CN106282130B - A kind of 4 type aviadenovirus of I group, inactivated vaccine and preparation method thereof - Google Patents
A kind of 4 type aviadenovirus of I group, inactivated vaccine and preparation method thereof Download PDFInfo
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Abstract
The present invention provides a kind of 4 type aviadenovirus of I group, inactivated vaccine and preparation method thereof, is related to bioengineering field.SD2015 plants of 4 type aviadenovirus of I group, deposit number are as follows: CCTCC NO:V201645.The present invention also provides a kind of 4 type aviadenovirus inactivated vaccines of I group, and described SD2015 plants of 4 type aviadenovirus of I group containing inactivation.The method for preparing 4 type aviadenovirus SD2015 strain virus liquid of I group obtains I group SD2015 plants of venom of 4 type aviadenovirus using described SD2015 plants of 4 type aviadenovirus of I group of SPF chicken embryo proliferation.SD2015 plants of 4 type aviadenovirus of I group of the present invention are cultivated in chicken embryo, and malicious valence is high, reach 105.80EID50/ 0.2ml or more, immunogenicity is good, safe to chicken and environment.4 type aviadenovirus inactivated vaccine of I group of the present invention, antibody level height, duration are long after being immunized, and protective rate is high.
Description
Technical field
The present invention relates to bioengineering fields, and in particular to a kind of 4 type aviadenovirus of I group, inactivated vaccine and its preparation side
Method.
Background technique
The hydropericardium hepatitis syndrome (HHS) as caused by 4 type of aviadenovirus I group is to encroach on a kind of acute infection of chicken
Disease, to have faint yellow hydrops in high incidence and high mortality, cavum pericardiale, liver enlargement matter is crisp, multiple focal necrosis, kidney
Dirty enlargement is characterized.The disease occurs in March, 1987 in Pakistan, then equal in many countries such as Mexico, India, Peru
There is generation, China is less to the relevant report of the disease.This disease has caused great economy to aviculture since being found
Loss.Most of report hydropericardium hepatitis syndromes are a kind of diseases of main harm broiler chicken, mainly encroach on 3~5 week old
The standby broiler chicken of broiler chicken and minor kind, but in recent years, it is also risen emphatically in terms of the high mortality of commodity egg
It acts on.Just there is fragmentary generation early in China in 2010, but do not form big fashion trend, starts to send out at early summer in 2015
Disease steeply rises, and first since Shandong District, rear various regions are reported successively increases.In the prior art, there is not the I group of commercialization also
4 type aviadenovirus inactivated vaccines.
Summary of the invention
The purpose of the present invention is to provide SD2015 plants of 4 type aviadenovirus of I group, deposit numbers are as follows: CCTCC NO:V201645,
The strain is proliferated in SPF chicken embryo, and viral level is more than or equal to 105.80EID50/ 0.2ml, and there is excellent immunogenicity.
It is a further object of the present invention to provide the preparation method of 4 type aviadenovirus SD2015 strain virus liquid of I group, this method letters
Single, safe and efficient, antigen viral titer is higher.
Another object of the present invention is to provide a kind of 4 type aviadenovirus inactivated vaccine of I group, the 4 type fowl gland of I group containing inactivation
SD2015 plants viral, which is proliferated in SPF chicken embryo, and viral level is more than or equal to 105.80EID50/ 0.2ml, above-mentioned 4 type of I group
Antibody level is high after aviadenovirus inactivated vaccine is immune, the duration is long, high to broiler chicken, laying hen and its filial generation protective rate, can be compared with
The generation and prevalence of fowl hydropericardium hepatitis syndrome disease are controlled well.
The present invention also provides the preparation methods of the 4 type aviadenovirus inactivated vaccine of I group, and this method is simple, safety, cost
It is low.
The purpose of the present invention adopts the following technical scheme that realization.
The present invention provides a kind of SD2015 plants of 4 type aviadenovirus of I group, deposit number are as follows: CCTCC NO:V201645.
The present invention also provides a kind of 4 type aviadenovirus inactivated vaccines of I group, the 4 type aviadenovirus of I group containing inactivation
SD2015 plants.
In preferred technical solution, the inactivated vaccine is oil emulsion inactivated vaccine.
The present invention also provides the methods for preparing 4 type aviadenovirus SD2015 strain virus liquid of I group, are proliferated institute using SPF chicken embryo
SD2015 plants of 4 type aviadenovirus of I group is stated, I group SD2015 plants of venom of 4 type aviadenovirus are obtained.
In preferred technical solution, by SD2015 plants of inoculation SPF chicken embryos of the 4 type aviadenovirus of I group, lesion chicken embryo is harvested
Allantoic fluid, chorioallantoic membrane and idiosome, homogenate, freeze thawing, it is sick to obtain SD2015 plants of 4 type aviadenovirus of I group centrifuging and taking supernatant
Venom.
