CN108558995A - A kind of Yolk antibody and preparation method thereof of the novel goose astrovirus of prevention - Google Patents
A kind of Yolk antibody and preparation method thereof of the novel goose astrovirus of prevention Download PDFInfo
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- CN108558995A CN108558995A CN201810494058.4A CN201810494058A CN108558995A CN 108558995 A CN108558995 A CN 108558995A CN 201810494058 A CN201810494058 A CN 201810494058A CN 108558995 A CN108558995 A CN 108558995A
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- yolk
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- astrovirus
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Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
- C07K16/10—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from RNA viruses
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/12—Antivirals
- A61P31/14—Antivirals for RNA viruses
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/02—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies from eggs
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/55—Medicinal preparations containing antigens or antibodies characterised by the host/recipient, e.g. newborn with maternal antibodies
- A61K2039/552—Veterinary vaccine
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
- C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
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- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- Virology (AREA)
- Medicinal Chemistry (AREA)
- Molecular Biology (AREA)
- General Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- Genetics & Genomics (AREA)
- Biophysics (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Immunology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Oncology (AREA)
- Communicable Diseases (AREA)
- General Chemical & Material Sciences (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Pharmacology & Pharmacy (AREA)
- Animal Behavior & Ethology (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
Abstract
The invention discloses a kind of Yolk antibody and preparation method thereof of the novel goose astrovirus of prevention, the Yolk antibody contains anti-goose astrovirus strain antibody；The deposit number of the goose astrovirus strain is CCTCC NO：V201808.The safety of Yolk antibody prepared by the present invention is good, does not occur any locally or systemically adverse reaction caused by Yolk antibody；And can effectively prevent and/or treat the infection of novel goose astrovirus, there is good commercialized development foreground.
Description
Technical field
The present invention relates to veterinary biologics technical fields, and in particular to a kind of yolk of the novel goose astrovirus of prevention is anti-
Body and preparation method thereof.
Background technology
Astrovirus (AStV) is a kind of single strand plus RNA virus without cyst membrane, 28-30nm spherical in shape, a diameter of, gene
There is group infectivity, the infection of long 6.4-7.9kb, the virus to have certain popularity, can cause enteritis, the abdomen of human or animal
It rushes down, some is with vomiting and abdominal pain.Astroviridae according to infection host difference can be divided into mammal Astrovirus and
Fowl Astrovirus, fowl astrovirus can cause birds that a variety of diseases occur, and clinical manifestation is because of virus stain, virulence or sense
Contaminate the different and variant of host.Goose astrovirus (Goose astrovirus, GAstV), it is main to encroach on young age young bird goose, the disease
Occur in multiple province prevalences, the death rate of goose is made to increase, the sound development of goose industry is supported in high risks China.
2~December in 2017, the ground such as China Shandong, Anhui, Jiangsu, Liaoning, Henan and Guangdong young bird gaggle have broken out one kind
Using gout as the communicable disease of main feature.The disease takes place mostly in the young goose of 5~20 ages in days, the death rate up to 50% with
On.Serious uric acid mineralization occurs for illness young bird goose body cavity and joint, and sleepingly tired dynamic, feeding is difficult, causes young goose slow-growing, expects
For meat than increasing, meat goose delivers qualification rate significant decrease for sale, and serious financial consequences are caused to meat goose cultivation industry.
It has been investigated that the outburst of the disease is caused by a kind of novel goose astrovirus infection, it there is no vaccine can at present
To be prevented, conventional antiviral and antibacterial therapy method is invalid.
Yolk antibody refers to the antibody for specific antigen extracted from immune eggs, due to only having IgG in yolk
Class antibody, therefore it is called Yolk immunoglobulin IgG (egg yolkimmunoglobulins), referred to as IgY.It is anti-with serum
Body is compared, and Yolk antibody has the advantages that many uniquenesses：(1) it is not necessarily to blood sampling, immune height need to be only acquired and exempt from egg yolk purifying i.e.
It can purify to obtain Yolk antibody；(2) there is good stability, it is heat-resisting acidproof, and remain to keep certain activity under room temperature；(3)
Therapeutic effect is apparent, high specificity, and is suitable for mass producing；(4) safe efficient, noresidue, mild environmental protection.But due to
The disease belongs in China to be reported for the first time, vaccine and the antibody listing of commercialization is there is no at present, for the novel goose astrovirus
It is urgent to prevent with the Yolk antibody of early infection treatment to be even more to have not been reported；And it due to containing a large amount of lipid in yolk, gives
Using many inconvenience are brought, this also constrains the development and application for the Yolk antibody for preventing the novel goose astrovirus.
Invention content
For the above-mentioned prior art, the object of the present invention is to provide a kind of Yolk antibody of the novel goose astrovirus of prevention,
Novel goose astrovirus infection for preventing and treating gaggle outburst, serious financial consequences caused by make up the disease.
It is a further object of the present invention to provide the preparation methods of the Yolk antibody of the novel goose astrovirus of the prevention.The preparation
Method is easily operated, is suitble to large-scale production；And Yolk antibody property stabilization, purity height, the potency prepared is high, specific
By force, drug residue free, mild environmental protection, the prevention to novel goose astrovirus infection and the great application value for the treatment of.
To achieve the above object, the present invention adopts the following technical scheme that：
The present invention is based on suffering from the novel goose astrovirus of isolated in goose body one plant, preservation from gout symptom
Number is CCTCC NO：V201808, on the basis of the separation strains, going out for the novel goose astrovirus can be prevented by having developed
Live vaccine, using the inactivated vaccine high intensity immune health laying hen, then egg yolk after separating immune, extraction prepares yolk and resists
Body.Specifically：
The first aspect of the present invention, provides a kind of Yolk antibody of the novel goose astrovirus of prevention, and the Yolk antibody contains
There is anti-goose astrovirus strain antibody；The deposit number of the goose astrovirus strain is CCTCC NO：V201808.
The second aspect of the present invention provides the preparation method of above-mentioned Yolk antibody, includes the following steps：
(1) it is CCTCC NO with deposit number：Inactivation is made as production of vaccine strain in the goose astrovirus of V201808
Vaccine；
(2) with the inactivated vaccine injecting immune laying hen prepared, high-immunity egg is prepared；
(3) yolk is collected by high-immunity egg, through once inactivating, acidizing extraction, secondary inactivation, coarse filtration, aseptic filtration, concentration and
After inactivating three times, it is prepared into the Yolk antibody for preventing novel goose astrovirus.
