CN105582533A - Combined inactivated vaccine for avian influenza virus and fowl adenovirus - Google Patents
Combined inactivated vaccine for avian influenza virus and fowl adenovirus Download PDFInfo
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Abstract
The invention provides a combined inactivated vaccine for an avian influenza virus and a fowl adenovirus. An H9 subtype avian influenza virus QDY strain and an I-group 4-type aviadenovirus YBAV-4 new strain used by the vaccine are high in TCID50/EID50 valence and good in immunogenicity and can well withstand attacks of the H9 avian virus and various local separate viruses. The vaccine prepared by the invention is good in safety, and free of local and whole-body adverse effects caused by the vaccine. Through analysis of characters, a safety test and efficacy test data in a storage-life test, the combined vaccine is free of an obvious difference from a single vaccine of a similar product in effect, and is stable and effective; the efficacy test result proves that the antibodies of the combined vaccine and two single vaccines are kept at a high level and are faster in antibody generation of similar products; and a control group antibody is negative.
Description
Technical field
The invention belongs to veterinary biologics technical field, be specifically related to a kind of avian influenza virus, aviadenovirus bivalent inactivated vaccine.
Background technology
Bird flu, by A type influenza virus causes, a kind of infectiousness, the death rate of Major Epidemic in animals and humans is high acute, hyperinfection disease, extensively betides all over the world, and often variation, very large to the mankind and aviculture harm.
By the epidemiology survey to I group I fowl adenovirus, this disease incidence of disease in China chicken group is higher, can propagate by two kinds of approach of horizontal and vertical, and be ascendant trend year by year. The host range of morbidity is also more and more wider, and white plumage broiler chicken, meat kind chicken, laying hen, yellow plumage chicken all can infection morbidities, particularly within 2010, present increase trend with sequela, all have popular in China. Many I group I fowl adenovirus can copy in healthy fowl body, symptom very slightly or not shows infection symptoms, but I group's 4 type aviadenovirus exceptions, can directly cause that chicken mass-sending is sick, major lesions shows as hydropericardium and liver, kidney enlargement, this disease occurred in the U.S. first in 1963, in succession occurring all over the world, was the common zymad of whole world poultry and wild fowl subsequently. 1976 IState TaiwanEconomize this disease occurs first, in all parts of the country afterwards all have the report that this disease occurs, and be ascendant trend year by year, brings serious harm to chicken aquaculture.
In the last few years, H9 subtype avian influenza was the important diseases of comparatively common serious threat poultry; And chicken group is more and more many to the infection of I group's 4 type aviadenovirus, this disease is easy to cause scabies secondary infection epidemic disease, bring a lot of worries to fowl industry raiser, and repeatedly use especially inactivated vaccine of vaccine, not only increase chicken house man power and material burden, repeatedly grab simultaneously chicken injection stress, also can affect production performance, cause chicken group to increase the neurological susceptibility of disease. In addition the continuous variation of strain in recent years, although make the vaccine of the multiple choices of releasing, but still there is losing control of the situation of epidemic situation development, deal with so need screening to obtain new epidemic isolates the new harm that variation causes.
Summary of the invention
The object of this invention is to provide a kind of avian influenza virus, aviadenovirus bivalent inactivated vaccine, thereby make up the deficiencies in the prior art.
Bivalent inactivated vaccine of the present invention, avian influenza virus and aviadenovirus that wherein antigen is deactivation;
Wherein avian influenza virus is preferably H9 subtype avian influenza virus QDY strain (Avianinfluenzavirus), be deposited in Wuhan, China on April 29th, 2015, the Chinese Typical Representative culture collection center of Wuhan University, deposit number is CCTCCNO:V201517.
Aviadenovirus is I group's 4 type aviadenovirus YBAV-4 strains, itsPreservationBe numbered CCTCCNo.V201541.
Above-mentioned inactivated vaccine, wherein the deactivation of virus adopts formalin-inactivated;
The preparation method of inactivated vaccine of the present invention is as follows:
1) oil phase preparation:
Get 95 parts, mineral oil, 1 part of aluminum stearate mixes and is heated to after 80 DEG C in oil phase preparation tank, then adds 5 parts of this 80 (Span-80) of department, in the time that temperature reaches 115 DEG C, maintains 40 minutes, completes oil phase preparation after cooling;
2) water preparation:
By the avian flu venom of deactivation, I group fowl adenopathyPoison poisonLiquid mixes; Get 95 parts of hybrid antigen liquid, 5 parts of Tween 80s of sterilizing, fully mix;
Wherein the quantity ratio of avian flu venom, I group I fowl adenovirus is preferably 1:2;
3) emulsification
Get 2 parts of oil phases and put into emulsion tank, add after 1 part of water, then stir and within 30～40 minutes, complete emulsification preparation with 3500r/min.
The TCID of the H9 subtype avian influenza virus QDY strain that vaccine of the present invention uses and the new strain of I group's 4 type aviadenovirus YBAV-4 strain50/EID50Height, the immunogenicity of tiring well also can be resisted the sick each place of H9 hypotype and aviadenovirus and separate malicious attack. The security of vaccine prepared by the present invention is good, does not occur any part and the systemic adverse reactions that are caused by vaccine. In storage life test, through the analysis of proterties, safety testing, potency test data, result is compared with single seedling of like product, and bigeminy seedling no significant difference is all effectively stable; Potency test result proves, bigeminy seedling and two kinds of single seedling antibody all keep high level, produce antibody fast, and control group antibody are negative than like product.