The present invention also provides a kind of preparation methods of 4 type aviadenovirus inactivated vaccine of I group, comprising:
(1) SD2015 plants of 4 type aviadenovirus of formalin-inactivated I group is used；
(2) injection white oil and Si Ben -80 are mixed according to volume ratio 94:6 and is mixed, as oil-phase solution；
(3) SD2015 plants of venom of 4 type aviadenovirus of I group and Tween-80 of inactivation are uniformly mixed according to volume ratio 96:4,
As aqueous phase solution；
(4) it emulsifies: oil-phase solution and aqueous phase solution being mixed evenly according to 2~3:1 of volume ratio, emulsified, that is, obtains the I
4 type aviadenovirus inactivated vaccines of group.
In preferred technical solution, the volume basis of formaldehyde in the 4 type aviadenovirus SD2015 strain virus liquid of I group after inactivation
Concentration is 0.15%~0.25%.
In preferred technical solution, the viral level of 4 type aviadenovirus SD2015 strain virus liquid of 0.2ml I group >=
105.80EID50。
The utility model has the advantages that SD2015 plants of 4 type aviadenovirus of I group of the present invention are cultivated in chicken embryo, malicious valence is high, reaches
105.80EID50/ 0.2ml or more, immunogenicity is good, safe to chicken and environment.4 type aviadenovirus inactivated vaccine of I group of the present invention,
Antibody level height, duration are long after immune, and protective rate is high, can preferably control the hair of fowl hydropericardium hepatitis syndrome disease
It is raw and popular, significantly improve the economic benefit of avian production.
Detailed description of the invention
Fig. 1 is the electrophoretogram of Hexon gene amplification product.
Specific embodiment
The screening and identification that SD2015 plants of 4 type aviadenovirus of 1 I group of embodiment
1. the separation of virus
Morbidity chicken group's clinical symptoms in Shandong District are taken hydropericardium occur, liver enlargement matter is crisp or downright bad, kidney enlargement
The liver organization of diseased chicken, with sterilizing PBS buffer solution mix with 1: 2 after grind, by 2000IU/ml addition penicillin, streptomysin, 4
DEG C after effect 1 hour, multigelation 3 times, 8000r/min is centrifuged 15 minutes, after 8 layers of filtered through gauze, takes filtrate with 0.22 μm of filter
Through 6 15 pieces of age in days SPF chicken embryo of yolk sac inoculation after device filtering, every embryo 0.2ml is sealed and is placed on 37.0 DEG C of incubations 10 days, discards
Dead germ in 48h after inoculation, takes 48h~240h death chicken embryo after inoculation, dead chicken embryo show as idiosome bleeding, curl, liver it is swollen
Big matter is crisp, hydropericardium occur in bleeding or necrosis, some dead germs.The allantoic fluid, chorioallantoic membrane and idiosome of dead chicken embryo is taken (to remove eye
Eyeball, beak and leg), homogenate is added penicillin, streptomysin after 4 DEG C act on 1 hour by 2000IU/ml and sets in the container of sterilizing and freeze
Melt 3 times, 8000r/min is centrifuged 15 minutes, is taken supernatant using 8 layers of filtered through gauze, is taken -20 DEG C of filtrate preservations, in case reflecting
Determine and is further purified.
2. the finally obtained supernatant of the present embodiment title 1 is inoculated with 10 28 age in days SPF chickens by animal Orthogonal Rotational Regressive Tests, often
0.2ml is subcutaneously injected in neck, is observed continuously 7 days, records result.As a result it falls ill within 3 days 10/10 after attacking poison, until the 5th day 10/10
Death, clinical symptoms show as down in spirits, loss of appetite, loose random, row's yellow green loose stool, serious person do not rise on sleeping ground until
It is dead.Dissect discovery, dead heart packet are full of faint yellow hydrops；Liver enlargement, matter are crisp, and there is necrotic spot at some edges；Kidney
Enlargement.Dead chicken liver is taken to prepare histotomy observation Histopathology lesion, the multifocal coagulation necrosis of liver cell, with list
Nucleus infiltration and basophilla intranuclear inclusion.Electronic Speculum inspection is carried out after taking pathologic liver to be homogenized, it is possible to find have in chicken liver cell
A large amount of typical hexagon adenovirions.
3. the identification of virus
3.1PCR detection and gene sequencing
Extract the DNA in above-mentioned isolated virus liquid (i.e. the finally obtained supernatant of the present embodiment title 1), specific steps
It is as follows: 400 μ l of virus liquid is taken, 12.5 μ l Proteinase K Solutions and 50 μ l concentration are added as 10% SDS solution, mix, 56 DEG C of works
Use 30min；200 μ l chloroforms and 200 μ l phenol are sequentially added, 15s is acutely shaken, is placed at room temperature for 5min；4 DEG C, 12000rpm, from
Heart 10min；Supernatant is taken, isometric chloroform is added and mixes, is placed at room temperature for 5min；4 DEG C, 12000rpm are centrifuged 10min；It takes
Clearly, the dehydrated alcohol and 1/10 volume, the sodium acetate solution that concentration is 3M of 2 times of volumes, -20 DEG C of placement 30min are added；4℃,
12000rpm is centrifuged 10min；Supernatant is abandoned, 75% 1000 μ l of ethanol solution washing is added；4 DEG C, 12000rpm are centrifuged 5min；
Abandon supernatant, dry 10min；30 μ l distilled waters dissolution precipitating is added and carries out PCR amplification as template.
It designs a pair of I group I fowl adenovirus Hexon gene region primer and carries out PCR identification.
