CN105770885A - Newcastle disease virus and aviadenovirus combined inactivate vaccine - Google Patents

Abstract

The invention provides a newcastle disease virus and aviadenovirus combined inactivate vaccine.I group 4 type aviadenovirus ybav-4 new strain used in the vaccine is high in TCID50 titer, good in immunogenicity and capable of resisting attacks of isolated viruses, in all regions, of the aviadenovirus.The prepared vaccine is good in safety, and no local or whole body untoward effect is caused by the vaccine.During a storage life test, character, safety test and potency test data is analyzed, a result is compared with a single vaccine of like products, and it is proved that the combined vaccine has no obvious difference and is stable and effective; it is proved through the potency test result that the combined vaccine and three single vaccines are kept at high level, and generate antibodies faster compared with the like products, wherein antibodies of a control group is negative.

Description

A kind of newcastle disease virus, aviadenovirus bivalent inactivated vaccine

Technical field

The invention belongs to veterinary biologics technical field, be specifically related to a kind of newcastle disease virus, aviadenovirus Bivalent inactivated vaccine.

Background technology

Newcastle disease has the highest sickness rate and a case fatality rate, and all over the world have a generation more, and once break out by Bring crushing blow to poultry breeding industry, be a kind of Infectious Diseases of harm aviculture.OIE is classified as A Class epidemic disease.

By the Epidemiological study to I group I fowl adenovirus, this disease sickness rate in China chicken group is higher, can Propagated by two kinds of approach of horizontal and vertical, and in ascendant trend year by year.The host range of morbidity is the most increasingly Extensively, white meat-type chickens, Breeder hens, laying hen, yellow plumage chicken all can infection morbidity, particularly 2010 with sequela Present increase trend, the most all have popular.Many I group I fowl adenovirus can replicate in healthy carcass, Symptom the most slightly or not shows infection symptoms, but I group 4 type aviadenovirus makes an exception, and can directly cause chicken to mass-send Disease, major lesions shows as pericardial effusion regulating liver-QI, renomegaly, and primary disease occurred in the U.S. first in 1963, In succession occur all over the world subsequently, be whole world poultry and the common zymad of wild fowl.China in 1976 There is primary disease in Taiwan Province, in all parts of the country afterwards all have the report that primary disease occurs first, and in ascendant trend year by year, Serious harm is brought to chicken aquaculture.

In the last few years, H9 subtype avian influenza was the important diseases of relatively conventional serious threat poultry；And chicken group Infection to I group 4 type aviadenovirus is the most, and this disease is easy to cause secondary infection epidemic disease, to fowl industry Raiser brings a lot of worry, and vaccine especially inactivated vaccine is used for multiple times, and not only increases chicken house manpower Bear with material resources, repeatedly grab simultaneously chicken injection stress, also can affect production performance, cause chicken group to disease Susceptibility increases.The most continuous variation of strain so that although the vaccine of the multiple choices released, But still there is losing control of the situation of epidemic situation development, tackle so needing screening to obtain new epidemic isolates The new harm that variation is caused.

Summary of the invention

It is an object of the invention to provide a kind of newcastle disease virus, aviadenovirus bivalent inactivated vaccine, thus make up The deficiencies in the prior art.

The bivalent inactivated vaccine of the present invention, wherein antigen is newcastle disease virus and the aviadenovirus of inactivation；

Wherein newcastle disease virus is preferably newcastle disease virus La Sota strain；

Aviadenovirus is I group 4 type aviadenovirus YBAV-4 strain, and its deposit number is CCTCC No.V201541。

Above-mentioned inactivated vaccine, wherein the inactivation of virus uses formalin-inactivated；

The preparation method of the inactivated vaccine of the present invention is as follows:

1) prepared by oil phase:

Take 95 parts of mineral oil, aluminium stearate 1 part, mix homogeneously after being heated to 80 DEG C in oil phase preparation tank, Add 5 parts of these 80 (Span-80) of department again, maintain 40 minutes when reaching 115 DEG C to temperature, after cooling, complete oil Prepare mutually；

2) prepared by aqueous phase:

By the Newcastle Disease venom of inactivation, the mixing of I group I fowl adenovirus venom；Take hybrid antigen liquid 95 parts, sterilizing 5 parts of Tween 80s, fully mix；

Wherein Newcastle Disease venom, I group I fowl adenovirus quantity than preferably 1:2；

3) emulsifying

Take oil phase 2 parts and put in emulsion tank, after adding aqueous phase 1 part, then stir 30～40 with 3500r/min Minute completing emulsifying prepares.

The TCID of the I group 4 type aviadenovirus new strain of YBAV-4 strain that the vaccine of the present invention is used₅₀Titer height, Immunogenicity well and can resist the attack that the separation of aviadenovirus disease each place is malicious.The peace of vaccine prepared by the present invention Full property is good, any locally and systemically untoward reaction caused by vaccine does not occurs.Storage life test is passed through Character, safety testing, the analysis of potency test data, result compared with single Seedling of like product, two Connection Seedling no significant difference, all stable effective；Efficacy test results proves, Combined vaccine and three kinds of single Seedling antibody are equal Keep high level, faster than like product produces antibody, and matched group antibody is negative.

