viruses breeding^-4Or 10^-5), inoculation 10 in allantoic cavity~ 11 age in days SPF chicken embryos, every embryo 0.1ml set 36~37 DEG C and continue to be incubated for.Harvest is inoculated with 72 hours not dead and infected chicken blastochyles (allantoic fluid and amniotic fluid), loaded in sterilization container.It will examine sterile and 1:512 is not less than to 1% chicken erythrocyte suspension agglutination titer The mixing of chicken embryo liquid, quantitative separating indicates harvest date, Virus passages etc., freezen protective.

The YBAV-4 plants of seed culture of viruses preparations of 1.3 I groups of 4 type aviadenovirus

1.3.1 the chicken liver cell to grow fine is selected in seed culture of viruses breeding, discards original fluid, and the dimension containing 1% seed culture of viruses is added Liquid is held, 37 DEG C is set and cultivates 36~48 hours, harvested 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 (ND, AI HI of 2.1 newcastle disease virus, avian influenza virus seedling material 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^{- 4}Or 10^{- 5}), press " fully automatic inoculating 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 be incubated for, Embryo need not be turned over.

3.1.2 after being incubated for and observing egg inoculation, every sunshine embryo 1 time is incubated for 96 hours, all takes out, according to embryo, discard Dead germ, 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 inhales Blastochyle is taken to be placed in sterilization container, sampling measurement erythrocyte agglutination valence, agglutination titer should be discarded lower than 1:256 person.The blastochyle of harvest It saves, should be no more than 5 at 2~8 DEG C before 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 works as red blood cell 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, formalin Ultimate density be 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 of guarantors It deposits, should be no more than 1 month.

3.2H9 subtype avian influenza virus antigen

3.2.1 inoculation takes production seed culture of viruses, makees appropriate dilution (such as 10 with sterilizing PBS^{- 4}Or 10^{- 5}), press " fully automatic inoculating 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 be incubated for, Embryo need not be turned over.

3.2.2 after being incubated for and observing egg inoculation, every sunshine embryo 1 time is incubated for 72 hours, all takes out, according to embryo, discard Dead germ, embryo gas chamber living is upright upwards, set 2~8 DEG C of coolings 12~24 hours.

3.2.3 it harvests and takes out cooling chicken embryo, by " full-automatic cropper operation instruction " requirement, harvest chicken embryo liquid.It inhales Blastochyle is taken to be placed in sterilization container, sampling measurement erythrocyte agglutination valence, agglutination titer should be discarded lower than 1:256 person.The blastochyle of harvest It saves, should be no more than 5 at 2~8 DEG C before inactivation.

3.2.4 it is concentrated by the blastochyle of harvest under the conditions of 2~8 DEG C, with the concentration of the machine of ultrafiltration concentration, sampling measurement at any time is red thin Born of the same parents' agglutination titer stops concentration when erythrocyte agglutination valence is not less than 1:1024.Keep sample, carry out the inspection of semifinished product, remaining blastochyle with Inactivated.

3.2.5 it inactivates and imports virus liquid in inactivation tank, metered 10% formalin is sufficiently mixed, formalin Ultimate density be 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 of guarantors It deposits, should be no more than 1 month.

3.3I group I fowl adenovirus antigen

3.3.1 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 is moved into, 10ml is housed In the centrifuge tube of culture solution, 1000r/min is centrifuged 5 minutes.With the culture solution suspension cell for containing 20% newborn bovine serum, 37 are set DEG C, 5%CO₂Incubator culture, when growing up to good single layer with pancreas enzyme -EDTA vitellophag.

3.3.2 prepared by antigen

3.3.2.1 the seed cell that cell monolayer culture will be enlarged by culture is inoculated into cell factory, 37 DEG C of cultures.

3.3.2.2 it connects poison and selects the chicken liver cell to grow fine, discard original fluid, the maintenance containing 1% seed culture of viruses is added Liquid sets 37 DEG C and continues to cultivate.

3.3.2.3 after observation connects poison with harvest, daily observation 2 times records cytopathy situation.When cytopathy is up to 80% It is harvested when above, freeze thawing 2 times, sampling carries out the inspection of semifinished product.- 15 DEG C of preservations should be no more than 30.

3.3.2.4 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, into The row inspection of semifinished product, remaining blastochyle are inactivated immediately.

3.3.2.5 it inactivates and imports virus liquid in inactivation tank, metered 10% formalin is sufficiently mixed, formaldehyde is molten The ultimate density of liquid is 0.2%.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 It saves, 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 measured by existing " Chinese veterinary pharmacopoeia " annex, Its 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^{- 9} 3 A dilution, 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, daily According to embryo 2 times, observe 5.Erythrocyte agglutination valence is measured by embryo, is not less than 1:128 person, is judged to infect, calculates EID₅₀.Every 0.1ml Viral level answers >=10^9.0EID₅₀。

4.1.3 steriling test takes the virus liquid after inactivation, tests by existing " Chinese veterinary pharmacopoeia " annex, answers sterile life It is long.

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 sets 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 surveyed respectively Determine erythrocyte agglutination valence, should all not be aggregated, by a blastochyle harvest blind passage generation again, still without agglutination titer when, it is complete to be judged to inactivation.

4.2H9 subtype avian influenza part

4.2.1 the measurement of erythrocyte agglutination valence takes the virus liquid before inactivation, carries out by existing " Chinese veterinary pharmacopoeia " annex, measurement Its erythrocyte agglutination valence should be not less than 1:1024.