Hexon upstream primer is 5 '-GTCTATACCAACACGAGCACC-3 ',
Hexon downstream primer is 5 '-GGTTAATGAAGTTATCCCTGAACCC.
The reaction system (20 μ l) of PCR amplification: 3 μ l, 2 × Premix Taq of template (has purchased from the raw work bioengineering in Shanghai
Limit company) 10 μ each 1 μ l of l, Hexon upstream and downstream primer, 5 μ l of distilled water.
PCR response procedures: 94 DEG C of initial denaturation 5min；Into circulation: 94 DEG C of 30s, 52 DEG C of 30s, 72 DEG C of 1min, 35 are followed
Ring；72 DEG C of extension 10min.
Pcr amplification product is detected with 1% agarose gel electrophoresis.Electrophoresis result (Fig. 1) shows that isolated strain expands
Corresponding target fragment, is consistent with expected target fragment size.The target fragment amplified is served into extra large English fine horse biology skill
Art Co., Ltd is sequenced, and clip size is 686bp as the result is shown, and sequence is as shown in SEQ ID NO:1.
It is analyzed using nucleotide sequence (SEQ ID NO:1) of the DNAStar software to the Hexon gene measured, and
It is compared respectively with the corresponding sequence of I group I fowl adenovirus strain in GenBank, finds separated virus and 4 type fowl gland of I group
Viral homology is 99%~100%.Therefore, PCR testing result proves that isolated virus is 4 type aviadenovirus of I group.
The inspection of 3.2 exogenous virus
Exogenous virus detection is carried out to the finally obtained supernatant of the present embodiment title 1.There is HA-HI test viral first
Detection, aseptic collection SPF chicken and duck anticoagulation 10ml, 1500rpm are centrifuged 10 minutes, wash 3 times with sterilizing PBS buffer solution, then
It is made into 1% red blood cell, 4 DEG C save backup.Whether the detection finally obtained supernatant of the present embodiment title 1 has according to a conventional method
It is aggregated the characteristic of chicken and duck red blood cell, while setting aviadenovirus EDSV-76 as agglutinating reaction positive control.As a result positive control at
It is vertical, and the isolated viral cannot be aggregated the red blood cell of 1% chicken and duck.Followed by infectious bursa of Fabricius virus (IBDV) and fowl infect
Property anemia virus (CAV) detection, synthesize referring to pertinent literature and (draw for detecting CAV (primer CAVPl and CAVP2) and IBDV
Object IBDVPl and IBDVP2) relevant primer, wherein the length of CAV primer amplification segment be 675bp, IBDV primer amplification segment
Length be 1300bp.
Sequence is as follows:
Primer CAVPl:5 '-GACTGTAAGATGGCAAGACGAGCTC-3 '；
Primer CAVP2:5 '-GGCTGAAGGATCCCTCATTC-3 '.
Primer I BDVPl:5 '-AGCCTTCTGATGCCAACAAC-3 '；
Primer I BDVP2:5 '-ATCTGTCAGTTCACTCAGGC-3 '.
Infectious bursa of Fabricius virus is not amplified using DNA in the finally obtained supernatant of the present embodiment title 1 as template
With the target fragment of avian infectious anemia virus.
These results suggest that in isolated virus without containing with HA-HI test virus and often with adenovirus mixed infection
Infectious bursa of Fabricius virus and avian infectious anemia virus.
3.3 steriling test
It tests according to existing " Chinese veterinary pharmacopoeia " annex.The finally obtained supernatant of the present embodiment title 1 is taken to be inoculated with
TG bottle, 37 DEG C are cultivated 3 days, asepsis growth.The TG tubule of transferred species, GP tubule, equal asepsis growth after GA inclined-plane culture 5 days.It connects
Mycoplasma species fluid nutrient medium, solid medium of transferring again, observation terminate negative.
The purifying of 4 type aviadenovirus limiting dilution assay of 4.I group
By the finally obtained supernatant of the present embodiment title 1 by 1: 10,1: 100,1: 1000 times of dilution, each dilution point
Not through 6 10 pieces of age in days SPF chicken embryo of yolk sac inoculation, every embryo 0.2ml is sealed and is placed on 37.0 DEG C of incubations 10 days, and record chicken embryo is dead
Die situation.The dead chicken embryo in 48h is discarded, 48~240h death chicken embryo is harvested.After choosing inoculation highest extension rate virus liquid
Dead in 48~240h and typical idiosome bleeding occur, curl, liver enlargement, matter is crisp, bleeding or necrosis and chorioallantoic membrane thicken
The chicken embryo of equal pathological changes (i.e. HHS characteristic lesion), takes its allantoic fluid, chorioallantoic membrane and idiosome (removing eyes, beak and leg), even
Slurry is added penicillin, streptomysin after 4 DEG C act on 1 hour by 2000IU/ml and sets freeze thawing 3 times, 8000r/ in the container of sterilizing
Min is centrifuged 15 minutes, is taken supernatant using 8 layers of filtered through gauze, is taken filtrate using the dilution of above-mentioned same procedure, inoculation, detection chicken
Embryo obtains E1 generation kind poison, is named as I groups until reaching 100% with disease incidence after batch virus liquid difference dilution inoculated into chick embryo
4 SD2015 plants of type aviadenovirus, and China typical culture collection center preservation (abbreviation CCTCC) is sent, depositary institution address is
Wuhan University, at August 24th 2016 preservation days, microbial preservation number is CCTCC NO:V201645.