Detailed description of the invention

Applicant's screening obtains the I group 4 type aviadenovirus of a strain novel variant, by this virus and newcastle disease Virus comes together to prepare combined vaccine, thus facilitates the present invention.

Below in conjunction with embodiment, the present invention is described in detail.

The screening of embodiment 1:YBAV-4 strain strain

1, Epidemiological study is since 2010, the part Breeder hens in the area such as Shandong, Jiangsu, laying hen And partridge chickens occurs in that a kind of high with mortality rate, dissection mainly shows as liver enlargement, hydropericardium is feature Disease, through clinical investigation and test in laboratory, tentative diagnosis is the pericardium that I group of C-4 type aviadenovirus causes Hydrops hepatitis syndrome.2010, inventor had inclusion body hepatitis and pericardium to amass from Shandong Zibo plant The chicken liver of dying of illness of water classical symptom is successfully separated 1 strain virus.

2, after virus purification takes the liver grinding of the chicken that dies of illness, sterile saline is added with in the ratio of 1:5 Make suspension；After multigelation 3 times, 3000r/min is centrifuged 30min, takes supernatant；Add penicillin and The each 10000IU/ml of streptomycin, 4 DEG C overnight, filters through micropore filter, saves backup after steriling test is qualified. By the virus liquid of above-mentioned preparation with the dosage of 0.2ml/ embryo, inoculate 6.5 age in days SPF Embryo Gallus domesticus through yolk sac approach, Abandon dead germ in 24h, take allantoic fluid and the fetus of dead Embryo Gallus domesticus in inoculating 48h～168h, process in aforementioned manners Rear continuous passage, observes the hepatic tissue of the 3rd generation dead germ, and Embryo Gallus domesticus shows as dead germ, idiosome is short and small, growth is slow Slow, fetus curling, liver enlargement and matter are crisp, and embryo is congested.Collection dead germ allantoic fluid and fetus ,-20 DEG C Preserve.

3, the qualification of virus

3.1 blood clotting CHARACTERISTICS IDENTIFICATION aseptic collection SPF chicken and Sanguis Anas domestica liquid 5～10ml, wash 3～5 times repeatedly, end Hemocyte mud is diluted to 0.8%, 1% and 2% concentration by secondary normal saline, and 4 DEG C save backup.Side routinely Whether method detection isolated strain has these erythrocytic characteristics of coagulation.Set III group I fowl adenovirus EDSV-76 simultaneously For agglutination positive control.Result: separate poison can not coagulation SPF chicken and duck erythrocyte, even if changing red The concentration of cell, can not be allowed to coagulation.III group I fowl adenovirus EDSV-76 can coagulation chicken, duck red carefully Born of the same parents.

The method that 3.2 physicochemical property inspections are introduced with reference to " animal virology ", virus liquid is respectively with 5-bromine Uracil-2 '-deoxynucleoside (BUDR), chloroform, ether, hydrochloric acid (pH3), sodium hydroxide (pH10), After temperature (60 DEG C, 1h) processes, inoculated into chick embryo (0.2ml/ embryo), separately set normal saline process group conduct Comparison.Embryo Gallus domesticus pathological changes is observed after inoculation 5d.Result: separate poison respectively through BUDR, sodium hydroxide (pH10) With 60 DEG C, after 1h processes, inoculated into chick embryo, Embryo Gallus domesticus acts normally, and PCR detects feminine gender.Show BUDR energy The duplication in Embryo Gallus domesticus of the suppression virus, the nucleic acid type of isolated strain is DNA, and virus is the most alkaline-resisting, to temperature-sensitive Sense, 60 DEG C, 1h can be inactivated.And the strain processed through ether, chloroform and hydrochloric acid (pH3), do not affect disease , there is obvious Embryo Gallus domesticus pathological changes in poison propagation in Embryo Gallus domesticus, and PCR testing result is positive.Show that virus does not has Lipid cyst membrane, has resistance to ether and chloroform, acidproof.

3.3 serological Identification

3.3.1 group specificity identification and utilization agar gel diffusion test (AGP) prepares agar gel flat board pair Isolated strain carries out group specificity qualification.After agar solidification, punching with card punch, perforation pattern is central authorities 1 Hole surrounding 6 hole, aperture 4mm, pitch-row is 4mm, and hole underseal closes.Virus to be checked is placed in interstitial hole, holes around Add I group I fowl adenovirus type strain, EDSV-76 capital 911 strain standard positive serum and negative serum.Fine jade is expanded plate Being positioned over 37 DEG C of effects in wet box of adding a cover, 24～48h see whether coagulation sedimentation line occur.Result: separate Poison antigen is only capable of obvious sediment line occur with I group I fowl adenovirus 4 type positive serum, and with III group I fowl adenovirus Precipitation line does not all occur between EDSV-76 capital 911 strain standard positive serum and negative serum.

