CN105664150A - Newcastle disease virus, avian influenza virus and avian adenovirus triple inactivated vaccine - Google Patents
Newcastle disease virus, avian influenza virus and avian adenovirus triple inactivated vaccine Download PDFInfo
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Abstract
The invention provides a newcastle disease virus, avian influenza virus and avian adenovirus triple inactivated vaccine. TCID50 of I-group 4-type avian adenovirus YBAV-4 new strains is high in tilter, good in immunogenicity and capable of resisting attack of isolated viruses of H9 sub-type and avian adenovirus diseases in various places. The prepared vaccine is good in safety, and no local or whole-body adverse effects caused by the vaccine occur. By means of analysis of character, safety test and potency test data in a preservation state test, compared with results of single vaccines of similar products, difference of the triple vaccine is not remarkable, and the triple vaccine is stable and effective; a potency test result proves that the triple vaccine and three kinds of single-vaccine antibodies are all kept at a high level, antibody generation is faster compared with similar products, and the antibody of a control group is negative.
Description
Technical field
The invention belongs to veterinary biologics technical field, be specifically related to a kind of newcastle disease virus, bird flu virus and aviadenovirus triple inactivated vaccine.
Background technology
Newcastle disease has significantly high sickness rate and case fatality rate, and all over the world have generation more, and once outburst will bring crushing blow to poultry breeding industry, is a kind of Infectious Diseases of harm aviculture. OIE is classified as A class epidemic disease.
Bird flu is caused by influenza A, Major Epidemic in a kind of infectiousness of animal and human's apoplexy due to endogenous wind, acute, hyperinfection disease that mortality rate is high, extensively betide all over the world, and often make a variation, the mankind and aviculture are endangered very big.
By the Epidemiological study to I group I fowl adenovirus, this disease sickness rate in China chicken group is higher, can pass through two kinds of approach of horizontal and vertical and propagate, and in ascendant trend year by year. The host range of morbidity is also increasingly wider, white meat-type chickens, Breeder hens, laying hen, yellow plumage chicken all can infection morbidity, particularly within 2010, present increase trend with sequela, all have popular in China. Many I group I fowl adenovirus can replicate in healthy carcass, symptom very slightly or not shows infection symptoms, but I group 4 type aviadenovirus makes an exception, can directly cause chicken mass-sending disease, major lesions shows as pericardial effusion regulating liver-QI, renomegaly, primary disease occurred in the U.S. first in 1963, in succession occurred all over the world subsequently, was whole world poultry and the common zymad of wild fowl. 1976 there is primary disease in Taiwan Province of China first, and in all parts of the country afterwards all have the report that primary disease occurs, and in ascendant trend year by year, brings serious harm to chicken aquaculture.
In the last few years, newcastle disease, H9 subtype avian influenza were 2 kinds of important diseases of relatively conventional serious threat poultry; And chicken group is more and more many to the infection of I group 4 type aviadenovirus, this disease is easy to cause secondary infection epidemic disease, to fowl, industry raiser brings a lot of worry, and repeatedly use vaccine especially inactivated vaccine, not only increase chicken house man power and material's burden, repeatedly grab simultaneously chicken injection stress, also can affect production performance, cause chicken group that the susceptibility of disease is increased.In addition the continuous of strain makes a variation in recent years so that although the vaccine of the multiple choices released, but still there is losing control of the situation of epidemic situation development, so needing the screening new epidemic isolates of acquisition to tackle the new harm that variation causes.
Summary of the invention
It is an object of the invention to provide a kind of newcastle disease virus, bird flu virus and aviadenovirus triple inactivated vaccine, thus making up the deficiencies in the prior art.
The triple inactivated vaccine of the present invention, wherein antigen is the newcastle disease virus of inactivation, bird flu virus and aviadenovirus;
Wherein newcastle disease virus is preferably NDV La Sota Strain;
Wherein bird flu virus is preferably H9 subtype avian influenza virus QDY strain (Avianinfluenzavirus), it is deposited in Wuhan, China on April 29th, 2015, the China typical culture collection center of Wuhan University, deposit number is CCTCCNO:V201517.
Aviadenovirus is I group 4 type aviadenovirus YBAV-4 strain, is deposited in Wuhan, China on October 15th, 2015, and the China typical culture collection center of Wuhan University, its deposit number is CCTCCNo.V201541.
Above-mentioned inactivated vaccine, wherein the inactivation of virus adopts 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 heating after 80 DEG C in oil phase preparation tank, then add 5 parts of these 80 (Span-80) of department, 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, avian influenza venom, the mixing of I group I fowl adenovirus venom; Take hybrid antigen liquid 95 parts, 5 parts of Tween 80s of sterilizing, fully mix;
The wherein quantity ratio preferably 1:1:2 of Newcastle Disease venom, avian influenza venom, I group I fowl adenovirus;
3) emulsifying
Take oil phase 2 parts and put in emulsion tank, add after aqueous phase 1 part, then within 30～40 minutes, complete emulsifying with 3500r/min stirring and prepare.