5. kind of malicious passage
After SD2015 plants of E1 generation kind viral disease venom of 4 type aviadenovirus of I group are carried out 1: 1000 times of dilution, through yolk bag approach
It is inoculated with 6 15 pieces of age in days SPF chicken embryos, every embryonic breeding kind 0.2ml.After inoculation, sets 37 DEG C and continue to be incubated for 10 days, discard after being inoculated in 48h
Dead germ takes 48~240h death after being inoculated with and the chicken embryo of typical cytopathic occurs, and typical cytopathic thickens for chorioallantoic membrane, idiosome bleeding,
It curls, liver enlargement, matter is crisp, bleeding or necrosis.Take typical cytopathic death chicken embryo allantoic fluid, chorioallantoic membrane and idiosome (go eyes,
Beak and leg), homogenate is added penicillin, streptomysin after 4 DEG C act on 1 hour by 2000IU/ml and sets freeze thawing 3 in the container of sterilizing
Secondary, 8000r/min is centrifuged 15 minutes, is taken supernatant using 8 layers of filtered through gauze, is taken filtrate to dispense, -20 save backup, and are labeled as
E2 generation kind poison.E15 generation is passed to repeatedly.
The toxicity test of SD2015 plants of 4 type aviadenovirus of 6.I group kind poison
Virulence of the 6.1E1 generation kind poison to chicken embryo
SD2015 plants of E1 generation kind poison of 4 type aviadenovirus of I group are made into 1: 1000 times of dilution, are inoculated with 6 ages in days through yolk bag approach
20 pieces of SPF chicken embryo, every embryo 0.2ml, sealing is placed on 37.0 DEG C and continues to be incubated for 10 days.Egg is shone in timing, discards the dead chicken in 48h
Embryo takes out dead embryo in time.The lesion situation of the death rate of 10 days chicken embryos and dead chicken embryo after observation inoculation.It the results are shown in Table 1, chicken
Whole death in 5~8 days, death time concentrate on 6~7 days after embryonic breeding kind, and dissect finds that dead chicken embryo chorioallantoic membrane occurs and thickens,
Idiosome bleeding is curled, the lesion of the typical fowl hydropericardium hepatitis syndrome such as liver enlargement, matter is crisp, bleeding or necrosis, inoculation
It is incubated for 10 days afterwards, the death rate and lesion rate are up to 100%.
Toxicity test result of 1 E1 of the table generation kind poison to chicken embryo
6.2 kinds of viral disease poison assays
Measure the viral level of each generation kind poison of SD2015 plants of 4 type aviadenovirus of I group.Poison will be planted with sterilizing PBS buffer solution
Make 10 times of dilutions, takes 10-4、10-5、10-6、10-7This 4 dilutions, each dilution are inoculated with 6 age in days SPF chickens through yolk bag approach
5 pieces of embryo, every embryonic breeding kind 0.2ml.It after inoculation, sets 37 DEG C and continues to be incubated for 10 days, dead germ in 48h is discarded after inoculation, after record inoculation
48~240h chicken embryo death situation, dead chicken embryo or embryo living show as chorioallantoic membrane and thicken, and idiosome bleeding is curled, liver enlargement matter
Crisp, bleeding or necrosis are judged to infect.According to chicken embryo death and lesion number, viral level is calculated by Reed-Muench method.Respectively
The viral level of generation kind poison is shown in Table 2.
Each generation kind viral disease poison assay result of table 2
Virulence of the 6.3E1 generation kind poison to chicken
By E1 generation kind poison make 1: 1000 times dilution, respectively neck be subcutaneously injected 1 age in days, 14 ages in days, 28 ages in days, 49 ages in days,
70 ages in days, 100 ages in days, 150 age in days SPF chickens, attacking toxic dose is 1000EID50/ 0.3ml/ only, observes statistics morbidity in 7 days after inoculation
Rate determines that infection standard has a weak yellow liquid for pericardium, and liver enlargement matter is crisp, bleeding or necrosis, kidney enlargement.The result shows that:
E1 generation kind poison has 1 age in days, 14 ages in days, 28 ages in days, 49 ages in days, 70 ages in days, 100 ages in days, 150 age in days SPF chickens different degrees of
Infectious and lethal, 1 age in days SPF chicken are stronger to SD2015 plants of 4 type aviadenovirus of I group of infectivity, after neck injection 48h,
100% falls ill and falls ill suddenly, and chicken down in spirits of falling ill does not rise sleepingly, row's yellow loose stool, and 72h is whole dead.Dissect occurs typical
Hydropericardium, liver enlargement, bleeding；Kidney enlargement.To the experimental infection of 28 ages in days, all fall ill in 72h after inoculation, institute
Have a morbidity chicken down in spirits, poor appetite or abolish, doze off roll up, feather it is fluffy, arrange yellow green mucoid excrement, serious person is sleeping
Ground does not rise, and the death time concentrates 48~96h after inoculation, dissect death chicken, and pericardium has different degrees of hydropericardium, and liver is swollen
Greatly, necrosis, kidney enlargement, Mortality chicken lungs oedema, glandular stomach nipple and muscular stomach glandular stomach intersection bleeding.E1 generation kind poison is right
100, there is different degrees of hydropericardium, liver in dead chicken after the experimental infection of 150 age in days SPF chickens finds all inoculations
Enlargement or necrosis, kidney enlargement these typical pathological changes, but some morbidity chickens are transient clinical onsets, such as
The depressed, poor appetite of spirit, rooster comb be thin out, the clinical symptoms such as sagging, and is not that every morbidity chicken occurs when dissect
Hydropericardium, liver enlargement or necrosis and kidney enlargement these three typical pathological changes.In addition the age in days of chicken is bigger, identical
The virus liquid of dosage is lower to its lethal, the results are shown in Table 3, table 4.