Preferably, in step (1), the inactivated vaccine is prepared by the following method：
It is CCTCC NO by deposit number：The goose astrovirus of V201808 is inoculated with SPF chicken embryos, collects dead in 24-120h
Chick embryo allantoic liquid obtains virus liquid；Virus liquid is concentrated into viral level >=105.0ELD50, the virus liquid after concentration goes out through formaldehyde
It is living, Tween-80 mixing is added and is used as water phase, is mixed using white oil, aluminum stearate and Span-80 as oil phase, by water phase and oil phase
By volume 1:Inactivated vaccine is made in 1 mixing, emulsification.
Preferably, in step (2), divided 4 times with the inactivated vaccine of preparation and laying hen is immunized, each immunization interval 2
Week.Immunization route is that neck subcutaneously or intramuscularly injects inactivated vaccine.
Preferably, in step (3), the primary inactivation is specially：By yolk liquid and water by volume 1:(0.5~1.5)
Mixing, yolk diluent is obtained after stirring evenly, and 25~35min is kept the temperature under the conditions of 60~65 DEG C.
Preferably, in step (3), the acidizing extraction is specially：The yolk diluent is added into yolk diluent
Filtrate is collected by filtration after stirring in the acetate buffer that 2.5~3.5 times of volume.It is further preferred that the acetate buffer
PH value be 4.8-5.2；More preferably 5.0.
Preferably, in step (3), the secondary inactivation is specially：Octanoic acid is added into the filtrate after acidizing extraction to end
Concentration 3.5~4.5% stirs 30-120min, is placed 4~8 hours in 2~8 DEG C after stirring.
Preferably, in step (3), the coarse filtration is specially：It is then clear to filtrate with membrane filtration again first with filter-cloth filtering
Clearly.It is further preferred that the filter cloth is polypropylene fibre 750B filter clothes.
Preferably, in step (3), the aseptic filtration is specially：With 0.22 μm of membrane filtration degerming.
Preferably, in step (3), the concentration is specially：Using the PES hollow fiber ultrafiltration membranes of 30~50KD 2~8
It is concentrated under the conditions of DEG C, until antibody titer is not less than 1:512.
Preferably, in step (3), the inactivation three times is specially：Formalin is added, makes the final concentration of of formalin
0.1%, 37 DEG C of inactivation 16h.
The third aspect of the present invention provides above-mentioned Yolk antibody and is preparing for preventing and/or treating goose astrovirus sense
Application in the product of dye.The deposit number of the goose astrovirus is CCTCC NO：V201808.
Beneficial effects of the present invention：
(1) for the present invention using isolated one plant of novel goose astrovirus out of morbidity young goose body, the novel goose is starlike
Strain has excellent immunogenicity, the passage that can stablize in chicken embryo, for current endemy based on gout
Want the novel goose astrovirus infection of symptom that there is better protectiveness.
(2) it is CCTCC NO with deposit number：The goose astrovirus of V201808 prepares inactivation epidemic disease as production of vaccine strain
Seedling carrys out Immune Laying Hens, can efficiently obtain Yolk antibody, and the safety of the Yolk antibody is good, does not occur being caused by Yolk antibody
Any locally or systemically adverse reaction；And the infection of novel goose astrovirus can be effectively prevented and treated, have very
Good commercialized development foreground.
(3) preparation process of Yolk antibody is optimized in the present invention, collects after height exempts from egg yolk and first carries out tentatively
Inactivation then carries out acidification with acetate buffer and extracts, then as inactivator and extracts the secondary inactivation of agent progress using octanoic acid,
It is filtered clarification, filtration sterilization on the basis of this, is most inactivated three times with formaldehyde after ultrafiltration concentration afterwards, yolk is finally obtained
Antibody-solutions.The content that lipid in final product is effectively reduced above by acidifying water-octanoic acid method, reduces adverse reaction
Risk, while step is concentrated by ultrafiltration and helps to ensure that antibody titer in solution, improve protective rate；Higher antibody titer will be in future
Higher extension rate is needed when clinical application, to reduce impurity content indirectly, and then promotes safety.
Description of the drawings
Fig. 1：The virus isolated strain N-AStV-SDPY of the present invention is inoculated with goose embryo lesion：Goose embryo under A normal growth states；B
For goose embryo lesion caused by SDPY separation strains.
Fig. 2：PCR qualification results；In figure, M is 2000bp Marker, and swimming lane 1 is blank control, and swimming lane 2 is AstV, swimming
Road 3 is AstV, and swimming lane 4 is AIV, and swimming lane 5 is NDV, and swimming lane 6 is ARV, and swimming lane 7 is GPV, and swimming lane 8 is GpoV, and swimming lane 9 is
GoCV。
Specific implementation mode
It is noted that following detailed description is all illustrative, it is intended to provide further instruction to the application.Unless another
It indicates, all technical and scientific terms used herein has usual with the application person of an ordinary skill in the technical field
The identical meanings of understanding.
As background technology is introduced, from 2017, the ground such as China Shandong, Anhui, Jiangsu, Liaoning, Henan and Guangdong
Young gaggle has been broken out a kind of using gout as the communicable disease of main feature.The astrovirus sense different from the past of the symptom of the disease
Dye, occurs the symptom of gout in gaggle, thus deduces, which may be to be drawn by novel astrovirus for the first time
It rises.It there is no the drug and method that can effectively control the novel astrovirus infection at present.Using astrovirus inactivated vaccine
Inoculation is to prevent the effective measures of disease infection and outburst, but on condition that need to detach the excellent starlike disease of goose of adaptive immune originality
Poison strain.
Since astrovirus is RNA virus, there are multiple segmentations, in antigenic structure, pathogenic, cell culture between different strains
It is had a certain difference on characteristic and host specificity, in genetic evolution, is easy to happen variation, traditional strain is caused to prepare
Vaccine cannot well prevention and control it is current popular using gout as the goose astrovirus infection of cardinal symptom.
Present inventor isolates one plant of new virus N- from the tissues such as liver, spleen and the kidney of the young goose that dies of illness
AStV-SDPY, identified new virus N-AStV-SDPY are goose astrovirus.The present invention carries out strain N-AStV-SDPY
Genome sequencing, and with the astrovirus of existing report homology analysis and phylogenetic analysis have been carried out, as a result, it has been found that,
The homology of each genetic fragments of strain N-AStV-SDPY is respectively less than 70%；Phylogenetic analysis shows strain N-AStV-SDPY
In a variation branch；It these results suggest that：Strain N-AStV-SDPY is different from other astrovirus, is the starlike disease of fowl
The new kind that poison belongs to.And the novel goose astrovirus is preserved in China typical culture collection center, deposit number is
CCTCC NO：V201808.