3.3.2 to identify that first I group I fowl adenovirus 1～12 type standard positive serum makees 1:10 dilute for type specificity Release, then by version " Chinese veterinary pharmacopoeia " annex fixed virus diluted blood therapy for clearing away heat in 2010, by I group I fowl adenovirus 1～12 type standard strains, separation poison carry out cross-neutralization to I group I fowl adenovirus 1～12 type standard positive serum Test, record neutralizes titer result.Result: separate poison and survey the neutralization titer (1:501) of 4 type standard positive serums The neutralization titer (1:537) surveying 4 type standard positive serums with 4 type standard strains is closer to；Isolated strain is surveyed The neutralization titer of other type standard positive serum is all at below 1:10.Show that separating strain is serum 4 type.

3.4 PCR detection and the aseptic grinding of gene sequencing disease chicken liver, multigelations 3 times, utilize pillar to move Thing DNA extraction kit extracts viral DNA, carries out PCR detection.1% agarose gel electrophoresis observed result. Positive is carried out Hexon gene sequencing, and carries out phylogenetic analysis.According to Hexon gene order Comparison and phylogenetic analysis result belong to same branch it can be seen that separate poison with I group I fowl adenovirus, with Serum 4 type homology is closest, but there is also the difference in sequence；With serum 6 type, 7 types, 8a and 8b Type homology is lower.

The China typical culture collection that this Strain is preserved in Wuhan Wuhan University on October 15th, 2015 Center, deposit number is CCTCC No.V201541.

The preparation of embodiment 2:YBAV-4 strain seed culture of viruses

(1) chicken liver cell optimal culture condition research

1, the impact of cell density cell growth by 5 kinds of different densities (1～50,000/ml, 5～100,000 Individual/ml, 10～150,000/ml, 15～200,000/ml, 20～250,000/ml) chicken liver cell connect respectively Plant with a batch of 25cm²Cell bottle, 225cm²In cell bottle, 3000ml rolling bottle, 10 layer cell factory, use Cultivating under the conditions of same with the DMEM nutritional solution of batch, each density inoculates 5 bottles/2, cultivate under the conditions of same, Observation of cell grows up to fine and close monolayer required time and the form of cell.

2, the impact new-born calf serum of same manufacturer production of new-born calf serum content cell growth, point Do not add in DMEM culture fluid in the ratio of 6%, 8%, 10%, 12%, cultivate with a collection of chicken liver cell, cell Whole density be 15～200,000/ml, the nutritional solution of every kind of serum-concentration inoculates same batch 25cm²Cell Bottle, 225cm²Each 5 bottles/2 of cell bottle, 3000ml rolling bottle, 10 layer cell factory, observation of cell grows up to densification Monolayer required time and cellular morphology.

3, cell dissociation buffer used by the impact of pancreatin cell growth is 0.02%EDTA-0.25% trypsin solution, After cell dissociation is good, a part of cell culture container goes Digestive system to add nutritional solution, another part cell Culture vessel does not discard Digestive system and is directly added into nutritional solution.Nutritional solution be pH value be 7.0～7.2, newborn containing 10% The DMEM nutritional solution of Ox blood serum, cell density is 15～200,000/ml.Inoculate same batch 25cm²Cell bottle, 225cm²Each 5 bottles/2 of cell bottle, 3000ml rolling bottle, 10 layer cell factory, cultivate under the conditions of same, observe Cell grows up to fine and close monolayer required time and cellular morphology.

4, nutritional solution pH value cell growth to affect other condition the most identical, only the pH value of nutritional solution is respectively Being adjusted to 6.8,7.0,7.2,7.4, the nutritional solution of different pH value inoculates 25cm respectively²Cell bottle, 225cm² Each 5 bottles/2 of cell bottle, 3000ml rolling bottle, 10 layer cell factory, observation battalion's nutrient solution color changes, cell is long Become fine and close monolayer required time and cellular morphology.

(2) I group 4 type aviadenovirus YBAV-4 strain optimal culture condition research

1, most preferably connect the determination of toxic agent amount respectively in different culture vessels with 0.1%, 0.5%, 1%, 2%, The YBAV-4 strain virus liquid inoculation chicken liver cell of 5% 5 various dose, observes and records appearance cytopathic Time and lesion degree, cells showed cytopathic (hereinafter referred to as CPE) the results virus liquid when more than 80%, After multigelation 2 times, measure its viral level (TCID respectively₅₀), with TCID₅₀The toxic agent amount that connects of soprano is Most preferably connect toxic agent amount.

2, the determination most preferably connecing the poison time breeds YBAV-4 strain virus with cell under three kinds of growth conditions Liquid, when chicken liver cell grows up to 60～70% monolayer, grows up to 70～80% monolayer and grow up to more than 90% monolayer inoculation disease Poison, connects poison by the amount of 1%, 37 DEG C, 5%CO₂Incubator is cultivated, and results during CPE occurs in the cell when more than 80% Virus liquid, after multigelation 2 times, measures its TCID respectively₅₀, with TCID₅₀The poison time that connects of soprano is optimal Connect the poison time.