H9 subtype avian influenza virus QDY strain that the vaccine of the present invention uses and the TCID of the I group 4 type aviadenovirus new strain of YBAV-4 strain50/EID50Titer is high, immunogenicity well and can resist H9 hypotype and the attack of aviadenovirus disease each place separation poison. The safety of vaccine prepared by the present invention is good, any locally and systemically untoward reaction caused by vaccine does not occur. In storage life test through character, safety testing, potency test data analysis, result with single Seedling of like product compared with, trigeminy vaccine no significant difference, all stable effective; Efficacy test results proves, trigeminy vaccine and three kinds of single Seedling antibody all 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, comes together to prepare combined vaccine by this virus and newcastle disease virus, bird flu virus, thus facilitating 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 occur in that a kind of high with mortality rate, dissection main manifestations is liver enlargement, hydropericardium is the disease of feature, through clinical investigation and test in laboratory, tentative diagnosis is the hydropericardium hepatitis syndrome that I group of C-4 type aviadenovirus causes. 2010, inventor had the chicken liver of dying of illness of inclusion body hepatitis and hydropericardium classical symptom from Shandong Zibo plant and is successfully separated 1 strain virus.
2, after virus purification takes the liver grinding of the chicken that dies of illness, suspension is made with adding sterile saline in the ratio of 1:5; After multigelation 3 times, 3000r/min is centrifuged 30min, takes supernatant; Adding penicillin and 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 the above-mentioned preparation dosage with 0.2ml/ embryo, 6.5 age in days SPF Embryo Gallus domesticus are inoculated through yolk sac approach, abandon dead germ in 24h, take allantoic fluid and the fetus of dead Embryo Gallus domesticus in inoculation 48h～168h, continuous passage after processing in aforementioned manners, observe the hepatic tissue of the 3rd generation dead germ, Embryo Gallus domesticus shows as that dead germ, idiosome be short and small, hypoevolutism, fetus are curling, liver enlargement and matter crisp, embryo is congested. Collect dead germ allantoic fluid and fetus ,-20 DEG C of preservations.
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, and hemocyte mud is diluted to 0.8%, 1% and 2% concentration by last normal saline, and 4 DEG C save backup. Whether detection isolated strain has these erythrocytic characteristics of coagulation according to a conventional method. Set III group I fowl adenovirus EDSV-76 as agglutination positive control simultaneously. Result: separate poison can not coagulation SPF chicken and duck erythrocyte, even if changing erythrocytic concentration, can not so as to coagulation. III group I fowl adenovirus EDSV-76 can coagulation chicken, duck erythrocyte.
The method that 3.2 physicochemical property inspections are introduced with reference to " animal virology ", virus liquid is respectively with 5-bromouracil-2 '-deoxynucleoside (BUDR), chloroform, ether, hydrochloric acid (pH3), sodium hydroxide (pH10), temperature (60 DEG C, 1h) process after, inoculated into chick embryo (0.2ml/ embryo), separately sets normal saline process group as comparison. Embryo Gallus domesticus pathological changes is observed after inoculation 5d. Result: separating poison respectively after BUDR, sodium hydroxide (pH10) and 60 DEG C, 1h process, inoculated into chick embryo, Embryo Gallus domesticus is acted normally, and PCR detects feminine gender. Showing that BUDR can suppress virus duplication in Embryo Gallus domesticus, the nucleic acid type of isolated strain is DNA, and virus is not alkaline-resisting, and to thermo-responsive, 60 DEG C, 1h can be inactivated. And through the strain that ether, chloroform and hydrochloric acid (pH3) process, do not affect virus propagation in Embryo Gallus domesticus, and obvious Embryo Gallus domesticus pathological changes occurs, PCR testing result is positive. Show that virus does not have lipid cyst membrane, ether and chloroform are had resistance, acidproof.
3.3 serological Identification
3.3.1 group specificity identification and utilization agar gel diffusion test (AGP) is prepared agar gel flat board isolated strain is carried out group specificity qualification. After agar solidification, punching with card punch, perforation pattern is central 1 hole surrounding 6 hole, and aperture 4mm, pitch-row is 4mm, and hole underseal closes. Virus to be checked is placed in interstitial hole, and holes around adds I group I fowl adenovirus type strain, EDSV-76 capital 911 strain standard positive serum and negative serum. Fine jade expanding plate and is positioned over 37 DEG C of effects in wet box of adding a cover, 24～48h sees whether coagulation sedimentation line occur. Result: separate poison antigen and be only capable of with I group I fowl adenovirus 4 type positive serum obvious sediment line occurring, and and between III group I fowl adenovirus EDSV-76 capital 911 strain standard positive serum and negative serum, precipitation line does not all occur.