It is long the time required to generating antibody after being immunized since vaccine immunity is to aim at prevention, be difficult to when gaggle falls ill into
Row in time, is quickly treated.Based on this, the present invention prepares vaccine immunity laying hen by the novel goose astrovirus of separation, acquisition
Height exempts from egg and prepares Yolk antibody, the urgent prevention for novel goose astrovirus and early infection treatment.
In one embodiment of the invention, the Yolk antibody of the novel goose astrovirus of prevention provided is by the following method
It is prepared：
(1) it takes novel goose astrovirus height to exempt from egg yolk, is uniformly mixing to obtain yolk liquid；
(2) step (1) the yolk liquid and water are pressed 1:(0.5~1.5) (V/V) ratio mixes, and is obtained after stirring evenly
Yolk diluent keeps the temperature 25~35min under the conditions of 60~65 DEG C；
(3) acetate buffer that the yolk liquid accumulates 2.5~3.5 times is added into yolk diluent, is filtered after stirring
Filtrate is collected by filtration in cloth；
(4) octanoic acid is added into step (3) described filtrate to final concentration 3.5~4.5% (V/V), in 2~8 DEG C after stirring
It places 4~8 hours；
(5) solution after taking step (4) to place is filtered to filtrate and is clarified, and collects clear filtrate；
(6) step (5) the clear filtrate filtration sterilization is taken, formaldehyde is then added to final concentration 0.1% (V/V), in 35
~37 DEG C inactivate 12~20 hours.
Preferably, the novel goose astrovirus height described in step (1), which exempts from egg, to be inactivated using novel goose astrovirus
Produced egg after 4 immune hens of vaccine point, each immunization interval 2 weeks；Further preferably, the immunization route is that neck is subcutaneous
Or the novel goose astrovirus inactivated vaccine of intramuscular injection.
Preferably, in step (1) the novel goose astrovirus high immunity egg yolk, novel goose astrovirus antibody titer
Not less than 1:1024.
Preferably, first exempting from egg to the height in step (1) carries out eggshell disinfection, yolk is then collected；It is further excellent
Choosing, the sterilization method is first to spray 75% alcohol again after eggshell drying with 1% bromogeramine solution soaking disinfection 15min
To eggshell surface.
Preferably, the water described in step (1), is prior to 80 DEG C holding 30min, is then cooled to 65 DEG C of notes below
Jetting.
Preferably, acetate buffer pH value described in step (2) is 4.8~5.2, more optimizedly 5.0.
Preferably, the stirring described in step (3), is persistently 30~120min, more optimizedly 90min.
Preferably, the filter cloth described in step (3) is polypropylene fibre 750B filter clothes.
Preferably, the stirring described in step (4), duration are 30~120min, more optimizedly 90min.
Preferably, the filtering described in step (5) is first with filter-cloth filtering, then clarified again with membrane filtration to filtrate；
It is further preferred that the filter cloth is polypropylene fibre 750B filter clothes.
Preferably, the filter sizes for being used for filtration sterilization in step (6) are 0.22 μm.
Preferably, novel goose astrovirus antibody titer is not less than 1 in clear filtrate after step (6) degerming:512.
Preferably, being first concentrated by ultrafiltration after degerming in step (6), formalin-inactivated is then added；Further preferably
, the ultrafiltration concentration is realized using the PES hollow fiber ultrafiltration membranes of 30~50KD；It is further preferred that the concentration
It is carried out under the conditions of 2~8 DEG C.
In above technical scheme, collects after height exempts from egg yolk and first tentatively inactivated, then with acetate buffer
It carries out acidification to extract, then the secondary inactivation of agent progress as inactivator and is extracted using octanoic acid, be filtered clarification, mistake on this basis
Bacterium is filtered out, is most inactivated three times with formaldehyde after ultrafiltration concentration afterwards, Yolk antibody solution is finally obtained.Above by acidification
Water-octanoic acid method effectively reduces the content of lipid in final product, reduces the risk of adverse reaction, while step is concentrated by ultrafiltration
It helps to ensure that antibody titer in solution, improves protective rate；Higher antibody titer needs higher when future clinical is applied
Extension rate to reduce impurity content indirectly, and then promotes safety.
In order to enable those skilled in the art can clearly understand the technical solution of the application, below with reference to tool
The technical solution of the application is described in detail in the embodiment of body.
The test material that test material is this field routine is not specifically described used in the embodiment of the present invention,
It can be commercially available by commercial channel.Specific experiment condition and method are not specified in the embodiment of the present invention, usually according to routine
Condition, such as J. Pehanorm Brooker chief editors, Science Press, 2002, Molecular Cloning:A Laboratory guide (third edition)；D.L. this peck
Top grade is edited, Science Press, and 2001, cell experiment guide；Or the condition suggested according to manufacturer.The embodiment of the present invention
In quantitative test, be respectively provided with three repeated experiments, results are averaged；% in the embodiment of the present invention, unless otherwise instructed,
It is mass percentage.
Embodiment 1：The discovery and identification of novel goose astrovirus
1. materials and methods
1.1 pathological material of diseases acquire and processing：
The tissues such as young Goose Liver, spleen and kidney of dying of illness are taken, according to 1:It is even that sterile physiological saline progress is added in 4 ratio
Slurry processing, refrigerator multigelation is placed in 3 times by homogenate, supernatant is taken after 6000r/min centrifugations 15min, through 0.22 μm of filter
It is saved backup for -20 DEG C after filter bacteria.
1.2 main agents：
Goose embryo is purchased from Distributions in Liaocheng of Shandong Province goose field, and it is century Co., Ltd, plain agar that RNA extracts kits, which are purchased from Beijing health,
Sugared gel reclaims kit is purchased from Beijing Quan Shi King Companies, and reverse transcription reagent box is purchased from precious biological (Dalian) Co., Ltd, 2 × Es
It is century Co., Ltd that Taq MasterMix, which are purchased from Beijing health, and DL2000Marker is purchased from precious biological (Dalian) Co., Ltd.
1.3 design of primers：
All astrovirus on the geneseq database Genbank established according to National Center for Biotechnology Information
The specific primer of gene order design amplification 601bp segments is a pair of, and by Beijing, Qing Kexin industry Bioisystech Co., Ltd synthesizes.
Sense primer：5′-TGG TGG TGY TTY CTC AAR A-3′；
Downstream primer：5′-GYC KGT CAT CMC CRT ARC A-3′.
Note：Y, K, M, R are degeneracy base.Wherein Y represents C/T；K represents G/T；M represents A/C；R represents A/G.