Detailed description of the invention
Applicant screens and has obtained a strainNovelI group's 4 type aviadenovirus of variation, come together to prepare combined vaccine by this virus and avian influenza virus, thereby have facilitated 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, epidemiology survey is since 2010, part meat kind chicken, laying hen and the numb chicken in the area such as Shandong, Jiangsu occurred a kind of high with the death rate, dissection main manifestations is the disease that liver enlargement, hydropericardium are feature, detect through clinical investigation and laboratory, tentative diagnosis is the hydropericardium hepatitis syndrome that I group C-4 type aviadenovirus causes. 2010, inventor was successfully separated to 1 strain virus from there is the chicken liver of dying of illness of inclusion body hepatitis and hydropericardium classical symptom in Shandong Zibo plant.
2, virus separates after the liver grinding of getting the chicken that dies of illness, and uses in the ratio of 1:5 and adds sterile saline to make suspension; After multigelation 3 times, the centrifugal 30min of 3000r/min, gets supernatant; Add the each 10000IU/ml of penicillin and streptomysin, 4 DEG C are spent the night, and filter through millipore filter, after steriling test is qualified, save backup. Dosage by the virus liquid of above-mentioned preparation with 0.2ml/ embryo, inoculate 6.5 age in days SPF chicken embryos through yolk bag approach, abandon dead germ in 24h, get allantoic fluid and the fetus of dead chicken embryo in inoculation 48h～168h, continuous passage after processing with said method, observe the 3rd generation dead germ hepatic tissue, chicken embryo shows as that dead germ, idiosome are short and small, hypoevolutism, fetus are curling, liver enlargement and matter crisp, embryo's hyperemia. 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 chickens and duck blood 5～10ml, wash 3～5 times repeatedly, and last is diluted to 0.8%, 1% and 2% concentration with physiological saline by haemocyte mud, and 4 DEG C save backup. Detect according to a conventional method isolated strain and whether there are these erythrocytic characteristics of aggegation. Establish III group I fowl adenovirus EDSV-76 is agglutinating reaction positive control simultaneously. Result: separating poison can not aggegation SPF chicken and duck red blood cell, even if change erythrocytic concentration, can not make it aggegation. III group I fowl adenovirus EDSV-76 can aggegation chicken, the red blood cell of duck.
The method that 3.2 physicochemical property inspections are introduced with reference to " animal virology ", after virus liquid is processed with 5-bromouracil-2 '-deoxyribonucleoside (BUDR), chloroform, ether, hydrochloric acid (pH3), NaOH (pH10), temperature (60 DEG C, 1h) respectively, inoculated into chick embryo (0.2ml/ embryo), separately establishes physiological saline processed group in contrast. After inoculation 5d, observe chicken embryo pathology. Result: separate poison respectively after BUDR, NaOH (pH10) and 60 DEG C, 1h process, inoculated into chick embryo, chicken embryo is acted normally, and PCR detects feminine gender. Show that BUDR can suppress virus copying in chicken embryo, 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 the strain of processing through ether, chloroform and hydrochloric acid (pH3) does not affect the propagation of virus in chicken embryo, there is obvious chicken embryo pathology, the PCR testing result positive. Show that virus does not have lipid cyst membrane, EC is had to 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 to group specificity qualification. After agar solidifies, with card punch punching, perforation pattern is central 1 hole surrounding 6 holes, aperture 4mm, and pitch-row is 4mm, hole underseal closes. Virus to be checked is placed in interstitial hole, and around hole adds I group I fowl adenovirus type strain, EDSV-76 capital 911 strain standard positive serum and negative serums. Fine jade is expanded to plate and be positioned over the 37 DEG C of effects in wet box of adding a cover, 24～48h observes whether occur coagulation sedimentation line. Result: separate malicious antigen and only can occur obvious sediment line with I group I fowl adenovirus 4 type positive serums, and and all do not occur precipitation line between III group I fowl adenovirus EDSV-76 capital 911 strain standard positive serums and negative serum.
3.3.2 first type specificity qualification 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 heat-clearing method in 2010, I group I fowl adenovirus 1～12 type standard strain, separation poison are carried out to cross neutralization test to I group I fowl adenovirus 1～12 type standard positive serum, and record neutralizes the result of tiring. Result: separate poison survey the neutralization of 4 type standard positive serums tire neutralization that (1:501) and 4 type standard strains survey 4 type standard positive serums tire (1:537) more approaching; Isolated strain is surveyed the neutralization of other type standard positive serum and is tired all below 1:10. Show that separated strain is serum 4 types.