3.3.2 type specificity identifies that first I group I fowl adenovirus 1～12 type standard positive serum does 1:10 dilution, again by version " Chinese veterinary pharmacopoeia " annex fixed virus diluted blood therapy for clearing away heat in 2010, to I group I fowl adenovirus 1～12 type standard positive serum, I group I fowl adenovirus 1～12 type standard strain, separation poison are carried out cross neutralization test, and record neutralizes titer result.Result: the neutralization titer (1:537) that the neutralization titer (1:501) of separation poison survey 4 type standard positive serums and 4 type standard strains survey 4 type standard positive serums is closer to; Isolated strain surveys the neutralization titer of other type standard positive serum all at below 1:10. Show that separating strain is serum 4 type.
3.4PCR detection and the aseptic grinding of gene sequencing disease chicken liver, multigelation 3 times, utilize pillar animal DNA to extract test kit and extract viral DNA, carry 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 it can be seen that separate poison to belong to same branch with I group I fowl adenovirus, closest with serum 4 type homology, but there is also the difference in sequence; Lower with serum 6 type, 7 types, 8a and 8b type homology.
The China typical culture collection center that this Strain is preserved in Wuhan Wuhan University on October 15th, 2015, deposit number is CCTCCNo.V201541.
The preparation of embodiment 2:YBAV-4 strain seed culture of viruses
(1) chicken liver cell optimal culture condition research
1, the chicken liver cell of 5 kinds of different densities (1～50,000/ml, 5～100,000/ml, 10～150,000/ml, 15～200,000/ml, 20～250,000/ml) is inoculated the 25cm of same batch by the impact of cell density cell growth respectively2Cell bottle, 225cm2In cell bottle, 3000ml rolling bottle, 10 layer cell factory, cultivating under same condition with the DMEM nutritional solution of same batch, each density inoculates 5 bottles/2, and cultivation under same condition, observation of cell grow up to the form of fine and close monolayer required time and cell.
2, the impact new-born calf serum of same manufacturer production of new-born calf serum content cell growth, add in DMEM culture fluid in the ratio of 6%, 8%, 10%, 12% respectively, cultivate with a collection of chicken liver cell, the whole density of cell is 15～200,000/ml, and the nutritional solution of every kind of serum-concentration inoculates same batch of 25cm2Cell bottle, 225cm2Each 5 bottles/2 of cell bottle, 3000ml rolling bottle, 10 layer cell factory, observation of cell grows up to fine and close 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, and another part cell culture container does not discard Digestive system and is directly added into nutritional solution. Nutritional solution is pH value is 7.0～7.2, DMEM nutritional solution containing 10% new-born calf serum, and cell density is 15～200,000/ml. Inoculate same batch of 25cm2Cell bottle, 225cm2Each 5 bottles/2 of cell bottle, 3000ml rolling bottle, 10 layer cell factory, cultivation under same condition, observation of cell grow up to fine and close monolayer required time and cellular morphology.
4, nutritional solution pH value cell growth to affect other condition all identical, only the pH value of nutritional solution is adjusted to 6.8,7.0,7.2,7.4 respectively, and the nutritional solution of different pH value inoculates 25cm respectively2Cell bottle, 225cm2Each 5 bottles/2 of cell bottle, 3000ml rolling bottle, 10 layer cell factory, observation battalion's nutrient solution color change, cell grow up to fine and close monolayer required time and cellular morphology.
(2) I group 4 type aviadenovirus YBAV-4 strain optimal culture condition research
1, the best connect the determination of toxic agent amount respectively in different culture vessels YBAV-4 strain virus liquid with 0.1%, 0.5%, 1%, 2%, 5% 5 various dose inoculate chicken liver cell, observe and record appearance cytopathic time and lesion degree, cells showed cytopathic (hereinafter referred to as CPE) when more than 80% gathers in the crops virus liquid, after multigelation 2 times, measure its viral level (TCID respectively50), with TCID50The toxic agent amount that connects of soprano is that the best connects toxic agent amount.
2, the best connects the determination of poison time and breeds YBAV-4 strain virus liquid under three kinds of growth conditions with cell, when chicken liver cell grows up to 60～70% monolayers, grows up to 70～80% monolayers and grows up to more than 90% monolayer virus inoculation, connects poison by the amount of 1%, 37 DEG C, 5%CO2Incubator is cultivated, results virus liquid when CPE occurs in the cell when more than 80%, after multigelation 2 times, measures its TCID respectively50, with TCID50The poison time that connects of soprano is that the best connects the poison time.