1.4 virus purification：
By the tissue sample treatment fluid of acquisition through 13 age in days goose embryo of chorioallantoic cavity approach aseptic inoculation, 0.2mL/ pieces；And
With sterile saline, negative control, 37 DEG C of incubations are set.Twice according to embryo daily, dead goose embryo in for 24 hours is discarded, is observed continuously 6
It.By dead goose embryo set 4 DEG C overnight processing after it is sterile collect allantoic fluid, and observe idiosome variation.Tissue treatment liquid blind passage three generations
Carry out subsequent detection.
1.5PCR identification：
1.5.1RNA extraction：
By the virus liquid harvested in 1.4 according to RNA extracts kit specification requirements, viral RNA is extracted, is placed in -20 DEG C
It saves backup.
1.5.2 reverse transcription obtains cDNA：
Reverse transcription reagent box used uses the article No. from precious biological (Dalian) Co., Ltd for RR036A's
PrimeScriptTMRT Master Mix sequentially add 5 × PrimeScript RT Master in 200 μ LPCR reaction tubes
The sample to be tested Total μ L of RNA~2 extracted in Mix × 2 μ L, step 1.5.1, use RNase Free dH2O is supplemented to 10 μ
L systems.It is placed in PCR instrument and is reacted, reaction condition is 37 DEG C of 15min；After 85 DEG C of 5s, 4 DEG C of preservations.
1.5.3PCR amplification：
It is expanded using 20 μ L systems：The μ L of template cDNA × 2, upstream and downstream primer is each × 1 μ L, 2 × Es
The μ L of TaqMasterMix × 10, using ddH2O is supplemented to 20 μ L systems.Mixing is simultaneously reacted in wink from PCR instrument is placed on, reaction
Condition is 95 DEG C, 5min, and 95 DEG C later, 45s, 68 DEG C, 30s, 72 DEG C, 45s carries out 30 and recycles, 72 DEG C, 4 DEG C after 10min
It saves backup.
2. result：
2.1 virus purification：
The positive hepatic of acquisition, the homogenate of kidney line and staff control were by processing, through chorioallantoic cavity approach aseptic inoculation 13 days
Age goose embryo, 0.2mL/ pieces；Sterile saline be used in combination that negative control, 37 DEG C of incubations are set.There is goose within 2-5 days in inoculation goose embryo
Embryo death (see Fig. 1) is normal with batch blank control goose embryo.
2.2PCR qualification result：
Product after carrying out PCR amplification with primer occurs big with desired value 601bp in 1% Ago-Gel after electrophoresis
The small specific band (Fig. 2) being consistent.Show that there are novel astrovirus (AstV) in the cell culture of separation strains.In addition right
The conventional goose source viral diagnosis that separation strains carry out, including：Avian influenza virus (AIV), newcastle disease virus (NDV), reovirus
(ARV), goose parvovirus (GPV), goose polyomavirus (GpoV) and goose circovirus (GoCV), and it is dirty that other viruses are not detected
It contaminates (Fig. 2).
Final separation obtains one plant of stable strain, which is named as N-AStV-SDPY.Using two generation sequencing technologies
Whole genome sequence measurement is carried out to strain N-AStV-SDPY, the whole genome sequence of the strain is as shown in SEQ ID NO.1.
Then sequence analysis and phylogenetic analysis are carried out with the astrovirus announced in the prior art.The result shows that strain N-
The homology of each genetic fragments of AStV-SDPY is respectively less than 70%；Phylogenetic analysis shows that strain N-AStV-SDPY is in one
A variation branch；It these results suggest that：Strain N-AStV-SDPY is different from other astrovirus, is fowl Astrovirus
One new kind.
The virus is subjected to preservation, deposit number is CCTCC NO：V201808.
Embodiment 2：The preparation of inactivated vaccine
(1) virus multiplication program：The novel goose astrovirus strain that embodiment 1 is detached is according to 0.2ml/ pieces of dose inoculation
9 age in days health SPF chicken embryos discard dead germ in for 24 hours, collect dead chick embryo allantoic liquid in 24~120h, obtain virus liquid, -20 DEG C
It preserves.
(2) virus liquid concentrates：By the virus liquid of harvest under the conditions of 2~8 DEG C, original volume is concentrated into ultrafiltration concentration machine
1/3, by existing《Chinese veterinary pharmacopoeia》Annex carries out steriling test, asepsis growth, and the malicious valence of standby survey that keeps sample.Virus liquid is per 0.1ml
Viral level answers >=105.0ELD50, the allantoic fluid after concentration inactivated immediately.
(3) it inactivates：Virus liquid after concentration is imported in inactivation tank, 10% formalin, which is added, keeps its final concentration of
0.1%.It is imported in another inactivation tank after adding formalin, fails to contact inactivator to avoid the virus that tank mouth nearby adheres to.37
DEG C sealing inactivation 24 hours (by temperature in tank reach 37 DEG C start in terms of), it is primary every stirring in 4~6 hours therebetween, after inactivation, set
It is saved backup in 2~8 DEG C.
(4) prepared by inactivated vaccine：
1) prepared by oil phase：94 parts of white oil for animals, 2 parts of aluminum stearate is taken to be placed in oil phase preparation tank after being heated to 80 DEG C, then
Add 6 parts of Span-80, until when temperature reaches 115 DEG C, maintains 30min, it is spare after cooling.
2) prepared by water phase：It will examine 96 parts of qualified virus liquid that 4 parts of sterilizing Tween-80 are added after inactivation, start refiner
20~30min is stirred, Tween-80 is made to be completely dissolved.
3) it emulsifies：Example is 1 to oil phase compared with water:1 (V/V), first imports colloid mill by oil phase, and 2500 revs/min of stirrings are delayed
It is slow that water phase is added, it is emulsified 5 minutes for 10000 revs/min after adding.Sulphur willow is added by vaccine total amount 0.01% before terminating emulsification
Mercury fully vibrates mixing.After emulsification, 10ml is taken, is centrifuged 15 minutes with 3000r/min, tube bottom is precipitated without water phase.
After aseptic subpackaged, roll lid and be stored in 2~8 DEG C.
Inactivated vaccine to preparation includes：Dosage form, centrifugal stability, viscosity and sterile quality inspection, specific side
Method refers to《Republic of China Veterinary Pharmacopoeia》(2015 editions).
As a result it is：The dosage form of inactivated vaccine prepared by the present invention is water-in-oil type (W/O)；Centrifugal stability, viscosity and nothing
Bacterium, which checks, to be met《Republic of China Veterinary Pharmacopoeia》The regulation of (2015 editions).