3, I group 4 type aviadenovirus YBAV-4 strain virus liquid is connect by the determination of optimum culturing temperature by the amount of 1% Plant the chicken liver cell growing up to good monolayer, be respectively placed in 34 DEG C, 36 DEG C, 37 DEG C, 38 DEG C of cultivations, observe thin The time of born of the same parents' pathological changes appearance and lesion degree, the harvesting venom when CPE occurs in 80% cell, multigelation 2 Its TCID is measured respectively after secondary₅₀, with TCID₅₀The cultivation temperature of soprano is optimum culturing temperature.

4, I group 4 type aviadenovirus YBAV-4 strain virus liquid is inoculated by the optimal determination receiving the poison time by the amount of 1% Grow up to the chicken liver cell of good monolayer, respectively at 37 DEG C, 5%CO₂Incubator cultivate, when cell occur 70%, 80%, After gathering in the crops virus liquid, multigelation 2 times during about 90% CPE, measure its TCID respectively₅₀, with TCID₅₀Soprano The receipts poison time be optimal to receive the poison times.

5, best maintained liquid serum content determine by I group 4 type aviadenovirus YBAV-4 strain virus liquid by 1% amount Inoculation grows up to the chicken liver cell of good monolayer, maintains new-born calf serum content in liquid to be respectively 1%, 2%, 3%, Respectively at 37 DEG C, 5%CO₂Incubator is cultivated, and the time that observation of cell pathological changes occurs, when CPE occurs in 80% cell Time harvesting venom, measure its TCID respectively after multigelation 2 times₅₀, with TCID₅₀The serum content of soprano For best maintained liquid serum content.

6, the checking above-mentioned result of the test of experimental evidence, we select most preferably to connect poison mode, most preferably connect toxic agent Amount, most preferably connect poison time, optimum culturing temperature, optimal receive the poison time and be prepared for 3 batches of virus liquids, by virus liquid After multigelation 2 times, measure the viral level of virus liquid.

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

1. Millipore is concentrated by ultrafiltration machine use, maintenance condition and using method, the use provided by producer Explanation is carried out.

2. concentrated effect detection

2.1 concentrated antigens effect inspections keep sample before concentrating mensuration concentrate antigen valence in provirus liquid (HA and /EID₅₀/TCID₅₀), sampling and measuring concentrated solution antigen valence (HA) at any time in concentration process, determine cycles of concentration, Concentrated solution after concentration keeps sample, and measures (the HA and/EID of antigen valence in concentrated solution₅₀/TCID₅₀)。

2.2 filter liquor Detection of antigen take concentration when closing to an end (now in concentrated solution, antigen concentration is maximum, The probability spilling antigen in filter liquor is maximum) filter liquor, be respectively adopted mensuration HA-HI test (HA), inoculation The ways such as SPF Embryo Gallus domesticus (observe and spill with or without live virus), inoculation SPF chicken (having detected whether that antigenic substance spills) Detection omission timber in footpath there is nonantigenic composition.

2.3 concentrate membranous type number selects varying in size of different virus antigen composition, cutting of different ultrafilter membranes Stay (filtration) aperture different, therefore select the ultrafiltration of different pore size model (1#, 2#, 3#, 4#, 5#) Film carries out rejection tests to different virus, and according to Detection of antigen result in filter liquor, concentrate effect inspection result and Concentrate required time, determine optimal concentration membranous type number used by concentration two-strain antigen respectively.

2.4 concentrated effect stability tests concentrate each 3 batches of membrance concentration NDV, FADV blastochyle by optimal model, The stability of detection concentrated effect.

2.5 cycles of concentration tests concentrate film by optimal model, and NDV, FADV blastochyle is respectively concentrated into substance Long-pending 1/2,1/3,1/4,1/5, measure the EID of different cycles of concentration₅₀/TCID₅₀, determine suitable concentration again Number.

2.6 concentrated cost analyses, according to material consumption when concentrating, calculate concentrated cost.

(1) malicious (bacterium) the required standard that should reach of planting:

Use newcastle disease virus La Sota strain, I group 4 type aviadenovirus YBAV-4 strain as antigen seed culture of viruses.

(2) antigen preparation and the inspection of semifinished product:

The preparation of 1 production seed culture of viruses

Prepared by 1.1 newcastle disease virus La Sota strain seeds culture of viruses:

Seed culture of viruses sterile saline or PBS are made suitably dilution (such as 10^-4Or 10^-5), inoculation in allantoic cavity 10 age in days SPF Embryo Gallus domesticus, every embryo 0.1ml.72～120 hours dead and obvious Embryo Gallus domesticus of lesion after choosing inoculation, Results Embryo Gallus domesticus liquid (allantoic fluid and amniotic fluid), are loaded in sterilization container respectively.To check aseptic, red to 1% chicken Cell agglutination valency is not less than the Embryo Gallus domesticus liquid mixing of 1:512 (micromethod), and quantitative separating, in aseptic bottle, indicates Harvest date, Virus passages and loading amount, freezen protective.