3, I group 4 type aviadenovirus YBAV-4 strain virus liquid is grown up to the chicken liver cell of good monolayer by the determination of optimum culturing temperature by the amount inoculation of 1%, it is respectively placed in 34 DEG C, 36 DEG C, 37 DEG C, 38 DEG C cultivations, the time of observation of cell pathological changes appearance and lesion degree, the harvesting venom when CPE occurs in 80% cell, measures its TCID after multigelation 2 times respectively50, with TCID50The cultivation temperature of soprano is optimum culturing temperature.
4, I group 4 type aviadenovirus YBAV-4 strain virus liquid is grown up to the chicken liver cell of good monolayer by the best determination receiving the poison time by the amount inoculation of 1%, respectively at 37 DEG C, 5%CO2Incubator is cultivated, when cell occur 70%, 80%, about 90% CPE time results virus liquid, after multigelation 2 times, measure its TCID respectively50, with TCID50The receipts poison time of soprano is the best receipts poison time.
5, best maintained liquid serum content is determined by the amount inoculation of 1%, I group 4 type aviadenovirus YBAV-4 strain virus liquid is grown up to the chicken liver cell of good monolayer, and new-born calf serum content respectively 1%, 2%, 3% in maintenance medium, respectively at 37 DEG C, 5%CO2Incubator is cultivated, the time that observation of cell pathological changes occurs, the harvesting venom when CPE occurs in 80% cell measures its TCID after multigelation 2 times respectively50, with TCID50The serum content of soprano is best maintained liquid serum content.
6, the result of the test that checking experimental evidence is above-mentioned, we select the best to connect, and poison mode, the best connect toxic agent amount, the best connects the poison time, optimum culturing temperature, best receive the poison time and be prepared for 3 batches of virus liquids, after virus liquid multigelation 2 times, measure the viral level of virus liquid.
Embodiment 3: newcastle disease, H9 subtype avian influenza, aviadenovirus (I group, 4 types) antigen preparation
1.Millipore is concentrated by ultrafiltration machine and uses, and maintains condition and using method, and the operation instruction provided by producer carries out.
2. concentrated effect detection
The mensuration that keeps sample before 2.1 concentrated antigen effect inspection concentrations concentrates the (HA and/EID of antigen valence in provirus liquid50/TCID50), sampling and measuring concentrated solution antigen valence (HA) at any time in concentration process, it is determined that cycles of concentration, the 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 when concentration closes to an end that (now in concentrated solution, antigen concentration is maximum, the probability spilling antigen in filter liquor is maximum) filter liquor, be respectively adopted in the approach detection omission timber such as mensuration HA-HI test (HA), inoculation SPF Embryo Gallus domesticus (observe spill with or without live virus), inoculation SPF chicken (having detected whether that antigenic substance spills) with or without antigen composition.
2.3 concentration membranous types number select varying in size of different virus antigen composition, different ultrafilter membranes retain (filtrations) aperture difference, therefore select the ultrafilter membrane of different pore size model (1#, 2#, 3#, 4#, 5#) that different virus is carried out rejection tests, and according to Detection of antigen result, concentration effect inspection result and concentration required time in filter liquor, determine concentration two-strain antigen best concentration membranous type number used respectively.
Best model each 3 batches of membrance concentration NDV, AIV, FADV blastochyle of concentration of 2.4 concentrated effect stability tests, the stability of detection concentrated effect.
NDV, AIV, FADV blastochyle, with best models concentration film, is respectively concentrated into the 1/2 of original volume, 1/3,1/4,1/5 by 2.5 cycles of concentration tests, measure the EID of different cycles of concentration50/TCID50, it is determined that suitable cycles of concentration.
Material consumption when 2.6 concentrated cost analyses are according to concentration, calculates concentrated cost.
(1) poison (bacterium) is planted and should be reached required standard:
Use NDV La Sota Strain, bird flu virus QDY 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 NDV La Sota Strain seeds culture of viruses:
Seed culture of viruses sterile saline or PBS are done suitably dilution (such as 10-4Or 10-5), inoculation 10 age in days SPF Embryo Gallus domesticus, every embryo 0.1ml in allantoic cavity. 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 the Embryo Gallus domesticus liquid mixing aseptic, 1% chicken red blood cell agglutination titer is not less than 1:512 (micromethod), quantitative separating, in aseptic bottle, indicates harvest date, Virus passages and loading amount, freezen protective.
Prepared by 1.2H9 subtype avian influenza virus QDY strain seed culture of viruses:
1.2.1 seed culture of viruses sterilizing PBS is done suitably dilution (such as 10 by seed culture of viruses breeding-4Or 10-5), in allantoic cavity, inoculation 10～11 age in days SPF Embryo Gallus domesticus, every embryo 0.1ml, put 36～37 DEG C and continue to hatch. 72 hours not dead and infected chicken blastochyles (allantoic fluid and amniotic fluid) of results inoculation, are loaded in sterilization container. By checking aseptic and that 1% chicken erythrocyte suspension agglutination titer is not less than 1:512 Embryo Gallus domesticus liquid mixing, quantitative separating, indicate harvest date, Virus passages etc., freezen protective.