4.2.2 10 times of the virus liquid progress taken out before inactivation is serially diluted by viral level measurement, takes 10^{- 7}、10^{- 8}、10^{- 9} 3 dilutions, each allantoic cavity is interior to be inoculated with 10~11 5 pieces of age in days SPF chicken embryos, and every embryo 0.1ml 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 1:16 person, is judged to infect, calculates EID₅₀.Every 0.1ml Viral level answers >=10^9.0EID₅₀。

4.2.3 steriling test takes the avian flu venom after inactivation, tests, answers by existing " Chinese veterinary pharmacopoeia " annex Asepsis growth.

4.2.4 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 sets 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 surveyed respectively Determine erythrocyte agglutination valence, should all not be aggregated, by a blastochyle harvest blind passage generation again, still without agglutination titer when, it is complete to be judged to inactivation.

4.3 I group I fowl adenovirus parts

4.3.1 viral level measurement takes the virus liquid before inactivation to be measured, and every 0.1ml viral level answers >= 10^7.3TCID₅₀。

4.3.2 steriling test takes the virus liquid after inactivation, tests by existing " Chinese veterinary pharmacopoeia " annex, answers sterile life It is long.

4.3.3 inactivation, which is examined, makees 10 times of dilutions for the virus liquid after inactivation, and being inoculated with the chicken liver cell that grows fine, (24 holes are thin Born of the same parents' plate) 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 DEG C Afterwards, then 5 parts of Jia Siben -80, spare after cooling until temperature maintains 40 minutes when reaching 115 DEG C.

The preparation of 2 water phases is by the Newcastle Disease venom, avian flu venom, I group I fowl adenovirus venom of inactivation with 1:1:2 ratio Mixing.95 parts of hybrid antigen liquid are taken, 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 1 part of water phase being added slowly Afterwards, then with 3500r/min stirring 30~40 minutes.After emulsification, takes vaccine 10ml to be added in centrifuge tube, be centrifuged with 3000r/min 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

1 age in days SPF chicken is taken 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.It takes 22 Age SPF chicken is divided into 3 groups, and every group 10,1401 batches of inactivated vaccines are subcutaneously injected in the 1st group of neck, and 0.5ml/ is only；2nd group of intramuscular injection 1401 batches of inactivated vaccines, 0.5ml/ is only；3rd group of intramuscular injection physiological saline 0.5ml/ is only compared, and is raised in isolator respectively, It is observed continuously 14.As a result neck subcutaneous injection and two kinds of approach of intramuscular injection do not cause obviously not injection site and whole body Good reaction, test chicken feeding drinking-water is normal within the entire observation period, dissects within 15 days after exempting from, injection site absorbs good, it was demonstrated that The vaccine is safe to SPF chicken through two kinds of injecting pathways.

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 triple 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 triple 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 triple 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 triple 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 table 2SPF chicken single dose 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 table 3SPF chicken single dose repeated inoculation

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 triple vaccines are subcutaneously injected in the 1st group of neck, Only, 1402 batches of triple vaccines are subcutaneously injected in the 2nd group of neck to 0.6ml/, and only, the 3rd group of neck is subcutaneously injected 1403 batches three 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 triple vaccines, 1.0ml/ only, the 2nd group of chest 1402 batches of triple vaccines of portion's intramuscular injection, 1.0ml/, the 3rd group of neck 1403 batches of triple 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 triple vaccines, and only, the 2nd group of neck is subcutaneously injected 1402 batches to 1.0ml/ Triple vaccine, only, 1403 batches of triple 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 triple vaccines are subcutaneously injected in 1st group of neck, and only, the 2nd group of chest muscle injects 1402 batches of triple vaccines, 1.0ml/ to 1.0ml/ Only, the 3rd group of neck 1403 batches of triple 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 three 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 triple 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.The part H9 hypotype AIV: H9 antibody in 21 days, 5 months after immune >= 8.0log2,9/10 or more virus purification is protected after attacking poison；6 months after immune, antibody is down to 7.5log2 hereinafter, attacking 9/10 after poison The above virus purification protection；Rather than it is 9/10~10/10 that immunized controls chicken, which attacks malicious restrovirus separation rate,.Aviadenovirus part: A small number of chickens on the 7th can detect that fine jade expands antibody after immune, and 21 days~6 months fine jades expand antibody equal 7/10 and with positive, are immunized after exempting from It attacks within 21 days afterwards, 5,6 months malicious immune group and reaches 8/10 or more protection；Control group attacks equal 8/10 or more morbidity after poison.From From the point of view of result above, triple vaccine is inoculated with 1 age in days SPF chicken (0.3ml/ is only) and still is able to reach ideal protecting effect afterwards in 5 months.

The 81 age in days SPF chicken immune phase of table tests

Moreover, viral disease inactivated vaccine prepared by the present invention is for YBAV-4 plants of the immune effect for attacking poison significantly better than commercially available Other I group of 4 type aviadenovirus vaccine, thus it is speculated that be morphed due to YBAV-4 pnca gene caused by.

Claims

1. a kind of inactivated vaccine, which is characterized in that the inactivated vaccine, antigen are the newcastle disease virus of inactivation, fowl stream Influenza Virus and aviadenovirus；

The newcastle disease virus is Sota plants of newcastle disease virus La；

The avian influenza virus, deposit number are CCTCC NO:V201517；

The deposit number of the aviadenovirus is CCTCC No.V201541.

2. 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 inactivated vaccine described in claim 1, which is characterized in that the preparation method includes following step It is rapid:

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, avian flu venom, I group I fowl adenovirus venom are mixed；95 parts of hybrid antigen liquid are taken, is gone out 5 parts of Tween 80s of bacterium, mix 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.