Prepared by 1.2 I groups of 4 type aviadenovirus YBAV-4 strain seeds culture of viruses

1.2.1 the chicken liver cell grown fine is selected in seed culture of viruses breeding, discards original fluid, adds containing 1% The maintenance liquid of seed culture of viruses, puts 37 DEG C and cultivates 36～48 hours, the results when cytopathy reaches more than 80%, freeze thawing 2 times, being sub-packed in sterilization container, sampling is identified.Indicate harvest date, Virus passages etc..

The selection of 2 seedling materials

2.1 Avian pneumo-encephalitis virus seedling the material well-developed 10～11 susceptible Embryo Gallus domesticus of age in days (ND HI antibody All≤1:4).

2.2 I group I fowl adenovirus seedling material chicken liver cells.

The preparation of 3 antigen for vaccine liquid

3.1 Avian pneumo-encephalitis virus antigens

3.1.1 inoculation takes production seed culture of viruses, make suitably dilution (such as 10 with sterilizing PBS^-4Or 10^-5), By " fully automatic inoculating machine operation instruction " requirement, inoculate instar chicken embryo on the 10th～11, inoculation in every embryo allantoic cavity 0.2ml, puts 36～37 DEG C and continues to hatch, it is not necessary to turn over embryo.

3.1.2, after hatching and observing egg inoculation, embryo 1 time, hatched to 96 hours, all took per sunshine Going out, according to embryo, discard dead germ, embryo air chamber of living is upwards upright, puts 2～8 DEG C and cools down 12～24 hours.

3.1.3 gather in the crops and the Embryo Gallus domesticus of cooling is taken out, by " full-automatic cropper operation instruction " requirement, receive Obtain Embryo Gallus domesticus liquid.Drawing blastochyle to be placed in sterilization container, sampling measures red cell agglutination valency, and agglutination titer is less than 1:256 Person should discard.2～8 DEG C of preservations before the blastochyle inactivation of results, should be less than 5 days.

3.1.4 concentrating by the blastochyle of results under the conditions of 2～8 DEG C, sampling at any time measures red cell agglutination valency, Stop when red cell agglutination valency is not less than 1:1024 concentrating.Keep sample, carry out the inspection of semifinished product, remaining blastochyle Inactivate immediately.

3.1.5 inactivateing and imported in inactivation tank by virus liquid, metering adds 10% formalin, is sufficiently mixed, The ultimate density of formalin is 0.1%.37 DEG C inactivate 16 hours (by temperature in tank reach 37 DEG C start in terms of Time) take out afterwards, put 2～8 DEG C of preservations, should be less than 1 month.

3.2 I group I fowl adenovirus antigens

3.2.1 cell preparation is taken out cryopreservation tube from liquid nitrogen container and is put thawing in 37 DEG C of water-baths, is moved into by cell In centrifuge tube equipped with 10ml culture fluid, 1000r/min is centrifuged 5 minutes.With containing 20% new-born calf serum Culture fluid suspension cell, puts 37 DEG C, 5%CO₂Incubator is cultivated, the pancreas enzyme-EDTA when growing up to good monolayer Peptic cell.

3.2.2 prepared by antigen

3.2.2.1 the seed cell that cell monolayer cultivation will be enlarged by cultivating is inoculated in cell factory, 37 DEG C of trainings Support.

3.2.2.2 connect poison and select the chicken liver cell grown fine, discard original fluid, add containing 1% poison The maintenance liquid planted, puts 37 DEG C and continues to cultivate.

3.2.2.3 observe after connecing poison with results, observe every day 2 times, record cytopathy situation.Work as cell Results when pathological changes reaches more than 80%, freeze thawing 2 times, sampling carries out the inspection of semifinished product.-15 DEG C of preservations, should not More than 30 days.

3.2.2.4 concentrate the venom of results under the conditions of 2～8 DEG C, concentrate 2～3 times with the machine of ultrafiltration concentration, Keeping sample, carry out the inspection of semifinished product, remaining blastochyle inactivates immediately.

3.2.2.5 inactivateing and imported in inactivation tank by virus liquid, metering adds 10% formalin, is sufficiently mixed, The ultimate density of formalin is 0.2%.37 DEG C inactivate 16 hours (by temperature in tank reach 37 DEG C start in terms of Time) take out afterwards, put 2～8 DEG C of preservations, should be less than 1 month.

4 inspections of semifinished product

4.1 newcastle parts

4.1.1 red cell agglutination valency measures the virus liquid before taking inactivation, attached by existing " Chinese veterinary pharmacopoeia " Record is measured, and its red cell agglutination valency should be not less than 1:1024.

4.1.2 viral level measures and the virus liquid taken out before inactivation is made 10 times of serial dilutions, takes 10^-7、10^-8、10^-93 dilution factors, in each allantoic cavity, inoculation 10～11 age in days SPF Embryo Gallus domesticus 5 pieces, every embryo 0.1m1, put 36～37 DEG C are continued to hatch, and per sunshine, embryo 2 times, observed 5.Measure red cell agglutination valency by embryo, be not less than 1:128 person, is judged to infect, calculates EID₅₀.Every 0.1ml viral level answers >=10⁹ _. ⁰EID₅₀。

4.1.3 the virus liquid after steriling test takes inactivation, is examined by existing " Chinese veterinary pharmacopoeia " annex Test, answer asepsis growth.