Prepared by 1.3 I groups of 4 type aviadenovirus YBAV-4 strain seeds culture of viruses
1.3.1 the chicken liver cell grown fine is selected in seed culture of viruses breeding, discards original fluid, adds the maintenance medium containing 1% seed culture of viruses, put 37 DEG C to cultivate 36～48 hours, gather in the crops 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
Well-developed 10～11 age in days susceptible Embryo Gallus domesticus of 2.1 Avian pneumo-encephalitis virus, bird flu virus seedling material (ND, AIHI 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, does 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, in every embryo allantoic cavity, inoculation 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 out per sunshine, according to embryo, discarded dead germ, and 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, gather in the crops Embryo Gallus domesticus liquid. Drawing blastochyle and be placed in sterilization container, sampling measures red cell agglutination valency, and agglutination titer should discard lower than 1:256 person. 2～8 DEG C of preservations before the blastochyle inactivation of results, should less than 5 days.
3.1.4 concentrating by the blastochyle of results under 2～8 DEG C of conditions, sampling at any time measures red cell agglutination valency, stops concentration when red cell agglutination valency is not less than 1:1024.Keeping sample, carry out the inspection of semifinished product, all the other blastochyles inactivate immediately.
3.1.5 inactivateing and imported in inactivation tank by virus liquid, metering adds 10% formalin, is sufficiently mixed, and the ultimate density of formalin is 0.1%. 37 DEG C inactivate 16 hours (reach 37 DEG C with temperature in tank and start timing) and take out afterwards, put 2～8 DEG C of preservations, should less than 1 month.
3.2H9 subtype avian influenza virus antigen
3.2.1 inoculation takes production seed culture of viruses, does 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, in every embryo allantoic cavity, inoculation 0.2ml, puts 36～37 DEG C and continues to hatch, it is not necessary to turn over embryo.
3.2.2 after hatching and observing egg inoculation, embryo 1 time, hatched to 72 hours, all took out per sunshine, according to embryo, discarded dead germ, and embryo air chamber of living is upwards upright, puts 2～8 DEG C and cools down 12～24 hours.
3.2.3 gather in the crops and the Embryo Gallus domesticus of cooling is taken out, by " full-automatic cropper operation instruction " requirement, gather in the crops Embryo Gallus domesticus liquid. Drawing blastochyle and be placed in sterilization container, sampling measures red cell agglutination valency, and agglutination titer should discard lower than 1:256 person. 2～8 DEG C of preservations before the blastochyle inactivation of results, should less than 5 days.
3.2.4 concentrating by the blastochyle of results under 2～8 DEG C of conditions, with the concentration of the machine of ultrafiltration concentration, sampling at any time measures red cell agglutination valency, stops concentration when red cell agglutination valency is not less than 1:1024. Keeping sample, carry out the inspection of semifinished product, all the other blastochyles inactivate immediately.
3.2.5 inactivateing and imported in inactivation tank by virus liquid, metering adds 10% formalin, is sufficiently mixed, and the ultimate density of formalin is 0.1%. 37 DEG C inactivate 16 hours (reach 37 DEG C with temperature in tank and start timing) and take out afterwards, put 2～8 DEG C of preservations, should less than 1 month.
3.3I group I fowl adenovirus antigen
3.3.1 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 the centrifuge tube equipped with 10ml culture fluid, centrifugal 5 minutes of 1000r/min. With the culture fluid suspension cell containing 20% new-born calf serum, put 37 DEG C, 5%CO2Incubator is cultivated, and uses pancreas enzyme-EDTA peptic cell when growing up to good monolayer.
3.3.2 prepared by antigen
3.3.2.1 the seed cell that cell monolayer cultivation will be enlarged by cultivating is inoculated in cell factory, 37 DEG C of cultivations.
3.3.2.2 connect poison and select the chicken liver cell grown fine, discard original fluid, add the maintenance medium containing 1% seed culture of viruses, put 37 DEG C and continue to cultivate.
3.3.2.3 observe after connecing poison with results, observe every day 2 times, record cytopathy situation. Gathering in the crops when cytopathy reaches more than 80%, freeze thawing 2 times, sampling carries out the inspection of semifinished product.-15 DEG C of preservations, should less than 30 days.
3.3.2.4 concentrating the venom of results under 2～8 DEG C of conditions, concentrate 2～3 times with the machine of ultrafiltration concentration, keep sample, carry out the inspection of semifinished product, all the other blastochyles inactivate immediately.