3.4PCR detects and the aseptic grinding of the sick chicken liver of gene sequencing, and multigelation 3 times utilizes pillar animal DNA to extract kit and extracts viral DNA, carries out PCR detection. 1% agarose gel electrophoresis observed result. Positive is carried out to Hexon gene sequencing, and carry out phylogenetic analysis. Can find out according to the comparison of Hexon gene order and phylogenetic analysis result, separate poison and belong to same branch with I group I fowl adenovirus, the most approaching with serum 4 type homologys, but also there is the difference in sequence; Lower with serum 6 types, 7 types, 8a and 8b type homology.
This Strain was on October 15th, 2015PreservationIn the Chinese Typical Representative culture of Wuhan Wuhan UniversityPreservationCenter,PreservationBe numbered 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 respectively the 25cm of same batch by the impact of cell density cell growth2Cell bottle, 225cm2In cell bottle, 3000ml rolling bottle, 10 confluent monolayer cells factories, cultivate under same condition with the DMEM nutrient solution of same batch, each density is inoculated 5 bottles/2, the form cultivate under same condition, observation of cell growing up to fine and close individual layer required time and cell.
2, the NBCS of same manufacturer production for the impact of NBCS content cell growth, add in DMEM nutrient solution in 6%, 8%, 10%, 12% ratio respectively, cultivate with a collection of chicken liver cell, the whole density of cell is 15～200,000/ml, the same batch of 25cm of nutrient solution inoculation of every kind of serum-concentration2Cell bottle, 225cm2Each 5 bottles/2 of cell bottle, 3000ml rolling bottle, 10 confluent monolayer cells factories, observation of cell grows up to fine and close individual layer required time and cellular morphology.
3, the impact of pancreatin cell growth cell dissociation buffer used is 0.02%EDTA-0.25% pancreatin solution, after cell dissociation is good, part cell culture container goes digestive juice to add nutrient solution, and another part cell culture container does not discard digestive juice and directly adds nutrient solution. Nutrient solution be pH value be 7.0～7.2, containing the DMEM nutrient solution of 10% NBCS, cell density is 15～200,000/ml. Inoculate same batch of 25cm2Cell bottle, 225cm2Each 5 bottles/2 of cell bottle, 3000ml rolling bottle, 10 confluent monolayer cells factories are cultivated, observation of cell grows up to fine and close individual layer required time and cellular morphology under same condition.
4, nutrient solution pH value cell growth to affect other condition all identical, only the pH value of nutrient solution is adjusted into respectively 6.8,7.0,7.2,7.4, the nutrient solution of different pH values is inoculated respectively 25cm2Cell bottle, 225cm2Each 5 bottles/2 of cell bottle, 3000ml rolling bottle, 10 confluent monolayer cells factories, observation battalion's nutrient solution change color, cell grow up to fine and close individual layer required time and cellular morphology.
(2) I group's 4 type aviadenovirus YBAV-4 strain optimal culture condition researchs
1, the best connects the YBAV-4 strain virus liquid inoculation chicken liver cell with 0.1%, 0.5%, 1%, 2%, 5% 5 various dose in different culture vessels of determining respectively of toxic agent amount, observe and record cytopathic time of appearance and lesion degree, when cytopathy (hereinafter to be referred as CPE) results virus liquid appears in more than 80% cell, after multigelation 2 times, measure respectively its viral level (TCID50), with TCID50Soprano's the toxic agent amount that connects is that the best connects toxic agent amount.
2, the best connects determining of poison time and under two kinds of growth conditions, breeds YBAV-4 strain virus liquid with cell, when chicken liver cell grows up to 60～70% individual layers, grows up to 70～80% individual layers and grows up to 90% above individual layer virus inoculation, connects poison, 37 DEG C, 5%CO by 1% amount2Incubator is cultivated, and gathers in the crops virus liquid in the time that CPE appears in more than 80% cell, after multigelation 2 times, measures respectively its TCID50, with TCID50Soprano's the poison time that connects is that the best connects the poison time.
3, determining of optimum culturing temperature inoculates by 1% amount the chicken liver cell that grows up to good individual layer by I group's 4 type aviadenovirus YBAV-4 strain virus liquid, be placed in respectively 34 DEG C, 36 DEG C, 37 DEG C, 38 DEG C cultivations, time and lesion degree that observation of cell pathology occurs, harvesting venom in the time that CPE appears in 80% cell, measures respectively its TCID after multigelation 2 times50, with TCID50Soprano's cultivation temperature is optimum culturing temperature.
4, the best chicken liver cell that I group's 4 type aviadenovirus YBAV-4 strain virus liquid is grown up to good individual layer by 1% amount inoculation of determining of receiving the poison time, respectively at 37 DEG C, 5%CO2Incubator is cultivated, and when virus liquid appears gathering in the crops when 70%, 80%, 90% left and right CPE in cell, after multigelation 2 times, measures respectively its TCID50, with TCID50Soprano's the receipts poison time is best receipts the poison time.
5, best maintained liquid serum content is determined the chicken liver cell that I group's 4 type aviadenovirus YBAV-4 strain virus liquid is grown up to good individual layer by 1% amount inoculation, and in maintenance medium, NBCS content is respectively 1%, 2%, 3%, respectively at 37 DEG C, 5%CO2Incubator is cultivated, and the time that observation of cell pathology occurs, harvesting venom in the time that CPE appears in 80% cell, measures respectively its TCID after multigelation 2 times50, with TCID50Soprano's serum content is best maintained liquid serum content.