4.1.4 inactivation inspection takes 10 age in days SPF Embryo Gallus domesticus 6 pieces, inoculation inactivation of viruses liquid, every embryo in allantoic cavity 0.2ml, puts 36～37 DEG C and continues to hatch, and per sunshine, embryo 2 times, observed 5, and Embryo Gallus domesticus nonspecific death should not surpass Cross 1 piece.All blastochyles are measured red cell agglutination valency respectively, coagulation all should be occurred without, by the blindest for blastochyle results Pass a generation, time still without agglutination titer, be judged to inactivation completely.

4.2 I group I fowl adenovirus parts

4.2.1 viral level measure take inactivation before virus liquid be measured, every 0.1ml viral level should >= 10⁷ _. ³TCID₅₀。

4.2.2 the virus liquid after steriling test takes inactivation, is examined by existing " Chinese veterinary pharmacopoeia " annex Test, answer asepsis growth.

4.2.3 the virus liquid after inactivation is made 10 times of dilutions by inactivation inspection, and the Hepar Gallus domesticus that inoculation is grown fine is thin Born of the same parents' (24 porocyte plate) 4 holes, every hole 0.2ml, supplementary maintenance liquid to 2.0ml；Set nonvaccinated chicken simultaneously Hepatocyte makees blank, 37 DEG C, 5%CO₂Incubator is cultivated, and observes 120 hours.Cell control well and Sample well all should occur without cytopathy.Culture is gathered in the crops a blind passage generation after multigelation, continues cultivation 120 Hour, when sample well still occurs without cytopathy, it is determined that for inactivation completely.

Embodiment 4: the preparation of vaccine

1 oil phase preparation takes 95 parts of mineral oil, aluminium stearate 1 part, mix homogeneously in oil phase preparation tank And after being heated to 80 DEG C, then Jia Siben-805 parts, maintain 40 minutes when reaching 115 DEG C to temperature, cooling The most standby.

The Newcastle Disease venom of inactivation, I group I fowl adenovirus venom are mixed by 2 aqueous phase preparations with 1:2 ratio. Take hybrid antigen liquid 95 parts, the tween 80 of sterilizing 5 parts, fully mix, make tween 80 be completely dissolved.

3 emulsifyings take oil phase 2 parts and put in emulsion tank, start motor, and slow rotation stirs, and blows slowly simultaneously After adding aqueous phase 1 part, then stir 30～40 minutes with 3500r/min.After emulsifying, take vaccine 10ml add from In heart pipe, being centrifuged 15 minutes with 3000r/min, the aqueous phase separated out at the bottom of pipe should be less than 0.5ml.

4 subpackage quantitative separatings, seal, and adhesive label, put 2～8 DEG C of preservations.

(4) safety test of vaccine

1. the safety test of a single dose inoculation of the various route of inoculation of pair target animals Take 1 age in days SPF chicken and be divided into 3 groups, often group 10,1401 batches of inactivated vaccines of the 1st group of cervical region subcutaneous injection, 0.3ml/ is only；1401 batches of inactivated vaccines of 2nd group of intramuscular injection, 0.3ml/ is only；3rd group of intramuscular injection physiology salt Water 0.3ml/ only compares.Taking 22 age in days SPF chickens and be divided into 3 groups, often group 10, the 1st group of cervical region is subcutaneous Injecting 1401 batches of inactivated vaccines, 0.5ml/ is only；1401 batches of inactivated vaccines of 2nd group of intramuscular injection, 0.5ml/ is only； 3rd group of intramuscular injection normal saline 0.5ml/ only compares, and raises respectively, Continuous Observation 14 in isolator Day.Injection site and whole body are not caused the most not by result cervical region subcutaneous injection and two kinds of approach of intramuscular injection Good reaction, within the whole observation period, test chicken is searched for food and is drunk water all normally, exempts from latter 15 days to dissect, and injection site is inhaled Receive good, it was demonstrated that this vaccine is safe through two kinds of injecting pathways to SPF chicken.

The different injecting pathway safety test to SPF chicken of table 1

Note: "-" represent chicken search for food, drink water, feces, spirit the most normal.

2. pair target animals single dose inoculation, single dose repeated inoculation, the safety test of an overdose inoculation

Single dose inoculation safety test

Take 1 age in days SPF chicken 20, be divided into 2 groups, often group 10.1st group of cervical region subcutaneous injection 1401 Criticizing Combined vaccine, 0.3ml/ is only；2nd group of cervical region subcutaneous injection normal saline, 0.3ml/ only, raises in isolator, Observe 14, the situation such as record is searched for food, drunk water, feces, exempt from latter 15 days anatomic observation injection site pathological changes And the absorbing state of vaccine.Take 22 age in days SPF chicken 20, be divided into 2 groups, often group 10.3rd group of neck 1401 batches of Combined vaccine of portion's subcutaneous injection, 0.5ml/ is only；4th group of cervical region subcutaneous injection normal saline, 0.5ml/ Only, raise in isolator, observe 14, the situation such as record is searched for food, drunk water, feces, exempt from latter 15 days solutions Cut open observation injection site pathological changes and the absorbing state of vaccine.The results are shown in Table 2.