Each group clinical onset and death record in 7 days after the infection of 31,28,49,70,100,150 age in days SPF chicken of table
41,28,49,70,100,150 age in days SPF chicken of table infects each organ disease result of dead chicken
7. immunogenicity: using conventional method with SD2015 plants of E3, E9, E15 generation kind poison preparation epidemic diseases of 4 type aviadenovirus of I group
Vaccine is subcutaneously injected 21 age in days SPF chicken each 10, every 0.3ml with neck, if compareing with age in days SPF chicken 10, is immunized by seedling
After 3 weeks, all immune chickens and control chicken carry out neck with I group SD2015 plants of hepatic tissue poison of 4 type aviadenovirus and subcutaneously attack poison, attack
Toxic dose is 0.3ml/ only (containing about 10ID50).After attacking poison, 7 days judgement results are observed.It as a result is all morbidities and 9/ of control group chicken
10 is dead, and it is 90%~100% that chicken protective rate, which is immunized,.The result shows that: I group SD2015 plants of inactivated vaccines of 4 type aviadenovirus are exempted from
Epidemic disease effect is fine, and seed culture of viruses immunogenicity within 15 generations is stablized.It the results are shown in Table 5.
5 I group of table SD2015 plants of each generation seed culture of viruses immunogenicity determinations of 4 type aviadenovirus
The preparation of 2 I group of embodiment, 4 SD2015 plants of inactivated vaccines of type aviadenovirus
1. what material was used in seedling selects well-developed 6~7 age in days SPF chicken embryo as seedling material.
2. the preparation of seedling virus liquid
2.1 inoculations take SD2015 plants of 4 type aviadenovirus of I group, are diluted with sterilizing PBS buffer solution according to 1:1000 times, yolk
Capsule is inoculated with 6~7 age in days SPF chicken embryos, and every embryo 0.2ml sets 37 DEG C of incubations, it is not necessary to egg-turning.
It is primary according to egg daily after 2.2 incubations and observation inoculation in 48h, discard dead embryo；It is inoculated with after 48h, it is every 8 small
When it is primary according to egg, take out dead embryo in time, set 2~8 DEG C.
Chicken embryo is all taken out, sets 2~8 DEG C overnight by 2.3 harvest hatchings to 16 ages in days.Eggshell gas chamber is sterilized with Iodophors
Position removes eggshell after 75% alcohol secondary sterilization with aseptic procedure, collect has HHS characteristic lesion chicken embryo respectively
Allantoic fluid, chorioallantoic membrane and idiosome (removing eyes, beak and leg).Chorioallantoic membrane and idiosome are homogenized with tissue mashing machine, used
Suspension is made as dilution in the chick embryo allantoic liquid of harvest, and penicillin, streptomysin is added by 2000IU/ml, and 4 DEG C act on 1 hour,
It after freeze thawing 3 times, sets in sterilization container, 8000 turns are centrifuged 15 minutes, take 8 layers of filtered through gauze of supernatant, filtrate is virus liquid.
Wherein, HHS characteristic lesion chicken embryo refers to: inoculation 48h later dead chicken embryo has following symptoms: chorioallantoic membrane increases
Thickness, idiosome bleeding are curled, and liver enlargement matter is crisp, bleeding or necrosis.
The measurement of 2.4 viral levels takes 4 type aviadenovirus SD2015 strain virus liquid of I group, by 1 viral level measurement side of example
Method is measured, viral level >=10 in 0.2ml virus liquid5.80EID50。
2.5 inactivate to qualified (viral level >=10 of inspection6.0EID50/ ml be qualification) virus liquid in, be added volume hundred
Dividing concentration is 10% formalin, and formaldehyde final concentration of 0.2% (concentration expressed in percentage by volume) in the virus liquid after making inactivation sets 37
Shake inactivates 36 hours (reaching 37 DEG C of beginning timing with the temperature in bottle) in DEG C constant-temperature table, and the virus liquid after inactivation sets 2~8
DEG C save.
3. the inspection of semifinished product
3.1 steriling tests are carried out referring to the steriling test of example 1.