Embodiment 3：The preparation of novel goose astrovirus Yolk antibody
1. using inactivated vaccine immunization laying hen：
Novel goose astrovirus inactivated vaccine Immune Laying Hens prepared by embodiment 2, every chicken neck of first immunisation are subcutaneous
2.0ml inactivated vaccines are injected, second of immune, every chicken neck hypodermic injection 2.0ml inactivated vaccine, after two exempt from is carried out after 14 days
It is immune to carry out within 14 days third time, 2.0ml inactivated vaccines are subcutaneously injected in every chicken neck, and three exempt to carry out booster immunization in latter 14 days, often
Chicken neck is subcutaneously injected 2.0ml inactivated vaccines, and 14 days after booster immunization, acquisition yolk measures novel goose astrovirus and neutralizes
Antibody titer answers >=1:1024.
2. Yolk antibody manufactures：
(1) eggshell sterilizes：Collection height exempts from egg and is soaked in 15min in 1% bromogeramine solution.After taking-up naturally dry or
After drying, spray 75% alcohol eggshell surface is sterilized it is spare.
(2) yolk detaches：It takes machinery to beat eggs mode, egg white, blastodisc and frenulum should be fully removed when beating eggs, collect ovum
It is yellow.
(3) I is inactivated：The yolk of collection is sufficiently stirred, it is in uniform paste to make yolk, starts peristaltic pump, by yolk liquid pump
Enter in interlayer retort, (water for injection is first sterilized through 80 DEG C of 30min, and is cooled to the isometric water for injection of yolk for addition
65 DEG C or less), after stirring and evenly mixing, 60~65 DEG C of heat preservation (inactivation) 30min.
(4) acidification extracts：The acetic acid that the pH value 5.0 for being equivalent to 3 times of volumes of former yolk is first added in retort is isolated is slow
Then yolk liquid is added in fliud flushing, open blender, abundant agitating 30 minutes.
(5) II is inactivated：The octanoic acid that final concentration (V/V) is 4% is added in yolk liquid liquid to make inactivator and extract agent, acutely stirs
90min is mixed, 2~8 DEG C are placed 4~8 hours.
(6) coarse filtration：It is filtered until clear after polypropylene fibre 750B filter-cloth filterings, then with column core filter.
(7) aseptic filtration：With 0.22 μm of membrane filtration degerming.2~8 DEG C of storages are set, should be no more than 14.Sample inspection simultaneously
Survey the neutralize antibody titers of novel goose astrovirus.
(8) it concentrates：As the neutralize antibody titers of the novel goose astrovirus of detection are less than 1:512, it should will be after aseptic filtration
Yolk antibody under the conditions of 2~8 DEG C, the ultrafiltration concentration of suitable multiple is carried out with the concentration film packet of 30~50KD, after concentration
Novel goose astrovirus neutralize antibody titers should be not less than 1:512.
(9) III is inactivated：Solution after concentration is imported in inactivation tank, metered 10% formalin opens stirring
Machine stirs, it is made to be sufficiently mixed, and the ultimate density (V/V) of formalin is 0.1%, and 37 DEG C inactivate 16 hours.
(10) packing storage：Aseptically the filtrate inactivated is sub-packed in sterile glass vials, covers rubber plug, is rolled
Aluminium lid, it is labelled, it saves backup, storage temperature is -20 DEG C.
Embodiment 4：Yolk antibody quality inspection
1. safety examination：
With the susceptible young goose of 1-3 ages in days (the equal ＜ of novel goose astrovirus maternal antibody 1:4) 10, each muscle branch injection is originally
Yolk antibody 2.0ml prepared by inventive embodiments 3 is observed 14, and susceptible young bird goose all health survivals illustrate the yolk of the present invention
The safety of antibody is good.
2. steriling test：
According to《Republic of China Veterinary Pharmacopoeia》(2015 editions) progress, result are：Without bacterium, mycoplasma and exogenous virus
Pollution.
3. efficacy test (Immunization method)：
The susceptible young goose of 5 ages in days (the equal ＜ of novel goose astrovirus maternal antibody 1:4) 20, it is randomly divided into tetra- groups, every group of A, B
10.Wherein, A is immune group, and neck subcutaneously or intramuscularly injects the Yolk antibody of the preparation of embodiment 3, every 0.5ml；B is to attack poison
Control group, subcutaneously or intramuscularly injecting normal saline, every 0.5ml.Isolated rearing.
After 16 hours, intramuscular injection does 10 times of diluted novel goose astrovirus to two groups of young bird geese of A, B with physiological saline respectively
(deposit number is CCTCC NO：V201808), every 0.5ml.It is observed 10 after attacking poison, records every group of young bird goose morbidity, dead feelings
Condition.
As a result：A groups attack 10/10 protection after poison；B groups attack poison and start to fall ill after 12 hours, dead 9 in 10 days.
Embodiment 5：The application of Yolk antibody
1. the application in preventing novel goose astrovirus
With the susceptible young goose of 3-5 ages in days (the equal ＜ of novel goose astrovirus maternal antibody 1:4) 30 are subjects, random point
It is three groups, every group 10.Wherein：
Test group：Neck subcutaneously or intramuscularly injects the Yolk antibody of the preparation of embodiment 3, every 0.5ml；
Control group：Laying hen is immunized with presently commercially available fowl astrovirus inactivated vaccine, and by the side of embodiment 3
Yolk antibody is prepared in method, and neck subcutaneously or intramuscularly injects the Yolk antibody, every 0.5ml；
Blank control group：Subcutaneously or intramuscularly injecting normal saline, every 0.5ml.
By above-mentioned three groups of young birds goose isolated rearing, after 16 hours, respectively intramuscular injection with physiological saline do 10 times it is diluted new
Type goose astrovirus, every 0.5ml.It is observed 10 after attacking poison, records every group of morbidity of young bird goose, death condition.It the results are shown in Table 1.
Table 1：
| Group | Attack poison strain | Prevention ＆ protection |
| Test group | Novel goose astrovirus | 10/10 |
| Control group | Novel goose astrovirus | 5/10 |
| Blank control group | Novel goose astrovirus | 1/10 |
Note：Prevention ＆ protection is the young goose number of health survival after attacking poison/young bird goose sum.
As can be seen from Table 1, Yolk antibody of the invention has splendid prevention ＆ protection effect, starlike for the novel goose
Virus is capable of providing 100% prevention ＆ protection, and Vaccine effectiveness is better than the Yolk antibody prepared using existing inactivated vaccine.