6, demonstration test is according to above-mentioned result of the test, we select the best to connect malicious mode, the best to connect toxic agent amount, the best and connect poison time, optimum culturing temperature, bestly receive the poison time and prepared 3 batches of virus liquids, by after virus liquid multigelation 2 times, measure the viral level of virus liquid.
The preparation of embodiment 3:H9 subtype avian influenza, aviadenovirus (I group, 4 types) antigen
1.Millipore ultrafiltration concentration machine uses, maintenance condition and using method, and the operation instruction providing by producer is carried out.
2. concentrated effect detects
Before 2.1 concentrated antigens effect inspections are concentrated, keep sample measure antigen valence in concentrated provirus liquid (HA and/EID50/TCID50), sampling and measuring concentrate antigen valence (HA) at any time in concentration process, determines cycles of concentration, the concentrate after concentrated keeps sample, measure antigen valence in concentrate (HA and/EID50/TCID50)。
2.2 filter liquor antigens detect (now antigen concentration maximum in concentrate while getting concentrated closing to an end, in filter liquor, spill the possibility maximum of antigen) filter liquor, adopt respectively the approach such as the blood clotting valency (HA) of measuring, inoculation SPF chicken embryo (observation has or not live virus to spill), inoculation SPF chicken (whether detect has antigenic substance to spill) to detect to spill in liquid, to have or not antigen composition.
2.3 concentrated film models are selected varying in size of different virus antigen composition, different milipore filters hold back (filtration) aperture difference, therefore select the milipore filter of different pore size model (1#, 2#, 3#, 4#, 5#) to carry out rejection tests to different virus, and according to antigen testing result, concentrated effect inspection result and concentrated required time in filter liquor, determine respectively the concentrated film model of concentrated two-strain antigen the best used.
Each 3 batches of concentrated NDV, AIV of the concentrated film of best model, FADV blastochyle for 2.4 concentrated effect stability tests, the stability of detection concentrated effect.
2.5 cycles of concentration tests concentrate film by best model, and AIV, FADV blastochyle are respectively concentrated into 1/2,1/3,1/4,1/5 of original volume,, measure the EID of different cycles of concentration50/TCID50, determine suitable cycles of concentration.
Material consumption when 2.6 concentrated costs analysis bases concentrate, calculates concentrated cost.
(1) poison (bacterium) is planted and should be reached required standard:
Use avian influenza virus QDY strain, I group's 4 type aviadenovirus YBAV-4 strains as antigen seed culture of viruses.
(2) antigen preparation and the inspection of semifinished product:
1 produces the preparation with seed culture of viruses
The preparation of 1.1H9 subtype avian influenza virus QDY strain seed culture of viruses:
1.1.1 seed culture of viruses breeding is done suitably dilution (as 10 by seed culture of viruses with sterilizing PBS-4Or 10-5), inoculation 10～11 age in days SPF chicken embryos in allantoic cavity, every embryo 0.1ml, puts 36～37 DEG C and continues to hatch. Not death and infected chicken blastochyle (allantoic fluid and amniotic fluid) of results inoculation 72 hours, is loaded in sterilization container. By aseptic inspection and the chicken blastochyle that 1% chicken erythrocyte suspension agglutination titer is not less than 1:512 is mixed, quantitative separating, indicates harvest date, Virus passages etc., freezing preservation.
1.2 I group 4 type aviadenovirus YBAV-4 strain seed culture of viruses preparations
1.2.1 the chicken liver cell growing fine is selected in seed culture of viruses breeding, discards original fluid, adds the maintenance medium that contains 1% seed culture of viruses, put 37 DEG C and cultivate 36～48 hours, gather in the crops freeze thawing 2 times when cytopathy reaches 80% when above, be sub-packed in sterilization container, sampling is identified. Dated harvest date, Virus passages etc.
The selection of 2 seedling materials
Well-developed 10～11 age in days susceptible chicken embryos of 2.1 avian influenza virus seedling materials (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
3.1H9 subtype avian influenza virus antigen
3.1.1 production seed culture of viruses is got in inoculation, does suitably to dilute (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, and needn't turn over embryo.
3.1.2 hatch and observe after egg inoculation, per sunshine, embryo 1 time, hatched to 72 hours, all took out, and according to embryo, discarded dead germ, the embryo air chamber of living is upwards upright, put 2～8 DEG C cooling 12～24 hours.
3.1.3 gather in the crops cooling chicken embryo is taken out, by " full-automatic cropper operation instruction " requirement, results chicken blastochyle. Draw blastochyle and be placed in sterilization container, RCA valency is measured in sampling, and agglutination titer should discard lower than 1:256 person. Before the blastochyle deactivation of results, 2～8 DEG C of preservations, should be no more than 5.
3.1.4 concentrate the blastochyle of results under 2～8 DEG C of conditions, concentrated with ultrafiltration concentration machine, the RCA of sampling mensuration at any time valency stops concentrating in the time that RCA valency is not less than 1:1024. Keep sample, carry out the inspection of semifinished product, all the other blastochyles are carried out deactivation immediately.