3.2 inactivations, which are examined, takes 6~7 age in days SPF chicken embryos 10, the virus liquid 0.2ml after every embryo yolk sac inoculation inactivation,
Continue to be incubated for 10 days, dissect, Ying Wu HHS lesion.The allantoic fluid, chorioallantoic membrane and idiosome of above-mentioned chicken embryo are taken, grinds, suspension is made,
6~7 age in days SPF chicken embryos 10 are inoculated, every embryo 0.2ml is incubated for 10 days, dissect, Ying Wu HHS lesion.
4. the preparation of oil adjuvant killed vaccine
4.1 oil mutually prepare 94 parts by volume injection white oils, 6 parts by volume Si Ben -80, are added in oily phase tank, heating stirring is mixed
It is even, it sterilizes 30 minutes through 121 DEG C, is cooled to room temperature, obtains oily phase.
The 4 type aviadenovirus SD2015 strain virus liquid of I group that the preparation of 4.2 water phases takes 96 parts by volume to inactivate, is added 4 parts by volume
Tween-80, be sufficiently stirred until Tween-80 be completely dissolved, obtain water phase.
3 parts by volume oil is mutually put into emulsion tank by 4.3 emulsifications, starting emulsion tank blender stirring, while 1 being added slowly
Parts by volume water phase continues 10~15min of stirring after adding, open homogenizer inlet and outlet switch, start homogenizer, pass through emulsion
Homogenizer enters another tank, emulsifies (general 6~8 times) for several times repeatedly, obtains SD2015 plants of 4 type aviadenovirus of I group inactivations
Vaccine.10ml inactivated vaccine is taken, with 3000r/min centrifugation 15 minutes, lamination should not occur.
The I group SD2015 plants of inactivated vaccines of 4 type aviadenovirus, the antigenic content of every plumage part (0.3ml) answer >=105.0EID50。
In actual production process, according to the practical viral level of virus liquid, the PBS buffer solution (concentration 0.1mol/L) of pH7.2 can be used
Dilution appropriate is carried out to virus liquid, as long as guaranteeing in final inactivated vaccine, the antigenic content of every plumage part (0.3ml) >=
105.0EID50?.
5. effect is comprehensive for preventing fowl hydropericardium hepatitis with SD2015 plants of inactivated vaccines of 4 type aviadenovirus of purposes I group
Sign.The above chicken of 14 ages in days can immunity inoculation, generation immunity on the 7th~14 after inoculation, duration of immunity be 6 months.
6. 0.3ml is subcutaneously injected in every chicken neck of usage and dosage.
Example 3 I group SD2015 plants of inactivated vaccine immuning effect tests of 4 type aviadenovirus
SD2015 plants of inactivated vaccine (lot numbers: 20151212, of 4 type aviadenovirus of I group are prepared by method in embodiment 2
20160116,20160201) efficacy test of finished product and is as follows carried out.4 type fowl adenopathy of I group in each lot number inactivated vaccine
Malicious SD2015 plants of viral level is 105.0EID50/ plumage.SD2015 plants of hepatic tissue poison of 4 type aviadenovirus of I group: I group 4 will be infected
The SPF that SD2015 plants of type aviadenovirus dies of illness the pathologic liver of chicken, grinds, presses after being mixed with sterilizing PBS buffer solution with 1:2
Penicillin, streptomysin is added in 2000IU/ml, and after 4 DEG C act on 1 hour, multigelation 3 times, 8000r/min is centrifuged 15 minutes, 8 layers
Filtrate is taken after filtered through gauze, (method is the same as embodiment 1 for progress Sterility testing (method is in embodiment 1) and exogenous virus detection
In), it should be without the pollution of other cause of diseases, -70 DEG C save backup.
1. serology efficacy test: every batch of seedling takes 21 age in days SPF chickens 20, and every neck subcutaneous injection 0.3ml inactivates epidemic disease
Seedling；Another 20 are only used as control group, are not immunized.Immune 7 days latter, 14 days, 21 days, January, 2 months, March, April, May, blood sampling in June, point
From serum, measure HHS neutralizing antibody and fine jade expands antibody (referring to existing " Chinese veterinary pharmacopoeia " method).It the results are shown in Table 6,7, three batches
Inactivated vaccine 7~14 days generation HHS neutralizing antibodies and fine jade after immune expand antibody, in 21 days to 6 months HHS and anti-after being immunized
Body average value is not less than 1:5000 (being qualification not less than 1: 5000), and fine jade expansion antibody average value is not less than 1: 2.0 and (is not less than
Be qualification at 1: 2.0).
HHS neutralizing antibody average value measurement result after 6 three batch I group of table, 4 type aviadenovirus SD2015 inactivated vaccine is immune
HHS fine jade expands antibody average value measurement result after 7 three batch inactivated vaccine of table is immune
SD2015 plants of inactivated vaccine Immunization Vaccine effectiveness of 4 type aviadenovirus of 2.I group are examined: being taken in this implementation title 1
Immune group SPF chicken and control SPF chicken each 10 after 21 days immune, are carried out with I group SD2015 plants of hepatic tissue poison of 4 type aviadenovirus
Neck subcutaneously attacks poison, and attacking toxic dose is 0.3ml/ only (containing about 10ID50).After attacking poison, 7 days judgement results are observed.It can be with from table 8
See, control group is all fallen ill, and 9/10 is dead, and dissect death chicken and morbidity survival chicken all have fowl hydropericardium hepatitis syndrome
Classical symptom, hydropericardium, liver enlargement, matter is crisp, bleeding or necrosis, kidney enlargement.Immune group protective rate 90%~
100%.The result shows that: the immune effect of 4 SD2015 plants of inactivated vaccines of type aviadenovirus of I group is fine.