2. the application in being treated to novel goose astrovirus
In novel goose astrovirus infection region of disease, symptom is ephritis, gout, is experiment with 30 similar in the course of disease young geese
Object, is randomly divided into 3 groups, every group 10, whole isolated rearings, wherein：
Test group：Neck subcutaneously or intramuscularly injects the Yolk antibody of the preparation of embodiment 3, every 1.0ml；
Control group：Laying hen is immunized with presently commercially available fowl astrovirus inactivated vaccine, and by the side of embodiment 3
Yolk antibody is prepared in method, and neck subcutaneously or intramuscularly injects the Yolk antibody, every 1.0ml；
Blank control group：Subcutaneously or intramuscularly injecting normal saline, every 1.0ml.
Observation 10 days, records every group of the young bird goose state of an illness, death condition.
As a result it is：Test group injected Yolk antibody after 2 days, and the feed intake of illness young bird goose starts to increase, and the state of mind is apparent
It improves, does not have young goose dead in 10 days；Wherein 1/2 young goose is slaughtered and dissected after 10 days, as a result, it has been found that illness young bird goose body cavity
And intra-articular uric acid mineralization disappears substantially.After control group injects Yolk antibody, the feed intake and the state of mind of illness young bird goose
There is not notable improvement, begins within 5th the goose death of illness young bird from injection Yolk antibody, the death rate of illness young bird goose is in 10 days
60%；It slaughters the 1/2 of the young goose of survival and dissects after 10 days, as a result, it has been found that illness young bird goose body cavity and intra-articular uric acid mineralization
Still exist on a small quantity.The death rate in the illness young bird goose 10 days of blank control group is up to 90%.
The above test results show that the Yolk antibody safety of the present invention is good, preventive effect is good, cure rate is high, Ke Yiyong
In the prevention of novel goose astrovirus infection, there is great economic and social benefit.
The foregoing is merely the preferred embodiments of the application, are not intended to limit this application, for the skill of this field
For art personnel, the application can have various modifications and variations.Within the spirit and principles of this application, any made by repair
Change, equivalent replacement, improvement etc., should be included within the protection domain of the application.
SEQUENCE LISTING
<110>Shandong Agricultural University
<120>A kind of Yolk antibody and preparation method thereof of the novel goose astrovirus of prevention
<130> 2018
<160> 1
<170> PatentIn version 3.5
<210> 1
<211> 7252
<212> DNA
<213>Goose astrovirus（Goose astrovirus）
<400> 1
tgtagcctct gtgttttcgc ttcctgcgca tcaaggcacg ccttctcacc caaattcata 60
aggtttgcac caatatcggg tgaccatgtt ggtttggtga catcatcggc gaaaaagaga 120
ccatacctcc cattgtcaac cccttttcct cacttgacac tcgtgaggaa aggaagactg 180
atgagattca gtcaggcctt gagaaggtct tccacttcaa tggtgttaat gaattataca 240
ccaagatgaa aggcttttat ggtcgcactc cagcctggag tgctctcatg aagtgtaatg 300
ccatctatct taaagattgg aaaacagcta taggggttga caatgggagg tatggtctct 360
ttttcgccga tgatgtcacc aaaccaacat ggtcacccga tattggtgca aaccttatga 420
atttgggtga gaaggcgtgc cttgatgcgc aggaagcgaa aacacagagg ctacaatgct 480
cgctgaaacg atgtggagct cttgtccaac aagttcttga gaagagcaaa caagcaaagg 540
aacaaatgca aaaagccgag gaattacaga agaagattga tcaaattaat gaaagcaaca 600
aggttctaca tcgcaaaatg caggagagaa atgttgagta ttctcagaag gtcataactc 660
gtattcatga gctgcaaaaa gacagagacc tgtggatgtc gcgtgctgct cattacgagc 720
aggagaacgt gcggctacgt attcaactgg ggacatatga tccgaacagg tctaagctga 780
tgactttcct agcatggatg tgtgttgcga tcattgcttt cagcttgatt caattggcgg 840
atgctagtga gattaaggat aacagaacag aaaattgttt cccaacatct ggctgtgttc 900
tctcagacgt tccttggact aagtcgtaca cctttatgga gctaatggac aagtgtgtga 960
ccggtactgc catcatacct aaaaacacat ttaatggtga agggcttgag ctggagtgta 1020
ttaagaattt tggcaagaaa tggtgcaaag gaagggttga gaagctcata ccgcattatt 1080
gtgatagaga taatactttt gatgccctct atcagtatta tgttgaaaca gtagcaattg 1140
ctgttgactt ctttaaagtt gcggtgcagt atcggattga caattggatt gtggctctct 1200
ttagtctagt attgactggg aagatggaga agtttcttaa gacgctaccc tttgtaattt 1260
tggcatggtg gtttaagttg cccatctttg ttatgagcat tgccacgaca atctttcctg 1320
tgcatgctct gccattcttg ttcttccagg tgtgctttcc aaactttgtc atcatcactg 1380
gttttctctt gtggatgacc tctatattag tggcttttta ttggtttgat gggactgcaa 1440
ttttggttga ggtttcttat gcggtattct atactattat cttctttgtg tggtctacag 1500
cattgacaat tatttcaact ttgtcattga cagtccctgt acagatttta ttattctgtg 1560
tttcattaac catttattgt ggcacaaagt ttgcgtgttc cactattaca gtcactaatc 1620
ctgacggtac tgtgtcgaaa tactcaagag ttgccaaaat gaaggatact gttgccatac 1680
agtgcaaaaa gatggtgagc tttgggcagg cccgtggtgt gataccagca actccagtga 1740
aggtgaattc cattgtcgtt gttgaaggaa aaaaaggttc aggggttggc ttccggttca 1800
tgaattatat aattacagca ggacacgtca cattaggctc tgactgggta acagtaaaac 1860
atgctggcac agtagccaaa acaaaagtcc tgaagcagat tgaggtgttt gagtgcgtcg 1920
atacaatagc ggttataaag ctgccccccg aattacagaa tttgaaaccc ataaaactcg 1980