3.3.2.5 inactivateing and imported in inactivation tank by virus liquid, metering adds 10% formalin, is sufficiently mixed, and the ultimate density of formalin is 0.2%. 37 DEG C inactivate 16 hours (reach 37 DEG C with temperature in tank and start timing) and take out afterwards, put 2～8 DEG C of preservations, should 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, is measured by existing " Chinese veterinary pharmacopoeia " annex, 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, inoculation 10～11 age in days SPF Embryo Gallus domesticus 5 pieces, every embryo 0.1m1, put 36～37 DEG C and continue to hatch in each allantoic cavity, and per sunshine, embryo 2 times, observed 5. Measure red cell agglutination valency by embryo, be not less than 1:128 person, be judged to infection, calculate EID50. Every 0.1ml viral level answers >=109.0EID50。
4.1.3 steriling test takes the virus liquid after inactivation, tests by existing " Chinese veterinary pharmacopoeia " annex, answers asepsis growth.
4.1.4 inactivation inspection takes 10 age in days SPF Embryo Gallus domesticus 6 pieces, and inoculation inactivation of viruses liquid, every embryo 0.2ml, put 36～37 DEG C and continue to hatch in allantoic cavity, and per sunshine, embryo 2 times, observed 5, and Embryo Gallus domesticus nonspecific death should less than 1 piece. All blastochyles are measured red cell agglutination valency respectively, coagulation all should be occurred without, blastochyle is gathered in the crops a blind passage generation again, time still without agglutination titer, be judged to inactivation completely.
4.2H9 subtype avian influenza part
4.2.1 red cell agglutination valency measures the virus liquid before taking inactivation, is undertaken by existing " Chinese veterinary pharmacopoeia " annex, measures its red cell agglutination valency, should be not less than 1:1024.
4.2.2 viral level measures and the virus liquid taken out before inactivation is carried out 10 times of serial dilutions, takes 10-7、10-8、10-93 dilution factors, inoculation 10～11 age in days SPF Embryo Gallus domesticus 5 pieces, every embryo 0.1ml, put 36～37 DEG C and continue to hatch in each allantoic cavity, and per sunshine, embryo 2 times, observed 5. Measure red cell agglutination valency by embryo, be not less than 1:16 person, be judged to infection, calculate EID50. Every 0.1ml viral level answers >=109.0EID50。
4.2.3 steriling test takes the avian influenza venom after inactivation, tests by existing " Chinese veterinary pharmacopoeia " annex, answers asepsis growth.
4.2.4 inactivation inspection takes 10 age in days SPF Embryo Gallus domesticus 6 pieces, and inoculation inactivation of viruses liquid, every embryo 0.2ml, put 36～37 DEG C and continue to hatch in allantoic cavity, and per sunshine, embryo 2 times, observed 5, and Embryo Gallus domesticus nonspecific death should less than 1 piece. All blastochyles are measured red cell agglutination valency respectively, coagulation all should be occurred without, blastochyle is gathered in the crops a blind passage generation again, time still without agglutination titer, be judged to inactivation completely.
4.3 I group I fowl adenovirus parts
4.3.1 the virus liquid that viral level measures before taking inactivation is measured, and every 0.1ml viral level answers >=107.3TCID50。
4.3.2 steriling test takes the virus liquid after inactivation, tests by existing " Chinese veterinary pharmacopoeia " annex, answers asepsis growth.
4.3.3 the virus liquid after inactivation is done 10 times of dilutions by inactivation inspection, chicken liver cell (24 porocyte plate) 4 holes grown fine of inoculation, every hole 0.2ml, and supplementary maintenance medium is to 2.0ml; Set nonvaccinated chicken liver cell simultaneously and make 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 to cultivate 120 hours, when sample well still occurs without cytopathy, it is determined that for inactivateing completely.
Embodiment 4: the preparation of vaccine
1 oil phase preparation takes 95 parts of mineral oil, aluminium stearate 1 part, mix homogeneously heating after 80 DEG C in oil phase preparation tank, then Jia Siben-805 parts, to temperature reach 115 DEG C time maintenance 40 minutes, standby after cooling.
The Newcastle Disease venom of inactivation, avian influenza venom, I group I fowl adenovirus venom are mixed by 2 aqueous phase preparations with 1:1:2 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 after slowly adding aqueous phase 1 part, then stir 30～40 minutes with 3500r/min simultaneously. After emulsifying, taking vaccine 10ml and add in centrifuge tube, be centrifuged 15 minutes with 3000r/min, the aqueous phase precipitated out at the bottom of pipe should 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 normal saline 0.3ml/ only compares. Take 22 age in days SPF chickens and be divided into 3 groups, often group 10,1401 batches of inactivated vaccines of the 1st group of cervical region subcutaneous injection, 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 days in isolator. Injection site and whole body are not caused obvious adverse reaction by result cervical region subcutaneous injection and two kinds of approach of intramuscular injection, within the whole observation period test chicken search for food drinking-water all normal, exempting from latter 15 days to dissect, injection site absorbs good, it was demonstrated that SPF chicken is safe through two kinds of injecting pathways by this vaccine.