Table 8 three batch I group SD2015 plants of inactivated vaccines of 4 type aviadenovirus attack malicious Vaccine effectiveness test result
Immune efficacy of the embodiment SD2015 plants of inactivated vaccines of 4 I group, 4 type aviadenovirus to laying hen and its filial generation
Detection is using I group SD2015 plants of inactivated vaccines of 4 type aviadenovirus of method preparation in embodiment 2 to laying hen and its son
The immune efficacy in generation, inactivated vaccine lot number are 20151212, and viral level is shown in embodiment 3.
18 week old sea is immunized in I group SD2015 plants of inactivated vaccines of 4 type aviadenovirus (from seedling) that lot number is 20151212
Blue laying breed 20, another 10 are not immunized, and as a control group, are only inoculated with according to from seedling dosage 0.3ml/, 7 days, 14 after being immunized
It, take a blood sample within 21 days, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, separate serum, measure HHS neutralizing antibody and fine jade
Expand antibody, evaluates immune effect of vaccine；1~6 month after immune, vaccinated flock hatching egg (male and female mixes group, flat to support this friendship) is collected,
Filial generation protest test is carried out after hatching, and the immune duration of vaccine is evaluated by the maternal antibody protection that filial generation obtains.
Neutralizing antibody level is not less than 1: 5000 with mean value, and fine jade expands antibody and is judged to qualification not less than 1: 2.0 with mean value.Malicious protection is attacked in filial generation
In test, carry out neck with I group SD2015 plants of hepatic tissue poison of 4 type aviadenovirus and subcutaneously attack poison, attack toxic dose be 0.3ml/ only (about
Containing 10ID50), after attacking poison, observes 7 days and determine that the infection of 8/10 or more control group is judged to close as a result, protecting with 8/10 or more immune group
Lattice.The result shows that: 6 months after the batch vaccine immunity, chicken group's serum neutralizing antibody is 1: 5024, and fine jade expands antibody 1:2.4, as a result
It is shown in Table 9.It is 90%~100% that malicious protective rate is attacked in filial generation, and control group all infects, and the results are shown in Table 10.Test result illustrates hen
1~6 month antibody generated can be provided with effect protection to filial generation after immune.
HHS is neutralized after 9 20151212 batches of SD2015 plants of inactivated vaccines of table are immune and fine jade expands antibody determination result
10 20151212 batches of SD2015 plants of inactivated vaccine Immune Laying Hens of table attack poison protection measurement result to filial generation
SD2015 plants of inactivated vaccine doubling dosages of 5 I group of embodiment, 4 type aviadenovirus are inoculated with safety test
SD2015 plants of inactivated vaccine (lot numbers: 20151212, of 3 batch I group, 4 type aviadenovirus are prepared by method in embodiment 2
20160116、20160201)。
Investigation object is 14 age in days SPF chickens (being purchased from Beijing Cimmeria Wei Tong experimental animal Co., Ltd) and the white plumage of 14 ages in days
Broiler chicken (is purchased from Nanjing Stone Buddha Temple chicken farm).After each batch of chicken is bought back, a couple of days is adapted to, the overall health of patients and clinical disease feelings of chicken are verified
Condition rejects weak chicken, and healthy chicken is selected to enter test.Three crowdes of I are subcutaneously injected to SPF chicken difference doubling dosage (0.6ml/ is only) neck
Group's SD2015 plants of inactivated vaccines of 4 type aviadenovirus, set 1 group and are not inoculated with, every time as control；Doubling dosage is distinguished to white meat-type chickens
Three batches of 4 type aviadenovirus SD2015 inactivated vaccines of I group are subcutaneously injected in (0.6ml/ is only) neck, set 1 group every time and are not inoculated with, as right
According to.Observed after inoculation whether occur in 14 days it is any as caused by vaccine locally and systemically react, 14 days after inoculation, 28 days each groups
Test chicken 5~10 are randomly selected, dissect injection site checks the absorbing state of vaccine.
As a result: after inoculation in 14 days, not observing clinical exception, feeding, drinking-water are normal, and health condition is good, does not find
It is any as caused by vaccine locally and systemically to react, specifically it is shown in Table 11.14 days after inoculation, each group takes 5~10 chicken dissect injections
Position, the visible a small amount of millet appearance size particles of 80% or more naked eyes do not absorb completely；28 days dissects after injection, 70% or more vaccine
Basic absorption, injection site have no the abnormal response as caused by vaccine inoculation, are specifically shown in Table 12.Therefore, 4 type fowl adenopathy of I group
Malicious SD2015 plants of inactivated vaccine doubling dosage inoculation is safe.