caaaacgtgt tgagtccaca tacttaacat tgacagcttt tgatcctaat tttcaaaatg 2040
ttgtgtcata cactggctgg tgtattattg atggaaattg gatcagtaat gcatttaata 2100
ctcaatttgg aaacagtggt ggcccatacg ttgatgcaga gggtaaattg gttgggattc 2160
atcttggaac tcagggagtt atgtcccaag gtattgttgt ggtggatgcc cttaaaacgc 2220
atttcacgct cgcagaccag tgcaaggatt ttgattatga tggcttcctg gagaaagtta 2280
ttgcaggaac aaagacatca catgaagcaa ttctcaaagg tattgaggaa ttgcgagagg 2340
acgtcgacac gctcaagaac aagtgcaaga attatgatga gtactggctt gttcaaacta 2400
tttttggcca gaagaaaaag ggcaaaacaa agaaaacagc ccgggggaat aaacatcttg 2460
taacaaagag attcctgagt aaaggccact tcatgaaact caaaatgctc accgatgagg 2520
aatataatca tatgattgag caaggtttca ccgctgatga gatacgtgag gcagtcaatg 2580
agctgagaga acaagcatgg ctcaactact gcattgataa tgatattgat gaagaaggtg 2640
cggaagagtg gtatgaagat atgattgaag atgaccaaat taatgaaaag attgatgaag 2700
ccattgagaa agcaatggaa gaaaggggcg agttttatca agtcaaaaaa aggaaaacat 2760
ttgcacaaca agcattatta cacctcatcc gtgtcagtaa agccaggagc caaactgcaa 2820
agcaagaggt tcagaaagaa aacgcagatc agctttacga gatgttcaaa tctgccgtaa 2880
aggatgaaca agttgatgaa ggcacaacct cttacgcact cttcgccaat ggtgatcaga 2940
ttgagatggt cgaaggaaag gaacttgatt ttgaaaaggt aaagcttatc cacgttgatg 3000
gtgaacaaaa aaatgagacc attccgggga cgaaagtcac tgagatttct actggccctg 3060
agaataagaa gaacattttg cgcaagaagg acactcaaat taatccagaa caagctcaac 3120
ctcttgagca gcgcaagagg atctgcaagt ggtgtgggtc gagccaaaag catgactttc 3180
gtgattgcaa gttccagcgt gagaaaaggt tttgtgtctt ctgtgcaaag atgcactcat 3240
tgtttgatgg tcatcaacgg gacgtcttgt gcgacgtttg tgggaagaag ttcaagagtg 3300
tagaggagct tgaacagcac gtaactcgtg aaacgtgtga aaaaaactag ataaggggga 3360
cgatgccatc tgggtccccc gcataccacc tgatgattct cacatttttt cttggcagga 3420
tgatattatt gagtgtgata agaaaatcac tttgcccgac tatatagata taattggttt 3480
tatacctgta gataaattag ttgtcagagc aaagaccatt catgacccct tattaaactt 3540
ggttgagcat tggcaacaag atgaatatgc tcctactaaa tgggactata aagcctatac 3600
caagatgttt gaaaaatttt tctatcatga acctcaggat tttgtaaaca attttccaga 3660
gctggtcatc ttaagtgata gtgttgtgtt agaagaacat tcttatatgg ccaattccaa 3720
tttaacaccc ataatggcaa ctaagaagaa tgtcgatagt acccctggtt atccaaaatt 3780
taagtacttt gacacagaga aggactatct tgaacaatgt ggctggggtg agtacttgaa 3840
ggctgtatca gatattgatg agactgtgca aaataggccc ctctggtggt gttttctcaa 3900
aaatgaggtc ctcaaaaaga agaaaatcat ggataatgat attcgtatga tcttgtgtgc 3960
tgatccagta tatacaagga ttggagccat gtttgagcag gaccagaatg agaaaatgaa 4020
gcaacagaca gaacggcggg ctgcacaagt tggttggaca cccttttttg gtggaataca 4080
tcagcgagta tctaggctca ttggtggtgg tgacaggttt tttgtagaga cggactggac 4140
gcgttatgat ggtacgttgc ccaagcctct cttctggcgg atacgacaga tgcgttactt 4200
tttcctgtcc aaccatcata agacaccaca gcttaagaaa ctctatgatt ggtatgtcaa 4260
aaacttagtt gaaaaaatta tattattacc aactggggag gtttgtacag ttaagaaggg 4320
aaatccaagt ggccaatatt caacaacagt ggacaacaat atgtgtaatg tctggctcac 4380
ccattttgag atagcttatc tttattggaa acagcatggg tcattgccga cgctcagatt 4440
actcagggat aatgtcacca tgatttgcta tggcgatgat aggcttgtgt caataaggaa 4500
gggttttatt aattacgaca cgaatgtcgt catagatatg tataaaaata tttttggtat 4560
gtgggttaag ccagaaaatg tcaaggtctc tgatgatatt gagggtatga ctttctgtgg 4620
tcttacgttg gttaaaagga gtgatggtag atttgttgga gttcccaatg ttgataagat 4680
attgtcaaca cttgaaaatc ctgttagaaa attacccaac ttagaatcac tctggggtaa 4740
attggtttct ttaaggatct tgtgcgaaaa tgcacccaat agtgttagag agtttcttga 4800
taaacaaatc aacacggttg aggagcacgc tgccagtgaa aatattaact tacctgaggt 4860
cgggcccgac ttcttttctc aaatctggtg agtggcggac cgaaataaaa tggcagacag 4920
ggcggtggcc ccgcgcgaga aggtgaccaa gaaggttaca aaagtagtca ccgttaagaa 4980
aaaacaccca aaaaagaaac caaagcagaa agtatataag ccccaaaaat tacccatgaa 5040
ggccgagagg aagcttgaga aagaagtgaa aggtttgaag aaaagagtag ctggaccacc 5100
cgttaatgac aaaatgacta ccacgataac acttggtcag atcactggga attcaacaga 5160
cacactcgac cggaagcata aatacttcac aaatccactc atgatgaaaa accaggaaaa 5220
tgggcaaaca gcaactcctc taagtataag ggcctcacaa tataacttgt ggaggatcag 5280
aaagctgcat atccgccttg ttccacttgc tggtagggcc aatattcttg gctcggttgt 5340
cttcctagat atagaacagg aagctaacac agctgggcca gagtccatag ataccataaa 5400
agctcggccg catcttgaac tcccgattgg ctcaaaacat ctttggaggg ttcaacccag 5460
gctgatgcag ggaccccggc aaggatggtg gaatgtagac cccggggatt cacctactga 5520
ctcacttggt ccagcaatca atatgtggac atatttgaaa acagtaaatg cactttcaac 5580
acgggcgcag gcacaacaag ttccctatac ttctgccctc ttcctggttg aagcaacggt 5640
cacttatgag ttctcgaact atggtccaaa gccagggctc tcactcatga ccagtgaaac 5700
actttcagca tcagggaaaa cggcaaccct tgtaaatacc caggatggtg cacttgcttt 5760
aacagttagt ggcgcattgc agagattcct cgatgagaag gagcaacaca ggcgcgtttc 5820
aaatgcgcag acctctggtg tcggtgaggt gttttgggct gtttccacag aggtggtcga 5880
gaccgtagcg agtgcacttg gaggctgggg ttggctcttg aaaggaggat ggttcgtgat 5940
cagaaaattg tttggtgctg cttcaaattc tggttccact tatctgatct attcctcagt 6000
ttcagacgcc cagatagaca gcaggattta tcagacagtt ccaccgaaca cgccactaca 6060
gctggctgca aatacagtta agcttgtaca gctaacacag ccaaatgtga atacaactgg 6120
acaaggtacc actgtccttt cacgggatgc cgattacctg cctctgccag tggcaccaat 6180
gcaggttact ccctcgcttg tgtacaactt ccagggtgaa aggcagagca ctacagaatc 6240
gtgctcattc ctggtgtttg gaataccaca ggcagaatcc aggtcaagat acaatgcaaa 6300
tataaccttc aatgttggct atcgtggaag gacttcaaca tcatttacac ttggaacaca 6360