The different injecting pathway safety test to SPF chicken of table 1
Note: "-" represent chicken search for food, drink water, feces, spirit all normal.
2. pair target animals single dose inoculation, single dose repeated inoculation, the inoculation of overdose safety test
Single dose inoculation safety test
Take 1 age in days SPF chicken 20, be divided into 2 groups, often group 10. 1401 batches of trigeminy vaccine of 1st group of cervical region subcutaneous injection, 0.3ml/ is only; 2nd group of cervical region subcutaneous injection normal saline, 0.3ml/ only, raises in isolator, observes 14, record search for food, drink water, the situation such as feces, exempt from the absorbing state of latter 15 days anatomic observation injection site pathological changes and vaccine. Take 22 age in days SPF chicken 20, be divided into 2 groups, often group 10. 1401 batches of trigeminy vaccine of 3rd group of cervical region subcutaneous injection, 0.5ml/ is only; 4th group of cervical region subcutaneous injection normal saline, 0.5ml/ only, raises in isolator, observes 14, record search for food, drink water, the situation such as feces, exempt from the absorbing state of latter 15 days anatomic observation injection site pathological changes and vaccine. Result is 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. 1401 batches of trigeminy vaccine of 1st group of cervical region subcutaneous injection, only, in immune latter 14 days, same dosage inoculated 1 time to 0.3ml/ again; 2nd group of cervical region subcutaneous injection normal saline, 0.3ml/ only, injects 1 time again in latter 14 days of injection same dosage again, raise in isolator, two exempt from after observe again 14, the situation such as record is searched for food, drunk water, feces, two exempt from the absorbing state of latter 15 days anatomic observation injection site pathological changes and vaccine. Take 22 age in days SPF chicken 20, be divided into 2 groups, often group 10. 1402 batches of trigeminy vaccine of 3rd group of cervical region subcutaneous injection, only, in immune latter 14 days, same dosage inoculated 1 time to 0.5ml/ again; 4th group of cervical region subcutaneous injection normal saline, 0.5ml/ only, injects 1 time again in latter 14 days of injection same dosage again, raise in isolator, two exempt from after observe again 14, the situation such as record is searched for food, drunk water, feces, two exempt from the absorbing state of latter 15 days anatomic observation injection site pathological changes and vaccine.Result is in Table 3.
Table 2, it is shown that after the inoculation of vaccine single dose, observe 14, do not cause obvious adverse reaction in injection site and whole body, exempts from latter 15 days to dissect, and injection site all absorbs well, without swelling, inflammation etc., within the whole observation period test chicken search for food drink water all normal. Table 3 is it is shown that after vaccine secondary inoculation, observe 14, do not cause obvious adverse reaction in injection site and whole body, and injection site all absorbs well, without swelling, inflammation etc. Within the whole observation period test chicken search for food drinking-water all normal.
The safety test of table 2SPF chicken single dose inoculation
Note: 1, "-" represent chicken search for food, drink water, feces, spirit all normal; Lower same. 2, immunization route adopts cervical region subcutaneous injection.
The safety test of table 3SPF 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 10,1401 batches of trigeminy vaccine of 1st group of cervical region subcutaneous injection, 0.6ml/, 1402 batches of trigeminy vaccine of the 2nd group of cervical region subcutaneous injection, 0.6ml/, 1403 batches of trigeminy vaccine of the 3rd group of cervical region subcutaneous injection, 0.6ml/ is only, 4th group of cervical region subcutaneous injection normal saline, 0.6ml/ only, raises in isolator, observes to 14 days, record is searched for food, is drunk water, before immunity and exempt from latter 15 days SPF chicken body weight etc., the absorbing state of anatomic observation injection site pathological changes and vaccine. With 7 age in days SPF chicken 40, it is divided into 4 groups, often group 10,1401 batches of trigeminy vaccine of 1st group of cervical region subcutaneous injection, only, the 2nd group of chest muscle injects 1402 batches of trigeminy vaccine to 1.0ml/, 1.0ml/, 1403 batches of trigeminy vaccine of the 3rd group of cervical region subcutaneous injection, 1.0ml/ is only, 4th group of cervical region subcutaneous injection normal saline, 0.6ml/ only, raises in isolator, observes to 14 days, record is searched for food, is drunk water, before immunity and exempt from latter 15 days SPF chicken body weight etc., the absorbing state of anatomic observation injection site pathological changes and vaccine. With 22 age in days SPF chicken 40, it is divided into 4 groups, often group 10,1st group of chest muscle 1401 batches of trigeminy vaccine of injection, 1.0ml/, 1402 batches of trigeminy vaccine of the 2nd group of cervical region subcutaneous injection, 1.0ml/, 1403 batches of