11 doubling dosage of table is inoculated with after SD2015 plants of inactivated vaccines of 4 type aviadenovirus of I group clinical observation result in 14 days
The absorption inspection result of table 12 I group SD2015 plants of inactivated vaccines of 4 type aviadenovirus
SEQUENCE LISTING
<110>Jiangsu Province Agriculture Science Institute
<120>a kind of 4 type aviadenovirus of I group, inactivated vaccine and preparation method thereof
<130> 20160930
<160> 1
<170> PatentIn version 3.3
<210> 1
<211> 686
<212> DNA
<213>SD2015 plants of 4 type aviadenovirus of I group
<400> 1
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tcgatggacc aacgcgcacc cacgttcgtg aacatgtcga cgacgttggt gagggggacg 120
cgcttgttca tgtactcgta ggtggtaggc gcgacgctgg tggcgtcgaa gccggagatg 180
ctaaacttgt aacggtcggg cagatactcg gcgatgttgg ccatgataaa gttgcgcctc 240
tgggtagcgc tgatatcgat ttcgtaggag ggtaccgtgc cgaagtagaa gttggtagtg 300
gcgttagcca cggcggtgtt tttgtcgttc gcggcggtgt tgttggtgta gagtttaatt 360
tgactgagat cggggccgta gccttctccc gcgcctaccg cttcggggtt gaaggcgtag 420
ctgggcgcgc cttcttcgta accgtcattg gagaagactc gcacctcggg gtcgtactgg 480
tccacggcct ggttccacag ggcgaaatag tgatggcggg acatcatgtc ggccagcatg 540
tactggtagc tgagctcggt attccggtcg ggcagctcga ccaccacgtt catgcccgaa 600
cgctccgagt tgagggtgcc cgagcacacg ccggagtcgt gatacagcag gttaatgaag 660
ttatccctga acccgatgta gttggg 686
Claims (8)
- SD2015 plants of 4 type aviadenovirus of 1.I group, deposit number are as follows: CCTCC NO:V201645.
- 2. a kind of 4 type aviadenovirus inactivated vaccine of I group, it is characterised in that: the 4 type aviadenovirus inactivated vaccine of I group contains inactivation Claim 1 described in SD2015 plants of 4 type aviadenovirus of I group.
- 3. 4 type aviadenovirus inactivated vaccine of I group according to claim 2, it is characterised in that the inactivated vaccine is oil emu Inactivated vaccine.
- 4. the method for preparing 4 type aviadenovirus SD2015 strain virus liquid of I group, it is characterised in that wanted using SPF chicken embryo proliferation right 1 described SD2015 plants of 4 type aviadenovirus of I group are asked, 4 type aviadenovirus SD2015 strain virus liquid of I group is obtained.
- 5. preparing the method for 4 type aviadenovirus SD2015 strain virus liquid of I group according to claim 4, it is characterised in that right It is required that 1 SD2015 plants of inoculation SPF chicken embryos of the 4 type aviadenovirus of I group, harvest the allantoic fluid of lesion chicken embryo, chorioallantoic membrane and Idiosome, homogenate, freeze thawing, centrifuging and taking supernatant obtain 4 type aviadenovirus SD2015 strain virus liquid of I group.
- 6. a kind of preparation method of 4 type aviadenovirus inactivated vaccine of the group of I described in Claims 2 or 3, comprising:(1) 4 type aviadenovirus SD2015 strain virus liquid of formalin-inactivated I group is used；(2) injection white oil and Si Ben -80 are mixed evenly according to volume ratio 94:6, as oil-phase solution；(3) 4 type aviadenovirus SD2015 strain virus liquid of the I group of inactivation and Tween-80 are uniformly mixed according to volume ratio 96:4, are made For aqueous phase solution；(4) it emulsifies: oil-phase solution and aqueous phase solution being mixed, emulsification according to 2 ~ 3:1 of volume ratio, that is, obtain the 4 type fowl gland of I group Viral inactivation vaccine.
- 7. the preparation method of 4 type aviadenovirus inactivated vaccine of I group according to claim 6, it is characterised in that: the I after inactivation The concentration expressed in percentage by volume of formaldehyde is 0.15% ~ 0.25% in 4 type aviadenovirus SD2015 strain virus liquid of group.
- 8. the preparation method of 4 type aviadenovirus inactivated vaccine of I group according to claim 7, it is characterised in that: 0.2ml I group 4 Viral level >=10 of type aviadenovirus SD2015 strain virus liquid5.80EID50。
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| CN107338226B (en) * | 2017-05-31 | 2020-09-08 | 河南农业大学 | Avian adenovirus type 4 strain, vaccine composition and application thereof |
| CN109207436B (en) * | 2017-07-07 | 2022-02-18 | 乾元浩生物股份有限公司 | Group I type 4 avian adenovirus strain and application thereof |
| CN108558989B (en) * | 2018-04-17 | 2019-07-02 | 扬州优邦生物药品有限公司 | 4 type aviadenovirus subunit vaccines of a kind of prevention and its preparation method and application |
| CN110079509B (en) * | 2019-05-31 | 2020-02-14 | 洛阳职业技术学院 | Group I11 d avian adenovirus strain, inactivated vaccine and preparation method thereof |
| CN111778217B (en) * | 2020-08-04 | 2022-08-09 | 江苏省农业科学院 | I-group 4 type avian adenovirus SD-F strain, inactivated vaccine and preparation method |
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