Single dose repeated inoculation safety testing

Take 1 age in days SPF chicken 20, be divided into 2 groups, often group 10.1st group of cervical region subcutaneous injection 1401 Criticizing Combined vaccine, only, in immune latter 14 days, same dosage inoculated 1 time to 0.3ml/ again；2nd group of cervical region skin Hemostasis normal saline, 0.3ml/ only, injects 1 time in latter 14 days of injection same dosage again, isolator again Interior raising, two exempt from after observe again 14, the situation such as record is searched for food, drunk water, feces, two exempt from latter 15 days to solve Cut open observation injection site pathological changes and the absorbing state of vaccine.Take 22 age in days SPF chicken 20, be divided into 2 groups, Often group 10.1402 batches of Combined vaccine of 3rd group of cervical region subcutaneous injection, 0.5ml/, in immune latter 14 days again Secondary same dosage inoculates 1 time；4th group of cervical region subcutaneous injection normal saline, 0.5ml/ only, after injection 14 Day, same dosage again injected 1 time again, raised in isolator, two exempt from after observe again 14, record searches for food, The situations such as drinking-water, feces, two exempt from latter 15 days anatomic observation injection site pathological changes and the absorbing state of vaccine.Knot Fruit is shown in Table 3.

Table 2 result shows, after the inoculation of vaccine single dose, observes 14, does not draw in injection site and whole body Playing obvious adverse reaction, exempt from latter 15 days to dissect, injection site all absorbs well, without swelling, and inflammation etc., In the whole observation period test chicken search for food drinking-water the most normal.Table 3 result shows, after vaccine secondary inoculation, observes 14, not causing obvious adverse reaction in injection site and whole body, injection site all absorbs well, without swollen Swollen, inflammation etc..Within the whole observation period test chicken search for food drinking-water the most normal.

The safety test of table 2 SPF chicken single dose inoculation

Note: 1, "-" represent chicken search for food, drink water, feces, spirit the most normal；Lower same.2, immunization route is adopted Use cervical region subcutaneous injection.

The safety test of table 3 SPF chicken single dose repeated inoculation

Overdose inoculation safety testing

With 1 age in days SPF chicken 40, it is divided into 4 groups, often group 10, the 1st group of cervical region subcutaneous injection 1401 Criticize Combined vaccine, 0.6ml/, 1402 batches of Combined vaccine of the 2nd group of cervical region subcutaneous injection, 0.6ml/, the 3rd group of neck 1403 batches of Combined vaccine of portion's subcutaneous injection, 0.6ml/, the 4th group of cervical region subcutaneous injection normal saline, 0.6ml/ Only, raising in isolator, observe to 14 days, record is searched for food, is drunk water, before immunity and exempt from latter 15 days SPF Chicken body weight etc., anatomic observation injection site pathological changes and the absorbing state of vaccine.With 7 age in days SPF chicken 40, It is divided into 4 groups, often group 10,1401 batches of Combined vaccine of the 1st group of cervical region subcutaneous injection, 1.0ml/, the 2nd group Chest muscle 1402 batches of Combined vaccine of injection, 1.0ml/, 1403 batches of Combined vaccine of the 3rd group of cervical region subcutaneous injection, Only, the 4th group of cervical region subcutaneous injection normal saline, 0.6ml/ only, raises in isolator, observes to 14 1.0ml/ Day, record is searched for food, is drunk water, before immunity and exempt from latter 15 days SPF chicken body weight etc., and anatomic observation injection site is sick Become and the absorbing state of vaccine.With 22 age in days SPF chicken 40, it is divided into 4 groups, often group 10, the 1st group Chest muscle 1401 batches of Combined vaccine of injection, 1.0ml/, 1402 batches of Combined vaccine of the 2nd group of cervical region subcutaneous injection, 1.0ml/, 1403 batches of Combined vaccine of the 3rd group of cervical region subcutaneous injection, 1.0ml/, the 4th group of cervical region subcutaneous injection Normal saline, 0.6ml/ only, raises in isolator, observes to 14 days, and record is searched for food, drunk water, before immunity And exempt from latter 15 days SPF chicken body weight etc., anatomic observation injection site pathological changes and the absorbing state of vaccine.With 270 Age in days SPF chicken 40, is divided into 4 groups, often group 10,1401 batches of Combined vaccine of the 1st group of cervical region subcutaneous injection, 1.0ml/ only, the 2nd group of chest muscle 1402 batches of Combined vaccine of injection, 1.0ml/, the 3rd group of cervical region subcutaneous injection 1403 batches of Combined vaccine, 1.0ml/, the 4th group of cervical region subcutaneous injection normal saline, 1.0ml/, in isolator Raise, observe 60, record clinical symptoms, drink water, search for food and laying rate situation.