3.1.5 deactivation imports virus liquid in deactivation tank, is metered into 10% formalin, fully mixes, and the ultimate density of formalin is 0.1%. 37 DEG C of deactivations are taken out after (reach 37 DEG C with temperature in tank and start timing) for 16 hours, put 2～8 DEG C of preservations, should be no more than 1 month.
3.2I group I fowl adenovirus antigen
3.2.1 cell preparation is taken out cryopreservation tube and is put in 37 DEG C of water-baths and melt from liquid nitrogen container, cell is moved into and is equipped with in the centrifuge tube of 10ml nutrient solution, centrifugal 5 minutes of 1000r/min. With the nutrient solution suspension cell containing 20% NBCS, put 37 DEG C, 5%CO2Incubator is cultivated, and uses pancreas enzyme-EDTA vitellophag in the time growing up to good individual layer.
3.2.2 antigen preparation
3.2.2.1 cell monolayer is cultivated and is inoculated in cell factory expanding the seed cell of cultivating, 37 DEG C of cultivations.
3.2.2.2 connect poison and select the chicken liver cell growing fine, discard original fluid, add the maintenance medium that contains 1% seed culture of viruses, put 37 DEG C and continue to cultivate.
3.2.2.3 observe with results and connect after poison, observe every day 2 times, record cytopathy situation. When cytopathy reaches 80% above time results, freeze thawing 2 times, the inspection of semifinished product is carried out in sampling.-15 DEG C of preservations, should be no more than 30.
3.2.2.4 concentrate the venom of results under 2～8 DEG C of conditions, with concentrated 2～3 times of ultrafiltration concentration machine, keep sample, carry out the inspection of semifinished product, all the other blastochyles are carried out deactivation immediately.
3.2.2.5 deactivation imports virus liquid in deactivation tank, is metered into 10% formalin, fully mixes, and the ultimate density of formalin is 0.2%. 37 DEG C of deactivations are taken out after (reach 37 DEG C with temperature in tank and start timing) for 16 hours, put 2～8 DEG C of preservations, should be no more than 1 month.
4 inspections of semifinished product
4.1H9 subtype avian influenza part
4.1.1 RCA valency is measured the virus liquid of getting before deactivation, is undertaken by existing " Chinese veterinary pharmacopoeia " annex, measures its RCA valency, should be not less than 1:1024.
4.1.2 viral level is measured the virus liquid taking out before deactivation is carried out to 10 times of serial dilutions, gets 10－7、10－8、10－93 dilution factors, 5 pieces of inoculation 10～11 age in days SPF chicken embryos in each allantoic cavity, every embryo 0.1ml, puts 36～37 DEG C and continues to hatch, and per sunshine, embryo 2 times, observed 5. Measure RCA valency by embryo, be not less than 1:16 person, be judged to infection, calculate EID50. Every 0.1ml viral level answers >=109.0EID50。
4.1.3 steriling test is got the avian flu venom after deactivation, tests by existing " Chinese veterinary pharmacopoeia " annex, answers asepsis growth.
4.1.4 6 pieces of 10 age in days SPF chicken embryos are got in deactivation inspection, inoculation inactivation of viruses liquid in allantoic cavity, and every embryo 0.2ml, puts 36～37 DEG C and continues to hatch, and per sunshine, embryo 2 times, observed 5, and chicken embryo nonspecific death should be no more than 1 piece. All blastochyles are measured respectively to RCA valency, all should not occur aggegation, blastochyle is gathered in the crops to a blind passage generation again, during still without agglutination titer, be judged to deactivation complete.
4.2 I group I fowl adenovirus part
4.2.1 viral level is measured the virus liquid of getting before deactivation and is measured, and every 0.1ml viral level should >=107.3TCID50。
4.2.2 steriling test is got the virus liquid after deactivation, tests by existing " Chinese veterinary pharmacopoeia " annex, answers asepsis growth.
4.2.3 the virus liquid after deactivation is done 10 times of dilutions by deactivation inspection, chicken liver cell (24 porocyte plate) 4 holes that inoculation is grown fine, and every hole 0.2ml, supplements maintenance medium to 2.0ml; Establish nonvaccinated chicken liver cell simultaneously and make blank, 37 DEG C, 5%CO2Incubator is cultivated, and observes 120 hours. All should not there is not cytopathy in cell control well and sample well. Culture is gathered in the crops to a blind passage generation after multigelation, continue to cultivate 120 hours, when sample well does not still occur cytopathy, be judged to be deactivation complete.
Embodiment 4: the preparation of vaccine
95 parts, mineral oil is got in 1 oil phase preparation, and 1 part of aluminum stearate mixes and is heated to after 80 DEG C in oil phase preparation tank, then Jia Siben-805 part, in the time that temperature reaches 115 DEG C, maintains 40 minutes, cooling rear for subsequent use.
2 waters are prepared the avian flu venom of deactivation, I group fowl adenopathyPoison poisonLiquid mixes with 1:1:2 ratio. Get 95 parts of hybrid antigen liquid, 5 parts of the Tween-80s of sterilizing, fully mix, and Tween-80 is dissolved completely.