trigeminy vaccine of the 3rd group of cervical region subcutaneous injection, 1.0ml/ is only, 4th group of cervical region subcutaneous injection normal saline, 0.6ml/ only, raises in isolator, observes to 14 days, record is searched for food, is drunk water, before immunity and exempt from latter 15 days SPF chicken body weight etc., the absorbing state of anatomic observation injection site pathological changes and vaccine. With 270 age in days SPF chicken 40, it is divided into 4 groups, often group 10,1401 batches of trigeminy vaccine of 1st group of cervical region subcutaneous injection, only, the 2nd group of chest muscle injects 1402 batches of trigeminy vaccine to 1.0ml/, 1.0ml/ is only, 1403 batches of trigeminy vaccine of 3rd group of cervical region subcutaneous injection, 1.0ml/, the 4th group of cervical region subcutaneous injection normal saline, 1.0ml/ is only, raise in isolator, 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 622 age in days SPF chicken
The safety test of overdose inoculation of table 7270 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, observe 14, do not cause obvious adverse reaction in injection site and whole body, vaccine injection group and the weightening finish of matched group SPF chicken are without too big change, anatomic observation, injection site vaccine absorbs good, without swelling, inflammation etc., within the whole observation period, test chicken drinking-water of searching for food is all normal.Laying hen result of the test shows, laying period laying hen is safe by trigeminy vaccine immunity within laying period, and laying rate, body weight are substantially unaffected.
(5) immune period test
The antibody dynamic regularity of 1 age in days and 22 age in days SPF chickens and the research of immune duration is carried out with 3 batches of Seedlings of laboratory trial-production. Trigeminy vaccine immunity 1 age in days SPF chicken, result of the test show, NDV part: in latter 21 days, 5 months of immunity ND antibody all >=6.0log2, after counteracting toxic substances, 10/10 protects; Latter 6 months of immunity, antibody minority is down to below 4log2,5/10～6/10 protection after counteracting toxic substances; But not equal 5/5 death after immunized controls chicken counteracting toxic substances. H9 hypotype AIV part: H9 antibody >=8.0log2 in immune latter 21 days, 5 months, more than 9/10 virus purification protection after counteracting toxic substances; Latter 6 months of immunity, antibody is down to below 7.5log2, more than 9/10 virus purification protection after counteracting toxic substances; But not immunized controls chicken counteracting toxic substances restrovirus separation rate is 9/10～10/10. Aviadenovirus part: latter 7 days minority chickens of immunity can detect that fine jade expands antibody, exempts from latter 21 days～6 months fine jades and expands antibody equal 7/10 and with positive, and latter 21 days, 5,6 months counteracting toxic substances immune group of immunity all reach 8/10 and protect above; After matched group counteracting toxic substances equal 8/10 and fall ill above. From the result above, trigeminy vaccine is inoculated 1 age in days SPF chicken (0.3ml/ only) and still is able to reach desirable protected effect in latter 5 months.
The table 81 age in days SPF chicken immune phase tests
And, viral disease inactivated vaccine prepared by the present invention for the immune effect of the counteracting toxic substances of YBAV-4 strain significantly better than other I group commercially available 4 type aviadenovirus vaccines, thus it is speculated that cause owing to YBAV-4 pnca gene morphs.
Claims (6)
1. an inactivated vaccine, it is characterised in that described inactivated vaccine, its antigen is the newcastle disease virus of inactivation, bird flu virus and aviadenovirus.
2. inactivated vaccine as claimed in claim 1, it is characterised in that described newcastle disease virus is NDV La Sota Strain.
3. inactivated vaccine as claimed in claim 1, it is characterised in that described bird flu virus, its deposit number is CCTCCNO:V201517.
4. inactivated vaccine as claimed in claim 1, it is characterised in that the deposit number of described aviadenovirus is CCTCCNo.V201541.
5. inactivated vaccine as claimed in claim 1, it is characterised in that the inactivation of described virus adopts formalin-inactivated.
6. the preparation method of the inactivated vaccine described in claim 1, it is characterised in that described preparation method comprises the following steps that
1) prepared by oil phase:
Take 95 parts of mineral oil, aluminium stearate 1 part, mix homogeneously heating after 80 DEG C in oil phase preparation tank, then add 5 parts of departments this 80, 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, avian influenza venom, the mixing of I group I fowl adenovirus venom; Take hybrid antigen liquid 95 parts, 5 parts of Tween 80s of sterilizing, fully mix;
3) emulsifying
Take oil phase 2 parts and put in emulsion tank, add after aqueous phase 1 part, then within 30～40 minutes, complete emulsifying with 3500r/min stirring and prepare.
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