The safety test of overdose inoculation of table 41 age in days SPF chicken

The safety test of overdose inoculation of table 57 age in days SPF chicken

The safety test of overdose inoculation of table 6 22 age in days SPF chicken

The safety test of overdose inoculation of table 7 270 age in days SPF laying hen

Note: often group experimental animal number is 10, injection dosage is 1.0ml/.

Table 4～7 result shows, after overdose inoculation of vaccine, observes 14, at injection site and whole body Obvious adverse reaction, vaccine injection group and the weightening finish of matched group SPF chicken is not caused without the biggest change, to dissect and see Examining, injection site vaccine absorbs good, and without swelling, inflammation etc., within the whole observation period, test chicken is searched for food drink Water is the most normal.Laying hen result of the test shows, Combined vaccine immunity within laying period is safety to laying period laying hen , laying rate, body weight are substantially unaffected.

(5) immune period test

1 age in days and the antibody dynamic regularity of 22 age in days SPF chickens and immunity is carried out with 3 batches of Seedlings of laboratory trial-production The research of duration.Combined vaccine immunity 1 age in days SPF chicken, result of the test shows, NDV part: after immunity 21 days, in 5 months ND antibody all >=6.0log2,10/10 protection after counteracting toxic substances；Latter 6 months of immunity, antibody Minority is down to below 4log2,5/10～6/10 protection after counteracting toxic substances；Rather than after immunized controls chicken counteracting toxic substances equal 5/5 Dead.Aviadenovirus part: latter 7 days minority chickens of immunity can detect that fine jade expands antibody, exempts from latter 21 days～6 The moon, fine jade expanded antibody equal 7/10 and with positive, and latter 21 days of immunity, 5,6 months counteracting toxic substances immune group all reach 8/10 Protection more than and；After matched group counteracting toxic substances equal 8/10 and fall ill above.From the point of view of result above, Combined vaccine is inoculated 1 age in days SPF chicken (0.3ml/ is only) still is able to reach preferable protected effect in latter 5 months.Combined vaccine immunity 22 age in days SPF chickens, result of the test shows, after vaccine immunity, 7 months indivedual chicken antibodies are down to threshold levels. After counteracting toxic substances, immune group laying rate is little with Normal group laying rate difference, and counteracting toxic substances matched group laying rate of chicken is bright Aobvious decline.As can be seen here, the Combined vaccine ND part protection period was up to 7 months.From the point of view of result above, two Connection Seedling is inoculated 22 age in days SPF chickens (0.5ml/ is only) and still is able to reach preferable protected effect in latter 7 months. In view of feeding environment and practical condition, for prevention newcastle disease, H9 subtype avian influenza, I group 4 The generation of type aviadenovirus, immunizing dose and duration of immunity be set to by we: 3 week old and within chicken, 0.3ml/ only, Duration of immunity is 4 months.The 3 above chickens of week old, only, duration of immunity is 6 months to 0.5ml/.

The table 81 age in days SPF chicken immune phase tests

And, inactivated vaccine prepared by the present invention is significantly better than city for the immune effect of the counteracting toxic substances of YBAV-4 strain The I group of 4 type aviadenovirus vaccine sold, thus it is speculated that cause owing to YBAV-4 pnca gene morphs.

Claims (5)

1. a newcastle disease virus, aviadenovirus bivalent inactivated vaccine, it is characterised in that described bigeminy Inactivated vaccine, its antigen used is newcastle disease virus and the aviadenovirus of inactivation.

2. bivalent inactivated vaccine as claimed in claim 1, it is characterised in that described newcastle disease virus For newcastle disease virus La Sota strain.

3. bivalent inactivated vaccine as claimed in claim 1, it is characterised in that described aviadenovirus is I Group's 4 type aviadenovirus YBAV-4 strains, its deposit number is CCTCC No.V201541.

4. bivalent inactivated vaccine as claimed in claim 1, it is characterised in that the inactivation of described virus is adopted Use formalin-inactivated.

5. the preparation method of bivalent inactivated vaccine as claimed in claim 1, it is characterised in that described side Method comprises the following steps that

1) prepared by oil phase:

Take 95 parts of mineral oil, aluminium stearate 1 part, mix homogeneously after being heated to 80 DEG C in oil phase preparation tank, Add 5 parts of department's bases 80 again, maintain 40 minutes when reaching 115 DEG C to temperature, complete oil phase after cooling and prepare；

2) prepared by aqueous phase:

By the Newcastle disease venom of inactivation, the mixing of aviadenovirus venom；Take hybrid antigen liquid 95 parts, sterilizing 5 parts of Tween 80s, fully mix；

3) emulsifying

Take oil phase 2 parts and put in emulsion tank, after adding aqueous phase 1 part, then stir 30～40 with 3500r/min Minute completing emulsifying prepares.