3 emulsifications are got 2 parts of oil phases and are put into emulsion tank, start motor, and slow rotation stirs, and slowly add after 1 part of water simultaneously, then stir 30～40 minutes with 3500r/min. After emulsification, get vaccine 10ml and add in centrifuge tube, with 3000r/min centrifugal 15 minutes, the water that separate out at the pipe end should be no more than 0.5ml.
4 packing 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 pair various route of inoculation of target animals
Get 1 age in days SPF chicken and be divided into 3 groups, 10 every group, 1401 batches of inactivated vaccines of the 1st group of neck hypodermic injection, 0.3ml/ is only; 1401 batches of inactivated vaccines of the 2nd group of intramuscular injection, 0.3ml/ is only; The 3rd group of intramuscular injection physiological saline 0.3ml/ only compares. Get 22 age in days SPF chickens and be divided into 3 groups, 10 every group, 1401 batches of inactivated vaccines of the 1st group of neck hypodermic injection, 0.5ml/ is only; 1401 batches of inactivated vaccines of the 2nd group of intramuscular injection, 0.5ml/ is only; The 3rd group of intramuscular injection physiological saline 0.5ml/ only compares, and raises respectively Continuous Observation 14 days in isolator. Two kinds of approach of the hypodermic injection of result neck and intramuscular injection do not cause obvious adverse reaction to injection site and whole body, within the whole observation period test chicken search for food drinking-water all normal, exempt to dissect for latter 15 days, injection site absorbs good, proves that this vaccine is safe through two kinds of injecting pathways to SPF chicken.
Table 1The safety test of different injecting pathways to SPF chicken
Note: "-" represents that chicken searches for food, drinking-water, ight soil, spirit are all normal.
2. the safety test of pair target animals single dose inoculation, single dose repeated inoculation, an overdose inoculation
Single dose inoculation safety test
Get 20 of 1 age in days SPF chickens, be divided into 2 groups, 10 every group. 1401 batches of bigeminy seedlings of the 1st group of neck hypodermic injection, 0.3ml/ is only; The 2nd group of neck hypodermic injection physiological saline, 0.3ml/ only, raises in isolator, observes 14, record search for food, the situation such as drinking-water, ight soil, exempt from the absorbing state of latter 15 days anatomic observation injection site pathologies and vaccine. Get 20 of 22 age in days SPF chickens, be divided into 2 groups, 10 every group. 1401 batches of bigeminy seedlings of the 3rd group of neck hypodermic injection, 0.5ml/ is only; The 4th group of neck hypodermic injection physiological saline, 0.5ml/ only, raises in isolator, observes 14, record search for food, the situation such as drinking-water, ight soil, exempt from the absorbing state of latter 15 days anatomic observation injection site pathologies and vaccine. The results are shown inTable 2。
Single dose repeated inoculation safety testing
Get 20 of 1 age in days SPF chickens, be divided into 2 groups, 10 every group. 1401 batches of bigeminy seedlings of the 1st group of neck hypodermic injection, 0.3ml/ only, in latter 14 days of immunity again same dosage inoculate 1 time; The 2nd group of neck hypodermic injection physiological saline, 0.3ml/ only, in latter 14 days of injection again same dosage inject again 1 time, in isolator, raise, two observe 14 after exempting from again, the situations such as record is searched for food, drinking-water, ight soil, and two exempt from the absorbing state of latter 15 days anatomic observation injection site pathologies and vaccine. Get 20 of 22 age in days SPF chickens, be divided into 2 groups, 10 every group. 1402 batches of bigeminy seedlings of the 3rd group of neck hypodermic injection, 0.5ml/ only, in latter 14 days of immunity again same dosage inoculate 1 time; The 4th group of neck hypodermic injection physiological saline, 0.5ml/ only, in latter 14 days of injection again same dosage inject again 1 time, in isolator, raise, two observe 14 after exempting from again, the situations such as record is searched for food, drinking-water, ight soil, and two exempt from the absorbing state of latter 15 days anatomic observation injection site pathologies and vaccine. The results are shown inTable 3。
Table 2Result shows, after the inoculation of vaccine single dose, observe 14, in injection site and whole body do not cause obvious adverse reaction, exempt to dissect for latter 15 days, injection site all absorbs well, without swelling, inflammation etc., within the whole observation period test chicken search for food drink water all normal.Table 3Result shows, after vaccine secondary inoculation, observe 14, in injection site and whole body do not cause obvious adverse reaction, injection site all absorbs well, without swelling, inflammation etc. Within the whole observation period test chicken search for food drinking-water all normal.
Table 2The safety test of SPF chicken single dose inoculation
Note: 1, "-" represents that chicken searches for food, drinking-water, ight soil, spirit are all normal; Lower same. 2, immunization route adopts neck hypodermic injection.