caattggtgg gctgttatga cactttcaca aacaggagta atttttgcac caccggctgt 6420
gggcacaggg gtctgtaata cattggctac agccatacaa cacttaaacc ctgagcttga 6480
aacagcagtc ctgcgtgtga ataccagtac aacatctact ggtgggctaa taacggaact 6540
caggaatcgg ctcaacatcg ctgatgggga ctatgtgatt tcaatgggtg atccgcaagg 6600
aaataggtca gcactgtact ttaggaattc agaccagaaa tgggtgtggc tctgggctgg 6660
cgactctaac cctggtgaaa ctttccaaag ttttaagatg ccagtcctaa ttaattggtc 6720
agtgtcagat tcccaggaac aatataatgc tagagtcagg atggtacagt atgctaatgc 6780
acaacagcag actttgacag accctgagga agatgatgat cccctctctg atgtcacttc 6840
gctttttgat ccaacagcgg aagatgagac tgacttccac ctagcagtct cactcaagac 6900
ctctgactac ttaaaagaag aggctgagta ctggaaagcg aaggcgcagg ccttgcttat 6960
ggagaaggca ctaagtgcac cacaagcagg gacagtccgc tttgagaagg gcggacatga 7020
gtgatctctt cactagcagc cgcggccacg ccgagtagga tcgagggtac agctgcacct 7080
tctcattagg cttgagggag aatcggccct ctctgcctta aattaacccg gtgacagtca 7140
ggtggtcacc cacacaaatc gcgtgcgggt acccctgacc gggtttttgt ttaaagaatc 7200
aaatgtgatt tttaagcatt taaaatcttt aaaaaaaaaa aaaaaaaaaa aa 7252
Claims (10)
1. a kind of Yolk antibody of the novel goose astrovirus of prevention, which is characterized in that the Yolk antibody contains the starlike disease of anti-goose
Strain antibody；The deposit number of the goose astrovirus strain is CCTCC NO：V201808.
2. the preparation method of Yolk antibody described in claim 1, which is characterized in that include the following steps：
(1) it is CCTCC NO with deposit number：Inactivated vaccine is made as production of vaccine strain in the goose astrovirus of V201808；
(2) with the inactivated vaccine injecting immune laying hen prepared, high-immunity egg is prepared；
(3) yolk is collected by high-immunity egg, through once inactivating, acidizing extraction, secondary inactivation, coarse filtration, aseptic filtration, concentration and three times
The Yolk antibody for preventing novel goose astrovirus is prepared into after inactivation.
3. preparation method according to claim 2, which is characterized in that in step (1), the inactivated vaccine is by the following method
It is prepared：
It is CCTCC NO by deposit number：The goose astrovirus of V201808 is inoculated with SPF chicken embryos, collects dead chicken embryo in 24-120h
Allantoic fluid obtains virus liquid；Virus liquid is concentrated into viral level >=105.0ELD50, virus liquid after concentration through formalin-inactivated,
Tween-80 mixing is added and is used as water phase, is mixed using white oil, aluminum stearate and Span-80 as oil phase, water phase and oil phase is pressed
Volume ratio 1:Inactivated vaccine is made in 1 mixing, emulsification.
4. preparation method according to claim 2, which is characterized in that in step (2), divided 4 times with the inactivated vaccine of preparation
Laying hen is immunized, each immunization interval 2 weeks.
5. preparation method according to claim 2, which is characterized in that in step (3), the primary inactivation is specially：It will
Yolk liquid and water by volume 1:(0.5~1.5) it mixes, yolk diluent is obtained after stirring evenly, under the conditions of 60~65 DEG C
Keep the temperature 25~35min.
6. preparation method according to claim 2, which is characterized in that in step (3), the acidizing extraction is specially：To
The acetate buffer of 2.5~3.5 times of the yolk diluent volume is added in yolk diluent, filter is collected by filtration after stirring
Liquid；Preferably, the pH value of the acetate buffer is 4.8-5.2；More preferably 5.0.
7. preparation method according to claim 2, which is characterized in that in step (3), the secondary inactivation is specially：To
Octanoic acid is added in filtrate after acidizing extraction to final concentration 3.5~4.5%, stirs 30-120min, in 2~8 DEG C of placements after stirring
4~8 hours；
Preferably, in step (3), the aseptic filtration is specially：With 0.22 μm of membrane filtration degerming.
8. preparation method according to claim 3, which is characterized in that in step (3), the concentration is specially：Utilize 30-
The PES hollow fiber ultrafiltration membranes of 50KD are concentrated under the conditions of 2-8 DEG C, until antibody titer is not less than 1:512.
9. Yolk antibody described in claim 1 is preparing the product for preventing and/or treating novel goose astrovirus infection
In application；The deposit number of the novel goose astrovirus is CCTCC NO：V201808.
10. deposit number is CCTCC NO：The goose astrovirus of V201808 is preparing treatment gout caused by goose astrovirus
Yolk antibody in application.
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| CN113214388A (en) * | 2021-05-12 | 2021-08-06 | 北京世华康源生物科技有限公司 | Goose astrovirus egg yolk antibody and preparation method thereof |
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| CN112341539A (en) * | 2020-10-22 | 2021-02-09 | 山东农业大学 | Yolk antibody for preventing and treating novel goose astrovirus with cross-species transmission capability and preparation method thereof |
| CN113214388A (en) * | 2021-05-12 | 2021-08-06 | 北京世华康源生物科技有限公司 | Goose astrovirus egg yolk antibody and preparation method thereof |
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