Table 3The safety test of SPF chicken single dose repeated inoculation
An overdose inoculation safety testing
With 40 of 1 age in days SPF chickens, be divided into 4 groups, 10 every group, 1401 batches of bigeminy seedlings of the 1st group of neck hypodermic injection, 0.6ml/, 1402 batches of bigeminy seedlings of the 2nd group of neck hypodermic injection, 0.6ml/, 1403 batches of bigeminy seedlings of the 3rd group of neck hypodermic injection, 0.6ml/ is only, the 4th group of neck hypodermic injection physiological 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 pathology and vaccine. With 40 of 7 age in days SPF chickens, be divided into 4 groups, 10 every group, 1401 batches of bigeminy seedlings of the 1st group of neck hypodermic injection, only, the 2nd group of chest muscle injected 1402 batches of bigeminy seedlings to 1.0ml/, 1.0ml/, 1403 batches of bigeminy seedlings of the 3rd group of neck hypodermic injection, 1.0ml/ is only, the 4th group of neck hypodermic injection physiological 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 pathology and vaccine. With 40 of 22 age in days SPF chickens, be divided into 4 groups, 10 every group, 1401 batches of bigeminy seedlings of the 1st group of chest muscle injection, 1.0ml/, 1402 batches of bigeminy seedlings of the 2nd group of neck hypodermic injection, 1.0ml/, 1403 batches of bigeminy seedlings of the 3rd group of neck hypodermic injection, 1.0ml/ is only, the 4th group of neck hypodermic injection physiological 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 pathology and vaccine. With 40 of 270 age in days SPF chickens, be divided into 4 groups, 10 every group, 1401 batches of bigeminy seedlings of the 1st group of neck hypodermic injection, only, the 2nd group of chest muscle injected 1402 batches of bigeminy seedlings to 1.0ml/, 1.0ml/ only, 1403 batches of bigeminy seedlings of the 3rd group of neck hypodermic injection, 1.0ml/, the 4th group of neck hypodermic injection physiological saline, 1.0ml/ only, in isolator, raise, observe 60, record clinical symptoms, drink water, search for food and laying rate situation.
Table 4The safety test of 1 age in days SPF chicken one time overdose inoculation
Table 5The safety test of 7 age in days SPF chicken one time overdose inoculations
Table 6The safety test of 22 age in days SPF chicken one time overdose inoculations
Table 7The safety test of 270 age in days SPF laying hen one time overdose inoculations
Note: every group of experimental animal number is 10, ID is 1.0ml/.
Table 4～7 results show, after an overdose inoculation of vaccine, observe 14, in injection site and whole body do not cause obvious adverse reaction, the weightening finish of vaccine injection group and control group SPF chicken is without too large variation, anatomic observation, injection site vaccine absorbs good, without swelling, inflammation etc., within the whole observation period test chicken search for food drinking-water all normal. Laying hen result of the test shows, bigeminy seedling within laying period immunity to laying period laying hen be safe, laying rate, body weight are substantially unaffected.
(5) immune period test
Carry out 1 age in days and the antibody Fluctuation of 22 age in days SPF chickens and the research of immune duration with 3 batches of seedlings of laboratory trial-production. Bigeminy seedling immunity 1 age in days SPF chicken, result of the test demonstration, H9 hypotype AIV part: H9 antibody >=8.1log2 in immune latter 21 days, 5 months, attacks the separation of rear more than 9/10 virus of poison and protects; Latter 6 months of immunity, antibody is down to below 7.4log2, attacks rear more than 9/10 virus of poison and separates protection; And nonimmune contrast chicken attack poison after virus isolated rate be 9/10～10/10. Aviadenovirus part: latter 7 days minority chickens of immunity can detect fine jade and expand antibody, exempts from latter 21 days～6 months fine jades and expands antibody equal 7/10 and positive above, and immunity is attacked malicious immune group for latter 21 days, 5,6 months and all reached 8/10 and protection above; Control group is attacked poison rear equal 8/10 and morbidity above. From above result, bigeminy seedling is inoculated 1 age in days SPF chicken (0.3ml/ only) still can reach desirable protection effect in latter 5 months. Bigeminy seedling immunity 22 age in days SPF chickens, result of the test demonstration, after vaccine immunity, 7 months indivedual chicken antibodies are down to threshold levels. Attack the rear immune group laying rate of poison and Normal group laying rate difference little, obviously decline and attack malicious control group laying rate of chicken. As can be seen here, the bigeminy seedling ND part protection period can reach 7 months. From above result, bigeminy seedling is inoculated 22 age in days SPF chickens (0.5ml/ only) still can reach desirable protection effect in latter 7 months. Considering feeding environment and practical condition, is the generation of prevention newcastle disease, H9 subtype avian influenza, I group's 4 type aviadenovirus, and we are decided to be immunizing dose and duration of immunity: 3 weeks age and with interior chicken, 0.3ml/, duration of immunity is 4 months. 3 week age above chicken, only, duration of immunity is 6 months to 0.5ml/.
Table 81 age in days SPF chicken immune phase tested
And viral disease inactivated vaccine prepared by the present invention is for the malicious immune effect of attacking of YBAV-4 strain significantly better than commercially available vaccine, supposition causes because YBAV-4 pnca gene morphs.
Claims (5)
1. avian influenza virus, an aviadenovirus bivalent inactivated vaccine, is characterized in that, described bigeminy is gone outLive vaccine, the avian influenza virus that the antigen of its use is deactivation and aviadenovirus.
2. bivalent inactivated vaccine as claimed in claim 1, is characterized in that, described avian influenza virus isH9 subtype avian influenza virus QDY strain, deposit number is CCTCCNO:V201517.
3. bivalent inactivated vaccine as claimed in claim 1, is characterized in that, described aviadenovirus is IGroup's 4 type aviadenovirus YBAV-4 strains, its deposit number is CCTCCNo.V201541.
4. bivalent inactivated vaccine as claimed in claim 1, is characterized in that, described viral deactivation is adoptedUse formalin-inactivated.
5. the preparation method of bivalent inactivated vaccine as claimed in claim 1, is characterized in that, described sideMethod comprises following step:
1) oil phase preparation:
Get 95 parts, mineral oil, 1 part of aluminum stearate mixes and is heated to after 80 DEG C in oil phase preparation tank,Add again 5 parts of departments this 80, in the time that temperature reaches 115 DEG C, maintain 40 minutes, complete oil phase preparation after cooling;
2) water preparation:
The avian flu venom of deactivation, aviadenovirus venom are mixed; Get 95 parts of hybrid antigen liquid, sterilizing5 parts of Tween 80s, fully mix;
3) emulsification
Get 2 parts of oil phases and put into emulsion tank, add after 1 part of water, then stir 30～40 with 3500r/minMinute complete emulsification preparation.
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107050448A (en) * | 2017-04-26 | 2017-08-18 | 广州博恒生物科技有限公司 | A kind of preparation method of avian influenza virus, aviadenovirus bivalent inactivated vaccine |
| CN107308447A (en) * | 2017-07-10 | 2017-11-03 | 广州博恒生物科技有限公司 | A kind of preparation method of triple inactivated vaccine |
| CN107412762A (en) * | 2017-08-09 | 2017-12-01 | 青岛易邦生物工程有限公司 | A kind of ewcastle disease, bird flu, the bursa of farbricius and aviadenovirus quadruple vaccine |
| CN110128545A (en) * | 2019-04-22 | 2019-08-16 | 肇庆大华农生物药品有限公司 | A kind of fusion, recombinant expression carrier, antigen and its preparation method and application |
| WO2020215350A1 (en) * | 2019-04-22 | 2020-10-29 | 肇庆大华农生物药品有限公司 | Avian influenza and fowl adenovirus serotype-4 combined genetic engineering subunit vaccine and preparation method therefor |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2007072916A1 (en) * | 2005-12-22 | 2007-06-28 | Juridical Foundation The Chemo-Sero-Therapeutic Research Institute | Vaccine for in ovo inoculation |
| CN104922663A (en) * | 2015-07-07 | 2015-09-23 | 青岛易邦生物工程有限公司 | New castle disease and H9 subtype bird flu bivalent vaccine |
-
2016
- 2016-02-23 CN CN201610099573.3A patent/CN105582533B/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2007072916A1 (en) * | 2005-12-22 | 2007-06-28 | Juridical Foundation The Chemo-Sero-Therapeutic Research Institute | Vaccine for in ovo inoculation |
| CN104922663A (en) * | 2015-07-07 | 2015-09-23 | 青岛易邦生物工程有限公司 | New castle disease and H9 subtype bird flu bivalent vaccine |
Non-Patent Citations (1)
| Title |
|---|
| 韦悠等: "I群禽腺病毒双价油乳剂灭活苗的研究", 《第四届中国兽药大会》 * |
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| CN107050448A (en) * | 2017-04-26 | 2017-08-18 | 广州博恒生物科技有限公司 | A kind of preparation method of avian influenza virus, aviadenovirus bivalent inactivated vaccine |
| CN107050448B (en) * | 2017-04-26 | 2020-06-23 | 广东渔跃生物技术有限公司 | Preparation method of avian influenza virus and avian adenovirus bivalent inactivated vaccine |
| CN107308447A (en) * | 2017-07-10 | 2017-11-03 | 广州博恒生物科技有限公司 | A kind of preparation method of triple inactivated vaccine |
| CN107412762A (en) * | 2017-08-09 | 2017-12-01 | 青岛易邦生物工程有限公司 | A kind of ewcastle disease, bird flu, the bursa of farbricius and aviadenovirus quadruple vaccine |
| CN107412762B (en) * | 2017-08-09 | 2020-08-14 | 青岛易邦生物工程有限公司 | Newcastle disease, avian influenza, bursa of fabricius and avian adenovirus quadruple vaccine |
| CN110128545A (en) * | 2019-04-22 | 2019-08-16 | 肇庆大华农生物药品有限公司 | A kind of fusion, recombinant expression carrier, antigen and its preparation method and application |
| WO2020215350A1 (en) * | 2019-04-22 | 2020-10-29 | 肇庆大华农生物药品有限公司 | Avian influenza and fowl adenovirus serotype-4 combined genetic engineering subunit vaccine and preparation method therefor |
| US11305006B2 (en) * | 2019-04-22 | 2022-04-19 | Zhaoqing Dahuanong Biology Medicine Co., Ltd | Avian influenza and fowl adenovirus type 4 bi-combined genetic engineering subunit vaccine and method for preparing the same |
| CN110128545B (en) * | 2019-04-22 | 2022-10-25 | 肇庆大华农生物药品有限公司 | Fusion gene, recombinant expression vector, antigen, preparation method and application thereof |
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